HK1242962A1 - Compositions and methods useful for stabilizing protein-containing formulations - Google Patents

Description

Compositions and methods useful for stabilizing protein-containing formulations

This application is a divisional application of PCT application PCT/US2011/029206 entitled "compositions and methods useful for stabilizing protein-containing formulations" filed on day 3/21 2011, which entered the national phase of china on day 24/9/2012 with application number 201180018192.3.

RELATED APPLICATIONS

This application is based on a non-provisional application filed at 37CFR 1.53(b) (1), the contents of which are incorporated herein by reference, based on the priority of 35USC 119(e) claiming provisional application number 61/316326 filed at 3/22/2010.

Technical Field

The present invention relates to the use of non-surfactant compounds, including for example Polyoxyethylene (POE) sorbitan and polyethylene glycol (PEG), for stabilising protein-containing formulations and for preventing aggregation of proteins in such formulations.

Background

Nonionic detergents (i.e., surfactants) are commonly used when stabilizers for protein formulations are needed to protect the protein from denaturation by shaking, stirring, shearing, and freezing-thawing, or from a quiescent state at the interface (see, e.g., U.S. Pat. No. 5,183,746). As an example the use of polysorbates in many protein containing products is exemplified. For example, polysorbates 20 and 80: (20 and80) in the formulation of biotherapeutic products, for preventing surface adsorption and as stabilizers against protein aggregation (Kerwin, J.Pharm. Sci.97(8): 2924-. Polysorbate is an amphiphilic, non-ionic surfactant consisting of fatty acid esters of Polyoxyethylene (POE) sorbitan, polysorbate 20 is polyoxyethylene sorbitan monolaurate and polysorbate 80 is polyoxyethylene sorbitan monooleate.

Unfortunately, however, polysorbates can be degraded by oxidation or hydrolysis. When polysorbate molecules degrade, a variety of degradation byproducts are produced, including, for example, fatty acids, POE sorbitan, PEG esters, and alkyl acids. Some of these by-products of polysorbate degradation, including free fatty acids, can lead to increased turbidity and protein aggregation in protein-containing formulations. Thus, while polysorbates are commonly used as protein stabilizers, the fatty acids and other degradation byproducts released over time from the degradation of the polysorbates can adversely affect the protective effect that the polysorbates exhibit in protein-containing formulations.

Thus, there is a need for additional compositions useful for preventing aggregation of proteins in aqueous formulations containing proteins.

Summary of The Invention

The present invention is based on the novel finding that Polyoxyethylene (POE) sorbitan and polyethylene glycol (PEG) present in aqueous formulations at certain concentrations are useful for stabilizing protein-containing or peptide-containing formulations, and for preventing aggregation of proteins in such formulations.

Thus, in one aspect, the invention relates to a composition of matter comprising an antibody or other protein or peptide, and POE sorbitan. In one embodiment, the polyoxyethylene sorbitan is present at a concentration of from about 20ppm to about 100,000 ppm. In another embodiment, the composition of matter comprising the antibody or other protein or peptide and the POE sorbitan is free of non-ionic surfactant, e.g., free of polysorbate. Optionally, the composition of matter may also be free of lyoprotectants (lyoprotectants), such as sugars, free amino acids, variants thereof, and the like. In certain embodiments, the composition of matter may be in aqueous or solid form.

In another aspect, the invention relates to a composition of matter comprising an antibody or other protein or peptide and polyethylene glycol. In certain embodiments, the polyethylene glycol is present in the composition of matter at a concentration of less than about 10,000ppm, preferably between about 20ppm and about 10,000 ppm. In another embodiment, the composition of matter optionally comprises at least one POE sorbitan, optionally present in the composition of matter at a concentration of from about 20ppm to about 100,000 ppm. The composition of matter may also optionally be free of nonionic surfactants, e.g., free of polysorbates. Optionally, the composition of matter may also be free of lyoprotectants (lyoprotectants), such as sugars, free amino acids, variants thereof, and the like. In certain embodiments, the composition of matter may be in aqueous or solid form.

In another aspect, the present invention relates to an article of manufacture comprising a container containing any of the compositions of matter described herein.

In another aspect, a method is provided for preparing a stable composition containing an antibody (or other protein or peptide) by co-mixing the antibody or other protein with a POE sorbitan or PEG.

