U.S. patent application number 10/291528 was filed with the patent office on 2003-07-24 for stable liquid pharmaceutical formulation of igg antibodies.
Invention is credited to Duvur, Shanti G., Gupta, Supriya, Kaisheva, Elizabet A., Subramanian, Malathy.
Application Number | 20030138417 10/291528 |
Document ID | / |
Family ID | 23320826 |
Filed Date | 2003-07-24 |
United States Patent
Application |
20030138417 |
Kind Code |
A1 |
Kaisheva, Elizabet A. ; et
al. |
July 24, 2003 |
Stable liquid pharmaceutical formulation of IgG antibodies
Abstract
This invention is directed to a stable liquid pharmaceutical
formulation comprising a high concentration, e.g. 50 mg/ml or more,
of antibody in about 20-60 mM succinate buffer or 30-70 mM
histidine buffer, having pH from about pH 5.5 to about pH 6.5,
about 0.01-0.1% polysorbate, and a tonicity modifier that
contributes to the isotonicity of the formulation. This liquid
formulation is stable at refrigerated temperature (2-8.degree. C.)
for at least 1 year, and preferably 2 years. This liquid
formulation is suitable for subcutaneous injection. The preferred
antibodies include Daclizumab, a humanized anti-IL-2 receptor
monoclonal antibody; HAIL-12, a humanized anti-IL-12 monoclonal
antibody; HuEP5C7, a humanized anti-L selectin monoclonal antibody;
and Flintozumab, a humanized anti-gamma interferon monoclonal
antibody.
Inventors: |
Kaisheva, Elizabet A.;
(Belmont, CA) ; Gupta, Supriya; (Sunnyvale,
CA) ; Duvur, Shanti G.; (Fremont, CA) ;
Subramanian, Malathy; (Fremont, CA) |
Correspondence
Address: |
HOWREY SIMON ARNOLD & WHITE, LLP
BOX 34
301 RAVENSWOOD AVE.
MENLO PARK
CA
94025
US
|
Family ID: |
23320826 |
Appl. No.: |
10/291528 |
Filed: |
November 8, 2002 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
60337509 |
Nov 8, 2001 |
|
|
|
Current U.S.
Class: |
424/130.1 |
Current CPC
Class: |
C07K 16/249 20130101;
A61K 47/02 20130101; A61K 47/26 20130101; A61K 39/39591 20130101;
A61K 47/12 20130101; C07K 16/246 20130101; A61P 37/06 20180101;
C07K 16/2854 20130101; C07K 2317/24 20130101; A61K 2039/505
20130101; C07K 16/244 20130101; A61K 9/0019 20130101 |
Class at
Publication: |
424/130.1 |
International
Class: |
A61K 039/395 |
Claims
What is claimed is:
1. A stable liquid pharmaceutical formulation comprising: about
20-60 mM succinate buffer having pH from about pH 5.5 to about pH
6.5, about 0.01%-0.1% polysorbate, a tonicity modifier that
contributes to isotonicity of the formulation, and greater than 50
mg/ml antibody.
2. A stable liquid pharmaceutical formulation comprising: about
30-70 mM histidine buffer having pH from about pH 5.5 to about pH
6.5, about 0.01%-0.1% polysorbate, a tonicity modifier that
contributes to isotonicity of the formulation, and greater than 50
mg/ml antibody.
3. The liquid pharmaceutical formulation according to claim 1 or 2,
wherein said antibody concentration is greater than 100 mg/ml.
4. The stable liquid pharmaceutical formulation according to claim
1 or 2, wherein said tonicity modifier is NaCl or MgCl.sub.2.
5. The stable liquid pharmaceutical formulation according to claim
4, wherein said NaCl is at 75-150 mM.
6. The stable liquid pharmaceutical formulation according to claim
4, wherein said MgCl.sub.2 is at 1-100 mM.
7. The stable liquid pharmaceutical formulation according to claim
1 or 2, wherein said pH is about pH 6.0 to 6.5.
8. The stable liquid pharmaceutical formulation according to claim
1 or 2, wherein said polysorbate is at a concentration of about
0.02-0.04%.
9. The stable liquid pharmaceutical formulation according to claim
1 or 2, further comprising 0.01 to 0.5% EDTA.
10. The stable liquid pharmaceutical formulation according to claim
1 or 2, wherein said formulation is stable at about 2-8.degree. C.
for at least one year.
11. The stable liquid pharmaceutical formulation according to claim
1 or 2, wherein said formulation is stable at about 23-27.degree.
C. for at least 6 months.
12. A liquid pharmaceutical formulation comprising: about 20-60 mM
succinate buffer having pH of from about pH 5.5 to about pH 6.5,
about 0.01%-0.05% polysorbate, about 75-150 mM sodium chloride, and
greater than 50 mg/ml antibody selected from the group consisting
of Daclizumab, Flintozumab, HAIL-12, and HuEP5C7.
13. A liquid pharmaceutical formulation comprising: about 30-70 mM
histidine buffer having pH of from about pH 5.5 to about pH 6.5,
about 0.01%-0.05% polysorbate, about 75-150 mM sodium chloride, and
greater than 50 mg/ml antibody selected from the group consisting
of Daclizumab, Flintozumab, HAIL-12, and HuEP5C7.
Description
[0001] This application claims the benefit of U.S. Provisional
Application No. 60/337,509 filed Nov. 8, 2001.
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, liquid, high concentration antibody
formulation. This invention is exemplified by a stabilized liquid
formulation of Daclizumab, an anti-IL2 receptor antibody; HAIL-12,
a humanized anti-IL-12 monoclonal antibody; and HuEP5C7, a
humanized anti-L selectin monoclonal antibody.
BACKGROUND OF THE INVENTION
[0003] Many protein preparations intended for human use require
stabilizers to prevent denaturation, aggregation and other
alternations to the proteins prior to the use of the preparation.
This instability is manifested in the formation of
soluble/insoluble particles, and is often increased when the
protein preparation is stored over time and during shipping. A
major aim in the development of protein drug formulations is to
maintain both protein solubility, stability and bioactivity.
[0004] Immunoglobulins, in particular, are recognized as possessing
characteristics that tend to form aggregates and particulates in
solution, and as such, may require filtration before use for
intravenous or subcutaneous injection. The formation of protein
aggregates and particulates has long been a problem in the
development of parenteral immunoglobulin products, especially when
the immunoglobulins are formulated at high concentrations.
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 (90%) and murine
(10%) antibody sequences. Synagis.TM. is supplied as a sterile
lyophilized product for reconstruction with sterile water for
injection. 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,
5.6% mannitol, and the active ingredient, IgG1 antibody, at a
concentration of 100 milligrams per vial. The reconstituted
Synagis.TM. is to be administered within 6 hours of
reconstitution.
