U.S. patent application number 15/423503 was filed with the patent office on 2017-05-25 for protein formulations and methods of making same.
The applicant listed for this patent is AbbVie Biotechnology Ltd. Invention is credited to Annika Bartl, Wolfgang Fraunhofer, Katharina Kaleta, Hans-Juergen Krause, Markus Tschoepe.
Application Number | 20170143828 15/423503 |
Document ID | / |
Family ID | 51391992 |
Filed Date | 2017-05-25 |
United States Patent
Application |
20170143828 |
Kind Code |
A1 |
Fraunhofer; Wolfgang ; et
al. |
May 25, 2017 |
PROTEIN FORMULATIONS AND METHODS OF MAKING SAME
Abstract
The invention provides an aqueous formulation comprising water
and a protein, and methods of making the same. The aqueous
formulation of the invention may be a high protein formulation
and/or may have low levels of conductivity resulting from the low
levels of ionic excipients. Also included in the invention are
formulations comprising water and proteins having low
osmolality.
Inventors: |
Fraunhofer; Wolfgang;
(Gurnee, IL) ; Bartl; Annika; (Ludwigshafen,
DE) ; Krause; Hans-Juergen; (Biblis, DE) ;
Tschoepe; Markus; (Hessheim, DE) ; Kaleta;
Katharina; (Ludwigshafen, DE) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
AbbVie Biotechnology Ltd |
Hamilton |
|
BM |
|
|
Family ID: |
51391992 |
Appl. No.: |
15/423503 |
Filed: |
February 2, 2017 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
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15096043 |
Apr 11, 2016 |
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15423503 |
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14796389 |
Jul 10, 2015 |
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15096043 |
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14506576 |
Oct 3, 2014 |
9085619 |
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14796389 |
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13774735 |
Feb 22, 2013 |
8883146 |
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14506576 |
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12325049 |
Nov 28, 2008 |
8420081 |
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13774735 |
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61004992 |
Nov 30, 2007 |
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2317/21 20130101;
A61K 39/3955 20130101; C07K 1/34 20130101; A61K 38/4893 20130101;
C07K 2317/94 20130101; A61K 38/21 20130101; A61K 39/39591 20130101;
A61K 2039/505 20130101; C07K 16/244 20130101; A61K 39/395 20130101;
A61K 9/0019 20130101; A61K 38/46 20130101; A61K 9/19 20130101; C07K
16/241 20130101; C07K 2317/76 20130101; A61K 38/385 20130101; A61K
9/08 20130101; A61K 38/50 20130101 |
International
Class: |
A61K 39/395 20060101
A61K039/395; C07K 1/34 20060101 C07K001/34; C07K 16/24 20060101
C07K016/24 |
Claims
1. An aqueous formulation comprising an antibody, or
antigen-binding fragment thereof, and water, wherein the
formulation has a conductivity of less than about 2.5 mS/cm and the
antibody, or antigen-binding fragment thereof, has a molecular
weight (Mw) greater than about 47 kDa
2. An aqueous formulation comprising an antibody, or
antigen-binding fragment thereof, at a concentration of at least
about 50 mg/mL and water, wherein the formulation has an osmolality
of no more than about 30 mOsmol/kg.
3. An aqueous formulation comprising water and a given
concentration of an antibody, or antigen-binding fragment thereof,
wherein the antibody, or antigen-binding fragment thereof, has a
hydrodynamic diameter (Dh) which is at least about 50% less than
the Dh of the antibody, or antigen-binding fragment thereof, in a
buffered solution at the given concentration.
4. An aqueous formulation comprising an antibody, or an
antigen-binding fragment, at a concentration of at least about 10
mg/mL and water, wherein the antibody, or antigen-binding fragment,
has a hydrodynamic diameter (Dh) of less than about 5 .mu.m.
5. The formulation of any one of claims 1-4, wherein the antibody,
or antigen-binding fragment thereof, is an anti-TNF.alpha.
antibody.
6. The formulation of any one of claims 1-4, wherein the
concentration of antibody, or antigen-binding fragment thereof, is
50 to 200 mg/ml.
7. The formulation of claim 1, further comprising a non-ionizable
excipient.
8. The formulation of claim 7, wherein the non-ionizable excipient
is selected from the group consisting of sugar alcohols and polyols
such as mannitol or sorbitol, a non-ionic surfactant, sucrose,
trehalose, raffinose, and maltose.
9. A method of treating a subject having a disorder, comprising
administering to the subject the formulation of any one of claims
1-4
10. A device comprising the formulation of any one of claims
1-4.
11. An article of manufacture comprising the formulation of any one
of claims 1-4.
12. A method of preparing an aqueous formulation comprising an
antibody, or antigen-binding fragment thereof, and water, the
method comprising: a) providing the antibody, or antigen-binding
fragment thereof, in a first solution; and b) subjecting the first
solution to diafiltration using water as a diafiltration medium
until at least a five-fold volume exchange with the water has been
achieved to thereby prepare the aqueous formulation.
13. An aqueous formulation prepared according to the method of
claim 12.
Description
RELATED APPLICATIONS
[0001] This application is a continuation of U.S. patent
application Ser. No. 15/096,043, filed on Apr. 11, 2016, which is a
continuation of U.S. patent application Ser. No. 14/796,389, filed
on Jul. 10, 2015, abandoned, which is a divisional of U.S. patent
application Ser. No. 14/506,576, filed on Oct. 3, 2014, now U.S.
Pat. No. 9,085,619, issued on Jul. 21, 2015, which is a
continuation of U.S. patent application Ser. No. 13/774,735, now
U.S. Pat. No. 8,883,146, issued on Nov. 11, 2014, which is a
continuation of U.S. patent application Ser. No. 12/325,049, now
U.S. Pat. No. 8,420,081, issued on Apr. 16, 2013, which claims the
benefit of priority to U.S. Provisional Application No. 61/004,992,
filed on Nov. 30, 2007. The entire contents of each of the
foregoing applications are hereby incorporated by reference
herein.
SEQUENCE LISTING
[0002] The instant application contains a Sequence Listing which
has been submitted in ASCII format via EFS-Web and is hereby
incorporated by reference in its entirety. Said ASCII copy, created
on Oct. 26, 2016, is named Seq_Listing_117813_26907 and is 4,507
bytes in size.
BACKGROUND OF THE INVENTION
[0003] A basic principle of pharmaceutical protein formulations is
that certain instabilities must be overcome. Degradation pathways
of proteins can be separated into two distinct classes, involving
chemical instability and physical instability. Chemical
instabilities lead to the modification of the protein through bond
formation or cleavage.
[0004] Examples of chemical instability problems include
deamidation, racemization, hydrolysis, oxidation, beta elimination
and disulfide exchange. Physical instabilities, on the other hand,
do not lead to covalent changes in proteins. Rather, they involve
changes in the higher order structure (secondary and above) of
proteins. These include denaturation, adsorption to surfaces,
aggregation and precipitation (Manning et al., Pharm. Res. 6, 903
(1989)).
[0005] It is generally accepted that these instabilities, which can
have great effect on the commercial viability and efficacy of
pharmaceutical protein formulations, can be overcome by including
additional molecules in the formulation. Protein stability can be
improved by including excipients that interact with the protein in
solution to keep the protein stable, soluble and unaggregated. For
example, salt compounds and other ionic species are very common
additives to protein formulations. They assist in fighting
denaturation of proteins by binding to proteins in a non-specific
fashion and increasing thermal stability. Salt compounds (e.g.,
NaCl, KCl) have been used successfully in commercial insulin
preparations to fight aggregation and precipitation (ibid. at 911).
Amino acids (e.g., histidine, arginine) have been shown to reduce
alterations in proteins' secondary structures when used as
formulation additives (Tian et al., Int'l J. Pharm. 355, 20
(2007)). Other examples of commonly used additives include
polyalcohol materials such as glycerol and sugars, and surfactants
such as detergents, both nonionic (e.g., Tween, Pluronic) and
anionic (sodium dodecyl sulfate). The near universal prevalence of
additives in all liquid commercial protein formulations indicates
that protein solutions without such compounds may encounter
challenges with degradation due to instabilities.
[0006] The primary goal of protein formulation is to maintain the
stability of a given protein in its native, pharmaceutically active
form over prolonged periods of time to guarantee acceptable
shelf-life of the pharmaceutical protein drug. Maintaining the
stability and solubility of proteins in solution, however, is
especially challenging in pharmaceutical formulations where the
additives are included into therapeutics. To date, biologic
formulations require additional excipients to maintain protein
stability. Typically, liquid pharmaceutical formulations contain
multiple additives for stability. For example, a liquid formulation
for patient self-administration of human growth hormone,
Norditropin SimpleXx.RTM., contains the additives mannitol (a sugar
alcohol), histidine and poloxamer 188 (a surfactant) to stabilize
the hormone.
[0007] Pharmaceutical additives need to be soluble, non-toxic and
used at particular concentrations that provide stabilizing effects
on the specific therapeutic protein. Since the stabilizing effects
of additives are protein- and concentration-dependent, each
additive being considered for use in a pharmaceutical formulation
must be carefully tested to ensure that it does not cause
instability or have other negative effects on the chemical or
physical make-up of the formulation. Ingredients used to stabilize
the protein may cause problems with protein stability over time or
with protein stability in changing environments during storage.
[0008] Typically, long shelf-life is achieved by storing the
protein in frozen from (e.g., at -80.degree. C.) or by subjecting
the protein to a lyophilization process, i.e., by storing the
protein in lyophilized form, necessitating a reconstitution step
immediately before use and thus posing a significant disadvantage
with regard to patient convenience. However, freezing a protein
formulation for storage may lead to localized high concentrations
of proteins and additives, which can create local extremes in pH,
degradation and protein aggregation within the formulation. In
addition, it is well known to those skilled in the art that
freezing and thawing processes often impact protein stability,
meaning that even storage of the pharmaceutical protein in frozen
form can be associated with the loss of stability due to the
freezing and thawing step. Also, the first process step of
lyophilization involves freezing, which can negatively impact
protein stability. In industry settings, a pharmaceutical protein
may be subjected to repeated freeze-thaw processing during Drug
Substance manufacturing (holding steps, storage, re-freeze and
re-thaw to increase timing and batch size flexibility in Drug
Product fill-finishing) and during subsequent Drug Product
fill-finishing (lyophilization). Since it is well known that the
risk of encountering protein instability phenomena increases with
increasing the number of freeze-thaw cycles a pharmaceutical
protein encounters, achieving formulation conditions that maintain
protein stability over repeated freeze-thaw processes is a
challenging task. There is a need in the biopharmaceutical industry
for formulations that can be frozen and thawed without creating
undesired properties in the formulations, especially gradients of
pH, osmolarity, density or protein or excipient concentration.
[0009] Often protein-based pharmaceutical products need to be
formulated at high concentrations for therapeutic efficacy. Highly
concentrated protein formulations are desirable for therapeutic
uses since they allow for dosages with smaller volumes, limiting
patient discomfort, and are more economically packaged and stored.
The development of high protein concentration formulations,
however, presents many challenges, including manufacturing,
stability, analytical, and, especially for therapeutic proteins,
delivery challenges. For example, difficulties with the
aggregation, insolubility and degradation of proteins generally
increase as protein concentrations in formulations are raised (for
review, see Shire, S. J. et al. J. Miami. Sci., 93, 1390 (2004)).
Previously unseen negative effects may be caused by additives that,
at lower concentrations of the additives or the protein, provided
beneficial effects. The production of high concentration protein
formulations may lead to significant problems with opalescence,
aggregation and precipitation. In addition to the potential for
nonnative protein aggregation and particulate formation, reversible
self-association may occur, which may result in increased viscosity
or other properties that complicate delivery by injection. High
viscosity also may complicate manufacturing of high protein
concentrations by filtration approaches.
[0010] Thus, pharmaceutical protein formulations typically
carefully balance ingredients and concentrations to enhance protein
stability and therapeutic requirements while limiting any negative
side-effects. Biologic formulations should include stable protein,
even at high concentrations, with specific amounts of excipients
reducing potential therapeutic complications, storage issues and
overall cost.
[0011] As proteins and other biomacromolecules gain increased
interest as drug molecules, formulations for delivering such
molecules are becoming an important issue. Despite the
revolutionary progress in the large-scale manufacturing of proteins
for therapeutic use, effective and convenient delivery of these
agents in the body remains a major challenge due to their intrinsic
physicochemical and biological properties, including poor
permeation through biological membranes, large molecular size,
short plasma half life, self association, physical and chemical
instability, aggregation, adsorption, and immunogenicity.
SUMMARY OF THE INVENTION
[0012] The invention is directed towards the surprising findings
that proteins formulated in water maintain solubility, as well as
stability, even at high concentrations, during long-term liquid
storage or other processing steps, such as freeze/thawing and
lyophilization.
[0013] The present invention relates to methods and compositions
for aqueous protein formulations which comprise water and a
protein, where the protein is stable without the need for
additional agents. Specifically, the methods and compositions of
the invention are based on a diafiltration process wherein a first
solution containing the protein of interest is diafiltered using
water as a diafiltration medium. The process is performed such that
there is at least a determined volume exchange, e.g., a five fold
volume exchange, with the water. By performing the methods of the
invention, the resulting aqueous formulation has a significant
decrease in the overall percentage of excipients in comparison to
the initial protein solution. For example, 95-99% less excipients
are found in the aqueous formulation in comparison to the initial
protein solution. Despite the decrease in excipients, the protein
remains soluble and retains its biological activity, even at high
concentrations. In one aspect, the methods of the invention result
in compositions comprising an increase in concentration of the
protein while decreasing additional components, such as ionic
excipients. As such, the hydrodynamic diameter of the protein in
the aqueous formulation is smaller relative to the same protein in
a standard buffering solution, such as phosphate buffered saline
(PBS).
[0014] The formulation of the invention has many advantages over
standard buffered formulations. In one aspect, the aqueous
formulation comprises high protein concentrations, e.g., 50 to 200
mg/mL or more. Proteins of all sizes may be included in the
formulations of the invention, even at increased concentrations.
Despite the high concentration of protein, the formulation has
minimal aggregation and can be stored using various methods and
forms, e.g., freezing, without deleterious effects that might be
expected with high protein formulations. Formulations of the
invention do not require excipients, such as, for example,
surfactants and buffering systems, which are used in traditional
formulations to stabilize proteins in solution. As a result of the
low level of ionic excipients, the aqueous formulation of the
invention has low conductivity, e.g., less than 2 mS/cm. The
methods and compositions of the invention also provide aqueous
protein formulations having low osmolality, e.g., no greater than
30 mOsmol/kg. In addition, the formulations described herein are
preferred over standard formulations because they have decreased
immunogenicity due to the lack of additional agents needed for
protein stabilization.
[0015] The methods and compositions of the invention may be used to
provide an aqueous formulation comprising water and any type of
protein of interest. In one aspect, the methods and compositions of
the invention are used for large proteins, including proteins which
are larger than 47 kDa. Antibodies, and fragments thereof,
including those used for in vivo and in vitro purposes, are another
example of proteins which may be used in the methods and
compositions of the invention.
[0016] Furthermore, the multiple step purification and
concentration processes that are necessary to prepare proteins and
peptide formulations often introduce variability in compositions,
such that the precise composition of a formulation may vary from
lot to lot. Federal regulations require that drug compositions be
highly consistent in their formulations regardless of the location
of manufacture or lot number. Methods of the invention can be used
to create solutions of proteins formulated in water to which
buffers and excipients are added back in precise amounts, allowing
for the creation of protein formulations with precise
concentrations of buffers and/or excipients.
[0017] In one embodiment, the invention provides an aqueous
formulation comprising a protein and water, wherein the formulation
has certain characteristics, such as, but not limited to, low
conductivity, e.g., a conductivity of less than about 2.5 mS/cm, a
protein concentration of at least about 10 .mu.g/mL, an osmolality
of no more than about 30 mOsmol/kg, and/or the protein has a
molecular weight (M.sub.w) greater than about 47 kDa. In one
embodiment, the formulation of the invention has improved
stability, such as, but not limited to, stability in a liquid form
for an extended time (e.g., at least about 3 months or at least
about 12 months) or stability through at least one freeze/thaw
cycle (if not more freeze/thaw cycles). In one embodiment, the
formulation is stable for at least about 3 months in a form
selected from the group consisting of frozen, lyophilized, or
spray-dried.
[0018] In one embodiment, proteins included in the formulation of
the invention may have a minimal size, including, for example, a
M.sub.w greater than about 47 kDa, a M.sub.w greater than about 57
kDa, a M.sub.w greater than about 100 kDa, a M.sub.w greater than
about 150 kDa, a M.sub.w greater than about 200 kDa, or a M.sub.w
greater than about 250 kDa. In one embodiment, the formulation of
the invention has a low conductivity, including, for example, a
conductivity of less than about 2.5 mS/cm, a conductivity of less
than about 2 mS/cm, a conductivity of less than about 1.5 mS/cm, a
conductivity of less than about 1 mS/cm, or a conductivity of less
than about 0.5 mS/cm.
[0019] In one embodiment, proteins included in the formulation of
the invention have a given concentration, including, for example, a
concentration of at least about 1 mg/mL, at least about 10 mg/mL,
at least about 50 mg/mL, at least about 100 mg/mL, at least about
150 mg/mL, at least about 200 mg/mL, or greater than about 200
mg/mL.
[0020] In one embodiment, the formulation of the invention has an
osmolality of no more than about 15 mOsmol/kg.
[0021] In one embodiment, the invention provides an aqueous
formulation comprising water and a given concentration of a
protein, wherein the protein has a hydrodynamic diameter (D.sub.h)
which is at least about 50% less than the D.sub.h of the protein in
a buffered solution at the given concentration. In one embodiment,
the D.sub.h of the protein is at least about 50% less than the
D.sub.h of the protein in phosphate buffered saline (PBS) at the
given concentration; the D.sub.h of the protein is at least about
60% less than the D.sub.h of the protein in PBS at the given
concentration; the D.sub.h of the protein is at least about 70%
less than the D.sub.h of the protein in PBS at the given
concentration.
[0022] In one embodiment, the invention provides an aqueous
formulation comprising a protein, such as, but not limited to, an
antibody, or an antigen-binding fragment, wherein the protein has a
hydrodynamic diameter (D.sub.h) of less than about 5 .mu.m. In one
embodiment, the protein has a D.sub.h of less than about 3
.mu.m.
[0023] Any protein may be used in the methods and compositions of
the invention. In one embodiment, the formulation comprises a
therapeutic protein. In one embodiment, the formulation comprises
an antibody, or an antigen-binding fragment thereof. Types of
antibodies, or antigen binding fragments, that may be included in
the methods and compositions of the invention include, but are not
limited to, a chimeric antibody, a human antibody, a humanized
antibody, and a domain antibody (dAb). In one embodiment, the
antibody, or antigen-binding fragment thereof, is an
anti-TNF.alpha., such as but not limited to adalimumab or
golimumab, or an anti-IL-12 antibody, such as but not limited to
J695. In addition, the formulation of the invention may also
include at least two distinct types of proteins, e.g., adalimumab
and J695.
[0024] In yet another embodiment of the invention, the formulation
may further comprise a non-ionizable excipient. Examples of
non-ionizable excipients include, but are not limited to, a sugar
alcohol or polyol (e.g, mannitol or sorbitol), a non-ionic
surfactant (e.g., polysorbate 80, polysorbate 20, polysorbate 40,
polysorbate 60), and/or a sugar (e.g, sucrose). Other non-limiting
examples of non-ionizable excipients that may be further included
in the formulation of the invention include, but are not limited
to, non-trehalose, raffinose, and maltose.
[0025] In one embodiment, the formulation does not comprise an
agent selected from the group consisting of a tonicity modifier, a
stabilizing agent, a surfactant, an antioxidant, a cryoprotectant,
a bulking agent, a lyroprotectant, a basic component, and an acidic
component.
[0026] The formulation of the invention may be suitable for any
use, including both in vitro and in vivo uses. In one embodiment,
the formulation of the invention is suitable for administration to
a subject via a mode of administration, including, but not limited
to, subcutaneous, intravenous, inhalation, intradermal,
transdermal, intraperitoneal, and intramuscular administration. The
formulation of the invention may be used in the treatment of a
disorder in a subject.
[0027] Also included in the invention are devices that may be used
to deliver the formulation of the invention. Examples of such
devices include, but are not limited to, a syringe, a pen, an
implant, a needle-free injection device, an inhalation device, and
a patch.
[0028] In one embodiment, the formulation of the invention is a
pharmaceutical formulation.
[0029] The invention also provides a method of preparing an aqueous
formulation comprising a protein and water, the method comprising
providing the protein in a first solution, and subjecting the first
solution to diafiltration using water as a diafiltration medium
until at least a five fold volume exchange with the water has been
achieved to thereby prepare the aqueous formulation. In one
embodiment, the protein in the resulting formulation retains its
biological activity.
[0030] The invention further provides a method of preparing an
aqueous formulation of a protein, the method comprising providing
the protein in a first solution; subjecting the first solution to
diafiltration using water as a diafiltration medium until at least
a five-fold volume exchange with the water has been achieved to
thereby prepare a diafiltered protein solution; and concentrating
the diafiltered protein solution to thereby prepare the aqueous
formulation of the protein. In one embodiment, the protein in the
resulting formulation retains its biological activity.
[0031] In one embodiment, the concentration of the diafiltered
protein solution is achieved via centrifugation.
[0032] In one embodiment, the diafiltration medium consists of
water.
[0033] In one embodiment, the first solution is subjected to
diafiltration with water until a volume exchange greater than a
five-fold volume exchange is achieved. In one embodiment, the first
solution is subjected to diafiltration with water until at least
about a six-fold volume exchange is achieved. In one embodiment,
the first solution is subjected to diafiltration with water until
at least about a seven-fold volume exchange is achieved.
[0034] In one embodiment, the aqueous formulation has a final
concentration of excipients which is at least about 95% less than
the first solution.
[0035] In one embodiment, the aqueous formulation has a final
concentration of excipients which is at least about 99% less than
the first solution.
[0036] In one embodiment, the first protein solution is obtained
from a mammalian cell expression system and has been purified to
remove host cell proteins (HCPs).
[0037] In one embodiment, the method of the invention further
comprises adding an excipient to the aqueous formulation.
BRIEF DESCRIPTION OF THE DRAWINGS
[0038] FIG. 1 shows the SEC chromatogram of Adalimumab reference
standard AFP04C (bottom line), Adalimumab DS (drug substance before
(middle line) and after DF/UF processing (top line).
[0039] FIG. 2 shows the impact of sorbitol (a non-ionizable
excipient) and NaCl (ionizable excipient) concentrations on the
hydrodynamic diameter (Dh) of Adalimumab monomer upon addition of
the excipient compound to DF/UF-processed Adalimumab monomer.
[0040] FIG. 3 shows the IEC profile of J695 reference standard
(bottom graph) and J695 DS, pH adjusted to pH 4.4 (top graph).
[0041] FIG. 4 shows the IEC profile of J695 after DF/UF with
Milli-Q water, pH 4.7 (top graph), and J695 DS before DF/UF, pH
adjusted to pH 4.4 (bottom curve).
[0042] FIG. 5 graphically depicts the correlation of hydrodynamic
diameter (z-average) and concentration of Adalimumab (dissolved in
WFI). X: determined with an SOP using 1.1 mPas as assumed sample
viscosity. y: determined with an SOP using 1.9 mPas as assumed
sample viscosity.
[0043] FIG. 6 graphically depicts the correlation of hydrodynamic
diameter (peak monomer) and concentration of Adalimumab (dissolved
in WFI). X: determined with an SOP using 1.1 mPas as assumed sample
viscosity. y: determined with an SOP using 1.9 mPas as assumed
sample viscosity
[0044] FIG. 7 graphically depicts the correlation of hydrodynamic
diameter (z-average) and concentration of J695 (dissolved in WFI).
X: determined with an SOP using 1.1 mPas as assumed sample
viscosity: determined with an SOP using 1.9 mPas as assumed sample
viscosity
[0045] FIG. 8 graphically depicts the correlation of hydrodynamic
diameter (peak monomer) and concentration of J695 (dissolved in
WFI). X: determined with an SOP using 1.1 mPas as assumed sample
viscosity. y: determined with an SOP using 1.9 mPas as assumed
sample viscosity.
[0046] FIG. 9 shows the sum of lysine 0, 1 and 2 of Adalimumab [%]
in dependence on Adalimumab concentration in water for
injection.
[0047] FIG. 10 shows the sum of peak 1 to 7 of J695 [%] in
dependence on J695 concentration in water for injection.
[0048] FIG. 11 shows the sum of acidic peaks of J695 [%] in
dependence on J695 concentration in water for injection.
[0049] FIG. 12 shows the sum of basic peaks of J695 [%] in
dependence on J695 concentration in water for injection (WFI).
[0050] FIG. 13 shows the efficiency of the dialysis performed in
Example 12, in terms of the reduction of components responsible for
osmolality and conductivity of the formulation (BDS, 74 mg/ml, 10
ml sample volume, SpectraPor7 MWCO10k).
[0051] FIG. 14 shows the stability of pH levels in dialyzed
Adalimumab Bulk Solutions. pH levels before and after dialysis
against deionized water (1:1,000,000) are shown. (BDS, 74 mg/ml, 10
ml sample volume, SpectraPor7 MWCO10k)
[0052] FIG. 15 shows bottle mapping density data for 250 mg/ml and
200 mg/ml low-ionic Adalimumab solutions after freeze thaw.
[0053] FIG. 16 shows bottle mapping pH data for 250 mg/ml and 200
mg/ml low-ionic Adalimumab solutions after freeze thaw.
[0054] FIG. 17 shows bottle mapping concentration data for 250
mg/ml and 200 mg/ml low-ionic Adalimumab solutions after freeze
thaw.
[0055] FIG. 18 shows bottle mapping osmolality data for 250 mg/ml
and 200 mg/ml low-ionic Adalimumab solutions after freeze thaw.
[0056] FIG. 19 shows bottle mapping conductivity data for 250 mg/ml
and 200 mg/ml low-ionic Adalimumab solutions after freeze thaw.
[0057] FIG. 20 shows SEC analysis of low-ionic Adalimumab (referred
to as D2E7 in FIG. 20) solutions that were either stored at
2-8.degree. C. for 8.5 months after DF/UF (bottom curve) or stored
at -80.degree. C. for 4.5 months after DF/UF (top curve).
[0058] FIG. 21 shows the stability of the monoclonal antibody 1D4.7
formulated in various solutions and in water before freeze-thaw
procedures (T0) and after each of four freeze-thaws (T1, T2, T3 and
T4).
[0059] FIG. 22 shows the stability of the monoclonal antibody
13C5.5 formulated in water and with various buffers before
freeze-thaw procedures (T0) and after each of four freeze-thaws
(T1, T2, T3 and T4). Blank=WFI control sample.
[0060] FIG. 23 shows the stability of the monoclonal antibody
13C5.5 formulated in water and with various excipients added,
before freeze-thaw procedures (T0) and after each of four
freeze-thaws (T1, T2, T3 and T4). Blank=WFI control sample.
[0061] FIG. 24 shows the impact of the concentration of Adalimumab
(WFI formulation) and solution pH on solution viscosity.
[0062] FIG. 25 shows turbidity data for Adalimumab solutions (WFI
formulations) of various concentrations and pH values.
[0063] FIG. 26 shows hydrodynamic diameter (Dh) data for Adalimumab
solutions (WFI formulations) at various pH values and
concentrations.
[0064] FIG. 27 shows a size distribution by intensity graph (Dh
measurements) for Adalimumab in water solutions, pH 5, at various
concentrations.
[0065] FIG. 28 shows size distribution by intensity for 100 mg/mL
Adalimumab in water at various pH levels.
[0066] FIG. 29 also shows size distribution by intensity for 100
mg/mL Adalimumab in water at various pH levels.
[0067] FIG. 30 shows monomer content (SEC) for Adalimumab in
water.
[0068] FIG. 31 shows aggregate content (SEC) for Adalimumab in
water.
[0069] FIG. 32 shows the viscosity of two J695 solutions (WFI
formulations) as a function of solution temperature.
[0070] FIG. 33 graphically depicts 1D4.7 antibody stability as
measured by subvisible particle (>1 .mu.m) during repeated
freeze/thaw (f/t) cycles for a number of different
formulations.
[0071] FIG. 34 graphically depicts 13C5.5 antibody stability as
measured by subvisible particle (>10 .mu.m) during repeated
freeze/thaw (f/t) cycles for a number of different
formulations.
[0072] FIG. 35 graphically depicts 13C5.5 antibody stability as
measured by subvisible particle (>1 .mu.m) during repeated
freeze/thaw (f/t) cycles for a number of different
formulations.
[0073] FIG. 36 graphically depicts 7C6 antibody stability as
measured by subvisible particle (>1 .mu.n) during repeated
freeze/thaw (f/t) cycles for a number of different
formulations.
DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
[0074] In order that the present invention may be more readily
understood, certain terms are first defined.
[0075] As used herein, the term "acidic component" refers to an
agent, including a solution, having an acidic pH, i.e., less than
7.0. Examples of acidic components include phosphoric acid,
hydrochloric acid, acetic acid, citric acid, oxalic acid, succinic
acid, tartaric acid, lactic acid, malic acid, glycolic acid and
fumaric acid. In one embodiment, the aqueous formulation of the
invention does not include an acidic component.
[0076] As used herein, the term "antioxidant" is intended to mean
an agent which inhibits oxidation and thus is used to prevent the
deterioration of preparations by the oxidative process. Such
compounds include by way of example and without limitation,
acetone, sodium bisulfate, ascorbic acid, ascorbyl palmitate,
citric acid, butylated hydroxyanisole, butylated hydroxytoluene,
hydrophosphorous acid, monothioglycerol, propyl gallate,
methionine, sodium ascorbate, sodium citrate, sodium sulfide,
sodium sulfite, sodium bisulfite, sodium formaldehyde sulfoxylate,
thioglycolic acid, sodium metabisulfite, EDTA (edetate), pentetate
and others known to those of ordinary skill in the art.
[0077] The term "aqueous formulation" refers to a solution in which
the solvent is water.
[0078] As used herein, the term "basic component" refers to an
agent which is alkaline, i.e., pH greater than 7.0. Examples of
basic components include potassium hydroxide (KOH) and sodium
hydroxide (NaOH) As used herein, the term "bulking agent" is
intended to mean a compound used to add bulk to the reconstitutable
solid and/or assist in the control of the properties of the
formulation during preparation. Such compounds include, by way of
example and without limitation, dextran, trehalose, sucrose,
polyvinylpyrrolidone, lactose, inositol, sorbitol,
dimethylsulfoxide, glycerol, albumin, calcium lactobionate, and
others known to those of ordinary skill in the art.
[0079] The term "conductivity," as used herein, refers to the
ability of an aqueous solution to conduct an electric current
between two electrodes. Generally, electrical conductivity or
specific conductivity is a measure of a material's ability to
conduct an electric current. In solution, the current flows by ion
transport. Therefore, with an increasing amount of ions present in
the aqueous solution, the solution will have a higher conductivity.
The unit of measurement for conductivity is mmhos (mS/cm), and can
be measured using a conductivity meter sold, e.g., by Orion
Research, Inc. (Beverly, Mass.). The conductivity of a solution may
be altered by changing the concentration of ions therein. For
example, the concentration of ionic excipients in the solution may
be altered in order to achieve the desired conductivity.
[0080] The term "cryoprotectants" as used herein generally includes
agents, which provide stability to the protein from
freezing-induced stresses. Examples of cryoprotectants include
polyols such as, for example, mannitol, and include saccharides
such as, for example, sucrose, as well as including surfactants
such as, for example, polysorbate, poloxamer or polyethylene
glycol, and the like. Cryoprotectants also contribute to the
tonicity of the formulations.
[0081] As used herein, the terms "ultrafiltration" or "UF" refers
to any technique in which a solution or a suspension is subjected
to a semi-permeable membrane that retains macromolecules while
allowing solvent and small solute molecules to pass through.
Ultrafiltration may be used to increase the concentration of
macromolecules in a solution or suspension. In a preferred
embodiment, ultrafiltration is used to increase the concentration
of a protein in water.
[0082] As used herein, the term "diafiltration" or "DF" is used to
mean a specialized class of filtration in which the retentate is
diluted with solvent and re-filtered, to reduce the concentration
of soluble permeate components. Diafiltration may or may not lead
to an increase in the concentration of retained components,
including, for example, proteins. For example, in continuous
diafiltration, a solvent is continuously added to the retentate at
the same rate as the filtrate is generated. In this case, the
retentate volume and the concentration of retained components does
not change during the process. On the other hand, in discontinuous
or sequential dilution diafiltration, an ultrafiltration step is
followed by the addition of solvent to the retentate side; if the
volume of solvent added to the retentate side is not equal or
greater to the volume of filtrate generated, then the retained
components will have a high concentration. Diafiltration may be
used to alter the pH, ionic strength, salt composition, buffer
composition, or other properties of a solution or suspension of
macromolecules.
[0083] As used herein, the terms "diafiltration/ultrafiltration" or
"DF/UF" refer to any process, technique or combination of
techniques that accomplishes ultrafiltration and/or diafiltration,
either sequentially or simultaneously.
[0084] As used herein, the term "diafiltration step" refers to a
total volume exchange during the process of diafiltration.
[0085] The term "excipient" refers to an agent that may be added to
a formulation to provide a desired consistency, (e.g., altering the
bulk properties), to improve stability, and/or to adjust
osmolality. Examples of commonly used excipients include, but are
not limited to, sugars, polyols, amino acids, surfactants, and
polymers. The term "ionic excipient" or "ionizable excipient," as
used interchangeably herein, refers to an agent that has a net
charge. In one embodiment, the ionic excipient has a net charge
under certain formulation conditions, such as pH. Examples of an
ionic excipient include, but are not limited to, histidine,
arginine, and sodium chloride. The term "non-ionic excipient" or
"non-ionizable excipient," as used interchangeably herein, refers
to an agent having no net charge. In one embodiment, the non-ionic
excipient has no net charge under certain formulation conditions,
such as pH. Examples of non-ionic excipients include, but are not
limited to, sugars (e.g., sucrose), sugar alcohols (e.g.,
mannitol), and non-ionic surfactants (e.g., polysorbate 80).
[0086] The term "first protein solution" or "first solution" as
used herein, refers to the initial protein solution or starting
material used in the methods of the invention, i.e., the initial
protein solution which is diafiltered into water. In one
embodiment, the first protein solution comprises ionic excipients,
non-ionic excipients, and/or a buffering system.
[0087] The term "hydrodynamic diameter" or "D.sub.h" of a particle
refers to the diameter of a sphere that has the density of water
and the same velocity as the particle. Thus the term "hydrodynamic
diameter of a protein" as used herein refers to a size
determination for proteins in solution using dynamic light
scattering (DLS). A DLS-measuring instrument measures the
time-dependent fluctuation in the intensity of light scattered from
the proteins in solution at a fixed scattering angle. Protein Dh is
determined from the intensity autocorrelation function of the
time-dependent fluctuation in intensity. Scattering intensity data
are processed using DLS instrument software to determine the value
for the hydrodynamic diameter and the size distribution of the
scattering molecules, i.e. the protein specimen.
[0088] The term "lyoprotectant" as used herein includes agents that
provide stability to a protein during water removal during the
drying or lyophilisation process, for example, by maintaining the
proper conformation of the protein. Examples of lyoprotectants
include saccharides, in particular di- or trisaccharides.
Cryoprotectants may also provide lyoprotectant effects.
[0089] The term "pharmaceutical" as used herein with reference to a
composition, e.g., an aqueous formulation, that it is useful for
treating a disease or disorder.
[0090] The term "protein" is meant to include a sequence of amino
acids for which the chain length is sufficient to produce the
higher levels of secondary and/or tertiary and/or quaternary
structure. This is to distinguish from "peptides" or other small
molecular weight drugs that do not have such structure. In one
embodiment, the proteins used herein have a molecular weight of at
least about 47 kD. Examples of proteins encompassed within the
definition used herein include therapeutic proteins. A
"therapeutically active protein" or "therapeutic protein" refers to
a protein which may be used for therapeutic purposes, i.e., for the
treatment of a disorder in a subject. It should be noted that while
therapeutic proteins may be used for treatment purposes, the
invention is not limited to such use, as said proteins may also be
used for in vitro studies. In a preferred embodiment, the
therapeutic protein is a fusion protein or an antibody, or
antigen-binding portion thereof. In one embodiment, the methods and
compositions of the invention comprise at least two distinct
proteins, which are defined as two proteins having distinct amino
acid sequences. Additional distinct proteins do not include
degradation products of a protein.
[0091] The phrase "protein is dissolved in water" as used herein
refers to a formulation of a protein wherein the protein is
dissolved in an aqueous solution in which the amount of small
molecules (e.g., buffers, excipients, salts, surfactants) has been
reduced by DF/UF processing. Even though the total elimination of
small molecules cannot be achieved in an absolute sense by DF/UF
processing, the theoretical reduction of excipients achievable by
applying DF/UF is sufficiently large to create a formulation of the
protein essentially in water exclusively. For example, with 6
volume exchanges in a continuous mode DF/UF protocol, the
theoretical reduction of excipients is .about.99.8% (ci=e.sup.-x,
with ci being the initial excipient concentration, and x being the
number of volume exchanges).
[0092] The term "pharmaceutical formulation" refers to preparations
which are in such a form as to permit the biological activity of
the active ingredients to be effective, and, therefore. may be
administered to a subject for therapeutic use.
[0093] A "stable" formulation is one in which the protein therein
essentially retains its physical stability and/or chemical
stability and/or biological activity upon storage.
[0094] Various analytical techniques for measuring protein
stability are available in the art and are reviewed in Peptide and
Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker,
Inc., New York, N.Y., Pubs. (1991) and Jones, A. Adv. Drug Delivery
Rev. 10: 29-90 (1993), for example. In one embodiment, the
stability of the protein is determined according to the percentage
of monomer protein in the solution, with a low percentage of
degraded (e.g., fragmented) and/or aggregated protein. For example,
an aqueous formulation comprising a stable protein may include at
least 95% monomer protein. Alternatively, an aqueous formulation of
the invention may include no more than 5% aggregate and/or degraded
protein.
[0095] The term "stabilizing agent" refers to an excipient that
improves or otherwise enhances stability. Stabilizing agents
include, but are not limited to, .alpha.-lipoic acid,
.alpha.-tocopherol, ascorbyl palmitate, benzyl alcohol, biotin,
bisulfites, boron, butylated hydroxyanisole (BHA), butylated
hydroxytoluene (BHT), ascorbic acid and its esters, carotenoids,
calcium citrate, acetyl-L-camitine, chelating agents, chondroitin,
chromium, citric acid, coenzyme Q-10, cysteine, cysteine
hydrochloride, 3-dehydroshikimic acid (DHS), EDTA
(ethylenediaminetetraacetic acid; edetate disodium), ferrous
sulfate, folic acid, fumaric acid, alkyl gallates, garlic,
glucosamine, grape seed extract, gugul, magnesium, malic acid,
metabisulfite, N-acetyl cysteine, niacin, nicotinomide, nettle
root, ornithine, propyl gallate, pycnogenol, saw palmetto,
selenium, sodium bisulfite, sodium metabisulfite, sodium sulfite,
potassium sulfite, tartaric acid, thiosulfates, thioglycerol,
thiosorbitol, tocopherol and their esters, e.g., tocopheral
acetate, tocopherol succinate, tocotrienal, d-.alpha.-tocopherol
acetate, vitamin A and its esters, vitamin B and its esters,
vitamin C and its esters, vitamin D and its esters, vitamin E and
its esters, e.g., vitamin E acetate, zinc, and combinations
thereof.
[0096] The term "surfactants" generally includes those agents that
protect the protein from air/solution interface-induced stresses
and solution/surface induced-stresses. For example surfactants may
protect the protein from aggregation. Suitable surfactants may
include, e.g., polysorbates, polyoxyethylene alkyl ethers such as
Brij 35..RTM., or poloxamer such as Tween 20, Tween 80, or
poloxamer 188. Preferred detergents are poloxamers, e.g., Poloxamer
188, Poloxamer 407; polyoxyethylene alkyl ethers, e.g., Brij
35..RTM., Cremophor A25, Sympatens ALM/230; and
polysorbates/Tweens, e.g., Polysorbate 20, Polysorbate 80, and
Poloxamers, e.g., Poloxamer 188, and Tweens, e.g., Tween 20 and
Tween 80.
[0097] As used herein, the term "tonicity modifier" is intended to
mean a compound or compounds that can be used to adjust the
tonicity of a liquid formulation. Suitable tonicity modifiers
include glycerin, lactose, mannitol, dextrose, sodium chloride,
magnesium sulfate, magnesium chloride, sodium sulfate, sorbitol,
trehalose, sucrose, raffinose, maltose and others known to those or
ordinary skill in the art. In one embodiment, the tonicity of the
liquid formulation approximates that of the tonicity of blood or
plasma.
[0098] The term "water" is intended to mean water that has been
purified to remove contaminants, usually by distillation or reverse
osmosis, also referred to herein as "pure water". In a preferred
embodiment, water used in the methods and compositions of the
invention is excipient-free. In one embodiment, water includes
sterile water suitable for administration to a subject. In another
embodiment, water is meant to include water for injection (WFI). In
one embodiment, water refers to distilled water or water which is
appropriate for use in in vitro assays. In a preferred embodiment,
diafiltration is performed in accordance with the methods of the
invention using water alone as the diafiltration medium.
[0099] The term "antibody" as referred to herein includes whole
antibodies and any antigen binding fragment (i.e., "antigen-binding
portion") or single chains thereof. An "antibody" refers to a
glycoprotein comprising at least two heavy (H) chains and two light
(L) chains inter-connected by disulfide bonds, or an antigen
binding portion thereof. Each heavy chain is comprised of a heavy
chain variable region (abbreviated herein as V.sub.H) and a heavy
chain constant region. The heavy chain constant region is comprised
of three domains, CH1, CH2 and CH3. Each light chain is comprised
of a light chain variable region (abbreviated herein as V.sub.L)
and a light chain constant region. The light chain constant region
is comprised of one domain, CL. The V.sub.H and V.sub.L regions can
be further subdivided into regions of hypervariability, termed
complementarity determining regions (CDR), interspersed with
regions that are more conserved, termed framework regions (FR).
Each V.sub.H and V.sub.L is composed of three CDRs and four FRs,
arranged from amino-terminus to carboxy-terminus in the following
order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. The variable regions
of the heavy and light chains contain a binding domain that
interacts with an antigen. The constant regions of the antibodies
may mediate the binding of the immunoglobulin to host tissues or
factors, including various cells of the immune system (e.g.,
effector cells) and the first component (Clq) of the classical
complement system.
[0100] The term "antigen-binding portion" of an antibody (or simply
"antibody portion"), as used herein, refers to one or more
fragments of an antibody that retain the ability to specifically
bind to an antigen (e.g., TNF.alpha., IL-12). It has been shown
that the antigen-binding function of an antibody can be performed
by fragments of a full-length antibody. Examples of binding
fragments encompassed within the term "antigen-binding portion" of
an antibody include (i) a Fab fragment, a monovalent fragment
consisting of the V.sub.L, V.sub.H, CL and CH1 domains; (ii) a
F(ab').sub.2 fragment, a bivalent fragment comprising two Fab
fragments linked by a disulfide bridge at the hinge region; (iii) a
Fd fragment consisting of the V.sub.H and C.sub.H1 domains; (iv) a
Fv fragment consisting of the V.sub.L and V.sub.H domains of a
single arm of an antibody, (v) a dAb fragment (Ward et al, (1989)
Nature 341:544-546), which consists of a V.sub.H or V.sub.L domain;
and (vi) an isolated complementarity determining region (CDR).
Furthermore, although the two domains of the Fv fragment, V.sub.L
and V.sub.H, are coded for by separate genes, they can be joined,
using recombinant methods, by a synthetic linker that enables them
to be made as a single protein chain in which the VL and VH regions
pair to form monovalent molecules (known as single chain Fv (scFv);
see e.g., Bird et al. (1988) Science 242:423-426; and Huston et al.
(1988) Proc. Natl. Acad. Sci. USA 85:5879-5883). Such single chain
antibodies are also intended to be encompassed within the term
"antigen-binding portion" of an antibody. These antibody fragments
are obtained using conventional techniques known to those with
skill in the art, and the fragments are screened for utility in the
same manner as are intact antibodies. In one embodiment of the
invention, the antibody fragment is selected from the group
consisting of a Fab, an Fd, an Fd', a single chain Fv (scFv), an
scFv.sub.a, and a domain antibody (dAb).
[0101] Still further, an antibody or antigen-binding portion
thereof may be part of a larger immunoadhesion molecule, formed by
covalent or noncovalent association of the antibody or antibody
portion with one or more other proteins or peptides. These other
proteins or peptides can have functionalities that allow for the
purification of antibodies or antigen-binding portions thereof or
allow for their association with each other or other molecules.
Thus examples of such immunoadhesion molecules include use of the
streptavidin core region to make a tetrameric single chain variable
fragment (scFv) molecules (Kipriyanov et al. (1995) Human
Antibodies and Hybridomas 6:93-101) and the use of a cysteine
residue, a marker peptide and a C-terminal polyhistidine tag to
make bivalent and biotinylated scFv molecules (Kipriyanov et al.
(1994) Mol. Immunol. 31:1047-1058). Antibody portions, such as Fab
and F(ab').sub.2 fragments, can be prepared from whole antibodies
using conventional techniques, such as papain or pepsin digestion,
respectively, of whole antibodies. Moreover, antibodies, antibody
portions and immunoadhesion molecules can be obtained using
standard recombinant DNA techniques.
[0102] Two antibody domains are "complementary" where they belong
to families of structures which form cognate pairs or groups or are
derived from such families and retain this feature. For example, a
VH domain and a VL domain of an antibody are complementary; two VH
domains are not complementary, and two VL domains are not
complementary. Complementary domains may be found in other members
of the immunoglobulin superfamily, such as the V.alpha. and V.beta.
(or gamma and delta) domains of the T-cell receptor.
[0103] The term "domain" refers to a folded protein structure which
retains its tertiary structure independently of the rest of the
protein. Generally, domains are responsible for discrete functional
properties of proteins, and in many cases may be added, removed or
transferred to other proteins without loss of function of the
remainder of the protein and/or of the domain. By single antibody
variable domain is meant a folded polypeptide domain comprising
sequences characteristic of antibody variable domains. It therefore
includes complete antibody variable domains and modified variable
domains, for example in which one or more loops have been replaced
by sequences which are not characteristic of antibody variable
domains, or antibody variable domains which have been truncated or
comprise N- or C-terminal extensions, as well as folded fragments
of variable domains which retain at least in part the binding
activity and specificity of the full-length domain.
[0104] Variable domains of the invention may be combined to form a
group of domains; for example, complementary domains may be
combined, such as VL domains being combined with VH domains
Non-complementary domains may also be combined. Domains may be
combined in a number of ways, involving linkage of the domains by
covalent or non-covalent means.
[0105] A "dAb" or "domain antibody" refers to a single antibody
variable domain (V.sub.H or V.sub.L) polypeptide that specifically
binds antigen.
[0106] As used herein, the term "antigen binding region" or
"antigen binding site" refers to the portion(s) of an antibody
molecule, or antigen binding portion thereof, which contains the
amino acid residues that interact with an antigen and confers on
the antibody its specificity and affinity for the antigen.
[0107] The term "epitope" is meant to refer to that portion of any
molecule capable of being recognized by and bound by an antibody at
one or more of the antibody's antigen binding regions. In the
context of the present invention, first and second "epitopes" are
understood to be epitopes which are not the same and are not bound
by a single monospecific antibody, or antigen-binding portion
thereof.
[0108] The phrase "recombinant antibody" refers to antibodies that
are prepared, expressed, created or isolated by recombinant means,
such as antibodies expressed using a recombinant expression vector
transfected into a host cell, antibodies isolated from a
recombinant, combinatorial antibody library, antibodies isolated
from an animal (e.g., a mouse) that is transgenic for human
immunoglobulin genes (see e.g., Taylor et al. (1992) Nucl. Acids
Res. 20:6287-6295) or antibodies prepared, expressed, created or
isolated by any other means that involves splicing of particular
immunoglobulin gene sequences (such as human immunoglobulin gene
sequences) to other DNA sequences. Examples of recombinant
antibodies include chimeric, CDR-grafted and humanized
antibodies.
[0109] The term "human antibody" refers to antibodies having
variable and constant regions corresponding to, or derived from,
human germline immunoglobulin sequences as described by, for
example, Kabat et al. (See Kabat, et al. (1991) Sequences of
Proteins of Immunological Interest, Fifth Edition, U.S. Department
of Health and Human Services, NIH Publication No. 91-3242). The
human antibodies of the invention, however, may include amino acid
residues not encoded by human germline immunoglobulin sequences
(e.g., mutations introduced by random or site-specific mutagenesis
in vitro or by somatic mutation in vivo), for example in the CDRs
and in particular CDR3.
[0110] Recombinant human antibodies of the invention have variable
regions, and may also include constant regions, derived from human
germline immunoglobulin sequences (See Kabat et al. (1991)
Sequences of Proteins of Immunological Interest, Fifth Edition,
U.S. Department of Health and Human Services, NIH Publication No.
91-3242). In certain embodiments, however, such recombinant human
antibodies are subjected to in vitro mutagenesis (or, when an
animal transgenic for human Ig sequences is used, in vivo somatic
mutagenesis) and thus the amino acid sequences of the VH and VL
regions of the recombinant antibodies are sequences that, while
derived from and related to human germline VH and VL sequences, may
not naturally exist within the human antibody germline repertoire
in vivo. In certain embodiments, however, such recombinant
antibodies are the result of selective mutagenesis or backmutation
or both.
[0111] The term "backmutation" refers to a process in which some or
all of the somatically mutated amino acids of a human antibody are
replaced with the corresponding germline residues from a homologous
germline antibody sequence. The heavy and light chain sequences of
a human antibody of the invention are aligned separately with the
germline sequences in the VBASE database to identify the sequences
with the highest homology. Differences in the human antibody of the
invention are returned to the germline sequence by mutating defined
nucleotide positions encoding such different amino acid. The role
of each amino acid thus identified as candidate for backmutation
should be investigated for a direct or indirect role in antigen
binding and any amino acid found after mutation to affect any
desirable characteristic of the human antibody should not be
included in the final human antibody. To minimize the number of
amino acids subject to backmutation those amino acid positions
found to be different from the closest germline sequence but
identical to the corresponding amino acid in a second germline
sequence can remain, provided that the second germline sequence is
identical and colinear to the sequence of the human antibody of the
invention for at least 10, preferably 12 amino acids, on both sides
of the amino acid in question. Backmuation may occur at any stage
of antibody optimization.
[0112] The term "chimeric antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species and constant region sequences from another species, such as
antibodies having murine heavy and light chain variable regions
linked to human constant regions.
[0113] The term "CDR-grafted antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from one
species but in which the sequences of one or more of the CDR
regions of VH and/or VL are replaced with CDR sequences of another
species, such as antibodies having murine heavy and light chain
variable regions in which one or more of the murine CDRs (e.g.,
CDR3) has been replaced with human CDR sequences.
[0114] The term "humanized antibody" refers to antibodies which
comprise heavy and light chain variable region sequences from a
non-human species (e.g., a mouse) but in which at least a portion
of the VH and/or VL sequence has been altered to be more
"human-like", i.e., more similar to human germline variable
sequences. One type of humanized antibody is a CDR-grafted
antibody, in which human CDR sequences are introduced into
non-human VH and VL sequences to replace the corresponding nonhuman
CDR sequences.
[0115] Various aspects of the invention are described in further
detail in the following subsections.
II. Methods of Invention
[0116] Generally, diafiltration is a technique that uses membranes
to remove, replace, or lower the concentration of salts or solvents
from solutions containing proteins, peptides, nucleic acids, and
other biomolecules. Protein production operations often involve
final diafiltration of a protein solution into a formulation buffer
once the protein has been purified from impurities resulting from
its expression, e.g., host cell proteins. The invention described
herein provides a means for obtaining an aqueous formulation by
subjecting a protein solution to diafiltration using water alone as
a diafiltration solution. Thus, the formulation of the invention is
based on using water as a formulation medium during the
diafiltration process and does not rely on traditional formulation
mediums which include excipients, such as surfactants, used to
solubilize and/or stabilize the protein in the final formulation.
