U.S. patent application number 10/206469 was filed with the patent office on 2003-06-19 for stable lyophilized pharmaceutical formulation of igg antibodies.
Invention is credited to Flores-Nate, Aleni, Gupta, Supriya, Kaisheva, Elizabet A..
Application Number | 20030113316 10/206469 |
Document ID | / |
Family ID | 23191544 |
Filed Date | 2003-06-19 |
United States Patent
Application |
20030113316 |
Kind Code |
A1 |
Kaisheva, Elizabet A. ; et
al. |
June 19, 2003 |
Stable lyophilized pharmaceutical formulation of IgG antibodies
Abstract
This invention is directed to a stable lyophilized
pharmaceutical formulation prepared by lyophilizing an aqueous
formulation comprising a high concentration, e.g. 50 mg/ml or more,
of an IgG antibody in about 5-25 mM histidine buffer having pH from
about 5.5 to about 6.5, about 0.005%-0.03% polysorbate, sucrose,
and optionally serine, and/or mannitol. This lyophilized
formulation is stable at room temperature for at least 6 months,
and preferably 1 year. This lyophilized formulation has a short
reconstitution time of less than 2 minutes, and is suitable for
parenteral administration such as intravenous, intramuscular,
intraperitoneal, or subcutaneous injection. This invention is
exemplified by the anti-IL2 receptor antibody.
Inventors: |
Kaisheva, Elizabet A.;
(Belmont, CA) ; Flores-Nate, Aleni; (Union City,
CA) ; Gupta, Supriya; (Sunnyvale, CA) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE, LLP
BOX 34
301 RAVENSWOOD AVE.
MENLO PARK
CA
94025
US
|
Family ID: |
23191544 |
Appl. No.: |
10/206469 |
Filed: |
July 25, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60307878 |
Jul 25, 2001 |
|
|
|
Current U.S.
Class: |
424/130.1 |
Current CPC
Class: |
A61K 9/0019 20130101;
C07K 16/2866 20130101; A61K 39/39591 20130101; A61P 43/00 20180101;
A61K 47/183 20130101; A61P 35/00 20180101; A61K 9/19 20130101; A61K
47/26 20130101 |
Class at
Publication: |
424/130.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A stable lyophilized formulation prepared by lyophilizing an
aqueous formulation comprising: about 5-25 mM histidine buffer
having a pH from 5.5 to about 6.5, about 0.005%-0.03% polysorbate,
about 100-300 mM sucrose, and greater than 50 mg/ml IgG antibody,
wherein said formulation is reconstitutable with a liquid to a
particle-free solution containing greater than 50 mg/ml IgG
antibody concentration within about 2 minutes or less.
2. The stable lyophilized formulation according to claim 1, wherein
said sucrose concentration is about 100-200 mM.
3 A stable lyophilized formulation prepared by lyophilizing an
aqueous formulation comprising: about 5-25 mM histidine buffer
having a pH from about 5.5 to about 6.5, about 0.005%-0.03%
polysorbate, about 110-130 mM sucrose, about 20-45 mM mannitol, and
greater than 50 mg/ml IgG antibody, wherein said lyophilized
formulation is reconstitutable with liquid to a particle-free
solution containing greater than 50 mg/ml IgG antibody
concentration within about 2 minutes or less.
4. A stable lyophilized formulation prepared by lyophilizing an
aqueous formulation comprising: about 5-25 mM histidine buffer
having a pH from about 5.5 to about 6.5, about 0.005% -0.03%
polysorbate, about 80-130 mM sucrose, about 7-55 mM serine, about
10-55 mM mannitol, and greater than 50 mg/ml IgG antibody, wherein
said lyophilized formulation is reconstitutable with liquid to a
particle-free solution containing greater than 50 mg/ml IgG
antibody concentration within about 2 minutes or less.
5. A stable lyophilized formulation prepared by lyophilizing an
aqueous formulation comprising: about 5-25 mM histidine buffer
having a pH from about 5.5 to about 6.5, about 0.005%-0.03%
polysorbate, about 100-130 mM sucrose, about 15-55 mM serine, and
greater than 50 mg/ml IgG antibody, wherein said lyophilized
formulation is reconstitutable with liquid to a particle-free
solution containing greater than 50 mg/ml IgG antibody
concentration within about 2 minutes or less.
6. The stable lyophilized formulation according to claim 1, 3, 4 or
5, wherein said IgG antibody is an anti-IL2 receptor antibody.
7. The stable lyophilized formulation according to claim 1, 3, 4 or
5, wherein said formulation is stable at about 22-28.degree. C. for
at least 3 months.
8. The stabilized lyophilized formulation according to claim 1, 3,
4 or 5, wherein said formulation is stable at about 2-8.degree. C.
for at least 1 year.
9. The stable lyophilized formulation according to claim 1, 3, 4,
or 5, wherein said formulation is suitable for subcutaneous
injection.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/307,878, filed Jul. 25, 2001, which is hereby
incorporated herein in its entirety by reference.
FIELD OF THE INVENTION
[0002] The present invention relates generally to the field of
pharmaceutical formulation of antibodies. Specifically, the present
invention relates to a stable, lyophilized, high concentration
antibody formulation. This invention is exemplified by a stabilized
lyophilized formulation of anti-IL.sub.2 receptor antibody.
BACKGROUND OF THE INVENTION
[0003] It is well known that many protein preparations intended for
administration to humans require stabilizers to prevent
denaturation, aggregation and other alternations to the proteins
prior to the use of the preparation. Many protein preparations are
particularly unstable in very dilute or highly concentrated
solutions. This instability is manifested in the formation of
soluble/insoluble aggregates, and is often increased when the
protein preparation is stored, or shipped. A major challenge that
exists in the field of protein drugs is in the development of
formulations that maintain both protein stability and activity.
[0004] Immunoglobulins, in particular, are recognized as possessing
characteristics that tend to form aggregates and particulates in
solution; thus requiring filtration of these formulations prior to
using them for intravenous injection. The formation of protein
aggregates and particulates has long been a problem in the
development of parenteral immunoglobulin products. There is a need
in the art for a stable pharmaceutical formulation comprising an
antibody.