[0005] WO 89/11297 discloses a lyophilized monoclonal antibody
formulation comprising a lyophilized formulation of 1-25 mg/ml IgG
monoclonal antibody, 2-10% maltose, and sodium acetate, phosphate,
or citrate buffer having a pH between 3.0 to 6.0.
[0006] WO 97/45140 discloses an aqueous preparation of anti-CD4
antibody concentrated to approximately 100 mg/ml in 100 mM sodium
citrate, 0.05 mM EDTA, pH 6.0. The application discloses a slight
rise in turbidity after concentration of the antibody, which likely
reflects protein aggregation. Removing this aggregation requires
addition of Polysorbate 80 and sterile filtration.
[0007] WO 90/11091 discloses injectable aqueous compositions
comprising about 5 mg/ml of IgM, 2.5-5% (w/v) human serum albumin,
in 8-20 mM phosphate buffer, 270 mM sodium chloride, pH
6.8-7.4.
[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, an
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] U.S. Patent Application Publication No. US 2001/0014326A1
discloses a pre-lyophilized antibody formulation containing 25
mg/ml anti-IgE antibody, 5 mM histidine, pH 6.0, 85 mM sucrose, and
0.01% polysorbate 20.
[0010] U.S. Pat. No. 5,744,132 discloses a composition comprising
1-1000 .mu.g/ml IL-12 antibody, 2% sucrose, 4.15% mannitol, 10 mM
sodium succinate, and about 0.02% Tween.RTM. 20, having a pH of
about 5.6.
[0011] U.S. Pat. No. 6,267,958 discloses a reconstituted
formulation of 100 mg/ml rhuMab E25, in 20 mM histidine, pH 6.0,
340 mM sucrose, 0.04% polysorbate 20, and 0.9% benzyl alcohol.
[0012] U.S. Pat. No. 6,165,467 discloses a process for stabilizing
a human monoclonal antibody composition produced by hybridoma cell
line having accession number HB8307, which comprises dialyzing the
human monoclonal antibody in a phosphate salt stabilized buffer
solution having a pH from 7.2 to 7.4, said solution comprising 1-20
mg of D-mannitol per mg of said monoclonal antibody, 0.005-0.2
millimole of glycine per mg of said monoclonal antibody, and an
amount of pH stabilizing phosphate salt to stabilize the pH of said
solution.
[0013] There is a need for a stable liquid antibody preparation,
wherein the antibody concentration is 50 mg/ml or greater; such
preparation is suitable for parenteral administration, including
intravenous, intramuscular, intraperitoneal, or subcutaneous
injection to a human.
SUMMARY OF THE INVENTION
[0014] This invention is directed to a stable liquid pharmaceutical
formulation comprising a high concentration, e.g., greater than 50
mg/ml, of an antibody in 20-60 mM succinate buffer or 30-70 mM
histidine buffer (pH from about pH 5.5 to about pH 6.5), a tonicity
modifier, and about 0.01-0.1% polysorbate. This formulation retains
the physical, chemical, and biological stability of antibody and
prevents the immunoglobulins intended for administration to human
subjects from forming aggregates and particulates in the final
product. Preferred antibodies of this invention include Daclizumab,
a humanized anti-1L-2 receptor monoclonal antibody; HAIL-12, a
humanized anti-IL-12 monoclonal antibody; and HuEP5C7, a humanized
anti-L selectin monoclonal antibody; and Flintozumab, a humanized
anti-gamma interferon monoclonal antibody.
[0015] The liquid antibody formulation is stable at refrigerated
temperature (2-8.degree. C.) for at least 1 year and preferably 2
years. This liquid formulation is also stable at room temperature
(23-27.degree. C.) for at least six months. This liquid formulation
is suitable for subcutaneous injection.
BRIEF DESCRIPTION OF THE FIGURES
[0016] FIG. 1A shows the percent clips formation, and FIG. 1B shows
the percent aggregates, at various pH levels following a four-week
incubation of the sample at 45.degree. C., as assessed by
SEC-HPLC.
[0017] FIG. 2 shows the percent of degradation obtained at various
pH levels as assessed by cIEF following a four-week incubation of
the sample at 45.degree. C.
[0018] FIG. 3 shows the percent of iso-aspartic acid formed at
various pH levels as assessed by the Promega IsoQuant kit following
a four-week incubation of the sample at 45.degree. C.
[0019] FIG. 4 shows the effect of different buffers over time on
potency following incubation at 37.degree. C.
DETAILED DESCRIPTION OF THE INVENTION
[0020] I. Definition
[0021] As used herein, the term "buffer" encompasses those agents
which maintain the solution pH in an acceptable range and may
include succinate (sodium), histidine, phosphate (sodium or
potassium), Tris (tris (hydroxymethyl) aminomethane),
diethanolamine, 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 phospate and other organic acid buffers.
[0022] "Pharmaceutically acceptable excipients" (vehicles,
additives) are those inert substances that can reasonably be
administered to a subject mammal and provide an effective dose of
the active ingredient employed. These substances are added to a
formulation to stabilize the physical, chemical and biological
structure of the antibody. The term also refers to additives that
may be needed to attain an isotonic formulation, suitable for the
intended mode of administration.
[0023] The term "pharmaceutical formulation" refers to preparations
which are in such form as to permit the biological activity of the
active ingredients to be unequivocally effective, and which contain
no additional components which are toxic to the subjects to which
the formulation would be administered.
[0024] A "stable" formulation is one in which the protein therein
essentially retains its physical stability, chemical stability, and
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.
[0025] A "stable" liquid antibody formulation is a liquid 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 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 (hydrolysis) is observed.
No more than 10%, preferably 5% of aggregation is formed.
[0026] 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-HPLC) and dynamic
light scattering. In addition the protein conformation is not
altered. 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.
[0027] 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.).
[0028] 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 ELISA
assay.
[0029] 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 osmotic pressure is 250-269 and
slightly hypertonic osmotic pressure is 328-350 mOsm. Osmotic
pressure can be measured, for example, using a vapor pressure or
ice-freezing type osmometer.
[0030] "Tonicity modifiers" are those pharmaceutically acceptable
inert substances that can be added to the formulation to provide an
isotonity of the formulation. Tonicity modifiers suitable for this
invention include salts and amino acids.
[0031] II. Analytical Methods
[0032] The following criteria are important in developing a stable
pharmaceutical antibody formulation. The antibody formulation
contains pharmaceutically acceptable excipients. The antibody
formulation is formulated such that the antibody retains its
physical, chemical and biological activity. The formulation is
preferably stable for at least 1 year at refrigerated temperature
(2-8.degree. C.) and 6 months at room temperature (23-27.degree.
