WO2004039337A2

WO2004039337A2 - Stable liquid pharmaceutical formulation of antibodies that are prone to isomerization

Info

Publication number: WO2004039337A2
Application number: PCT/US2003/034950
Authority: WO
Inventors: Elizabet A. Kaisheva; Supriya Gupta; Aleni Flores-Nate
Original assignee: Protein Design Labs, Inc.
Priority date: 2002-10-31
Filing date: 2003-10-31
Publication date: 2004-05-13
Also published as: AU2003291689A8; AU2003291689A1; WO2004039337A3

Abstract

This invention is directed to a stable liquid pharmaceutical formulation comprising an antibody that is prone to isomerization, and 10-120 mM MgCl2. The antibody comprises at least two aspartic acid residues or one aspartic acid and one glutamic acid residue in its primary sequence. The aspartic acid tends to form a succinimide intermediate and isoAsp, thus causes the antibody to lose stability. The addition of MgCl2 in the antibody formulation slows down the isomerization and stabilizes the antibody. This invention is exemplified by a stabilized liquid formulation of HuMv833, a monoclonal antibody against vascular endothelial growth factor (VEGF).

Description

STABLE LIQUID PHARMACEUTICAL FORMULATIONS OF ANTIBODIES THAT ARE PRONE TO ISOMERIZATION

FIELD OF THE INVENTION The present invention relates generally to the field of pharmaceutical formulation of antibodies. Specifically, the present invention relates to an antibody formulation that slows down the isomerization process of the antibody. This invention is exemplified by a stabilized liquid formulation of HuMv833, a monoclonal antibody against vascular endothelial growth factor (VEGF).

BACKGROUND OF THE INVENTION

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. Two general approaches can be used to address the stability issues of proteins. Improved stability can be achieved by modifying its covalent structure or its primary sequence, or by using excipients that improve protein stability. The first approach is generally not feasible in the case of drug applications as it results in producing a new protein with possibly altered pharmacokinetics and pharmacodynamics profile. The second method is often more successful, however, it is limited by the current availability of a small number of approved excipients, particularly when the nature of instability is related to chemical degradation pathways such as deamidation, isomerization, and/or hydrolysis.

The formation of succinimide intermediates and their hydrolysis products (Aspartate and isoaspartate), represents a common source of microheterogeneity in therapeutic proteins. For an antibody that tends to form succinimide and subsequently degrade, attaining the desired shelf life in the liquid formulation is a problem yet to be resolved. There is a need in the art for developing stable aqueous pharmaceutical formulation for an antibody suitable for intravenous or subcutaneous injection.

Gietz, et al. (Pharmaceutical Res., 16:1626 (1999)) report the interactions of zinc and rHir and show site-specific inhibition of succinimide formation with an increase in the shelf life of rHir in the Zn-rHir suspension. In this case, zinc chloride induced precipitation of recombinant hirudin HV1 (rHir), an anticoagulant protein. Einspahr, et al, (Met. Ions. Bio Syst., Vol. 17, pp. 51-97, ed. Sigel) disclose the interaction of calcium ions with carboxylate groups of aspartate and glutamate residues in protein. Chakrabarti (Biochemistry, 28:6081-6085 (1989)) discloses that in many proteins, carbonyl groups that are one, two, or three residues apart along the polypeptide chain bind to the same metal ions such as calcium.

To applicant's knowledge, there are no published reports using Mg⁺² ions in stabilizing an antibody formulation by slowing down the isomerization of antibody in an aqueous phase. Applicants provide herewith a novel antibody formulation that reduces isomerization in the antibody

SUMMARY OF THE INVENTION The present invention provides a stable liquid antibody formulation comprising an antibody that is prone to isomerization, and 10-120 mM MgCl₂. The isomerization of aspartate residues to isoaspartate residues are widespread spontaneous reaction that can alter antibody's structure, function and stability.