The invention provides a method for transferring a protein into
pure water for use in a stable formulation, wherein the protein
remains in solution and is able to be concentrated at high levels
without the use of other agents to maintain its stability.
[0117] Prior to diafiltration or DF/UF in accordance with the
teachings herein, the method includes first providing a protein in
a first solution. The protein may be formulated in any first
solution, including formulations using techniques that are well
established in the art, such as synthetic techniques (e.g.,
recombinant techniques, peptide synthesis, or a combination
thereof). Alternatively, the protein used in the methods and
compositions of the invention is isolated from an endogenous source
of the protein. The initial protein solution may be obtained using
a purification process whereby the protein is purified from a
heterogeneous mix of proteins. In one embodiment, the initial
protein solution used in the invention is obtained from a
purification method whereby proteins, including antibodies,
expressed in a mammalian expression system are subjected to
numerous chromatography steps which remove host cell proteins
(HCPs) from the protein solution. In one embodiment, the first
protein solution is obtained from a mammalian cell expression
system and has been purified to remove host cell proteins (HCPs).
Examples of methods of purification are described in US application
Ser. No. 11/732,918 (US 20070292442), incorporated by reference
herein. It should be noted that there is no special preparation of
the first protein solution required in accordance with the methods
of the invention.
[0118] Proteins which may be used in the compositions and methods
of the invention may be any size, i.e., molecular weight (M.sub.w).
For example, the protein may have a M.sub.w equal to or greater
than about 1 kDa, a M.sub.w equal to or greater than about 10 kDa,
a M.sub.w equal to or greater than about 47 kDa, a M.sub.w equal to
or greater than about 57 kDa, a M.sub.w equal to or greater than
about 100 kDa, a M.sub.w equal to or greater than about 150 kDa, a
M.sub.w equal to or greater than about 200 kDa, or a M.sub.w equal
to or greater than about 250 kDa. Numbers intermediate to the above
recited M.sub.w, e.g., 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57,
58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74,
75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91,
92, 93, 94, 95, 96, 97, 98, 99, 100, 101, 102, 103, 104, 105, 106,
107, 108, 109, 110, 111, 112, 113, 114, 115, 116, 117, 118, 119,
120, 121, 122, 123, 124, 125, 126, 127, 128, 129, 130, 131, 132,
133, 134, 135, 136, 137, 138, 139, 140, 141, 142, 143, 144, 145,
146, 147, 148, 149, 150, 151, 153, 153, 154, 155, 156, 157, 158,
159, 160, 161, 162, 163, 164, 165, 166, 167, 168, 169, 170, and so
forth, as well as all other numbers recited herein, are also
intended to be part of this invention. Ranges of values using a
combination of any of the above recited values as upper and/or
lower limits are intended to be included in the scope of the
invention. For example, proteins used in the invention may range in
size from 57 kDa to 250 kDa, from 56 kDa to 242 kDa, from 60 kDa to
270 kDa, and so forth.
[0119] The methods of the invention also include diafiltration of a
first protein solution that comprises at least two distinct
proteins. For example, the protein solution may contain two or more
types of antibodies directed to different molecules or different
epitopes of the same molecule.
[0120] In one embodiment, the protein that is in solution is a
therapeutic protein, including, but not limited to, fusion proteins
and enzymes. Examples of therapeutic proteins include, but are not
limited to, Pulmozyme (Dornase alfa), Regranex (Becaplermin),
Activase (Alteplase), Aldurazyme (Laronidase), Amevive (Alefacept),
Aranesp (Darbepoetin alfa), Becaplermin Concentrate, Betaseron
(Interferon beta-1b), BOTOX (Botulinum Toxin Type A), Elitek
(Rasburicase), Elspar (Asparaginase), Epogen (Epoetin alfa), Enbrel
(Etanercept), Fabrazyme (Agalsidase beta), Infergen (Interferon
alfacon-1), Intron A (Interferon alfa-2a), Kineret (Anakinra),
MYOBLOC (Botulinum Toxin Type B), Neulasta (Pegfilgrastim), Neumega
(Oprelvekin), Neupogen (Filgrastim), Ontak (Denileukin diftitox),
PEGASYS (Peginterferon alfa-2a), Proleukin (Aldesleukin), Pulmozyme
(Dornase alfa), Rebif (Interferon beta-1a), Regranex (Becaplermin),
Retavase (Reteplase), Roferon-A (Interferon alfa-2), TNKase
(Tenecteplase), and Xigris (Drotrecogin alfa), Arcalyst
(Rilonacept), NPlate (Romiplostim), Mircera (methoxypolyethylene
glycol-epoetin beta), Cinryze (C1 esterase inhibitor), Elaprase
(idursulfase), Myozyme (alglucosidase alfa), Orencia (abatacept),
Naglazyme (galsulfase), Kepivance (palifermin) and Actimmune
(interferon gamma-1b).
[0121] The protein used in the invention may also be an antibody,
or antigen-binding fragment thereof. Examples of antibodies that
may be used in the invention include chimeric antibodies, non-human
antibodies, human antibodies, humanized antibodies, and domain
antibodies (dAbs). In one embodiment, the antibody, or
antigen-binding fragment thereof, is an anti-TNF.alpha. and/or an
anti-IL-12 antibody (e.g., it may be a dual variable domain (DVD)
antibody). Other examples of antibodies, or antigen-binding
fragments thereof, which may be used in the methods and
compositions of the invention include, but are not limited to,
1D4.7 (anti-IL-12/IL-23 antibody; Abbott Laboratories), 2.5(E)mg1
(anti-IL-18; Abbott Laboratories), 13C5.5 (anti-IL-13 antibody;
Abbott Laboratories), J695 (anti-IL-12; Abbott Laboratories),
Afelimomab (Fab 2 anti-TNF; Abbott Laboratories), Humira
(adalimumab) Abbott Laboratories), Campath (Alemtuzumab), CEA-Scan
Arcitumomab (fab fragment), Erbitux (Cetuximab), Herceptin
(Trastuzumab), Myoscint (Imciromab Pentetate), ProstaScint
(Capromab Pendetide), Remicade (Infliximab), ReoPro (Abciximab),
Rituxan (Rituximab), Simulect (Basiliximab), Synagis (Palivizumab),
Verluma (Nofetumomab), Xolair (Omalizumab), Zenapax (Daclizumab),
Zevalin (Ibritumomab Tiuxetan), Orthoclone OKT3 (Muromonab-CD3),
Panorex (Edrecolomab), Mylotarg (Gemtuzumab ozogamicin), golimumab
(Centocor), Cimzia (Certolizumab pegol), Soliris (Eculizumab), CNTO
1275 (ustekinumab), Vectibix (panitumumab), Bexxar (tositumomab and
I.sup.131 tositumomab), an anti-IL-17 antibody Antibody 7 as
described in International Application WO 2007/149032 (Cambridge
Antibody Technology), the entire contents of which are incorporated
by reference herein, the anti-IL-13 antibody CAT-354 (Cambridge
Antibody Technology), the anti-human CD4 antibody CE9y4PE
(IDEC-151, clenoliximab) (Biogen IDEC/Glaxo Smith Kline), the
anti-human CD4 antibody IDEC CE9.1/SB-210396 (keliximab) (Biogen
IDEC), the anti-human CD80 antibody IDEC-114 (galiximab) (Biogen
IDEC), the anti-Rabies Virus Protein antibody CR4098 (foravirumab),
and the anti-human TNF-related apoptosis-inducing ligand receptor 2
(TRAIL-2) antibody HGS-ETR2 (lexatumumab) (Human Genome Sciences,
Inc.), and Avastin (bevacizumab).
[0122] Techniques for the production of antibodies are provided
below.
Polyclonal Antibodies
[0123] Polyclonal antibodies generally refer to a mixture of
antibodies that are specific to a certain antigen, but bind to
different epitopes on said antigen. Polyclonal antibodies are
generally raised in animals by multiple subcutaneous (sc) or
intraperitoneal (ip) injections of the relevant antigen and an
adjuvant. It may be useful to conjugate the relevant antigen to a
protein that is immunogenic in the species to be immunized, e.g.,
keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or
soybean trypsin inhibitor using a bifunctional or derivatizing
agent, for example, maleimidobenzoyl sulfosuccinimide ester
(conjugation through cysteine residues), N-hydroxysuccinimide
(through lysine residues), glutaraldehyde, succinic anhydride,
SOCl.sub.2, or R.sub.1NCNR, where R and R.sub.1 are different alkyl
groups. Methods for making polyclonal antibodies are known in the
art, and are described, for example, in Antibodies: A Laboratory
Manual, Lane and Harlow (1988), incorporated by reference
herein.
Monoclonal Antibodies
[0124] A "monoclonal antibody" as used herein is intended to refer
to a hybridoma-derived antibody (e.g., an antibody secreted by a
hybridoma prepared by hybridoma technology, such as the standard
Kohler and Milstein hybridoma methodology). For example, the
monoclonal antibodies may be made using the hybridoma method first
described by Kohler et al., Nature, 256:495 (1975), or may be made
by recombinant DNA methods (U.S. Pat. No. 4,816,567). Thus, a
hybridoma-derived dual-specificity antibody of the invention is
still referred to as a monoclonal antibody although it has
antigenic specificity for more than a single antigen.
[0125] Monoclonal antibodies are obtained from a population of
substantially homogeneous antibodies, i.e., the individual
antibodies comprising the population are identical except for
possible naturally occurring mutations that may be present in minor
amounts. Thus, the modifier "monoclonal" indicates the character of
the antibody as not being a mixture of discrete antibodies.
[0126] In a further embodiment, antibodies can be isolated from
antibody phage libraries generated using the techniques described
in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al.,
Nature, 352:624-628 (1991) and Marks et al., J. Mol. Biol.,
222:581-597 (1991) describe the isolation of murine and human
antibodies, respectively, using phage libraries. Subsequent
publications describe the production of high affinity (nM range)
human antibodies by chain shuffling (Marks et al., Bio/Technology,
10:779-783 (1992)), as well as combinatorial infection and in vivo
recombination as a strategy for constructing very large phage
libraries (Waterhouse et al., Nuc. Acids. Res., 21:2265-2266
(1993)). Thus, these techniques are viable alternatives to
traditional monoclonal antibody hybridoma techniques for isolation
of monoclonal antibodies.
[0127] Antibodies and antibody fragments may also be isolated from
yeast and other eukaryotic cells with the use of expression
libraries, as described in U.S. Pat. Nos. 6,423,538; 6,696,251;
6,699,658; 6,300,065; 6,399,763; and 6,114,147. Eukaryotic cells
may be engineered to express library proteins, including from
combinatorial antibody libraries, for display on the cell surface,
allowing for selection of particular cells containing library
clones for antibodies with affinity to select target molecules.
After recovery from an isolated cell, the library clone coding for
the antibody of interest can be expressed at high levels from a
suitable mammalian cell line.
[0128] Additional methods for developing antibodies of interest
include cell-free screening using nucleic acid display technology,
as described in U.S. Pat. Nos. 7,195,880; 6,951,725; 7,078,197;
7,022,479, 6,518,018; 7,125,669; 6,846,655; 6,281,344; 6,207,446;
6,214,553; 6,258,558; 6,261,804; 6,429,300; 6,489,116; 6,436,665;
6,537,749; 6,602,685; 6,623,926; 6,416,950; 6,660,473; 6,312,927;
5,922,545; and 6,348,315. These methods can be used to transcribe a
protein in vitro from a nucleic acid in such a way that the protein
is physically associated or bound to the nucleic acid from which it
originated. By selecting for an expressed protein with a target
molecule, the nucleic acid that codes for the protein is also
selected. In one variation on cell-free screening techniques,
antibody sequences isolated from immune system cells can be
isolated and partially randomized polymerase chain reaction
mutagenesis techniques to increase antibody diversity. These
partially randomized antibody genes are then expressed in a
cell-free system, with concurrent physical association created
between the nucleic acid and antibody.
[0129] The DNA also may be modified, for example, by substituting
the coding sequence for human heavy- and light-chain constant
domains in place of the homologous murine sequences (U.S. Pat. No.
4,816,567; Morrison, et al., Proc. Natl. Acad. Sci. USA, 81:6851
(1984)), or by covalently joining to the immunoglobulin coding
sequence all or part of the coding sequence for a
non-immunoglobulin polypeptide.
[0130] Typically such non-immunoglobulin polypeptides are
substituted for the constant domains of an antibody, or they are
substituted for the variable domains of one antigen-combining site
of an antibody to create a chimeric bivalent antibody comprising
one antigen-combining site having specificity for an antigen and
another antigen-combining site having specificity for a different
antigen.
[0131] Chimeric or hybrid antibodies also may be prepared in vitro
using known methods in synthetic protein chemistry, including those
involving crosslinking agents. For example, immunotoxins may be
constructed using a disulfide-exchange reaction or by forming a
thioether bond. Examples of suitable reagents for this purpose
include iminothiolate and methyl-4-mercaptobutyrimidate.
Humanized Antibodies
[0132] Methods for humanizing non-human antibodies are well known
in the art. Generally, a humanized antibody has one or more amino
acid residues introduced into it from a source which is non-human.
These non-human amino acid residues are often referred to as
"import" residues, which are typically taken from an "import"
variable domain. Humanization can be essentially performed
following the method of Winter and co-workers (Jones et al.,
Nature, 321:522-525 (1986); Riechmann et al., Nature, 332:323-327
(1988); Verhoeyen et al., Science, 239:1534-1536 (1988)), by
substituting non-human (e.g., rodent) CDRs or CDR sequences for the
corresponding sequences of a human antibody. Accordingly, such
"humanized" antibodies are chimeric antibodies (U.S. Pat. No.
4,816,567), wherein substantially less than an intact human
variable domain has been substituted by the corresponding sequence
from a non-human species. In practice, humanized antibodies are
typically human antibodies in which some CDR residues and possibly
some framework (FR) residues are substituted by residues from
analogous sites in rodent antibodies. Additional references which
describe the humanization process include Sims et al., J. Immunol.,
151:2296 (1993); Chothia et al., J. Mol. Biol., 196:901 (1987);
Carter et al., Proc. Natl. Acad. Sci. USA, 89:4285 (1992); Presta
et al., J. Immunol., 151:2623 (1993), each of which is incorporated
by reference herein.
Human Antibodies
[0133] Alternatively, it is now possible to produce transgenic
animals (e.g., mice) that are capable, upon immunization, of
producing a full repertoire of human antibodies in the absence of
endogenous immunoglobulin production. For example, it has been
described that the homozygous deletion of the antibody heavy-chain
joining region (J.sub.H) gene in chimeric and germ-line mutant mice
results in complete inhibition of endogenous antibody production.
Transfer of the human germ-line immunoglobulin gene array in such
germ-line mutant mice will result in the production of human
antibodies upon antigen challenge. See, e.g., Jakobovits et al.,
Proc. Natl. Acad. Sci. USA, 90:2551 (1993); Jakobovits et al.,
Nature, 362:255-258 (1993); Bruggermann et al., Year in Immuno.,
7:33 (1993). Human antibodies can also be derived from
phage-display libraries (Hoogenboom et al., J. Mol. Biol., 227:381
(1991); Marks et al., J. Mol. Biol., 222:581-597 (1991)).
[0134] In one embodiment, the formulation of the invention
comprises an antibody, or antigen-binding portion thereof, which
binds human TNF.alpha., including, for example, adalimumab (also
referred to as Humira, adalimumab, or D2E7; Abbott Laboratories).
In one embodiment, the antibody, or antigen-binding fragment
thereof, dissociates from human TNF.alpha. with a K.sub.d of
1.times.10.sup.-8 M or less and a K.sub.off rate constant of
1.times.10.sup.-3 s.sup.-1 or less, both determined by surface
plasmon resonance, and neutralizes human TNF.alpha. cytotoxicity in
a standard in vitro L929 assay with an IC.sub.50 of
1.times.10.sup.-7 M or less. Examples and methods for making human,
neutralizing antibodies which have a high affinity for human
TNF.alpha., including sequences of the antibodies, are described in
U.S. Pat. No. 6,090,382 (referred to as D2E7), incorporated by
reference herein.
[0135] In one embodiment, the human neutralizing, antibody, or an
antigen-binding portion thereof, having a high affinity for human
TNF.alpha. is an isolated human antibody, or an antigen-binding
portion thereof, with a light chain variable region (LCVR) having a
CDR3 domain comprising the amino acid sequence of SEQ ID NO:3, or
modified from SEQ ID NO:3 by a single alanine substitution at
position 1, 4, 5, 7 or 8, and with a heavy chain variable region
(HCVR) having a CDR3 domain comprising the amino acid sequence of
SEQ ID NO:4, or modified from SEQ ID NO:4 by a single alanine
substitution at position 2, 3, 4, 5, 6, 8, 9, 10 or 11. Preferably,
the LCVR further has a CDR2 domain comprising the amino acid
sequence of SEQ ID NO:5 (i.e., the D2E7 VL CDR2) and the HCVR
further has a CDR2 domain comprising the amino acid sequence of SEQ
ID NO: 6 (i.e., the D2E7 VH CDR2). Even more preferably, the LCVR
further has CDR1 domain comprising the amino acid sequence of SEQ
ID NO:7 (i.e., the D2E7 VL CDR1) and the HCVR has a CDR1 domain
comprising the amino acid sequence of SEQ ID NO:8 (i.e., the D2E7
VH CDR1).
[0136] In another embodiment, the human neutralizing, antibody, or
an antigen-binding portion thereof, having a high affinity for
human TNF.alpha. is an isolated human antibody, or an
antigen-binding portion thereof, with a light chain variable region
(LCVR) comprising the amino acid sequence of SEQ ID NO:1 (i.e., the
D2E7 VL) and a heavy chain variable region (HCVR) comprising the
amino acid sequence of SEQ ID NO:2 (i.e., the D2E7 VH). In certain
embodiments, the antibody comprises a heavy chain constant region,
such as an IgG1, IgG2, IgG3, IgG4, IgA, IgE, IgM or IgD constant
region. Preferably, the heavy chain constant region is an IgG1
heavy chain constant region or an IgG4 heavy chain constant region.
Furthermore, the antibody can comprise a light chain constant
region, either a kappa light chain constant region or a lambda
light chain constant region. Preferably, the antibody comprises a
kappa light chain constant region.
[0137] In one embodiment, the formulation of the invention
comprises an antibody, or antigen-binding portion thereof, which
binds human IL-12, including, for example, the antibody J695
(Abbott Laboratories; also referred to as ABT-874) (U.S. Pat. No.
6,914,128). J695 is a fully human monoclonal antibody designed to
target and neutralize interleukin-12 and interleukin-23. In one
embodiment, the antibody, or antigen-binding fragment thereof, has
the following characteristics: it dissociates from human
IL-1.alpha. with a K.sub.D of 3.times.10.sup.-7 M or less;
dissociates from human IL-1.beta. with a K.sub.D of
5.times.10.sup.-5 M or less; and does not bind mouse IL-1.alpha. or
mouse IL-1.beta.. Examples and methods for making human,
neutralizing antibodies which have a high affinity for human IL-12,
including sequences of the antibody, are described in U.S. Pat. No.
6,914,128, incorporated by reference herein.
[0138] In one embodiment, the formulation of the invention
comprises an antibody, or antigen-binding portion thereof, which
binds human IL-18, including, for example, the antibody 2.5(E)mg1
(Abbott Bioresearch; also referred to as ABT-325) (see U.S. Patent
Application No. 2005/0147610, incorporated by reference
herein).
[0139] In one embodiment, the formulation of the invention
comprises an anti-IL-12/anti-IL-23 antibody, or antigen-binding
portion thereof, which is the antibody 1D4.7 (Abbott Laboratories;
also referred to as ABT-147) (see WO 2007/005608 A2, published Jan.
11, 2007, incorporated by reference herein).
[0140] In one embodiment, the formulation of the invention
comprises an anti-IL-13 antibody, or antigen-binding portion
thereof, which is the antibody 13C5.5 (Abbott Laboratories; also
referred to as ABT-308) (see. PCT/US2007/19660 (WO 08/127271),
incorporated by reference herein).
[0141] In one embodiment, the formulation of the invention
comprises an antibody, or antigen-binding portion thereof, which is
the antibody 7C6, an anti-amyloid .beta. antibody (Abbott
Laboratories; see PCT publication WO 07/062852, incorporated by
reference herein).
Bispecific Antibodies
[0142] Bispecific antibodies (BsAbs) are antibodies that have
binding specificities for at least two different epitopes. Such
antibodies can be derived from full length antibodies or antibody
fragments (e.g., F(ab')2 bispecific antibodies).
[0143] Methods for making bispecific antibodies are known in the
art. Traditional production of full length bispecific antibodies is
based on the coexpression of two immunoglobulin heavy chain-light
chain pairs, where the two chains have different specificities
(Millstein et al., Nature, 305:537-539 (1983)). Because of the
random assortment of immunoglobulin heavy and light chains, these
hybridomas (quadromas) produce a potential mixture of 10 different
antibody molecules, of which only one has the correct bispecific
structure. Purification of the correct molecule, which is usually
done by affinity chromatography steps, is rather cumbersome, and
the product yields are low. Similar procedures are disclosed in WO
93/08829 and in Traunecker et al., EMBO J., 10:3655-3659
(1991).
[0144] According to a different approach, antibody variable domains
with the desired binding specificities (antibody-antigen combining
sites) are fused to immunoglobulin constant domain sequences.
[0145] The fusion preferably is with an immunoglobulin heavy chain
constant domain, comprising at least part of the hinge, CH2, and
CH3 regions. It is preferred to have the first heavy-chain constant
region (CH1) containing the site necessary for light chain binding,
present in at least one of the fusions. DNAs encoding the
immunoglobulin heavy chain fusions and, if desired, the
immunoglobulin light chain, are inserted into separate expression
vectors, and are co-transfected into a suitable host organism. This
provides for great flexibility in adjusting the mutual proportions
of the three polypeptide fragments in embodiments when unequal
ratios of the three polypeptide chains used in the construction
provide the optimum yields. It is, however, possible to insert the
coding sequences for two or all three polypeptide chains in one
expression vector when the expression of at least two polypeptide
chains in equal ratios results in high yields or when the ratios
are of no particular significance.
[0146] In a preferred embodiment of this approach, the bispecific
antibodies are composed of a hybrid immunoglobulin heavy chain with
a first binding specificity in one arm, and a hybrid immunoglobulin
heavy chain-light chain pair (providing a second binding
specificity) in the other arm. It was found that this asymmetric
structure facilitates the separation of the desired bispecific
compound from unwanted immunoglobulin chain combinations, as the
presence of an immunoglobulin light chain in only one half of the
bispecific molecule provides for a facile way of separation. This
approach is disclosed in WO 94/04690 published Mar. 3, 1994. For
further details of generating bispecific antibodies see, for
example, Suresh et al., Methods in Enzymology, 121:210 (1986).
[0147] Bispecific antibodies include cross-linked or
"heteroconjugate" antibodies. For example, one of the antibodies in
the heteroconjugate can be coupled to avidin, the other to biotin.
Such antibodies have, for example, been proposed to target immune
system cells to unwanted cells (U.S. Pat. No. 4,676,980), and for
treatment of HIV infection (WO 91/00360, WO 92/200373, and EP
03089). Heteroconjugate antibodies may be made using any convenient
cross-linking methods. Suitable cross-linking agents are well known
in the art, and are disclosed in U.S. Pat. No. 4,676,980, along
with a number of cross-linking techniques.
[0148] Techniques for generating bispecific antibodies from
antibody fragments have also been described in the literature. The
following techniques can also be used for the production of
bivalent antibody fragments which are not necessarily bispecific.
For example, Fab' fragments recovered from E. coli can be
chemically coupled in vitro to form bivalent antibodies. See,
Shalaby et al., J. Exp. Med., 175:217-225 (1992).
[0149] Various techniques for making and isolating bivalent
antibody fragments directly from recombinant cell culture have also
been described. For example, bivalent heterodimers have been
produced using leucine zippers. Kostelny et al., J. Immunol.,
148(5): 1547-1553 (1992). The leucine zipper peptides from the Fos
and Jun proteins were linked to the Fab' portions of two different
antibodies by gene fusion. The antibody homodimers were reduced at
the hinge region to form monomers and then re-oxidized to form the
antibody heterodimers. The "diabody" technology described by
Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993)
has provided an alternative mechanism for making
bispecific/bivalent antibody fragments. The fragments comprise a
heavy-chain variable domain (VH) connected to a light-chain
variable domain (VL) by a linker which is too short to allow
pairing between the two domains on the same chain. Accordingly, the
VH and VL domains of one fragment are forced to pair with the
complementary VL and VH domains of another fragment, thereby
forming two antigen-binding sites. Another strategy for making
bispecific/bivalent antibody fragments by the use of single-chain
Fv (sFv) dimers has also been reported. See Gruber et al., J.
Immunol., 152:5368 (1994).
[0150] In one embodiment, the formulation of the invention
comprises an antibody which is bispecific for IL-1 (including
IL-1.alpha. and IL-1.beta.). Examples and methods for making
bispecific IL-1 antibodies can be found in U.S. Provisional Appln.
No. 60/878,165, filed Dec. 29, 2006.
[0151] Diafiltration/Ultrafiltration (also generally referred to
herein as DF/UF) selectively utilizes permeable (porous) membrane
filters to separate the components of solutions and suspensions
based on their molecular size. A membrane retains molecules that
are larger than the pores of the membrane while smaller molecules
such as salts, solvents and water, which are permeable, freely pass
through the membrane. The solution retained by the membrane is
known as the concentrate or retentate. The solution that passes
through the membrane is known as the filtrate or permeate. One
parameter for selecting a membrane for concentration is its
retention characteristics for the sample to be concentrated. As a
general rule, the molecular weight cut-off (MWCO) of the membrane
should be 1/3rd to 1/6th the molecular weight of the molecule to be
retained. This is to assure complete retention. The closer the MWCO
is to that of the sample, the greater the risk for some small
product loss during concentration. Examples of membranes that can
be used with methods of the invention include Omega.TM. PES
membrane (30 kDa MWCO, i.e. molecules larger than 30 kDa are
retained by the membrane and molecules less than 30 kDa are allowed
to pass to the filtrate side of the membrane) (Pall Corp., Port
Washington, N.Y.); Millex.RTM.-GV Syringe Driven Filter Unit, PVDF
0.22 .mu.m (Millipore Corp., Billerica, Mass.); Millex.RTM.-GP
Syringe Driven Filter Unit, PES 0.22 .mu.m; Sterivex.RTM.0.22 .mu.m
Filter Unit (Millipore Corp., Billerica, Mass.); and Vivaspin
concentrators (MWCO 10 kDa, PES; MWCO 3 kDa, PES) (Sartorius Corp.,
Edgewood, N.Y.). In order to prepare a low-ionic protein
formulation of the invention, the protein solution (which may be
solubilized in a buffered formulation) is subjected to a DF/UF
process, whereby water is used as a DF/UF medium. In a preferred
embodiment, the DF/UF medium consists of water and does not include
any other excipients.
[0152] Any water can be used in the DF/UF process of the invention,
although a preferred water is purified or deionized water. Types of
water known in the art that may be used in the practice of the
invention include water for injection (WFI) (e.g., HyPure WFI
Quality Water (HyClone), AQUA-NOVA.RTM. WFI (Aqua Nova)),
UltraPure.TM. Water (Invitrogen), and distilled water (Invitrogen;
Sigma-Aldrich).
[0153] There are two forms of DF/UF, including DF/UF in
discontinuous mode and DF/UF in continuous mode. The methods of the
invention may be performed according to either mode.
[0154] Continuous DF/UF (also referred to as constant volume DF/UF)
involves washing out the original buffer salts (or other low
molecular weight species) in the retentate (sample or first protein
solution) by adding water or a new buffer to the retentate at the
same rate as filtrate is being generated. As a result, the
retentate volume and product concentration does not change during
the DF/UF process. The amount of salt removed is related to the
filtrate volume generated, relative to the retentate volume. The
filtrate volume generated is usually referred to in terms of
"diafiltration volumes". A single diafiltration volume (DV) is the
volume of retentate when diafiltration is started. For continuous
diafiltration, liquid is added at the same rate as filtrate is
generated. When the volume of filtrate collected equals the
starting retentate volume, 1 DV has been processed.
[0155] Discontinuous DF/UF (examples of which are provided below in
the Examples section) includes two different methods, discontinuous
sequential DF/UF and volume reduction discontinuous DF/UF.
Discontinuous DF/UF by sequential dilution involves first diluting
the sample (or first protein solution) with water to a
predetermined volume.
[0156] The diluted sample is then concentrated back to its original
volume by UF. Discontinuous DF/UF by volume reduction involves
first concentrating the sample to a predetermined volume, then
diluting the sample back to its original volume with water or
replacement buffer. As with continuous DF/UF, the process is
repeated until the level of unwanted solutes, e.g., ionic
excipients, are removed.
[0157] DF/UF may be performed in accordance with conventional
techniques known in the art using water, e.g, WFI, as the DF/UF
medium (e.g., Industrial Ultrafiltration Design and Application of
Diafiltration Processes, Beaton & Klinkowski, J. Separ. Proc.
Technol., 4(2) 1-10 (1983)). Examples of commercially available
equipment for performing DF/UF include Millipore Labscale.TM. TFF
System (Millipore), LV Centramate.TM. Lab Tangential Flow System
(Pall Corporation), and the UniFlux System (GE Healthcare).
[0158] For example, in a preferred embodiment, the Millipore
Labscale.TM. Tangential Flow Filtration (TFF) system with a 500 mL
reservoir is used to perform a method of the invention to produce a
diafiltered antibody solution. The DF/UF procedure is performed in
a discontinuous manner, with 14 process steps used to produce a
high concentration antibody formulation in water. For additional
exemplary equipment, solution and water volumes, number of process
steps, and other parameters of particular embodiments of the
invention, see the Examples section below.
[0159] Alternative methods to diafiltration for buffer exchange
where a protein is re-formulated into water in accordance with the
invention include dialysis and gel filtration, both of which are
techniques known to those in the art. Dialysis requires filling a
dialysis bag (membrane casing of defined porosity), tying off the
bag, and placing the bag in a bath of water. Through diffusion, the
concentration of salt in the bag will equilibrate with that in the
bath, wherein large molecules, e.g., proteins that cannot diffuse
through the bag remain in the bag. The greater the volume of the
bath relative to the sample volume in the bags, the lower the
equilibration concentration that can be reached. Generally,
replacements of the bath water are required to completely remove
all of the salt. Gel filtration is a non-adsorptive chromatography
technique that separates molecules on the basis of molecular size.
In gel filtration, large molecules, e.g., proteins, may be
separated from smaller molecules, e.g., salts, by size
exclusion.
[0160] In a preferred embodiment of the invention, the first
protein solution is subjected to a repeated volume exchange with
the water, such that an aqueous formulation, which is essentially
water and protein, is achieved. The diafiltration step may be
performed any number of times, depending on the protein in
solution, wherein one diafiltration step equals one total volume
exchange. In one embodiment, the diafiltration process is performed
1, 2, 3, 4, 5, 6, 7, 8, 9, or up to as many times are deemed
necessary to remove excipients, e.g., salts, from the first protein
solution, such that the protein is dissolved essentially in water.
A single round or step of diafiltration is achieved when a volume
of water has been added to the retentate side that is equal to the
starting volume of the protein solution.
[0161] In one embodiment, the protein solution is subjected to at
least 2 diafiltration steps. In one embodiment, the diafiltration
step or volume exchange with water may be repeated at least four
times, and preferably at least five times. In one embodiment, the
first protein solution is subjected to diafiltration with water
until at least a six-fold volume exchange is achieved. In another
embodiment, the first protein solution is subjected to
diafiltration with water until at least a seven-fold volume
exchange is achieved. Ranges intermediate to the above recited
numbers, e.g., 4 to 6 or 5 to 7, are also intended to be part of
this invention. For example, ranges of values using a combination
of any of the above recited values as upper and/or lower limits are
intended to be included.
[0162] In a preferred embodiment, loss of protein to the filtrate
side of an ultrafiltration membrane should be minimized. The risk
of protein loss to the filtrate side of a particular membrane
varies in relation to the size of the protein relative to the
membrane's pore size, and the protein's concentration. With
increases in protein concentration, risk of protein loss to the
filtrate increases. For a particular membrane pore size, risk of
protein loss is greater for a smaller protein that is close in size
to the membrane's MWCO than it is for a larger protein. Thus, when
performing DF/UF on a smaller protein, it may not be possible to
achieve the same reduction in volume, as compared to performing
DF/UF on a larger protein using the same membrane, without
incurring unacceptable protein losses. In other words, as compared
to the ultrafiltration of a solution of a smaller protein using the
same equipment and membrane, a solution of a larger protein could
be ultrafiltered to a smaller volume, with a concurrent higher
concentration of protein in the solution. DF/UF procedures using a
particular pore size membrane may require more process steps for a
smaller protein than for a larger protein;
[0163] a greater volume reduction and concentration for a larger
protein permits larger volumes of water to be added back, leading
to a larger dilution of the remaining buffer or excipient
ingredients in the protein solution for that individual process
step. Fewer process steps may therefore be needed to achieve a
certain reduction in solutes for a larger protein than for a
smaller one. A person with skill in the art would be able to
calculate the amount of concentration possible with each process
step and the number of overall process steps required to achieve a
certain reduction in solutes, given the protein size and the pore
size of the ultrafiltration device to be used in the procedure.
[0164] As a result of the diafiltration methods of the invention,
the concentration of solutes in the first protein solution is
significantly reduced in the final aqueous formulation comprising
essentially water and protein. For example, the aqueous formulation
may have a final concentration of excipients which is at least 95%
less than the first protein solution, and preferably at least 99%
less than the first protein solution. For example, in one
embodiment, to dissolve a protein in WFI is a process that creates
a theoretical final excipient concentration, reached by constant
volume diafiltration with five diafiltration volumes, that is equal
or approximate to Ci e.sup.-5=0.00674, i.e., an approximate 99.3%
maximum excipient reduction. In one embodiment, a person with skill
in the art may perform 6 volume exchanges during the last step of a
commercial DF/UF with constant volume diafiltration, i.e., Ci would
be C.sub.i e.sup.6=0.0025. This would provide an approximate 99.75%
maximum theoretical excipient reduction. In another embodiment, a
person with skill in the art may use 8 diafiltration volume
exchanges to obtain a theoretical .about.99.9% maximum excipient
reduction.
[0165] The term "excipient-free" or "free of excipients" indicates
that the formulation is essentially free of excipients. In one
embodiment, excipient-free indicates buffer-free, salt-free,
sugar-free, amino acid-free, surfactant-free, and/or polyol free.
In one embodiment, the term "essentially free of excipients"
indicates that the solution or formulation is at least 99% free of
excipients. It should be noted, however, that in certain
embodiments, a formulation may comprise a certain specified
non-ionic excipient, e.g., sucrose or mannitol, and yet the
formulation is otherwise excipient free. For example, a formulation
may comprise water, a protein, and mannitol, wherein the
formulation is otherwise excipient free. In another example, a
formulation may comprise water, a protein, and polysorbate 80,
wherein the formulation is otherwise excipient free. In yet another
example, the formulation may comprise water, a protein, a sorbitol,
and polysorbate 80, wherein the formulation is otherwise excipient
free.
[0166] When water is used for diafiltering a first protein solution
in accordance with the methods described herein, ionic excipients
will be washed out, and, as a result, the conductivity of the
diafiltered aqueous formulation is lower than the first protein
solution. If an aqueous solution conducts electricity, then it must
contain ions, as found with ionic excipients. A low conductivity
measurement is therefore indicative that the aqueous formulation of
the invention has significantly reduced excipients, including ionic
excipients.
[0167] Conductivity of a solution is measured according to methods
known in the art. Conductivity meters and cells may be used to
determine the conductivity of the aqueous formulation, and should
be calibrated to a standard solution before use. Examples of
conductivity meters available in the art include MYRON L Digital
(Cole Parmer.RTM.), Conductometer (Metrohm AG), and Series
3105/3115 Integrated Conductivity Analyzers (Kemotron). In one
embodiment, the aqueous formulation has a conductivity of less than
3 mS/cm. In another embodiment, the aqueous formulation has a
conductivity of less than 2 mS/cm. In yet another embodiment, the
aqueous formulation has a conductivity of less than 1 mS/cm. In one
aspect of the invention, the aqueous formulation has a conductivity
of less than 0.5 mS/cm. Ranges intermediate to the above recited
numbers, e.g., 1 to 3 mS/cm, are also intended to be encompassed by
the invention. For example, ranges of values using a combination of
any of the above recited values as upper and/or lower limits are
intended to be included. In addition, values that fall within the
recited numbers are also included in the invention, e.g., 0.5, 0.6,
0.7, 0.8, 0.9, 1.0, 1.1, 1.2, 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9,
2.0, 2.1, 2.2, 2.3, 2.4, 2.5, 2.6, 2.7, 2.8, 2.9, 3.0 and so
forth.
[0168] An important aspect of the invention is that the diafiltered
protein solution (solution obtained following the diafiltration
process of the first protein solution) can be concentrated. By
following this process, it has been discovered that high
concentrations of protein are stable in water. Concentration
following diafiltration results in an aqueous formulation
containing water and an increased protein concentration relative to
the first protein solution. Thus, the invention also includes
diafiltering a protein solution using water as a diafiltration
medium and subsequently concentrating the resulting aqueous
solution. Concentration of the diafiltered protein solution may be
achieved through means known in the art, including centrifugation.
For example, following diafiltration, the water-based diafiltrated
protein solution is subjected to a centrifugation process which
serves to concentrate the protein via ultrafiltration into a high
concentration formulation while maintaining the water-based
solution. Means for concentrating a solution via centrifugation
with ultrafiltration membranes and/or devices are known in the art,
e.g., with Vivaspin centrifugal concentrators (Sartorius Corp.
Edgewood, N.Y.).
[0169] The methods of the invention provide a means of
concentrating a protein at very high levels in water without the
need for additional stabilizing agents. The concentration of the
protein in the aqueous formulation obtained using the methods of
the invention can be any amount in accordance with the desired
concentration. For example, the concentration of protein in an
aqueous solution made according to the methods herein is at least
about 10 .mu.g/mL; at least about 1 mg/mL; at least about 10 mg/mL;
at least about 20 mg/mL; at least about 50 mg/mL; at least about 75
mg/mL; at least about 100 mg/mL; at least about 125 mg/mL; at least
about 150 mg/mL; at least about 175 mg/mL; at least about 200
mg/mL; at least about 220 mg/mL; at least about 250 mg/mL; at least
about 300 mg/mL; or greater than about 300 mg/mL. Ranges
intermediate to the above recited concentrations, e.g., at least
about 113 mg/mL, at least about 214 mg/mL, and at least about 300
mg/mL, are also intended to be encompassed by the invention. In
addition, ranges of values using a combination of any of the above
recited values (or values between the ranges described above) as
upper and/or lower limits are intended to be included, e.g., 100 to
125 mg/mL, 113 to 125 mg/mL, and 126 to 200 mg/mL or more.
[0170] The methods of the invention provide the advantage that the
resulting formulation has a low percentage of protein aggregates,
despite the high concentration of the aqueous protein formulation.
In one embodiment, the aqueous formulations comprising water and a
high concentration of a protein, e.g., antibodies, contains less
than about 5% protein aggregates, even in the absence of a
surfactant or other type of excipient. In one embodiment, the
formulation comprises no more than about 7.3% aggregate protein;
the formulation comprises no more than about 5% aggregate protein;
the formulation comprises no more than about 4% aggregate protein;
the formulation comprises no more than about 3% aggregate protein;
the formulation comprises no more than about 2% aggregate protein;
or the formulation comprising no more than about 1% aggregate
protein. In one embodiment, the formulation comprises at least
about 92%, at least about 93%, at least about 94%, at least about
95%, at least about 96%, at least about 97%, at least about 98%, or
at least about 99% monomer protein. Ranges intermediate to the
above recited concentrations, e.g., at least about 98.6%, no more
than about 4.2%, are also intended to be part of this invention. In
addition, ranges of values using a combination of any of the above
recited values as upper and/or lower limits are intended to be
included.
[0171] Many protein-based pharmaceutical products need to be
formulated at high concentrations. For example, antibody-based
products increasingly tend to exceed 100 mg/mL in their Drug
Product (DP) formulation to achieve appropriate efficacy and meet a
typical patient usability requirement of a maximal .about.1 mL
injection volume. Accordingly, downstream processing steps, such as
diafiltration into the final formulation buffer or ultrafiltration
to increase the protein concentration, are also conducted at higher
concentrations.
[0172] Classic thermodynamics predicts that intermolecular
interactions can affect the partitioning of small solutes across a
dialysis membrane, especially at higher protein concentrations, and
models describing non-ideal dialysis equilibrium and the effects of
intermolecular interactions are available (Tanford Physical
chemistry or macromolecules. New York, John Wiley and Sons, Inc.,
p. 182, 1961; Tester and Modell Thermodynamics and its
applications, 3.sup.rd ed. Upper Saddle River, NL, Prentice-Hall,
1997). In the absence of the availability of detailed thermodynamic
data in the process development environment, which is necessary to
apply these type of models, intermolecular interactions rarely are
taken into account during the design of commercial DF/UF
operations. Consequently, DP excipient concentrations may differ
significantly from the concentration labeled. Several examples of
this discrepancy in commercial and development products are
published, e.g., chloride being up to 30% lower than labeled in an
IL-1 receptor antagonist, histidine being 40% lower than labeled in
a PEG-sTNF receptor, and acetate being up to 200% higher than
labeled in a fusion conjugate protein (Stoner et al., J. Pharm.
Sci., 93, 2332-2342 (2004)). There are several reasons why the
actual DP may be different from the composition of the buffer the
protein is diafiltered into, including the Donnan effect (Tombs and
Peacocke (1974) Oxford; Clarendon Press), non-specific interactions
(Arakawa and Timasheff, Arch. Biochem. Biophys., 224, 169-77
(1983); Timasheff, Annu. Rev. Biophys. Biomol. Struct., 22, 67-97
(1993)), and volume exclusion effects. Volume exclusion includes
most protein partial specific volumes are between 0.7 and 0.8
mL/g..sup.5 Thus, for a globular protein at 100 mg/mL, protein
molecules occupy approx. 7.5% of the total solution volume. No
significant intermolecular interactions assumed, this would
translate to a solute molar concentration on the retentate side of
the membrane that is 92.5% of the molar concentration on the
permeate side of the membrane. This explains why basically all
protein solution compositions necessarily change during
ultrafiltration processing. For instance, at 40 mg/mL the protein
molecules occupy approx. 3% of the total solution volume, and an
ultrafiltration step increasing the concentration to 150 mg/mL will
necessarily induce molar excipient concentrations to change by more
than 8% (as protein at 150 mg/mL accounts for more than 11% of
total solution volume). Ranges intermediate to the above recited
percentages are also intended to be part of this invention. In
addition, ranges of values using a combination of any of the above
recited values as upper and/or lower limits are intended to be
included.
[0173] In accordance with the methods and compositions of the
invention, buffer composition changes during DF/UF operations can
be circumvented by using pure water as diafiltration medium. By
concentrating the protein .about.20% more than the concentration
desired in the final Bulk DS, excipients could subsequently be
added, for instance, via highly concentrated excipient stock
solutions. Excipient concentrations and solution pH could then be
guaranteed to be identical as labeled.
[0174] The aqueous formulation of the invention provides an
advantage as a starting material, as it essentially contains no
excipient. Any excipient(s) which is added to the formulation
following the diafiltration in water can be accurately calculated,
i.e., pre-existing concentrations of excipient(s) do not interfere
with the calculation. Examples of pharmaceutically acceptable
excipients are described in Remington's Pharmaceutical Sciences
16th edition, Osol, A. Ed. (1980), incorporated by reference
herein. Thus, another aspect of the invention includes using the
aqueous formulation obtained through the methods described herein,
for the preparation of a formulation, particularly a pharmaceutical
formulation, having known concentrations of excipient(s), including
non-ionic excipient(s) or ionic excipient(s). One aspect of the
invention includes an additional step where an excipient(s) is
added to the aqueous formulation comprising water and protein.
Thus, the methods of the invention provide an aqueous formulation
which is essentially free of excipients and may be used as a
starting material for preparing formulations comprising water,
proteins, and specific concentrations of excipients.
[0175] In one embodiment, the methods of the invention may be used
to add non-ionic excipients, e.g., sugars or non-ionic surfactants,
such as polysorbates and poloxamers, to the formulation without
changing the characteristics, e.g., protein concentration,
hydrodynamic diameter of the protein, conductivity, etc.
[0176] Additional characteristics and advantages of aqueous
formulations obtained using the above methods are described below
in section III. Exemplary protocols for performing the methods of
the invention are also described below in the Examples.
III. Formulations of Invention
[0177] The invention provide an aqueous formulation comprising a
protein and water which has a number of advantages over
conventional formulations in the art, including stability of the
protein in water without the requirement for additional excipients,
increased concentrations of protein without the need for additional
excipients to maintain solubility of the protein, and low
osmolality. The formulations of the invention also have
advantageous storage properties, as the proteins in the formulation
remain stable during storage, e.g., stored as a liquid form for
more than 3 months at 7.degree. C. or freeze/thaw conditions, even
at high protein concentrations and repeated freeze/thaw processing
steps. In one embodiment, formulations of the invention include
high concentrations of proteins such that the aqueous formulation
does not show significant opalescence, aggregation, or
precipitation.
[0178] The aqueous formulation of the invention does not rely on
standard excipients, e.g., a tonicity modifier, a stabilizing
agent, a surfactant, an anti-oxidant, a cryoprotectant, a bulking
agent, a lyroprotectant, a basic component, and an acidic
component. In other embodiments of the invention, the formulation
contains water, one or more proteins, and no ionic excipients
(e.g., salts, free amino acids).
[0179] In certain embodiments, the aqueous formulation of the
invention comprises a protein concentration of at least 50 mg/mL
and water, wherein the formulation has an osmolality of no more
than 30 mOsmol/kg. Lower limits of osmolality of the aqueous
formulation are also encompassed by the invention. In one
embodiment the osmolality of the aqueous formulation is no more
than 15 mOsmol/kg. The aqueous formulation of the invention may
have an osmolality of less than 30 mOsmol/kg, and also have a high
protein concentration, e.g., the concentration of the protein is at
least 100 mg/mL, and may be as much as 200 mg/mL or greater. Ranges
intermediate to the above recited concentrations and osmolality
units are also intended to be part of this invention. In addition,
ranges of values using a combination of any of the above recited
values as upper and/or lower limits are intended to be
included.
[0180] The concentration of the aqueous formulation of the
invention is not limited by the protein size and the formulation
may include any size range of proteins. Included within the scope
of the invention is an aqueous formulation comprising at least 50
mg/mL and as much as 200 mg/mL or more of a protein, which may
range in size from 5 kDa to 150 kDa or more. In one embodiment, the
protein in the formulation of the invention is at least about 15 kD
in size, at least about 20 kD in size; at least about 47 kD in
size; at least about 60 kD in size; at least about 80 kD in size;
at least about 100 kD in size; at least about 120 kD in size; at
least about 140 kD in size; at least about 160 kD in size; or
greater than about 160 kD in size. Ranges intermediate to the above
recited sizes are also intended to be part of this invention. In
addition, ranges of values using a combination of any of the above
recited values as upper and/or lower limits are intended to be
included.
[0181] The aqueous formulation of the invention may be
characterized by the hydrodynamic diameter (D.sub.h) of the
proteins in solution. The hydrodynamic diameter of the protein in
solution may be measured using dynamic light scattering (DLS),
which is an established analytical method for determining the
D.sub.h of proteins. Typical values for monoclonal antibodies,
e.g., IgG, are about 10 nm. Low-ionic formulations, like those
described herein, may be characterized in that the D.sub.h of the
proteins are notably lower than protein formulations comprising
ionic excipients. It has been discovered that the D.sub.h values of
antibodies in aqueous formulations made using the DF/UF process
using pure water as an exchange medium, are notably lower than the
D.sub.h of antibodies in conventional formulations independent of
protein concentration. In one embodiment, antibodies in the aqueous
formulation of the invention have a D.sub.h of less than 4 nm, or
less than 3 nm.
[0182] In one embodiment, the D.sub.h of the protein in the aqueous
formulation is smaller relative to the D.sub.h of the same protein
in a buffered solution, irrespective of protein concentration.
Thus, in certain embodiments, protein in an aqueous formulation
made in accordance with the methods described herein, will have a
D.sub.h which is at least 25% less than the D.sub.h of the protein
in a buffered solution at the same given concentration. Examples of
buffered solutions include, but are not limited to phosphate
buffered saline (PBS). In certain embodiments, proteins in the
aqueous formulation of the invention have a D.sub.h that is at
least 50% less than the D.sub.h of the protein in PBS in at the
given concentration; at least 60% less than the D.sub.h of the
protein in PBS at the given concentration; at least 70% less than
the D.sub.h of the protein in PBS at the given concentration; or
more than 70% less than the D.sub.h of the protein in PBS at the
given concentration. Ranges intermediate to the above recited
percentages are also intended to be part of this invention, e.g.,
55%, 56%, 57%, 64%, 68%, and so forth. In addition, ranges of
values using a combination of any of the above recited values as
upper and/or lower limits are intended to be included, e.g., 50% to
80%.
[0183] Protein aggregation is a common problem in protein
solutions, and often results from increased concentration of the
protein. The instant invention provides a means for achieving a
high concentration, low protein aggregation formulation.
Formulations of the invention do not rely on a buffering system and
excipients, including surfactants, to keep proteins in the
formulation soluble and from aggregating. Formulations of the
invention can be advantageous for therapeutic purposes, as they are
high in protein concentration and water-based, not relying on other
agents to achieve high, stable concentrations of proteins in
solution.
[0184] The majority of biologic products (including antibodies) are
subject to numerous degradative processes which frequently arise
from non-enzymatic reactions in solution. These reactions may have
a long-term impact on product stability, safety and efficacy. These
instabilities can be retarded, if not eliminated, by storage of
product at subzero temperatures, thus gaining a tremendous
advantage for the manufacturer in terms of flexibility and
availability of supplies over the product life-cycle. Although
freezing is often the safest and most reliable method of biologics
product storage, it has inherent risks. Freezing can induce stress
in proteins through cold denaturation, by introducing ice-liquid
interfaces, and by freeze-concentration (cryoconcentration) of
solutes when the water crystallizes.
[0185] Cryoconcentration is a process in which a flat, uncontrolled
moving ice front is formed during freezing that excludes solute
molecules (small molecules such as sucrose, salts, and other
excipients typically used in protein formulation, or macromolecules
such as proteins), leading to zones in which proteins may be found
at relatively high concentration in the presence of other solutes
at concentrations which may potentially lead to local pH or ionic
concentration extremes. For most proteins, these conditions can
lead to denaturation and in some cases, protein and solute
precipitation. Since buffer salts and other solutes are also
concentrated under such conditions, these components may reach
concentrations high enough to lead to pH and/or redox changes in
zones within the frozen mass. The pH shifts observed as a
consequence of buffer salt crystallization (e.g., phosphates) in
the solutions during freezing can span several pH units, which may
impact protein stability.
[0186] Concentrated solutes may also lead to a depression of the
freezing point to an extent where the solutes may not be frozen at
all, and proteins will exist within a solution under these adverse
conditions. Often, rapid cooling may be applied to reduce the time
period the protein is exposed to these undesired conditions.
However, rapid freezing can induce a large-area ice-water
interface, whereas slow cooling induces smaller interface areas.
For instance, rapid cooling of six model proteins during one
freeze/thaw step was shown to reveal a denaturation effect greater
than 10 cycles of slow cooling, demonstrating the great
destabilization potential of hydrophobic ice surface-induced
denaturation.