C.).
[0033] The analytical methods for evaluating the product stability
include size exclusion chromatography (SEC-HPLC), dynamic light
scattering test (DLS), differential scanning calorimetery (DSC),
iso-asp quantification, potency, UV at 340 nm, and UV spectroscopy.
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 and clips.
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.
[0034] The iso-Asp content in the samples is measured using the
Isoquant Isoaspartate Detection kit (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.
[0035] The potency or bioactivity 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).
[0036] III. Preparation of Antibody
[0037] The invention herein relates to a stable aqueous formulation
comprising an antibody. The antibody in the formulation is prepared
using techniques available in the art for generating antibodies,
exemplary methods of which are described in more detail in the
following sections.
[0038] 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.
[0039] 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-P;
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-12; receptors to interleukins
IL-1 to IL-12; selectins such as L, E, and P-selectin; 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; integrins 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.
[0040] 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.
[0041] 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 .UPSILON..sub.1, .UPSILON..sub.2, or
.UPSILON..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 .UPSILON..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 precipitation are also available depending on the antibody
to be recovered.
[0042] Preferred antibodies encompassed by the present invention
include Daclizumab (USAN, United States Adopted Names), a humanized
anti-IL-2 receptor antibody. Daclizumab is currently being marketed
as Zenapax.RTM. for the prevention of organ rejection after renal
transplantation and is administered through the intravenous route.
Daclizumab is also useful for treating psoriasis, for which, the
subcutaneous delivery is the preferred route of administration. For
a subcutaneous delivery of antibody, high concentration of antibody
is preferred. 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-NSO 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.
[0043] Another preferred antibody is anti-interleukin 12 (IL-12)
antibody. IL-12 is a cytokine synthesized by antigen presenting
cells. It is composed of two subunits (p35 and p40), both must be
present for functional activity. Functional IL-12 is also called
IL-12p70. This cytokine preferentially acts on T helper cell type 1
(Th1) lymphocytes and natural killer cells by increasing their
proliferative rate. One downstream effect is the secretion of
interferon gamma (IFNg) by Th1 cells. Both of these functions
(proliferative and IFNg production) can be easily assayed for and
were used to detect IL-12 activity in samples. Certain antibodies
to IL-12 have been shown to "neutralize" the above activities.
Since Th1 cells have been implicated in playing a pivotal role in a
variety of diseases, an antibody with neutralizing characteristics
would have potential therapeutic value. 16G2 (Hoffman La Roche) is
a murine antibody raised against IL-12p70. 16G2 has been shown to
act in near stoichiometric amounts to IL-12 in a functional
assay-the inhibition of proliferation of activated T cells from
human peripheral blood (PBMC). This is an important characteristic
because p40 dimers of IL-12 exist in serum and antibodies raised to
the p40 subunit need to be used in excess amounts to neutralize the
proliferative capacity of a given amount of IL-12. 16G2 was
humanized at Protein Design Labs. (Fremont, Calif.) to give rise to
HAIL-12 (humanized anti-IL-12, an IgG1 antibody).
[0044] Another preferred antibody is anti-L selectin antibody.
Selectins, such as L, E, and P-selectin have been found to be
associated with tissue damage during the course of ischemia and
reperfusion. Neutrophils play an important role in this connection.
It is assumed that selectin is required for the recruitment of
neutrophils. L-selectin is important for the complete development
of damage in skeletal muscle as well as in the lung (Seekamp, et
al., Am. J. Pathol. 11:592-598 (1994). Mulligan, et al., J,
Immunol. 151:832-840 (1994). HuEP5C7 (SMART Anti-L Selectin) is a
humanized anti-L selectin monoclonal antibody, that contains mutant
IgG2 Fc, cross reacts with both human E and P selectin antigens. It
is currently being developed by Protein Design Labs, Inc. for
various indications such as asthma, stroke, trauma, and certain
autoimmune diseases.
[0045] Another preferred antibody is Flintozumab, an anti-gamma
interferon antibody. Flintozumab is an IgG1 humanized monoclonal
antibody developed by Protein Design Labs, Inc. for the treatment
of immune disorders mediated by interferon-gamma (IFN-g), a
proinflammatory cytokine. IFN-g induces the expression of major
histocompatibility complex (MHC) class I and/or class II (HLA-DR)
antigens, enhances the cytolytic activity of natural killer cells,
activates macrophages, and modulates the immunoglobulin isotype
profile of the humoral response. As a lymphokine, IFN-g also
enhances the development of T helper cell type 1 (Th1), while
suppressing the development of T helper cell type 2 (Th2) cells.
Aberrations in the Th1/Th2 ratio have been implicated in a variety
of autoimmune conditions.
[0046] IV. Preparation of the Formulation
[0047] After the antibody of interest is prepared as described
above, a 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 stability, and optimizing the concentration of the screened
excipients using an I-optimal experimental design (Statistics for
Experimenters: An Introduction to Design, Data Analysis, and Model
Building, Box, George E. P. et al., John Wiley and Sons, Inc.,
1978).
[0048] The compositions of this invention minimize the formation of
antibody aggregates and particulates and insure that the antibody
maintains its bioactivity over time. The composition is a
pharmaceutically acceptable liquid formulation containing a high
concentration of an antibody in a buffer having a neutral or
slightly acidic pH (pH 5.5-6.5), a surfactant, and a tonicity
modifier.
[0049] The antibody in the composition is a high concentration of
50 mg/ml or greater, preferably 100 mg/ml or greater. A preferred
composition of this invention contains Daclizumab, a humanized
anti-IL2 receptor antibody; HAIL12, a humanized anti-IL-12
antibody; HaEP5C7, a humanized anti-L selectin antibody; and
Flintozumab, a humanized anti-gamma interferon antibody.
[0050] A buffer of pH 5.5-6.5 is used in the composition. A buffer
of pH 6.0-6.5 is preferred. Examples of buffers that control the pH
in this range include succinate (such as sodium succinate),
gluconate, histidine, citrate, phosphate, and other organic acid
buffers. Succinate (pKa 5.63) is a preferred buffer for
subcutaneous injection. Histidine (PK 5.97) is less preferred
because of its susceptibility to oxidization, although such
oxidation can be retarded by replacing the vial headspace with
N.sub.2 or adding an antioxidant. Citrate and phosphate buffers are
much less preferred because it causes a painful reaction when
injected subcutaneously. A preferred buffer contains about 20-60 mM
sodium succinate. Another preferred buffer is 30-70 mM histidine
buffer overlaid with N.sub.2.