[0005] WO 89/11297 discloses a lyophilized composition comprising a
monoclonal immunoglobulin antibody of 1-25 mg/ml, 2-10% maltose,
and sodium acetate, phosphate, or citrate buffer having a pH
between 3.0 to 6.0.
[0006] Synagis.TM. (MedImmune) is a humanized monoclonal IgG1
antibody produced by recombinant DNA technology, directed to an
epitope in the A antigenic site of the T protein of respiratory
syncytial virus (RSV). Synagis.TM. is a composite of human (95%)
and murine (5%) antibody sequences. Synagis.TM. is supplied as a
sterile lyophilized product for reconstruction with sterile water
for injection. Reconstituted Synagis.TM. is required to stand at
room temperature for a minimum of 20 minutes until the solution
clarifies. Reconstituted Synagis.TM. is to be administered by
intramuscular injection only. Upon reconstitution, Synagis.TM.
contains the following excipients: 47 mM histidine, 3.0 mM glycine
and 5.6% mannitol and the active ingredient, IgG1 antibody, at a
concentration of 100 milligrams per vial. (See Physicians' Desk
Reference.RTM., Medical Economic Company, Inc., Montvale, N.J.)
[0007] U.S. Patent Application Publication No. U.S. 2001/0014326A1
discloses a prelyophilized antibody formulation containing 25 mg/ml
anti-IgE antibody, 5 mM histidine, pH 6.0, 85 mM sucrose, and 0.01%
polysorbate 20.
[0008] U.S. Pat. No. 6,171,586 discloses a stable aqueous
pharmaceutical formulation comprising a therapeutically effective
amount of an antibody not subjected to prior lyophilization, and
acetate buffer from about pH 4.8 to about 5.5, a surfactant, and a
polyol, wherein the formulation lacks a tonicifying amount of
sodium chloride.
[0009] WO 97/45140 discloses a monoclonal antibody preparation of a
humanized antibody against the CDw52 antigen, having a
concentration of 100 mg/ml or greater, wherein the preparation is
substantially free from aggregates.
[0010] Cleland, et al. (J. Pharm. Sci., 90:310-321 (2001)) disclose
that a 360:1 molar ratio of lyoprotectant to protein is required
for storage stability of a lyophilized monoclonal antibody.
[0011] There is a need for a stable, highly concentrated
lyophilized antibody preparation for administration to a human,
such antibody can be reconstituted within a short time and is
suitable for parenteral administration, including intravenous,
intramuscular, intraperitoneal, or subcutaneous injection.
SUMMARY OF THE INVENTION
[0012] This invention is directed to a stable lyophilized
pharmaceutical formulation prepared from an aqueous formulation
comprising a high concentration, e.g., greater than 50 mg/ml of an
IgG antibody in about 5-25 mM histidine buffer (pH from about 5.5
to about 6.5), about 0.005%-0.03% polysorbate, and sucrose,
optionally in combination with serine, and/or mannitol. This
formulation retains the stability of the IgG antibody, and prevents
the immunoglobulins intended for administration to human subjects
from forming aggregates/particulates in the final product. The
lyophilized formulation is reconstituted with a liquid to a
clarified solution containing greater than 50 mg/ml IgG antibody
concentration within about 2 minutes or less.
[0013] This lyophilized formulation is stable at room temperature
for at least 3 months, preferably 6 months, and more preferably 1
year. The lyophilized formulation is also stable at 2-8.degree. C.
for 1 year, preferably 2 years. This lyophilized formulation has a
short reconstitution time of less than 2 minutes, and is suitable
for parenteral administration such as intravenous, intramuscular,
intraperitoneal, or subcutaneous injection.
BRIEF DESCRIPTION OF THE FIGURES
[0014] FIG. 1 shows the effect of excipients on antibody stability
by testing (A) the liquid after storage at 55.degree. C. for 10
days and (B) the lyophile at 40.degree. C. for 12 days.
[0015] FIGS. 2A and 2B show the comparison of model predictions and
experimental observation for the test samples 1-15. FIG. 2A shows
the % drop in monomer. FIG. 2B shows the reconstruction time.
[0016] FIGS. 3A and 3B show the comparison of model predictions and
experimental observation for the control samples 16-20. FIG. 3A
shows the % drop in monomer. FIG. 3B shows the reconstitution
time.
[0017] FIG. 4 shows the model coefficients of (A) main effect, and
(B) interaction effect.
[0018] FIGS. 5A and 5B show the representative model simulations of
mannitol vs. sucrose, [serine]=0. FIG. 5A shows the % monomer drop.
FIG. 5B shows the reconstitution time.
[0019] FIGS. 6A and 6B show the representative model simulations of
mannitol vs. serine, [sucrose]=0. FIG. 6A shows the % monomer drop.
FIG. 6B shows the reconstitution time.
[0020] FIGS. 7A and 7B show the representative model simulations of
sucrose vs. serine, [mannitol]=0. FIG. 7A shows the % monomer drop.
FIG. 7B shows the reconstitution time.
[0021] FIGS. 8A and 8B show the representative model simulations of
mannitol vs. serine, [sucrose]=100 mM. FIG. 8A shows the % monomer
drop. FIG. 8B shows the reconstitution time.
[0022] FIGS. 9A-9C show percent monomer in Formulations I, II and
II as a function of time at (A) 5.degree. C., (B) 25.degree. C.,
and (C) 40.degree. C.
[0023] FIGS. 10A-C show percent aggregates in Formulations I, II
and III as a function of time at (A) 5.degree. C., (B) 25.degree.
C., and (C) 40.degree. C.
[0024] FIGS. 11A-C shows percent clips in Formulations I, II and
III as a function of time at (A) 5.degree. C., (B) 25.degree. C.,
and (C) 40.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0025] It is an object of the present invention to provide a
lyophilized highly concentrated antibody formulation that is stable
upon storage and delivery. It is a further object to provide a
lyophilized highly concentrated antibody formulation that can be
reconstituted in a short time prior to administration to a
patient.