[0187] The aqueous formulation of the invention has advantageous
stability and storage properties. Stability of the aqueous
formulation is not dependent on the form of storage, and includes,
but is not limited to, formulations which are frozen, lyophilized,
or spray-dried. Stability can be measured at a selected temperature
for a selected time period. In one aspect of the invention, the
protein in the aqueous formulations is stable in a liquid form for
at least 3 months; at least 4 months, at least 5 months; at least 6
months; at least 12 months. Ranges intermediate to the above
recited time periods are also intended to be part of this
invention, e.g., 9 months, and so forth. In addition, ranges of
values using a combination of any of the above recited values as
upper and/or lower limits are intended to be included. Preferably,
the formulation is stable at room temperature (about 30.degree. C.)
or at 40.degree. C. for at least 1 month and/or stable at about
2-8.degree. C. for at least 1 year, or more preferably stable at
about 2-8.degree. C. for at least 2 years. Furthermore, the
formulation is preferably stable following freezing (to, e.g.,
-80.degree. C.) and thawing of the formulation, hereinafter
referred to as a "freeze/thaw cycle."
[0188] Stability of a protein can be also be defined as the ability
to remain biologically active. A protein "retains its biological
activity" in a pharmaceutical formulation, if the protein in a
pharmaceutical formulation is biologically active upon
administration to a subject. For example, biological activity of an
antibody is retained if the biological activity of the antibody in
the pharmaceutical formulation is within about 30%, about 20%, or
about 10% (within the errors of the assay) of the biological
activity exhibited at the time the pharmaceutical formulation was
prepared (e.g., as determined in an antigen binding assay).
[0189] Stability of a protein in an aqueous formulation may also be
defined as the percentage of monomer, aggregate, or fragment, or
combinations thereof, of the protein in the formulation. A protein
"retains its physical stability" in a formulation if it shows
substantially no signs of aggregation, precipitation and/or
denaturation upon visual examination of color and/or clarity, or as
measured by UV light scattering or by size exclusion
chromatography. In one aspect of the invention, a stable aqueous
formulation is a formulation having less than about 10%, and
preferably less than about 5% of the protein being present as
aggregate in the formulation.
[0190] Another characteristic of the aqueous formulation of the
invention is that, in some instances, diafiltering a protein using
water results in an aqueous formulation having improved viscosity
features in comparison to the first protein solution (i.e., the
viscosity of the diafiltered protein solution is reduced in
comparison to the first protein solution.) A person with skill in
the art will recognize that multiple methods for measuring
viscosity can be used in the preparation of formulations in various
embodiments of the invention. For example, kinematic viscosity data
(cSt) may be generated using capillaries. In other embodiments,
dynamic viscosity data is stated, either alone or with other
viscosity data. The dynamic viscosity data may be generated by
multiplying the kinematic viscosity data by the density.
[0191] In one embodiment, the invention also provides a method for
adjusting a certain characteristic, such as the osmolality and/or
viscosity, as desired in high protein concentration-water
solutions, by adding non-ionic excipients, such as mannitol,
without changing other desired features, such as non-opalescence.
As such, it is within the scope of the invention to include
formulations which are water-based and have high concentrations of
protein, where, either during or following the transfer of the
protein to water or during the course of the diafiltration,
excipients are added which improve, for example, the osmolality or
viscosity features of the formulation. Thus, it is also within the
scope of the invention that such non-ionic excipients could be
added during the process of the transfer of the protein into the
final low ionic formulation. Examples of non-ionizable excipients
which may be added to the aqueous formulation of the invention for
altering desired characteristics of the formulation include, but
are not limited to, mannitol, sorbitol, a non-ionic surfactant
(e.g., polysorbate 20, polysorbate 40, polysorbate 60 or
polysorbate 80), sucrose, trehalose, raffinose, and maltose.
[0192] The formulation herein may also contain more than one
protein. With respect to pharmaceutical formulations, an
additional, distinct protein may be added as necessary for the
particular indication being treated, preferably those with
complementary activities that do not adversely affect the other
protein. For example, it may be desirable to provide two or more
antibodies which bind to TNF or IL-12 in a single formulation.
Furthermore, anti-TNF or anti-IL12 antibodies may be combined in
the one formulation. Such proteins are suitably present in
combination in amounts that are effective for the purpose
intended.
[0193] Examples of proteins that may be included in the aqueous
formulation include antibodies, or antigen-binding fragments
thereof. Examples of different types of antibodies, or
antigen-binding fragments thereof, that may be used in the
invention include, but are not limited to, a chimeric antibody, a
human antibody, a humanized antibody, and a domain antibody (dAb).
In one embodiment, the antibody used in the methods and
compositions of the invention is an anti-TNF.alpha. antibody, or
antigen-binding portion thereof, or an anti-IL-12 antibody, or
antigen binding portion thereof. Additional examples of an
antibody, or antigen-binding fragment thereof, that may be used in
the invention includes, but is not limited to, 1D4.7
(anti-IL-12/anti-IL-23; Abbott Laboratories), 2.5(E)mg1
(anti-IL-18; Abbott Laboratories), 13C5.5 (anti-Il-13; Abbott
Laboratories), J695 (anti-IL-12; Abbott Laboratories), Afelimomab
(Fab 2 anti-TNF; Abbott Laboratories), Humira (adalimumab (D2E7);
Abbott Laboratories), Campath (Alemtuzumab), CEA-Scan Arcitumomab
(fab fragment), Erbitux (Cetuximab), Herceptin (Trastuzumab),
Myoscint (Imciromab Pentetate), ProstaScint (Capromab Pendetide),
Remicade (Infliximab), ReoPro (Abciximab), Rituxan (Rituximab),
Simulect (Basiliximab), Synagis (Palivizumab), Verluma
(Nofetumomab), Xolair (Omalizumab), Zenapax (Daclizumab), Zevalin
(Ibritumomab Tiuxetan), Orthoclone OKT3 (Muromonab-CD3), Panorex
(Edrecolomab), and Mylotarg (Gemtuzumab ozogamicin) golimumab
(Centocor), Cimzia (Certolizumab pegol), Soliris (Eculizumab), CNTO
1275 (ustekinumab), Vectibix (panitumumab), Bexxar (tositumomab and
I.sup.131 tositumomab) and Avastin (bevacizumab).
[0194] In one alternative, the protein is a therapeutic protein,
including, but not limited to, Pulmozyme (Dornase alfa), Regranex
(Becaplermin), Activase (Alteplase), Aldurazyme (Laronidase),
Amevive (Alefacept), Aranesp (Darbepoetin alfa), Becaplermin
Concentrate, Betaseron (Interferon beta-1b), BOTOX (Botulinum Toxin
Type A), Elitek (Rasburicase), Elspar (Asparaginase), Epogen
(Epoetin alfa), Enbrel (Etanercept), Fabrazyme (Agalsidase beta),
Infergen (Interferon alfacon-1), Intron A (Interferon alfa-2a),
Kineret (Anakinra), MYOBLOC (Botulinum Toxin Type B), Neulasta
(Pegfilgrastim), Neumega (Oprelvekin), Neupogen (Filgrastim), Ontak
(Denileukin diftitox), PEGASYS (Peginterferon alfa-2a), Proleukin
(Aldesleukin), Pulmozyme (Dornase alfa), Rebif (Interferon
beta-1a), Regranex (Becaplermin), Retavase (Reteplase), Roferon-A
(Interferon alfa-2), TNKase (Tenecteplase), and Xigris (Drotrecogin
alfa), Arcalyst (Rilonacept), NPlate (Romiplostim), Mircera
(methoxypolyethylene glycol-epoetin beta), Cinryze (C1 esterase
inhibitor), Elaprase (idursulfase), Myozyme (alglucosidase alfa),
Orencia (abatacept), Naglazyme (galsulfase), Kepivance (palifermin)
and Actimmune (interferon gamma-1b).
[0195] Other examples of proteins which may be included in the
methods and compositions described herein, include mammalian
proteins, including recombinant proteins thereof, such as, e.g.,
growth hormone, including human growth hormone and bovine growth
hormone; growth hormone releasing factor; parathyroid hormone;
thyroid stimulating hormone; lipoproteins; .alpha.-1-antitrypsin;
insulin A-chain; insulin B-chain; proinsulin; follicle stimulating
hormone; calcitonin; luteinizing hormone; glucagon; clotting
factors such as factor VIIIC, factor IX, tissue factor, and von
Willebrands factor; anti-clotting factors such as Protein C; atrial
natriuretic factor; lung surfactant; a plasminogen activator, such
as urokinase or tissue-type plasminogen activator (t-PA);
bombazine; thrombin; tumor necrosis factor-.alpha. and -.beta.
enkephalinase; RANTES (regulated on activation normally T-cell
expressed and secreted); human macrophage inflammatory protein
(MIP-1-.alpha.); serum albumin such as human serum albumin;
mullerian-inhibiting substance; relaxin A-chain; relaxin B-chain;
prorelaxin; mouse gonadotropin-associated peptide; DNase; inhibin;
activin; vascular endothelial growth factor (VEGF); receptors for
hormones or growth factors; an integrin; protein A or D; rheumatoid
factors; a neurotrophic factor such as bone-derived neurotrophic
factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT4, NT-5, or
NT-6), or a nerve growth factor such as NGF-.beta.;
platelet-derived growth factor (PDGF); fibroblast growth factor
such as aFGF and bFGF; epidermal growth factor (EGF); transforming
growth factor (TGF) such as TGF.alpha. and TGF-.beta., including
TGF-.beta. 1, TGF-.beta. 2, TGF-.beta. 3, TGF-..beta. 4, or
TGF-.beta. 5; insulin-like growth factor-I and -II (IGF-I and
IGF-II); des(1-3)-IGF-I (brain IGF-I); insulin-like growth factor
binding proteins; CD proteins such as CD3, CD4, CD8, CD19 and CD20;
erythropoietin (EPO); thrombopoietin (TPO); osteoinductive factors;
immunotoxins; a bone morphogenetic protein (BMP); an interferon
such as interferon-.alpha., -.beta.., and -.gamma..; colony
stimulating factors (CSFs), e.g., M-CSF, GM-CSF, and G-CSF;
interleukins (ILs), e.g., IL-1 to IL-10; superoxide dismutase;
T-cell receptors; surface membrane proteins; decay accelerating
factor (DAF); a viral antigen such as, for example, a portion of
the AIDS envelope; transport proteins; homing receptors;
addressins; regulatory proteins; immunoadhesins; antibodies; and
biologically active fragments or variants of any of the
above-listed polypeptides.
IV. Uses of Invention
[0196] The formulations of the invention may be used both
therapeutically, i.e., in vivo, or as reagents for in vitro or in
situ purposes.
Therapeutic Uses
[0197] The methods of the invention may also be used to make a
water-based formulation having characteristics which are
advantageous for therapeutic use. The aqueous formulation may be
used as a pharmaceutical formulation to treat a disorder in a
subject.
[0198] The formulation of the invention may be used to treat any
disorder for which the therapeutic protein is appropriate for
treating. A "disorder" is any condition that would benefit from
treatment with the protein. This includes chronic and acute
disorders or diseases including those pathological conditions which
predispose the mammal to the disorder in question. In the case of
an anti-TNF.alpha. antibody, a therapeutically effective amount of
the antibody may be administered to treat an autoimmune disease,
such as rheumatoid arthritis, an intestinal disorder, such as
Crohn's disease, a spondyloarthropathy, such as ankylosing
spondylitis, or a skin disorder, such as psoriasis. In the case of
an anti-IL-12 antibody, a therapeutically effective amount of the
antibody may be administered to treat a neurological disorder, such
as multiple sclerosis, or a skin disorder, such as psoriasis. Other
examples of disorders in which the formulation of the invention may
be used to treat include cancer, including breast cancer, leukemia,
lymphoma, and colon cancer.
[0199] The term "subject" is intended to include living organisms,
e.g., prokaryotes and eukaryotes. Examples of subjects include
mammals, e.g., humans, dogs, cows, horses, pigs, sheep, goats,
cats, mice, rabbits, rats, and transgenic non-human animals. In
specific embodiments of the invention, the subject is a human.
[0200] The term "treatment" refers to both therapeutic treatment
and prophylactic or preventative measures. Those in need of
treatment include those already with the disorder, as well as those
in which the disorder is to be prevented.
[0201] The aqueous formulation may be administered to a mammal,
including a human, in need of treatment in accordance with known
methods of administration. Examples of methods of administration
include intravenous administration, such as a bolus or by
continuous infusion over a period of time, intramuscular,
intraperitoneal, intracerobrospinal, subcutaneous, intra-articular,
intrasynovial, intrathecal, intradermal, transdermal, oral,
topical, or inhalation administration.
[0202] In one embodiment, the aqueous formulation is administered
to the mammal by subcutaneous administration. For such purposes,
the formulation may be injected using a syringe, as well as other
devices including injection devices (e.g., the Inject-ease and
Genject devices); injector pens (such as the GenPen); needleless
devices (e.g., MediJector and Biojectorr 2000); and subcutaneous
patch delivery systems. In one embdodiment, the device, e.g., a
syringe, autoinjector pen, contains a needle with a gauge ranging
in size from 25 G or smaller in diameter. In one embodiment, the
needle gauge ranges in size from 25G to 33 G (including ranges
intermediate thereto, e.g., 25sG, 26, 26sG, 27G, 28G, 29G, 30G,
31G, 32G, and 33G). In a preferred embodiment, the smallest needle
diameter and appropriate length is chosen in accordance with the
viscosity characteristics of the formulation and the device used to
deliver the formulation of the invention.
[0203] One advantage of the methods/compositions of the invention
is that they provide large concentrations of a protein in a
solution which may be ideal for administering the protein to a
subject using a needleless device. Such a device allows for
dispersion of the protein throughout the tissue of a subject
without the need for an injection by a needle. Examples of
needleless devices include, but are not limited to, Biojectorr 2000
(Bioject Medical Technologies), Cool.Click (Bioject Medical
Technologies), Iject (Bioject Medical Technologies), Vitajet 3,
(Bioject Medical Technologies), Mhi500 (The Medical House PLC),
Injex 30 (INJEX--Equidyne Systems), Injex 50 (INJEX--Equidyne
Systems), Injex 100 (INJEX-Equidyne Systems), Jet Syringe
(INJEX--Equidyne Systems), Jetinjector (Becton-Dickinson), J-Tip
(National Medical Devices, Inc.), Medi-Jector VISION (Antares
Pharma), MED-JET (MIT Canada, Inc.), DermoJet (Akra Dermojet),
Sonoprep (Sontra Medical Corp.), PenJet (PenJet Corp.), MicroPor
(Altea Therapeutics), Zeneo (Crossject Medical Technology),
Mini-Ject (Valeritas Inc.), ImplaJect (Caretek Medical LTD),
Intraject (Aradigm), and Serojet (Bioject Medical
Technologies).
[0204] Also included in the invention are delivery devices that
house the aqueous formulation. Examples of such devices include,
but are not limited to, a syringe, a pen (such as an autoinjector
pen), an implant, an inhalation device, a needleless device, and a
patch. An example of an autoinjection pen is described in U.S.
application Ser. No. 11/824,516, filed Jun. 29, 2007.
[0205] The invention also includes methods of delivering the
formulations of the invention by inhalation and inhalation devices
containing said formulation for such delivery. In one embodiment,
the aqueous formulation is administered to a subject via inhalation
using a nebulizer or liquid inhaler. Generally, nebulizers use
compressed air to deliver medicine as wet aerosol or mist for
inhalation, and, therefore, require that the drug be soluble in
water. Types of nebulizers include jet nebulizers (air-jet
nebulizers and liquid-jet nebulizers) and ultrasonic
nebulizers.
[0206] Examples of nebulizers include Akita.TM. (Activaero GmbH)
(see US2001037806, EP1258264). Akita.TM. is a table top nebulizer
inhalation system (Wt: 7.5 kg, B.times.W.times.H:
260.times.170.times.270) based on Paris LC Star that provides full
control over patient's breathing pattern. The device can deliver as
much as 500 mg drug in solution in less than 10 min with a very
high delivery rates to the lung and the lung periphery. 65% of the
nebulized particles are below 5 microns and the mass median
aerodynamic diameter (MMAD) is 3.8 microns at 1.8 bar. The minimum
fill volume is 2 mL and the maximum volume is 8 mL. The inspiratory
flow (200 mL/sec) and nebulizer pressure (0.3-1.8 bar) are set by
the smart card. The device can be individually adjusted for each
patient on the basis of a lung function test.
[0207] Another example of a nebulizer which may be used with
compositions of the invention includes the Aeroneb.RTM. Go/Pro/Lab
nebulizers (AeroGen). The Aeroneb.RTM. nebulizer is based on
OnQ.TM. technology, i.e., an electronic micropump (3/8 inch in
diameter and wafer-thin) comprised of a unique dome-shaped aperture
plate that contains over 1,000 precision-formed tapered holes,
surrounded by a vibrational element. Aeroneb.RTM. Go is a portable
unit for home use, whereas Aeroneb.RTM. Pro is a reusable and
autoclavable device for use in hospital and ambulatory clinic, and
Aeroneb.RTM. Lab is a device for use in pre-clinical aerosol
research and inhalation studies. The features of the systems
include optimization and customization of aerosol droplet size;
low-velocity aerosol delivery with a precisely controlled droplet
size, aiding targeted drug delivery within the respiratory system;
flexibility of dosing; accommodation of a custom single dose
ampoule containing a fixed volume of drug in solution or
suspension, or commercially available solutions for use in general
purpose nebulizers; continuous, breath-activated or programmable;
and adaptable to the needs of a broad range of patients, including
children and the elderly; single or multi-patient use.
[0208] Aerocurrent.TM. (AerovertRx corp) may also be used with
compositions of the invention (see WO2006006963). This nebulizer is
a portable, vibrating mesh nebulizer that features a disposable,
pre-filled or user filled drug cartridge.
[0209] Staccato.TM. (Alexza Pharma) may also be used with
compositions of the invention (see WO03095012). The key to
Staccato.TM. technology is vaporization of a drug without thermal
degradation, which is achieved by rapidly heating a thin film of
the drug. In less than half a second, the drug is heated to a
temperature sufficient to convert the solid drug film into a vapor.
The inhaler consists of three core components: a heating substrate,
a thin film of drug coated on the substrate, and an airway through
which the patient inhales. The inhaler is breath-actuated with
maximum dose delivered to be 20-25 mg and MMAD in the 1-2 micron
range.
[0210] AERx.RTM. (Aradigm) may also be used with compositions of
the invention (see WO9848873, U.S. Pat. No. 5,469,750, U.S. Pat.
No. 5,509,404, U.S. Pat. No. 5,522,385, U.S. Pat. No. 5,694,919,
U.S. Pat. No. 5,735,263, U.S. Pat. No. 5,855,564). AERx.RTM. is a
hand held battery operated device which utilizes a piston mechanism
to expel formulation from the AERx.RTM. Strip. The device monitors
patients inspiratory air flow and fires only when optimal breathing
pattern is achieved. The device can deliver about 60% of the dose
as emitted dose and 50-70% of the emitted dose into deep lung with
<25% inter-subject variability.
[0211] Another example of a nebulizer device which may also be used
with compositions of the invention includes Respimat.RTM.
(Boehringer). Respimat.RTM. is a multi-dose reservoir system that
is primed by twisting the device base, which is compressed a spring
and transfers a metered volume of formulation from the drug
cartridge to the dosing chamber. When the device is actuated, the
spring is released, which forces a micro-piston into the dosing
chamber and pushes the solution through a uniblock; the uniblock
consists of a filter structure with two fine outlet nozzle
channels. The MMAD generated by the Respimat.RTM. is 2 um, and the
device is suitable for low dose drugs traditionally employed to
treat respiratory disorders.
[0212] Compositions of the invention may also be delivered using
the Collegium Nebulizer.TM. (Collegium Pharma), which is a
nebulizer system comprised of drug deposited on membrane. The
dosage form is administered to a patient through oral or nasal
inhalation using the Collegium Nebulizer after reconstitution with
a reconstituting solvent.
[0213] Another example of a nebulizer device which may also be used
with compositions of the invention includes the Inspiration.RTM.
626 (Respironics). The 626 is a compressor based nebulizer for home
care. The 626 delivers a particle size between 0.5 to 5
microns.
[0214] Nebulizers which can be used with compositions of the
invention may include Adaptive Aerosol Delivery.RTM. technology
(Respironics), which delivers precise and reproducible inhaled drug
doses to patients regardless of the age, size or variability in
breathing patterns of such patients. AAD.RTM. systems incorporate
electronics and sensors within the handpiece to monitor the
patient's breathing pattern by detecting pressure changes during
inspiration and expiration. The sensors determine when to pulse the
aerosol delivery of medication during the first part of
inspiration. Throughout the treatment, the sensors monitor the
preceding three breaths and adapt to the patient's inspiratory and
expiratory pattern. Because AAD.RTM. systems only deliver
medication when the patient is breathing through the mouthpiece,
these devices allow the patient to take breaks in therapy without
medication waste. Examples of AAD.RTM. system nebulizers include
the HaloLite.RTM. AAD.RTM., ProDose.RTM. AAD.RTM., and I-Neb.RTM.
AAD.RTM..
[0215] The HaloLite.RTM. Adaptive Aerosol Delivery (AAD).RTM.
(Respironics) is a pneumatic aerosolisation system powered by a
portable compressor. The AAD.RTM. technology monitors the patient's
breathing pattern (typically every ten milliseconds) and, depending
upon the system being used, either releases pulses of aerosolized
drug into specific parts of the inhalation, or calculates the dose
drawn during inhalation from a "standing aerosol cloud" (see EP
0910421, incorporated by reference herein).
[0216] The ProDos AAD.RTM. (Respironics) is a nebulizing system
controlled by "ProDose Disc.TM." system. (Respironics). ProDos
AAD.RTM. is a pneumatic aerosol system powered by a portable
compressor, in which the dose to be delivered is controlled by a
microchip-containing disc inserted in the system that, among other
things, instructs the system as to the dose to deliver. The ProDose
Disc.TM. is a plastic disc containing a microchip, which is
inserted into the ProDose AAD.RTM. System and instructs it as to
what dose to deliver, the number of doses, which may be delivered
together with various control data including drug batch code and
expiry date (see EP1245244, incorporated by reference herein).
Promixin.RTM. can be delivered via Prodose AAD.RTM. for management
of pseudomonas aeruginosa lung infections, particularly in cystic
fibrosis. Promixin.RTM. is supplied as a powder for nebulization
that is reconstituted prior to use.
[0217] The I-neb AAD.RTM. is a handheld AAD.RTM. system that
delivers precise and reproducible drug doses into patients'
breathing patterns without the need for a separate compressor
("I-Neb"). The I-neb AAD.RTM. is a miniaturized AAD.RTM. inhaler
based upon a combination of electronic mesh-based aerosolisation
technology (Omron) and AAD.RTM. technology to control dosing into
patients' breathing patterns. The system is approximately the size
of a mobile telephone and weighs less than 8 ounces. I-neb AAD.RTM.
has been used for delivery of Ventavis.RTM. (iloprost)
(CoTherix/Schering AG).
[0218] Another example of a nebulizer which may be used with
compositions of the invention is Aria.TM. (Chrysalis). Aria is
based on a capillary aerosol generation system. The aerosol is
formed by pumping the drug formulation through a small,
electrically heated capillary. Upon exiting the capillary, the
formulation rapidly cooled by ambient air to produce an aerosol
with MMAD ranging from 0.5-2.0 um.
[0219] In addition the TouchSpray.TM. nebulizer (Odem) may be used
to deliver a composition of the invention. The TouchSpray.TM.
nebulizer is a hand-held device which uses a perforate membrane,
which vibrates at ultrasonic frequencies, in contact with the
reservoir fluid, to generate the aerosol cloud. The vibration
action draws jets of fluid though the holes in the membrane,
breaking the jets into drug cloud. The size of the droplets is
controlled by the shape/size of the holes as well as the surface
chemistry and composition of the drug solution. This device has
been reported to deliver 83% of the metered dose to the deep lung.
Details of the TouchSpray.TM. nebulizer are described in U.S. Pat.
No. 6,659,364, incorporated by reference herein.
[0220] Additional nebulizers which may be used with compositions of
the invention include nebulizers which are portable units which
maximize aerosol output when the patient inhales and minimize
aerosol output when the patient exhales using two one-way valves
(see PARI nebulizers (PARI GmbH). Baffles allow particles of
optimum size to leave the nebulizer. The result is a high
percentage of particles in the respirable range that leads to
improved drug delivery to the lungs. Such nebulizers may be
designed for specific patient populations, such a patients less
than three years of age (PARI BABY.TM.) and nebulizers for older
patients (PARI LC PLUS.RTM. and PARI LC STAR.RTM.).
[0221] An additional nebulizer which may be used with compositions
of the invention is the e-Flow.RTM. nebulizer (PARI GmbH) which
uses vibrating membrane technology to aerosolize the drug solution,
as well as the suspensions or colloidal dispersions (,
TouchSpray.TM.; ODEM (United Kingdom)). An e-Flow.RTM. nebulizer is
capable of handling fluid volumes from 0.5 ml to 5 ml, and can
produce aerosols with a very high density of active drug, a
precisely defined droplet size, and a high proportion of respirable
droplets delivered in the shortest possible amount of time. Drugs
which have been delivered using the e-Flow.RTM. nebulizer include
aztreonam and lidocaine. Additional details regarding the
e-Flow.RTM. nebulizer are described in U.S. Pat. No. 6,962,151,
incorporated by reference herein.
[0222] Additional nebulizers which may be used with compositions of
the invention include a Microair.RTM. electronic nebulizer (Omron)
and a Mystic.TM. nebulizer (Ventaira). The Microair.RTM. nebulizer
is extremely small and uses Vibrating Mesh Technology to
efficiently deliver solution medications. The Microair device has 7
mL capacity and produces drug particle MMAD size around 5 microns.
For additional details regarding the Microair.RTM. nebulizer see US
patent publication no. 2004045547, incorporated by reference
herein. The Mystic.TM. nebulizer uses strong electric field to
break liquid into a spray of nearly monodispersed, charged
particles. The Mystic.TM. system includes a containment unit, a
dose metering system, aerosol generation nozzles, and voltage
converters which together offer multi-dose or unit-dose delivery
options.
[0223] The Mystic.TM. device is breath activated, and has been used
with Corus 1030.TM. (lidocaine HCl), Resmycin.RTM. (doxorubicin
hydrochloride), Acuair (fluticasone propionate), NCE with
ViroPharm, and NCE with Pfizer. Additional details regarding the
Mystic.TM. nebulizer may be found in U.S. Pat. No. 6,397,838,
incorporated by reference herein.
[0224] Additional methods for pulmonary delivery of the formulation
of the invention are provided in U.S. application Ser. No.
12/217,972, incorporated by reference herein.
[0225] The appropriate dosage ("therapeutically effective amount")
of the protein will depend, for example, on the condition to be
treated, the severity and course of the condition, whether the
protein is administered for preventive or therapeutic purposes,
previous therapy, the patient's clinical history and response to
the protein, the type of protein used, and the discretion of the
attending physician. The protein is suitably administered to the
patient at one time or over a series of treatments and may be
administered to the patient at any time from diagnosis onwards. The
protein may be administered as the sole treatment or in conjunction
with other drugs or therapies useful in treating the condition in
question.
[0226] The formulations of the invention overcome the common
problem of protein aggregation often associated with high
concentrations of protein, and, therefore, provide a new means by
which high levels of a therapeutic protein may be administered to a
patient. The high concentration formulation of the invention
provides an advantage in dosing where a higher dose may be
administered to a subject using a volume which is equal to or less
than the formulation for standard treatment. Standard treatment for
a therapeutic protein is described on the label provided by the
manufacturer of the protein. For example, in accordance with the
label provided by the manufacturer, infliximab is administered for
the treatment of rheumatoid arthritis by reconstituting lyophilized
protein to a concentration of 10 mg/mL. The formulation of the
invention may comprise a high concentration of infliximab, where a
high concentration would include a concentration higher than the
standard 10 mg/mL. In another example, in accordance with the label
provided by the manufacturer, Xolair (omalizumab) is administered
for the treatment of asthma by reconstituting lyophilized protein
to a concentration of 125 mg/mL. In this instance, the high
concentration formulation of the invention would include a
concentration of the antibody omalizumab which is greater than the
standard 125 mg/mL.
[0227] Thus, in one embodiment, the formulation of the invention
comprises a high concentration which is at least about 10%, at
least about 20%, at least about 30%, at least about 40%, at least
about 50%, at least about 60%, at least about 70%, at least about
80%, at least about 90%, at least about 100%, at least about 110%,
at least about 120%, at least about 130%, at least about 140%, at
least about 150%, at least about 175%, at least about 200%, at
least about 225%, at least about 250%, at least about 275%, at
least about 300%, at least about 325%, at least about 350%, at
least about 375%, at least about 400%, and so forth, greater than
the concentration of a therapeutic protein in a known, standard
formulation.
[0228] In another embodiment, the formulation of the invention
comprises a high concentration which is at least about 2 times
greater than, at least about 3 times greater than, at least about 4
times greater than, at least about 5 times greater than, at least
about 6 times greater than, at least about 7 times greater than, at
least about 8 times greater than, at least about 9 times greater
than, at least about 10 times greater than and so forth, the
concentration of a therapeutic protein in a known, standard
formulation.
[0229] Characteristics of the aqueous formulation may be improved
for therapeutic use. For example, the viscosity of an antibody
formulation may be improved by subjecting an antibody protein
solution to diafiltration using water without excipients as the
diafiltration medium. As described above in Section II, excipients,
such as those which improve viscosity, may be added back to the
aqueous formulation such that the final concentration of excipient
is known and the specific characteristic of the formulation is
improved for the specified use. For example, one of skill in the
art will recognize that the desired viscosity of a pharmaceutical
formulation is dependent on the mode by which the formulation is
being delivered, e.g., injected, inhaled, dermal absorption, and so
forth. Often the desired viscosity balances the comfort of the
subject in receiving the formulation and the dose of the protein in
the formulation needed to have a therapeutic effect. For example,
generally acceptable levels of viscosity for formulations being
injected are viscosity levels of less than about 100 mPas,
preferentially less than 75 mPas, even more preferentially less
than 50 mPas. As such, viscosity of the aqueous formulation may be
acceptable for therapeutic use, or may require addition of an
excipient(s) to improve the desired characteristic.
[0230] In one embodiment, the invention provides an aqueous
formulation comprising water and a human TNF.alpha. antibody, or
antigen-binding portion thereof, wherein the formulation is
excipient-free, wherein the formulation has viscosity which makes
it advantageous for use as a therapeutic, e.g., low viscosity of
less than 40 cP at 8.degree. C., and less than 25 cP at 25.degree.
C. when the protein concentration is about 175 mg/mL. In one
embodiment, the concentration of the antibody, or antigen-binding
portion thereof, in a formulation having improved viscosity is at
least about 50 mg/mL. In one embodiment, the formulation of the
invention has a viscosity ranging between about 1 and about 2
mPas.
Non-Therapeutic Uses
[0231] The aqueous formulation of the invention may also be used
for non-therapeutic uses, i.e., in vitro purposes.
[0232] Aqueous formulations described herein may be used for
diagnostic or experimental methods in medicine and biotechnology,
including, but not limited to, use in genomics, proteomics,
bioinformatics, cell culture, plant biology, and cell biology. For
example, aqueous formulations described herein may be used to
provide a protein needed as a molecular probe in a labeling and
detecting methods. An additional use for the formulations described
herein is to provide supplements for cell culture reagents,
including cell growth and protein production for manufacturing
purposes.
[0233] Aqueous formulations described herein could be used in
protocols with reduced concern regarding how an excipient in the
formulation may react with the experimental environment, e.g.,
interfere with another reagent being used in the protocol. In
another example, aqueous formulations containing high
concentrations of proteins may be used as a reagent for laboratory
use. Such highly concentrated forms of a protein would expand the
current limits of laboratory experiments.
[0234] Another alternative use for the formulation of the invention
is to provide additives to food products. Because the aqueous
formulation of the invention consists essentially of water and
protein, the formulation may be used to deliver high concentrations
of a desired protein, such as a nutritional supplement, to a food
item. The aqueous formulation of the invention provides a high
concentration of the protein in water, without the concern for
excipients needed for stability/solubility which may not be
suitable for human consumption. For example, whey- and soy-derived
proteins are lending versatility to foods as these proteins have an
ability to mimic fat's mouthfeel and texture. As such, whey- and
soy-derived proteins may be added to foods to decrease the overall
fat content, without sacrificing satisfaction. Thus, an aqueous
formulation comprising suitable amounts of whey- and soy-derived
proteins may be formulated and used to supplement food
products.
Articles of Manufacture
[0235] In another embodiment of the invention, an article of
manufacture is provided which contains the aqueous formulation of
the present invention and provides instructions for its use. The
article of manufacture comprises a container. Suitable containers
include, for example, bottles, vials (e.g., dual chamber vials),
syringes (such as dual chamber syringes), autoinjector pen
containing a syringe, and test tubes. The container may be formed
from a variety of materials such as glass, plastic or
polycarbonate. The container holds the aqueous formulation and the
label on, or associated with, the container may indicate directions
for use. For example, the label may indicate that the formulation
is useful or intended for subcutaneous administration. The
container holding the formulation may be a multi-use vial, which
allows for repeat administrations (e.g., from 2-6 administrations)
of the aqueous formulation. The article of manufacture may further
comprise a second container. The article of manufacture may further
include other materials desirable from a commercial and user
standpoint, including other buffers, diluents, filters, needles,
syringes, and package inserts with instructions for use.
[0236] The contents of all references, patents and published patent
applications cited throughout this application are incorporated
herein by reference
[0237] This invention is further illustrated by the following
examples which should not be construed as limiting.
EXAMPLES
[0238] The following examples describe experiments relating to an
aqueous formulation comprising water as the solution medium. It
should be noted that in some instances, decimal places are
indicated using European decimal notation. For example, in Table 31
the number "0,296" is synonymous with "0.296".
Example 1: Diafiltration/Ultrafiltration with Adalimumab and
J695
Materials and Methods
[0239] Adalimumab and J695 were diafiltered using pure water. After
an at least 5-fold volume exchange with pure water, the protein
solutions were ultrafiltered to a final target concentration of at
least 150 mg/mL. Osmolality, visual inspection and protein
concentration measurements (OD280) were performed to monitor the
status of the proteins during DF/UF processing.
[0240] Size exclusion chromatography and ion exchange
chromatography were used to characterize protein stability in each
final DF/UF product as compared to the starting formulation, e.g.,
drug substance (DS) starting material and protein standard. Drug
substance or "DS" represents the active pharmaceutical ingredient
and generally refers to a therapeutic protein in a common bulk
solution. [0241] Adalimumab Drug Substance, (Adalimumab extinction
coefficient 280 nm: 1.39 mL/mg cm). Drug Substance did not contain
polysorbate 80. DS composition: 5.57 mM sodium phosphate monobasic,
8.69 mM sodium phosphate dibasic, 106.69 mM sodium chloride, 1.07
mM sodium citrate, 6.45 mM citric acid, 66.68 mM mannitol. [0242]
Adalimumab solution used for dynamic light scattering (DLS)
measurements: Adalimumab solution that was diafiltered using pure
water as exchange medium was adjusted to 1 mg/mL concentration by
diluting the Adalimumab solution with Milli-Q water and excipient
stock solutions (excipients dissolved in Milli-Q water),
respectively. [0243] J695 Drug Substance, (J695 extinction
coefficient 280 nm: 1.42 mL/mg cm). DS composition: Histidine,
Methionine, Mannitol, pH 5.8, and polysorbate 80. [0244] Millipore
Labscale.TM. Tangential Flow Filtration (TFF) system, equipped with
a 500 mL reservoir. The Labscale TFF system was operated in
discontinuous mode at ambient temperature according to Millipore
Operating Instructions. Stirrer speed was set to approx. 1.5, and
the pump speed was set to approximately 3. The target inlet and
outlet pressures were 15 mm psig approximately 50 mm psig,
respectively. [0245] Minimate.TM. Tangential Flow Filtration
capsule, equipped with an Omega.TM. PES membrane, 30 kDa cut-off.
The capsule was rinsed for 30 min with 0.1 N NaOH and for another
30 min with Milli-Q water. [0246] 780 pH meter, Metrohm, equipped
with pH probe Pt1000, No. 6.0258.010, calibrated with buffer
calibration solutions VWR, pH 4.00 buffer solution red, Cat. No.
34170-127, and pH 7.00 buffer solution yellow, Cat. No. 34170-130.
[0247] Varian 50 Bio UV visible spectrophotometer, AI 9655, with a
fixed Cary 50 cell was used for protein concentration measurements
(280 nm wavelength). A 100 .mu.L protein sample was diluted with
water (Milli-Q water for HPLC) to a final volume of 50.00 mL for
protein concentration measurements of all J695 samples and the
Adalimumab solution after DF/UF. Concentration of all other
Adalimumab samples was monitored by diluting 40 .mu.L sample
solution with 1960 .mu.L Milli-Q water. Disposable UV cuvettes, 1.5
mL, semi-micro, Poly(methyl methacrylate) (PMMA), were used for
concentration measurements, Milli-Q water was used as OD 280 blank.
[0248] Milli-Q water for HPLC grade was used as DF/UF medium.
[0249] A Malvern Zetasizer Nano ZS, Instrument No. AI 9494 was used
for DLS measurements. [0250] Helima precision cells, suprasil, Type
No. 105.251-QS, light path 3 mm, center 8.5, were used for DLS
measurements (filled with 75 .mu.L sample, Malvern Mastersizer Nano
ZS, Item No. AI 9494). [0251] Knauer Osmometer Automatic, Instr.
No. 83963, Berlin, Germany, was used for osmolality measurement
(calibrated with 400 mOsmol/kg NaCl calibration solution, Art. No.
Y1241, Herbert Knauer GmbH, Berlin, Germany) [0252] 250 mL Corning
cell culture flasks, 75 cm.sup.2, polystyrene, sterile, Corning,
N.Y., USA, were used for storage of the protein solutions after the
DF/UF operation. [0253] Sodium chloride: J. T. Baker was used for
preparing a 2M NaCl stock solution. The stock solution was used to
prepare 1 mg/mL Adalimumab solution in pure water with various
concentrations of NaCl (10, 20, 30, and 50 mM) [0254] D-sorbitol,
Sigma Chemical Co., St. Louis, Mo. 63178 was used for preparing a
200 mg/mL sorbitol stock solution. The stock solution was used to
prepare 1 mg/mL Adalimumab solution in pure water with various
concentrations of sorbitol (10, 20, 30, and 40 mg/mL).
HPLC Methods
[0254] [0255] Adalimumab, SEC analysis: Sephadex 200 column
(Pharmacia Cat. No. 175175-01, S/N 0504057). Mobile phase 20 mM
sodium phosphate, 150 mM sodium chloride, pH 7.5, 0.5 mL/min flow
rate, ambient temperature, detection UV 214 nm and 280 nm. Each
sample was diluted to 1.0 mg/mL with Milli-Q water, sample
injection load 50 .mu.g (duplicate injection). [0256] Adalimumab,
IEC analysis: Dionex, Propac WCX-10 column (p/n 054993) along with
a corresponding guard column (p/n 054994). Separation conditions:
mobile phase A: 10 mM sodium phosphate, pH 7.5; mobile phase B 10
mM Sodium phosphate, 500 mM Sodium chloride, pH 5.5. 1.0 mL/min
flow rate, ambient temperature. Each sample was diluted to 1.0
mg/mL with Milli-Q water, sample injection load 100 .mu.g,
duplicate injection. [0257] J695, SEC analysis: Tosoh Bioscience
G3000swxl, 7.8 mm.times.30 cm, 5 .mu.m (Cat. No. 08541). Mobile
phase 211 mM Na.sub.2SO.sub.4/92 mM Na.sub.2HPO.sub.4, pH 7.0. 0.3
mL/min flow rate, ambient temperature, detection UV 214 nm and 280
nm. Each sample was diluted to 2.5 mg/mL with Milli-Q water, sample
injection load 50 .mu.g (duplicate injection). [0258] J695, IEC
analysis: Dionex, Propac WCX-10 column (p/n 054993) along with a
corresponding guard column (p/n 054994). Separation conditions:
mobile phase A: 10 mM Na.sub.2HPO.sub.4, pH 6.0; mobile phase B 10
mM Na.sub.2HPO.sub.4, 500 mM NaCl, pH 6.0. 1.0 mL/min flow rate,
35.degree. C. temperature. Each sample was diluted to 1.0 mg/mL
with Milli-Q water, sample injection load 100 .mu.g. J695 Reference
standard 29001BF was run in triplicate as a comparison and was
diluted to 1 mg/ml in Milli-Q water based on the concentration from
the Certificate of Analysis.
Calculation of the Protein Concentration
Calculation Formula:
[0259] E = - lg ( I I 0 ) = c d .fwdarw. c = E .times. d
##EQU00001##
.epsilon.--absorption coefficient c--concentration d--length of
cuvette that the light has to pass E--absorbance I.sub.0--initial
light intensity I--light intensity after passing through sample
Adalimumab = 1.39 mL mg .times. cm ##EQU00002## J 695 = 1.42 mL mg
.times. cm ##EQU00002.2## HSA = 1.042 mL mg .times. cm
##EQU00002.3##
1.1: DF/UF Processing of Adalimumab
[0260] DF/UF experiments are carried out following the standard
operating procedures of the DF/UF equipment manufacturers. For
example, the Millipore Labscale.TM. TFF system was equipped with a
500 mL reservoir and the system operated in discontinuous mode at
ambient temperature, in accord with Millipore operating
instructions. Stirrer speed was set to approximately 1.5, and the
pump speed was set to approximately 3. The target inlet and outlet
pressures were 15 mm psig and approximately 50 mm psig,
respectively, and the target pressures were monitored to ensure
that they were not exceeded.
[0261] A Minimate.TM. Tangential Flow Filtration capsule equipped
with an Omega.TM. PES membrane (Pall Corp., Port Washington, N.Y.),
30 kDa MWCO, was used. The capsule was rinsed for 30 min with 0.1 N
NaOH and for another 30 min with Milli-Q water before use.
[0262] Approximately 500 mL of Adalimumab solution were placed into
the TFF reservoir and DF/UF processing was started in discontinuous
mode. Table 1 provides details on the In-Process-Control (IPC) data
characterizing the DF/UF process.
TABLE-US-00001 TABLE 1 Overview on Adalimumab DF/UF Processing
Volume of Approx. volume of Adalimumab Adalimumab Milli-Q
Adalimumab solution concentration concentration Process water added
in retentate of retentate Osmolality of permeate Step (mL) (mL)
(mg/mL) (mOsmol/kg) (mg/mL) 1 500 54.66 305 -- 2 400 68.33 297 3.15
3 300 -- -- -- 4 250 550 43.73 169 1.39 5 300 4.45 6 250 550 47.27
93 2.58 7 250 -- -- -- 8 250 500 -- -- -- 9 250 -- -- -- 10 250 500
-- -- -- 11 250 -- -- -- 12 250 500 52.24 9 1.24 13 300 90.27 7.5
-- 14 130 213.87 -- 4.08 Fields filled with "--" indicate that no
IPC samples were pulled at that step.
[0263] The DF/UF processing was stopped after an approximate 5-fold
volume exchange (1 volume exchange accounting for approx. 250 mL
diafiltration medium). Assuming an ideal 100% excipient membrane
permeability, the theoretical final excipient concentration reached
by the experiment parameters applied is
C.sub.i(250/500).sup.5=0.03125*Ci, with Ci being the initial
concentration. The maximum excipient reduction was therefore
96.875% (if constant volume diafiltration would have been used, the
theoretical excipient reduction with 5 diafiltration volumes would
have been C.sub.i e.sup.-5=0.00674, i.e. an approximate 99.3%
maximum excipient reduction). Adalimumab solution was drained from
the TFF system to a 250 mL cell culture flask (low-volume rinse of
the TFF system was performed using WFI yielding a 175.05 mg/mL
concentration; without the rinse, the retentate concentration was
213.87 mg/mL). Samples were pulled for determination of pH,
osmolality and Adalimumab concentration. Additionally, samples were
pulled for characterization by SEC and IEC. Characteristic
parameters of the Adalimumab solution before and after DF/UF
processing, respectively, are listed in Table 2.
TABLE-US-00002 TABLE 2 Impact of DF/UF processing on Adalimumab
solution solution solution parameter before DF/UF after DF/UF pH
5.19 5.22 concentration (mg/mL) 54.66 175.05 osmolality (mOsmol/kg)
305 24 *SEC data (% aggregate, 0.26 00.50 monomer, 99.74 99.50
fragment) 0.00 0.00 *IEC data (acidic regions, 13.89 14.07 lys 0,
62.05 61.97 lys 1, 19.14 18.51 lys 2, %) 4.83 4.73 *samples were
subjected to one freeze/thaw step (-80.degree. C./25.degree. C.)
before analysis via SEC and IEC
[0264] In the course of DF/UF processing, Adalimumab concentration
exceeded 210 mg/mL. Throughout the experiment, the protein solution
remained clear, and no solution haziness or protein precipitation,
which would have indicated Adalimumab solubility limitations, was
observed. Compared to the original Adalimumab DS solution
(.about.55 mg/mL), Adalimumab solution diafiltered by using pure
water as DF/UF exchange medium revealed lower opalescence, despite
a more than 3-fold increase in protein concentration (.about.175
mg/mL).
1.2: Adalimumab Characterization Via Chromatography
[0265] FIG. 1 shows a SEC chromatogram of an Adalimumab reference
standard (Adalimumab standard (bottom line)) compared to the
Adalimumab drug standard solution before (middle line) and after
(top line) the DF/UF processing procedure. Note that all samples
were frozen at -80.degree. C. prior to analysis.
[0266] Table 3 also contains the IEC chromatogram data (note all
samples were frozen at -80.degree. C. prior to analysis).
TABLE-US-00003 TABLE 3 IEC Data of Various Adalimumab Samples %
Acidic % Acidic % 0 % 1 % 2 Sample Name Region 1 Region 2 Lys Lys
Lys Reference standard 2.69 11.66 60.77 19.42 5.40 Adalimumab DS
2.51 11.38 62.05 19.14 4.83 Adalimumab, after 2.26 11.81 61.97
18.51 4.73 DF/UF
1.3: Impact of Excipients on Adalimumab Hydrodynamic Diameter
D.sub.h)
[0267] It was previously determined that the hydrodynamic diameter
of J695, as determined by dynamic light scattering (DLS)
measurements, was notably decreased when formulating J695 into pure
water. J695 in WFI had a D.sub.h of .about.3 nm, far below the
values that are expected for immunoglobulins. Upon addition of low
amounts of ionizable NaCl, the Dh values increased to .about.10 nm
(independent of the NaCl concentration). Addition of non-ionizable
mannitol increased J695 solution osmolality, but had no effect on
J695 Dh.
[0268] In order to assess the impact of excipients on the
hydrodynamic diameter of Adalimumab that had been processed
according to the above DF/UF procedure, the Adalimumab solution
from the DF/UF experiment was used to formulate Adalimumab
solutions in pure water, but with varying levels of NaCl (0-50 mM)
and sorbitol (0-40 mg/mL), respectively. The impact of sorbitol (a
non-ionizable excipient) and NaCl (ionizable excipient)
concentrations on Dh of Adalimumab monomer is displayed in FIG.
2.
[0269] The hydrodynamic diameter of Adalimumab monomer in pure
water was 2.65 nm. The Adalimumab Dh response to salt and non-ionic
excipients was identical to the J695 response seen previously.
Adalimumab Dh was virtually not impacted by the presence of
sorbitol. Low concentrations of NaCl induced the monomer
hydrodynamic diameter to increase to expected levels of .about.11
nm. These findings demonstrate that protein hydrodynamic diameters
as measured by dynamic light scattering are crucially impacted by
the presence of ionizable excipients. Absence of ionizable
excipients also is linked to solution viscosities.
[0270] These findings have implications for high-concentrated
protein solutions: the lower the hydrodynamic diameter, the lower
the spatial volume proteins occupy. In high-concentration
scenarios, the viscosities of protein solutions that are prepared
by using water as DF/UF exchange medium will be substantially lower
than the viscosities of traditional protein formulations containing
considerable amounts of ionizable buffer excipients. The Adalimumab
data confirmed this, as viscosities of 200 mg/mL Adalimumab
solutions in water for injection were found to be well below 50
mPas, independent of pH (e.g. pH 4, pH 5, and pH 6). More data on
the effect of pH on D.sub.h can be found in Example 17 below.
[0271] Overall, these findings are useful in high-concentration
protein formulation activities, where viscosity related
manufacturing and dosing/delivery problems are well known.
Furthermore, these findings show that the osmolality values of
final Drug Product can be adjusted with non-ionizable excipients
such as sugars or sugar alcohols as desired without inducing an
increase in protein Dh and solution viscosity, respectively.
1.4: DF/UF Processing of J695 (Anti-IL12 Antibody)
[0272] Approximately 200 mL of J695 solution were adjusted to pH
4.4 with 1 M phosphoric acid and filled into the TFF reservoir (pH
adjustment was made to ensure a positive zeta-potential of J695
monomers and thus avoid a potential impact of uncharged protein
monomer on data). Then, 300 mL of Milli-Q water were added to the
TFF reservoir, and DF/UF processing was started in discontinuous
mode. 250 mL reservoir volume, 250 mL of Milli-Q water were added,
and DF/UF processing was started again. The DF/UF processing was
stopped after a total of 5 volume exchange steps were performed (1
volume exchange accounting for approx. 250 mL).
[0273] Assuming an ideal 100% excipient membrane permeability, the
theoretical final excipient concentration reached by the experiment
parameters applied is Ci(250/500)5=0.03125*Ci, with Ci being the
initial concentration. The maximum excipient reduction is therefore
96.875% (if constant volume diafiltration would have been used, the
theoretical excipient reduction with 5 diafiltration volumes would
have been Ci e-5=0.00674, i.e. an approx. 99.3% maximum excipient
reduction). J695 solution was drained from the TFF system to a 250
mL cell culture flask (no rinse of the TFF system was performed).
Samples were pulled for determination of pH, osmolality and J695
concentration. Additionally, samples were pulled for
characterization by SEC and IEC. Characteristic parameters of the
J695 solution before and after DF/UF processing, respectively, are
listed in Table 4.
TABLE-US-00004 TABLE 4 Impact of DF/UF Processing on J695 Solution
solution solution parameter before DF/UF after DF/UF pH 4.40 4.70
concentration (mg/mL) 122.9 192.8 osmolality (mOsmol/kg) 265 40 SEC
data (% aggregate, 0.41 0.69 monomer, 98.42 98.11 fragment) 1.18
1.21 IEC data (sum of isoforms, 92.00 92.11 acidic species, 5.17
5.30 basic species, %) 2.83 2.59
[0274] As with Adalimumab, the DF/UF experiments on J695
substantiate the principal possibility of processing and
formulating J695 by using pure water as exchange medium in DF/UF
operations. Both SEC and IEC data suggest no substantial impact on
J695 stability while DF/UF processing for an overall period of 1.5
days (process interruption overnight) at ambient temperature using
Milli-Q water as diafiltration medium. Throughout the experiment,
the protein solution remained clear, indicating no potential J695
solubility limitations.
1.5: J695 Characterization
[0275] Table 5 describes the percentages for aggregate, monomer and
fragment content for the three solutions as determined by SEC
chromatogram.
TABLE-US-00005 TABLE 5 Data from SEC chromatogram % Aggregate %
Monomer % Fragment Sample Name content content content Reference
standard 0.45 98.00 1.56 J695 before DF/UF 0.41 98.42 1.18 J695
after DF/UF 0.69 98.11 1.21
[0276] FIG. 3 shows the IEC profile of J695 reference standard
(bottom graph) and J695 DS, pH adjusted to pH 4.4 (top graph).
[0277] Only a small increase in aggregate content was observed in
the J695 samples after DF/UF processing.