[0051] A surfactant is also 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 adsorption. The surfactant may be
present in the formulation in an amount from about 0.005% to about
0.5%, preferably from about 0.01% to about 0.1%, more preferably
from about 0.01% to about 0.05%, and most preferably from about
0.02% to about 0.04%.
[0052] A tonicity modifier, which contributes to the isotonicity of
the formulations, is added to the present composition. The tonicity
modifier useful for the present invention includes salts and amino
acids. Salts that are pharmaceutically acceptable and suitable for
this invention include sodium chloride, sodium succinate, sodium
sulfate, potassuim chloride, magnesium chloride, magnesium sulfate,
and calcium chloride. Preferred salts for this invention are NaCl
and MgCl.sub.2. MgCl.sub.2 may also improve the antibody stability
by protecting the protein from deamidation. A preferred
concentration of NaCl is about 75-150 mM. A preferred concentration
of MgCl.sub.2 is about 1-100 mM. Amino acids that are
pharmaceutically acceptable and suitable for this invention include
proline, alanine, L-arginine, asparagine, L-aspartic acid, glycine,
serine, lysine, and histidine. A preferred amino acid for this
invention is proline. A preferred concentration of proline is than
200 mM.
[0053] EDTA, which is commonly used to stabilize a protein
formulation, may also be included in the formulation. EDTA, as a
chelating agent, may inhibit the metal-catalyzed oxidation of the
sulfhydryl groups, thus reducing the formation of disulfide-linked
aggregates. A preferred concentration of EDTA is 0.01-0.2%.
[0054] Exemplary liquid compositions are formulations comprising
antibody at about 100 mg/ml or greater, about 20-60 mM sodium
succinate (pH 6), about 0.01-0.1% polysorbate 20 or 80, and about
75-150 mM NaCl. This formulation retains the stability of
biological activity of the monoclonal antibody, and prevents the
immunoglobulins intended for administration to human subjects from
physical, chemical and biological degradation in the final
product.
[0055] The liquid antibody formulation of this invention is
suitable for parenteral administration such as intravenous,
intramuscular, intraperitoneal, or subcutaneous injection;
particularly suitable for subcutaneous injection.
[0056] 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
Optimization of pH
[0057] To identify the optimum formulation for pH range and to
identify major degradation pathways, a pH profile study was
conducted. Sample formulations contained 5.0 mg/ml anti-IL2
receptor antibody (Daclizumab) in one of three buffers: 50 mM
sodium acetate buffer at pH 4.0 or 5.0, 50 mM histidine at pH 5.5,
6.0, or 6.5, or 50 mM sodium phosphate buffer at pH 7.0 or 8. 5.
Independent formulations were incubated at either 5.degree. C. or
45.degree. C. with 100 RPM shaking for 4 weeks. The physical and
chemical stability of each sample was assessed at 0 and 4 weeks by
analytical methods including: pH and visual analysis, UV
spectroscopy at 340 nm, size exclusion chromatography (SEC-HPLC),
fluorescence spectroscopy, dynamic light scattering (DLS),
differential scanning calorimetry (DSC), Promega IsoQuant Assay,
capillary isoelectric focusing (cIEF), SDS-PAGE (reduced and
non-reduced), and bioactivity assessments (ELISA).
[0058] SEC-HPLC performed on samples after four weeks of incubation
at 45.degree. C. showed that clipping is a major degradation
pathway for the liquid formulation, as shown in FIG. 1A by the
percent of clips recovered at various pH levels with SEC. Both the
percent of clips and the percent of aggregates (FIG. 1B) determined
by SEC were reduced at the midrange pH values of 5.5 to 6.5.
[0059] FIG. 2 shows the percent of degradation obtained at various
pH levels as assessed by cIEF following a four-week incubation of
the sample at 45.degree. C. Minimal degradation was obtained at a
pH value of about 5.5.
[0060] FIG. 3 shows the percent of iso-aspartic acid formed at
various pH levels as assessed by the Promega IsoQuant kit following
a four-week incubation of the sample at 45.degree. C. Iso-aspartic
acid formation (deamidation) was minimized at pH values of 6 and
6.5, and increased sharply at pH 8.0.
[0061] The results from this experiment indicate that pH 5.5 to 6.5
and preferable pH 6.0 to 6.5, are the optimal pH which minimize
antibody degradation and aggregation.
Example 2
Optimization of Buffers
[0062] In this experiment, independent formulations contained 5.0
mg/ml Daclizumab antibody in 50 mM sodium succinate, pH 6.0; and 50
mM histidine, pH 6.0, with and without N.sub.2 gassing. Sodium
citrate buffer was not included because of reports of pain on
subcutaneous injection. The bioactivity (potency) at time 0, and
after 4, 8, and 12 weeks of incubation at 37.degree. C. was
measured by ELISA using microplates coated with recombinant human
IL2 alpha receptor (IL-2 sRa) antigen, and goat anti-human IgG-HRP
conjugate.
[0063] FIG. 4 shows the effect of different buffers over time on
potency following incubation at 37.degree. C. Highest stability of
the antibody formulation was achieved through 8 weeks with 50 mM
sodium succinate buffer at pH 6.0. Formulations in histidine alone
rapidly (less than 8 weeks) lost their potency as the buffer
oxidized. Potency of the formulation remained greater than 80% for
at least 12 weeks in either sodium succinate buffer or histidine
buffer gassed with N.sub.2 to prevent oxidation.
Example 3
Screening of Excipients
[0064] Objectives
[0065] This study was conducted to screen various excipients for
the formulation of Daclizumab antibody at 50 mg/mL. From the pH
optimization study conducted earlier (Example 1), the formulation
stability was maximized in the pH range of 6.0-6.5. Therefore in
this study, excipients were screened in two buffers; 50 mM
phosphate, pH 6.5 and 50 mM succinate, pH 6.0. The stability of
antibody was monitored in the two buffers for 3 weeks at 5.degree.
C. and 45.degree. C. with shaking at 100 RPM at a concentration of
50 mg/mL. The excipients examined included: surfactants (Tween
80.RTM. and Tween 20.RTM.), salts (NaCl and MgCl.sub.2),
antioxidants (EDTA and methionine), amino acids (glycine, lysine,
serine and proline), and co-solvents (glycerol and ethanol).
Various analytical techniques (clarity, pH, SEC-HPLC, UV-Vis, and
cIEF) were used to characterize the excipient-containing
formulations.
[0066] Sample Preparation
[0067] The Daclizumab antibody was in a 67 mM sodium phosphate
formulation (without Tween.RTM. 80) at a concentration of 6.6
mg/mL. This material was concentrated to about 30 mg/mL in the
Pellicon II (Millipore) unit, and subsequently, buffer exchanged
into two selected buffers (50 mM sodium phosphate pH 6.5, and 50 mM
sodium succinate pH 6.0) using the 50 mL amicon stir cell
(Millipore). During the third and final buffer exchange step, the
material was also concentrated to a final concentration of 125
mg/mL. Finally, the antibody was filtered through 0.8 .mu.m
membrane (Uniflo). The post filtration protein concentration was
determined to be approximately 100 mg/mL for the phosphate buffer
sample and 97 mg/mL for the succinate buffer sample.