[0026] I. Definition
[0027] The term "bulking agents" comprise agents that provide the
structure of the freeze-dried product. Common examples used for
bulking agents include mannitol, glycine, lactose and sucrose. In
addition to providing a pharmaceutically elegant cake, bulking
agents may also impart useful qualities in regard to modifying the
collapse temperature, providing freeze-thaw protection, and
enhancing the protein stability over long-term storage. These
agents can also serve as tonicity modifiers.
[0028] The term "buffer" encompasses those agents which maintain
the solution pH in an acceptable range prior to lyophilization and
may include succinate (sodium or potassium), histidine, phosphate
(sodium or potassium), Tris(tris(hydroxymethyl)aminomethane),
diethanolamine, citrate (sodium) and the like. The buffer of this
invention has a pH in the range from about 5.5 to about 6.5; and
preferably has a pH of about 6.0. Examples of buffers that will
control the pH in this range include succinate (such as sodium
succinate), gluconate, histidine, citrate and other organic acid
buffers.
[0029] The term "cryoprotectants" generally includes agents which
provide stability to the protein against freezing-induced stresses,
presumably by being preferentially excluded from the protein
surface. They may also offer protection during primary and
secondary drying, and long-term product storage. Examples are
polymers such as dextran and polyethylene glycol; sugars such as
sucrose, glucose, trehalose, and lactose; surfactants such as
polysorbates; and amino acids such as glycine, arginine, and
serine.
[0030] The terms "lyophilization," "lyophilized," and
"freeze-dried" refer to a process by which the material to be dried
is first frozen and then the ice or frozen solvent is removed by
sublimation in a vacuum environment. An excipient may be included
in pre-lyophilized formulations to enhance stability of the
lyophilized product upon storage.
[0031] The term "lyoprotectant" includes agents that provide
stability to the protein during the drying or `dehydration` process
(primary and secondary drying cycles), presumably by providing an
amorphous glassy matrix and by binding with the protein through
hydrogen bonding, replacing the water molecules that are removed
during the drying process. This helps to maintain the protein
conformation, minimize protein degradation during the
lyophilization cycle and improve the long-term product stability.
Examples include polyols or sugars such as sucrose and
trehalose.
[0032] The term "pharmaceutical formulation" refers to preparations
which are in such form as to permit the active ingredients to be
effective, and which contains no additional components which are
toxic to the subjects to which the formulation would be
administered.
[0033] "Pharmaceutically acceptable" excipients (vehicles,
additives) are those which can reasonably be administered to a
subject mammal to provide an effective dose of the active
ingredient employed.
[0034] "Reconstitution time" is the time that is required to
rehydrate a lyophilized formulation with a solution to a
particle-free clarified solution.
[0035] 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. 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).
Stability can be measured at a selected temperature for a selected
time period.
[0036] A "stable" lyophilized antibody formulation is a lyophilized
antibody formulation with no significant changes observed at a
refrigerated temperature (2-8.degree. C.) for at least 12 months,
preferably 2 years, and more preferably 3 years; or at room
temperature (23-27.degree. C.) for at least 3 months, preferably 6
months, and more preferably 1 year. The criteria for stability are
as follows. No more than 10%, preferably 5%, of antibody monomer is
degraded as measured by SEC-HPLC. The rehydrated solution is
colorless, or clear to slightly opalescent by visual analysis. The
concentration, pH and osmolality of the formulation have no more
than +/-10% change. Potency is within 70-130, preferably 80-120% of
the control. No more than 10%, preferably 5% of clipping is
observed. No more than 10%, preferably 5% of aggregation is
formed.
[0037] An antibody "retains its physical stability" in a
pharmaceutical formulation if it shows no significant increase of
aggregation, precipitation and/or denaturation upon visual
examination of color and/or clarity, or as measured by UV light
scattering, size exclusion chromatography (SEC) and dynamic light
scattering. The changes of protein conformation can be evaluated by
fluorescence spectroscopy, which determines the protein tertiary
structure, and by FTIR spectroscopy, which determines the protein
secondary structure.
[0038] An antibody "retains its chemical stability" in a
pharmaceutical formulation, if it shows no significant chemical
alteration. Chemical stability can be assessed by detecting and
quantifying chemically altered forms of the protein. Degradation
processes that often alter the protein chemical structure include
hydrolysis or clipping (evaluated by methods such as size exclusion
chromatography and SDS-PAGE), oxidation (evaluated by methods such
as by peptide mapping in conjunction with mass spectroscopy or
MALDI/TOF/MS), deamidation (evaluated by methods such as
ion-exchange chromatography, capillary isoelectric focusing,
peptide mapping, isoaspartic acid measurement), and isomerization
(evaluated by measuring the isoaspartic acid content, peptide
mapping, etc.).
[0039] An antibody "retains its biological activity" in a
pharmaceutical formulation, if the biological activity of the
antibody at a given time is within a predetermined range of the
biological activity exhibited at the time the pharmaceutical
formulation was prepared. The biological activity of an antibody
can be determined, for example, by an antigen binding assay.
[0040] The term "isotonic" means that the formulation of interest
has essentially the same osmotic pressure as human blood. Isotonic
formulations will generally have an osmotic pressure from about
270-328 mOsm. Slightly hypotonic pressure is 250-269 and slightly
hypertonic pressure is 328-350 mOsm. Osmotic pressure can be
measured, for example, using a vapor pressure or ice-freezing type
osmometer.
[0041] Tonicity Modifiers: Salts (NaCl, KCl, MgCl.sub.2,
CaCl.sub.2, etc) are used as tonicity modifiers to control osmotic
pressure. In addition, cryprotecants/lyoprotectants and/or bulking
agents such as sucrose, mannitol, glycine etc. can serve as
tonicity modifiers.
[0042] II. Analytical Methods
[0043] The analytical methods for evaluating the product stability
include size exclusion chromatography (SEC), dynamic light
scattering test (DLS), differential scanning calorimetery (DSC),
iso-asp quantification, potency, UV at 340 nm, UV spectroscopy, and
FTIR. SEC (J. Pharm. Scien., 83:1645-1650, (1994); Pharm. Res.,
11:485 (1994); J. Pharm. Bio. Anal., 15:1928 (1997); J. Pharm. Bio.