[0278] FIG. 4 shows the IEC profile of J695 after DF/UF with
Milli-Q water, pH 4.7 (top graph), and J695 DS before DF/UF, pH
adjusted to pH 4.4 (bottom curve). As depicted in FIG. 4, the DF/UF
step had no notable impact on J695 stability when monitored by IEC.
The differences between the two J695 reference standards (refer to
FIG. 3) can be attributed to differences in the manufacturing
processes between the 3000 L and 6000 L DS campaigns. Table 6
highlights more details on IEC data.
TABLE-US-00006 TABLE 6 IEC Data of Various J695 Samples other
Sample Name 0 glu (1) isoforms acidic basic Reference standard
43.57 50.06 4.47 1.90 J695 35.74 56.26 5.17 2.83 J695 after DF/UF
36.59 55.52 5.30 2.59
1.6: Conclusion
[0279] The above example provides a diafiltration/ultrafiltration
(DF/UF) experiment where water (Milli-Q water for HPLC) was used as
diafiltration medium for monoclonal antibodies Adalimumab and
J695.
[0280] Adalimumab was subjected to DF/UF processing by using pure
water as DF/UF exchange medium and was formulated at pH 5.2 at high
concentrations (>200 mg/mL) without inducing solution haziness,
severe opalescence or turbidity formation. Upon one subsequent
freeze/thaw step, SEC and IEC data suggested no notable difference
between Adalimumab solution formulated in water via DF/UF
processing and the original Adalimumab DS.
[0281] J695 also was also subjected to DF/UF processing by using
pure water as DF/UF exchange medium and was formulated at pH 4.7
without impacting J695 stability (visual inspection, SEC, IEC).
[0282] When formulated using such a DF/UF processing, the
hydrodynamic diameter (Dh) of Adalimumab monomer was approx. 2.65
nm. The presence of non-ionic excipients such as sorbitol in
concentrations up to 40 mg/mL was shown to have no impact on Dh
data, whereas ionic excipients such as NaCl already in low
concentrations were demonstrated to induce the Adalimumab monomer
Dh to increase to approx. 11 nm (such Dh data are commonly
monitored for IgG). Similar findings were made earlier for
J695.
[0283] In conclusion, processing and formulating proteins using
pure water as DF/UF exchange medium is feasible. Assuming an ideal
100% excipient membrane permeability, the theoretical final
excipient concentration reached by the constant volume
diafiltration with 5 diafiltration volumes would be Ci e-5=0.00674,
i.e. an approx. 99.3% maximum excipient reduction. Using 6
diafiltration volume exchanges, an theoretical .about.99.98%
maximum excipient reduction would result.
[0284] Examples 2 to 5 describe experimental execution with respect
to three different proteins which were concentrated into an aqueous
formulation, and Examples 6 to 11 describe analysis of each of the
aqueous formulations.
Materials and Methods for Examples 2-11
[0285] Adalimumab protein solution (10 mg/mL) in water for
injection, Protein Drug Substance (DS) Material (49.68 mg/mL), DS
contains Tween 80, Adalimumab, Adalimumab Drug Product (DP) (40 mg,
solution for injection, filtered solution from commercial
production). Protein absorption coefficient 280 nm: 1.39. [0286]
J695 protein solution (10 mg/mL) in water for injection, Protein
Drug Substance (DS) (54 mg/mL), DS contains Tween 80. Absorption
coefficient 280 nm: 1.42 [0287] HSA protein solution (10 mg/mL) in
water for injection, DP without Tween 80, Grifols Human Serum
Albumin Grifols.RTM., 20%, 50 mL. Absorption coefficient 280 nm:
1.042 [0288] 6 R vial and 10R vial [0289] vial preparation: the
vials were washed and autoclaved [0290] stoppers, 19 mm, West,
4110/40/grey [0291] sample repositories (e.g., Eppendorf sample
repository, Safe-Lock or simple snap-fit, 1-2 mL) [0292] single-use
syringes, sterile, 20 mL; NormJect, 10 mL [0293] single use filter
units (filter Millex.RTM.-GV, Syringe Driven Filter Unit, PVDF 0.22
.mu.m; Millex.RTM.-GP, Syringe Driven Filter Unit, PES 0.22 .mu.m,
Sterivex.RTM.0.22 .mu.m Filter Unit) [0294] Vivaspin concentrators
(cut off 10 kDa, PES; cut off 3 kDa, PES) [0295] Pipettes (e.g.,
Eppendorf, max.: 1000 .mu.L) [0296] Water for injection [0297]
Centrifuge (Eppendorf) and Centrifuge No. 1
(temperature-controlled) [0298] Diafiltration equipment: Millipore
Labscale.TM. TFF System, Millipore diafiltration membrane:
Adalimumab: Polyethersulfone; J695: Polyethersulfone; HSA:
regenerated cellulose [0299] pH probe (Metrohm pH-Meter,
protein-suitable probe, biotrode 46) [0300] Laminar-Air-Flow bench,
Steag LF-Werkbank, Mp.-No. 12.5 [0301] NaCl; mannitol [0302] Gemini
150 Peltier Plate Rheometer, Malvern [0303] Rheometer MCR 301
[temperature-controlled P-PTD 200 (plate with Peltiertempering)]
and cone/plate measurement system CP50/0.5.degree. Ti as well as
CP50/1.degree. (stainless steel); Anton Paar [0304] Capillary
viscometer, Schott, capillaries: type 537 20, type 537 10, type 537
13 [0305] 1M hydrochloric acid (J. T. Baker)
Analytics
[0305] [0306] UV/VIS spectrophotometry (OD 280 nm); Photon
Correlation Spectroscopy (PCS): for approximately 10 mg/mL and
approximately 20 mg/mL: 1.1 mPas, 3 runs, 30 s, one measurement,
25.degree. C., from approximately 30 mg/mL and above: 1.9 mPas, 30
s, 3 runs, one measurement, 25.degree. C. [0307] Size Exclusion
Chromatography (SEC) and Ion Exchange Chromatography (IEC), as
described below. [0308] viscosity measurement: different
viscometers with individual and different set-ups were used
Calculation of the Protein Concentration
Calculation Formula:
[0309] E = - lg ( I I 0 ) = c d .fwdarw. c = E .times. d
##EQU00003##
.epsilon.--absorption coefficient c--concentration d--length of
cuvette that the light has to pass E--absorbance I.sub.0--initial
light intensity I--light intensity after passing through sample
Adalimumab = 1.39 mL mg .times. cm ##EQU00004## J 695 = 1.42 mL mg
.times. cm ##EQU00004.2## HSA = 1.042 mL mg .times. cm
##EQU00004.3##
Viscosity Data and Calculation for Adalimumab
[0310] Adalimumab commercial formulation (approximately 194 mg/mL)
density:
.rho. = 1 , 05475 g cm 3 ##EQU00005##
Adalimumab commercial formulation (approximately 194 mg/mL)
kinematic viscosity: K--constant of the capillary t--the time the
solution needs for passing the capillary [s] v--kinematic
viscosity
v = K t = 0 , 03159 mm 2 s 2 279 , 36 s = 8 , 825 mm 2 s
##EQU00006##
Adalimumab commercial formulation (approximately 194 mg/mL) dynamic
viscosity: .eta.--dynamic viscosity .rho.--density
.eta. = v .rho. = 8 , 825 mm 2 s 1 , 05475 g cm 3 = 9 , 308 mPas
##EQU00007##
Viscosity Data and Calculation for Human Serum Albumin
[0311] HSA commercial formulation (approximately 200 mg/mL)
density:
.rho. = 1 , 05833 g cm 3 ##EQU00008##
HSA commercial formulation (app. 200 mg/mL) kinematic viscosity:
K--constant of the capillary t--the time the solution needs for
passing the capillary [s] v--kinematic viscosity
v = K t = 0 , 01024 mm 2 s 2 337 , 69 s = 3 , 46 mm 2 s
##EQU00009##
HSA commercial formulation (approximately 200 mg/mL) dynamic
viscosity: .eta.--dynamic viscosity .rho.--density
.eta. = v .rho. = 3 , 46 mm 2 s 1 , 05833 g cm 3 = 3 , 662 mPas
##EQU00010##
HSA in WFI (approximately 180 mg/mL) density:
.rho. = 1 , 07905 g cm 3 ##EQU00011##
HSA in WFI (approximately 180 mg/mL) kinematic viscosity:
K--constant of the capillary t--the time the solution needs for
passing the capillary [s] v--kinematic viscosity
v = K t = 0 , 09573 mm 2 s 2 185 , 3 s = 17 , 72 mm 2 s
##EQU00012##
HSA in WFI (approximately 180 mg/mL) dynamic viscosity:
.eta.--dynamic viscosity .rho.--density
.eta. = v .rho. = 17 , 72 mm 2 s 1 , 07905 g cm 3 = 19 , 121 mPas
##EQU00013##
General Experimental Execution for Arriving at High Concentration
Formulation
[0312] Generally, the process of the invention for arriving at a
high concentration, salt-free, protein formulation includes
diafiltration of the initial drug substance material, followed by a
procedure to increase the concentration of the drug substance in
the solution. This may be done in separate procedures or may be
done in separate or coinciding steps within the same procedure.
Diafiltration
[0313] A sufficient amount of Drug Substance (DS) material
(depending on protein concentration of DS) was subjected to
diafiltration. Prior to diafiltration, the DS material was diluted
with water for injection .about.10 mg/ml. Note that in total
approximately 540 mL of a 10 mg/mL solution was needed for the
experiment.
[0314] Water for injection was used as diafiltration medium. The
number of diafiltration steps performed was 5 to 7 (one
diafiltration step equals one total volume exchange).
In-Process-Control (IPC) samples were pulled prior to diafiltration
and after diafiltration step (200 .mu.L for osmolality, 120 .mu.L
for SEC).
[0315] Diafiltration with TFF equipment is performed by applying
the following parameters: [0316] stirrer: position 2 [0317] pump:
position 2 [0318] pressure up-stream/inlet: max 20-30 psi [0319]
pressure down-stream/outlet: max 10 psi
[0320] (Parameters used in this experiment were derived from
manufacturer's recommendations. One with skill in the art would be
able to alter the parameters of equipment operation to accommodate
a particular protein or variances in equipment, formulation,
viscosity, and other variables.)
[0321] After diafiltration, protein concentration was assessed by
means of OD280. If protein concentration was >10 mg/mL, the
protein concentration was adjusted to 10 mg/mL by appropriately
diluting the solution with water for injection.
Concentration
[0322] 20 mL of diafiltrated protein solution (e.g., Adalimumab,
J695, HSA) were put into a Vivaspin 20 Concentrator. The
concentrator was closed and put into the centrifuge. The protein
solution was centrifuged at maximum speed (5000 rpm).
Sample Pulls
[0323] Samples of the concentrated solutions were pulled at: 10
mg/mL and then every 10 mg/mL (20, 30, 40 mg/ml etc.) or until the
protein aggregates visibly, and samples were analyzed as follows:
[0324] The protein solution was homogenized in the Vivaspin
concentrator and filled in an adequate vial. [0325] Optical
appearance was inspected directly in the vial. [0326] 300 .mu.L was
used for UV spectroscopy, 160 .mu.L for PCS, 120 .mu.L, for SEC and
300 .mu.L for IEX. [0327] the samples for SEC and IEX were stored
at -80.degree. C.
Dynamic Light Scattering (DLS) Protocol
[0328] Dynamic light scattering was performed using the Zetasizer
Nano ZS (Malvern Instruments, Southborough, Mass.) equipped with
Hellma precision cells (Plainview, N.Y.), suprasil quartz, type
105.251-QS, light path 3 mm, center Z8.5 mm, with at least 60 .mu.L
sample fill, using protein samples as is and placed directly in
measurement cell. Prior to measurement, the cell window was checked
to verify that the solution was free of air bubbles or
particles/dust/other contaminants that may impact DLS measurement.
Measurements were taken under standard operating procedures
("general purpose" mode, 25.degree. C., refractive index set to
1.45, measurement mode set to "manual", 1 run per measurement, each
comprising 3 measurements of 30 s each, type of measurement set to
"size"). Dispersion Technology Software, version 4.10b1, Malvern
Instruments, was used to analyze data. About 70 .mu.L of a sample
solution were filled in precision cell for analysis of hydrodynamic
diameters (Dh). Default sample viscosity was set 1.1 mPas for low
concentrated protein solutions (e.g. <5 mg/mL). Underlying
measurement principles concluded that minimal differences between
real viscosity values of the sample solution to be measured and the
use of default viscosities does not impact DLS data readout
substantially. This was verified by performing DLS measurements of
low protein concentration solutions (<5 mg/mL) where solution
viscosities were determined and taken into account in subsequent
DLS measurements. For all samples with higher protein
concentration, viscosities were determined and taken into account
during DLS measurements.
Example 2: Formulation Comprising High TNF.alpha. Antibody
Concentration
2.1: Diafiltration
[0329] Prior to diafiltration, Adalimumab (49.68 mg/mL) was diluted
with water for injection to a concentration of approximately 15
mg/mL. Therefore 140.8 mL Adalimumab solution (49.68 mg/mL) were
filled in a 500 mL volumetric flask. The flask was filled up to the
calibration mark with water for injection. The volumetric flask was
closed and gently shaken for homogenization of the solution. The
TFF labscale system was flushed with water. Then the membrane (PES)
was adapted and was also flushed with 1 L distilled water. Next,
the TFF labscale system and the membrane were flushed with
approximately 300 mL of water for injection. The diluted Adalimumab
solution was then filled in the reservoir of the TFF. A sample for
an osmolality measurement (300 .mu.L), UV spectrophotometry (500
.mu.L) and a sample for SEC analysis (120 .mu.L) were pulled. The
system was closed and diafiltration was started. The DF/UF
(diafiltration/ultrafiltration) was finished after 5 volume
exchanges and after an osmolality value of 3 mosmol/kg was reached.
The pH-value of the Adalimumab solution after diafiltration was pH
5.25.
[0330] Diafiltration with TFF equipment was performed by applying
the following parameters: [0331] stirrer: position 2 [0332] pump:
position 2 [0333] pressure up-stream/inlet: max 20-30 psi [0334]
pressure down-stream/outlet: max 10 psi
[0335] After diafiltration, protein concentration was assessed by
means of OD280. The concentration was determined to be 13.29
mg/mL.
[0336] The Adalimumab solution was sterile filtered.
[0337] The TFF and the membrane were flushed with approximately 1 L
distilled water and then with 500 mL 0.1M NaOH. The membrane was
stored in 0.1M NaOH, the TFF was again flushed with approximately
500 mL distilled water.
2.2: Protein Concentration
[0338] Prior to concentrating the antibody, the protein
concentration was again assessed by means of OD280. Adalimumab
concentration was determined to be 13.3 mg/mL. The Adalimumab
solution was then diluted to 10 mg/mL. 375.94 mL of Adalimumab
solution (13.3 mg/mL) was filled in a 500 mL volumetric flask and
the flask was filled up to the calibration mark with water for
injection (WFI). 75.19 mL of Adalimumab solution (13.3 mg/mL) was
also filled in a 100 mL volumetric flask, and filled up to the
calibration mark with pure water, i.e., water for injection (WFI).
Both flasks were gently shaken for homogenization. The solutions
from both flasks were placed in a 1 L PETG bottle. The bottle was
gently shaken for homogenization.
[0339] Four Vivaspin 20 concentrators (10 kDa) were used. In three
Vivaspins, 20 mL of Adalimumab solution (10 mg/mL) were filled (in
each). In the fourth Vivaspin device, water was filled as
counterbalance weight while centrifuging. The concentrators were
closed and put into the centrifuge. The Adalimumab solution was
centrifuged applying 4500.times.g centrifugation force (in a swing
out rotor).
2.3: Sample Pull
[0340] Samples of the concentrated Adalimumab solution were pulled
when they reached a concentration of 10 mg/mL and at each
subsequent 10 mg/mL concentration increment increase (at 20 mg/mL,
30 mg/mL, 40 mg/mL etc. until approximately 200 mg/mL). At 40
minute intervals, the concentrators were taken out of the
centrifuge, the solution was homogenized, and the solution and the
centrifuge adapters were cooled for approximately 10 min on ice.
After each 10 mg/mL concentrating increment, the solutions in the
concentrators were homogenized, the optical appearance was checked
and samples were pulled for analysis via UV (300 .mu.L), PCS (160
.mu.L), SEC (120 .mu.L) and IEC (300 .mu.L). After sample pulls,
the concentrators were filled up to approximately 20 mL with
Adalimumab solution (10 mg/mL).
[0341] Visual Inspection and PCS analysis of protein precipitation
were used to determine the solubility limit of Adalimumab protein
(i.e. isoforms) in the solution.
[0342] At a concentration of approximately 80 mg/mL it became
obvious that the Adalimumab solution was not opalescent anymore,
opalescence being a known characteristic of Adalimumab solutions
having a high amount of fragment. Therefore, it was suspected that
fragmentation might have occurred during experiment execution. For
further analysis, a sample of Adalimumab solution (approximately 80
mg/mL) was analyzed by SEC. The remainder of the solution in each
Vivaspin, as well as the rest of Adalimumab solution (10 mg/mL),
was removed to 50 mL falcon tubes and stored at -80.degree. C. The
SEC analysis showed a purity of 99.6% monomer.
[0343] The solution was thawed in water bath at 25.degree. C. and
sterile filtered. Afterwards the solutions from 3 falcon tubes were
place into each Vivaspin. The concentrators were filled to
approximately 20 mL and the concentration was continued. The
experiment was finished as a concentration of approximately 200
mg/mL was reached.
[0344] All SEC and IEC samples were stored at -80.degree. C. until
further analysis. UV and PCS were measured directly after sample
pull. The rest of the concentrated Adalimumab solution was placed
in Eppendorf repositories and stored at -80.degree. C.
[0345] Table 7 shown below describes calculation of volumes of
protein solution to be refilled into the concentrators while
concentrating Adalimumab solution. The scheme was calculated before
experiment execution to define at which volume samples have to be
pulled. The duration of centrifugation is shown in Table 8.
TABLE-US-00007 TABLE 7 Centrifugation Scheme volume volume
concentration 10 mg/ml step [ml] [mg/ml] protein solution 0 20 10
conc. 1 10 20 sampling 2 9 20 filling 3 20 14.5 11 conc. 4 9.66 30
sampling 5 8.66 30 filling 6 20 18.66 11.34 conc. 7 9.33 40
sampling 8 8.33 40 filling 9 20 22.49 11.67 conc. 10 8.99 50
sampling 11 7.99 50 filling 12 20 25.98 12.01 conc. 13 8.66 60
sampling 14 7.66 60 filling 15 20 29.15 12.34 conc. 16 8.32 70
sampling 17 7.32 70 filling 18 20 31.96 12.68 conc. 19 7.99 80
sampling 20 6.99 80 filling 21 20 34.46 13.01 conc. 22 7.65 90
sampling 23 6.65 90 filling 24 20 36.6 13.35 conc. 25 7.32 100
sampling 26 6.32 100 filling 27 20 38.44 13.68 conc. 28 6.98 110
sampling 29 5.98 110 filling 30 20 39.9 14.02 conc. 31 6.65 120
sampling 32 5.65 120 filling 33 20 41.07 14.35 conc. 34 6.31 130
sampling 35 5.31 130 filling 36 20 41.86 14.69 conc. 37 5.98 140
sampling 38 4.98 140 conc. 39 4.64 150 154.14 174.14
TABLE-US-00008 TABLE 8 Centrifugation times required for
concentrating the Adalimumab solution concentration [from -> to]
[mg/mL] time [min] 10 to 20 15 20 to 30 20 30 to 40 27 40 to 50 30
50 to 60 40 60 to 70 50 70 to 80 60 80 to 90 67 90 to 100 80 100 to
110 100 110 to 200 206
Results from the concentration of Adalimumab are also shown below
in Table 12.
2.4: Viscosity Measurement
[0346] Adalimumab solutions comprising either 50 mg/mL in WFI or
200 mg/mL in WFI were measured for viscosity. 50 mg/mL and 200
mg/mL in WFI were measured using a Gemini 150 Peltier Plate
Rheometer, Malvern, and the 200 mg/mL in WFI solution was also
measured via rheometer MCR 301 [temperature-controlled P-PTD 200
(plate with Peltier tempering)] and cone/plate measurement system
CP50/1 (stainless steel); Anton Paar).
[0347] Adalimumab solutions (200 mg/mL) in repository tubes were
thawed and homogenized in a 6R vial. 1 mL Adalimumab (200 mg/mL)
was diluted with 3 mL WFI to obtain the diluted solution (for a 50
mg/ml Adalimumab solution).
[0348] For the rheometer Gemini 150 approximately 2 mL were needed
and for the MCR 301 less than 1 mL was needed for measurement.
[0349] Adalimumab (approximately 194 mg/mL) in the commercial
formulation was obtained by using Vivaspin tubes. The tubes were
filled with Adalimumab solution in commercial buffer and
centrifugation was applied until a 194 mg/mL concentration was
reached. Viscosity was measured with the capillary viscometer
Schott.
2.5: Summary
[0350] In sum, Adalimumab was concentrated from 50 mg/mL to
approximately 194 mg/mL in Vivaspin 20 tubes in four different
tubes. At the beginning, 20 mL of Adalimumab solution (50 mg/mL)
were in every tube (four tubes). At the end of the concentration, 5
mL of Adalimumab solution (approximately 194 mg/mL) were in every
tube. The concentration step was performed at 5000 rpm
(approximately 4500 g). After every hour, the oblong beakers and
the protein solution in the Vivaspin tubes were cooled in crushed
ice for approximately 10 to 15 min. The density was measured with
density measurement device DMA 4500, Anton Paar. Further analysis
of the high Adalimumab concentration formulation is provided in
Examples 5 to 11.
Example 3: Formulation Comprising High Concentration Il-12
Antibody
3.1: Diafiltration
[0351] Prior to diafiltration, IL-12 antibody J695 (54 mg/mL) was
diluted with water for injection to a concentration of
approximately 15 mg/mL. This was done by placing 150 mL J695
solution (54 mg/mL) in a 500 mL volumetric flask and filling the
flask to the calibration mark with water for injection. The
volumetric flask was closed and gently shaken for homogenization of
the solution. The TFF labscale system was flushed with water. Then
the polyethersulfone membrane (PES) was adapted and was also
flushed with 1 L of distilled water. Afterwards the TFF labscale
system and the membrane were flushed with approximately 300 mL of
water for injection. Next, the diluted J695 solution was placed in
the reservoir of the TFF. A sample for osmolality measurement (300
.mu.L), UV spectrophotometry (500 .mu.L) and a sample for SEC
analysis (120 .mu.L) were pulled. The system was closed and
diafiltration was started. After 200 mL of processing the DF
volume, the diafiltration was stopped and another sample for UV
measurement was pulled. The DF/UF was stopped after 1800 mL
diafiltration volume (approximately factor 3.5 volume exchange),
reaching an osmolality value of 4 mosmol/kg. The pH-value of the
J695 solution after diafiltration was pH 6.48.
[0352] Diafiltration with TFF equipment was performed by applying
the following parameters: [0353] stirrer: position 2 [0354] pump:
position 2 [0355] pressure up-stream/inlet: max 20-30 psi [0356]
pressure down-stream/outlet: max 10 psi
[0357] After diafiltration, the protein concentration was assessed
by means of OD280. The concentration was determined to be 16.63
mg/mL.
[0358] The J695 solution was sterile filtered.
[0359] The TFF instrument and the membrane were flushed with
approximately 1 L of distilled water and then with 500 mL 0.1M
NaOH. The membrane was stored in 0.1M NaOH and the TFF was again
flushed with approximately 500 mL of distilled water.
3.2: Concentrating
[0360] Prior to concentrating, the J695 solution was diluted to 10
mg/mL: 316 mL of J695 solution (16.63 mg/mL) was placed in a 500 mL
volumetric flask and the flask was filled to the calibration mark
with water for injection (WFI). Additionally, 64 mL of J695
solution (16.63 mL) was placed in a 100 mL volumetric flask and
filled to the calibration mark with WFI. Both flasks were gently
shaken for homogenization. The solutions from both flasks were
placed in a 1 L PETG bottle. The bottle was gently shaken for
homogenization.
[0361] Four Vivaspin 20 concentrators (10 kDa cut-off) were used.
20 mL of J695 solution (10 mg/mL) were place in each of three
Vivaspins. In the fourth Vivaspin concentrator device, water was
filled as counterbalance weight while centrifuging. The
concentrators were closed and put into the centrifuge. The J695
solution was centrifuged applying 4500.times.g centrifugation force
(in a swing out rotor).
3.3: Sample Pull
[0362] Samples of the concentrated J695 solution were pulled when
they reached a concentration of 10 mg/mL and at each subsequent 10
mg/mL concentration increment increase (at 20 mg/mL, 30 mg/mL, 40
mg/mL etc. until 200 mg/mL). After every 40 minutes, the
concentrators were taken out of the centrifuge, the solution was
homogenized, and the solution and the centrifuge adapters were
cooled for approximately 10 min on ice. After every 10 mg/mL
concentration increase, the solutions in the concentrators were
homogenized, the optical appearance was checked and samples were
pulled for UV (300 .mu.L), PCS (160 .mu.L), SEC (120 .mu.L) and IEC
analysis (300 .mu.L). After sample pulls, the concentrators were
filled up to approximately 20 mL with J695 solution (10 mg/mL).
[0363] Visual Inspection and PCS analysis were used to determine
the solubility (i.e., to check for potential precipitation) and
stability of J695.
[0364] At the conclusion of the experiment, a concentration of
approximately 200 mg/mL was reached.
[0365] All SEC and IEC samples were stored at -80.degree. C. for
further analysis (see below). UV spectrophotometry and PCS
measurements were taken directly after each sample pull. The rest
of the concentrated J695 solution was placed in Eppendorf
repositories and stored at -80.degree. C.
[0366] Details regarding the centrifugation scheme are provided
above in Table 7. The duration of the J695 centrifugation are
provided in Table 9.
TABLE-US-00009 TABLE 9 Centrifugation Times used to Concentrate the
J695 Solution concentration [from -> to] [mg/mL] time [min] 10
to 20 13 20 to 30 22 30 to 40 27 40 to 50 38 50 to 60 45 60 to 70
80 70 to 80 90 80 to 90 105 90 to 100 165 100 to 200 270
3.4: Impact of Excipients on the Hydrodynamic Diameter of J695
[0367] In this experiment the impact of sodium chloride and
mannitol, separately, on the hydrodynamic diameter of J695 was
analyzed. For this purpose, stock solutions of sodium chloride (12
mg/mL) and of mannitol (120 mg/mL) were prepared. 1.2 g NaCl was
weighed in a beaker, which was filled with approximately 70 mL of
WFI, and 12.002 g of Mannitol was weighed in a beaker which was
filled with approximately 70 mL of WFI. The two solutions were
stirred for homogenization. Each solution was placed in a
volumetric flask, which was filled to the calibration mark with
WFI. The flasks were gently shaken for homogenization.
[0368] Approximately 8 mL of J695 solution (approximately 200
mg/mL) was thawed at 37.degree. C. The solution was filled in a 10R
vial and homogenized. Seven 2R vials were filled with 500 .mu.L
J695 solution (approximately 200 mg/mL) each. The filling scheme is
described in Table 10 below.
TABLE-US-00010 TABLE 10 Filling Scheme for Preparation of J695
Solutions Containing Different Concentrations of NaCl or Mannitol
volume vol. NaCl vol. mannitol ABT-874 stock solution stock
solution concentration (200 mg/ml) (12 mg/mL) (12 mg/mL) vol. WFI
excipient excipient [.mu.L] [.mu.L] [.mu.L] [.mu.L] -- -- 500 -- --
500 NaCl 2 mg/mL 500 167 -- 333 NaCl 4 mg/mL 500 333 -- 167 NaCl 6
mg/mL 500 500 -- -- mannitol 20 mg/mL 500 -- 167 333 mannitol 40
mg/mL 500 -- 333 167 mannitol 60 mg/mL 500 -- 500 --
The 2R vials were gently homogenized via shaking. Thereafter, PCS
and osmolality measurements were taken of the different J695
solutions (100 mg/mL).
[0369] To prepare samples for PCS analysis, the cuvettes were first
flushed with 50 .mu.L of the sample. Then measurements were taken
using 100 .mu.L of the sample.
[0370] Further analysis of the high J695 concentration formulation
is provided in Examples 5 to 11.
Example 4: High Concentration Human Serum Albumin (HSA)
Formulation
4.1: Diafiltration
[0371] Prior to diafiltration, HSA solution (200 mg/mL, commercial
formulation) was diluted with water for injection to a
concentration of 15.29 mg/mL. To achieve this, 38 mL HSA (200
mg/mL) were filled in a 500 mL volumetric flask. The flask was
filled to the calibration mark with water for injection. The
volumetric flask was closed and gently shaken for homogenization of
the solution. The TFF labscale system was flushed with water. Then
the membrane (regenerated cellulose) was adapted and was also
flushed with 1 L of distilled water. Afterwards the TFF labscale
system and the membrane were flushed with approximately 300 mL
water for injection. Next, the diluted HSA solution was filled in
the reservoir of the TFF. Samples for osmolality measurement (300
.mu.L), UV spectrophotometry (500 .mu.L) and a sample for SEC
analysis (120 .mu.L) were pulled. The system was closed and
diafiltration was started. After diafiltration of approximately 300
mL of volume, a UV measurement of the permeate was taken. The
permeate revealed a concentration of 2.74 mg/mL, indicating that
protein was passing through the membrane. The diafiltration was
stopped after approximately 500 mL of DF, and another sample for UV
measurement was pulled (HSA concentration 11.03 mg/mL). The DF/UF
was finished after 950 mL of diafiltration volume (approximately 2
volume exchanges) and after reaching an osmolality value of 4
mosmol/kg. The pH-value of the HSA solution after diafiltration was
pH 7.13.
[0372] UV spectrophotometric measurement of the permeate was
performed three times (n=3).
[0373] Diafiltration with TFF equipment was performed by applying
the following parameters: [0374] stirrer: position 2 [0375] pump:
position 2 [0376] pressure up-stream/inlet: max 20-30 psi [0377]
pressure down-stream/outlet: max 10 psi
[0378] After diafiltration, protein concentration was assessed by
means of OD280. The concentration was determined 9.41 mg/mL.
[0379] The HSA solution was sterile filtered. The TFF and the
membrane were flushed with approximately 1 L of distilled water.
Afterwards an integrity test was done (see Operating Instructions
Labscale.TM. TFF System, page 5-3 to 5-5, 1997). The volume flow
was 1.2 mL/min, thus, the integrity test was passed (acceptable
maximal limit 3 mL/min). The membrane was once more flushed with
500 mL of distilled water and then with 500 mL of 0.05 M NaOH. The
membrane was stored in 0.05 M NaOH, the TFF was again flushed with
approximately 500 mL of distilled water.
4.2: Concentration Process
[0380] Prior to concentrating the HSA protein solution, the
concentration was assessed by means of OD280 and was determined to
be 9.52 mg/mL. Four Vivaspin 20 concentrators (10 kDa) were used.
20 mL of HSA solution (9.52 mg/mL) were placed in each of 3
Vivaspin concentrators. In the fourth Vivaspin, water was filled as
counterweight balance while centrifuging. The concentrators were
closed and put into the centrifuge. The HSA solution was
centrifuged applying 4500.times.g centrifugation force (in a swing
out rotor).
4.3: Sample Pull
[0381] Samples of the concentrated HSA solution were pulled when
the concentration reached 10 mg/mL and subsequently after every 10
mg/mL concentration increment increase (at 20 mg/mL, 30 mg/mL, 40
mg/mL etc. until approximately 180 mg/mL). Every 40 minutes the
concentrators were taken out of the centrifuge, the solution was
homogenized, and the solution and the centrifuge adapters were
cooled for approximately 10 min on ice. After every 10 mg/mL
concentration increment increase, the solutions in the
concentrators were homogenized, the optical appearance was checked
and samples were pulled for analysis via UV (300 .mu.L), PCS (160
.mu.L), SEC (120 .mu.L) and IEC (300 .mu.L). After the sample pull,
HSA solution (9.52 mg/mL) was added to the concentrators, up to
approximately 20 mL.
[0382] When the projected concentration for the HSA solution in the
concentrator reached approximately 20 mg/mL, permeate was measured
via OD280, revealing a concentration of 0.5964 mg/mL. The
concentration of the HSA solution was only 15.99 mg/mL, which was
less than expected. A sample of concentrated HSA in WFI (10 mg/mL)
was analyzed via SEC to scrutinize for potential fragmentation. The
HSA solution (15.99 mg/mL) in the Vivaspins was placed in falcon
tubes and stored at -80.degree. C. The remainder of the original
HSA solution (9.52 mg/mL) used to fill the concentrators was also
stored at -80.degree. C.
[0383] SEC analysis was performed to determine whether the HSA
protein underwent degradation, producing small fragments that could
pass through the membrane. The SEC analysis, however, revealed a
monomer amount of 92.45% for 10 mg/mL HSA in WFI with virtually no
fragments.
[0384] The solutions that were stored at -80.degree. C. were thawed
at 25.degree. C. and sterile filtered. The solutions in the falcon
tubes were transferred in one Vivaspin 20 concentrator each (3 kDa
cut-off). The Vivaspins were filled up with HSA solution (9.52
mg/mL) and centrifuged (refer to 3.2 concentration process
described above).
[0385] Visual Inspection and PCS analysis were used to determine
the solubility limit of HSA.
[0386] At the completion of the experiment, a concentration of
approximately 180 mg/mL HSA was reached.
[0387] All SEC and IEC samples were stored at -80.degree. C. until
further analysis. UV and PCS measurements were performed directly
after sample pull. The rest of the concentrated HSA solution was
placed in Eppendorf repositories and stored at -80.degree. C.
[0388] An overview of the centrifugation scheme is described above
in Table 7. The duration of the centrifugation used to concentrate
HSA is described in Table 11.
TABLE-US-00011 TABLE 11 Centrifugation Times Necessary to
Concentrate HSA Solution concentration [from -> to] [mg/mL] time
[min] 10 to 20 9 20 to 30 30 30 to 40 40 40 to 50 50 50 to 60 80 60
to 70 90 70 to 80 110 80 to 90 130 90 to 100 170 100 to 180 360
4.4: Impact of the pH Value on the Hydrodynamic Diameter of HSA
[0389] The following part of the experiment was performed to
evaluate a potential impact of pH on the hydrodynamic diameter of
HSA when the protein is dissolved in WFI. Four 6R vials were filled
with 5.09 mL HSA solution (9.83 mg/mL), and pH values from 3 to 6
were set up with 1M HCl (actual pH: 3.04, 3.99, 5.05, 6.01). These
solutions were each transferred to a separate 10 mL volumetric
flask. The flasks were then filled to the calibration mark and
gently shaken for homogenization.
[0390] The HCl solutions were placed in 10R vials and analyzed via
PCS. The solutions were sterile filtered and measured again via
PCS. Also, 5.09 mL HSA solution (9.83 mg/mL) were transferred in a
10 mL volumetric flask and this was filled with WFI to the
calibration mark. The flask was gently shaken for homogenization
and then the solution was sterile filtered and measured via PCS.
Sample preparation for PCS measurement:
[0391] The cuvettes were flushed with 50 .mu.L of sample.
Measurement was performed with 100 .mu.L of sample volume.
4.5: Viscosity Measurement
[0392] For HSA in commercial formulation (200 mg/mL) and for HSA in
WFI (approximately 180 mg/mL) the viscosity was measured with a
capillary viscometer (Schott, MP.-No. 33.2).
[0393] A 15 mL aliquot was pulled from a 50 mL bottle of commercial
formulation HSA. HSA in WFI was thawed at approximately 20.degree.
C. and approximately 9 mL were aliquotted in a Falcon tube. The
density was measured with density measurement device DMA 4500,
Anton Paar.
[0394] Further analysis of the high HSA concentration formulation
is provided in Examples 5 to 11.
Example 5: Analysis of High Protein Formulations--Optical
Appearance
[0395] In contrast to Adalimumab in the commercial formulation,
Adalimumab in WFI did not reveal opalescence. J695 also did not
reveal any opalescence phenomena when dissolved in WFI. Despite the
fact that the protein concentration of Adalimumab was 80 mg/mL and
200 mg/mL in WFI, there was virtually no opalescence observed. In
contrast, the commercial formulation comprising 50 mg/mL Adalimumab
revealed notable opalescence in the commercial formulation. Thus,
the use of pure water, i.e., WFI, as a dissolution medium had a
positive effect on protein solution opalescence.
[0396] It was a surprising observation that (in addition to being
soluble at all at such a high protein concentration) Adalimumab in
WFI appeared to have a low viscosity, even at higher concentrations
such as 200 mg/mL.
[0397] Depending on the concentration, the optical
characteristics/color of HSA solutions changed from clear and
slightly yellow (10 mg/mL in WFI) to clear and yellow
(approximately 180 mg/mL in WFI).
[0398] During the concentration process, no precipitation was
observed for the Adalimumab solution and HSA solution.
Precipitation would have been an indication for solubility
limitations. The solutions stayed clear until the experiment was
finalized. It is to be highlighted that the experiments were not
finished because potential solubility limits were approached and
precipitation occurred, but were finished because the solution
volumes remaining in the concentrators were not sufficient to
proceed with concentrating (i.e. lack of material). It appears very
likely that the solubility limits of Adalimumab, J695, and HSA are
well beyond 220 mg/mL.
[0399] In the J695 solution a crystal like precipitate was observed
when the high-concentrated solution was stored over night at
2-8.degree. C. in the concentrators (approximately 120 mg/mL). The
crystal like precipitate redissolved after approximately 3-5 min
when the solution was stored at ambient temperature. Thus the
environment created by dissolving J695 at high concentration in
pure water provides conditions where protein crystallization might
be performed by mere temperature cycling (e.g., from ambient
temperature to 2-8.degree. C.).
Example 6: Analysis of High Protein Formulations--Protein
Concentration
[0400] The calculation of the protein concentrations is provided
above in the Materials and Methods section.
[0401] An overview of the concentration of Adalimumab, J695, and
HSA into pure water, high protein formulation is provided below in
Tables 12-14:
TABLE-US-00012 TABLE 12 Concentrations of Adalimumab as Assessed
Via OD280 during the Concentration Process absor- average concen-
sample name bance value dilution tration Adalimumab in WFI 10 mg/mL
0.680 0.650 20 9.35 0.695 0.575 Adalimumab in WFI 20 mg/mL 1 1.064
0.813 40 23.40 0.688 0.687 Adalimumab in WFI 20 mg/mL 2 0.781 0.788
40 22.68 0.793 0.791 Adalimumab in WFI 20 mg/mL 3 0.870 0.824 40
23.71 0.883 0.719 Adalimumab in WFI 30 mg/mL 1 0.817 0.807 60 34.84
0.812 0.793 Adalimumab in WFI 30 mg/mL 2 0.839 0.827 60 35.69 0.813
0.829 Adalimumab in WFI 30 mg/mL 3 0.770 0.744 60 32.10 0.729 0.732
Adalimumab in WFI 40 mg/mL 1 0.494 0.491 100 35.35 0.493 0.488
Adalimumab in WFI 40 mg/mL 2 0.499 0.501 100 36.06 0.516 0.489
Adalimumab in WFI 40 mg/mL 3 0.495 0.512 100 36.81 0.523 0.517
Adalimumab in WFI 50 mg/mL 1 0.574 0.585 100 42.11 0.579 0.603
Adalimumab in WFI 50 mg/mL 2 0.671 0.634 100 45.63 0.630 0.601
Adalimumab in WFI 50 mg/mL 3 0.579 0.574 100 41.27 0.574 0.568
Adalimumab in WFI 60 mg/mL 1 0.838 0.837 100 60.21 0.833 0.840
Adalimumab in WFI 60 mg/mL 2 0.793 0.777 100 55.89 0.767 0.770
Adalimumab in WFI 60 mg/mL 3 0.802 0.780 100 56.10 0.759 0.779
Adalimumab in WFI 70 mg/mL 1 0.911 0.878 100 63.15 0.866 0.857
Adalimumab in WFI 70 mg/mL 2 1.012 0.996 100 71.68 0.976 1.001
Adalimumab in WFI 70 mg/mL 3 0.879 0.871 100 62.66 0.874 0.861
Adalimumab in WFI 80 mg/mL 1 0.512 0.510 200 73.45 0.489 0.531
Adalimumab in WFI 80 mg/mL 2 0.542 0.526 200 75.64 0.519 0.517
Adalimumab in WFI 80 mg/mL 3 0.551 0.531 200 76.42 0.511 0.531
Adalimumab in WFI 90 mg/mL 1 0.550 0.547 200 78.64 0.550 0.539
Adalimumab in WFI 90 mg/mL 2 0.549 0.548 200 78.80 0.551 0.543
Adalimumab in WFI 90 mg/mL 3 0.532 0.534 200 76.81 0.533 0.537
Adalimumab in WFI 100 mg/mL 1 0.640 0.628 200 90.36 0.621 0.623
Adalimumab in WFI 100 mg/mL 2 0.748 0.747 200 107.41 0.735 0.757
Adalimumab in WFI 100 mg/mL 3 0.625 0.621 200 89.39 0.616 0.623
Adalimumab in WFI 110 mg/mL 1 0.674 0.669 200 96.19 0.671 0.660
Adalimumab in WFI 110 mg/mL 2 0.693 0.668 200 96.05 0.690 0.620
Adalimumab in WFI 110 mg/mL 3 0.604 0.640 200 92.05 0.664 0.652
Adalimumab in WFI 200 mg/mL 1 0.863 0.698 400 201.00 0.612 0.621
Adalimumab in WFI 200 mg/mL 2 1.055 0.791 400 227.53 0.658 0.659
Adalimumab in WFI 200 mg/mL 3 0.732 0.665 400 191.44 0.648
0.615
TABLE-US-00013 TABLE 13 Concentrations of J695 as Assessed Via
OD280 during the Concentration Process absor- average concen-
sample name bance value dilution tration ABT-874 in WFI 10 mg/mL
0.715 0.703 20 9.90 0.708 0.705 ABT-874 in WFI 20 mg/mL 1 0.686 --
40 19.31 ABT-874 in WFI 20 mg/mL 2 0.684 -- 40 19.26 ABT-874 in WFI
20 mg/mL 3 0.685 -- 40 19.29 ABT-874 in WFI 30 mg/mL 1 0.700 -- 60
29.59 ABT-874 in WFI 30 mg/mL 2 0.703 -- 60 29.70 ABT-874 in WFI 30
mg/mL 3 0.684 -- 60 28.91 ABT-874 in WFI 40 mg/mL 1 0.539 -- 100
37.97 ABT-874 in WFI 40 mg/mL 2 0.540 -- 100 38.02 ABT-874 in WFI
40 mg/mL 3 0.520 -- 100 36.65 ABT-874 in WFI 50 mg/mL 1 0.698 --
100 49.15 ABT-874 in WFI 50 mg/mL 2 0.653 -- 100 45.95 ABT-874 in
WFI 50 mg/mL 3 0.623 -- 100 43.87 ABT-874 in WFI 60 mg/mL 1 0.834
-- 100 58.75 ABT-874 in WFI 60 mg/mL 2 0.781 -- 100 55.02 ABT-874
in WFI 60 mg/mL 3 0.778 -- 100 54.76 ABT-874 in WFI 70 mg/mL 1
1.103 -- 100 77.69 ABT-874 in WFI 70 mg/mL 2 1.102 -- 100 77.62
ABT-874 in WFI 70 mg/mL 3 1.110 -- 100 78.13 ABT-874 in WFI 80
mg/mL 1 0.671 -- 200 94.45 ABT-874 in WFI 80 mg/mL 2 0.746 -- 200
105.06 ABT-874 in WFI 80 mg/mL 3 0.664 -- 200 93.45 ABT-874 in WFI
90 mg/mL 1 0.826 -- 200 116.37 ABT-874 in WFI 90 mg/mL 2 0.809 --
200 113.92 ABT-874 in WFI 90 mg/mL 3 0.804 -- 200 113.27 ABT-874 in
WFI 100 mg/mL 1 0.861 -- 200 121.21 ABT-874 in WFI 100 mg/mL 2
0.993 -- 200 139.80 ABT-874 in WFI 100 mg/mL 3 0.985 -- 200 138.73
ABT-874 in WFI 200 mg/mL 1 0.681 0.805 400 226.67 0.864 0.869
ABT-874 in WFI 200 mg/mL 2 0.690 0.767 400 216.10 0.828 0.784
ABT-874 in WFI 200 mg/mL 3 0.708 0.745 400 209.83 0.789 0.738
Tables 14a and 14b: Concentrations of HSA as Assessed Via OD280
during the Concentration Process
TABLE-US-00014 TABLE 14a sample name absorbance dilution
concentration HSA in WFI 10 mg/mL 0.515 20 9.88 HSA in WFI 30 mg/mL
1 0.398 60 22.94 HSA in WFI 30 mg/mL 2 0.395 60 22.73 HSA in WFI 30
mg/mL 3 0.400 60 23.00 HSA in WFI 40 mg/mL 1 0.383 100 36.78 HSA in
WFI 40 mg/mL 2 0.389 100 37.33 HSA in WFI 40 mg/mL 3 0.368 100
35.29 HSA in WFI 50 mg/mL 1 0.479 100 45.97 HSA in WFI 50 mg/mL 2
0.496 100 47.61 HSA in WFI 50 mg/mL 3 0.465 100 44.61 HSA in WFI 60
mg/mL 1 0.609 100 58.47 HSA in WFI 60 mg/mL 2 0.653 100 62.69 HSA
in WFI 60 mg/mL 3 0.568 100 54.52 HSA in WFI 70 mg/mL 1 0.645 100
61.89 HSA in WFI 70 mg/mL 2 0.623 100 59.76 HSA in WFI 70 mg/mL 3
0.618 100 59.28 HSA in WFI 80 mg/mL 1 0.393 200 75.37 HSA in WFI 80
mg/mL 2 0.436 200 83.69 HSA in WFI 80 mg/mL 3 0.363 200 69.67 HSA
in WFI 90 mg/mL 1 0.484 200 92.90 HSA in WFI 90 mg/mL 2 0.439 200
84.22 HSA in WFI 90 mg/mL 3 0.419 200 80.50 HSA in WFI 100 mg/mL 1
0.604 200 115.93 HSA in WFI 100 mg/mL 2 0.573 200 110.00 HSA in WFI
100 mg/mL 3 0.585 200 112.30
TABLE-US-00015 TABLE 14b average sample name absorbance value
dilution concentration HSA in WFI 180 mg/mL 1 0.946 0.952 200
182.79 0.950 0.961 HSA in WFI 180 mg/mL 2 0.994 0.929 200 178.24
0.906 0.886 HSA in WFI 180 mg/mL 3 0.843 0.896 200 172.05 0.963
0.884
[0402] All three proteins evaluated remained soluble in the
concentration ranges evaluated (i.e. >200 mg/mL for Adalimumab
and J695, >175 mg/mL for HSA). No indications of insolubility,
e.g., the clouding phenomena or precipitation occurring in the
solution, were observed. For Adalimumab, the results indicate that,
over the concentration range evaluated, all Adalimumab isoforms
(i.e. lysine variants) remained soluble, as no precipitation
occurred at all. This observation is also consistent with ion
exchange chromatography data described in Example 11, which
describes that the sum of lysine variants stayed virtually
consistent regardless of Adalimumab concentration.
Example 7: Analysis of High Protein Formulations--Viscosity
7.1: Adalimumab Viscosity
[0403] The viscosity of Adalimumab (approximately 50 mg/mL) in
water for injection was determined to be around 1.5-2 mPas. For
Adalimumab (approximately 200 mg/mL) in WFI, two values were
determined. The one value determined with cone/plate rheometer from
Malvern (Gemini 150) was approximately 6-6.5 mPas, while the other
value (measured with cone/plate rheometer from Anton Paar, MCR 301)
was approximately 12 mPas.
Adalimumab commercial formulation (approximately 194 mg/mL)
viscosity: K--constant of the capillary t--the time the solution
needs for passing the capillary [s] v--kinematic viscosity
.eta.--dynamic viscosity .rho.--density
TABLE-US-00016 time [s] K [mm2/s2] v [mm2/s] .rho. [g/cm3] .eta.
[mPas] 279.36 0.03159 8.825 1.05475 9.31
[0404] The viscosity of Adalimumab in WFI (approximately 200 mg/mL)
was determined to be approximately 12 mPas with the viscosimeter
from Anton Paar and approximately 6 mPas determined with the
viscometer from Malvern. In contrast, the viscosity of Adalimumab
in the commercial formulation (approximately 194 mg/mL) is higher,
at 9.308 mPas (measured with the capillary viscometer from
Schott).
7.2: Human Serum Albumin Viscosity
[0405] HSA commercial formulation (approximately 200 mg/mL)
viscosity: K--constant of the capillary t--the time the solution
needs for running through the capillary [s] v--kinematic viscosity
dynamic viscosity p--density
TABLE-US-00017 time [s] K [mm2/s2] v [mm2/s] .rho. [g/cm3] .eta.
[mPas] 337.69 0.01024 3.46 1.05475 3.66
HSA in WFI (approximately 180 mg/mL) viscosity: K--constant of the
capillary t--the time the solution needs for running through the
capillary [s] v--kinematic viscosity dynamic viscosity
p--density
TABLE-US-00018 time [s] K [mm2/s2] v [mm2/s] .rho. [g/cm3] .eta.
[mPas] 185.3 0.09573 17.72 1.07905 19.12
[0406] The viscosity of HSA in WFI (approximately 180 mg/mL) was
determined to be approximately 19.121 mPas. The viscosity of HSA in
the commercial formulation (approximately 194 mg/mL) was determined
to be 9.308 mPas (measured with the capillary viscometer from
Schott).
7.3: Analysis of the Viscosities of Adalimumab and HSA
[0407] The dynamic viscosity of Adalimumab 50 mg/mL in WFI was
lower than the viscosity of Adalimumab 200 mg/mL in WFI and in
commercial buffer, respectively. For HSA the dynamic viscosity for
a concentration of 180 mg/mL in WFI was about six-fold higher than
for a concentration of 200 mg/mL in commercial buffer. Thus, it
seems that the intensity of viscosity change (i.e. increase and
decrease, respectively) due to effects conveyed by pure water as
dissolution medium may depend on the individual protein.
Example 8: Analysis of the Hydrodynamic Diameters of High Protein
Formulations--Photon Correlation Spectroscopy (PCS)
[0408] The following example provides an analysis of the
hydrodynamic diameter (Dh) (the z-average of the mean hydrodynamic
molecule diameter) of various proteins in aqueous formulations
obtained using the DF/UF methods of the invention.
8.1: Adalimumab Hydrodynamic Diameter
[0409] As shown in FIGS. 5 and 6, a trend can be observed where the
hydrodynamic diameter (D.sub.h) increases with increasing
Adalimumab concentration. FIG. 5 shows the correlation between
hydrodynamic diameter (z-average) and the concentration of
Adalimumab in WFI. FIG. 6 shows the correlation between
hydrodynamic diameter (peak monomer) and the concentration of
Adalimumab in WFI.
[0410] The difference between the D.sub.h determined from the 23.27
mg/mL sample compared to the 34.20 mg/mL sample exists because of
assumptions made in the Standard Operating Procedure (SOP) for
hydrodynamic diameter measurement. For Adalimumab samples having
<23.27 mg/mL concentration, PCS measurements were performed with
a SOP that assumes a 1.1 mPas value for the viscosity of the
samples. For Adalimumab samples having >34.20 mPas, a SOP
assuming a 1.9 mPas sample viscosity was used. It is known that PCS
data are strongly influenced by the given viscosity of the sample
solution, as PCS data is based on random Brownian motion of the
sample specimen, which is impacted by sample viscosity. Thus, the
increase in the hydrodynamic diameter with increasing protein
concentration can be explained, as increasing protein concentration
raises the viscosity of the solution (higher viscosity leads to
lower Brownian motion and higher calculated D.sub.h data). The
protein molecules experience a lower random Brownian motion and
thus, for a given viscosity, the hydrodynamic diameters of the
sample specimen are calculated higher. Overall, the z-average based
D.sub.h values and the D.sub.h values of the monomer match well.