In another aspect, the invention relates to a method of increasing the stability of an antibody or other protein or peptide in an aqueous formulation, comprising admixing the antibody or other protein or peptide with a stabilizing amount of a POE sorbitan, wherein the POE sorbitan increases the stability of the antibody or other protein or peptide. In one embodiment, the POE sorbitan is present at a concentration of from about 20ppm to about 100,000ppm, optionally free of non-ionic surfactants (e.g., free of polysorbates), or lyoprotectants, such as sugars, free amino acids, variants thereof, and the like.

In another aspect, the invention relates to a method of increasing the stability of an antibody or other protein or peptide in an aqueous formulation comprising mixing the antibody or other protein or peptide with a stabilizing amount of polyethylene glycol, wherein the polyethylene glycol increases the stability of the antibody or other protein or peptide. In one embodiment, the polyethylene glycol is present at a concentration of less than about 10,000ppm, preferably between about 20ppm to about 10,000 ppm. In another embodiment, the composition of matter optionally comprises at least one POE sorbitan, which may optionally be present at a concentration of from about 20ppm to about 100,000ppm, and may optionally be free of nonionic surfactants (e.g., free of polysorbates), or lyoprotectants, such as sugars, free amino acids, variants thereof, and the like.

In another aspect, the invention relates to a method of preventing or reducing aggregation of an antibody or other protein or peptide in an aqueous formulation comprising admixing the antibody or other protein or peptide with POE sorbitan. In one embodiment, the POE sorbitan is present at a concentration of from about 20ppm to about 100,000 ppm. The aqueous formulation optionally does not contain a non-ionic surfactant (e.g., does not contain polysorbate), or a lyoprotectant, such as a sugar, a free amino acid, variants thereof, and the like. In another embodiment, aggregation of the antibody or other protein is induced by agitation of the aqueous solution.

In another aspect, the invention relates to a method of preventing or reducing aggregation of an antibody or other protein or peptide in an aqueous formulation comprising mixing the antibody or other protein or peptide with polyethylene glycol. In one embodiment, the polyethylene glycol is present at a concentration of less than about 10,000ppm, preferably between about 20ppm to about 10,000 ppm. In another embodiment, the aqueous formulation optionally comprises at least one POE sorbitan, optionally present at a concentration of from about 20ppm to about 100,000ppm, and may optionally be free of nonionic surfactants (e.g., free of polysorbates), or lyoprotectants, such as sugars, free amino acids, variants thereof, and the like. In another embodiment, aggregation of the antibody or other protein is induced by agitation of the aqueous solution.

In a method of purifying a protein, peptide or antibody from a recombinant cellular protein or other contaminating protein, embodiments of the present invention relate to improvements that include the addition of POE sorbitan or polyethylene glycol to the protein, peptide or antibody during the purification process.

Another aspect of the present invention is a method of making a non-aggregating aqueous solution of another self-aggregating antibody or other protein or peptide by mixing at least one POE sorbitan or polyethylene glycol in an aqueous solution containing the self-aggregating antibody or other protein or peptide, and then concentrating the aqueous solution.

Other features and advantages of the present invention will be apparent from the following detailed description and examples, which, however, should not be taken to be limiting. The contents of all references, patents, and published patent applications cited throughout this application are expressly incorporated by reference herein.

Brief description of the drawings

Figure 1 shows the results obtained from turbidity analysis of anti-IL 13 antibodies in combination with various additives including polysorbate 20(PS), Lauric Acid (LA) or POE sorbitan 20 (POE).

Figure 2 shows the results obtained from the analysis of protein concentrations of anti-IL 13 antibody in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 3 shows the results obtained from the analysis of the protein concentration of anti-IgE antibodies in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 4 shows the results obtained from turbidity analysis of anti-IL 13 antibody in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 5 shows the results obtained from turbidity analysis of anti-IgE antibodies in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 6 shows the results obtained from particle size analysis (particles larger than 2 μm) of anti-IL 13 antibody in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 7 shows the results obtained from particle size analysis (particles larger than 2 μm) of anti-IgE antibodies in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 8 shows the results obtained from particle size analysis (particles larger than 10 μm) of anti-IL 13 antibody in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 9 shows the results obtained from particle size analysis (particles larger than 10 μm) of anti-IgE antibodies in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 10 shows the results obtained from particle size analysis (particles larger than 50 μm) of anti-IL 13 antibody in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Figure 11 shows the results obtained from particle size analysis (particles larger than 50 μm) of anti-IgE antibodies in combination with various additives, including POE sorbitan 20, PEG 1000 or PEG 6000, all at various concentrations.