Anal., 14:1133-1140 (1986)) measures percent monomer in the product
and gives information of the amount of soluble aggregates. DSC
(Pharm. Res., 15:200 (1998); Pharm. Res., 9:109 (1982)) gives
information of protein denaturation temperature and glass
transition temperature. DLS (American Lab., November (1991))
measures mean diffusion coefficient, and gives information of the
amount of soluble and insoluble aggregates. UV at 340 nm measures
scattered light intensity at 340 nm and gives information about the
amounts of soluble and insoluble aggregates. UV spectroscopy
measures absorbance at 278 nm and gives information of protein
concentration. FTIR (Eur. J Pharm. Biopharm., 45:231 (1998); Pharm.
Res., 12:1250 (1995); J. Pharm. Scien., 85:1290 (1996); J. Pharm.
Scien., 87:1069 (1998)) measures IR spectrum in the amide one
region, and gives information of protein secondary structure.
[0044] The iso-asp content in the samples is measured using the
Isoquant Isoaspartate Detection System (Promega). The kit uses the
enzyme Protein Isoaspartyl Methyltransferase (PIMT) to specifically
detect the presence of isoaspartic acid residues in a target
protein. PIMT catalyzes the transfer of a methyl group from
S-adenosyl-L-methionine to isoaspartic acid at the .alpha.-carboxyl
position, generating S-adenosyl-L-homocystei- ne (SAH) in the
process. This is a relatively small molecule, and can usually be
isolated and quantitated by reverse phase HPLC using the SAH HPLC
standards provided in the kit.
[0045] The potency or bioidentity of an antibody can be measured by
its ability to bind to its antigen. The specific binding of an
antibody to its antigen can be quantitated by any method known to
those skilled in the art, for example, an immunoassay, such as
ELISA (enzyme-linked immunosorbant assay).
[0046] III. Preparation of Antibody
[0047] The invention herein relates to a stable formulation
comprising an antibody. The antibody is prepared using techniques
available in the art for generating antibodies, exemplary methods
of which are described in more detail in the following
sections.
[0048] The antibody is directed against an antigen of interest.
Preferably, the antigen is a biologically important polypeptide and
administration of the antibody to a mammal may prevent or treat a
disorder. However, antibodies directed against nonpolypeptide
antigens (such as tumor-associated glycolipid antigens; see U.S.
Pat. No. 5,091,178) are also contemplated.
[0049] Where the antigen is a polypeptide, it may be a
transmembrane molecule (e.g. receptor) or ligand such as a growth
factor. Exemplary antigens include molecules such as renin; a
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 human urine or tissue-type plasminogen activator
(t-PA); bombesin; thrombin; hemopoietic growth factor; tumor
necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated
on activation normally T-cell expressed and secreted); human
macrophage inflammatory protein (MIP-1-alpha); a serum albumin such
as human serum albumin; Muellerian-inhibiting substance; relaxin
A-chain; relaxin B-chain; prorelaxin; mouse gonadotropin-associated
peptide; a microbial protein, such as beta-lactamase; DNase; IgE; a
cytotoxic T-lymphocyte associated antigen (CTLA), such as CTLA-4;
inhibin; activin; vascular endothelial growth factor (VEGF);
receptors for hormones or growth factors; 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..sub.1, TGF-.beta..sub.2, TGF-.beta..sub.3,
TGF-.beta..sub.4, or TGF-.beta..sub.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; 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; receptors to interleukins
IL-1 to IL-10; superoxide dismutase; T-cell receptors; surface
membrane proteins; decay accelerating factor; viral antigen such
as, for example, a portion of the AIDS envelope; transport
proteins; homing receptors; addressins; regulatory proteins;
integrns such as CD11a, CD11b, CD11c, CD18, an ICAM, VLA-4 and
VCAM; a tumor associated antigen such as HER2, HER3 or HER4
receptor; and fragments of any of the above-listed
polypeptides.
[0050] When using recombinant techniques, the antibody can be
produced intracellularly, in the periplasmic space, or directly
secreted into the medium. If the antibody is produced
intracellularly, as a first step, the particulate debris, either
host cells or lysed cells, is removed, for example, by
centrifugation or ultrafiltration. Where the antibody is secreted
into the medium, supernatants from such expression systems are
generally first concentrated using a commercially available protein
concentration filter, for example, an Amicon or Millipore Pellicon
ultrafiltration unit. A protease inhibitor such as PMSF may be
included in any of the foregoing steps to inhibit proteolysis and
antibiotics may be included to prevent the growth of adventitious
contaminants.
[0051] The antibody composition prepared from the cells can be
purified using, for example, hydroxylapatite chromatography, gel
electrophoresis, dialysis, and affinity chromatography, with
affinity chromatography being the preferred purification technique.
The suitability of protein A as an affinity ligand depends on the
species and isotype of any immunoglobulin Fc domain that is present
in the antibody. Protein A can be used to purify antibodies that
are based on human .gamma..sub.1, .gamma..sub.2, or .gamma..sub.4
heavy chains (Lindmark et al., J. Immunol. Meth. 62:1-13 (1983)).
Protein G is recommended for all mouse isotypes and for human
.gamma..sub.3 (Guss et al., EMBO J. 5:1567-1575 (1986)). The matrix
to which the affinity ligand is attached is most often agarose, but
other matrices are available. Mechanically stable matrices such as
controlled pore glass or poly(styrenedivinyl)benzene allow for
faster flow rates and shorter processing times than can be achieved
with agarose. Where the antibody comprises a C.sub.H3 domain, the
Bakerbond ABX.TM. resin (J. T. Baker, Phillipsburg, N.J.) is useful
for purification. Other techniques for protein purification such as
fractionation on an ion-exchange column, ethanol precipitation,
Reverse Phase HPLC, chromatography on silica, chromatography on
heparin SEPHAROSET.TM. chromatography on an anion or cation
exchange resin (such as a polyaspartic acid column),
chromatofocusing, SDS-PAGE, and ammonium sulfate precipiation are
also available depending on the antibody to be recovered.