Additionally, no increase in D.sub.h indicative for protein
insolubility is observed with increasing concentration (i.e. high
molecular weight aggregates and precipitate (if present) would
induce a substantial increase in D.sub.h).
8.2: J695 Hydrodynamic Diameter
[0411] FIGS. 7 and 8 show that the hydrodynamic diameter of J695
was relatively independent from the protein concentration until a
114.52 mg/mL concentration was reached. Increasing the J695
concentration from 114.52 mg/mL to 133.25 mg/mL induced a rapid
increase in D.sub.h. The hydrodynamic diameter at the 217.53 mg/mL
concentration was higher than at 114.52 mg/mL. This finding was not
surprising as both protein solutions were measured using the same
SOP (assuming same viscosity of 1.9 mPas), when in reality the
viscosity increases as the protein concentration increases. The
strong increase from 114.52 mg/mL to 133.25 mg/mL thus can be
explained as an artifact.
8.3: Human Serum Albumin Hydrodynamic Diameter
[0412] The hydrodynamic diameter of HSA in WFI was found to
decrease as concentrations rose from 9.88 mg/mL to 112.74 mg/mL.
From 112.74 mg/mL to 177.69 mg/mL, however, the hydrodynamic
diameter was found to increase.
[0413] HSA showed a general tendency of increasing hydrodynamic
diameters (peak monomer) with increasing protein concentration
which is in-line with underlying theoretical principles. The
D.sub.h decrease from 9.88 mg/mL to 22.89 mg/mL is caused by a
change in the measurement SOP (switching from assumed viscosity of
1.1 mPas to an assumed viscosity of 1.9 mPas).
[0414] Numerical data describing the above is provided in Appendix
A.
Example 9: J695: Impact of Excipients of the Hydrodynamic
Diameter
[0415] Having found the surprising result that proteins can be
dissolved in high concentrations in pure water, the impact of
ionizable and non-ionizable excipients typically used in parenteral
formulations on the hydrodynamic diameter was evaluated. J695 was
used as a model protein.
[0416] Table 15 shows that the solution osmolality is directly
proportional to the concentration of sodium chloride. The
osmolality in the protein solution rises along with the NaCl
concentration (an almost linear correlation). Interestingly, the
hydrodynamic diameter of J695 protein increased with increasing
salt concentration. NaCl is an ionic excipient and dissociated into
positively charged sodium ions and negatively charged chloride ions
which might adsorb at the surface of the protein. Without salt
being present, the hydrodynamic diameter of J695 was dramatically
lower than what normally is expected for J695 (usually values
around 10 nm are determined).
[0417] As illustrated in Table 15, the osmolality increased
linearly with an increase in concentration of mannitol in the
protein solution. In contrast, the hydrodynamic diameter did not
show a dependence on mannitol concentration. Mannitol is a
non-ionic sugar alcohol/polyol. Mannitol or polyols are used as
stabilizers during parenteral formulation development and in final
formulations. Mannitol stabilizes the protein by preferential
exclusion. As other osmolytes, mannitol is preferentially excluded
from the surface of the protein and it is outside of the hydrate
shell of the protein. Thus the folded state of the protein is
stabilized because the unfolded state, which has a larger surface,
becomes thermodynamically less favorable (Foster, T. M., Thermal
instability of low molecular weight urokinase during heat
treatment. III. Effect of salts, sugars and Tween 80, 134
International Journal of Pharmaceutics 193 (1996); Singh, S. and
Singh, J., Effect of polyols on the conformational stability and
biological activity of a model protein lysozyme, 4 AAPS
PharmSciTech, Article 42 (2003)). However, it is interesting that
the osmolality can be adjusted basically as desired--which would be
an important feature of the protein findings described
herein--without impacting the D.sub.h of the protein. These
findings may be useful in high-concentration protein formulation,
where viscosity related manufacturing and dosing issues may be
present, as the osmolality adjustment with mannitol is not mirrored
by an increase in protein D.sub.h (meaning viscosity is expected to
remain constant).
TABLE-US-00019 TABLE 15 Impact of Excipients on J695 Osmolality and
Z-Average osmolality z-average [mosmol/kg] [nm] NaCl concentration
[mg/mL] 0 16 4.19 2 92 12.2 4 158 16.2 6 230 17 mannitol
concentration [mg/mL] 0 16 4.19 20 148 5.49 40 276 3.22 60 432
3.54
Example 10: Analysis of High Protein Formulations with Size
Exclusion Chromatography (SEC)
[0418] For the SEC analysis, samples of Adalimumab, J695 and HSA
were diluted to 2 mg/mL before injection. The injection volume for
Adalimumab was 20 .mu.L. For J695 and HSA, a 10 .mu.L injection
volume was used.
10.1: SEC Analysis of Adalimumab
[0419] The amount of monomer of Adalimumab tended to slightly
decrease from 99.4% to 98.8% while concentrating from 9.35 mg/mL to
206.63 mg/mL. That decrease of monomer is associated with an
increase in the amount of aggregate in Adalimumab solution from
0.4% to 1.1% while concentrating from 23.27 mg/mL to 206.62 mg/mL,
respectively. The amount of fragment remained constant at 0.1%,
independent of protein concentration (see table in Appendix B).
Thus, Adalimumab was stable in WFI.
[0420] Overall, the increase in protein aggregation with increasing
protein concentration is deemed only minor. A similar trend in
monomer decrease would be expected when either the protein was
formulated in a buffer system and when additionally surfactants are
added. Adalimumab protein appears to be surprisingly stable when
formulated in pure water.
10.2: SEC Analysis of J695
[0421] The amount of J695 monomer slightly decreased from 99.4% to
98.6% with increasing protein concentration from 9.99 mg/mL to
217.53 mg/mL. The decrease of monomer was associated with an
increase in aggregate from about 0.4% to about 1.2% with increasing
protein concentration from 9.99 mg/mL to 217.53 mg/mL. Independent
from protein concentration, the amount of fragment was almost
constant with 0.17% to 0.23% with increasing protein concentration
from 9.99 mg/mL to 217.53 mg/mL.
[0422] Overall, the increase in protein aggregation with increasing
protein concentration was deemed only minor. A similar trend in
monomer decrease would be expected when the protein is formulated
in buffer systems and when additional surfactants are added. Thus,
J695 protein appears to be surprisingly stable when formulated in
pure water.
10.3: SEC Analysis of HSA
[0423] The amount of monomer HSA decreased from 95.9% to 92.75%
while concentrating from 9.88 mg/ml to 112.74 mg/mL. For the sample
with 177.69 mg/mL, an increase in monomer up to 94.5% was
determined. The decrease of the amount of monomer goes along with
an increase in protein aggregate from 4.1% to 7.25% while
concentrating from 9.88 mg/mL to 112.74 mg/mL. Thus, HSA protein
also appears to be stable when formulated in pure water.
[0424] Numerical data describing the above-mentioned SEC
experiments is provided in Appendix B.
Example 11: Analysis of High Protein
Formulations--Ion-Exchange-Chromatography (IEC)
[0425] For the IEC analysis the samples of Adalimumab, J695 and HSA
were diluted to 1 mg/mL before injection. The injection volume for
all proteins was 100 .mu.L.
11.1: IEC Analysis of Adalimumab
[0426] As shown in FIG. 9, Adalimumab was stable in WFI. FIG. 9
shows a slight trending which may be interpreted as indicating that
the sum of lysine variants (lysine 0, 1 and 2) decreases with an
increase concentration of Adalimumab in WFI. Overall, however, the
percentage of lysine variants varied less than 0.25%.
11.2: IEC Analysis of J695
[0427] FIG. 10 shows that the sum of the J695 peaks 1 to 7 is
slightly decreasing with increasing J695 concentration. With the
decrease in peak 1-7 the sum of acidic and basic peaks is slightly
increasing, with the increase in the acidic peaks being a little
more pronounced (see FIGS. 11 and 12). The sum of acidic peaks
slightly increases from approximately 10.2% to 10.6% and the sum of
basic peaks from 0.52% to 0.59%, respectively.
[0428] Overall, it can be stated that no major instability effects
or insolubility effects of J695 formulations in pure water were
observed via IEC.
[0429] Numerical data describing the above IEC experiments is
provided in Appendix C.
Summary of Findings in Examples 2-11
[0430] It was initially thought that transferring proteins, such as
antibodies, into WFI would likely induce protein precipitation by
concentrating the protein beyond its solubility limit in pure
water. The above studies demonstrate that proteins, including
antibodies, not only can be transferred into pure WFI at lower
concentrations without encountering any precipitation phenomena and
solubility limitations, but that, surprisingly, Adalimumab (as well
as the other two test proteins) can be concentrated in pure water
to ultra-high concentrations beyond 200 mg/mL using UF/DF and
centrifugation techniques (e.g., TFF equipment, Vivaspin devices).
In addition, Adalimumab opalescence was unexpectedly found to be
substantially reduced when the protein was formulated in WFI.
Osmolality was monitored to ensure that the Adalimumab buffer
medium was completely exchanged by pure, salt free water (i.e.
WFI). Moreover, freeze-thaw processing was performed during sample
preparation for analysis, and virtually no instability phenomena
were observed with SEC and IEC analysis.
[0431] The approach of formulating proteins, e.g., Adalimumab, at
high concentrations in WFI revealed the potential to reduce
viscosity phenomena, which often impedes straightforward Drug
Product development at high protein concentrations.
[0432] Finally, the hydrodynamic diameter (determined via photon
correlation spectroscopy, PCS) of Adalimumab was found to be
notably lower in WFI than in commercial buffer (indicative of lower
viscosity proneness).
[0433] Overall, it was concluded that the findings of antibodies
and the globular model protein HSA being soluble in pure water in
ultra-high concentrations have potential to provide new insight
into fundamental protein regimes and to potentially offer new
approaches in protein drug formulation and manufacturing, for
instance by: [0434] reducing opalescence of high-concentrated
protein formulations [0435] reducing viscosity of high-concentrated
protein formulations [0436] enabling to adjust osmolality as
desired in protein-WFI solutions by adding non-ionic excipients
such as mannitol without changing features such as viscosity and
non-opalescence (it was demonstrated for J695 that hydrodynamic
diameter and opalescence did not change when mannitol was added,
but increased dramatically when NaCl was added) [0437] providing a
new paradigm in Drug Substance formulation and processing, as it
was demonstrated that proteins can be subjected to operations such
as DF/UF for concentrating the protein in WFI to ultra-high
concentrations and to freeze and thaw without substantial stability
implications. Given the background that it is well-known that
during DF/UF the composition of protein formulations, especially
during processing to high concentrations, necessarily changes
(Stoner, M. et al., Protein-solute interactions affect the outcome
of ultrafiltration/diafiltration operations, 93 J. Pharm. Sci. 2332
(2004)), these new findings could beneficially be applied by either
adjusting the Drug Substance concentration by DF/UF of the protein
in pure water, and add excipients at high DS concentrations
subsequently (by this avoiding the risk of DS formulation changes
during process unit operations). Alternatively, the excipients
could be added to Drug Substance during final Drug Product
fill-finishing.
Example 12: Preparation of Adalimumab in Water Formulation
[0438] The following example illustrates the scaling up the DF/UF
procedures resulting in large scale production of adalimumab in
water.
12.1: Evaluation of Process Parameters
[0439] Dialysis process evaluation studies were performed on a
laboratory scale to define suitable parameters for the dialysis of
Bulk Adalimumab Drug Solution formulated in a phosphate/citrate
buffer system containing other excipients, e.g., mannitol and
sodium chloride (FIGS. 13 and 14).
[0440] Conductivity measurements may be taken with any commercially
available conductivity meter suitable for conductivity analysis in
protein solutions, e.g. conductivity meter Model SevenMulti, with
expansion capacity for broad pH range (Mettler Toledo,
Schwerzenbach, Switzerland). The instrument is operated according
to the manufacturers instructions (e.g., if the conductivity sensor
is changed in the Mettler Toledo instrument, calibration must be
performed again, as each sensor has a different cell constant;
refer to Operating Instructions of Model SevenMulti conductivity
meter). If the instructions are followed, conductivity measurements
can be taken by directly immersing the measuring probe into the
sample solution.
[0441] FIG. 13 shows the efficiency of the dialysis procedure, in
terms of the reduction of components responsible for the osmolality
and the conductivity of the formulation containing adalimumab at 74
mg/ml. After a reduction of the solutes in the antibody solution by
a factor of 100, osmolality and conductivity measurements largely
stabilized at levels far below the original measurements of these
parameters from the commercial formulation.
[0442] FIG. 14 shows the stability of pH in dialyzed Adalimumab
bulk solutions. pH levels before and after dialysis against
deionized water (1:1,000,000) are shown for Adalimumab solutions
with a range of different initial pH readings. pH levels remained
nearly the same in the retentate before and after dialysis.
12.2: Production of High-Concentrated Adalimumab in Water Bulk Drug
Solution
[0443] In a first step, the formulated bulk drug solution
(Phosphate/Citrate Buffer system containing other excipients, e.g.,
Mannitol and Sodium Chloride) was up-concentrated by
Ultrafiltration/Diafiltration to a concentration of approximately
100 mg/ml (12 L scale, Millipore Pellicon 2 Mini Bio-A MWCO 10k
columns). In a second step, the up-concentrated solution was
dialyzed against deionized water (SpectraPor7 MWCO10k, dilution
factor 1:100,000). As a third step the dialyzed solution was
up-concentrated by Ultrafiltration/Diafiltration to a concentration
of approximately 250 mg/ml using Millipore Pellicon 2 Mini Bio-A
MWCO 10k columns.
[0444] Table 16a shows the results of analysis of highly
concentrated Adalimumab in water (DF/UF processed) bulk drug
solutions after Step 3 of the procedure.
TABLE-US-00020 TABLE 16a Osmolality and Conductivity Data for DF/UF
Bulk-Processed Adalimumab osmolality conductivity density
Adalimumab mosm/kg mS/cm pH g/cm3 conc mg/ml 62 0.95 5.28 1.0764
277.8
12.3: Freeze/Thaw (F/T) Procedure Simulating Manufacturing
Conditions
[0445] Freezing was conducted using an ultra-low temperature
freezer (Revco Ultima II, 20 cu. ft.) with a manufacturing scale
load of 47 kg of liquid to be frozen at a temperature below
-50.degree. C., typically -70.degree. C. to -80.degree. C. The
liquid was packaged in individual bottles of 1.6 kg fill weight
(e.g., Nalgene 2 L PETG square media). Freezing was completed after
48 hours. Thawing was conducted in a circulating water bath (e.g.,
Lindergh/Blue) with a manufacturing scale load of 24 kg at a
temperature between 20.degree. C. and 40.degree. C., typically
30.degree. C., until the material was completely thawed.
12.4: Bottle Mapping During Freeze and Thaw
[0446] Individual horizontal solution layers in the bottle volume
were isolated and analyzed. At protein concentrations of 250 mg/ml
and 200 mg/ml, only minimal gradient formation was detected in the
Adalimumab water solution, as seen in FIGS. 15 through 19. Freezing
and thawing of formulated Adalimumab solutions (solutions with a
Phosphate/Citrate Buffer system containing other excipients, e.g.,
Mannitol and Sodium Chloride) at 250 mg/ml and 200 mg/ml, however,
led to the formation of precipitate on the bottom of the
bottle.
12.5: Gradient Formation in Commercial and Low-Ionic Formulations
of Adalimumab
[0447] The formation of gradients by freeze thaw procedures in
commercial and low-ionic (water) formulations of Adalimumab was
compared. Table 16b shows the results of visual inspection of
commercial Adalimumab solutions of various concentrations after a
f/t step. The formation of precipitates indicates that instability
was created in the solution by the f/t procedure. Above 100 mg/ml,
significant precipitate formation was observed. Table 17 shows the
analytical data of two 50 mg/ml solutions and one 100 mg/ml
low-ionic formulation before the freeze-thaw experiment.
TABLE-US-00021 TABLE 16b Observed Precipitation in Commercial
Adalimumab Solutions after F/T 250 mg/ml 220 mg/ml 200 mg/ml 150
mg/ml 120 mg/ml 100 mg/ml 60 mg/ml Commericial Precipitate
Precipitate Precipitate Precipitate Partial Clear Clear freezing
process Precipitate in ultra low temp freezer: -70.degree.
C./23.degree. C.
TABLE-US-00022 TABLE 17 Solution Analytical Data before Freeze-Thaw
Protein Density Osmolality conc formulation pH (g/cm.sup.3)
(mOsmol/kg) (mg/mL) E167 130 01 low-ionic 5.18 1.0121 5 49.3 CL 50
mg/mL in water E167 140 01 Commercial 5.20 1.0224 280 48.7 CL 50
mg/mL formulation in buffer 100 mg/mL low-ionic 5.32 1.0262 12 99.8
in water
[0448] About 1600 mL (50 mg/ml solutions) or 800 ml (100 mg/ml
solution) of each formulation were placed into PETG bottles and
subjected to a conventional freeze (-80.degree. C.) thaw
(23.degree. C., water bath) procedures. Samples were then pulled
from top, center and bottom of the PETG bottles and analyzed for
pH, density, osmolality, and protein concentration. Analysis
results are shown in Table 18.
TABLE-US-00023 TABLE 18 Analysis of Bottle-Mapped Layers from
Frozen/Thawed Solutions protein content density osmolality
(volumetric) sample pH g/cm3 mOsmol/kg mg/mL 50 mg/mL in water top
5.20 1.0119 6 48.72 middle 5.19 1.0120 8 49.35 bottom 5.17 1.0120 6
49.76 commercial formulation top 5.16 1.0165 236 37.9 middle 5.13
1.0221 306 45.58 bottom 5.12 1.0257 368 55.48 100 mg/mL in water
top 5.29 1.0259 13 98.7 middle 5.3 1.0262 16 99.9 bottom 5.28
1.0262 14 101.2
[0449] The commercial formulation of Adalimumab revealed
significant gradients upon freeze/thaw with regard to density
(indicating heterogeneities/gradients of protein and excipients),
osmolality (indicating excipient gradients), and protein content.
In contrast, no gradients were found in the 50 mg/ml low-ionic
Adalimumab formulation upon freeze/thaw.
[0450] At higher protein concentrations, gradient formation may
sometimes be expected to become worse. However, no gradients were
found in the 100 mg/ml low-ionic Adalimumab formulation upon
freeze/thaw with regard to pH, density, osmolality and protein
concentration.
Example 13: Stability of J695 after DF/UF
[0451] The following example provides data on the stability of J695
after DF/UF processing in accordance with the methods of the
invention.
[0452] Protein samples from J695 in normal DS buffer were analyzed,
either after pH adjustment or after diafiltration. pH was adjusted
to pH 4.4 with 0.1M phosphoric acid, protein concentration 112
mg/mL. For concentrated samples in WFI, protein samples were
diafiltered (DF/UF) against water for approximately 1.5 days at
ambient temperature, using a TFF equipped with a 30 kDa RC
membrane. The protein concentration after DF/UF was determined
approx. 192 mg/mL. pH 4.7.
13.1: Size Exclusion Analysis (SEC) Experimental Procedures
[0453] A size exclusion method was developed for the purity
assessment of J695. Size exclusion chromatography (SEC) separates
macromolecules according to molecular weight. The resin acts as a
sieving agent, retaining smaller molecules in the pores of the
resin and allowing larger molecules to pass through the column.
Retention time and resolution are functions of the pore size of the
resin selected.
[0454] Each sample was diluted to 2.5 mg/mL with purified water
(Milli-Q) based on the stated concentration. 50 .mu.g of each
sample was injected onto the column in duplicate. A Tosoh
Bioscience G3000swxl, 7.8 mm.times.30 cm, 5 .mu.m (Cat #08541) SEC
column is used for separation. For Buffer A, 211 mM
Na.sub.2SO.sub.4/92 mM Na.sub.2HPO.sub.4, pH 7.0 was used.
Detection was performed at 280 nm and 214 nm. The column was kept
at room temperature with a flow rate of 0.3 mL/min
[0455] This chromatography utilized an isocratic gradient with a
100% mobile phase A solvent for 50 minutes duration.
13.2: SEC Data
[0456] Table 19 describes data from size exclusion chromatography
experiments.
TABLE-US-00024 TABLE 19 SEC Analysis Data for J695 Reference
Standard, DS and Post-DF/UF (in water) ABT-874 load HM Monom Frag
BF Ref Std 50 u 0.49 97.9 1.28 0.26 BF Ref Std dup 50 u 0.41 98.0
1.29 0.27 BF Ref std avg 0.45 98.0 1.29 0.27 std 0.06 0.05 0.01
0.01 % RS 12.5 0.05 0.55 2.67 DS in Buffer pH 4.4 50 u 0.42 98.3
1.04 0.16 DS in Buffer pH 4.4 dup 50 u 0.40 98.4 1.01 0.13 DS in
Buffer pH 4.4 avg 0.41 98.4 1.03 0.15 std 0.01 0.06 0.02 0.02 % RS
3.45 0.06 2.07 14.6 DS UF/DF in Water pH 4.7 50 u 0.69 98.1 1.04
0.14 UF/DF in Water pH 4.7 dup 50 u 0.69 98.0 1.07 0.16 DS UF/DF in
Water pH 4.7 avg 0.69 98.1 1.06 0.15 std 0.00 0.04 0.02 0.01 % RS
0.00 0.04 2.01 9.43
13.3: SEC Analysis Conclusions
[0457] The data in Table 19 shows that the commercial formulation
of J695 (DS PFS, pH=4.4) has comparable levels of fragments and
aggregate as the J695 reference standard. There was a difference
noted in the aggregate amount between the commercial formulation
J695 control and the J695 that had undergone DF/UF, in water (DS in
H2O, pH=4.7, 192 mg/ml): an increase from 0.4% to 0.7% in
aggregation was seen. This was not a significant increase and may
be due to time spent at room temperature during the UF/DF. There is
no change to the fragments.
13.4: IEC (WCX-10) Experimental Procedure
[0458] A cation exchange method was developed for the assessment of
the heterogeneity of J695 using the Dionex WCX-10 column.
Generally, cation exchange chromatography separates protein
isoforms according to the apparent pI and the surface charge
interaction with the resin. The protein of interest is bound to the
column under specific low salt starting conditions and is eluted
from the column by increasing the salt concentration through a
gradient. Proteins with lower apparent pI bind less tightly to a
cation exchange column and are the first to elute and proteins with
a higher apparent pI bind tighter and are the last to elute.
[0459] Cation exchange chromatography using WCX-10 was used in
quality control as a lot release assay. The assay conditions were
modified to improve separation of known J695 isoforms.
[0460] The sample was diluted to 1.0 mg/mL with purified water
(Milli-Q). Reference standard was run in triplicate as a comparison
and was diluted to 1 mg/ml in purified water (Milli-Q).
[0461] Dionex Propac WCX-10 columns (p/n 054993), along with
corresponding guard columns (p/n 054994), were used for separation.
Buffers used in the procedure included Buffer A (10 mM
Na.sub.2HPO.sub.4, pH=6.0) and Buffer B (10 mM Na.sub.2HPO.sub.4,
500 mM NaCl, pH=6.0). Column temperature was maintained at
35.degree. C. and column flow rate was 1 mL/min. Injection volumes
were 100 .mu.l for a 100 .mu.g load and detection was performed at
280 nm. Buffer gradients over the course of the chromatographic
separation are provided in Table 20.
TABLE-US-00025 TABLE 20 Buffer Gradients used in IEC Analysis of
J695 Time (min) % MPA % MPB 0 75 25 3 60 40 33 40 60 36 0 100 41 0
100 43 75 25 48 75 25
13.5: IEC Data
[0462] Table 21 provides results from experiments comparing the
J695 Reference Standard to J695 in commercial buffer (DS pH=4.4),
as well as a comparison of the commercial buffer formulation to
J695 after DF/UF (DF/UF H2O, pH=4.7).
TABLE-US-00026 TABLE 21 IEC Data for J695 Reference Standard,
Commercial Formulation (DS) and after DF/UF (in water) 0 glu (1) 0
glu (2 + 2a) 1 glu (3) 1 glu (4) 1 glu (5) + (5a) 2 glu (6) 2 glu
(7) acidic basic ref std 43.77 7.55 8.00 21.87 4.28 4.82 3.75 4.05
1.92 ref std dup 43.49 7.49 7.98 21.70 4.26 4.81 3.75 4.61 1.90 ref
std dup 43.44 7.49 8.00 21.65 4.24 4.81 3.74 4.75 1.89 ref std avg
43.57 7.51 7.99 21.74 4.26 4.81 3.75 4.47 1.90 SD 0.20 0.04 0.01
0.12 0.01 0.01 0.00 0.40 0.01 % RSD 0.45 0.56 0.18 0.55 0.33 0.15
0.00 8.86 0.74 ABT-874 DS pH = 4.4 35.65 14.74 7.26 18.06 6.76 5.32
3.98 5.55 2.70 ABT-874 DS pH = 4.4 Dup 35.82 14.73 7.29 18.14 6.82
5.39 4.06 4.79 2.95 ABT-874 DS pH = 4.4 avg 35.74 14.74 7.28 18.10
6.79 5.36 4.02 5.17 2.83 SD 0.12 0.01 0.02 0.06 0.04 0.05 0.06 0.54
0.18 % RSD 0.34 0.05 0.29 0.31 0.62 0.92 1.41 10.39 6.26 ABT-874
DF/UF H2O pH = 4.7 36.57 14.51 7.26 18.09 6.57 5.22 3.91 5.28 2.61
ABT-874 DF/UF H2O pH = 4.7 Dup 36.60 14.43 7.25 18.02 6.66 5.18
4.00 5.31 2.56 ABT-874 DF/UF H2O pH = 4.7 36.59 14.47 7.26 18.06
6.62 5.20 3.96 5.30 2.59 SD 0.02 0.06 0.01 0.05 0.06 0.03 0.06 0.02
0.04 % RSD 0.06 0.39 0.10 0.27 0.96 0.54 1.61 0.40 1.37
13.6: IEC Analysis Conclusions
[0463] There were some differences noted between the J695 Reference
Standard and the commercial formulation (DS, pH 4.4). These
differences were noted in the initial run of the DS engineering run
sample and are attributed to differences in the manufacturing
processes between the 3000 L and 6000 L campaigns. There were no
notable differences between the DS, pH 4.4 control and the J695 in
H.sub.2O pH=4.7, 192 mg/ml sample.
Example 14: Stability of Adalimumab after DF/UF and Long-Term
Storage at 2-8.degree. C.
[0464] The following example provides data showing the stability of
Adalimumab in an aqueous formulation in accordance with the methods
of the invention, after 22.5 months storage at 2-8.degree. C.
[0465] Adalimumab samples for SEC and WCX-10 analysis were
diafiltered against water and concentrated to about 177 mg/mL.
Samples were stored and analyzed at various time points for
stability.
[0466] Standard Adalimumab solution (DS, pH approx. 5.2) in
commercial Humira buffer was used as a starting material for
generating a concentrated solution in water. Protein solution
samples were diafiltered (DF/UF) against water for approximately
1.5 days at ambient temperature, using a TFF equipped with a 30 kDa
RC membrane. Protein concentration after DF/UF was determined
approximately 177 mg/mL, pH 5.2. The sample was stored at
2-8.degree. C. for 22.5 months before analysis.
14.1: SEC Experimental Procedure
[0467] A size exclusion method was previously developed to check
for the presence of antibody fragments and aggregates. Size
exclusion chromatography (SEC) separates macromolecules according
to molecular weight. The resin acts as a sieving agent, retaining
smaller molecules in the pores of the resin and allowing larger
molecules to pass through the column. Retention time and resolution
are functions of the pore size of the resin selected.
[0468] Each sample was diluted to 1.0 mg/mL with milli Q water and
50 .mu.g of each sample was injected onto the column. For SE-HPLC,
a Sephadex 200 column (Pharmacia cat #175175-01, S/N 0504057) or a
TSK gel G3000SW (cat #08541; for analysis of 22.5 month samples)
were used. The mobile phase of the column comprised 20 mM Sodium
phosphate and 150 mM Sodium chloride, pH 7.5. Detection was
performed at 280 nm and 214 nm. Columns were kept at ambient
temperature and the flow rate was 0.5 mL/min (Sephadex column), or
0.3 mL/min (TSK column).
14.2: SEC Data
[0469] FIG. 20 and Table 22 contain results of the analysis of a
low-ionic Adalimumab solution stored as a liquid at 2-8.degree. C.
for 8.5 months compared to the same solution stored at -80.degree.
C. Table 23 contains analysis data for a low-ionic Adalimumab
solution stored at 2-8.degree. C. for 22.5 months compared to a
reference standard sample of Adalimumab.
TABLE-US-00027 TABLE 22 SEC Analysis Data Comparing Adalimumab from
Frozen Storage versus Adalimumab from Long-Term Refrigerated
Storage % % % Sample Load HMW Monomer LMW DF/UF against water, 177
mg/mL, 50 .mu.g 0.1 99.6 0.3 4.5 months at -80.degree. C. DF/UF
against water, 177 mg/mL, 50 .mu.g 0.2 99.5 0.3 9 months at
2-8.degree. C.
TABLE-US-00028 TABLE 23 SEC Analysis Data Comparing Adalimumab
Reference Standard against Adalimumab from Long-Term Refrigerated
Storage % % % Sample Load HMW Monomer LMW Reference Std. Adalimumab
50 .mu.g 0.31 98.85 0.84 DF/UF against water, 177 mg/mL, 50 .mu.g
1.42 97.59 0.98 22.5 months at 2-8.degree. C.
[0470] As can be seen in Table 22, SEC analysis revealed that
adalimumab in water was stable even after 9 months at 2-8.degree.
C. or for 4.5 months at -80.degree. C., as the percent aggregate (%
HMW) and percent fragment (% LMW) were minimal over time.
14.3: SEC Analysis Conclusions
[0471] After 8.5 months storage at 2-8.degree. C., the Adalimumab
solution (DF/UF against water) revealed a small fraction of high
molecular weight (HMW) species (0.2%) and a small fraction of
fragment (0.3%). Storage for 4.5 months at -80.degree. C. and
subsequent thaw (water bath, 23.degree. C.) did not impact
Adalimumab stability (0.1% aggregate, 0.3% fragment).
[0472] Analysis of a sample stored for 22.5 months at 2-8.degree.
C. also shows comparable fragment content to Adalimumab reference
standard (Table 23). However, the aggregate levels detected in the
22.5 month stability sample (1.66%) are somewhat higher than
aggregate levels detected in the reference standard.
[0473] It is known that self-association of antibodies is highly
dependent on the antibody concentration, i.e. the formation of
non-covalent aggregate and associate complexes is most pronounced
at high protein concentration. This self-association is reversible,
and dilution with buffer solution results in reduced
self-association tendencies (Liu, J. et al., 94 Journal of
Pharmaceutical Sciences 1928 (2004)).
[0474] Thus, it is likely that differences in sample preparations
and different lag-times between Adalimumab solution dilution (from
177 mg/mL to 1 mg/mL) and subsequent sample analysis by SEC are the
reason for the differences in aggregate content of the 8.5 month
and the 9 month stability samples.
14.4: IEC Experimental Procedure
[0475] A cation exchange method was developed for the assessment of
antibody charge heterogeneity using the Dionex WCX-10 column.
Cation exchange chromatography separates protein isoforms according
to the apparent pI and the surface charge interaction with the
resin. The protein of interest is bound to the column under
specific low salt starting conditions and is eluted from the column
by increasing the salt concentration through a gradient. Proteins
with lower apparent pI bind less tightly to a cation exchange
column and are the first to elute and proteins with a higher
apparent pI bind tighter and are the last to elute.
[0476] Before the procedure, samples were diluted to 1.0 mg/mL with
milli Q water. Dionex Propac WCX-10 columns (p/n 054993), along
with a corresponding guard columns (p/n 05499), were used for
separation. Two mobile phase buffers were prepared, 10 mM Sodium
phosphate, pH 7.5 (Buffer A) and 10 mM Sodium phosphate, 500 mM
Sodium chloride, pH 5.5 (Buffer B). Columns were kept at ambient
temperature and the flow rate was 1.0 mL/min Injection volumes were
100 .mu.l for a 100 .mu.g load and detection was performed at 280
nm. Buffer gradients over the course of the chromatographic
separation are provided in Table 24.
TABLE-US-00029 TABLE 24 Buffer Gradients used in IEC Analysis of
Adalimumab Time (min) % MPA % MPB 0.05 94 6 20 84 16 22 0 100 26 0
100 28 94 6 34 94 6 35 94 6
14.5: Ion Exchange Data
[0477] Table 25 shows the ion exchange chromatographic data for the
Adalimumab reference standard, commercial formulation (150 mg/ml)
and post-DF/UF low-ionic solution before storage. Table 26 shows
data for the reference standard compared to the low-ionic solution
after 22.5 months of storage at 2-8.degree. C.
TABLE-US-00030 TABLE 25 IEC Analysis Data of Adalimumab Reference
Standard, DS/Commercial Formulation and After DF/UF (in water) %
Acidic % Acidic % 0 % 1 % 2 Sample Name Region 1 Region 2 Lys Lys
Lys Adalimumab Ref. Std. 2.69 11.66 60.77 19.42 5.40 Adalimumab DS
150 2.51 11.38 62.05 19.14 4.83 mg/ml Adalimumab diafiltered 2.26
11.81 61.97 18.51 4.73 against water, 177 mg/ml
TABLE-US-00031 TABLE 26 IEC Analysis Data Comparing Reference
Standard to DF/UF Sample from Long-Term Refrigerated Storage %
Acidic % Acidic % 0 % 1 % 2 Sample Name Region 1 Region 2 Lys Lys
Lys Adalimumab Ref. Std. 2.1 10.9 63.8 18.4 4.6 Adalimumab DF/UF
2.7 13.4 62 16.7 4.1 against water, 177 mg/mL, 22.5 months at
2-8.degree. C.
14.6: Ion Exchange Analysis Conclusions
[0478] For the T0 samples, data show no significant difference in
the percentage of acidic region 1, 2, 0 Lys, 1 Lys, or 2 Lys (i.e.,
charge heterogeneity) between reference standard Adalimumab,
commercial formulation Adalimumab (used as DS to formulate
Adalimumab into water by DF/UF), and Adalimumab diafiltered against
water and concentrated to 177 mg/ml (Table 25).
[0479] Also, after 22.5 months storage of the 177 mg/mL Adalimumab
sample in water, only slight differences in 0 Lys, 1 Lys and 2 Lys
fractions can be seen when compared to the Adalimumab reference
standard. In summary, no significant chemical instability
tendencies are observed when Adalimumab is formulated into water by
DF/UF processing and stored for 22.5 months at 2-8.degree. C. at a
concentration of 177 mg/mL.
Example 15: Freeze/Thaw Stability of Low-Ionic 1D4.7 Solution
[0480] 1D4.7 protein (an immunoglobulin G1) anti-IL 12/anti-IL 23
was formulated in water by dialysis (using slide-a-lyzer cassettes,
used according to operating instructions of the manufacturer,
Pierce, Rockford, Ill.) was demonstrated to be stable during
repeated freeze/thaw (f/t) processing (-80.degree. C./25.degree. C.
water bath) at 2 mg/mL concentration, pH 6. Data were compared with
routine formulations (2 mg/mL, pH 6), and it was found that the
stability of 1D4.7 formulated in water exceeded the stability of
1D4.7 formulated in routinely screened buffer systems (e.g. 20 mM
histidine, 20 mM glycine, 10 mM phosphate, 10 mM citrate) and even
exceeded the stability of 1D4.7 formulations based on universal
buffer (10 mM phosphate, 10 mM citrate) with a variety of
excipients that are commonly used in protein formulation, e.g. 10
mg/mL mannitol, 10 mg/mL sorbitol, 10 mg/mL sucrose, 0.01%
polysorbate 80, 20 mM NaCl.
[0481] SEC, DLS and particle counting was performed to monitor
protein stability, and particle counting was performed by using a
particle counting system with a 1-200 .mu.m measurement range (e.g.
particle counter Model Syringe, Markus Klotz GmbH, Bad Liebenzell,
Germany). Experiment details are as follows: [0482] 1D4.7
formulated in water compared with formulations listed above [0483]
4 freeze/thaw cycles applied [0484] 30 mL PETG repository, about 25
mL fill, 2 mg/mL, pH 6 [0485] sampling at T0, T1 (i.e. after one
f/t step), T2, T3, and T4 [0486] analytics: visual inspection, SEC,
DLS, subvisible particle measurement
[0487] FIG. 21 shows 1D4.7 stability during repeated f/t cycling
(-80.degree. C./25.degree. C.), mirrored by formation of subvisible
particles >1 .mu.m. 1D4.7 was formulated in universal buffer (10
mM citrate, 10 mM phosphate) and then the following excipient
variations were tested: sorbitol (10 mg/mL), mannitol (10 mg/mL),
sucrose (10 mg/mL), NaCl (100 mM), and polysorbate 80 (0.01%).
1D4.7 was also formulated in water (by dialysis) with no excipients
added at all. Water for injection was also subjected to f/t cycling
and subvisible particle testing to evaluate a potential impact of
material handling, f/t, and sample pull on particle load.
[0488] The stability of 1D4.7 formulated in water upon f/t exceeded
the stability of 1D4.7 solutions formulated with excipients
typically used in protein formulations. Mannitol, sucrose, and
sorbitol are known to act as lyoprotectant and/or cryoprotectant,
and polysorbate 80 is a non-ionic excipient prevalently known to
increase physical stability of proteins upon exposure to
hydrophobic-hydrophilic interfaces such as air-water and ice-water,
respectively. Thus, 1D4.7 solutions formulated in water appeared to
be stable when analyzed with other methodologies applied (e.g. SEC,
visual inspection, etc.).
Example 16: Freeze/Thaw Stability of Low-Ionic 13C5.5 Antibody
Solution
[0489] 13C5.5 anti IL-13 protein formulated in water was
demonstrated to be stable during repeated freeze/thaw processing
(-80.degree. C./25.degree. C. water bath) at 2 mg/mL concentration,
pH 6. Data were compared with routine formulations (2 mg/mL, pH 6),
and it was found that the stability of 13C5.5 formulated in water
exceeded the stability of 13C5.5 formulated in routinely screened
buffer systems (e.g. 20 mM histidine, 20 mM glycine, 10 mM
phosphate, 10 mM citrate) and even exceeded the stability of 13C5.5
formulations based on universal buffer (10 mM phosphate, 10 mM
citrate) with a variety of excipients that are commonly used in
protein formulation (e.g. 10 mg/mL mannitol, 10 mg/mL sorbitol, 10
mg/mL sucrose, 0.01% polysorbate 80, 20 mM NaCl, 200 mM NaCl).
[0490] Sample preparation, experiment processing, sample pull and
sample analysis was performed in the same way as outlined in
Example 15 for 1D4.7. [0491] 13C5.5 formulated in water compared
with formulations listed above [0492] 4 freeze/thaw cycles applied
[0493] 30 mL PETG repository [0494] 2 mg/mL, pH 6 [0495] sampling
at T0, T1, T2, T3, and T4 [0496] analytics: visual inspection, SEC,
DLS, subvisible particle measurement
[0497] FIG. 22 shows 13C5.5 stability during repeated f/t cycling
(-80.degree. C./25.degree. C.), mirrored by formation of subvisible
particles >10 .mu.m. 13C5.5 was formulated in either 10 mM
phosphate buffer, 10 mM citrate buffer, 20 mM glycine buffer, and
20 mM histidine buffer. 13C5.5 was also formulated in water (by
dialysis) with no excipients added at all. Water for injection was
also subjected to f/t cycling and subvisible particle testing to
evaluate a potential impact of material handling, f/t, and sample
pull on particle load (blank) The stability of 13C5.5 formulated in
water upon f/t exceeded the stability of 13C5.5 solutions
formulated in buffers typically used in protein formulations. No
instabilities of 13C5.5 solutions formulated in water have been
observed with other analytical methodologies applied (e.g. SEC,
visual inspection, etc.)
[0498] FIG. 23 shows 13C5.5 stability during repeated f/t cycling
(-80.degree. C./25.degree. C.), mirrored by formation of subvisible
particles >1 .mu.m. 13C5.5 was formulated in universal buffer
(10 mM citrate, 10 mM phosphate) and then the following excipient
variations were tested: sorbitol (10 mg/mL), mannitol (10 mg/mL),
sucrose (10 mg/mL), NaCl (200 mM), NaCl (20 mM) and polysorbate 80
(0.01%). 13C5.5 was also formulated in water (by dialysis) with no
excipients added at all for comparison (pure water). Water for
injection was also subjected to f/t cycling and subvisible particle
testing to evaluate a potential impact of material handling, f/t,
and sample pull on particle load.
[0499] The stability of 13C5.5 formulated in water upon f/t
exceeded the stability of 13C5.5 solutions formulated with
excipients typically used in protein formulations. Mannitol,
sucrose, and sorbitol are known to act as lyoprotectant and/or
cryoprotectant, and polysorbate 80 is a non-ionic excipient
prevalently known to increase physical stability of proteins upon
exposure to hydrophobic-hydrophilic interfaces such as air-water
and ice-water, respectively.
[0500] No instabilities of 13C5.5 solutions formulated in water
have been observed with other analytical methodologies applied,
(e.g. SEC, visual inspection, etc.).
[0501] DLS analysis of 13C5.5 solutions after f/t procedures was
performed as described above. An 13C5.5 solution with 0.01%
Tween-80 contained significant high molecular weight (HMW)
aggregate forms after only 1 f/t step, whereas 13C5.5 in water
contained no HMW aggregate forms, even after 3 f/t steps.
Example 17: Impact of Solution pH on Adalimumab In WFI
[0502] The following experiments were performed to determine the
impact of solution pH on physico-chemical characteristics of highly
concentrated Adalimumab formulated in WFI. The following
concentrations were tested: 2 mg/mL, 50 mg/mL, 100 mg/mL, 150
mg/mL, 200 mg/mL, and 250 mg/mL.
Materials
[0503] Adalimumab Drug Substance (DS), commercial material [0504]
25.degree. C. water bath (circulating) used for thawing [0505]
Diafiltration equipment: Sartorius Sartocon Slice, membrane: PES 50
kD, 1000 cm.sup.2 [0506] Diafiltration equipment: Millipore
Labscale.TM. TFF System, membrane: PLCTK 30 kD, regenerated
Cellulose, size: 50 cm.sup.2 [0507] Eppendorf Centrifuge 5810 R
[0508] Amicon Ultra-15 repositories for centrifugation,
Ultracel-30k, Regenerated Cellulose 30,000 MWCO [0509] Millex GV
0.22 .mu.m, Millipore for sterile filtration of samples [0510]
Sample repositories (Eppendorf sample repository 1.5 mL, Roth
cryovials 5 mL, PETG bottle 125 mL)
[0511] Analytics: [0512] pH measurement using Biothrode [0513]
Density measurement [0514] Osmolality measurement [0515] UV/VIS
spectrophotometer for protein concentration measurement [0516]
Photon Correlation Spectroscopy (PCS) [0517] Viscosity measurement
[0518] Turbidity measurement [0519] Size Exclusion Chromatography
(SEC) [0520] Fourier transform mid infrared spectroscopy
(FT-M-IR)
17.1 Overview of Preparation for DF/UF of Adalimumab Commercial
Formulation
[0521] The Adalimumab DS solution (120 mg/mL) was divided into 7
volume portions which were adjusted to pH3, pH4, pH5, pH6, pH7,
pH8, pH9 with 0.25N NaOH and 0.25N HCl, respectively. Then the
samples were diluted with Adalimumab buffer of the respective pH to
100 mg/mL. The solutions revealed a slight cloudyness that
disappeared after sterile filtration (0.22 .mu.m, PVDF sterile
filter). After dilution, the pH value were monitored again (see
Table 27 below).
[0522] The following samples of the 100 mg/mL solutions were pulled
from each solution: [0523] 4 mL for turbidity and subsequent
zetapotential measurement [0524] 1 mL for viscosity measurement
(using dropping-ball viscometer) [0525] 0.15 mL for osmolality
measurement [0526] 2 mL for density measurement [0527] 0.15 mL for
PCS (sample viscosity taken into account for measurements) [0528] 1
mL for FT-M-IR [0529] 2 mL for viscosity and static light
scattering measurements
[0530] The samples for zetapotential, viscosity and static light
scattering measurements were frozen (-80.degree. C.). The remaining
volumes of pH 4, pH 5, pH 6, pH 7, and pH 8 solutions were
subjected to continuous mode diafiltration using water for
injection as exchange medium. The samples were first frozen at
-80.degree. C. Before DF/UF, the samples were thawed at 25.degree.
C. in a Julabo water bath.
17.2 DF/UF and Concentration Procedures
[0531] Adalimumab solutions in commercial formulation, with
concentrations of 100 mg/ml, with pH levels of 4, 5, 6, 7 and 8,
were subject to DF/UF processing and further subject to
concentration process with UF in a centrifuge. This section
describes the processing of the pH 6 Adalimumab solution as an
example. Processing for the other solutions was done in a similar
manner.
[0532] The Adalimumab solution (100 mg/mL, pH 6) was thawed in a
water bath at 25.degree. C. and then homogenized. Then, the
solution was subjected to diafiltration using water for injection
as exchange medium with TFF equipment M.P. 33.4 by applying the
following parameters: [0533] stirrer: speed 2 [0534] pump: speed 1
[0535] pressure up-stream/inlet: 2-2.4 bar [0536] pressure
down-stream/outlet: 0.6-0.8 bar [0537] membrane: regenerated
Cellulose, cut off 30 kD [0538] continuous mode DF/UF [0539] about
6-fold volume exchange applied during DF/UF operation
[0540] After applying 6-volume exchange steps, the concentration of
Adalimumab was determined by means of OD280, photometer M.P. 9.7.
The osmolality of permeate and retentate was checked.
[0541] concentration: 125.1 mg/mL
[0542] osmolality permeaet: 57 mOsmol/kg
[0543] osmolality retentate: 12 mOsmol/kg
[0544] The Adalimumab solution in water after DF was diluted with
water for injection to 100 mg/mL and sterile filtered. The
following samples were pulled from 100 mg/mL solution after the
DF/UF process: [0545] 4 mL for turbidity and subsequent
zetapotential measurement [0546] 1 mL for viscosity measurement
[0547] 0.15 mL for osmolality measurement [0548] 2 mL for density
measurement [0549] 0.15 mL for PCS (viscosity taken into account
during measurement) [0550] 0.15 mL for SEC [0551] pH--measurement
[0552] 1 mL for FT-M-IR [0553] 2 mL for viscosity and static light
scattering measurements
[0554] A portion of the 100 mg/mL Adalimumab solution was diluted
with water for injection to create 50 mg/mL and 2 mg/mL solutions.
The following samples were pulled from both solutions: [0555] 4 mL
for turbidity and subsequent zetapotential measurement [0556] 2 mL
for viscosity measurement [0557] 0.15 mL for osmolality measurement
[0558] 2 mL for density measurement [0559] 0.15 mL for PCS
(viscosity taken into account) [0560] pH- measurement
[0561] Adalimumab solutions (pH 6, 100 mg/mL) in water were
subjected to concentration experiments using centrifugation.
Centrifugation was performed with Eppendorf Centrifuge (5810R M.P.
33.57). Each centrifugation step was applied for 15 min. at 4000
rpm. After that, homogenization of sample solution in the
centrifuge concentration device was performed by gentle upside-down
rotation in order to homogenized the solution and thereby to avoid
gel formation in areas immediately adjacent to the membrane.
Temperature during concentration was 15.degree. C. The
centrifugation was performed to about 250 mg/mL. The concentration
was determined by means of measuring OD280, photometer M.P. 9.7.
The Adalimumab solutions were then diluted to concentrations of 250
mg/mL, 200 mg/mL and 150 mg/mL.
[0562] The following samples were pulled after the concentration
procedure and after each individual step of dilution. Sample
volumes pulled from 250 mg/mL and 150 mg/mL solutions were: [0563]
2 mL for viscosity-measurement [0564] 0.15 mL for PCS (viscosity
taken into account) [0565] 0.15 mL for osmolality measurement
[0566] 0.15 mL for SEC [0567] pH- measurement Sample volumes pulled
from the 200 mg/mL solution were: [0568] 4 mL for turbidity and
subsequent zetapotential measurement [0569] 1 mL for viscosity
measurement [0570] 0.15 mL for osmolality measurement [0571] 2 mL
for density measurement [0572] 0.15 mL for PCS (viscosity taken
into account) [0573] 0.15 mL for SEC [0574] pH- measurement [0575]
2 mL for analytical work to be performed at ABC (viscosity and
static light scattering measurements)
[0576] The concentration processing of Adalimumab solution in water
was halted at approximately 250 mg/mL at each pH value because the
viscosity of Adalimumab solution in water at higher concentrations,
and especially at pH values close to the pI (about pH 8.5 for
Adalimumab), increased dramatically (viscosities approaching gel
formation).
17.3 Visual Inspection of Adalimumab Solutions
[0577] After DF/UF and concentration to 250 mg/mL, the Adalimumab
solutions in water at various pH appeared less opalescent than the
Adalimumab solution in buffer (commercial formulation). All of the
Adalimumab solutions in water appeared as clear solutions at each
pH value. None of the Adalimumab solutions revealed opalescence
after dilution. Overall, during concentration and dilution
procedures, no precipitation was observed in Adalimumab solutions
in water.
17.4 Viscosity
[0578] The viscosity measurements were performed taking into
account the density of pH 5 Adalimumab solutions at each of the
respective concentrations. A dropping-ball viscometer was used.
Viscosities higher than 200 mPa*s were measured using capillary
viscometer.
[0579] FIG. 24 provides an overview of viscosity data of Adalimumab
solutions in water with pH ranging from 4 to 8, at various
concentrations (2 mg/mL to 250 mg/mL, in 50 mg/mL concentration
steps). There is a clear correlation between solution pH,
concentration and viscosity. The viscosity increases with increases
of protein concentration, independent of the solution pH. At
solution pH values close to the pI of Adalimumab (i.e. pH 7 and pH
8), increases in solution viscosity were most pronounced,
especially at higher protein concentrations (i.e. 200 mg/mL, 250
mg/mL).
17.5 Turbidity
[0580] As seen in FIG. 25, the same trend was found for turbidity
data, (i.e., the turbidity increased with increasing concentration
and with increasing pH). All samples were sterile filtered (0.22
.mu.m) before turbidity measurement.
17.6 Hydrodynamic diameter (PCS)
[0581] The PCS measurements were performed taking into account the
viscosity for each sample, at each concentration and at each pH
value. Solutions at 200 mg/mL and 250 mg/mL were measured but were
outside the testing parameters of the Zetasizer nano series
(Malvern Instruments) equipment, and consequently the data from
these measurements was not analyzed.
[0582] The hydrodynamic diameter (Dh) was found to be notably
decreased when Adalimumab was formulated in water (Dh about 2 nm at
50 mg/mL, pH 5) in comparison to Adalimumab formulated into
commercial formulation (Dh about 7 nm). FIG. 26 illustrates the PCS
data (also found in Table 39). Corresponding data tables are shown
below in part 17.11.
[0583] As shown in FIG. 26, for solutions at pH values of 4, 5 and
6, the Dh of Adalimumab monomer decreased constantly with increased
protein concentration. In contrast, solutions with pH values closer
to the pI of Adalimumab (i.e., at pH 7 and pH8) showed considerable
increases in Dh as concentration increased from 2 mg/mL to 50
mg/mL. As concentrations rose beyond 50 mg/mL in pH 7 and 8
solutions, however, Dh decreased. At a concentration of 150 mg/mL,
all of the solutions had lower Dh values than the corresponding pH
solution at 2 mg/mL. FIG. 27 shows Dh size distributions for pH 5
solutions of various concentrations. FIG. 28 shows Dh size
distributions for five Adalimumab solutions formulated in water,
each having a 100 mg/mL protein concentration and a different pH
value. FIG. 29 shows data similar to data in FIG. 28, except that
the five Adalimumab solutions were formulated in buffer.