Antibodies, which have at least two aspartic acid residues or one aspartic acid and one glutamic acid residue in the vicinity of each other, exposed to the polar media, tend to form succinimide intermediates and isoAsp, but that can be stabilized by the addition of MgCl₂. When antibodies have at least two aspartic acid residues or one aspartic acid and one glutamic acid residue in the vicinity of each other in their CDR's (complementary determining region), it affects the antibody bioactivity (potency). Applicants have found that in the presence of MgCl₂, the loss in potency of an antibody due to isomerization of aspartic acid residues (present in the CDR region) is considerably slowed down. The present formulation optionally comprises a surfactant such as polysorbate, and a salt to contribute to the isotonicity of the formulation. An exemplary formulation comprises an antibody at 1-100 mg/mL concentration, about 20-60 mM sodium citrate/phosphate (pH 6.5-7.5), about 0.01-0.1% polysorbate 20 or 80, about 20-120 mM NaCl, and 20-150 mM MgCl₂, wherein said antibody has improved stability against isomerization.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 depicts the mechanism of isomerization.

Figure 2 shows the rate of antibody potency loss as a function of pH. Figure 3 shows the effects of cations on antibody potency loss. Figure 4 shows stability results of HuMv833 antibody in a formulation without MgCl₂.

Figure 5 shows stability results of HuMv833 in different formulations.

DETAILED DESCRIPTION OF THE INVENTION I. Definition

As used herein, the term "buffer" encompasses those agents that 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 6.5 to about 7.5; and preferably has a pH of about 7.0. Examples of buffers that will control the pH in this range include succinate (such as sodium succinate), gluconate, histidine, citrate, phosphate and other organic acid buffers. The term "Complementary-Determining Regions (CDR)" refer to hypervariable regions of antibodies. Detailed comparison of the amino acid sequences of VL (variable region of the light chain) and VH (variable region of the heavy chain) domains of antibodies reveals that the sequence variability is concentrated in several hypervariable (HV) regions. Three such hypervariable regions are present in the heavy and light chain. The remainders of the VL and VH domains exhibit far less variation and are called the framework regions (FRs). The hypervariable regions form the antigen-binding site of the antibody molecule. Because antigen-binding sites are complementary to the structure of the epitope, hence they are also called complementary-determining regions (CDR's). "Pharmaceutically acceptable excipients" (vehicles, additives) are those inert substances which can reasonably be administered to a subject mammal to provide an effective dose of the active ingredient employed. These 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.

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.

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. A "stable" liquid antibody formulation is a liquid antibody formulation with no significant changes observed at a refrigerated temperature (2-8 °C) for at least 12 months, preferably 2 years, and more preferably 3 years; or at room temperature (23 - 27 °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. 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 UN light scattering, size exclusion chromatography (SEC) 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.

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.).

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.

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.

II. Analytical Methods

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°C) and 6 months at room temperature (23- 27°C).

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 340nm, 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., Nov. (1991)) measures mean diffusion coefficient, and gives information of the amount of soluble and insoluble aggregates. UV at 340nm measures scattered light intensity at 340nm and gives information about the amounts of soluble and insoluble aggregates. UV spectroscopy measures absorbance at 278nm and gives information of protein concentration.

The iso-Asp content in the samples is measured using the Isoquant Isoaspartate Detection kit (Promega). The kit uses the enzyme Protein Isoaspartyl Methyltransferase (PEVIT) 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 α-carboxyl position, generating S- adenosyl-L-homocysteine (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.

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).

III. Preparation of Antibody

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. 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. 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 VIQC, factor DC, 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-β; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factor (TGF) such as TGF- and TGF-β, including TGF- βi, TGF-β₂, TGF-β₃, TGF-β₄, or TGF-β₅; insulin-like growth factor-I and -II (IGF-I and IGF-II); des(l -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-α, -β, and -γ; 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; integrals such as CD1 la, CD1 lb, CD1 lc, 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.

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. 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 Yi, Y₂, or Y₄ heavy chains (Lindmark et al, J. Immunol. Meth. 62:1-13 (1983)). Protein G is recommended for all mouse isotypes and for human Y₃ (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_H3 domain, the Bakerbond ABX™ 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™ 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.