[0052] A preferred antibody encompassed by the present invention is
an IgG antibody. This invention is exemplified by an anti-IL-2
receptor antibody such as Daclizumab. Daclizumab is a recombinant
humanized monoclonal antibody, subclass IgG1. The molecule is
composed of two identical heavy chain and two identical light chain
subunits. Disulfide bridges link the four chains. Daclizumab
monomer is approximately 150,000 daltons in molecular weight.
Daclizumab binds to the p55 subunit of the IL-2 receptor expressed
on activated T cells. The antigen target is designated CD25.
Daclizumab is produced from a GS-NS0 cell line containing the heavy
and light chain genes by fed- batch fermentation culture.
Bioreactor harvests are processed to remove cells and debris and
purified using a combination of ion-exchange and gel filtration
chromatography and a series of ultrafiltration and filtration
techniques to produce drug substance containing greater than 95%
monomeric species.
[0053] IV. Preparation of the Formulation
[0054] After the antibody of interest is prepared as described
above, the pharmaceutical formulation comprising the antibody is
prepared. The formulation development approach is as follows:
selecting the optimum solution pH, selecting buffer type and
concentration, evaluating the effect of various excipients of the
liquid and lyophilized stability, and optimizing the concentration
of the screened excipients using an I-optimal experimental design
(Statistics for Experimental, Box, George E. P. John Wiley and
Sons, Inc., 1978).
[0055] The following criteria are important in developing stable
lyophilized protein products. Protein unfolding during
lyophilization should be minimized. Various degradation pathways
should be minimized. Glass transition temperature (Tg) should be
greater than the product storage temperature. Residual moisture
should be low (<1% by mass). A strong and elegant cake structure
should be obtained. A preferred shelf life should be at least 3
months, preferably 6 months, more preferably 1 year at room
temperature (22 to 28.degree. C.). A reconstitution time should be
short, for example, less than 5 minutes, preferably less than 2
minutes, and more preferably less than 1 minute. When the
lyophilized product is reconstituted, the reconstituted sample
should be stable for at least 48 hours at 2-8.degree. C.
[0056] The compositions of this invention minimize the formation of
protein aggregates and particulates in reagents containing
immunoglobulin antibodies and insure that the antibody in solution
maintains its immunoreactivity over time. The composition comprises
a sterile, pharmaceutically acceptable lyophilized formulation
prepared from an aqueous pre-lyophilized formulation comprising an
antibody in a buffer having a neutral or acidic pH (pH 5.5-6.5), a
surfactant, and a polyol. The preferred composition additionally
contains a bulking agent, and/or a tonicity modifier.
[0057] The antibody in the pre-lyophilized formulation has a high
concentration of 50 mg/ml or greater. A preferred antibody is an
IgG antibody, preferably a monoclonal IgG antibody.
[0058] A buffer of pH 5.5-6.5 is used in the composition. Examples
of buffers that control the pH in this range include succinate
(such as sodium succinate), gluconate, histidine, citrate and other
organic acid buffers. Histidine is a preferred buffer for
subcutaneous, intramuscular and peritoneal injection. Sodium
succinate buffer is less preferred because it does not have a good
buffer capacity at low strength. To increase the buffer strength of
sodium succinate, the amount of the excipients will have to be
decreased in order to maintain the osmolarity in a desired range.
If the lyophile is to be reconstituted with half of the fill
volume, then the desired osmolarity of the pre-lyophilized (fill)
liquid is about 140-160 mOsm. The advantage of histidine buffer is
that 1 mmole of the histidine buffer only contributes 1 mOsm,
whereas 1 mmole of the sodium succinate buffer contributes 3 mOsm.
Because histidine buffer contributes less to the osmolarity, it
allows more stabilizing excipients to be added to the formulation.
Citrate buffer is also less preferred because it causes a painful
reaction when injected subcutaneously. A preferred buffer contains
about 5-25 mM histidine. A more preferred buffer contains about
10-20 mM histidine.
[0059] A surfactant is added to the antibody formulation. Exemplary
surfactants include nonionic surfactants such as polysorbates (e.g.
polysorbates 20, 80, such as Tween.RTM.20, Tween.RTM.80) or
poloxamers (e.g. poloxamer 188). The amount of surfactant added is
such that it reduces aggregation of the formulated antibody and/or
minimizes the formation of particulates in the formulation and/or
reduces protein adsorption onto the container. The surfactant also
reduces the reconstitution time of the lyophilized formulation. For
example, the surfactant is present in the formulation in an amount
from about 0.001% to about 0.5%, preferably from about 0.005% to
about 0.1% and most preferably from about 0.01% to about 0.05%.
[0060] A polyol, which acts as a tonicifying agent and a
cryoprotector/lyoprotector, is included in the formulation. In a
preferred embodiment, the polyol is a nonreducing sugar, such as
sucrose or trehalose. In the present invention, the polyol such as
sucrose is the primary stabilizer against antibody aggregation, and
it also plays an important role in reducing the reconstitution time
of the lyophilized formulation to a particle-free solution. The
polyol is added to the formulation in an amount that may vary with
respect to the desired tonicity of the formulation. Preferably the
lyophilized formulation after reconstitution is isotonic; however,
hypertonic or hypotonic formulations may also be suitable. Suitable
concentrations of the polyol such as sucrose in the pre-lyophilized
formulation are in the range from about 100-300 mM, preferably in
the range from about 100-200 mM.
[0061] A bulking agent that provides good lyophilized cake
properties, such as serine, glycine, mannitol, can be optionally
added to the present composition. These agents also contribute to
the tonicity of the formulations and may provide protection to the
freeze-thaw process and improve long-term stability. A preferred
bulking agent is serine at a concentration about 15-55 mM, and
preferably about 20-30 mM. Another preferred bulking agent is
mannitol, at a concentration about 10-55 mM, and preferably about
20-45 mM. The addition of serine or mannitol to the pre-lyophilized
formulation reduces the concentration of polyol required for
stabilizing the antibody, for example, to 30-180 mM and preferably
80-130 mM.