[0068] The target concentration of the excipients at which they
were screened is shown in Table 1. The formulations were prepared
by either weighing the required amount of the excipients directly
into the vial (e.g. all amino acids) or by preparing concentrated
stock solutions of the excipients. The excipients were added to 0.5
mL of the appropriate buffer solution and the pH adjusted to the
desired value with either 1N HCl or 10% NaOH. Subsequently, 0.5 mL
of the concentrated antibody solution in the appropriate buffer
(.about.100 mg/mL) was added to attain the target concentration of
50 mg/mL. This procedure was adopted to prevent protein degradation
due to direct contact with concentrated excipients. The 1 mL
solution was split into two vials with 0.5 mL fill each. One vial
was used for initial T=0 analysis and then stored at 2-8.degree. C.
for the 3 week time point analysis at 2-8.degree. C. The other vial
was incubated at 45.degree. C. with shaking at 100 RPM for 3 weeks
and analyzed at the end of that time period.
1TABLE 1 List of excipients and their concentrations as used in the
study. Target # Excipient Conc. 1 Tween 80 0.05% 2 EDTA 0.05% 3
NaCl 150 mM 4 Methionine 100 mM 5 Glycine 200 mM 6 Serine 200 mM 7
Proline 200 mM 8 Lysine 200 mM 9 MgCl.sub.2 100 mM 10 Tween 20
0.05% 11 Glycerol 5.0% 12 Ethanol 5.0%
[0069] Analytical Methods
[0070] At each of the two time points, the samples were analyzed
using various analytical techniques. Solution clarity was visually
examined by holding the sample vials up against a black background
under fluorescent lighting. The solution was inspected for
insoluble species and color changes were recorded. Size exclusion
chromatography was performed using a Perkin Elmer HPLC unit with
diode array detection and two Tosohaas columns connected in series.
The samples were diluted approximately 5 fold with the
corresponding buffer to bring the concentration to about 1 mg/mL
and 100 .mu.L of the sample was injected onto the column. The
sample concentration was measured by UV spectroscopy using the
Perkin Elmer Lambda Bio 40 spectrophotometer.
[0071] The samples from the 3-week time point were analyzed by
Capillary Isoelectric Focusing on the BioRAD CE (BioFocus 3000)
System. All the samples were diluted to 0.25 mg/mL with water and a
1:1 dilution (to a final concentration of 0.125 mg/mL) was made
with the pharmalyte solution containing TEMED and two internal pI
markers, 8.4 and 10.1. The capillary used was an eCAP with neutral
coating (Beckmann, 56 cm length, 50 um ID).
[0072] The potency of samples formulated in the succinate buffer
with the excipients, Tween-80, EDTA, NaCl and MgCl.sub.2 was tested
after 3 weeks of incubation at 5 and 45.degree. C. It was a
bio-assay involving KIT-225-K6 cells.
[0073] Results
[0074] There were 24 samples at time point T=0 as 12 different
excipients were monitored in two different buffers. At the 3 week
time point, there were 48 samples to be analyzed (12 different
excipients.times.2 temperatures.times.2 buffers=48). Assays
performed include concentration determination by UV-Vis, pH,
Clarity, SEC-HPLC, and CIEF.
[0075] (a) Sample Clarity
[0076] Sample appearance is indicated in Table 2. All samples were
clear in both the buffers at the initial time point T=0. At the 3
week time point, all samples in the phosphate buffer except the one
containing lysine were clear at 5.degree. C. In the same buffer, at
45.degree. C., the samples containing amino acids (glycine, serine,
proline and lysine) appeared clear but had some thread like
floaties in the vials. The sample with MgCl.sub.2 had clear
crystals settled in the bottom of the vial.
[0077] In the succinate buffer, all samples except the amino acid
containing formulations were clear after three weeks of incubation
at 5.degree. C. The samples with proline and lysine were the most
turbid. At 45.degree. C., all samples in succinate buffer were
clear at the 3 week time point.
2TABLE 2 Sample clarity determined by fluorescence light at T = 0
and T = 4 weeks at 5 and 45.degree. C. in the Na-succinate (pH 6.0)
and Na-phosphate (pH 6.5) buffers. Phosphate Phosphate Phosphate
Succinate Succinate Succinate T = 0 T = 3 wks T = 3 wks T = 0 T = 3
wks T = 3 wks Sample Clarity Clarity, 5.degree. C. Clarity,
45.degree. C. Clarity Clarity, 5.degree. C. Clarity, 45.degree. C.
Tween-80 Clear Clear Clear Clear Clear Clear EDTA Clear Clear Clear
Clear Clear Clear NaCl Clear Clear Clear Clear Clear Clear
Methionine Clear Clear Clear Clear Turbid Clear Glycine Clear Clear
Clear Clear Turbid Clear Serine Clear Clear Clear Clear Turbid
Clear Proline Clear Clear Clear Clear Turbid Clear Lysine Clear
Turbid Clear Clear Turbid Clear MgCl.sub.2 Clear Clear Clear Clear
Clear Clear Tween-20 Clear Clear Clear Clear Clear Clear Glycerol
Clear Clear Clear Clear Clear Clear Ethanol Clear Clear Clear Clear
Clear Clear
[0078] (b) SEC-HPLC
[0079] Results of SEC-HPLC are tabulated in Table 3(A-C). Table 3A
indicates the % monomer for all samples being investigated in this
study. The % monomer at T=0 for all samples was >99%. At the
three weeks time point, no significant change was observed in the %
monomer for the 5.degree. C. samples in both buffers. However, at
45.degree. C., all samples indicated a slight drop in the % monomer
(<5%). For samples formulated in the phosphate buffer, the %
monomer varied from 94.08 (methionine) to 97.29 (proline), while
for the samples formulated in the succinate buffer, the % monomer
varied from 95.86 (methionine) to 97.55 (Tween-80). In both the
buffers, the methionine and glycine containing formulations showed
the most significant drop in % monomer. The decrease in % monomer
was mostly due to clip formation.