17.7 pH- Measurement
[0584] Measurements of solution pH were performed at 100 mg/mL
before and after DF/UF using water (i.e., performed on Adalimumab
formulated in buffer and in water, respectively). Table 27 shows
the results. The pH values stay constant at pH 5, pH 6 and pH 7
before and after DF/UF. The solution pH does not change because of
a medium change. The pH value at pH 4 slightly increases and at pH
8 slightly decreases after DF/UF using water.
TABLE-US-00032 TABLE 27 pH values before and after DF/UF with water
pH 4 pH 5 pH 6 pH 7 pH 8 Adalimumab 100 mg/mL in 4.00 4.99 6.00
7.03 8.00 buffer Adalimumab 100 mg/mL in 4.29 4.98 5.98 7.02 7.67
water
17.8 Osmolality Measurements
[0585] During DF/UF of the pH 5 solution samples, solution
osmolality was measured after each volume exchange step (i.e, after
100 mL permeate, 200 mL permeate, etc.) to check whether a 5-fold
volume exchange is sufficient to reduce osmolality to values below
15 mOsmol/kg. Table 28 shows the results.
TABLE-US-00033 TABLE 28 Osmolality change during DF/UF using water,
pH 5 solution Volume exchange Retentate Permeate step in mL
mOsmol/kg mOsmol/kg 100 96 166 200 28 115 300 29 89 400 12 67 500
15 49
[0586] At pH 4, pH 6, pH 7 and pH 8, the osmolality was measured at
the end of the DF/UF process only. Table 29 shows the osmolality
results (in mOsmol/kg units) for each pH.
TABLE-US-00034 TABLE 29 Osmolality at various pH values, before and
after DF/UF with water pH 4 pH 5 pH 6 pH 7 pH 8 Adalimumab 100
mg/mL in 287 298 297 286 279 buffer Adalimumab 100 mg/mL in 40 13
11 5 5 water
[0587] The osmolality measurements were performed with a freezing
point viscometer.
17.9 Fragmentation (SEC)
[0588] The SEC data show a relative pronounced fragmentation of the
protein in ph 4 solutions over the whole concentration range
(100-250 mg/mL), while there almost no fragmentation detected at pH
ranging from 5 to 8 over the same concentration range.
Consequently, the monomer content of pH 4 solutions decreased
accordingly (FIG. 30). Aggregate values were found to increase with
increasing pH values (from pH 4 to pH 8), independent of the
concentration (FIG. 31).
17.10 Conclusions
[0589] This experiment was designed to examine the impact of
solution pH and protein concentration on viscosity and Dh
(hydrodynamic diameter) of Adalimumab solutions formulated in water
by DF/UF processing. Such solutions are referred to as low-ionic
solutions. A pH range of 4-8 was evaluated, and protein
concentrations tested were in a range between 2 and 250 mg/mL.
[0590] With regard to viscosity (Section 17.4), it was found that
low-ionic Adalimumab solutions have the same characteristics as
Adalimumab solutions formulated in the presence of ions (i.e. ionic
excipients such as organic buffer components or salts): [0591] The
higher the protein concentration, the higher solution viscosity.
This concentration-viscosity correlation was more pronounced for
solutions with pH values close to the Adalimumab pI (i.e., pH 7 and
pH 8). Conversely, for solutions at a constant concentration,
viscosity correlated with the closeness of the solution's pH value
to the pI of Adalimumab.
[0592] With regard to DLS data (Section 17.6), the following
conclusions can be drawn: [0593] Adalimumab Dh values determined by
DLS of low ionic Adalimumab solutions were found to be lower than
Dh values measured in Adalimumab commercial formulations,
especially at very low solution pH. [0594] The lower the solution
pH, the lower Dh values determined by DLS. [0595] The higher the
protein concentration, the lower the Dh values in low-ionic
Adalimumab solutions of a given pH.
[0596] The explanation for this behavior is that the ionic strength
(i.e. the presence of ions and ionizable excipients) in protein
solutions is crucial for the extent of protein-protein
interactions. Especially at lower solution pH, charge-charge
repulsions are more pronounced in low ionic Adalimumab solutions.
When a protein is formulated in water by using water as exchange
medium in DF/UF processing, the amount of ionizable counter ions
present that can compose both the Helmholtz layer and the
Gouy-Chapman layer is notably reduced. Consequently, intermolecular
charge-charge interactions (due to the charges of amino acid
residues present at the protein's surface) may be more pronounced
than in an environment where ionizable counter ions (e.g. ionizable
excipients) are abundant, and charge-charge repulsion between
protein monomers (leading to molecule motion in case of
charge-charge repulsion) and random Brownian motion contribute to
the mobility/motion of the protein molecule measured by DLS. In DLS
experiments, greater molecule mobilities are translated into
greater molecular diffusion coefficients, which usually are
assigned to molecules with smaller hydrodynamic sizes via using the
Stokes-Einstein equation. This can explain why the hydrodynamic
diameter of proteins is reduced in low-ionic formulations.
[0597] Charge-charge interactions between antibody molecules can be
repulsive (at lower solution pH) and attractive (at higher solution
pH close to the protein's pI).
17.11 Data Tables
TABLE-US-00035 [0598] TABLE 30 Adalimumab 100 mg/mL in buffer
before DF versus water pH 3 pH 4 pH 5 pH 6 pH 7 pH 8 pH 9 turbity
(NTU) 9.9 15.4 28.5 36.3 45.0 48.4 46.5 viscosity (mPa*s) 2.5197
2.7935 3.2062 3.1512 3.5116 3.5494 3.5844 viscosity (mm2/s) 2.4366
2.6991 3.0969 3.0444 3.3893 3.4261 3.4589 density (g/cm3) 1.0341
1.0350 1.0353 1.0351 1.0361 1.0360 1.0363 osmolality (mOsmol/kg)
293 287 298 297 286 279 285 Z-Ave d (nm) PCS 4.3 4.3 6.0 7.3 7.7
8.0 7.8 pH 4.00 4.99 6.00 7.03 8.00 9.03
TABLE-US-00036 TABLE 31 Adalimumab after DF versus water, before
concentration, diluted with water to pH 4 pH 4 pH 4 2 mg/mL 50
mg/mL 100.5 mg/mL turbity (NTU) 0.296 1.46 3.30 viscosity (mPa*s)
0.9653 1.4471 2.2411 viscosity (mm2/s) 0.9665 1.4298 2.1834 density
(g/cm3) osmolality 40 (mOsmol/kg) Z-Ave d (nm) PCS 3.37 2.24 1.81
pH 4.29 Adalimumab after concentration and dilution with water to
pH 4 pH 4 pH 4 150.5 mg/mL 219.0 mg/mL 251.8 mg/mL turbity (NTU)
3.56 viscosity (mPa*s) 4.0283 13.304 48.642 viscosity (mm2/s)
3.8712 12.614 45.567 density (g/cm3) osmolality 64 96 141
(mOsmol/kg) Z-Ave d (nm) PCS 1.32 0.458 0.162 pH 4.32 4.54
Adalimumab after DF versus water, before concentration, diluted
with water to pH 5 pH 5 pH 5 2 mg/mL 50 mg/mL 97.5 mg/mL turbity
(NTU) 0.02 1.66 3.54 viscosity (mPa*s) 1.0563 1.6664 2.8661
viscosity (mm2/s) 1.0576 1.6465 2.7924 density (g/cm3) 0.9988
1.0121 1.0264 osmolality 13 (mOsmol/kg) Z-Ave d (nm) PCS 157 32.4
1.3 pH 4.55 4.83 4.98 Adalimumab after concentration and dilution
with water to pH 5 pH 5 pH 5 150.7 mg/mL 200.2 mg/mL 253.0 mg/mL
turbity (NTU) 7.24 viscosity (mPa*s) 7.0866 19.539 79.272 viscosity
(mm2/s) 6.8102 18.525 74.26 density (g/cm3) 1.0406 1.0547 1.0675
osmolality 78 80 96 (mOsmol/kg) Z-Ave d (nm) PCS 0.727 0.335 0.255
pH 5.03 5.05 5.08
TABLE-US-00037 TABLE 32 Adalimumab after DF versus water, before
concentration, diluted with water to pH 6 pH 6 pH 6 2 mg/mL 50
mg/mL 100 mg/mL turbity (NTU) 0.458 2.24 2.95 viscosity (mPa*s)
1.0696 1.8003 3.1147 viscosity (mm2/s) 1.0708 1.7789 3.0385 density
(g/cm3) 0.9989 1.012 1.0251 osmolality 3 + 11 = 14:2 = 7 27 11
(mOsmol/kg) Z-Ave d (nm) PCS 30.8 2.78 2.48 pH 5.72 5.95 5.98
TABLE-US-00038 TABLE 33 Adalimumab after concentration and diluted
with water to pH 6 pH 6 pH 6 146.6 mg/mL 201.8 mg/mL 248.5 mg/mL
turbity (NTU) 9.29 viscosity (mPa*s) 9.0193 32.352 126.06 viscosity
(mm2/s) 8.6775 30.709 118.07 density (g/cm3) 1.0394 1.0535 1.0677
osmolality 37 58 95 (mOsmol/kg) Z-Ave d (nm) PCS 0.989 0.355 0.108
pH 5.92 6.05 6.03
TABLE-US-00039 TABLE 34 Adalimumab after DF versus water, before
concentration, diluted with water to pH 7 pH 7 pH 7 2 mg/mL 50
mg/mL 103.2 mg/mL turbity (NTU) 0.1 7.13 14.9 viscosity (mPa*s)
1.1252 1.6898 4.2257 viscosity (mm2/s) 1.1268 1.6688 4.1146 density
(g/cm3) 0.9986 1.0126 1.027 osmolality (mOsmol/kg) 0 2 5 Z-Ave d
(nm) PCS 3.31 4.16 2.89 pH 6.63 6.93 7.02
TABLE-US-00040 TABLE 35 Adalimumab after concentration and diluted
with water to pH 7 pH 7 pH 7 143.0 mg/mL 203.4 mg/mL 251.7 mg/mL
turbity (NTU) 19.3 viscosity (mPa*s) 14.024 74.987 343.881
viscosity (mm2/s) 13.492 70.928 321.144 density (g/cm3) 1.0571
1.0708 osmolality 65 106 160 (mOsmol/kg) Z-Ave d (nm) PCS 1.27
0.346 0.0876 pH 6.9 7.01 7.2
TABLE-US-00041 TABLE 36 Adalimumab after DF versus water, before
concentration, diluted with water to pH 8 pH 8 pH 8 2 mg/mL 50
mg/mL 96.1 mg/mL turbity (NTU) 0.41 12.10 28.300 viscosity (mPa*s)
1.261 1.8444 4.3486 viscosity (mm2/s) 1.2625 1.8224 4.2368 density
(g/cm3) osmolality (mOsmol/kg) 5 Z-Ave d (nm) PCS 5.59 5.62 4.28 pH
7.67
TABLE-US-00042 TABLE 37 Adalimumab after concentration and dilution
with water to pH 8 pH 8 pH 8 148.5 mg/mL 200.6 mg/mL 230.7 mg/mL
turbity (NTU) 32.5 viscosity (mPa*s) 20.102 85.5 233.14 viscosity
(mm2/s) 19.318 81.066 218.04 density (g/cm3) osmolality (mOsmol/kg)
Z-Ave d (nm) PCS 1.42 0.398 0.168 pH 7.6
TABLE-US-00043 TABLE 38 PCS data: Adalmumab in buffer Z-Ave d nm
PDI Pk1 d nm Pk1 Area % Pk2 d nm Pk2 Area % Pk3 d nm Pk3 Area % pH
3 100 mg/mL 4.23 0.283 4.43 86.4 54.1 13.6 0 0 pH 4 100 mg/mL 4.3
0.101 4.81 100 0 0 0 0 pH 5 100 mg/mL 6.01 0.065 6.5 100 0 0 0 0 pH
6 100 mg/mL 7.25 0.063 7.82 100 0 0 0 0 pH 7 100 mg/mL 7.64 0.094
8.53 100 0 0 0 0 pH 8 100 mg/mL 7.95 0.099 8.88 100 0 0 0 0 pH 9
100 mg/mL 7.7 0.133 8.98 100 0 0 0 0
TABLE-US-00044 TABLE 39 PCS data: Adalmumab in water Z-Ave d nm PDI
Pk1 d nm Pk1 Area % Pk2 d nm Pk2 Area % Pk3 d nm Pk3 Area % pH 4 2
mg/ml 3.37 0.219 3.39 88.8 73.3 11.2 0 0 pH 4 50 mg/ml 2.24 0.194
2.65 97.7 3300 2.3 0 0 pH 4 100.5 mg/ml 1.81 0.172 2.02 97.4 3390
2.6 0 0 pH 4 150.5 mg/ml 1.32 0.181 1.64 100 0 0 0 0 pH 4 219.0
mg/ml 0.458 0.217 4070 62 0.621 38 0 0 pH 4 251.8 mg/ml 0.162 0.263
0 0 0 0 0 0 pH 5 2 mg/ml 157 0.468 1.88 84.3 181 10.7 17 5 pH 5 50
mg/ml 32.4 0.17 1.6 87.7 15.5 4.8 186 4.7 pH 5 97.4 mg/ml 1.32
0.183 1.52 97.4 3290 2.6 0 0 pH 5 150.7 mg/ml 0.931 0.209 1.36 98.7
3710 1.3 0 0 pH 5 200.2 mg/ml 0.335 0.203 0 0 0 0 0 0 pH 5 253.0
mg/ml 0.107 0.255 0 0 0 0 0 0 pH 6 2 mg/ml 30.8 0.382 2.78 60.9 273
30.2 5070 5 pH 6 50 mg/ml 2.78 0.247 2.68 86.4 1600 7.8 114 5.8 pH
6 100 mg/ml 2.01 0.171 2.48 100 0 0 0 0 pH 6 146.6 mg/ml 0.989
0.219 1.32 96.9 3770 301 0 0 pH 6 201.8 mg/ml 0.355 0.231 0 0 0 0 0
0 pH 6 248.5 mg/ml 0.108 0.301 0 0 0 0 0 0 pH 7 2 mg/ml 3.31 0.211
3.58 93.9 1250 6.1 0 0 pH 7 50 mg/ml 4.16 0.132 4.84 100 0 0 0 0 pH
7 103.2 mg/ml 2.89 0.141 3.39 100 0 0 0 0 pH 7 143.3 mg/ml 1.27
0.212 1.68 100 0 0 0 0 pH 7 203.4 mg/ml 0.346 0.306 0 0 0 0 0 0 pH
7 251.7 mg/ml 0.0876 0.497 0 0 0 0 0 0 pH 8 2 mg/ml 5.59 0.365 3.15
67.4 244 30.2 26.5 2.4 pH 8 50 mg/ml 5.62 0.174 7 100 0 0 0 0 pH 8
96.1 mg/ml 4.28 0.192 4.81 96.9 3640 3.1 0 0 pH 8 148.5 mg/ml 1.43
0.253 1.68 93.9 2910 6.1 0 0 pH 8 200.6 mg/ml 0.398 0.246 4920 100
0 0 0 0 pH 8 230.7 mg/ml 0.168 0.3 0 0 0 0 0 0
TABLE-US-00045 TABLE 40 SEC data conc. % % % Area pH mg/mL
aggregate monomer fragmente (mVs) 4 100 0.28 67.95 31.76 45195.348
4 150 0.26 66.07 33.68 44492.803 4 200 0.30 64.59 35.11 52558.050 4
250 0.29 64.40 35.31 48491.299 5 100 1.46 98.44 0.11 48127.249 5
150 1.33 98.56 0.11 43226.397 5 200 1.39 98.50 0.11 43634.282 5 250
1.38 98.52 0.11 41643.062 6 100 2.00 97.90 0.10 44338.373 6 150
2.52 97.37 0.11 41899.182 6 200 2.52 97.37 0.11 43869.183 6 250
2.39 97.50 0.11 34969.456 7 100 2.78 97.12 0.10 46194.824 7 150
4.24 95.65 0.11 47443.014 7 200 3.61 96.29 0.10 41916.220 7 250
3.39 96.50 0.11 38185.208 8 100 3.24 96.65 0.12 42334.491 8 150
3.64 96.18 0.18 40305.890 8 200 3.63 96.25 0.13 40280.342 8 250
3.76 96.05 0.19 32067.297
Example 18: Impact of pH on J695 Viscosity
[0599] Viscosity data were generated for J695 after DF/UF
processing using water as exchange medium. J695 DS (see Example 1)
was diafiltered against water, applying at least 5 DF/UF steps.
Viscosity was then determined at various temperatures using a
plate-plate viscometer, 100 rpm shear rate, 150 .mu.m gap, 60 mm
plate diameter (equipment: Bohlin Geminim viscometer (Malvern
Instruments, Southborough, Mass.), temperature range evaluated
8-25.degree. C.).
[0600] As seen in FIG. 32, at concentrations of 179 mg/mL and 192
mg/mL, respectively, J695 solution viscosities were below 70 cP at
12.degree. C., below 40 cP at 20.degree. C., and below 30 cP at
25.degree. C.
Example 19: Pharmacokinetics (PK) of an Antibody in Pure Water
[0601] The goal of this study was to evaluate potential impact of
formulation parameters (i.e. low ionic protein formulation
containing water vs conventional protein formulations using ionic
excipients such as buffers and salts) on local tolerability and PK
after subcutaneous (s.c.) dosing with Afelimomab. In addition,
systemic toxicity and toxicokinetic data of the formulations was
investigated. Protein concentrations used ranged from 50 mg/mL to
200 mg/mL and ionic strengths ranged from 3 mOsm/kg to 300
mOsm/kg.
[0602] A single (s.c.) dose feasibility study was carried out with
Afelimomab (MAK195F--mouse anti human TNF F(ab')2 (Abbott
Laboratories)) in male Sprague-Dawley rats to assess the local
tolerance and toxicity of Afelimomab in rats following s.c.
administration of liquid formulations at 50 and 200 mg/kg. Single
s.c. doses were followed by an observation/recovery period. Limited
blood sampling was carried out to measure circulating Afelimomab
levels and assess absorption and half-life. The administered dose
volume was 1 mL/kg body weight. The experimental groups included
the following:
[0603] Experimental Groups
TABLE-US-00046 01 Control (vehicle) 02 50 mg/ml Afelimomab, liquid,
standard formulation 07 200 mg/ml Afelimomab, liquid, water
formulation Group A Observation period 2 days Group B Observation
period 7 days Group C Observation period 14 days
Grouping and Rat Identification (N=1 Per Group)
TABLE-US-00047 [0604] Animal number Group Group A Group B Group C
01 1 2 3 02 4 5 6 07 19 20 21
[0605] The animals were repeatedly observed for clinical signs and
mortality on day 1 at 15 min, 1, 3, 5, and 24 hours past
administration and at least once daily afterwards. Body weights
were measured on the days of dosing (day 1) and necropsy (day 3, 15
or 21, respectively) and twice weekly, if applicable. Blood samples
for drug analysis were collected on Day 1 (4 hours past
administration), and on Days 2, 3, 5, 8, and 15 as applicable.
Prior to necropsy blood was collected and hematological and
clinical chemistry parameters were evaluated. Blood smears were
prepared of each animal prior to necropsy. At necropsy, macroscopy
of body cavities was performed. Organ weight measurement was
performed on liver, kidneys, thymus, spleen, and lymph nodes.
Preliminary histopathology was performed on the injection site and
on liver, kidneys, thymus, spleen, and lymph nodes.
[0606] All animals survived the study until scheduled necropsy. The
rat administered the water formulation showed crusts in the
cervical region from Day 14 to 15. No test item-related effect on
body weight was observed. Hematology and clinical chemistry values
were variable. No clearly test item-related changes were identified
in haematology or clinical chemistry. No test item-related changes
were noted in urinalysis. Measurement of organ weights resulted in
high variability and no clearly test item-related changes in organ
weights.
[0607] At gross observation reddening of the subcutis at the area
of injection was noted in the rat receiving the water formulation
at Day 3. All other changes belonged to the spectrum of spontaneous
findings commonly seen in Sprague-Dawley rats of this strain and
age.
[0608] Microscopic Findings were as follows: [0609] No findings in
Groups 01, 02 [0610] Minimal diffuse subcutaneous inflammation in
Group 07 [0611] Focal subcutaneous hemorrhage correlating with
reddening on gross pathology in Group 07 (Day 3), thought to be
administration related [0612] Preliminary immunohistochemistry
results of pan-T, suppressor/cytotoxic T cells/natural killer
cells, pan-B cells and pan-macrophage markers on the local
reactions indicate mainly macrophages and natural killer cells
involved in the subcutaneous inflammations/infiltrations. Thus, so
far there are no hints for a local immunogenic response to the
formulations used.
[0613] All other changes belonged to the spectrum of spontaneous
findings commonly seen in Sprague-Dawley rats of this strain and
age.
[0614] Following subcutaneous administration Afelimomab absorption
appeared to be fast with maximum serum levels reached 0.2-3 days
after injection. The absolute levels of Afelimomab in all samples
tested were low. Large variability was observed between the
samples, likely because of the limited sampling frequency and the
low number of animals used. In most samples, no Afelimomab could be
detected in serum after 5-8 days. This drop in serum levels is
probably due to the high clearance of the F(ab').sub.2. The
observed T1/2 for most samples were in the range of 1-2 days in
agreement with previous observations. The longer half-life of the
low-ionic formulation (7.8 d) may represent a protracted absorption
of the sample. Data are presented in Tables 41 and 42.
TABLE-US-00048 TABLE 41 Plasma exposure levels of MAK195F Time
Concentration (.mu.g/ml) Average (day) Rat 4 Rat 5 Rat 6 (.mu.g/ml)
STD 50 mg/kg 0.167 1.40 1.17 1.38 1.32 0.13 liquid 2 0.76 0.97 0.66
0.80 0.16 standard 3 0.45 0.67 0.47 0.53 0.12 formulation 5 LLOQ
LLOQ LLOQ 8 LLOQ LLOQ LLOQ 15 LLOQ LLOQ Time Concentration
(.mu.g/ml) Average (day) Rat 19 Rat 20 Rat 21 (.mu.g/ml) STD 200
mg/kg 0.167 1.27 3.01 3.17 2.48 1.05 water 2 0.17 1.57 1.53 1.09
0.80 formulation 3 LLOQ 1.54 1.56 1.55 0.02 5 0.64 0.66 0.65 0.02 8
0.40 0.37 0.38 0.02 15 0.25 0.25 0.00 LLOQ = below quantitation
limit
[0615] There were no aggregation state findings for liquids,
neither Afelimomab or control substance.
[0616] For the low-ionic strength formulation, minimal diffuse s.c.
injection site inflammation was seen. Inflammation, either minimal
to slight, or slight to moderate, was seen with increased protein
concentration (50 mg/mL and 200 mg/mL, respectively). Some local
s.c. hemorrhage was seen, correlating with reddening on gross
pathology; this was considered to be the consequence of blood
vessel puncture during injection. Some subcutaneous reddening at
the injection site was observed at Day 3 for the water formulation,
but was not considered detrimental. Overall, the formulation was
tolerated locally.
[0617] In Table 42 below, PK data of conventional liquid
formulation vs. the water formulation is presented.
TABLE-US-00049 TABLE 42 Pharmacokinetic parameters of MAK 195F
after subcutaneous dosing in different formulations. Last Mean Time
of Last Detectable Dose Half-life Tmax Cmax AUC Residence
Detectable Conc. (mg/kg) Formulation (day) (day) (.mu.g/ml)
(day*.mu.g/ml) Time (day) Conc. (day) (.mu.g/ml) 50 liquid 0.5 0.2
1.32 3.3 1.5 3 0.53 200 water 5.9 0.2 2.48 11.8 7.5 15 0.25
formulation
An increase in duration of detectable serum levels was seen with
low ionic formulation (i.e. Afelimomab formulated in water), as
seen in Table 42.
[0618] In this study, the observed absolute levels of MAK195F in
low ionic solution (water) provided a better exposure, longer
detectable serum levels and `half-life` than in conventional
MAK195F liquid formulation.
[0619] Afelimomab half-lives were in the range of 1-2 days in
standard formulation in agreement with previous observations for
F(ab').sub.2 molecules. However, a seemingly longer half-life was
observed for the low-ionic formulation (7.8 d). Accordingly, the
mean residence time of MAK 195F in this formulation appeared to be
longer compared to the standard formulation tested.
Example 20: DF/UF of 2.5(E)mg1 (Anti IL-18 Antibody)
[0620] Diafiltration/ultrafiltration (continuous mode) of 2.5(E)mg1
bulk solution (59.6 mg/mL) was performed, applying an about 4-fold
volume exchange using water for injection (in the following
referred to as "water"). The DF/UF operation was controlled by
monitoring turbidity, protein concentration (OD280), pH and
osmolarity of retentate, and DLS measurements. During DF/UF,
permeate osmolarities were also monitored to control the excipient
reduction of the 2.5(E)mg1 bulk solutions.
Materials and Methods
[0621] -2.5(E)mg1 Bulk Drug Substance (methionine, histidine, free
of polysorbate 80) (Abbott Bioresearch Center, Worcester, Mass.): 2
PETG bottles with a total of 589.12 g solution, solution
concentration 59.6 mg/mL. [0622] Ampuwa (water for injection)
(Fresenius Medical Care, Waltham, Mass.). [0623] Millipore Labscale
TFF DF/UF unit including 2.times. Pellicon XL filter cassettes,
Millipore, PLCTK 30 kDa membrane, regenerated cellulose [0624]
UV/VIS spectrophotometer, Specord 50 using 280 nm wavelength [0625]
Metrohm pH-meter, type 744 with Biotrode probe No. 57 [0626]
Osmometer: Knauer, K-7400 [0627] density measurements using
equipment of Paar, DMA 4100 [0628] Laminar air flow box Hereaus
[0629] turbidity measurements: Hach, 2100AN [0630] viscometer:
Paar, AMVn [0631] scales: Mettler Toledo, AT261 and 33.45 [0632]
filters: Millex AP 20 (fiberglass) and Minisart High Flow Filter
(celluloseacetate), 0.20 .mu.m pore size.
20.1 Experimental Procedures
[0633] Thawing of 2.5(E)mg1 DS samples: 2 L PETG bottles containing
frozen DS were thawed within 2 hrs using a circulating water bath
at 23.degree. C. The thawed DS was clear, slightly opalescent, and
free from visible particles.
[0634] Concentration of DS by DF/UF: due to the DF/UF unit
reservoir volume limit of 530 mL, the 2.5(E)mg1 DS was concentrated
to a final volume of 525 mL.
[0635] DF/UF using water (buffer exchange): the DS (methionine,
histidine, 2.5(E)mg1) was subjected to DF/UF, applying a 4-fold
volume exchange. Table 43 gives the amounts of water that were used
throughout the experiment and Table 44 provides the experimental
parameters.
TABLE-US-00050 TABLE 43 DF/UF water volume exchanges Volume of
water used Volume exchange (cumulative) 1- fold 576 mL 2- fold 1152
mL 3- fold 1728 mL 4- fold (end of experiment) 2304 mL
TABLE-US-00051 TABLE 44 DF/UF procedure parameters Labscale TFF DF
settings Pump speed 1.5-2 Pressure of pump 20-30 psi Stirring speed
~3 Experiment duration 8 hrs
[0636] Osmolarity measurement of permeate was performed at about
every 200 mL of permeate processed.
[0637] After DF/UF against water, the volume of the 2.5(E)mg1
solution was 450 mL and the protein concentration 76.6 mg/mL. This
solution, containing 2.5(E)mg1 dissolved essentially in water, was
then concentrated.
TABLE-US-00052 TABLE 45 DF/UF process parameters for solution
concentration Labscale TFF UF settings Pump speed 1.5-2 Pressure of
pump max. 30 psi Stirring speed ~3 Experiment duration 51 min.
Final weight of solution: 257.83 g
[0638] The concentrated solution (.about.130 mg/mL) was subjected
to 0.2 .mu.m filtration. The solution was cooled to 2-8.degree. C.
and then stored at -80.degree. C.
20.2 Data Collected During DF/UF of 2.5(E)mg1
TABLE-US-00053 [0639] TABLE 46 In Process control data Temperature
solution/Room Volume temperature turbidity Osmolality conc DF steps
[mL] time [.degree. C.] [NTU] pH [mOsmol/kg] [mg/ml] 2.5(E)mg1 14.5
5.91 150 59.6 .sub. 0.sup.1 0 08:00 19.0/24.1 N/A 5.91 125 65.2 1
575 10:02 24.4/24.4 10.1 5.92 50 70.1 2 1150 11:50 24.3/24.7 6.67
5.94 16 72.8 3 1730 13:50 25.0/24.8 6.55 5.97 6 74.6 ca. 4 2200
15:35 25.8/25.5 10.1 5.97 5 76.7
TABLE-US-00054 TABLE 47 Osmolalty of permeate (fractionated and
measured during process) Temperature solution/Room Permeat No. Of
Sample temperature Permeate cumulative DF/UF osmolality no. time
[.degree. C.] [ml] [ml] steps [mOsmol/kg] 0.sup.3 07:35 N/A 90 N/A
N/A 125 1 08:00 19.0/24.1 200 200 0.3 124 2 08:50 23.0/24.1 200 400
0.7 82 3 09:27 24.0/24.2 200 600 1.0 53 4 10:12 24.4/24.4 200 800
1.4 37 5 10:50 24.5/24.3 200 1000 1.7 25 6 11:25 24.6/24.3 200 1200
2.1 16 7 12:10 24.7/24.3 230 1430 2.5 7 8 12:55 24.7/24.4 170 1600
2.8 4 9 13:25 24.8/24.4 200 1800 3.1 2 10 14:15 25.1/24.8 200 2000
3.5 0 11 14:55 25.8/25.5 200 2200 3.8 1
TABLE-US-00055 TABLE 48 Concentration of 2.5(E)mg1 solution after
buffer exchange Solution Volume in reservoir temperature (i.e.
retentate) time [.degree. C.] [ml] pH 15:54 25.9 450 5.94 16:02
26.1 400 5.96 16:07 26.1 375 5.96 16:20 26.2 350 5.96 16:28 26.3
300 5.98 16:35 26.5 275 5.98 16:45 26.5 250 5.99
TABLE-US-00056 TABLE 49 Analytical characterization of concentrated
2.5(E)mg1 solution (before and after 0.2 .mu.m filtration): lot
parameter before filtration after filtration turbidity [NTU] 15.4
9.58 osmolality [mOsmol/kg] 6 N/A density [g/ml] 1.0346 N/A pH 5.99
N/A Dyn. Viscosity (25.degree. C.) N/A 7.9998 [mPas]
TABLE-US-00057 TABLE 50 Dynamic light scattering data
(determination of Dh of monomer and z-average value of Dh = Dh of
all specimen present in solution) during DF/UF Sample pull DV DV DV
DV After con- After DLS data 1- fold 2- fold 3- fold 4- fold
centration filtration Peak 1 diameter 4.32 3.68 3.54 3.48 2.03 2.13
monomer intensity 100.0 100.0 100.0 89.6 87.0 100.0 [%] Z-Average
3.95 3.28 3.20 3.53 2.12 1.89 [nm] Pdl 0.077 0.106 0.094 0.245
0.287 0.113 Peak 2 diameter N/A N/A N/A 984 411 N/A [nm] intensity
10.4 11.8 [%] Peak 3 diameter N/A N/A N/A N/A 4260 N/A [nm]
intensity 1.2 [%]
20.3 Discussion
[0640] The experiment demonstrated that 2.5(E)mg1 (buffered in
methionine, histidine) can be formulated in essentially water at
higher concentration (no solubility limitations observed at 130
mg/mL). After 3 volume exchanges using water osmolality of permeate
and retentate were below 10 mOsmol/kg, demonstrating that buffer
excipients have been effectively reduced. The opalescence of the
2.5(E)mg1 solution was reduced during DF/UF using water (optimal
appearance), mirrored also by reduces turbidity values
(nephelometric turbidity units (NTU) of DS starting solution 14.5,
after 3-fold volume exchange 6.55, after 4-fold volume exchange
10.5.
[0641] As seen with other antibodies, the hydrodynamic diameter as
determined by DLS decreased due to excipient reduction
(intermolecular charge-charge repulsion adding to random Brownian
motion, resulting in higher molecule mobility, translates to lower
Dh values calculated). The pH of the 2.5(E)mg1 solution was
basically the same before (pH 5.94) and after (pH 5.99) the DF/UF
operation.
[0642] As shown by DLS monitoring, the 2.5(E)mg1 remained stable
during the DF/UF operation. No substantial increase in high
molecular weight specimen was detected.
Example 21: Preparation of Adalimumab Formulated in Water and
Stability Studies Thereof
[0643] The following example describes the stability of a
formulation comprising adalimumab originating from processes
described in the above examples, i.e., adalimumab was successfully
dialyzed into water.
Materials and Methods
[0644] 3323.6 g Adalimumab solution (71.3 mg/mL) were diafiltered
using pure water. After a 7-fold volume exchange with pure water
(theoretical excipients reduction, 99.9%), the protein solution was
diluted/ultrafiltered to final target concentrations of 220 and 63
mg/mL, respectively. PH, osmolality, viscosity, conductivity, PCS,
visual inspection and protein concentration measurements (OD280)
were performed to monitor the status of the protein after DF/UF
processing.
[0645] After DF/UF processing, the protein solutions were sterile
filtered (0.22 .mu.m Millipak-60 and Millipak-200 membrane filters)
and subsequently filled into BD HyPak SCF.TM. 1 mL long syringes,
equipped with 27.5G RNS needles and sterile BD HyPak BSCF 4432/50
stoppers. The filling volume was around 0.825 mL per syringe.
[0646] After filling the syringes were stored at 2-8.degree. C.,
25.degree. C. and 40.degree. C., respectively, and analyzed as
indicated in the sample pull scheme depicted below. [0647]
Adalimumab Drug Substance (Adalimumab extinction coefficient 280
nm: 1.39 mL/mg cm): Drug Substance did not contain polysorbate 80.
DS buffer, pH 5.38. [0648] Sortorius Sartocon Slice diafiltration
system, equipped with Ultrasert PES membrane cassettes (50 kDa and
30 kDa cutoff). The Sartocon Slice system was operated in
continuous mode at ambient temperature according to Sartorius
Operating Instructions. [0649] pH electrodes [0650] PerkinElmer UV
visible spectrophotometer, Lambda 35, was used for protein
concentration measurements (280 nm wavelength). Disposable UV
cuvettes, 1.5 mL, semi-micro, Poly(methyl methacrylate) (PMMA),
were used for the concentration measurements. [0651] Sterilized
water for injection Ph. Eur./USP was used as DF/UF medium. [0652] A
Vogel Osmometer OM815, was used for osmolality measurements
(calibrated with 400 mOsmol/kg NaCl calibration solution, Art. No.
Y1241, Herbert Knauer GmbH, Berlin, Germany) [0653] Anton Paar
Microviscosimeter, type AWVn, was used for viscosity assessment of
the protein solutions according to Anton Paar Operating
Instructions. Viscosity was assessed at 20.degree. C. [0654] An
InoLab Cond Level2 WTW device was used for conductivity
measurements normalized to 25.degree. C. [0655] A Malvern
Instruments Zetasizer nano ZS, was used for determination of
Z-average values, applying a standard method. Measurements were
performed at 25.degree. C., using viscosity data obtained by
falling ball viscosimetry (Anton Paar Microviscosimeter, type AWVn,
at 25.degree. C.).
[0656] HPLC Methods [0657] Adalimumab, SEC analysis: Sephadex 200
column (Pharmacia Cat. No. 175175-01). Mobile phase 20 mM sodium
phosphate, 150 mM sodium chloride, pH 7.5, 0.5 mL/min flow rate,
ambient temperature, detection UV 214 nm and 280 nm. Each sample
was diluted to 1.0 mg/mL with Milli-Q water, sample injection load
50 .mu.g (duplicate injection). [0658] Adalimumab, IEC analysis:
Dionex, Propac WCX-10 column (p/n 054993) along with a
corresponding guard column (p/n 054994). Separation conditions:
mobile phase A: 10 mM sodium phosphate, pH 7.5; mobile phase B 10
mM Sodium phosphate, 500 mM Sodium chloride, pH 5.5. 1.0 mL/min
flow rate, ambient temperature. Each sample was diluted to 1.0
mg/mL with Milli-Q water, sample injection load 100 .mu.g,
duplicate injection.
[0659] Calculation of the Protein Concentration
[0660] Calculation formula:
E = - l g ( I I 0 ) = c d -> c = E d ##EQU00014## [0661]
.epsilon.--absorption coefficient [0662] c--concentration [0663]
d--length of cuvette that the light has to pass [0664]
E--absorbance [0665] I.sub.0--initial light intensity [0666]
I--light intensity after passing through sample
[0666] Adalimumab = 1.39 mL mg cm ##EQU00015##
[0667] Sample Pull Scheme [0668] Samples of the prepared solutions
are stored at the temperatures listed below and pulled (x) at the
indicated time points after study start.
TABLE-US-00058 [0668] Temp. T0 1 m 3 m 5.degree. C. -- x x
25.degree. C. x x x 40.degree. C. -- x x
TABLE-US-00059 Test parameter Test method Visible particles
analogous DAC (EA 4.43) Subvisible particles analogous Ph. Eur./USP
EA 4.44 Turbidity analogous Ph. Eur. (EA 4.42) Color (visual) Ph.
Eur. (EA 4.50) pH Ph. Eur. (EA 4.24) Size exclusion HPLC Desribed
in the text above Cation exchange HPLC Desribed in the text
above
DF/UF Processing of Adalimumab
[0669] Table 51 describes the adalimumab characteristics after
diafiltration.
TABLE-US-00060 TABLE 51 Protein PCS Concentration Osmolality
Viscosity Conductivity [Z-average/ Sample [mg/mL] pH [mosmol/kg]
[cP] Visual Inspection [.mu.S/cm] d nm] High 220 5.57 26 27.9
Slightly opalescent, 1167 0.34 concentration essentially free from
visible particles Low 63 5.44 5 1.8 Slightly opalescent, 522 1.85
concentration essentially free from visible particles
[0670] Adalimumab characterization upon storage, including clarity
and opalescence, degree of coloration of liquids, SEC, at different
temperature degrees is described in Appendix D.
Conclusion
[0671] The above example provides a diafiltration/ultrafiltration
(DF/UF) experiment where water (sterilized water for injection Ph.
Eur./USP) was used as diafiltration medium for the monoclonal
antibody Adalimumab.
[0672] Adalimumab was subjected to DF/UF processing by using pure
water as DF/UF exchange medium and was formulated at pH 5.57 at
high concentration (220 mg/mL) and at pH 5.44 at lower
concentration (63 mg/mL) without inducing solution haziness, severe
opalescence or turbidity formation.
[0673] Adalimumab from the DF/UF experiments was stored in SCF
syringes at 2-8.degree. C., 25.degree. C. and 40.degree. C. for 3
months. Data obtained points at favorable overall stability of the
protein.
[0674] In conclusion, processing and formulating proteins using
pure water as DF/UF exchange medium is feasible. Assuming an ideal
100% excipient membrane permeability, an approx. 99.9% maximum
excipient reduction can be estimated.
Example 22: Stability Studies of Adalimumab Formulated in Water
Using Non-Ionic Excipients
[0675] The following example describes stability studies of a
formulation containing an antibody, i.e., adalimumab, in water with
additional non-ionic excipients.
Materials and Methods
[0676] Adalimumab material was the same as in example 21 (DF/UF
processing). After DF/UF processing, the protein solutions were
formulated as denoted in Table 52. Mannitol was chosen as example
from the group of sugar alcohols, like mannitol, sorbitol, etc.
Sucrose was chosen as example from the group of sugars, like
sucrose, trehalose, raffinose, maltose, etc. Polysorbate 80 was
chosen as example from the group of non-ionic surfactants, like
polysorbate 80, polysorbate 20, pluronic F68, etc. .about.10.7 mL
were prepared for any formulation. Osmolality, viscosity and PCS
measurements were performed for any formulation after
preparation.
TABLE-US-00061 TABLE 52 Final protein Mannitol Sucrose Polysorbate
80 Sample concentration (mg/mL) (mg/mL) (% w/w) Name 50 mg/mL 50 --
-- LI50/01 50 mg/mL -- 80 -- LI50/02 50 mg/mL 50 -- 0.01 LI50/03 50
mg/mL -- 80 0.01 LI50/04 50 mg/mL 50 -- 0.1 LI50/05 50 mg/mL -- 80
0.1 LI50/06 50 mg/mL -- -- 0.01 LI50/07 50 mg/mL -- -- 0.1 LI50/08
200 mg/mL 50 -- -- LI200/01 200 mg/mL -- 80 -- LI200/02 200 mg/mL
50 -- 0.01 LI200/03 200 mg/mL -- 80 0.01 LI200/04 200 mg/mL 50 --
0.1 LI200/05 200 mg/mL -- 80 0.1 LI200/06 200 mg/mL -- -- 0.01
LI200/07 200 mg/mL -- -- 0.1 LI200/08 Polysorbate 80 stock solution
0.5% and 5% in sterile water for injection: Addition in 1:50 ratio
(210 .mu.L to 10.5 mL Adalimumab solution, addition of 210 .mu.L
water for injection to samples formulated without surfactant to
assure equal protein concentration in all samples) Addition of
mannitol/sucrose in solid form (525 mg/840 mg, respectively).
[0677] The preparations were sterile filtered (Millex GV,
Millipore, 0.22 .mu.m, O 33 mm, Art. SLGV033RS) and subsequently
filled into BD HyPak SCF.TM. 1 mL long syringes, equipped with
27.5G RNS needles and sterile BD HyPak BSCF 4432/50 stoppers. The
filling volume was around 0.6 mL per syringe.
[0678] After filling the syringes were stored at 2-8.degree. C.,
25.degree. C. and 40.degree. C., respectively, and analyzed as
indicated in the sample pull scheme depicted below. [0679]
Adalimumab Drug Substance (Adalimumab extinction coefficient 280
nm: 1.39 mL/mg cm): Drug Substance did not contain polysorbate 80.
DS buffer, pH 5.38. [0680] PH electrodes [0681] Sterilized water
for injection Ph. Eur./USP was used as DF/UF medium. [0682]
Mannitol, polysorbate 80, and sucrose, all matching Ph. Eur.
quality [0683] A Vogel Osmometer OM815, was used for osmolality
measurements (calibrated with 400 mOsmol/kg NaCl calibration
solution, Art. No. Y1241, Herbert Knauer GmbH, Berlin, Germany)
[0684] Anton Paar Microviscosimeter, type AWVn, was used for
viscosity assessment of the protein solutions according to Anton
Paar Operating Instructions. Viscosity was assessed at 20.degree.
C. [0685] Fluostar Optima, BMG Labtech (absorption measurement at
344 nm in well plates, assessment of turbidity) [0686] A Malvern
Instruments Zetasizer nano ZS, was used for determination of
Z-average values, applying a standard method. Measurements were
performed at 25.degree. C., using viscosity data obtained by
falling ball viscosimetry (Anton Paar Microviscosimeter, type AWVn,
at 25.degree. C.).
HPLC Methods
[0686] [0687] Adalimumab, SEC analysis: Sephadex 200 column
(Pharmacia Cat. No. 175175-01). Mobile phase 20 mM sodium
phosphate, 150 mM sodium chloride, pH 7.5, 0.5 mL/min flow rate,
ambient temperature, detection UV 214 nm and 280 nm. Each sample
was diluted to 1.0 mg/mL with Milli-Q water, sample injection load
50 .mu.g (duplicate injection). [0688] Adalimumab, IEC analysis:
Dionex, Propac WCX-10 column along with a corresponding guard
column. Separation conditions: mobile phase A: 10 mM sodium
phosphate, pH 7.5; mobile phase B 10 mM Sodium phosphate, 500 mM
Sodium chloride, pH 5.5. 1.0 mL/min flow rate, ambient temperature.
Each sample was diluted to 1.0 mg/mL with Milli-Q water, sample
injection load 100 .mu.g, duplicate injection.
Sample Pull Scheme
[0689] Samples of the prepared solutions were stored at 5.degree.
C., 25.degree. C., and 40.degree. C. and pulled at either 1 minute
(5.degree. C. and 40.degree. C.) or at T0 and 1 minute (25.degree.
C.) after study start. Test parameters were measured according to
appropriate methods, e.g., color was determined visually, turbidity
was determined at an absorption at 344 nm.
Initial Formulation Characterization
[0690] Table 53 described the initial formulation osmolalities and
viscosities.
TABLE-US-00062 TABLE 53 osmolarity viscosity Lot. comp. [mosmol]
[mPas] LI 50/01 50 mg/mL mannitol 309 1.9796 LI 50/02 80 mg/mL
sucrose 272 2.1284 LI 50/03 50 mg/mL mannitol; 0.01% Tween 307
1.9843 80 LI 50/04 80 mg/mL sucrose; 0.01% Tween 269 2.1194 80 LI
50/05 50 mg/mL mannitol; 0.1% Tween 307 1.9980 80 LI 50/06 80 mg/mL
sucrose; 0.1% Tween 272 2.1235 80 LI 50/07 0.01% Tween 80 8 1.7335
LI 50/08 0.1% Tween 80 8 1.8162 LI 200/01 50 mg/mL mannitol 396
21.395 LI 200/02 80 mg/mL sucrose 351 21.744 LI 200/03 50 mg/mL
mannitol; 0.01% Tween 387 21.233 80 LI 200/04 80 mg/mL sucrose;
0.01% Tween 350 21.701 80 LI 200/05 50 mg/mL mannitol; 0.1% Tween
387 21.592 80 LI 200/06 80 mg/mL sucrose; 0.1% Tween 355 21.943 80
LI 200/07 0.01% Tween 80 27 21.296 LI 200/08 0.1% Tween 80 28
21.889
[0691] All formulations of one concentration demonstrated equal
viscosities. Those of sucrose containing formulations were slightly
higher. The reduced viscosities of the highly concentrated
formulations in comparison to the highly concentrated formulation
in water (example A, viscosity 27.9 cP) is explained by sample
dilution with polysorbate 80 stock solutions or plain water,
leading to a final concentration of .about.215 mg/mL vs. 220 mg/mL
in example 21.
[0692] Table 54 describes the PCS data determined for each
sample.
TABLE-US-00063 TABLE 54 PCS Sample [Z-average/d nm] LI50/01 2.58
LI50/02 2.22 LI50/03 2.13 LI50/04 2.22 LI50/05 2.25 LI50/06 2.55
LI50/07 2.87 LI50/08 1.94 LI200/01 0.50 LI200/02 0.43 LI200/03 0.36
LI200/04 0.38 LI200/05 0.37 LI200/06 0.41 LI200/07 0.35 LI200/08
0.36
[0693] The data provided in Table 54 shows that z-average values do
not significantly differ from the values obtained from Adalimumab
solutions in non-ionic excipient free systems (63 mg/mL, 1.85 dnm,
220 mg/mL, 0.34 dnm, example 21).
Adalimumab Characterization Upon Storage
[0694] Appendix E provides data on Adalimumab stability upon
storage.
Conductivity of Placebo Solutions
[0695] Table 55 describes the influence of non-ionic excipients on
the conductivity of the various adalimumab formulations. All
placebo solutions were prepared using sterilized water for
injection Ph. Eur./USP.
TABLE-US-00064 TABLE 55 Mannitol Sucrose Polysorbate 80
Conductivity (mg/mL) (mg/mL) (% w/w) (.mu.S/cm) -- -- -- 1.1 50 --
-- 1.2 -- 80 -- 2.2 50 -- 0.01 2.3 -- 80 0.01 1.4 50 -- 0.1 2.6 --
80 0.1 3.6 -- -- 0.01 1.2 -- -- 0.1 2.6
Conclusion
[0696] The preparations were stored in SCF syringes at 2-8.degree.
C., 25.degree. C. and 40.degree. C. for 1 month. Data obtained from
the storage study showed that there was overall stability of the
protein in all formulations tested. The stability data was
comparable to the stability of samples from example 21. Measurement
of the conductivity of non-ionic excipient containing placebo
solutions demonstrates a marginal increase of conductivity for some
excipients in the range of some .mu.S/cm. PCS measurements
demonstrate no significant increase in hydrodynamic diameters in
comparison to non-ionic excipient free systems.
[0697] In conclusion, processing proteins using pure water as DF/UF
exchange medium and formulation with non-ionic excipients is
feasible. Adalimumab was also assessed by PCS in a buffer of
following composition: 10 mM phosphate buffer, 100 mM sodium
chloride, 10 mM citrate buffer, 12 mg/mL mannitol, 0.1% polysorbate
80, pH 5.2. The Adalimumab concentration was 50 mg/mL and 200
mg/mL, respectively. The z-average values were 11.9 dnm for the 50
mg/mL formulation and 1.01 dnm for the 200 mg/mL formulation,
respectively. Thus, it was clearly demonstrated that hydrodynamic
diameters at a given protein concentration are dependent on the
ionic strength (clearly higher diameters in salt containing
buffers).
Example 23: Preparation of J695 Formulated in Water with Non-Ionic
Excipients
[0698] The following example describes the preparation of a
formulation containing an antibody, i.e., adalimumab, in water with
additional non-ionic excipients. The example also describes the
stability (as measured for example by SE-HPLC and IEC) of J695
formulated in water with additional non-ionic excipients.
Materials and Methods
[0699] 2.times.30 mL J695 solution (.about.125 mg/mL) at different
pH were dialyzed using pure water applying Slide-A-Lyzer dialysis
cassettes. Dialysis of the samples was performed for 3 times
against 3 L pure water, respectively (theoretical excipients
reduction, 1:1,000,000). The protein solutions were ultrafiltered
to final target concentrations of 200 mg/mL, by using Vivaspin 20
concentrators. PH, osmolality, viscosity, conductivity, PCS, visual
inspection, HPLC and protein concentration measurements (OD280)
were performed to monitor the status of the protein during and
after processing.
[0700] After processing, the protein solutions were formulated as
denoted in the following. Mannitol was chosen as an example to use
from the group of sugar alcohols, like mannitol, sorbitol, etc.
Sucrose was chosen as an example to use from the group of sugars,
like sucrose, trehalose, raffinose, maltose, etc. Polysorbate 80
was chosen as an example to use from the group of non-ionic
surfactants, like polysorbate 80, polysorbate 20, pluronic F68,
etc. A volume of 0.5 mL was prepared for each of these
formulations. PH, osmolality, visual inspection, and HPLC analysis
were performed to monitor the status of the protein after sample
preparation.
TABLE-US-00065 TABLE 56 Description of various J695 formulations
Final protein Mannitol Sucrose Polysorbate 80 Sample concentration
(mg/mL) (mg/mL) (% w/w) Name* 200 mg/mL 50 -- -- LI200/01/5 200
mg/mL -- 80 -- LI200/02/5 200 mg/mL 50 -- 0.01 LI200/03/5 200 mg/mL
-- 80 0.01 LI200/04/5 200 mg/mL 50 -- 0.1 LI200/05/5 200 mg/mL --
80 0.1 LI200/06/5 200 mg/mL -- -- 0.01 LI200/07/5 200 mg/mL -- --
0.1 LI200/08/5 200 mg/mL 50 -- -- LI200/01/6 200 mg/mL -- 80 --
LI200/02/6 200 mg/mL 50 -- 0.01 LI200/03/6 200 mg/mL -- 80 0.01
LI200/04/6 200 mg/mL 50 -- 0.1 LI200/05/6 200 mg/mL -- 80 0.1
LI200/06/6 200 mg/mL -- -- 0.01 LI200/07/6 200 mg/mL -- -- 0.1
LI200/08/6 *The term "/5" or "/6" is added to any sample name to
differentiate between samples at pH 5 and 6. Polysorbate 80 stock
solution 0.5% and 5% in sterile water for injection: Addition in
1:50 ratio (10 .mu.L to 0.5 mL J695 solution, addition of 10 .mu.L
water for injection to samples formulated without surfactant to
assure equal protein concentration in all samples) Addition of
mannitol/sucrose in solid form (25 mg/40 mg, respectively).