[0062] Tonicity modifiers such as salts (e.g., NaCl, KCl,
MgCl.sub.2, CaCl.sub.2) can be added to the formulation to control
osmotic pressure.
[0063] Exemplary pre-lyophilized compositions are formulations
comprising an IgG antibody at about 50 mg/ml or greater, about
10-20 mM histidine (pH 5.5-6.5), about 0.005-0.03 % polysorbate 20
or 80, and one of the following combinations of excipients: (a)
100-200 mM sucrose, (b) 110-130 mM sucrose and 20-45 mM mannitol,
(c) 100-130 mM sucrose and 15-55 mM serine, and (d) 7-55 mM serine,
80-130 mM sucrose, and 10-55 mM mannitol. The above pre-lyophilized
formulation is lyophilized to form a dry, stable powder, which can
be easily reconstituted to a particle-free solution suitable for
administering to humans.
[0064] Lyophilization is a freeze drying process that is often used
in the preparation of pharmaceutical products to preserve their
biological activity. The liquid composition is prepared, then
lyophilized to form a dry cake-like product. The process generally
involves drying a previously frozen sample in a vacuum to remove
the ice, leaving the non-water components intact, in the form of a
powdery or cake-like substance. The lyophilized product can be
stored for prolonged periods of time, and at elevated temperatures,
without loss of biological activity, and can be readily
reconstituted into a particle-free solution by the addition of an
appropriate diluent. An appropriate diluent can be any liquid which
is biologically acceptable and in which the lyophilized powder is
completely soluble. Water, particularly sterile, pyrogen-free
water, is a preferred diluent, since it does not include salts or
other compounds which may affect the stability of the antibody. The
advantage of lyophilization is that the water content is reduced to
a level that greatly reduce the various molecular events which lead
to instability of the product upon long-term storage. The
lyophilized product is also more readily able to withstand the
physical stresses of shipping. The reconstituted product is
particle free, thus it can be administered without prior
filtration.
[0065] The liquid formulation can be lyophilized using appropriate
drying parameters. The following drying parameters are preferred: a
primary drying phase temperature of about -20.degree. C. to
-50.degree. C. and pressure between about 80 mTorr to about 120
mTorr; and a secondary drying phase at ambient temperature, and
pressure between about 80 mTorr to 120 mTorr.
[0066] This lyophilized product retains the stability of
immunological activity of the monoclonal antibody, and prevents the
immunoglobulins intended for administration to human subjects from
physical and chemical degradation in the final product.
[0067] The lyophilized product is rehydrated at the time of use in
a diluent (e.g., sterile water or saline) to yield a particle-free
solution. The reconstituted antibody solution is particle-free even
after prolonged storage of the lyophilized cake at ambient
temperature. The reconstituted solution is administered
parenterally, preferably intravenously or subcutaneously, to the
subject.
[0068] An important characteristic of the lyophilized product is
the reconstitution time or the time taken to rehydrate the product.
To enable very fast and complete rehydration, it is important to
have a cake with a highly porous structure. The cake structure is a
function of a number of parameters including the protein
concentration, excipient type and concentration, and the process
parameters of the lyophilization cycle. Generally the
reconstitution time increases as the protein concentration
increases, and thus, a short reconstitution time is an important
goal in the development of high concentration lyophilized antibody
formulations. A long reconstitution time can deteriorate the
product quality due to the longer exposure of the protein to a more
concentrated solution. In addition, at the user end, the product
cannot be administered until the product is completely rehydrated.
This is to ensure that the product is particulate-free, the correct
dosage is administered, and its sterility is unaffected. Thus,
quick rehydration offers more convenience to the patients and the
physicians.
[0069] In lyophilized products, the desired dosage can be obtained
by lyophilizing the formulation at the target protein concentration
and reconstituting the product with the same volume as that of the
starting fill volume. The desired dosage can also be obtained by
lyophilizing a larger volume of a diluted formulation, and
reconstituting it with a less volume. For example, if a desired
product dosage is 100 mg of protein in 1 mL of the formulation, the
formulations can be lyophilized with the following liquid
configurations: 1 mL of 100 mg/mL, 2 mL of 50 mg/ml, or 4 mL of 25
mg/mL protein formulation. In all cases, the final product can be
reconstituted with 1 mL diluent to obtain the target protein
concentration of 100 mg/mL. However, as the protein concentration
in the pre-lyophilized formulation is reduced, the fill volume
increases proportionately. This correspondingly increases the
length of the lyophilization cycle (especially the primary drying
time), and thus significantly adds to the cost of the product. For
example, if 1 mL fill volume (1 mm height in vial) of frozen
material takes approximately 1 hour to sublimate its free water,
then 10 mL fill volume (10 mm height) of frozen product will take
approximately 10 hours of primary drying time. Therefore, it is
advantageous to have a concentrated pre-lyophilized formulation
(with antibody greater than 50 mg/mL) such that the lyophilization
process will be more efficient.
[0070] The present invention provides a highly concentrated
pre-lyophilized antibody formulation (greater than 50 mg/mL), which
is lyophilized efficiently and effectively to a dry formulation
that retains the biological, physical and chemical stability of the
antibody. The dry formulation is stable for storage at least for 3
months, preferably 6 months, at room temperature. The dry
formulation can be reconstituted within a short time of less than 2
minutes to a particle-free solution containing greater than 50
mg/mL antibody. Such highly concentrated antibody solution is ready
for parenteral administration such as intravenous, intramuscular,
intraperitoneal, or subcutaneous injection.
[0071] The invention is illustrated further by the following
examples, which are not to be construed as limiting the invention
in scope of the specific procedures described in them.
EXAMPLES
Example 1
Procedures of Lyophilization
[0072] Vial configuration: 2 mL fill in 5 mL Wheaton vial;
[0073] Lyophilization Cycle:
[0074] 1. Freezing:
[0075] Temp: -40.degree. C.