Preferred antibodies encompassed by the present invention include HuMV833, a humanized monoclonal antibody (IgG4) against Vascular Endothelial Growth Factor (VEGF). The antibody against VEGF was developed for IV administration as adjunct therapy (used in combination with chemotherapy) to lengthen survival time in cancer patients with refractory/relapsed solid tumors (small cell lung cancer, HER-2 negative breast cancer, ovarian carcinoma and renal cell carcinoma). HuMV833 is produced from a Sp2/0 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 affinity ion-exchange and gel filtration chromatography and a series of ultrafiltration and filtration techniques to produce drug substance containing greater than 95% monomeric species.

IV. Isomerization of Antibody The formation of succinimide intermediates and their hydrolysis products

(aspartate and isoaspartate), at the aspartic acid sites of an antibody represents a stability problem. When isomerization occurs, the chemical structure of the antibody alters. Applicants have found that when the isomerization site is in the CDR of an antibody light chain, the isomerization results in a significant drop in the bioactivity of the protein at the intended storage temperature of 2-8°C and at elevated temperatures. For an antibody that tends to form succinimide and subsequently degrades, attaining satisfactory shelf life of the antibody is a significant problem yet to be resolved in the development of aqueous formulations.

Briefly, the mechanism of the isomerization reaction entails the following steps (see Figure 1):

1. Deprotonation of the nitrogen on the residue n+1.

2. Unhindered approach of the nitrogen anion towards the side chain carbonyl of residue n to form a five-membered cyclic imide intermediate (succinimide), and the corresponding protonation of the -OH leaving group. 3. Hydrolysis of the cyclic imide at either of the two C-N bonds to produce α- or β-carbonyl groups, which generates isoaspartate or aspartate residues in the ratio of 3:1.

Results of published studies have suggested that the hydrolysis of succinimide intermediate is quite rapid once it is formed, and the crucial rate-determining steps for isomerization of the aspartate residues are the initial deprotonation of the peptide- bond nitrogen atom and the protonation of the -OH group (Geiger et. al, J. Biol Chem., 262:785-794 (1987); Lura et. al, Biochemistry, 27:7671-7677 (1988)). Thus, conditions and/or excipients that can interfere with the deprotonation of the nitrogen atom, or reduce the protonation affinity of the carbonyl -OH group, or sterically interfere with the approach of the nitrogen anion towards the carbonyl group can slow down the overall isomerization reaction. Antibodies suitable for this invention include those antibodies that are prone to isomerization and can be stabilized by the present invention. Based on this invention, these antibodies have at least two aspartic acids (D) or one aspartic acid and one glutamic acid (E) in their primary sequence. The first D and the second D or E are in the vicinity of each other such that their carboxyl groups are sterically close to interact with each other to form first a succinimide intermediate and then an isoaspartate product. "Vicinity" mean that the first D and the second D or E are immediately next to each other or have only one, two or three amino acid residues in between them. For example, antibodies suitable for this invention include those that comprise D* -amino acid-D/E or D/E-amino acid-D* in their primary sequence, where * indicates the site of isomerization. An antibody suitable for this invention is HuMV833, a humanized monoclonal antibody against vascular endothelial growth factor, which comprises ITSNDIDZ DMN in its CDR's (The italicized Asp is a residue 30 from the N-terminal end of the light chain). In the presence of MgCl₂, the loss of antibody stability due to isomerization of aspartic acid residues is slowed down, possibly due to the interaction of MgCl₂ with the antibody, which impedes one of the key steps in the process of isomerization reaction. Applicants have found that when isomerization is in the CDR of an antibody, the addition of MgCl₂ to the antibody formulation slows down the loss of antibody potency.