[0701] J695 Drug Substance (J695 extinction coefficient 280 nm:
1.42 mL/mg cm): Drug Substance did not contain polysorbate 80. DS
buffer, pH 6.29. [0702] pH electrodes [0703] Demineralized and
sterile filtered water was used as dialysis medium. [0704]
Mannitol, polysorbate 80, and sucrose, all matching Ph. Eur.
quality [0705] A Vogel Osmometer OM815, was used for osmolality
measurements (calibrated with 400 mOsmol/kg NaCl calibration
solution, Art. No. Y1241, Herbert Knauer GmbH, Berlin, Germany)
[0706] Anton Paar Microviscosimeter, type AWVn, was used for
viscosity assessment of the protein solutions according to Anton
Paar Operating Instructions. Viscosity was assessed at 20.degree.
C. [0707] Fluostar Optima, BMG Labtech (absorption measurement at
344 nm in well plates, assessment of turbidity) [0708] Eppendorf
Centrifuge 5810 R [0709] Slide-A-Lyzer dialysis cassettes, Pierce
Biotechnology (Cat No 66830) [0710] Vivaspin 20 concentrators, 10
KDa PES membranes (Vivascience, Product number VS2001), used
according to standard Operating Instructions [0711] PerkinElmer UV
visible spectrophotometer, Lambda 35, was used for protein
concentration measurements (280 nm wavelength). Disposable UV
cuvettes, 1.5 mL, semi-micro, Poly(methyl methacrylate) (PMMA),
were used for the concentration measurements. [0712] An InoLab Cond
Level2 WTW device was used for conductivity measurements normalized
to 25.degree. C. [0713] A Malvern Instruments Zetasizer nano ZS,
was used for determination of Z-average values, applying a standard
method. Measurements were performed at 25.degree. C., using
viscosity data obtained by falling ball viscosimetry (Anton Paar
Microviscosimeter, type AWVn, at 25.degree. C.).
HPLC Methods
[0713] [0714] J695, SEC analysis: Tosoh Bioscience G3000swxl, 7.8
mm.times.30 cm, 5 .mu.m (Cat. No. 08541). Mobile phase 211 mM
Na.sub.2SO.sub.4/92 mM Na.sub.2HPO.sub.4, pH 7.0. 0.25 mL/min flow
rate, ambient temperature, detection UV 214 nm and 280 nm. Each
sample was diluted to 2.0 mg/mL with Milli-Q water, sample
injection load 20 .mu.g (duplicate injection). [0715] J695, IEC
analysis: Dionex, Propac WCX-10 column (p/n 054993) along with a
corresponding guard column (p/n 054994). Separation conditions:
mobile phase A: 10 mM Na.sub.2HPO.sub.4, pH 6.0; mobile phase B 10
mM Na.sub.2HPO.sub.4, 500 mM NaCl, pH 6.0. 1.0 mL/min flow rate,
35.degree. C. temperature. Each sample was diluted to 1.0 mg/mL
with Milli-Q water, sample injection load 100 .mu.g.
Calculation of the Protein Concentration
[0716] Calculation formula:
E = - l g ( I I 0 ) = c d -> c = E d ##EQU00016## [0717]
.epsilon.--absorption coefficient [0718] c--concentration [0719]
d--length of cuvette that the light has to pass [0720]
E--absorbance [0721] I.sub.0--initial light intensity [0722]
I--light intensity after passing through sample
[0722] Adalimumab = 1.42 mL mg cm ##EQU00017##
Processing of J695
[0723] J695 in water was characterized prior to the addition of any
non-ionic excipients. Table 57 provides details of the J695
characterization during dialysis/ultrafiltration.
TABLE-US-00066 TABLE 57 Protein PCS Concentration Osmolality
Viscosity Conductivity [Z-average/ Sample [mg/mL] PH [mosmol/kg]
[cP] Visual Inspection [.mu.S/cm] d nm] Starting ~125 mg/mL 6.29
(for N/A N/A Slightly opalescent, N/A N/A material low pH
essentially free samples, from visible adjusted to particles 4.77
with 0.01M hydrochloric acid) After dialysis, 42.5 mg/mL 5.21 7
1.60 Slightly opalescent, 602 1.5 low pH essentially free from
visible particles After dialysis, 56.9 mg/mL 6.30 6 2.11 Slightly
opalescent, 500 2.7 high pH essentially free from visible particles
After 206 mg/mL 5.40 50 39.35 Slightly opalescent, 1676 0.21
concentration, essentially free low pH from visible particles After
182 mg/mL 6.46 39 47.76 Slightly opalescent, 1088 0.21
concentration, essentially free high pH from visible particles
Characterization of Formulations with Non-Ionic Excipients
[0724] Following the addition of the various non-ionic excipients
to the J695 formulation (see description in Table 56), each
formulation was analysed. Results from osmolality and visual
inspection, and pH are described below in Table 58.
TABLE-US-00067 TABLE 58 Osmolality Sample pH [mosmol/kg] Visual
Inspection LI200/01/5 5.39 473 Slightly opalescent, essentially
free from visible particles LI200/02/5 5.38 402 Slightly
opalescent, essentially free from visible particles LI200/03/5 5.37
466 Slightly opalescent, essentially free from visible particles
LI200/04/5 5.37 397 Slightly opalescent, essentially free from
visible particles LI200/05/5 5.37 458 Slightly opalescent,
essentially free from visible particles LI200/06/5 5.37 396
Slightly opalescent, essentially free from visible particles
LI200/07/5 5.36 50 Slightly opalescent, essentially free from
visible particles LI200/08/5 5.36 48 Slightly opalescent,
essentially free from visible particles LI200/01/6 6.43 428
Slightly opalescent, essentially free from visible particles
LI200/02/6 6.42 405 Slightly opalescent, essentially free from
visible particles LI200/03/6 6.43 348 Slightly opalescent,
essentially free from visible particles LI200/04/6 6.43 383
Slightly opalescent, essentially free from visible particles
LI200/05/6 6.42 432 Slightly opalescent, essentially free from
visible particles LI200/06/6 6.42 402 Slightly opalescent,
essentially free from visible particles LI200/07/6 6.43 38 Slightly
opalescent, essentially free from visible particles LI200/08/6 6.43
39 Slightly opalescent, essentially free from visible particles
HPLC Data
[0725] Each of the non-ionic excipient containing J695 formulations
were also examined using SE-HPLC and IEX. The data from these
analyses are provided in Tables 59 and 60 and provide an overview
of J695 stability during processing and formulation.
TABLE-US-00068 TABLE 59 SE-HPLC results of various J695
formulations Sum Sum Aggregates Monomer Fragments sample name [%]
[%] [%] pH 5 0.608 98.619 0.773 125 mg/mL 0.619 98.598 0.783
starting mat. 0.614 98.608 0.778 pH 6 0.427 98.809 0.764 125 mg/mL
0.392 99.005 0.603 starting mat. 0.409 98.907 0.683 pH 5 0.654
98.604 0.742 42.5 mg/mL 0.677 98.560 0.763 after dialysis 0.666
98.582 0.753 pH 6 0.748 98.541 0.711 56.9 mg/mL 0.739 98.597 0.665
after dialysis 0.743 98.569 0.688 pH 5 0.913 98.416 0.671 206 mg/mL
0.923 98.356 0.721 in Water 0.918 98.386 0.696 pH 5 0.928 98.312
0.760 50 mg/mL mannitol 0.926 98.339 0.736 LI 200/01/5 0.927 98.325
0.748 pH 5 0.925 98.319 0.755 80 mg/mL sucrose 0.929 98.332 0.738
LI 200/02/5 0.927 98.326 0.747 pH 5, 50 mg mannitol 0.942 98.326
0.732 0.01% Tween 80 0.942 98.300 0.758 LI 200/03/5 0.942 98.313
0.745 pH 5, 80 mg sucrose 0.944 98.315 0.741 0.01% Tween 80 0.944
98.339 0.717 LI 200/04/5 0.944 98.327 0.729 pH 5, 50 mg mannitol
0.941 98.348 0.711 0.1% Tween 80 0.967 98.299 0.734 LI 200/05/5
0.954 98.323 0.722 pH 5, 50 mg mannitol 0.944 98.346 0.710 0.1%
Tween 80 0.948 98.340 0.712 LI 200/06/5 0.946 98.343 0.711 pH 5, 50
mg mannitol 0.946 98.348 0.706 0.1% Tween 80 0.953 98.328 0.719 LI
200/07/5 0.949 98.343 0.713 pH 5, 50 mg mannitol 0.987 98.313 0.701
0.1% Tween 80 0.994 98.283 0.723 LI 200/08/5 0.991 98.298 0.712 pH
6 1.091 98.169 0.739 182 mg/mL 1.075 98.221 0.703 in Water 1.083
98.195 0.721 pH 6 0.998 98.350 0.652 50 mg/mL mannitol 1.002 98.364
0.634 Li 200/01/6 1.000 98.357 0.643 pH 6 1.028 98.243 0.729 80
mg/mL sucrose 0.983 98.355 0.662 LI 200/02/6 1.006 98.299 0.695 pH
6, 50 mg mannitol 1.005 98.322 0.673 0.01% Tween 80 1.008 98.317
0.676 LI 200/03/6 1.006 98.299 0.695 pH 6, 80 mg sucrose 0.987
98.363 0.649 0.01% Tween 80 0.987 98.321 0.692 LI 200/04/6 0.987
98.342 0.671 pH 6, 50 mg mannitol 0.996 98.326 0.678 0.1% Tween 80
0.996 98.338 0.666 LI 200/03/6 0.996 98.332 0.672 pH 6, 80 mg
sucrose 0.998 98.305 0.697 0.1% Tween 80 0.984 98.345 0.671
LI200/06/6 0.991 98.325 0.684 pH 6 1.000 98.325 0.675 0.01% Tween
80 0.994 98.347 0.659 LI200/07/6 0.997 98.336 0.667 pH 6 1.003
98.314 0.682 0.01% Tween 80 0.998 98.338 0.664 LI200/08/6 1.001
98.326 0.673
TABLE-US-00069 TABLE 60 IEX results of various J695 formulations
Sum Sum Acicid Sum Basic Peaks Glutamine Peaks sample name [%] [%]
[%] pH 5 4.598 3.303 125 mg/mL 4.599 3.177 starting mat. 4.599
92.162 3.240 pH 6 4.597 3.159 125 mg/mL 4.629 3.156 starting mat.
4.613 92.229 3.158 pH 5 4.706 3.177 42.5 mg/mL 4.725 3.205 after
dialyhsis 4.715 92.094 3.191 pH 6 4.739 3.182 56.9 mg/mL 4.752
3.167 after dialysis 4.746 92.080 3.174 pH 5 4.655 3.167 206 mg/mL
4.676 3.210 in Water 4.666 92.146 3.189 pH 5 4.721 3.321 50 mg/mL
mannitol 4.733 3.356 LI 200/01/5 4.727 91.935 3.338 pH 5 4.715
3.299 80 mg/mL sucrose 4.687 3.338 LI 200/02/5 4.701 91.981 3.318
pH 5, 50 mg mannitol 4.767 3.246 0.01% Tween 80 4.736 3.253 Li
200/03/5 4.752 91.989 3.250 pH 5, 80 mg sucrose 4.751 3.257 0.01%
Tween 80 4.742 3.229 LI 200/04/5 4.746 92.011 3.243 pH 5, 50 mg
mannitol 4.780 3.420 0.1% Tween 80 4.720 3.394 LI 200/05/5 4.750
91.843 3.407 pH 5, 80 mg sucrose 4.756 3.421 0.1% Tween 80 4.894
3.375 LI 200/06/5 4.825 91.777 3.398 pH 5 4.813 3.425 0.01% Tween
80 4.757 3.413 LI 200/07/5 4.785 91.798 3.419 pH 5 4.769 3.361 0.1%
Tween 80 4.842 3.335 LI 200/08/5 4.806 91.846 3.348 pH 6 4.882
3.452 182 mg/mL 4.886 3.451 in Water 4.884 91.664 3.451 pH 6 4.843
3.456 50 mg/mL mannitol 4.833 3.393 LI 200/01/6 4.838 91.737 3.425
pH 6 4.923 3.407 80 mg/mL sucrose 4.896 3.491 LI 200/02/6 4.909
91.642 3.449 pH 6, 50 mg mannitol 4.864 3.423 0.01% Tween 80 4.899
3.392 LI 200/03/6 4.882 91.711 3.408 pH 6, 80 mg sucrose 4.870
3.320 0.01% Tween 80 4.928 3.369 LI 200/04/6 4.899 91.756 3.345 pH
6, 50 mg mannitol 4.905 3.385 0.1% Tween 80 4.922 3.489 LI 200/05/6
4.914 91.649 3.437 pH 6, 80 mg sucrose 4.973 3.443 0.1% Tween 80
4.962 3.335 LI200/06/6 4.968 91.644 3.389 pH 6 4.934 3.413 0.01%
Tween 80 4.899 3.392 LI 200/07/6 4.916 91.681 3.402 pH 6 4.884
3.410 0.1% Tween 80 4.934 3.366 LI 200/08/6 4.909 91.783 3.388
Conclusion
[0726] The above example provides an experiment where water
(demineralised and sterile filtered water) was used as dialysis
medium for the monoclonal antibody J695.
[0727] J695 was subjected to dialysis and concentration processing
by using pure water as exchange medium and was formulated at pH
5.40 as well as 6.46 at high concentration (206 and 182 mg/mL,
respectively) without inducing solution haziness, severe
opalescence or turbidity formation.
[0728] J695 from the processing experiment was characterized, and
formulated with various non-ionic excipients. Data obtained points
at favorable overall stability of the protein in the formulations
tested.
[0729] In conclusion, processing proteins using pure water as
exchange medium and formulation with non-ionic excipients is
feasible. Assuming an ideal 100% excipient membrane permeability,
an approx. 99.9% maximum excipient reduction can be estimated.
Example 24: Syringeability of Adalimumab Formulated in Water
[0730] The formulations from example 21 (63 and 220 mg/mL
Adalimumab) were subjected to force measurements upon syringe
depletion. 220 mg/mL samples of Adalimumab were diluted to 200
mg/mL, 150 mg/mL and 100 mg/mL, respectively, and were also
assessed. A Zwick Z2.5/TN1S was used at a constant feed of 80
mm/min Finally, viscosity data of the formulations was assessed
using an Anton Paar Microviscosimeter, type AWVn, at 20.degree. C.
The following data collection suggests that both needle and syringe
diameters have a significant effect on the gliding forces upon
syringe depletion. Surprisingly, the highly concentrated solution
at 220 mg/mL (viscosity 27.9 cP at 20.degree. C.) can be delivered
by applying equivalent depletion forces as with the lower
concentrated formulation at 63 mg/mL (viscosity 1.8 cP at
20.degree. C.).
TABLE-US-00070 TABLE 61 Gliding Force Values obtained for
Adalimumab solutions in different packaging systems. BD HyPak D
HyPak SCF .TM. 1 mL long SCF .TM. 1 mL syringes, equipped with 1 mL
Soft-Ject .RTM. long syringes, 25 G .times. 26 G .times. 28 G
.times. Tuberkulin equipped with 5/8'' 1/2'' 1/2'' syringes
(smaller 27.5 G RNS needles needles needles diameter than BD
needles (Sterican) (Sterican) (Sterican) HyPak syringes), BD HyPak
BD BD BD equipped with Adaliumab BSCF HyPak HyPak HyPak 27 G
.times. 1/2'' Concentration 4432/50 4432/50 4432/50 4432/50 needles
(Viscosity) stoppers stoppers stoppers stoppers (Sterican) 63 mg/mL
39N -- -- -- -- (1.8 cP) 100 mg/mL 3.30N 1.02N 1.33N 1.69N 1.00N
(2.9 cP) 150 mg/mL 4.63N 1.16N 1.58N 2.93N 1.33N (7.4 cP) 200 mg/mL
7.25N 2.16N 3.24N 6.25N 2.55N (15.7 cP) 220 mg/mL 14.5N 2.99N 3.97N
9.96N 3.16N (27.9 cP)
[0731] The above suggests that even with high concentrations of
protein, such formulations are conducive to administration using a
syringe, e.g., subcutaneous.
Examples 25-28
[0732] Examples 25-28 describe freeze/thaw stability experiments of
various antibody formulations containing the antibody formulated in
water (referred to in examples 26-28 as low-ionic strength protein
formulations). The freeze thaw behavior of a number of antibodies
was evaluated by cycling various protein formulations up to 4 times
between the frozen state and the liquid state. Freezing was
performed by means of a temperature controlled -80.degree. C.
freezer, and thawing was performed by means of a 25.degree. C.
temperature controlled water bath. About 25 mL of antibody solution
each were filled in 30 mL PETG repositories for these experiment
series.
[0733] Formation of subvisible particles presents a major safety
concern in pharmaceutical protein formulations. Subvisible protein
particles are thought to have the potential to negatively impact
clinical performance to a similar or greater degree than other
degradation products, such as soluble aggregates and chemically
modified species that are evaluated and quantified as part of
product characterization and quality assurance programs (Carpenter,
J F et al. Commentary: Overlooking subvisible particles in
therapeutic protein products: baps that may compromise product
quality. J. Pharm. Sci., 2008). As demonstrated in the examples
listed below, a number of antibodies were surprisingly
stable--especially with regard to subvisible particle
formation--when formulated in the formulation of invention.
Example 25: Freeze/Thaw Stability of Adalimumab Formulated in Water
and with Non-Ionic Excipients
[0734] The following example describes the stability of an
antibody, e.g., adalimumab, in a water formulation and in water
formulations in which non-ionic excipients have been added.
Aliquots of samples from examples 21 and 22 were subjected to
freeze/thaw experiments and analyzed by SE-HPLC. Data was compared
to SE-HPLC results derived from freeze/thaw experiments using
Adalimumab in a buffer of the following composition: 10 mM
phosphate buffer, 100 mM sodium chloride, 10 mM citrate buffer, 12
mg/mL mannitol, 0.1% polysorbate 80, pH 5.2. Adalimumab in this
latter buffer was used at 50 mg/mL and 200 mg/mL, respectively.
Freeze/thaw cycles were performed in Eppendorf caps, by freezing to
-80.degree. C. and storage in the freezer for 8 hours, followed by
thawing at room temperature for 1 hour and subsequent sample pull.
Each formulation was subjected to 5 cycles, i.e., cycles 0, 1, 2,
3, 4, and 5 described in the tables below.
HPLC Method
[0735] Adalimumab, SEC analysis: Sephadex 200 column (Pharmacia
Cat. No. 175175-01). Mobile phase 20 mM sodium phosphate, 150 mM
sodium chloride, pH 7.5, 0.5 mL/min flow rate, ambient temperature,
detection UV 214 nm and 280 nm. Each sample was diluted to 1.0
mg/mL with Milli-Q water, sample injection load 50 .mu.g (duplicate
injection).
Adalimumab Characterization Upon Freeze/Thaw Cycling
[0736] Table 62 describes Adalimumab purity during the freeze/thaw
experiments. For sample composition, refer to examples 21 and
22.
TABLE-US-00071 TABLE 62 Freeze/thaw + Low Ionic Adalimumag-50 mg/mL
Fraction monomere [%] cycle LI 50/01 LI 50/02 LI 50/03 LI 50/04 LI
50/05 LI 50/06 LI 50/07 LI 50/08 0 99.605 99.629 99.632 99.626
99.619 99.626 99.653 99.655 1 99.743 99.768 99.739 99.759 99.731
99.725 99.686 99.669 2 99.715 99.726 99.571 99.661 99.721 99.721
99.601 99.619 4 99.668 99.689 99.632 99.678 99.523 99.724 99.491
99.542 5 99.627 99.771 99.539 99.772 99.525 99.773 99.357 99.445
Fraction aggregate [%] cycle LI 50/01 LI 50/02 LI 50/03 LI 50/04 LI
50/05 LI 50/06 LI 50/07 LI 50/08 0 0.106 0.104 0.119 0.136 0.139
0.145 0.158 0.159 1 0.149 0.134 0.153 0.150 0.149 0.166 0.206 0.201
2 0.192 0.178 0.320 0.242 0.178 0.184 0.319 0.261 4 0.213 0.185
0.239 0.187 0.357 0.170 0.393 0.353 5 0.301 0.151 0.384 0.150 0.398
0.150 0.568 0.484 Fraction fragmente [%] cycle LI 50/01 LI 50/02 LI
50/03 LI 50/04 LI 50/05 LI 50/06 LI 50/07 LI 50/08 0 0.289 0.267
0.249 0.238 0.242 0.229 0.189 0.186 1 0.108 0.098 0.108 0.091 0.120
0.108 0.107 0.130 2 0.093 0.097 0.108 0.097 0.100 0.094 0.080 0.119
4 0.119 0.126 0.130 0.135 0.120 0.106 0.116 0.105 5 0.072 0.078
0.078 0.078 0.077 0.077 0.075 0.071 Freeze/thaw - Low ionic
Adalimumab-200 mg/mL Fraction monomere [%] cycle LI 200/01 LI
200/02 LI 200/03 LI 200/04 LI 200/05 LI 200/06 LI 200/07 LI 200/08
0 99.294 99.296 99.348 99.333 99.313 99.349 99.320 99.290 1 99.286
99.267 99.259 99.256 99.120 99.254 98.999 99.126 2 99.305 99.311
99.249 99.214 99.296 99.288 99.149 99.128 4 99.303 99.272 99.261
99.301 99.296 99.283 99.004 99.061 5 99.320 99.322 99.330 99.331
99.327 99.333 98.939 98.949 Fraction aggregate [%] cycle LI 200/01
LI 200/02 LI 200/03 LI 200/04 LI 200/05 LI 200/06 LI 200/07 LI
200/08 0 0.489 0.509 0.491 0.484 0.492 0.488 0.515 0.575 1 0.590
0.574 0.584 0.586 0.680 0.582 0.785 0.718 2 0.604 0.604 0.616 0.630
0.607 0.607 0.731 0.736 4 0.591 0.592 0.612 0.581 0.604 0.596 0.868
0.836 5 0.593 0.586 0.583 0.596 0.597 0.589 0.985 0.981 Fraction
fragmente [%] cycle LI 200/01 LI 200/02 LI 200/03 LI 200/04 LI
200/05 LI 200/06 LI 200/07 LI 200/08 0 0.218 0.196 0.161 0.183
0.195 0.163 0.165 0.135 1 0.124 0.159 0.157 0.159 0.200 0.164 0.216
0.157 2 0.091 0.085 0.135 0.156 0.097 0.105 0.120 0.136 4 0.106
0.136 0.127 0.118 0.100 0.121 0.128 0.103 5 0.087 0.092 0.087 0.073
0.075 0.078 0.076 0.070 Freeze/thaw - Adalimumab Commerical and in
water Fraction monomer [%] from example A, from example A, cycle
low conc. high conc. Standard, 50 mg/mL Standard, 200 mg/mL 0
99.733 99.286 99.374 99.227 1 99.689 99.212 99.375 99.215 2 99.614
99.130 99.370 99.218 4 99.489 99.029 99.361 99.196 5 99.430 98.945
99.362 99.177 Fraction aggregates [%] from example A, from example
A, cycle low conc. high conc. Standard, 50 mg/mL Standard, 200
mg/mL 0 0.186 0.635 0.358 0.502 1 0.226 0.706 0.359 0.516 2 0.304
0.780 0.364 0.513 4 0.428 0.888 0.372 0.535 5 0.485 0.971 0.373
0.553 Fraction fragments [%] from example A, from example A, cycle
low conc. high conc. Standard, 50 mg/mL Standard, 200 mg/mL 0 0.080
0.079 0.268 0.272 1 0.085 0.083 0.266 0.269 2 0.082 0.090 0.266
0.269 4 0.083 0.083 0.267 0.270 5 0.085 0.085 0.265 0.269
Conclusion
[0737] The above example provides an experiment where Adalimumab
DF/UF processed into water (Sterilized water for injection Ph.
Eur./USP) and formulated with various non-ionic excipients was
subjected to freeze/thaw cycling. Data obtained (described in Table
62) indicates favorable overall stability of the protein in all
formulations tested. All formulations contained above 98.5%
monomeric species after 5 freeze/thaw cycles, with minimal amounts
of aggregate or fragments as cycles continued.
Example 26: Freeze/Thaw Stability of Low-Ionic 1D4.7 Solutions
[0738] 1D4.7 protein (an anti-IL 12/anti-IL 23 IgG1) was formulated
in water by dialysis (using slide-a-lyzer cassettes, used according
to operating instructions of the manufacturer, Pierce, Rockford,
Ill.) and was demonstrated to be stable during repeated freeze/thaw
(f/t) processing (-80.degree. C./25.degree. C. water bath) at 2
mg/mL concentration, pH 6. Data were compared with data of various
formulations (2 mg/mL protein, pH 6) using buffers and excipients
commonly used in parenteral protein formulation development. It was
found that the stability of 1D4.7 formulated in water exceeded the
stability of 1D4.7 formulated in established buffer systems (e.g.
20 mM histidine, 20 mM glycine, 10 mM phosphate, or 10 mM citrate)
and even exceeded the stability of 1D4.7 formulations based on
universal buffer (10 mM phosphate, 10 mM citrate) combined with a
variety of excipients that are commonly used to stabilize protein
formulations, e.g. 10 mg/mL mannitol, 10 mg/mL sorbitol, 10 mg/mL
sucrose, 0.01% polysorbate 80, or 20 mM NaCl.
[0739] SEC, DLS and particle counting analysis were applied to
monitor protein stability, and particle counting was performed
using a particle counting system with a 1 200 .mu.m measurement
range (particle counter Model Syringe, Markus Klotz GmbH, Bad
Liebenzell, Germany) Experiment details are as follows: [0740]
--1D4.7 formulated in water compared with formulations listed above
[0741] 4 freeze/thaw cycles applied [0742] 30 mL PETG repository,
about 20 mL fill, 2 mg/mL protein, pH 6 [0743] sampling at T0, T1
(i.e. after one f/t step), T2, T3, and T4 [0744] analytics: visual
inspection, SEC, DLS, subvisible particle measurement
[0745] FIG. 33 shows 1D4.7 stability during repeated f/t cycling
(-80.degree. C./25.degree. C.), mirrored by formation of subvisible
particles >1 .mu.m. 1D4.7 was formulated in universal buffer (10
mM citrate, 10 mM phosphate) and then the following excipient
variations were tested: sorbitol (10 mg/mL), mannitol (10 mg/mL),
sucrose (10 mg/mL), NaCl (100 mM), and polysorbate 80 (0.01%).
1D4.7 was also formulated in water (by dialysis) with no excipients
added at all ("water" in FIG. 33). Water for injection was also
subjected to f/t cycling and subvisible particle testing to
evaluate a potential impact of material handling, f/t, and sample
pull on particle load.
[0746] The stability of 1D4.7 formulated in water upon f/t exceeded
the stability of 1D4.7 solutions formulated with excipients
typically used in protein formulations. Mannitol, sucrose, and
sorbitol are known to act as lyoprotectant and/or cryoprotectant,
and polysorbate 80 is a non-ionic excipient prevalently known to
increase physical stability of proteins upon exposure to
hydrophobic-hydrophilic interfaces such as air-water and ice-water,
respectively.
[0747] In summary, 1D4.7 solutions formulated in water appeared to
be surprisingly stable when analyzed with various analytical
methodologies typically applied to monitor stability of
pharmaceutical proteins upon freeze-thaw processing (e.g. SEC,
visual inspection, dynamic light scattering, and especially light
obscuration).
Example 27: Freeze/Thaw Stability of Low-Ionic 13C5.5 Solutions
[0748] 13C5.5 (an anti IL-13 IgG1) formulated in water was
demonstrated to be stable during repeated freeze/thaw processing
(-80.degree. C./25.degree. C. water bath) at 2 mg/mL concentration,
pH 6. Data were compared with other formulations (2 mg/mL protein,
pH 6), and it was found that the stability of 13C5.5 formulated in
water exceeded the stability of 13C5.5 formulated in buffer systems
often used in parenteral protein formulations (e.g. 20 mM
histidine, 20 mM glycine, 10 mM phosphate, or 10 mM citrate) and
even exceeded the stability of 13C5.5 formulations based on
universal buffer (10 mM phosphate, 10 mM citrate) that has been
combined with a variety of excipients that are commonly used in
protein formulation (e.g. 10 mg/mL mannitol, 10 mg/mL sorbitol, 10
mg/mL sucrose, 0.01% polysorbate 80, 20 mM NaCl, 200 mM NaCl).
[0749] Sample preparation, experiment processing, sample pull and
sample analysis was performed in the same way as outlined in the
above examples. [0750] 13C5.5 formulated in water compared with
formulations listed above [0751] 4 freeze/thaw cycles applied
[0752] 30 mL PETG repository [0753] 2 mg/mL, pH 6 [0754] sampling
at T0, T1, T2, T3, and T4 [0755] analytics: visual inspection, SEC,
DLS, subvisible particle measurement
[0756] FIG. 34 shows 13C5.5 stability during repeated f/t cycling
(-80.degree. C./25.degree. C.), mirrored by formation of subvisible
particles >10 .mu.m. 13C5.5 was formulated in either 10 mM
phosphate buffer, 10 mM citrate buffer, 20 mM glycine buffer, and
20 mM histidine buffer. 13C5.5 was also formulated in the
formulation of invention (by dialysis) with no excipients added at
all. Water for injection was also subjected to f/t cycling and
subvisible particle testing to evaluate a potential impact of
material handling, f/t, and sample pull on particle load (referred
to as blank).
[0757] The stability of 13C5.5 formulated in water upon f/t
exceeded the stability of 13C5.5 solutions formulated in buffers
typically used in protein formulations. No instabilities of 13C5.5
solutions formulated in water have been observed with other
analytical methodologies applied (e.g. SEC, visual inspection,
etc.)
[0758] FIG. 35 shows 13C5.5 stability during repeated f/t cycling
(-80.degree. C./25.degree. C.), mirrored by formation of subvisible
particles >1 .mu.m. 13C5.5 was formulated in universal buffer
(10 mM citrate, 10 mM phosphate) and in universal buffer combined
with the following excipient variations were tested: sorbitol (10
mg/mL), mannitol (10 mg/mL), sucrose (10 mg/mL), NaCl (200 mM),
NaCl (20 mM) and polysorbate 80 (0.01%). 13C5.5 was also formulated
in water (by dialysis) with no excipients added at all for
comparison (pure water). Water for injection was also subjected to
f/t cycling and subvisible particle testing to evaluate a potential
impact of material handling, f/t, and sample pull on particle
load.
[0759] The stability of 13C5.5 formulated in water upon f/t
exceeded the stability of 13C5.5 solutions formulated with
excipients typically used in protein formulations. Mannitol,
sucrose, and sorbitol are known to act as lyoprotectant and/or
cryoprotectant, and polysorbate 80 is a non-ionic excipient
prevalently known to increase physical stability of proteins upon
exposure to hydrophobic-hydrophilic interfaces such as air-water
and ice-water, respectively. The low number of subvisible particles
in 13C5.5 samples formulated into the formulation of invention was
found to be at surprisingly low levels, demonstrating the high
safety and stability potential of such formulations.
[0760] No instabilities of 13C5.5 solutions formulated in water
have been observed with other analytical methodologies applied,
(e.g. SEC, visual inspection, etc.).
[0761] DLS analysis of 13C5.5 solutions after f/t procedures was
performed as described above. Results from the DLS analysis showed
that an 13C5.5 solution with 0.01% Tween-80 contained significant
high molecular weight (HMW) aggregate forms after only 1 f/t step,
whereas 13C5.5 in water contained no HMW aggregate forms, even
after 3 f/t steps applied.
[0762] In summary, 13C5.5 solutions formulated in water appeared to
be surprisingly stable when analyzed with various analytical
methodologies typically applied to monitor stability of
pharmaceutical proteins upon freeze-thaw processing (e.g. SEC,
visual inspection, dynamic light scattering, and especially light
obscuration).
Example 28: Freeze/Thaw Stability Of Low-Ionic 7C6 Solutions
[0763] 7C6 (an anti amyloid beta IgG1) formulated in water was
demonstrated to be stable during repeated freeze/thaw processing
(-80.degree. C./30.degree. C. water bath) at 2 mg/mL concentration,
pH 6. Data were compared with other formulations (2 mg/mL protein,
pH 6), and it was found that the stability of 7C6 formulated in
water exceeded the stability of 7C6 formulated in buffer systems
often used in parenteral protein formulations and even exceeded the
stability of 7C6 formulations based on universal buffer (10 mM
phosphate, 10 mM citrate) that has been combined with a variety of
excipients that are commonly used in protein formulation.
[0764] The following solution compositions were evaluated for their
potential to maintain 7C6 physical stability during freeze/thaw
experiments: [0765] Phosphate buffer, 15 mM [0766] Citrate buffer,
15 mM [0767] Succinate buffer, 15 mM [0768] Histidine buffer, 15 mM
[0769] Arginine buffer, 15 mM [0770] Low ionic protein formulation,
no excipients added [0771] Universal buffer, sorbitol (10 mg/mL)
[0772] Universal buffer, mannitol (10 mg/mL) [0773] Universal
buffer, sucrose (10 mg/mL) [0774] Universal buffer, trehalose (10
mg/mL) [0775] Universal buffer, 0.01% (w/w) polysorbate 80
[0776] Sample preparation, experiment processing, sample pull and
sample analysis was performed in very similar way as outlined in
Examples 26 and 27. [0777] 7C6 formulated in water compared with
formulations listed above [0778] 4 freeze/thaw cycles applied
[0779] 30 mL PETG repository, approx. 20 mL fill [0780] 2 mg/mL, pH
6 [0781] sampling at T0, T1, T2, T3, and T4 [0782] Analytics:
A.beta.-antibody stability was assessed by the following methods:
[0783] Visual inspection of the protein solution was performed in
polypropylene round-bottom tubes wherein the samples were filled
for light obscuration measurements. It was carefully inspected
against both a black and a white background for signs indicating
protein physical instability such as haziness, turbidity and
particle formation. [0784] Dynamic light scattering (eZetasizer
Nano ZS, Malvern Instruments, AI9494; equipped with Hellma
precision cells, suprasil quartz, type 105.251-QS, light path 3 mm,
center Z8.5 mm, at least 60 .mu.L sample fill, protein sample
remaining from light obscuration measurements in PP round-bottom
tubes were used for DLS measurements). Automated measurements (1
measurement per sample) were performed. [0785] Light obscuration
analysis. 3.5 mL of sample were filled in 5 mL round-bottom tube
under laminar air flow conditions, measurement was performed in n=3
mode (0.8 mL per single measurement) after an initial 0.8 mL rinse.
[0786] Size-exclusion chromatography, combined with
UV.sub.214/UV.sub.280 and multi-angle light scattering. Mobile
phase: 100 mM Na2HPO4/200 mM Na2SO4, pH 7.0 (49.68 g anhydrous
disodium hydrogen phosphate and 99.44 g anhydrous sodium sulfate
were dissolved in approx. 3300 mL Milli-Q water, the pH was
adjusted to 7.0 using 1 M phosphoric acid, filled to a volume of
3500 mL with Milli-Q water, and the solution was filtered through a
membrane filter). SEC column, TSK gel G3000SW (cat. no. 08541) 7.8
mm.times.30 cm, 5 .mu.m combined with a TSK gel guard (cat. no.
08543) 6.0 mm.times.4.0 cm, 7 .mu.m. Flow 0.3 mL/min, injection
volume 20 .mu.L (equivalent to 20 .mu.g sample), column temperature
room temperature, autosampler temp. 2 to 8.degree. C., run time 50
minutes, gradient isocratic, piston rinsing with 10% isopropyl
alcohol, detection via UV absorbance, diode array detector:
wavelength 214 nm, peak width >0.1 min, band width: 8 nm,
reference wavelength 360 nm, band width 100 nm
[0787] FIG. 36 shows 7C6 stability during repeated f/t cycling
(-80.degree. C./25.degree. C.), mirrored by formation of subvisible
particles >1 .mu.m. The stability of 7C6 formulated in water
upon f/t for many formulations exceeded the stability of 7C6
solutions formulated in buffers typically used in protein
formulations. No instabilities of 7C6 solutions formulated in water
have been observed with other analytical methodologies applied
(e.g. SEC, visual inspection, dynamic light scattering)
[0788] Surprisingly, the stability of 7C6 formulated in water upon
f/t exceeded the stability of 7C6 solutions formulated with
excipients typically used in protein formulations. Mannitol,
sucrose, and sorbitol are known to act as lyoprotectant and/or
cryoprotectant, and polysorbate 80 is a non-ionic excipient
prevalently known to increase physical stability of proteins upon
exposure to hydrophobic-hydrophilic interfaces such as air-water
and ice-water, respectively. The low number of subvisible particles
in 7C6 samples formulated into the formulation of invention was
found to be at surprisingly low levels, demonstrating the high
safety and stability potential of such formulations.
[0789] In summary, 7C6 solutions formulated in water appeared to be
surprisingly stable when analyzed with various analytical
methodologies typically applied to monitor stability of
pharmaceutical proteins upon freeze-thaw processing (e.g. SEC,
visual inspection, dynamic light scattering, and especially light
obscuration).
Example 29: Preparation of J695 Formulated in Water and Stability
Studies Thereof
Materials and Methods
[0790] 427.1 g (80 mg/mL) of J695 were diluted to 40 mg/mL and
diafiltered using purified water. After a 5-fold volume exchange
with purified water (theoretical excipients reduction, 99.3%), the
protein solution was ultrafiltered to target concentration of 100
mg/mL. pH, osmolality, density, visual inspection and protein
concentration measurements (OD280) were performed to monitor the
status of the protein after DF/UF processing.
[0791] After DF/UF processing, the protein solution was sterile
filtered (0.22 .mu.m Sterivex GV membrane filter) into a 60 mL PETG
bottle (Nalgene) and subsequently stored at -80.degree. C. for 3
months.
[0792] After thawing at 37.degree. C., the solution was sterile
filtered (0.22 .mu.m Sterivex GV membrane filter) and filled into
sterile BD Hypak Physiolis SCF.TM. 1 mL long syringes 29G, 1/2
inch, 5-bevel, RNS TPE and closed with sterile BD Hypak SCF.TM. 1
ml W4023/50 Flur Daikyo stoppers. The filling volume was 1.000 mL
per syringe.
[0793] After filling, the syringes were stored at 2-8.degree. C.
and 40.degree. C., respectively, and analyzed as indicated in the
sample pull scheme depicted below. [0794] J695 Drug Substance
(extinction coefficient at 280 nm: 1.42 mL/mg cm): Drug Substance,
pH 6.0, did not contain polysorbate 80. [0795] Sartorius Sartocon
Slice diafiltration system, equipped with Ultrasert PES membrane
cassettes (50 kDa cutoff). The Sartocon Slice system was operated
in continuous mode at ambient temperature according to Sartorius
Operating Instructions. [0796] pH electrodes [0797] Perkin Elmer
UV/Vis spectrophotometer, Lambda 25, was used for protein
concentration measurements (280 nm wavelength). Disposable UV
cuvettes, 1.5 mL, semi-micro, were used for the concentration
measurements. [0798] 0.22 .mu.m filtered purified water was used as
DF/UF medium. [0799] Anton Paar Density Meter DMA 4100 was used for
density measurements [0800] A Knauer Osmometer Type ML, was used
for osmolality measurements (calibrated with 400 mOsmol/kg NaCl
calibration solution, Art. No. Y1241, Herbert Knauer GmbH, Berlin,
Germany).
[0801] Analytical Methods [0802] J695, SEC analysis: Superdex 200
column (Pharmacia). Mobile phase 92 mM di-sodium hydrogen
phosphate, 211 mM sodium sulfate, pH 7.0, 0.75 mL/min flow rate,
ambient temperature, detection UV 214 nm. Each sample was diluted
to 2.0 mg/mL with mobile phase, sample injection load 20 .mu.g
(duplicate injection). [0803] J695, IEC analysis: Dionex, Propac
WCX-10 column along with a corresponding guard column. Separation
conditions: mobile phase A: 20 mM di-sodium hydrogen phosphate and
20 mM sodium acetate, pH 7.0; mobile phase B 20 mM di-sodium
hydrogen phosphate, 400 mM Sodium chloride, pH 5.0. 1.0 mL/min flow
rate, ambient temperature. Each sample was diluted to 1.0 mg/mL
with Milli-Q water, sample injection load 100 .mu.g (duplicate
injection). [0804] J695, SDS-PAGE analysis: Novex acryl amide slab
gels (8-16% for non-reducing conditions, 12% for reducing
conditions, Invitrogen), Coomassie staining (Invitrogen).
Separation under reducing (.beta.-mercaptoethanol) and non-reducing
conditions using Tris-Glycine buffer made of 10.times. stock
solution (Invitrogen). [0805] J695, quantitation of buffer
components: [0806] Mannitol: separation per ReproGel Ca column (Dr.
Maisch, Germany) and RI detection, mobile phase: deionized water,
0.6 mL/min flow rate, 20 .mu.L sample injection. Quantitation was
performed using external calibration standard curve. [0807]
Histidine and Methionine: fluorescence labelling of the amino acids
with OPA (ortho-phthalic aldehyde) and HPLC separation per ReproSil
ODS-3 column (Dr. Maisch, Germany) and fluorescence detection at
420 nm (extinction at 330 nm), mobile phase A: 70% citric acid
(10.51 g/L) buffer, pH 6.5, 30% methanol, mobile phase B: methanol,
1.0 mL/min flow rate, 20 .mu.L sample injection. Quantitation was
performed using external calibration standard curve. [0808] J695,
PCS analysis: was performed undiluted at 100 mg/mL in single-use
plastic cuvettes at 25.degree. C. using a A Malvern Instruments
Zetasizer nano ZS at 173.degree. angle assuming solution viscosity
of 4.3875 mPas, refractive index of the protein of 1.450 and
refractive index of the buffer solution of 1.335. The averaged
results of 20 scans, 20 seconds each, are reported.
[0809] Calculation of the Protein Concentration
[0810] Calculation formula:
E = - l g ( I I 0 ) = c d -> c = E d ##EQU00018## [0811]
.epsilon.--absorption coefficient [0812] c--concentration [0813]
d--length of cuvette that the light has to pass [0814]
E--absorbance [0815] I.sub.0--initial light intensity [0816]
I--light intensity after passing through sample
[0816] J 695 = 1.42 mL mg cm ##EQU00019##
Sample Pull Scheme
[0817] Samples of the prepared solutions were stored at the
temperatures listed below and pulled (x) at the indicated time
points after study start (Table 63). Test parameters and methods
are described in Table 64.
TABLE-US-00072 TABLE 63 Temp. T0 1 m 3 m 6 m 5.degree. C. x x x x
40.degree. C. x x x
TABLE-US-00073 TABLE 64 Test parameter Test method Appearance
Visual inspection Visible particles analogous DAC (EA 4.43)
Sub-visible particles analogous Ph.Eur./USP EA 4.44 Clarity Ph.Eur.
(EA 4.42) Color (visual) Ph.Eur. (EA 4.50) pH Ph.Eur. (EA 4.24)
Size exclusion HPLC See above Cation exchange HPLC See above
SDS-PAGE See above PCS See above
[0818] DF/UF Processing of J695
[0819] Table 65 provides the J695 status after diafiltration.
TABLE-US-00074 TABLE 65 Protein Concentration Osmolality Sample
[mg/mL] pH [mosmol/kg] Visual Inspection after 107 6.4 10 Slightly
opalescent, DF/UF slightly yellow essentially free from visible
particles
[0820] After DF/UF the concentrations of the originating buffer
components were quantitatively monitored to assess the DF
effectiveness. All results were found to be below the practical
detection limits (see Table 66) of the corresponding analytical
methods (HPLC with RI for Mannitol and fluorescence detection for
the methionine and histidine after OPA labeling, respectively).
TABLE-US-00075 TABLE 66 Methionine Histidine Mannitol Sample
[mg/mL] [mg/mL] [mg/mL] before DF/UF 0.669 0.586 18.36 after DF/UF
<0.13 <0.14 <3.20
[0821] J695 Characterization Upon Storage
[0822] Table 67 below supports the stability of J695 DF/UF at 100
mg/mL upon storage.
TABLE-US-00076 Duration Storage conditions of testing [.degree.
C./%, RH] Test criteria Specification [months] +5 +40/75 Appearance
solution Initial complies 1 complies complies 3 complies complies 6
complies complies Clarity Report Results, Initial .ltoreq.RSII
Compare to reference 1 -- -- suspensions acc. to 3 .ltoreq.RSII
.ltoreq.RSII Ph.Eur. 6 .ltoreq.RSII .ltoreq.RSIII Particulate
Report Result Initial 2.0 (1) contamination Visual Score 1 2.0 (1)
3.0 (1) Visible particles (number of samples 3 1.6 (5) 1.0 (5)
tested) 6 1.1 (9) 1.6 (9) Particulate .gtoreq.10 .mu.m:
.ltoreq.6000 particles initial contamination Per container
.gtoreq.10 .mu.m 290 Subvisible particles .gtoreq.25 .mu.m:
.ltoreq.600 particles .gtoreq.25 .mu.m 16 per container 1
.gtoreq.10 .mu.m -- -- .gtoreq.25 .mu.m 3 .gtoreq.10 .mu.m -- --
.gtoreq.25 .mu.m 6 .gtoreq.10 .mu.m 124 54 .gtoreq.25 .mu.m 1 3
Size Exclusion HPLC Report Results (%) for initial Aggregates (A) A
0.9 Monomer (M) M 98.9 Fragments (F) F 0.2 1 A 0.8 2.3 M 99.0 97.1
F 0.1 0.6 3 A 1.0 3.4 M 98.8 95.1 F 0.1 1.4 6 A 1.4 5.5 M 98.4 85.6
F 0.1 8.9 SDS-PAGE The predominant banding Initial complies
(Non-reducing pattern is comparable to 1 complies complies
conditions) that of the reference 3 complies complies standard. 6
complies complies SDS-PAGE The predominant banding Initial complies
(reducing conditions) pattern is comparable to 1 complies complies
that of the reference 3 complies complies standard. 6 complies
complies PCS Report Results for initial 0.9 Z-Average [nm] and 0.23
PDI 1 0.9 1.0 0.23 0.23 3 0.9 1.1 0.23 0.24 6 0.9 1.3 0.23 0.29
Cation Exchange Report Results (%) for initial HPLC Acidic Species
(A) A 5.9 Main Isoforms (M) M 91.5 Basic Species (B) B 2.5 1 A 5.6
10.6 M 92.0 98.9 B 2.4 0.5 3 A 5.7 14.4 M 92.1 85.0 B 2.1 0.6 6 A
6.0 29.6 M 91.6 69.4 B 2.3 1.0
Conclusion
[0823] The above example provides a diafiltration/ultrafiltration
(DF/UF) experiment where water (0.22 .mu.m filtered purified water)
was used as diafiltration medium for the monoclonal antibody
J695.
[0824] J695 was subjected to DF/UF processing using pure water as
DF/UF exchange medium and was formulated at about pH 6.4 at high
concentration (100 mg/mL) without inducing solution haziness,
severe opalescence or turbidity formation.
[0825] J695 from the DF/UF experiments was stored in SCF syringes
at 2-8.degree. C. and 40.degree. C. for up to 6 months. Data
obtained points to a favourable overall stability of the
protein.
[0826] In conclusion, processing and formulating proteins using
pure water as DF/UF exchange medium is feasible. Assuming an ideal
100% excipient membrane permeability, an approx. 99.3% maximum
excipient reduction can be estimated. Evidence is given by specific
methods that after DF/UF the excipient concentration is below the
practical detection limits.
Example 30: Freeze/Thaw Characteristics and Stability Testing of
High Concentration Adalimumab Water Solution--Homogeneity and
Physical Stability
Preparation of Low-Ionic Adalimumab Solutions
[0827] 1.6 L of Drug Substance (DS) material in 2 L PETG bottle was
thawed at 25.degree. C. in a water bath, homogenized and subjected
to DF/UF using water for injection as a diafiltration exchange
medium. Diafiltration was performed in continuous mode with
Sartorius Sartocon Slice equipment by applying the following
parameter: [0828] Pump output: 8% [0829] Pressure inlet: max 1 bar
(0.8 bar) [0830] Membrane: 2.times.PES, cut off 50 kD [0831] During
the diafiltration 5-fold volume exchange was sufficient to reduce
osmolality to 8 mOsmol/kg.
[0832] In-Process-Control (IPC) samples were pulled prior to
diafiltration (SEC, protein concentration by means of OD280, pH,
osmolality and density) and after diafiltration (protein
concentration by means of OD280, pH, osmolality and density). The
IPC-samples were not sterile.
[0833] After diafiltration the .about.70 mg/mL Adalimumab
formulated in water was diluted to 50 mg/mL with water for
injection and the pH value was adjusted to 5.2.
[0834] 1.6 L of the Adalimumab 50 mg/mL formulated in water pH 5.2
was refilled in 2 L PETG bottle. The remaining volume of Adalimumab
solution was subjected to DF/UF to increase the concentration to
100 mg/mL.
[0835] The Adalimumab 100 mg/mL formulated in water pH 5.3 was
sterile filtered and 0.8 L of them was filled in 1 L PETG
bottle.
Analytics
[0836] Size exclusion chromatography (SEC) [0837] pH- measurement
[0838] Osmolality measurement [0839] Density measurement [0840]
Protein concentration by means of OD280 [0841] Optical appearance
[0842] Ion exchange chromatography (IEC)
Freeze/Thaw Experiment of Adalimumab 1 L Containers
[0843] Adalimumab 100.degree. mg/mL formulated in water in 1 L PETG
containers was precooled to 2-8.degree. C. and than froze at
-80.degree. C., freezing cycle >12 hrs. The frozen samples in 1
L PETG bottles were successively thawed at 25.degree. C. in a water
bath. During thawing the bottles of the frozen solutions dipped in
the water bath up to liquid level. The following samples were
pulled just after thawing without homogenization and after
homogenization by 15 and 30 turn top over end.
TABLE-US-00077 TABLE 68 Sample pull scheme: Turns of each bottle
Sample Analytical tests 1 0 5 mL top protein content, osmolality, 2
0 5 mL middle pH, density, SEC 3 0 5 mL bottom 4 15 5 mL top
protein content, osmolality, 5 15 5 mL middle pH, density, SEC 6 15
5 mL bottom 7 30 5 mL top protein content, osmolality, 8 30 5 mL
middle pH, density, SEC and 9 30 5 mL bottom subvisible
particles
Characterization of Adalimumab Solutions
[0844] Adalimumab 50 mg/mL and 100.degree. mg/mL formulated in
water appeared every time clearly, light yellow, not opalescent and
without wave pattern after gentle movement.
[0845] Also after freezing and thawing the Adalimumab formulated in
water did not change the appearance (just after thawing and also
after 15 and 30 times turn top over end).
[0846] A slight wave patterns were seen after gentle movement of
the bottle just after thawing and dipping the needle into the
solution during sample pull just after thawing.
[0847] In contrast to similar experiments with Adalimumab in
commercial buffer the Adalimumab solution 50 mg/mL in water did not
show any gradient of protein concentration, density and
osmolality.
[0848] The Adalimumab solution 100 mg/mL did also not show any
gradient of protein concentration, density, osmolality.
[0849] Stability was assessed after 6 months storage at -30.degree.