[0076] Rate: 2.degree. C./min
[0077] Freezing time: 3 hrs
[0078] 2. Primary drying:
[0079] Temp: -20.degree. C.
[0080] Rate: 1.degree. C./min
[0081] Duration: 12 hrs
[0082] Pressure: 150 mTorr
[0083] 3. Secondary drying:
[0084] Temp:20.degree. C.
[0085] Rate: 1.degree. C./min
[0086] Duration: 10 hrs
[0087] Pressure: 150 mTorr
Example 2
Preliminary Screening of Excipients
[0088] In this experiment, the formulation matrix contains 10 mg/ml
anti-IL2 receptor antibody, 10 mM histidine, pH 6.0, and 0.015%
Tween.RTM.80. The excipients screened includes (a)
cryoprotectors/lyoprotectors such as sucrose, trehalose,
polyethylene glycol (PEG), and polyvinylpyrrolidone (PVP); (b)
bulking agents and tonicity modifiers such as mannitol, glycine,
and serine; and (c) Tg enhancers such as dextran.
[0089] Two mL of each formulation was filled into a vial, and
lyophilized by a conservative lyophilization cycle according to
Example 1. Each lyophile was reconstituted with 2 mL of sterile
water.
[0090] The accelerated stability test of each liquid formulation
and each lyophile was performed at 55 or 40.degree. C. The amounts
of soluble aggregates was determined by SEC. The % monomer drop of
different excipients was shown in FIG. 1.
[0091] The results indicated that PEG, dextran, and glycine
decreased the pre-lyophilization liquid stability. The effect of
all other excipients was comparable to the control formulation with
no excipients.
[0092] The results also indicated PVP, dextran, and glycine caused
significant protein aggregation in the lyophilized formulation.
Relative to the control formulation, sucrose, mannitol and serine
each stabilized the formulation against aggregation.
Example 3
Optimization of Sucrose, Mannitol and Serine Concentration
[0093] The effect of sucrose, mannitol and serine on the protein
stability was investigated using an I-Optimal (Hardin-Sloane)
experimental design approach.
[0094] In this experiment, the formulation matrix contains 50 mg/ml
anti-IL2 receptor antibody, 10 mM histidine, pH 6.0, and 0.015%
Tween.RTM.80. The excipients screened include serine (0-100 mM),
sucrose (0-120 mM) and mannitol (0-170 mM). The I-Optimal Design is
shown as Table 1. Samples 1-15 are test formulations, and samples
16-20 are control formulations.
[0095] Two mL of each sample and control formulation was filled
into a vial, and lyophilized according to the procedures described
in Example 2. Each lyophile was reconstituted with 1 mL of sterile
water. As the lyophile is reconstituted with only half the solution
volume, osmolality and protein concentration are doubled.
1TABLE 1 I-Optimal Design Table Sample Serine Sucrose Mannitol 1
0.029 -0.029 -0.080 2 0.236 1.000 1.000 3 1.000 0.196 0.333 4 1.000
-1.000 1.000 5 -0.044 0.044 -1.000 6 -1.000 1.000 0.167 7 1.000
1.000 -0.883 8 -1.000 -1.000 -0.883 9 -0.196 -1.000 0.333 10 1.000
-1.000 -1.000 11 -1.000 -0.236 1.000 12 -1.000 1.000 -1.000 13
0.028 -0.028 -0.080 14 1.000 -1.000 1.000 15 -1.000 1.000 -1.000 16
-1.000 0.500 -0.333 17 0.200 0.500 -1.000 18 -0.400 0.000 -0.333 19
0.200 -0.500 -0.333 20 -1.000 -1.000 -1.000
[0096] The lyophile product stability test was performed at
37.degree. C. for 4 weeks. The formulations were distinguishable.
The analytic methods of SEC and reconstitution time show
statistically significant responses among the formulations.
[0097] FIG. 2 shows the comparison of model predictions and
experimental observation for the test samples. FIG. 3 shows the
comparison of model predictions and experimental observation for
the controls. FIG. 4 shows the model coefficients of main effect
and interaction effect.
[0098] FIG. 5 shows the representative model simulations of
mannitol vs. sucrose, [serine]=0. The results indicate that sucrose
and mannitol stabilize the protein against aggregation
(sucrose>mannitol). Sucrose has a favorable effect in reducing
the reconstitution time. Using high concentrations of sucrose and
mannitol, formulation stability is enhanced and cakes with short
reconstitution time are obtained.
[0099] FIG. 6 shows the representative model simulations of
mannitol vs. serine, [sucrose]=0. The results indicate that serine
and mannitol stabilize the protein against aggregation
(mannitol>serine). At high concentrations, serine increases the
reconstitution time. Formulation stability is enhanced using high
concentrations of serine and mannitol, and their combination
significantly reduces the reconstitution time.
[0100] FIG. 7 shows the representative model simulations of sucrose
vs. serine, [mannitol]=0. The results indicate that combinations of
sucrose and serine can effectively stabilize the formulation
against aggregation and form cakes with a short reconstitution
time.
[0101] FIG. 8 shows the representative model simulations of
mannitol vs. serine, [sucrose]=100 mM. Model simulations allow
selection of conditions that provide isotonic formulations. Data
supports maximizing the sucrose concentration. At 100 mM sucrose,
the model provides conditions for optimizing serine and/or
mannitol.
[0102] The conclusions for this experiment are as follows.
[0103] High concentrations of sucrose and mannitol provided maximum
stability to the lyophilized formulation. However, osmolarity
constraints precluded the addition of high concentrations of both
excipients.
[0104] Sucrose had the strongest stabilizing effect on the lyophile
stability, it also provides a cake with a short reconstitution
time.
Example 4
Stability Data of Two Formulations
[0105] Formulations 1 and 2 were prepared and lyophilized according
to procedures similar to those described in Example 1. The
lyophilized formulation was incubated at 37.degree. C. for 2.5
months. The lyophilized formulation at different time point was
reconstituted with 1 mL water and tested by analytical methods.