[0080] Table 3B lists the % aggregate formation in all samples
being investigated in this study. It is clear from these results
that the increase in aggregate formation during the 3-week duration
is minimal for all samples at 5.degree. C. in both buffers. After 3
weeks of incubation at 45.degree. C., samples in the phosphate
buffer showed an increase in % aggregate ranging from 0.40% (EDTA)
to 2.40% (glycine). In the succinate buffer, the aggregate
formation was slightly lower; ranging from 0.7% (methionine) to
1.09% (glycine) after the 3 week incubation period. One of the
hypotheses that supports these results is that if aggregate
formation is due to oxidation, it may be slowed down in the
succinate buffer due to the metal chelating properties of the
succinate buffer.
[0081] Table 3C lists the % clip formation in all samples being
investigated in this study.
[0082] At the initial time point, the % clipping ranged from
.about.0.2-0.4% in all samples. For all samples incubated at
5.degree. C., the % increase in clips was insignificant over the
3-week period. At 45.degree. C., a significant increase in the rate
of clip formation was observed. For samples formulated in the
phosphate buffer, the % clipping varied from 4.74 (methionine) to
1.5% (proline, glycerol and ethanol), while in the succinate
buffer, the range was 1.48%(Tween-80) to 3.44 (methionine). In
general, an increase in the clip formation was observed in the
amino acid containing formulations. Further, the rate of clip
formation appears to be higher in the phosphate buffer. This may be
attributed to the pH difference of the Na-succinate and
Na-phosphate buffers (pH 6.0 and 6.5, respectively), indicating
base catalyzed hydrolysis as being the primary reason for clip
formation
3TABLE 3A % Monomer as determined by SEC at T = 0 and 3 weeks at
5.degree. C. and 45.degree. C. in the Na-succinate (pH 6.0) and
Na-phosphate (pH 6.5) buffers. Phosphate Phosphate Phosphate
Succinate Succinate Succinate T = 0 T = 3 wks T = 3 wks T = 0 T = 3
wks T = 3 wks Sample % Mono % Mono, 5.degree. C. % Mono, 45.degree.
C. % Mono % Mono, 5.degree. C. % Mono, 45.degree. C. Tween-80 99.36
99.48 96.71 99.43 99.51 97.55 EDTA 99.37 99.42 96.43 99.42 99.53
97.51 NaCl 99.37 99.41 96.84 99.42 99.53 97.31 Methionine 99.42
99.42 94.08 99.47 99.53 95.86 Glycine 99.41 99.42 95.90 99.46 99.53
96.46 Serine 99.41 99.45 96.15 99.45 99.53 97.29 Proline 99.40
99.43 97.29 99.45 99.52 97.06 Lysine 99.34 99.62 95.45 99.45 99.57
96.28 MgCl.sub.2 99.37 99.44 97.12 99.47 99.53 96.62 Tween-20 99.17
99.53 96.33 99.44 99.53 97.27 Glycerol 99.41 99.59 96.32 99.43
99.48 97.46 Ethanol 99.41 99.42 97.24 99.31 99.19 97.42
[0083]
4TABLE 3B % Aggregate as determined by SEC at T = 0 and 3 weeks at
5.degree. C. and 45.degree. C. in the Na-succinate (pH 6.0) and
Na-phosphate (pH 6.5) buffers. Phosphate Succinate Phosphate
Phosphate T = 3 wks Succinate Succinate T = 3 wks T = 0 T = 3 wks %
Agg, T = 0 T = 3 wks % Agg, Sample % Agg % Agg, 5.degree. C.
45.degree. C. % Agg % Agg, 5.degree. C. 45.degree. C. Tween-80 0.41
0.00 1.61 0.36 0.36 0.96 EDTA 0.39 0.43 0.40 0.35 0.35 0.96 NaCl
0.40 0.43 1.23 0.33 0.34 0.85 Methionine 0.36 0.41 1.18 0.32 0.34
0.70 Glycine 0.38 0.42 2.40 0.33 0.35 1.09 Serine 0.38 0.40 2.15
0.32 0.33 0.91 Proline 0.38 0.41 1.14 0.35 0.34 0.86 Lysine 0.39
0.36 1.50 0.32 0.30 0.64 MgCl.sub.2 0.38 0.42 0.60 0.32 0.34 0.82
Tween-20 0.40 0.44 1.55 0.34 0.34 1.00 Glycerol 0.37 0.40 2.13 0.35
0.32 0.94 Ethanol 0.37 0.43 1.26 0.28 0.38 0.91
[0084]
5TABLE 3C % Clipping as determined by SEC at T = 0 and 3 weeks at
5.degree. C. and 45.degree. C. in the Na-succinate (pH 6.0) and
Na-phosphate (pH 6.5) buffers. Phosphate Succinate Phosphate
Phosphate T = 3 wks Succinate Succinate T = 3 wks T = 0 T = 3 wks %
Clip, T = 0 T = 3 wks % Clip, Sample % Clip % Clip, 5.degree. C.
45.degree. C. % Clip % Clip, 5.degree. C. 45.degree. C. Tween-80
0.21 0.52 1.67 0.22 0.11 1.48 EDTA 0.22 0.15 2.00 0.22 0.12 1.53
NaCl 0.24 0.16 1.93 0.21 0.12 1.85 Methionine 0.21 0.16 4.74 0.21
0.13 3.44 Glycine 0.20 0.15 1.70 0.21 0.12 2.41 Serine 0.21 0.14
1.69 0.23 0.12 1.81 Proline 0.22 0.16 1.58 0.21 0.13 2.08 Lysine
0.24 0.02 3.05 0.23 0.12 3.09 MgCl.sub.2 0.21 0.14 2.28 0.21 0.13
2.55 Tween-20 0.44 0.03 2.12 0.22 0.11 1.73 Glycerol 0.23 0.01 1.54
0.22 0.20 1.61 Ethanol 0.22 0.14 1.51 0.41 0.40 1.67
[0085] (c) Capillary Electrophoresis
[0086] All the samples from this study were analyzed by capillary
electrophoresis (cIEF) on the BioRAD system. A typical cIEF profile
of Daclizumab shows four peaks. Typically on accelerated aging at
high temperatures, the area of the main isoform peak decreases
followed by an increase in the other isoform peaks, which indicates
the conversion of one isoform to another isoform. The % degradation
is calculated by percent decrease in the peak area of the main
isoform: 1 % Degradation = [ Peak Area at T = 0 - Peak Area at 45 C
. ] .times. 100 % [ Peak Area at T = 0 ]
[0087] Our results indicate that the 45.degree. C. samples are more
degraded in the phosphate buffer (pH 6.5) when compared with
similar samples in the succinate buffer (pH 6.0). The best
electropherograms were seen for the excipients, EDTA, NaCl, lysine
and MgCl.sub.2. The % degradation after 3 weeks for the 5.degree.
C. versus the 45.degree. C. could not be calculated for the samples
containing Tween 80, Tween 20, serine and proline as their
electropherograms were very collapsed and the peaks
indistinguishable.