Description of The Preferred Embodiment

The present invention may be understood more readily by reference to the following detailed description of specific embodiments and the examples included herein.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are described herein. All publications mentioned herein are incorporated by reference in their entirety.

Aggregation of antibodies and other proteins is primarily caused by hydrophobic interactions, ultimately leading to denaturation. When the hydrophobic region of a partially or completely unfolded protein is exposed to water, a thermodynamically unfavorable situation results because the normally embedded hydrophobic interior is now exposed to a hydrophilic, aqueous environment. Thus, the reduction in entropy from the structural water molecules surrounding the hydrophobic region drives the denatured protein to aggregate, mainly through the exposed hydrophobic region. Thus, the solubility of the protein is also compromised. In some cases, protein subunits may self-associate (whether native or misfolded) under certain conditions, which may lead to precipitation and loss of activity.

Factors that affect aggregation of proteins in solution generally include protein concentration, pH, temperature, other excipients, and mechanical stress. Some factors (e.g., temperature) are easier to control than others (e.g., mechanical stress) during purification, compounding, production, storage and use. Formulation studies will specify the proper choice of pH and excipients that do not induce aggregation and/or actually help prevent aggregation. The protein concentration is dictated by the desired therapeutic dose and depending on how much of that concentration will determine whether there is a possibility of a more highly correlated state (dimer, tetramer, etc.), which may lead to aggregation in solution. Careful research must be performed during formulation development to determine what factors affect protein aggregation and how those factors can then be eliminated or controlled.

The need to identify stable solution preparations of antibodies or other proteins for parenteral or other administration may lead to the development of test methods for assessing the effect of various additives on physical stability. Based on known factors affecting protein aggregation and requirements for such applications, mechanical methods involving stirring or rotating the protein solution can be used to assess physical stability. Physical stress test methods for identifying the ability of various additives to prevent aggregation may involve exposure to agitation or stirring in a horizontal plane, or rotation x cm from an axle rotating in a vertical plane at n rev/min. The turbidity caused by aggregation is usually determined as a function of time by visual observation or light scattering analysis. Alternatively, the decrease in soluble protein content due to precipitation can be quantified as a function of time as determined by HPLC.

The present invention is based on the novel finding that certain concentrations of Polyoxyethylene (POE) sorbitan and polyethylene glycol (PEG) present in liquid formulations are useful for stabilizing protein-containing formulations, and for preventing aggregation of proteins in such formulations.

Thus, in one aspect, the invention describes a composition of matter comprising both high or low concentrations of antibody or other protein and POE sorbitan. As used herein, "polyoxyethylene sorbitan" or "POE sorbitan" refers to a non-surfactant compound having the following chemical structure:

wherein a + b + c + d is preferably from about 6 to about 80, more preferably from about 8 to about 60, more preferably from about 10 to about 40, and more preferably from about 10 to about 20. As noted above, it will be understood by those skilled in the art that chemical synthesis of a compound (e.g., POE sorbitan as described herein) results in a somewhat heterogeneous mixture of compounds, rather than a fully homologous preparation. It follows that when it is described herein that a + b + c + d is, for example, preferably from about 6 to about 80, it is understood that the definition refers to the major component of the heterogeneous mixture resulting from its chemical synthesis.

POE sorbitan can be used alone as a stabilizer for antibodies or other proteins, or can be used in combination with other POE sorbitan for stabilizing antibodies or other proteins in aqueous solution. POE sorbitan can be used as a stabilizing (or anti-aggregating) agent for antibodies or other proteins in aqueous solutions over a wide range of concentrations. In certain embodiments of the invention, the POE sorbitan (if used as a single stabilizer) or POE sorbitan (if used in combination) can be present in an aqueous formulation containing an antibody or other protein at a concentration of from about 20ppm to about 100,000ppm, more preferably from 100ppm to about 50,000ppm, more preferably from 150ppm to about 10,000ppm, more preferably from 200ppm to about 5,000ppm, more preferably from 200ppm to about 1,000 ppm.