At neutral or near-neutral pH such as 7.0, the carboxyl groups of the aspartate residues are completely deprotonated. For antibody having two aspartate residues or one aspartate and one glutamate residues flanking the isomerization site; the aspartate or glutamate residues are in the flexible solvent-exposed region (β-turns) of the antibody. It is known that metal binding to amino acid residues is favored in sites such as the β-sheets where the carbonyls are not tied up in the regular secondary structure features and are free to interact with metals (Chakrabarti, Biochemistry 29:6510658 (1990)). It is thus possible that the Mg²⁺ ions form a bridge between the deprotonated carboxyl groups of the two aspartate residues or one aspartate and one glutamate residues, thus inhibiting the succinimide ring formation and stabilizing the antibody against isomerization. In order for the above stabilization mechanism to work, the two aspartate residues or one aspartate and one glutamate residues do not have to be immediately next to each other, but they have to be in the vicinity, such that it is sterically feasible to have the Mg²⁺ ions to form a bridge between the two carboxyl groups of the aspartate/aspartate or aspartate/glutamate. Other divalent metals, such as Ca ⁺ or Zn²⁺, tend to cause antibody precipitation when included in the antibody formulation, thus are not suitable to stabilize an aqueous antibody formulation.

V. Preparation of the Formulation

MgCl₂ salt interacts with the antibody and slows down one of the key steps in the process of isomerization reaction. The compositions of this invention minimize the formation of succinimide intermediate and isoAsp isomers of an antibody and insure that the antibody maintains its bioactivity over time. The composition comprises a pharmaceutically acceptable liquid formulation containing an antibody that is prone to isomerization, a buffer having a close to neutral pH (pH 6-8), a surfactant, a salt, and MgCl₂. A buffer of pH 6.0-8.0 is used in the composition. A buffer of pH 6.5-7.5 is preferred. Examples of suitable buffers include succinate, gluconate, histidine, citrate, phosphate, and other organic acid buffers. For example, citrate, such as 10-50 mM sodium citrate, is a preferred buffer. Phosphate buffers are much less preferred because phosphates accelerate succinimide formation. A surfactant can be added to the antibody formulation. Exemplary surfactants include nonionic surfactants such as polysorbates (e.g. polysorbates 20, 80, such as Tween^® 20, Tween^® 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%.

A salt is added to the present composition to contribute to the isotonicity of the formulations. An exemplary salt is NaCl at a concentration about 75 to 150 mM. MgCl₂, which inhibits the formulation of succinimide intermediates is key to the formulation. MgCl₂, 20-150 mM, preferably 30-100 mM, and more preferably 40- 60 mM is included in the composition. An exemplary formulation comprises antibody at any concentration (for example, 1-100 mg/mL), about 20-60 mM sodium citrate/phosphate (pH 6.5-7.5), about 0.01-0.1% polysorbate 20 or 80, about 20-120 mM NaCl, and 20-150 mM MgCl₂. This formulation slows down the isomerization and degradation of the antibody, and maintains the physical, chemical and biological stability of the antibody during storage.

The liquid antibody formulation of this invention is suitable for parenteral administration such as intravenous, intramuscular, intraperitoneal, or subcutaneous injection. 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. Factors evaluated to slow down the isomerization process.

Objective

To develop a liquid formulation to slow down the rate of potency loss over time and to achieve the maximum shelf life at 2-8 °C.

Sample

10 mg/mL HuMV833 antibody in 20 mM Na Citrate, 120 mM NaCl, 0.01%

Tween® 80, pH 6.

Results

The effect of different excipients was evaluated after one- week incubation of the sample at 45 °C in a shaker/incubator. During this incubation, the degraded sample showed no change in the secondary and tertiary structure or aggregation/clipping rate. The only effect accelerated by the temperature was the rate of potency loss. The peptide map of the light chain also showed a change in the peptide 'LI ' that corresponded to the following sequence (from the N-terminal end): 'DIOMTOSPSSLSASVGDRVTITZITSNDroflDMNWYOO- . The underlined residues are in the CDR region; Asp 30 (italicized) underwent isomerization. 1. Effect of the dielectric constant

To investigate the effect of the dielectric constant on the rate of potency loss, the effects of benzyl alcohol, ethanol, PEG or PG were evaluated. The addition of any of the above co-solvent did not slow down the observed potency loss.