[0088] (d) Potency
[0089] Based on the results of this study, the Na-succinate buffer
appears to be more promising than the Na-phosphate buffer. Thus,
potency assessments were done for the most stabilizing excipients
in the Na-succinate buffer only. This included the formulations
containing Tween-80, EDTA, NaCl and MgCl.sub.2, subject to three
weeks of incubation at 5 and 45.degree. C. Results (Table 4) showed
that the potency of all formulations was within specifications,
indicating that the underlying chemical and physical degradation
processes are not significantly altering the protein activity.
6TABLE 4 Potency results of selected formulations in succinate
buffer at T = 3 weeks at 5 and 45.degree. C. Sample pH % Potency
Tween-80, 5.degree. C. 6.0 105 Tween-80, 45.degree. C. 6.0 80 EDTA,
5.degree. C. 6.0 103 EDTA, 45.degree. C. 6.0 74 NaCl, 5.degree. C.
6.0 105 NaCl, 45.degree. C. 6.0 98 MgCl.sub.2, 5.degree. C. 6.0 112
MgCl.sub.2, 45.degree. C. 6.0 96
[0090] Discussion
[0091] Based on the results of this study, the stability of
formulation was higher in the Na-succinate buffer at pH 6.0,
compared with the Na-phosphate buffer at pH 6.5. This is primarily
due to base-catalyzed hydrolysis that is accelerated at the higher
pH of 6.5, causing an increase in the rate of clip formation. Thus,
the Na-succinate buffer at pH 6.0 is the selected buffer for all
future studies. Results of this study also clearly indicated that
in both buffers, the amino acids (glycine, lysine, serine, proline,
and methionine) did not have a stabilizing effect on the protein
stability. As shown by the data on sample clarity, all amino acid
containing formulations indicated the formation of insoluble
aggregates at 45.degree. C.
[0092] The excipient MgCl.sub.2 was selected in this study based on
the hypothesis that it might protect the protein against
dimidiation. While MgCl.sub.2 precipitated in the Na-phosphate
buffer; in the Na-succinate buffer, based on the cIEF data,
MgCl.sub.2 has a stabilizing effect on the protein. Ethanol was
also included as an excipient to test if it stabilized the protein
against deamidation by lowering the dielectric constant of the
solution. The results, however, do not support this hypothesis.
Finally, Tween-80, EDTA, and NaCl, the excipients most commonly
used to stabilize protein formulations, did not show any
destabilizing effect on the protein in either buffer.
[0093] Further experiments were conducted in the Na-succinate
buffer at pH 6.0; the effect of the excipients (MgCl.sub.2,
Tween-80, NaCl, and EDTA) was further examined on the protein
stability. The results indicate that to formulate an antibody at
100 mg/mL with 100 mM NaCl, the optimal concentration of Tween 80
falls in the range of 0.02-0.03%. Results also indicate that
increasing the salt concentration (100-150 mM) could further
stabilize the formulation. Thus, the concentration of NaCl should
be maximized while maintaining the tonicity requirements. The
results also indicate that the stability of the Tween 80 and NaCl
containing formulation could be enhanced by adding EDTA in the
concentration range of 0.35-0.5%. The addition of MgCl.sub.2 in the
concentration range of 0-50 mM also could have a favorable effect.
The results also indicate that the excipient concentrations for the
most stable formulation are: 150 mM NaCl, 0.05% Tween 80,
0.03-0.04% EDTA and 60-70 mM MgCl.sub.2, however, these conditions
are not practical because they do not provide isotonic
conditions.
Example 4
Stability Data of Two Daclizumab Antibody Formulations in Succinate
Buffer
[0094] Formulations 1 and 2 were prepared according to Example
3.
[0095] Formulation 1: 100 mg/ml Daclizumab antibody, 30 mM sodium
succinate (pH 6.0) 100 mM NaC1 and 0.03% Tween -80.
[0096] Formulation 2: same as Formulation 1, plus 0.05% EDTA.
[0097] The stability results of Formulations 1 and 2 at T=0, 2
weeks, 4 weeks, 8 weeks, and 12 weeks are shown as follows at 5,
25, and 37.degree. C. (Table 5).
7TABLE 5 Stability results of Formulations 1 and 2. % Sample
Clarity Monomer % Clip % Aggregate % Potency T = 0 F1 Clear 98.27
0.77 0.96 100 F2 Clear 98.27 0.77 0.96 90 T = 2 Weeks F1-5C Clear
98.31 0.73 0.95 NA F1-25C Clear 98.03 0.82 1.14 NA F1-37C Clear
97.11 1.21 1.69 NA F2-5C Clear 98.20 0.92 0.90 NA F2-25C Clear
97.90 1.09 1.06 NA T = 4 Weeks F1-5C Clear 98.30 0.74 0.96 93
F1-25C Clear 97.80 0.92 1.28 88 F1-37C Clear 96.20 1.77 2.03 84
F2-5C Clear 98.30 0.77 0.93 94 F2-25C Clear 97.85 0.95 1.20 92
F2-37C Clear 96.30 1.83 1.87 80 T = 8 Weeks F1-5C Clear 98.24 0.73
0.95 96 F1-25C Clear 97.51 0.82 1.14 96 F1-37C Clear 94.76 1.21
1.69 90 F2-5C Clear 98.34 0.78 0.88 90 F2-25C Clear 97.42 1.20 1.38
90 F2-37C Clear 94.63 3.06 2.31 85 T = 12 Weeks F1-5C Clear 98.25
0.73 1.02 98 F1-25C Clear 97.07 1.26 1.62 90 F1-37C Clear 93.31
3.88 2.81 84 F2-5C Clear 98.30 0.70 1.00 94 F2-25C Clear 97.22 1.30
1.48 88 F2-37C Clear 92.88 4.05 1.54 82
Example 5
Stability Data of Two Daclizumab Formulations in Histidine
Buffer
[0098] Formulations 3 and 4 are prepared according to Example
3.
[0099] Formulation 3: 100 mg/ml Daclizumab antibody, 50 mM
histidine (pH 6.0), 115 mM NaCl, 0.03% Tween.RTM.-80, purged with
nitrogen.
[0100] Formulation 4: same as Formulation 3, plus 0.05% EDTA.
[0101] The stability results of Formulations 3 and 4 at T=0, 2
weeks, 4 weeks, 8 weeks, and 12 weeks are shown as follows at 5,
25, and 37.degree. C. (Table 6).