In another aspect, the invention features a composition of matter that includes an antibody or other protein, whether at a high or low concentration, and polyethylene glycol.

As used herein, "polyethylene glycol," "PEG," and similar terms are intended to encompass polyethylene glycol and various derivatives thereof, such as methoxy-PEG-amine, diamine-PEG, and the like. More specifically, in certain embodiments of the present invention, the term "polyethylene glycol" or "PEG" refers to a non-surfactant compound having the following chemical structure:

wherein n is from about 5 to about 240 and may optionally contain some degree of unsaturation. The PEG encompassed in the use of the invention may be branched or linear, preferably linear. As noted above, one skilled in the art will appreciate that chemical synthesis of a compound (e.g., PEG as described herein) results in a somewhat heterogeneous mixture of compounds, rather than a completely homogeneous preparation. It follows that when n is described herein as preferably from about 5 to 240, it is understood that the definition refers to the major component of the heterogeneous mixture resulting from its chemical synthesis.

PEG may be used singly as a stabilizer for antibodies or other proteins, or may be used in combination with other PEGs for stabilizing antibodies or other proteins in aqueous solutions. PEG can be used as a stabilizing (or anti-aggregating) agent for antibodies or other proteins in aqueous solutions over a wide range of concentrations. In certain embodiments of the invention, PEG (if used as a single stabilizer) or PEG (if used in combination) can be present in a concentration of less than about 10,000ppm, preferably from about 20ppm to about 10,000ppm, more preferably from about 200ppm to about 5,000ppm, more preferably from about 200ppm to about 1,000ppm, more preferably from about 200ppm to about 500ppm, in an aqueous formulation containing an antibody or other protein.

Preferred PEGs include polymers having a molecular weight of about 200 to 12,000, but higher molecular weight polymers are also within the scope of the invention. PEG includes linear and branched polymers, star molecules and PEG block copolymers formed by coupling at least two different PEG polymers to form a higher molecular weight polymer, all as generally known in the art.

By "polypeptide" or "protein" is meant an amino acid sequence having a chain length sufficient to produce higher levels of tertiary and/or quaternary structure. Thus, proteins are distinct from "peptides", which are also amino acid-based molecules, but do not have such structures. Generally, the proteins used herein have a molecular weight of at least about 5-20kD, alternatively at least about 15-20kD, preferably at least about 20 kD. "peptide" means an amino acid sequence that does not generally exhibit a higher level of tertiary and/or quaternary structure. Peptides generally have a molecular weight of less than about 5 kD.

Examples of polypeptides encompassed within the definition herein include mammalian proteins, such as renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; a lipoprotein; alpha-1-antitrypsin; an insulin a chain; insulin B chain; proinsulin; follicle stimulating hormone; a calcitonin; luteinizing hormone; glucagon; coagulation factors such as factor VIIIC, factor IX, tissue factor and von Willebrands factor; anti-coagulation factors such as protein C; atrial natriuretic factor; a pulmonary surfactant; plasminogen activators, such as urokinase or human urine or tissue-like plasminogen activator (t-PA); bombesin; thrombin; a hematopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalinase; RANTES (secretion regulated by activation expressed by normal T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin such as human serum albumin; muellian inhibitory substances; a relaxin a chain; a relaxin B chain; prorelaxin; mouse gonadotropin-related peptides; microbial proteins, such as beta-lactamases; a DNA enzyme; IgE; cytotoxic T lymphocyte-associated antigens (CTLA), such as CTLA-4; a statin; an activin; vascular Endothelial Growth Factor (VEGF); receptors for hormones or growth factors; protein A or D; rheumatoid factor; a neurotrophic factor such as bone-derived neurotrophic factor (BDNF), neurotrophic factor-3, -4, -5, or-6 (NT-3, NT-4, NT-5, or NT-6), or a nerve growth factor such as NGF-beta; platelet Derived Growth Factor (PDGF); fibroblast growth factors such as aFGF and bFGF; epidermal Growth Factor (EGF); transforming Growth Factors (TGF), such as TGF-alpha and TGF-beta, including TGF-beta 1, TGF-beta 2, TGF-beta 3, TGF-beta 4, or TGF-beta 5; insulin-like growth factors-I and-II (IGF-I and IGF-II); des (1-3) -IGF-I (brain IGF-I), insulin-like growth factor binding protein (IGFBP); CD proteins such as CD3, CD4, CD8, CD19, and CD 20; erythropoietin; an osteoinductive factor; an immunotoxin; bone Morphogenetic Protein (BMP); interferons such as interferon alpha, beta and gamma; colony Stimulating Factors (CSF), such as M-CSF, GM-CSF, and G-CSF; interleukins (IL), such as IL-1 through IL-10; superoxide dismutase; a T-cell receptor; surface membrane proteins; a decay accelerating factor; viral antigens, such as, for example, part of the AIDS envelope; a transporter protein; a homing receptor; an address element; a regulatory protein; integrins such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4 and VCAM; a tumor associated antigen such as CA125 (ovarian cancer antigen) or HER2, HER3, or HER4 receptor; an immunoadhesin; and fragments and/or variants of any of the above-listed proteins, as well as antibodies, including antibody fragments, that bind to any of the above-listed proteins.