2. Effect of pH.

The potency loss was calculated as:

_n/ „ , . "control 5C^~ "sample45C _{1 ΛΛ} % Potency drop= x 100 co«tro/5 where, P control 5 C : potency at 5°C

P control 45 C- potency after one week at 45°C

The potency loss decreased (from 80 to 27%) as the pH increased from 4.5 to

7.5. The change in the pH did not affect the percentage aggregates of the molecule from pH 5 to 7.5 (See Figure 2). The clipping rate was also significantly decreased (from 9 to 1%) as the pH increased from 4.0 to 5.5 as indicated by the low molecule weight molecule.

3. Effect of Amino Acids

100 mM of each amino acid (aspartic acid, serine, lysine, glycine and proline) was added to a formulation containing 20 mM NaCitrate, 120 mM NaCl, 0.01 % Tween® 80 at pH 7, and the potency was measured after one week incubation at 45 °C in a shaker/incubator. Results indicated that the rate of potency loss was unaffected in the presence of the above selected amino acids.

4. Effect of Buffer's Type and Strength

20 mM NaCitrate, 20 mM histidine and 20 mM phosphate buffers were tested. The rate of potency loss in histidine and the phosphate buffers was found to be slightly higher compared with the NaCitrate formulation.

Two different strengths of the NaCitrate buffer were tested: 20 and 50 mM. It was found that 50 mM had a higher rate of potency loss compared to the 20 mM NaCitrate buffer. Based on the above results, the HuMV833 formulation were further evaluated in 20 mM NaCitrate buffer, pH 7.0. The low buffering capacity of the citrate buffer at this pH was compensated by the inherent buffering capacity of this protein.

5. Effect of Solvent Viscosity

The effect of the addition of 5 and 14% sucrose; 25% trehalose and 25% glycerol on the rate of potency loss was investigated (formulation: 20 mM NaCitrate buffer, 120 mM NaCl, 0.01% Tween® 80, pH 7.0). The addition of sucrose or trehalose did not show a positive effect. However, the addition of 25% glycerol slows down the rate of potency loss by ~ 40% after one week incubation at 45 °C, relative to the control formulation at pH 6.0.

6. Effect of Cations

(a) Monovalent: NaCl The addition of 120 mM NaCl to the formulation has a small positive effect on decreasing the rate of potency loss.

(b) Divalent:

The addition of 50 and 100 mM CaCl₂ to the formulation resulted in antibody precipitation.

ZnCl₂ at concentrations of 10, 25, 100 and 150 mM also resulted in antibody precipitation.

Addition of 100 mM MgCl₂ to a formulation containing 20 mM

NaCitrate, 120 mM NaCl, 0.01 % Tween® 80 at pH 7 decreased the rate of potency loss by 60% after one week incubation at 45 °C relative to the control formulation at pH 6.0. However, the addition of 150 mM MgCl₂ had a negative effect (See Figure 3).

7. Combined Effect of MgCl₂ and Glycerol The addition of 100 mM MgCl₂ to a formulation containing 25% (or 50%) glycerol did not have a positive effect on decreasing the rate of potency loss over time. Example 2. Effect of ZnCl₂ on different monoclonal antibodies.

Study Design

HuMV833, HuM291, HulDIO, and Dregg55 at 10 mg/mL in 20 mM Na- Citrate, 0.01% Tween® 80, pH 6.0, 120 mM NaCl, were selected as the starting samples. These antibodies were selected to represent different antibody isotypes; HuMV833 and Dregg55 represent IgG4, HulDIO represents IgGl, and HuM291 represents IgG2M3.