[0106] Formulation 1 (prior lyophilization): 50 mg/mL anti IL.sub.2
receptor antibody in 10 mM histidine buffer and 0.015%
Tween.RTM.80, pH=6, 25 mM Serine, 4% sucrose (117 mM), 0.25%
Mannitol (13.7 mM)
2 Recon time % % Moisture pH (Seconds) Monomer Aggregates (% w/w)
Pre-lyo 6.1 N/A 98.1 1.1 N/A T0 6.1 40 98.2 0.9 0.75 T = 2.5 mo 6.1
36 97 1.9 --
[0107] Formulation 2 (prior lyophilization): 50 mg/mL drug in 10 mM
histidine buffer and 0.015% Tween.RTM.80, pH=6, 4% Sucrose (117 mM)
and 0.5% Mannitol (27.4 mM)
3 Recon time % % Moisture pH (Seconds) Monomer Aggregates (% w/w)
Pre-lyo 6.1 N/A 98.1 1.1 N/A T0 6.0 32 98.2 1.0 0.8 T = 2.5 months
6.1 39 96.7 1.8 -- *The concentration of all excipients doubles
after reconstitution of the cake with 1 mL water (half of the fill
volume) for injection.
[0108] Pre-lyo: prior the lyophilization process
[0109] T0: The formulation has been lyophilized and immediately
after this reconstituted with 1 mL water for injection
[0110] T=2.5 months: The formulation was lyophilized; the cake was
incubated at 37.degree. C. for two and half months, then
reconstituted with 1 mL water for injection (WFI).
Example 5
Long-term Stability Study of Three Lyophilized Daclizumab
Formulations
[0111] Study Description
[0112] Long-term stability of the following three formulations are
tested at 5, 25 and 40.degree. C. The stability of these samples is
monitored over 24 months at T.sub.o, 1 month, 3 months, 6 months,
12 months, and 24 months. The lyophilized formulation at different
time point is reconstituted with water for injection (WFI) and
tested by analytical methods.
[0113] 1. Formulation I (FORM-I): 50 mg/mL anti-IL2 receptor
antibody, 20 mM Histidine, 4% (117 mM) sucrose, 0.015% Tween.RTM.
80, pH 6.0. Vial configuration: 2 mL fill in 2 mL vial.
Reconstituted with 1 mL WFI. Protein concentration
post-reconstitution=100 mg/mL.
[0114] 2. Formulation II (FORM-II): 80 mg/mL protein, 20 mM
Histidine, 6.5% (190 mM) sucrose, 0.025% Tween 80, pH 6.0. Vial
configuration: 1.25 mL fill in 2 mL vial, Reconstituted with 1 mL
WFI. Protein concentration post-reconstitution=100 mg/mL.
[0115] 3. Formulation III (FORM-III): 80 mg/mL protein, 20 mM
Histidine, 4% sucrose, 0.015% Tween 80, pH 6.0. Vial configuration:
2 mL fill in 2 mL vial. Reconstituted with 1 mL WFI. Protein
concentration post-reconstitution=160 mg/mL.
[0116] Summary of Results
[0117] FIG. 9 shows the % monomer measured by size exclusion
chromatography for the three formulations as a function of time and
temperature. At 5.degree. C., over the 3 month duration, no
significant change is observed in the monomer content of the three
formulations. At 25 and 40.degree. C., over the 3 month duration,
less than 3% drop in the monomer content is observed for all three
formulations compared with To.
[0118] FIG. 10 shows the % aggregates measured by size exclusion
chromatography for the three formulations as a function of time and
temperature. No significant increase in aggregation is observed at
5.degree. C. At 25.degree. C. for 3 months, the increase in
aggregation for FORM-I and FORM-II is <1% and .about.2% for
FORM-III. At 40.degree. C. for 3 months, the increase in
aggregation for FORM-I and FORM-II is about 2.5% and <4% for
FORM-III.
[0119] FIG. 11 shows the % clips measured by size exclusion
chromatography for the three formulations as a function of time and
temperature. Minimal changes in the % clips are observed for all
three formulations over the three month duration at all
temperatures. Thus, hydrolysis, which was a significant stability
concern in the aqueous state, has been successfully curtailed in
the lyophilized formulation.
[0120] In addition, the protein secondary structure in the
lyophilized and the reconstituted formulations at the initial time
point (T=0) and after 3 months at 40.degree. C. is measured by
Fourier Transform Infrared Spectroscopy (FTIR). For all
formulations, no significant changes are observed in the secondary
structure with time. Furthermore, the protein structure in the
lyophilized and the reconstituted formulation appear unchanged.
Generally, changes in the secondary structure between the
lyophilized and the reconstituted liquid states, correlate with
protein aggregation upon long-term storage.
[0121] The isoform profile of the three formulations at T=0 and T=3
months as a function of temperature is compared by cIEF analysis.
For all formulations, minimal changes in the isoform profile are
observed over the three month duration. Furthermore, no changes are
seen as a function of temperature. Generally, isoform changes
result due to chemical degradation processes such as deamidation
and hydrolysis. This data is thus indicative of the chemical
stability of the monitored formulations at temperatures as high as
40.degree. C.
[0122] Table 2 lists the potency of the three formulations at T=0
and at T=3 months measured by an ELISA based binding assay. The
bioactivity of the samples is preserved during the lyophilization
cycle, and is unchanged upon storage for 3 months at temperatures
as high as 40.degree. C.
4TABLE 2 3 3 3 T.sub.0 Pre- T.sub.0 Months Months Months Sample
lyophilization Reconstituted 5.degree. C. 25.degree. C. 40.degree.
C. FORM-I 99 89 84 76 80 FORM-II 99 94 81 83 77 FORM-III 95 96 78
78 80
[0123] The invention, and the manner and process of making and
using it, are now described in such full, clear, concise and exact
terms as to enable any person skilled in the art to which it
pertains, to make and use the same. It is to be understood that the
foregoing describes preferred embodiments of the present invention
and that modifications may be made therein without departing from
the scope of the present invention as set forth in the claims. To
particularly point out and distinctly claim the subject matter
regarded as invention, the following claims conclude this
specification.
* * * * *