The formulated protein is preferably substantially pure and ideally substantially homogeneous (i.e., free of contaminating proteins). By "substantially pure" protein is meant a composition comprising at least about 90% by weight, preferably at least about 95% by weight, of protein, based on the total weight of the composition. By "substantially homogeneous" protein is meant a composition comprising at least about 99% by weight of protein, based on the total weight of the composition.

In certain embodiments, the protein is an antibody. Antibodies are herein directed against the "antigen" of interest. Preferably, the antigen is a biologically important protein and administration of the antibody to a mammal suffering from a disease or disorder can result in a therapeutic benefit in the mammal. However, antibodies directed against non-protein antigens (e.g., tumor-associated glycolipid antigens; see U.S. Pat. No. 5,091,178) are also contemplated. When the antigen is a protein, it may be a transmembrane molecule (e.g., receptor) or a ligand, such as a growth factor. Exemplary antigens include the proteins described above. Preferred molecular targets for antibodies encompassed by the invention include CD polypeptides, such as CD3, CD4, CD8, CDl9, CD20 and CD 34; a HER receptor family member such as the EGF receptor (HER1), HER2, HER3 or HER4 receptor; cell adhesion molecules such as LFA-1, Mac1, p150, 95, VLA-4, ICAM-1, VCAM, and av/b3 integrin (including its a or b subunit) (e.g., anti-CD 11a, anti-CD 18, or anti-CD 11b antibodies); growth factors such as VEGF; IgE; blood group antigens; flk2/flt3 receptor; obesity (OB) receptors; an mpl receptor; CTLA-4; polypeptide C, and the like. Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for generating antibodies. For transmembrane molecules, such as receptors, fragments thereof (e.g., the extracellular domain of the receptor) can be used as immunogens. Alternatively, cells expressing transmembrane molecules can be used as immunogens. Such cells may be derived from a natural source (e.g., cancer cell lines), or may be cells transformed by recombinant techniques to express the transmembrane molecule.