8TABLE 6 Stability results of Formulations 3 and 4. % Sample
Clarity Monomer % Clip % Aggregate % Potency T = 0 F3 Clear 99.24
0.43 0.33 79 F4 Clear 99.01 0.68 0.32 89 T = 2 Weeks F3-5C Clear
99.24 0.38 0.38 ND F3-25C Clear 99.09 0.47 0.44 ND F3-37C Clear
98.32 1.01 0.67 ND F4-5C Clear 99.19 0.44 0.37 ND F4-25C Clear
99.11 0.47 0.42 ND F4-37C Clear 98.41 0.93 0.66 ND T = 4 Weeks
F3-5C Clear 99.26 0.37 0.35 91 F3-25C Clear 98.99 0.56 0.45 76
F3-37C Clear 97.96 1.42 0.62 83 F4-5C Clear 99.28 0.38 0.34 81
F4-25C Clear 99.00 0.56 0.44 85 F4-37C Clear 97.94 1.44 0.63 79 T =
8 Weeks pH 6.0 F3-5C Clear 99.24 0.38 0.38 86 F3-25C Clear 98.74
0.72 0.54 82 F3-37C Clear 96.87 2.37 0.76 75 F4-5C Clear 99.23 0.39
0.38 97 F4-25C Clear 98.71 0.75 0.54 92 F4-37C Clear 96.90 2.34
0.76 86 T = 12 Weeks pH 6.0 F3-5C Clear 98.89 0.63 0.49 99 F3-25C
Clear 98.04 1.21 0.75 96 F3-37C Clear 94.79 4.06 1.17 90 F4-5C
Clear 98.92 0.60 0.48 91 F4-25C Clear 98.06 1.23 0.72 87 F4-37C
Clear 95.02 3.83 1.15 78
Example 6
Stability Data of Daclizumab Formulation at Room Temperature for
One Year
[0102] A liquid antibody formulation of 100 mg/ml Daclizumab in 30
mM sodium succinate, pH 6, 100 mM NaCl, and 0.03% Tween.RTM. 80 was
tested for stability after one year storage at 25.degree. C. The
stability results indicate that the formulation is stable for at
least one year at 25.degree. C. (Table 7).
9TABLE 7 Stability Results of Daclizumab formulation after One-Year
Storage at 25.degree. C. % % Sample Clarity % Monomer Clip
Aggregate % Potency T = 0 Clear 98.27 0.77 0.96 100 T = 1 year
Clear 94.32 3.14 2.53 86
Example 7
Stability Data of Daclizumab Formulation at 5.degree. C. for 18
Months
[0103] A liquid antibody formulation of 100 mg/ml Daclizumab in 30
mM sodium succinate, pH 6, 100 mM NaCl, and 0.03% Tween.RTM. 80 was
incubated at 5.degree. C. (2-8.degree. C.) and tested for stability
at different time points. The stability results indicate that the
formulation is stable for at least 18 months at refrigerated
temperature (Table 8).
10TABLE 8 Stability Results of Daclizumab at 5.degree. C. Time
(Month) % Monomer % Aggregate 0 99.0 N/A 3 99.1 0.2% 6 99.1 0.2% 9
98.8 0.2% 12 98.9 0.2% 18 98.6 0.2%
Example 8
Stability data of HAIL-12 (Histidine Buffer)
[0104] HAIL-12 (anti-IL12 antibody, 50 mg/mL) was formulated in 50
mM Histidine buffer, 120 mM sodium chloride, 0.03% Tween 80, pH
6.0. The on-going stability testing indicates that the formulation
is stable at 5.degree. C. for at least 9 months (Table 9).
11TABLE 9 Stability Results of HAIL-12 at 5.degree. C. % % Sample
Clarity Monomer Clip % Aggregate % Potency T = 0 Clear 99.47 0.18
0.35 95 T = 7 months Clear 98.90 0.65 0.45 -- T = 8 months -- -- --
-- 100 T = 9 months -- 98.52 -- -- --
Example 9
Stability data of HAIL-12 (Succinate Buffer)
[0105] HAIL-12 (50 and 100 mg/mL) was formulated in 40 mM
Na-succinate buffer, 100 mM NaCl, and 0.03% Tween-80, pH 6.0. The
ongoing stability testing indicates that the formulation is stable
at 5, 25, and 37.degree. C. for at least 12 weeks (Tables 10 and
11).
12TABLE 10 Stability Results of HAIL-12 (50 mg/mL)at various
temperatures. % Sample Clarity Monomer % Clip % Aggregate % Potency
T = 0 5.degree. C. Clear 99.27 0.27 0.47 99 T = 12 Weeks 5.degree.
C. Clear 99.00 0.34 0.67 109 25.degree. C. Clear 98.05 0.92 1.04 76
37.degree. C. Clear 93.86 4.25 1.90 75 T = 6 Months 5.degree. C.
Clear 98.63 0.61 0.76 97 25.degree. C. Clear 97.1 1.67 1.22 78
[0106]
13TABLE 11 Stability Results of HAIL-12 (100 mg/ml) at various
temperatures. % Sample Clarity Monomer % Clip % Aggregate T = 0
5.degree. C. Clear 99.2 0.31 0.49 T = 12 Weeks 5.degree. C. Clear
98.9 0.31 0.78 25.degree. C. Clear 97.67 0.95 1.38 37.degree. C.
Clear 93.26 4.14 2.6
Example 11
Stability data of HuEP5C7
[0107] HuEP5C7 (anti-L selectin antibody, 50 and 100 mg/mL) was
formulated in 50 mM histidine buffer, 125 mM sodium chloride, 0.01%
Tween 80, pH 6.0. The on-going stability testing indicates that the
formulation is stable for three months at 25.degree. C. and
45.degree. C. and for at least 9 months at 5.degree. C. The results
of the 9-month stability testing at 5.degree. C. is shown in Table
12. The results of the 3-month accelerated stability testing is
shown in Table 13.
14TABLE 12 Stability Results of HuEP5C7 at 5.degree. C. % Sample
Monomer % Clip % Aggregate % Potency 50 mg/mL T = 0 98.54 0.30 1.17
83 T = 9 months 99.08 0 0.91 99 100 mg/mL T = 0 98.56 0.23 1.21 79
T = 9 months 98.05 0.03 1.47 90
[0108]
15TABLE 13 Stability Results of HuEP5C7 at various temperatures. %
Sample Monomer % Clip % Aggregate % Potency T = 3 months 50 mg/mL -
5.degree. C. 99.48 0.14 0.39 121 50 mg/mL - 25.degree. C. 98.81
0.31 0.88 72 50 mg/mL - 45.degree. C. 98.26 0.99 0.76 107 100 mg/mL
- 5.degree. C. 99.03 0 0.97 93 100 mg/mL - 25.degree. C. 98.56 0.40
1.06 78 100 mg/mL - 45.degree. C. 97.88 0.92 1.20 91
[0109] 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.
* * * * *