ZnCl₂ was added to the formulations at 10, 25 and 150 mM by directly weighing the appropriate amount into the vialed samples. After the addition of ZnCl₂, the solution pH was adjusted to 7.0 for each sample using 0.1 N NaOH. Formulation placebos with 10, 25 and 150 mM ZnCl₂ were also included in the study to ensure that the observed precipitate was protein-specific and not related to the other formulation components. The samples were incubated for 1 week at 45°C and the solution clarity was visually observed.

Results

At T=0, the addition of 25 and 150 mM ZnCl₂ caused protein precipitation for all the antibody formulations. At T=0, the addition to antibody formulation does not cause protein precipitation. However, all samples with 10 mM ZnCl₂ were clear at T = 0. After 1 week incubation at 45°C, the 10 mM ZnCl₂ formulation for HuMV833 and HuM291 precipitated, while the Dregg55 and HulDIO formulations were clear.

Based on the results of this study, higher concentrations of ZnCl₂ (25 and 150 mM) destabilize and cause protein precipitation for all antibodies. Lower concentrations of ZnCl₂ affect antibodies differently; causing some to precipitate.

Example 3. Stability Results of HuMv833 Antibody in a Formulation Without MgCl₂.

Antibody HuMv833 was formulated at 10 mg/mL in 20 mM Na citrate, 120 mM NaCl, and 0.01% Tween^® 80, pH 6. The antibody formulation was incubated at 2-8°C for 30 months. Figure 4 shows the percent potency of antibody at different time points. After 20 months, the antibody lost more than 30% potency. Therefore, this antibody formulation does not provide a 2-year shelf life at refrigerated temperatures. The same formulation was also incubated at an elevated temperature of 45°C. After one week of incubation, the antibody lost about 40% potency. No change in the secondary and tertiary structure of antibody was observed. Also, the aggregation and clipping were minimal.

Example 4. Stability Results of Antibody in Different Formulations.

Antibody HuMv833 was formulated at 10 mg/mL in 20 mM Na citrate, 80 mM NaCl, 0.01% Tween^® 80, at (a) pH 6.0, (b) pH 7.0, and (c) pH 7.0, plus 40 mM MgCl₂. The three antibody formulations were incubated at room temperature (25°C) for 4 months. The percent potency of antibody was determined at T = 0, 1 , 2, 3 and 4 months (Figure 5). The accelerated stability results show that the potency drop of antibody is considerably slowed down by the addition of MgCl₂ and pH adjustment.

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.

Claims

What is claimed is:

1. A stable liquid antibody formulation comprising: an antibody that is prone to isomerization, and 10-120 mM MgCl₂-

2. The stable liquid antibody formulation according to Claim 1, wherein said antibody comprises at least two aspartic acid residues or one aspartic acid and one glutamic acid residue in the vicinity of each other.

3. The stable liquid antibody formulation according to Claim 2, wherein said two aspartic acid residues or one aspartic acid and one glutamic acid are next to each other.

4. The stable liquid antibody formulation according to Claim 2, wherein said two aspartic acid residues or one aspartic acid and one glutamic acid are separated by one amino acid residue.

5. The stable liquid antibody formulation according to Claim 2, wherein the CDR of said antibody comprises at least two aspartic acid residues or one aspartic acid and one glutamic acid residue in the vicinity of each other.

6. The stable liquid antibody formulation according to Claim 1 or 2, wherein said formulation has a pH about pH 6.5 to 7.5.

7. The stable liquid antibody formulation according to Claim 1 or 2, further comprising a polysorbate at a concentration of about 0.01-0.1 %.

8. The stable liquid antibody formulation according to Claim 1 or 2, wherein said MgCl₂ is 40-60 mM.

9. The stable liquid antibody formulation according to Claim 1 or 2, further comprising a salt, which contributes to isotonicity of said formulation.

10. The stable liquid antibody formulation according to Claim 1 or 2, wherein said formulation is stable at about 2-8°C for at least one year.

11. The stable liquid antibody formulation according to Claim 1 or 2, wherein said formulation is stable at about 23-27°C for at least 6 months.

12. The stable liquid antibody formulation according to Claim 1 or 2, wherein said antibody is HuMv833.