C. and -80.degree. C., respectively.
In the following the respective analytical data are outlined:
TABLE-US-00078 TABLE 69 Adalimumab 50 and 100 mg/mL, before
freeze/thaw processing protein content density osmolality
(gravimertic) subvisible particles pH g/cm3 mOsmol/kg mg/mL 1 mL
>= 1 .mu.m 1 mL >= 10 .mu.m 1 mL >= 25 .mu.m 50 mg/mL in
5.18 1.0121 5 49.3 7953 5 0 water 100 mg/mL in 5.32 1.0262 12 99.8
154 4 2 water
TABLE-US-00079 TABLE 70 Adalimumab 50 mg/mL, pH 5.2 formulated in
water, after freeze/thaw processing protein purity density
osmolality content (SEC) subvisible particles turn sample pH g/cm3
mOsmol/kg mg/mL % 1 mL >= 1 .mu.m 1 mL >= 10 .mu.m 1 mL >=
25 .mu.m 0 top 5.20 1.0119 6 48.7 99.597 -- -- -- 0 middle 5.19
1.0120 8 49.4 99.576 -- -- -- 0 bottom 5.17 1.0120 6 49.8 99.649 --
-- -- 15 top 5.20 1.0120 4 49.7 99.649 -- -- -- 15 middle 5.18
1.0120 5 49.2 99.678 -- -- -- 15 bottom 5.17 1.0120 4 49.1 99.637
-- -- -- 30 top 5.19 1.0120 5 49.7 99.647 1280 4 0 30 middle 5.17
1.0120 3 50.4 99.637 2055 13 0 30 bottom 5.18 1.0120 6 48.9 99.611
3889 37 11
TABLE-US-00080 TABLE 71 Adalimumab 100 mg/mL, pH 5.2 formulated in
water, after freeze/thaw processing protein purity density
osmolality content (SEC) subvisible particles turn sample pH g/cm3
mOsmol/kg mg/mL % 1 mL >= 1 .mu.m 1 mL >= 10 .mu.m 1 mL >=
25 .mu.m 0 top 5.29 1.0259 13 98.7 99.424 -- -- -- 0 middle 5.3
1.0262 16 99.9 99.468 -- -- -- 0 bottom 5.28 1.0262 14 101.2 99.48
-- -- -- 15 top 5.27 1.0261 13 98.9 99.511 -- -- -- 15 middle 5.27
1.0261 16 97.7 99.466 -- -- -- -- bottom 5.28 1.0261 15 97.0 99.483
-- -- -- 30 top 5.29 1.0261 16 96.6 99.439 231 58 49 30 middle 5.28
1.0261 16 97.0 99.467 169 21 9 30 bottom 5.28 1.0261 16 99.3 99.476
131 3 1
TABLE-US-00081 TABLE 72 Adalimumab 100 mg/mL, pH 5.2 formulated in
water, stability after storage SEC aggregates IEC monomer sum of
lysin visual subvisible particles (1 mL) Testing time point
fragments isoforms appearance >=1 .mu.m >=10 .mu.m >=25
.mu.m T 0 0.55 85.523 clear, 155 3 1 99.40 no particular 0.05
matter T 6 months, -80.degree. C. 0.47 82.124 clear, 210 8 5 99.39
no particular 0.14 matter T 6 months, -30.degree. C. 1.28 81.61
clear, 171 71 51 98.58 no particular 0.14 matter
Conclusion
[0850] No significant instabilities of Adalimumab formulated in
water at 50 and 100 mg/mL after freeze/thaw processing and after
storage at -30.degree. C. or -80.degree. C. for up to 6 months have
been observed with the analytical methodologies applied.
Example 31: Freezing and Thawing Process of Adalimumab in Low-Ionic
Formulation--Process Design Space Including Protein Content
Preparation of Solution
[0851] Adalimumab BDS (Bulk Drug Substance) was thawed in a
23.degree. C. circulating water bath. The solution was
up-concentrated to a target concentration of 100 mg/ml for the
purpose of volume reduction using a Ultrafiltration/Diafiltration
(UF/DF) method (Pellicon "Mini" 2). Two cassettes of Millipore
Pellicon 2 tangential flow mini-cassettes with Biomax 10K
polyethersulfone were installed in the Pellicon 2 unit. At process
start the flow rate was measured at 60 ml/min and feed pressure was
21 psi. The process was stopped at 111.3 mg/ml protein
concentration.
[0852] Spectra/Por molecularporous membrane tubing was used for
dialysis (diameter 48 mm, 18 ml/cm volume, 75 cm length). A volume
of 8 L of Adalimumab 100 mg/ml at pH 5.2 were transferred to 8
dialysis tubes. Each tube was filled with 1 L of Adalimumab 100
mg/ml. Four tubes equal to 4 L of solution were placed in a
container with 36 L of water for injection, i.e. a solution
exchange factor of 1:10 was accomplished. The solution was allowed
to reach equilibrium before the volume was exchanged against fresh
water for injection. The solution exchange was repeated 5 times
until a total solution exchange factor of 1:100,000 was
reached.
[0853] After the solution was completely exchanged by dialysis it
was up-concentrated by the second UF/DF step. The second UF/DF step
was performed like the first step. A final concentration of 247.5
mg/ml Adalimumab in low-ionic formulation was achieved. The UF/DF
was performed with starting material that already contained
polysorbate 80. It could be expected that polysorbate 80
accumulated in the final protein solution resulting in a higher
polysorbate content than 0.1%.
[0854] The up-concentrated bulk solution of 247.5 mg/ml Adalimumab
was diluted with WFI to lower protein concentration levels as
needed--200 mg/ml, 175 mg/ml, 150 mg/ml, 140 mg/ml, 130 mg/ml, 120
mg/ml, 100 mg/ml, 80 mg/ml, 50 mg/ml, 40 mg/ml, and 25 mg/ml. The
bottle fill volume was 1600 ml for all experiments.
Freezing Procedures
[0855] A series of increasing freeze rates was used in this
evaluation: Ultra-low temperature freezer bottom shelf
<Ultra-low temperature freezer middle shelf <Ultra-low
temperature freezer top shelf <<Dry ice.
[0856] A<-70.degree. C. freezer was used for the experiments
(Capacity: 20.2 Cu. Ft. (572 liters). Three shelves were used. Each
was loaded with nine 2 L PETG bottles. The bottles were stored at
room temperature before being placed in the freezer. Freezing
continues for at least 48 hours. For the design space evaluations,
three positions with increasing freeze rates were chosen. A front
position on the bottom shelf was used for the slowest freeze rate.
Faster freeze rates were accomplished at the center position on the
middle shelf. The fastest freeze rate in the freezer setup was
performed in the back/right position on the top shelf.
[0857] For freezing by dry ice, one bottle was completely
surrounded by dry ice for at least 8 hours. In a Styrofoam box, the
bottom was covered with a layer of dry ice (approx. 3 to 5 cm
thick). One bottle was placed standing on top of the dry ice layer.
Consequently, the space between the bottle and the inner walls of
the styrofoam box was filled with dry ice until every surface but
the cap was covered. After freezing time, the bottle was removed
and thawed immediately or placed in a -70.degree. C. freezer for
storage.
Thawing Procedures
[0858] A series of thawing rates was used in this evaluation:
Cooled air at 4.degree. C. <<Water bath 23.degree. C.
<Water bath 37.degree. C.
Analytics
[0859] The following analytics were performed to characterize the
samples: [0860] Osmolality [0861] Conductivity [0862] pH [0863]
Density [0864] Protein concentration by direct UV (280 nm)
[0865] For the concentration test, samples were diluted with water
until an absorbance <1.2 was reached. The absorbance coefficient
for the Adalimumab molecule at 280 nm of 1.39 was used.
Characterization of Adalimumab solutions
[0866] Bottle mapping studies revealed a slight tendency towards
gradient formation in the bottle volume. Especially for the slower
freeze and thaw rates, higher protein concentrations were detected
near the bottle bottom. This phenomenon was also reflected in
conductivity, density, and osmolality data. The pH appears
practically constant in all tested conditions.
[0867] In previous investigations regarding the Freeze and Thaw
design space for the bottle based system in ultra-low temperature
freezers, the appearance of sedimentation was found to be the main
failure mode determining the boundaries of the allowable operating
range. In this study, this boundary was not observed although the
investigated design space covered very wide ranges. The unique
behavior of this product is also reflected in the very low tendency
to form concentration gradients during this freezing and thawing
process. In prior studies it was concluded that the product and
process inherent gradient formation is the cause for the appearance
of precipitate under certain process conditions. As a result, it
was determined that from a process standpoint this system is
feasible for Adalimumab in low-ionic formulation pH 5 up to a bulk
drug substance concentration of 247.5 mg/ml. The investigated
Adalimumab water formulation surprisingly demonstrated superior
performance in comparison to other tested Adalimumab
formulations.
TABLE-US-00082 TABLE 73 Distribution of Protein Concentration,
Conductivity, Osmolarity, Density, and pH in the Freshly Thawed
(23.degree. C. water bath) 100 mg/ml Adalimumab in Low Ionic
Formulation Containing Bottles conduc- sample volume osmolarity
tivity density Adalimumab name ml mosm/kg mS/cm pH g/cm.sup.3 conc
mg/ml Freeze & Thaw Conditions: -70 C. Top/23 C. Thaw 1 40 11
0.61 5.43 1.021 78.0 2 210 14 0.67 5.43 1.024 92.2 3 225 15 0.70
5.43 1.0253 98.1 4 200 17 0.72 5.46 1.0259 103.9 5 175 18 0.73 5.43
1.0268 100.2 6 180 18 0.74 5.43 1.0275 100.9 7 230 20 0.80 5.46
1.0284 109.1 8 180 22 0.81 5.45 1.0294 111.2 9 150 21 0.82 5.44
1.0307 118.0 Freeze & Thaw Conditions: -70 C. Middle/23 C. Thaw
1 30 8 0.54 5.43 1.0174 65.3 2 175 18 0.68 5.44 1.0235 91.3 3 200
17 0.70 5.44 1.0245 92.1 4 185 17 0.72 5.44 1.0249 102.9 5 200 18
0.71 5.43 1.0248 95.6 6 200 20 0.73 5.44 1.0262 96.6 7 175 20 0.74
5.44 1.0283 107.5 8 180 20 0.77 5.45 1.0306 116.1 9 200 26 0.82
5.44 1.0346 131.1 Freeze & Thaw Conditions: -70 C. Bottom/23 C.
Thaw 1 35 9 0.60 5.41 1.0195 73.2 2 200 13 0.68 5.41 1.0231 89.6 3
225 16 0.70 5.41 1.0241 93.2 4 180 15 0.71 5.41 1.0246 96.8 5 200
15 0.72 5.40 1.0249 95.7 6 200 19 0.73 5.41 1.0259 96.4 7 185 21
0.75 5.42 1.0272 102.6 8 200 26 0.79 5.41 1.0309 116.8 9 175 31
0.85 5.42 1.0372 141.5
TABLE-US-00083 TABLE 74 Distribution of Protein Concentration,
Conductivity, Osmolarity, Density, and pH in the Freshly Thawed
(23.degree. C. water bath) 140 mg/ml Adalimumab in Low Ionic
Formulation Containing Bottles conduc- sample volume osmolarity
tivity density Adalimumab name ml mosm/kg mS/cm pH g/cm.sup.3 conc
mg/ml Freeze & Thaw Conditions: -70 C. Top/23 C. Thaw 1 50 36
0.37 5.43 1.0338 130.0 2 215 42 0.90 5.43 1.0354 139.9 3 170 54
0.91 5.43 1.0362 144.9 4 210 41 0.84 5.44 1.0365 141.5 5 200 40
0.93 5.43 1.0364 157.7 6 200 41 0.92 5.43 1.0364 140.0 7 190 41
0.92 5.43 1.0363 143.4 8 180 44 0.82 5.43 1.037 150.1 9 140 45 0.95
5.41 1.038 148.3 Freeze & Thaw Conditions: -70 C. Middle/23 C.
Thaw 1 25 32 0.81 5.45 1.0284 112.0 2 175 34 0.84 5.44 1.0307 122.4
3 175 38 0.88 5.44 1.033 133.9 4 200 40 0.90 5.43 1.0342 134.6 5
220 40 0.92 5.43 1.0351 140.9 6 185 45 0.94 5.43 1.0369 143.6 7 210
47 0.97 5.43 1.0384 149.8 8 175 47 0.99 5.43 1.0399 160.3 9 190 48
1.01 5.43 1.0435 168.3 Freeze & Thaw Condition: -70 C.
bottom/23 C. Thaw 1 75 28 0.75 5.45 1.0257 88.7 2 180 34 0.82 5.46
1.029 111.6 3 175 34 0.84 5.44 1.0313 123.5 4 220 37 0.86 5.44
1.0322 118.8 5 165 38 0.89 5.45 1.0337 126.3 6 215 44 0.95 5.45
1.0374 137.6 7 210 49 1.00 5.45 1.0407 149.6 8 150 53 1.03 5.43
1.0429 154.9 9 180 60 1.06 5.44 1.0501 183.2
TABLE-US-00084 TABLE 75 Distribution of Protein Concentration,
Conductivity, Osmolarity, Density, and pH in the Freshly Thawed
(37.degree. C. water bath) 200 mg/ml Adalimumab in Low Ionic
Formulation Containing Bottles conduc- sample volume osmolarity
tivity density Adalimumab name ml mosm/kg mS/cm pH g/cm3 conc mg/ml
Freeze & Thaw Conditions: -70 C. Top/37 C. Thaw 1 40 37 0.89
5.26 1.0573 197.5 2 210 35 0.96 5.24 1.0573 195.3 3 175 34 0.96
5.22 1.0578 200.3 4 210 36 0.88 5.27 1.0579 193.9 5 210 39 0.89
5.24 1.058 210.6 6 190 39 0.84 5.27 1.058 213.8 7 200 41 0.88 5.27
1.058 206.7 8 170 41 0.88 5.24 1.0581 196.6 9 160 39 0.89 5.29
1.0595 201.9 Freeze & Thaw Conditions: -70 C. Center/37 C. Thaw
1 10 31 0.85 5.29 1.0485 170.2 2 185 35 0.89 5.31 1.0505 183.3 3
215 37 0.90 5.33 1.0518 191.7 4 185 38 0.89 5.27 1.0519 191.8 5 200
36 0.90 5.32 1.0528 196.3 6 200 48 0.90 5.28 1.0533 189.3 7 170 37
0.90 5.23 1.0536 193.1 8 215 39 0.91 5.33 1.0552 202.1 9 180 48
0.92 5.31 1.0813 225.5 Freeze & Thaw Conditions: -70 C.
Bottom/37 C. Thaw 1 50 22 0.96 5.27 1.0361 107.1 2 185 29 0.83 5.26
1.0422 163.0 3 180 38 0.88 5.27 1.0522 201.2 4 185 41 0.90 5.24
1.0535 198.9 5 180 44 0.92 5.28 1.0552 201.4 6 195 40 0.91 5.32
1.0558 201.7 7 180 40 0.91 5.32 1.0560 199.6 8 175 41 0.85 5.26
1.0568 206.2 9 190 48 0.91 5.3 1.0619 229.3
TABLE-US-00085 TABLE 76 Distribution of Protein Concentration,
Conductivity, Osmolarity, Density, and pH in the Freshly Thawed
(23.degree. C. water bath) 247.5 mg/ml Adalimumab in Low Ionic
Formulation Containing Bottles conduc- sample volume osmolarity
tivity density Adalimumab name ml mosm/kg mS/cm pH g/cm.sup.3 conc
mg/ml Freeze & Thaw Conditions: -70 C. Top/23 C. Thaw 1 65 46
0.98 5.28 1.0755 260.9 2 190 72 0.97 5.28 1.0751 270.8 3 190 56
0.97 5.28 1.0751 314.7 4 200 49 0.96 5.27 1.0751 274.8 5 200 58
0.96 5.27 1.0752 278.4 6 210 57 0.97 5.28 1.0752 275.0 7 210 76
0.96 5.28 1.0748 276.5 8 175 75 0.96 5.27 1.0754 274.5 9 150 62
0.97 5.28 1.0763 276.3 Freeze & Thaw Conditions: -70 C.
Middle/23 C. Thaw 1 80 37 0.95 5.29 1.0671 250.0 2 200 59 0.95 5.32
1.0704 251.3 3 175 51 0.97 5.31 1.0722 262.7 4 215 56 0.98 5.31
1.073 327.1 5 200 48 0.99 5.31 1.0739 267.7 6 200 67 0.98 5.31
1.0744 270.6 7 230 59 0.95 5.32 1.0753 273.2 8 175 70 0.96 5.32
1.0771 273.3 9 175 83 0.96 5.32 1.0825 289.6 Freeze & Thaw
Conditions: -70 C. Bottom/23 C. Thaw 1 50 32 0.92 5.24 1.0632 215.3
2 220 59 0.95 5.27 1.069 221.7 3 175 72 0.96 5.27 1.0708 268.1 4
180 58 0.96 5.27 1.0725 260.7 5 210 83 0.96 5.27 1.0729 266.8 6 150
89 0.96 5.28 1.0744 280.3 7 225 50 0.96 5.29 1.0762 280.3 8 200 68
0.95 5.28 1.0789 288.6 9 180 70 0.95 5.29 1.0846 293.0
TABLE-US-00086 TABLE 77 Distribution of Protein Concentration,
Conductivity, Osmolarity, Density, and pH in the Freshly Thawed
(23.degree. C. water bath) 247.5 mg/ml Adalimumab in Low Ionic
Formulation Containing Bottles After Dry Ice Freezing conduc-
sample volume osmolarity tivity density Adalimumab name ml mosm/kg
mS/cm pH g/cm.sup.3 conc mg/ml Freeze & Thaw Conditions: Dry
Ice Freeze/23 C. Thaw 1 50 51 0.94 5.28 1.0643 258.9 2 210 68 0.94
5.29 1.0683 261.9 3 180 50 0.95 5.29 1.0702 251.7 4 190 69 0.95
5.29 1.0732 262.2 5 210 72 0.96 5.31 1.0738 274.4 6 225 63 0.95 5.3
1.0746 265.7 7 160 57 0.95 5.3 1.0747 261.9 8 190 63 0.95 5.31
1.0749 270.9 9 200 50 0.95 5.31 1.075 271.4 Freeze & Thaw
Conditions: Dry Ice Freeze/2-8 C. Thaw 1 50 44 0.96 5.29 1.0665
263.1 2 190 53 0.96 5.31 1.0684 258.1 3 200 56 0.96 5.30 1.0691
247.6 4 200 58 0.96 5.30 1.0693 262.2 5 190 64 0.95 5.31 1.0695
243.2 8 200 61 0.95 5.3 1.0695 266.8 7 175 49 0.96 5.32 1.0697
256.2 8 200 50 0.95 5.31 1.0697 261.2 9 175 48 0.96 5.32 1.0704
247.1
TABLE-US-00087 APPENDIX A: PCS DATA Adalimumab peak concentration
z-average monomer [mg/mL] average peak average concentration
averagne z-average value monomer value [mg/mL] value [nm] [nm] [nm]
[nm] 9.35 9.35 2.08 2.08 2.55 2.55 23.40 23.27 2.30 2.47 2.81 2.87
22.70 2.77 3.01 23.70 2.36 2.78 34.80 34.20 1.56 1.55 1.85 1.87
35.70 1.54 1.82 32.10 1.56 1.93 35.40 36.10 1.61 1.63 1.92 1.92
36.10 1.64 1.93 36.80 1.63 1.92 42.10 43.00 1.75 1.75 2.12 2.12
45.60 1.78 2.15 41.30 1.74 2.10 60.20 57.40 2.06 2.02 2.27 2.37
55.90 2.04 2.45 56.10 1.98 2.39 63.20 65.87 2.11 2.24 2.52 2.67
71.70 2.49 2.89 62.70 2.13 2.61 73.40 75.13 2.38 2.41 2.83 2.89
75.60 2.51 3.01 76.40 2.35 2.82 78.60 78.07 2.53 2.55 2.99 2.99
78.80 2.62 3.01 76.80 2.50 2.96 90.40 95.73 2.80 2.85 3.35 3.41
107.40 2.99 3.55 89.40 2.76 3.33 96.20 94.77 2.88 2.86 3.50 3.50
96.00 2.91 3.61 92.10 2.80 3.38 201.00 206.63 4.52 4.82 5.22 5.74
227.50 5.04 6.12 191.40 4.89 5.89 J695 9.99 9.99 2.28 2.28 1.66
1.66 19.31 19.29 2.05 2.12 1.81 1.81 19.26 2.30 1.79 19.29 2.02
1.84 29.59 29.40 1.78 1.62 1.10 1.16 29.7 1.51 1.15 28.91 1.56 1.22
37.97 37.55 1.51 1.56 1.22 1.23 38.02 1.67 1.22 36.65 1.49 1.24
49.15 46.32 1.64 1.58 1.31 1.29 45.95 1.57 1.30 43.87 1.53 1.26
58.75 56.18 1.60 1.61 1.49 1.47 55.02 1.71 1.38 54.76 1.53 1.53
77.69 77.81 2.64 2.43 2.73 2.61 77.62 2.31 2.57 78.13 2.35 2.52
94.45 97.65 2.11 2.07 2.05 2.05 105.06 2.14 2.05 93.45 1.97 2.04
116.37 114.52 3.69 2.69 1.95 2.00 113.92 2.25 2.06 113.27 2.13 1.99
121.21 133.25 9.78 9.49 11.50 11.00 139.8 9.63 11.10 138.73 9.06
10.40 226.67 217.53 4.94 5.34 4.72 4.84 216.1 6.01 5.25 209.83 5.06
4.55 Human Serum Albumin 9.88 9.88 14.90 14.90 2.32 2.32 22.94
22.89 8.26 8.29 1.2 1.18 22.73 8.28 1.18 23.00 8.33 1.17 36.78
36.47 7.40 7.44 1.22 1.23 37.33 7.80 1.24 35.29 7.12 1.22 45.97
46.06 7.09 6.92 1.27 1.25 47.61 6.54 1.24 44.61 7.13 1.25 58.47
58.56 5.94 6.13 1.3 1.31 62.69 6.04 1.31 54.52 6.41 1.32 61.89
60.31 5.83 6.14 1.33 1.32 59.76 6.57 1.34 59.28 6.01 1.29 75.37
76.24 5.58 5.46 1.4 1.40 83.69 5.14 1.45 69.67 5.67 1.36 92.90
85.87 5.30 5.14 1.49 1.47 84.22 5.05 1.49 80.50 5.08 1.43 115.93
112.74 4.78 4.94 1.68 1.61 110.00 5.04 1.58 112.30 4.99 1.57 182.79
177.69 9.85 9.13 2.27 2.19 178.24 9.29 2.21 172.05 8.26 2.08
TABLE-US-00088 APPENDIX B SEC DATA mean mean mean mean conc.
monomer aggregate fragment conc. [mg/mL] [mg/mL] monomer [%] [%]
aggregate [%] [%] fragment [%] [%] Adalimumab 9.35 9.35 99.40 99.40
0.50 0.50 0.10 0.10 23.40 23.27 99.60 99.57 0.40 0.40 0.10 0.10
22.70 99.50 0.40 0.10 23.70 99.60 0.40 0.10 34.80 34.20 99.50 99.47
0.50 0.47 0.10 0.10 35.70 99.40 0.50 0.10 32.10 99.50 0.40 0.10
35.40 36.10 99.40 99.40 0.60 0.53 0.10 0.10 36.10 99.40 0.50 0.10
36.80 99.40 0.50 0.10 42.10 43.00 99.40 99.33 0.50 0.57 0.10 0.10
45.60 99.30 0.60 0.10 41.30 99.30 0.60 0.10 60.20 57.40 99.30 99.30
0.60 0.60 0.10 0.10 55.90 99.30 0.60 0.10 56.10 99.30 0.60 0.10
63.20 65.87 99.30 99.27 0.60 0.67 0.10 0.10 71.70 99.20 0.70 0.10
62.70 99.30 0.70 0.10 73.40 75.13 99.20 99.23 0.70 0.70 0.10 0.10
75.60 99.20 0.70 0.10 76.40 99.30 0.70 0.10 78.60 78.07 99.30 99.30
0.60 0.60 0.10 0.10 78.80 99.30 0.60 0.10 76.80 99.30 0.60 0.10
90.40 95.73 99.20 99.13 0.80 0.80 0.10 0.10 107.40 99.10 0.80 0.10
89.40 99.10 0.80 0.10 96.20 94.77 99.10 99.03 0.80 0.87 0.10 0.10
96.00 99.00 0.90 0.10 92.10 99.00 0.90 0.10 201.00 206.63 98.80
98.80 1.10 1.10 0.10 0.10 227.50 98.80 1.10 0.10 191.40 98.80 1.10
0.10 J695 9.99 9.99 99.39 99.39 0.44 0.44 0.17 0.17 19.31 19.29
99.38 99.38 0.44 0.44 0.18 0.19 19.26 99.37 0.44 0.20 19.29 99.38
0.44 0.18 29.59 29.40 99.31 99.33 0.51 0.50 0.18 0.18 29.70 99.32
0.50 0.18 28.91 99.35 0.48 0.17 37.97 37.55 99.31 99.29 0.52 0.52
0.17 0.19 38.02 99.27 0.52 0.21 36.65 99.30 0.51 0.19 49.15 46.32
99.19 99.20 0.60 0.60 0.21 0.20 45.95 99.20 0.60 0.20 43.87 99.20
0.61 0.19 58.75 56.18 99.16 99.16 0.64 0.64 0.21 0.21 55.02 99.17
0.64 0.20 54.76 99.15 0.63 0.22 77.69 77.81 99.11 99.10 0.70 0.70
0.19 0.20 77.62 99.09 0.69 0.22 78.13 99.10 0.70 0.20 94.45 97.65
99.05 99.06 0.72 0.71 0.23 0.22 105.06 99.06 0.72 0.21 93.45 99.07
0.70 0.23 116.37 114.52 98.94 98.91 0.85 0.88 0.21 0.22 113.92
98.91 0.88 0.22 113.27 98.89 0.90 0.22 121.21 133.25 98.87 98.89
0.91 0.90 0.22 0.22 139.80 98.89 0.89 0.22 138.73 98.90 0.89 0.21
226.67 217.53 98.58 98.57 1.19 1.21 0.24 0.23 216.10 98.58 1.18
0.24 209.83 98.54 1.25 0.21 Human Serum Albumin Peak 1 Peak 2 Peak
3 Peak 4 (HSA) sample Area [mVs] Area [%] Area [mVs] Area [%] Area
[mVs] Area [%] Area [mVs] Area [%] sample 1 59.710 2.312 2.975
0.115 43.159 1.671 2477.282 95.902 c = 9.88 mg/ml sample 2 102.785
2.685 7.859 0.205 73.588 1.923 3643.350 95.187 c = 22.94 mg/ml
sample 3 124.226 3.071 11.038 0.273 83.310 2.059 3826.908 94.597 c
= 22.73 mg/ml sample 4 138.353 3.266 14.525 0.343 88.429 2.087
3994.990 94.304 c = 23.00 mg/ml sample 5 147.465 3.459 14.537 0.341
91.304 2.141 4010.385 94.059 c = 36.78 mg/ml sample 6 153.956 3.552
14.707 0.339 94.093 2.171 4071.680 93.938 c = 37.33 mg/ml sample 7
171.478 3.608 16.064 0.338 105.244 2.214 4459.830 93.839 c = 35.29
mg/ml sample 8 180.027 3.675 17.392 0.355 109.717 2.239 4592.102
93.731 c = 45.97 mg/ml sample 9 193.325 3.719 19.206 0.370 116.474
2.241 4868.705 93.670 c = 47.61 mg/ml sample 10 191.512 3.799
19.167 0.380 112.261 2.227 4718.554 93.594 c = 44.61 mg/ml sample
11 215.044 4.026 17.870 0.335 118.481 2.218 4989.978 93.421 c =
58.47 mg/ml sample 12 218.072 4.037 20.088 0.372 122.251 2.263
5041.542 93.328 c = 62.69 mg/ml sample 13 228.014 4.053 19.957
0.355 126.583 2.250 5251.513 93.343 c = 54.52 mg/ml sample 14
231.235 4.085 22.518 0.398 127.330 2.250 5279.038 93.267 c = 61.89
mg/ml sample 15 237.894 4.100 22.939 0.395 130.352 2.246 5411.384
93.258 c = 59.76 mg/ml sample 16 202.103 4.139 17.178 0.352 108.780
2.228 4554.912 93.282 c = 59.28 mg/ml sample 17 230.552 4.196
18.565 0.338 123.207 2.242 5122.467 93.224 c = 75.37 mg/ml sample
18 215.365 4.162 18.136 0.351 110.152 2.129 4830.372 93.358 c =
83.69 mg/ml sample 21 233.866 4.316 21.951 0.405 116.325 2.147
5046.183 93.132 c = 84.22 mg/ml sample 22 221.816 4.461 18.940
0.381 111.006 2.232 4620.655 92.926 c = 80.50 mg/ml sample 23
223.187 4.783 16.684 0.358 104.116 2.231 4322.732 92.629 c = 115.93
mg/ml sample 24 209.281 4.718 18.745 0.423 96.430 2.174 4111.363
92.686 c = 110.00 mg/ml sample 25 172.657 4.537 15.457 0.406 80.850
2.125 3536.192 92.932 c = 112.30 mg/ml sample 26 178.208 4.950
15.254 0.424 80.906 2.247 3325.648 92.379 c = 182.79 mg/ml sample
27 194.516 4.814 17.323 0.429 90.433 2.238 3738.717 95.520 c =
178.24 mg/ml sample 28 79.605 2.103 12.876 0.340 74.965 1.981
3617.238 95.576 c = 172.05 mg/ml
TABLE-US-00089 APPENDIX C IEC DATA Adalimumab mean conc. sum Lysin
mean sum conc. [mg/mL] [mg/mL] [%] [%] 9.35 9.35 86.09 86.09 23.40
23.27 86.15 86.13 22.70 86.12 23.70 86.13 34.80 34.20 86.15 86.11
35.70 86.11 32.10 86.06 35.40 36.10 86.03 86.04 36.10 86.06 36.80
86.03 42.10 43.00 85.98 85.96 45.60 85.95 41.30 85.95 60.20 57.40
85.97 85.96 55.90 85.94 56.10 85.97 63.20 65.87 85.96 85.94 71.70
85.97 62.70 85.90 73.40 75.13 85.99 85.97 75.60 85.98 76.40 85.95
78.60 78.07 86.00 85.97 78.80 85.97 76.80 85.94 90.40 95.73 85.96
85.92 107.40 85.97 89.40 85.83 96.20 94.77 85.93 85.88 96.00 85.87
92.10 85.84 201.00 206.63 85.88 85.90 227.50 85.97 191.40 85.84
J695 mean sum mean sum acidic mean sum mean conc. sum peak 1-7 peak
1-7 sum acidic peaks sum basic basic peaks conc. [mg/mL] [mg/mL]
[%] [%] peaks [%] [%] peaks [%] [%] 9.99 9.99 89.24 89.24 10.24
10.24 0.52 0.52 19.31 19.29 89.32 89.28 10.19 10.21 0.50 0.51 19.26
89.23 10.26 0.52 19.29 89.30 10.19 0.51 29.59 29.40 89.33 89.30
10.14 10.17 0.54 0.53 29.70 89.26 10.20 0.54 28.91 89.32 10.16 0.52
37.97 37.55 89.32 89.30 10.13 10.15 0.56 0.55 38.02 89.27 10.18
0.55 36.65 89.31 10.15 0.55 49.15 46.32 89.07 89.10 10.40 10.37
0.53 0.53 45.95 89.12 10.34 0.54 43.87 89.12 10.36 0.53 58.75 56.18
89.13 89.17 10.36 10.31 0.52 0.53 55.02 89.21 10.27 0.52 54.76
89.18 10.29 0.54 77.69 77.81 89.22 89.17 10.25 10.29 0.53 0.54
77.62 89.09 10.36 0.55 78.13 89.20 10.26 0.55 94.45 97.65 89.20
89.16 10.28 10.30 0.52 0.54 105.06 89.12 10.33 0.55 93.45 89.16
10.29 0.55 116.37 114.52 89.03 89.08 10.41 10.36 0.56 0.55 113.92
89.15 10.31 0.54 113.27 89.06 10.37 0.56 121.21 133.25 89.26 89.13
10.20 10.33 0.54 0.55 139.80 89.07 10.38 0.56 138.73 89.05 10.40
0.55 226.67 217.53 88.72 88.78 10.69 10.63 0.59 0.59 216.10 88.82
10.60 0.58 209.83 88.81 10.60 0.59
TABLE-US-00090 APPENDIX D Duration of 63 mg/mL 220 mg/mL Test Item
Component Testing 5.degree. C. 5.degree. C. Clarity and Turbidity
Initial 3.6 8.0 opalescence 1 month 3.5 8.0 3 month 3.5 7.4 Degree
of B scale Initial <B9 =B9 coloration of 1 month <B9 <B8
liquids 3 month <B9 <B7 pH Single value Initial 5.3 5.4 1
month 5.3 5.4 3 month 5.3 5.4 Particulate visual score Initial 2.2
0.2 contamination: 1 month 2.2 0.4 visible particles 3 month 2.1
0.2 Particulate Particles >= 10 Initial 181 357 contamination:
.mu.m [/Container] 1 month 423 290 subvisible 3 month 216 1762
particles Particles >= 25 Initial 15 3 .mu.m [/Container] 1
month 11 18 3 month 2 50 Size exclusion Principal peak Initial 0.2
0.5 chromatography (aggregate) [%] 1 month 0.2 0.6 (SE-HPLC) 3
month 0.2 0.7 Principal peak Initial 99.8 99.4 (monomer) [%] 1
month 99.7 99.3 3 month 99.7 99.2 Principal peak Initial 0.1 0.1
(fragment) [%] 1 month 0.1 0.1 3 month 0.0 0.0 Cation exchange 1st
acidic region Initial 2.2 2.2 HPLC (CEX- [%] 1 month 2.2 2.2 HPLC)
3 month 2.1 2.0 2nd acidic Initial 10.4 10.3 region [%] 1 month
10.2 10.0 3 month 10.4 10.2 Sum of lysine Initial 86.0 86.1
variants [%] 1 month 85.9 85.9 3 month 86.2 86.1 Peak between
Initial 0.8 0.8 lysine 1 und 1 month 1.0 1.0 lysine 2 [%] 3 month
0.8 0.8 Peaks after Initial 0.5 0.6 Lysin 2 [%] 1 month 0.7 0.9 3
month 0.5 0.8 Duration of 63 mg/mL 220 mg/mL Test Item Component
Testing 25.degree. C./60% R.H. 25.degree. C./60% R.H. Clarity and
Turbidity Initial -- -- opalescence 1 month 3.51 8.55 3 month 3.70
7.56 Degree of B scale Initial -- -- coloration of 1 month <B9
<B8 liquids 3 month <B9 <B7 pH Single value Initial -- --
1 month 5.4 5.4 3 month 5.3 5.4 Particulate visual score Initial --
-- contamination: 1 month 2.5 0.7 visible particles 3 month 3.4 0.0
Particulate Particles >=10 Initial -- -- contamination: .mu.m
[/Container] 1 month 412 490 subvisible 3 month 277 4516 particles
Particles >= 25 Initial -- -- .mu.m [/Container] 1 month 10 14 3
month 7 128 Size exclusion Principal peak Initial -- --
chromatography (aggregate) [%] 1 month 0.3 0.8 (SE-HPLC) 3 month
0.4 1.1 Principal peak Initial -- -- (monomer) [%] 1 month 99.6
99.0 3 month 99.4 98.6 Principal peak Initial -- -- (fragment) [%]
1 month 0.2 0.2 3 month 0.2 0.2 Cation exchange 1st acidic region
Initial -- -- HPLC (CEX- [%] 1 month 2.5 2.4 HPLC) 3 month 3.4 3.2
2nd acidic Initial -- -- region [%] 1 month 11.7 11.4 3 month 15.3
14.9 Sum of lysine Initial -- -- variants [%] 1 month 83.6 83.8 3
month 79.2 79.2 Peak between Initial -- -- lysine 1 und 1 month 1.2
1.3 lysine 2 [%] 3 month 1.3 1.3 Peaks after Initial -- -- Lysin 2
[%] 1 month 0.9 1.1 3 month 0.8 1.4 Duration of 63 mg/mL 220 mg/mL
Test Item Component Testing 40.degree. C./75% R.H. 40.degree.
C./75% R.H. Clarity and Turbidity Initial -- -- opalescence 1 month
3.93 7.80 3 month 3.70 8.10 Degree of B scale Initial -- --
coloration of 1 month =B9 =B8 liquids 3 month <B8 <B7 pH
Single value Initial -- -- 1 month 5.3 5.4 3 month 5.3 5.4
Particulate visual score Initial -- -- contamination: 1 month 6.7
0.5 visible particles 3 month 17.5 0.4 Particulate Particles
>=10 Initial -- -- contamination: .mu.m [/Container] 1 month
1088 518 subvisible 3 month 166 612 particles Particles >= 25
Initial -- -- .mu.m [/Container] 1 month 16 14 3 month 11 30 Size
exclusion Principal peak Initial -- -- chromatography (aggregate)
[%] 1 month 0.4 1.4 (SE-HPLC) 3 month 0.8 2.5 Principal peak
Initial -- -- (monomer) [%] 1 month 99.0 98.0 3 month 97.8 96.0
Principal peak Initial -- -- (fragment) [%] 1 month 0.6 0.6 3 month
1.4 1.5 Cation exchange 1st acidic region Initial -- -- HPLC (CEX-
[%] 1 month 6.7 6.8 HPLC) 3 month 17.5 17.4 2nd acidic Initial --
-- region [%] 1 month 25.1 23.6 3 month 40.9 38.6 Sum of lysine
Initial -- -- variants [%] 1 month 64.5 62.0 3 month 36.0 36.0 Peak
between Initial -- -- lysine 1 und 1 month 2.2 2.5 lysine 2 [%] 3
month 2.9 3.1 Peaks after Initial -- -- Lysin 2 [%] 1 month 1.5 5.2
3 month 1.7 4.8
TABLE-US-00091 APPENDIX E Duration of LI 50 LI 50 LI 50 LI 50 LI 50
LI 50 LI 50 LI 50 LI 200 LI 200 LI 200 LI 200 LI 200 LI 200 LI 200
LI 200 Test Item Component Testing 01*.sup.2 02*.sup.2 03*.sup.2
04*.sup.2 05*.sup.2 06*.sup.2 07*.sup.2 08*.sup.2 01*.sup.2
02*.sup.2 03*.sup.2 04*.sup.2 05*.sup.2 06*.sup.2 07*.sup.2
08*.sup.2 2-8.degree. C. Clarity and Absorption (340 nm) Initial
0.096 0.096 0.095 0.100 0.104 0.105 0.099 0.107 0.181 0.187 0.182
0.192 0.184 0.197 0.191 0.199 coalescence 1 month 0.102 0.102 0.093
0.094 0.099 0.101 0.097 0.100 0.181 0.185 0.189 0.181 0.190 0.180
0.192 0.192 Degree of visual Initial clear and colorless coloration
1 month clear and colorless pH Single value Initial 5.4 5.4 5.4 5.4
5.4 5.4 5.4 5.4 5.6 5.6 5.6 5.6 5.6 5.5 5.6 5.5 1 month 5.4 5.5 5.5
5.4 5.5 5.4 5.5 5.5 5.6 5.6 5.5 5.5 5.6 5.5 5.6 5.6 Size exclusion
Principal peak Initial 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.5 0.5 0.5
0.5 0.5 0.5 0.5 0.6 chromatography (aggregat) [%] 1 month 0.1 0.1
0.1 0.1 0.1 0.1 1.7 0.2 0.6 0.6 0.6 0.6 0.6 0.6 0.7 0.7 (SE-HPLC)
Principal peak Initial 99.6 99.6 99.6 99.6 99.6 99.6 99.7 99.7 99.3
99.3 99.3 99.3 99.3 99.3 99.3 99.3 (monomer) [%] 1 month 99.7 99.7
99.7 99.7 99.7 99.7 99.7 99.7 99.3 99.3 99.2 99.3 99.3 99.2 99.2
99.2 Principal peak Initial 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2 0.2 0.1 (fragment) [%] 1 month 0.2 0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 Cation exchange 1st
acidic region Initial 3.8 3.7 3.7 3.7 3.7 3.9 3.8 3.8 3.7 3.8 3.6
4.5 3.9 2.7 2.7 2.8 HPLC [%] 1 month 3.4 3.4 3.4 3.4 3.4 3.5 3.4
3.4 3.4 3.4 3.5 3.5 3.5 3.5 3.5 3.5 (CEX-HPLC) 2nd acidic region
Initial 10.9 10.7 10.4 10.5 10.4 10.1 10.3 10.2 10.4 10.2 9.8 10.1
9.5 11.6 11.5 11.3 [%] 1 month 9.2 9.2 9.2 9.2 9.2 9.2 9.2 9.3 9.1
9.0 9.1 9.1 9.0 9.1 9.1 9.1 Sum of Initial 83.8 84.2 84.4 84.3 84.4
84.6 84.4 84.6 84.4 84.6 85.2 83.9 85.2 84.4 84.3 84.5 lysine
variants [%] 1 month 86.0 86.0 86.0 86.0 86.0 85.9 86.1 86.0 86.3
86.1 86.1 86.2 86.0 86.0 86.1 86.1 Peak between lysine 1 Initial
0.9 8.3 0.8 0.9 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 and
lysine 2 [%] 1 month 0.9 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.6 0.8 0.6
0.6 0.8 0.7 0.6 0.6 Peaks after Initial 0.6 0.6 0.6 0.6 0.6 0.6 0.6
0.6 0.6 0.6 0.6 0.7 0.6 0.6 0.6 0.6 Lysin 2 [%] 1 month 0.6 0.5 0.5
0.5 0.5 0.5 0.5 0.5 0.7 0.7 0.7 0.7 0.7 0.7 0.8 0.7 25.degree. C.
Clarity and Absorption (340 nm) Initial 0.096 0.096 0.095 0.100
0.104 0.105 0.099 0.107 0.181 0.187 0.182 0.192 0.184 0.197 0.191
0.199 coalescence 1 month 0.106 0.109 0.096 0.099 0.104 0.104 0.096
0.105 0.178 0.177 0.198 0.189 0.200 0.194 0.194 0.172 Degree of
visual Initial clear and colorless coloration 1 month clear and
colorless pH Single value Initial 5.4 5.4 5.4 5.4 5.4 5.4 5.4 5.4
5.6 5.6 5.6 5.6 5.6 5.5 5.6 5.5 1 month 5.4 5.4 5.4 5.4 5.4 5.4 5.4
5.4 5.5 5.6 5.6 5.5 5.5 5.5 5.5 5.5 Size exclusion Principal peak
Initial 0.1 0.1 0.1 0.1 0.1 0.1 0.2 0.2 0.5 0.5 0.5 0.5 0.5 0.5 0.5
0.6 chromatography (aggregat) [%] 1 month 0.2 0.2 0.2 0.2 0.2 ? 0.2
0.2 0.8 0.8 0.8 0.8 0.8 0.8 0.9 0.9 (SE-HPLC) Principal peak
Initial 99.6 99.6 99.6 99.6 99.6 99.6 99.7 99.7 99.3 99.3 99.3 99.3
99.3 99.3 99.3 99.3 (monomer) [%] 1 month 99.6 99.6 99.6 99.6 99.6
? 99.5 99.5 99.0 99.0 99.0 99.0 99.0 99.0 98.9 98.9 Principal peak
Initial 0.3 0.3 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2 0.2
0.1 (fragment) [%] 1 month 0.2 0.2 0.2 0.2 0.2 ? 0.2 0.2 0.2 0.2
0.2 0.2 0.2 0.2 0.2 0.2 Cation exchange 1st acidic region Initial
3.8 3.7 3.7 3.7 3.7 3.9 3.8 3.8 3.7 3.8 3.6 4.5 3.9 2.7 2.7 2.8
HPLC [%] 1 month 4.1 4.2 4.1 4.1 4.2 4.2 4.1 4.2 4.0 4.0 4.1 4.1
4.0 4.1 4.1 4.0 (CEX-HPLC) 2nd acidic region Initial 10.9 10.7 10.4
10.5 10.4 10.1 10.3 10.2 10.4 10.2 9.8 10.1 9.5 11.6 11.5 11.3 [%]
1 month 10.9 10.9 11.0 10.9 11.0 11.0 11.0 11.1 10.6 10.7 10.7 10.7
10.7 10.7 10.7 10.9 Sum of Initial 83.8 84.2 84.4 84.3 84.4 84.6
84.4 84.6 84.4 84.6 85.2 83.9 85.2 84.4 84.3 84.5 lysine variants
[%] 1 month 83.3 83.4 83.3 83.3 83.2 83.2 83.3 83.1 83.4 83.6 83.4
83.6 83.6 83.5 83.3 83.4 Peak between lysine 1 Initial 0.9 8.3 0.8
0.9 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 0.8 and lysine 2
[%] 1 month 1.0 0.9 1.0 1.0 1.0 0.9 0.9 1.0 1.0 0.8 0.8 0.7 0.7 0.8
0.9 0.8 Peaks after Initial 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6
0.6 0.7 0.6 0.6 0.6 0.6 Lysin 2 [%] 1 month 0.7 0.6 0.7 0.7 0.7 0.6
0.6 0.6 0.9 0.9 0.9 0.9 0.9 0.9 0.9 0.9 40.degree. C. Clarity and
Absorption (340 nm) Initial 0.096 0.096 0.095 0.100 0.104 0.105
0.099 0.107 0.181 0.187 0.182 0.192 0.184 0.197 0.191 0.199
coalescence 1 month 0.100 0.110 0.099 0.101 0.111 0.116 0.105 0.113
0.204 0.202 0.198 0.205 0.199 0.216 0.209 0.224 Degree of visual
Initial clear and colorless coloration 1 month clear and colorless
pH Single value Initial 5.40 5.41 5.41 5.40 5.40 5.40 5.41 5.41
5.57 5.56 5.55 5.56 5.55 5.54 5.55 5.52 1 month 5.42 5.42 5.43 5.43
5.44 5.43 5.44 5.44 5.54 5.55 5.55 5.55 5.55 5.55 5.53 5.54 Size
exclusion Principal peak Initial 0.106 0.104 0.119 0.136 0.139
0.145 0.158 0.159 0.489 0.509 0.491 0.484 0.492 0.488 0.515 0.575
chromatography (aggregat) [%] 1 month 0.324 0.336 0.322 0.334 0.347
0.349 0.411 0.444 1.308 1.343 1.331 1.340 1.341 1.374 1.453 1.521
(SE-HPLC) Principal peak Initial 99.605 99.629 99.632 99.626 99.619
99.626 99.653 99.655 99.294 99.296 99.348 99.333 99.313 99.349
99.320 99.290 (monomer) [%] 1 month 98.924 98.914 98.931 98.914
98.895 98.894 98.845 98.782 97.920 97.876 97.892 97.901 97.898
97.861 97.752 97.701 Principal peak Initial 0.289 0.267 0.249 0.238
0.242 0.229 0.189 0.186 0.218 0.196 0.161 0.183 0.195 0.163 0.165
0.135 (fragment) [%] 1 month 0.752 0.751 0.747 0.752 0.758 0.757
0.744 0.773 0.773 0.781 0.777 0.759 0.762 0.765 0.794 0.779 Cation
exchange 1st acidic region Initial 3.8 3.7 3.7 3.7 3.7 3.9 3.8 3.8
3.7 3.8 3.6 4.5 3.9 2.7 2.7 2.8 HPLC [%] 1 month 5.4 5.8 5.3 5.8
5.4 5.8 5.4 5.5 5.3 5.6 5.3 5.6 5.3 5.6 5.3 5.4 (CEX-HPLC) 2nd
acidic region Initial 10.9 10.7 10.4 10.5 10.4 10.1 10.3 10.2 10.4
10.2 9.8 10.1 9.5 11.6 11.5 11.3 [%] 1 month 29.8 29.8 29.7 29.7
29.8 29.8 30.2 30.7 28.6 28.9 28.5 28.7 28.6 28.9 29.1 29.2 Sum of
Initial 83.8 84.2 84.4 84.3 84.4 84.6 84.4 84.6 84.4 84.6 85.2 83.9
85.2 84.4 84.3 84.5 lysine variants [%] 1 month 61.2 61.0 61.3 61.0
61.2 60.9 60.9 60.5 62.0 61.7 62.0 61.8 62.0 61.6 61.5 61.4 Peak
between lysine 1 and Initial 0.9 8.3 0.8 0.9 0.8 0.8 0.8 0.8 0.8
0.8 0.8 0.8 0.8 0.8 0.8 0.8 lysine 2 [%] 1 month 2.3 2.1 2.3 2.2
2.2 2.2 2.1 2.0 2.2 2.0 2.2 2.0 2.2 2.0 2.0 2.0 Peaks after Initial
0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.6 0.7 0.6 0.6 0.6 0.6
Lysin 2 [%] 1 month 1.3 1.3 1.3 1.3 1.4 1.3 1.4 1.4 1.9 1.9 2.0 1.9
1.9 1.9 2.0 2.0
EQUIVALENTS
[0868] Those skilled in the art will recognize, or be able to
ascertain using no more than routine experimentation, many
equivalents to the specific embodiments of the invention described
herein. Such equivalents are intended to be encompassed by the
following claims. The contents of all references, patents and
published patent applications cited throughout this application are
incorporated herein by reference
Sequence CWU 1
1
81107PRTArtificial SequenceDescription of Artificial Sequence
Synthetic variable light chain polypeptide 1Asp Ile Gln Met Thr Gln
Ser Pro Ser Ser Leu Ser Ala Ser Val Gly 1 5 10 15 Asp Arg Val Thr
Ile Thr Cys Arg Ala Ser Gln Gly Ile Arg Asn Tyr 20 25 30 Leu Ala
Trp Tyr Gln Gln Lys Pro Gly Lys Ala Pro Lys Leu Leu Ile 35 40 45
Tyr Ala Ala Ser Thr Leu Gln Ser Gly Val Pro Ser Arg Phe Ser Gly 50
55 60 Ser Gly Ser Gly Thr Asp Phe Thr Leu Thr Ile Ser Ser Leu Gln
Pro 65 70 75 80 Glu Asp Val Ala Thr Tyr Tyr Cys Gln Arg Tyr Asn Arg
Ala Pro Tyr 85 90 95 Thr Phe Gly Gln Gly Thr Lys Val Glu Ile Lys
100 105 2121PRTArtificial SequenceDescription of Artificial
Sequence Synthetic variable heavy chain polypeptide 2Glu Val Gln
Leu Val Glu Ser Gly Gly Gly Leu Val Gln Pro Gly Arg 1 5 10 15 Ser
Leu Arg Leu Ser Cys Ala Ala Ser Gly Phe Thr Phe Asp Asp Tyr 20 25
30 Ala Met His Trp Val Arg Gln Ala Pro Gly Lys Gly Leu Glu Trp Val
35 40 45 Ser Ala Ile Thr Trp Asn Ser Gly His Ile Asp Tyr Ala Asp
Ser Val 50 55 60 Glu Gly Arg Phe Thr Ile Ser Arg Asp Asn Ala Lys
Asn Ser Leu Tyr 65 70 75 80 Leu Gln Met Asn Ser Leu Arg Ala Glu Asp
Thr Ala Val Tyr Tyr Cys 85 90 95 Ala Lys Val Ser Tyr Leu Ser Thr
Ala Ser Ser Leu Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Leu Val Thr
Val Ser Ser 115 120 39PRTArtificial SequenceDescription of
Artificial Sequence Synthetic variable light chain CDR3 peptide
3Gln Arg Tyr Asn Arg Ala Pro Tyr Xaa 1 5 412PRTArtificial
SequenceDescription of Artificial Sequence Synthetic variable heavy
chain CDR3 peptide 4Val Ser Tyr Leu Ser Thr Ala Ser Ser Leu Asp Xaa
1 5 10 57PRTArtificial SequenceDescription of Artificial Sequence
Synthetic variable light chain CDR2 peptide 5Ala Ala Ser Thr Leu
Gln Ser 1 5 617PRTArtificial SequenceDescription of Artificial
Sequence Synthetic variable heavy chain CDR2 peptide 6Ala Ile Thr
Trp Asn Ser Gly His Ile Asp Tyr Ala Asp Ser Val Glu 1 5 10 15 Gly
711PRTArtificial SequenceDescription of Artificial Sequence
Synthetic variable light chain CDR1 peptide 7Arg Ala Ser Gln Gly
Ile Arg Asn Tyr Leu Ala 1 5 10 85PRTArtificial SequenceDescription
of Artificial Sequence Synthetic variable heavy chain CDR1 peptide
8Asp Tyr Ala Met His 1 5
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