Examples of antibodies to be purified herein include, but are not limited to: HER2 antibodies, including trastuzumab (trastuzumab)(Carter et al, Proc. Natl. Acad. Sci. USA, 89: 4285-^TM) (WO 01/00245); CD20 antibody (see below); IL-8 antibodies (St John et al, Chest, 103: 932(1993), and International publication No. WO 95/23865); VEGF or VEGF receptor antibodies, including humanized and/or affinity matured VEGF antibodies, e.g., humanized VEGF antibody huA4.6.1 bevacizumabAnd ranibizumab (ranibizumab)(Kim et al, Growth Factors, 7: 53-64 (1992); WO 96/30046 and International publication No. WO 98/45331, 10, 15, 1998); PSCA antibodies (WO 01/40309); CD11a antibodies, including efacizumab (efalizumab)(U.S. Pat. No. 6,037,454, U.S. Pat. No. 5,622,700, WO 98/23761, Stoppa et al, Transplantation Intl.4: 3-7(1991) and Hourmant et al, Transplantation 58: 377-380 (1994)); IgE-binding antibodies, including omalizumab(Presta et al, J.Immunol.151: 2623-2632(1993) and International publication No. WO 95/19181; U.S. patent No. 5,714,338 published on 3.3.1998 or U.S. patent No. 5,091,313 published on 25.2.1992; WO 93/04173 published on 4.3.1993 or International application No. PCT/US98/13410 published on 30.6.30.1998; U.S. patent No. 5,714,338); CD18 antibody (U.S. patent No. 5,622,700 published on 22.4.1997 or WO 97/26912 published on 31.7.1997); Apo 2 receptor antibody (WO 98/51793 published on 19.11.1998); Tissue Factor (TF) antibody (European patent No. 0420937B 1 published on 9.11.1994); α)₄-α₇Integrin antibodies (WO 98/06248 published on 19/2/1998); EGFR antibodies (e.g., chimeric or humanized 225 antibody, cetuximab (cetuximab), as in WO 96/40210 published 12, 19/1996) (ii) a CD3 antibodies, such as OKT3 (U.S. patent No. 4,515,893, 5/7 in 1985); CD25 or Tac antibodies, e.g. CHI-621And(see U.S. Pat. No. 5,693,762 issued on 12/2/1997); CD4 antibodies, such as the cM-7412 antibody (Choy et al, Arthritis Rheum 39 (1): 52-56 (1996)); CD52 antibodies, such as CAMPATH-1H (ILEX/Berlex) (Riechmann et al, Nature 332: 323-337 (1988)); fc receptor antibodies, such as M22 antibody directed against Fc γ RI (e.g., RI in Graziano et al, J.Immunol.155 (10): 4996-5002 (1995)); carcinoembryonic antigen (CEA) antibodies, such as hMN-14(Sharkey et al, Cancer Res.55(23 Suppl): 5935s-5945s (1995)); antibodies against mammary epithelial cells (including huBrE-3, hu-Mc 3 and CHL6) (Ceriani et al, Cancer Res.55 (23): 5852s-5856s (1995); and Richman et al, Cancer Res.55(23 Supp): 5916s-5920s (1995)); antibodies that bind colon cancer cells such as C242(Litton et al, Eur J. Immunol.26 (1): l-9 (1996)); CD38 antibodies, such as AT 13/5(Ellis et al, J.Immunol.155 (2): 925-937 (1995)); CD33 antibodies, such as Hu M195(Jurcic et al, Cancer Res 55(23 Suppl): 5908s-5910s (1995)) and CMA-676 or CDP 771; EpCAM antibodies, e.g. 17-1A(ii) a GpIIb/IIIa antibodies, e.g. abciximab or c7E3 Fab(ii) a RSV antibodies, e.g. MEDI-493(ii) a CMV antibodies, e.g.(ii) a HIV antibodies, such as PRO 542; hepatitis antibodies, e.g. Hep B antibodiesCA125 antibodies including anti-MUC 16(WO 2007/001851; Yin, BWT and Lloyd, KO, J.biol.chem.276: 27371-27375(2001)) and OvaRex, idiotypic GD3 epitope antibody BEC 2; α v β 3 antibodies (for example,(ii) a Medimmune); human renal cell carcinoma antibodies, such as ch-G250; ING-1; anti-human 17-1An antibody (3622W 94); anti-human colorectal tumor antibody (a 33); anti-human melanoma antibody R24 directed against GD3 ganglioside; anti-human squamous cell carcinoma (SF-25); human Leukocyte Antigen (HLA) antibodies such as Smart ID10 and anti-HLA DR antibody Oncolym (Lym-1); CD37 antibodies, such as TRU016 (trubiun); IL-21 antibody (Zymogenetics/Novo Nordisk); anti-B cell antibodies (Impheron); b cell targeting MAb (Immunogen/Aventis); 1D09C3 (Morphosys/GPC); LymphoRad 131 (HGS); lym-1 antibodies, such as Lym-1Y-90(USC) or anti-Lym-1 Oncolym (USC/Peregrine); LIF 226 (EnhancedLifesci.); BAFF antibodies (e.g., WO 03/33658); BAFF receptor antibodies (see, e.g., WO 02/24909); a BR3 antibody; blys antibodies, such as belimumab (belimumab); LYMPHOSTAT-B^TM(ii) a ISF 154 (UCSD/Roche/Tragen); gomilixima (Idec 152; Biogen Idec); IL-6 receptor antibodies, e.g. atlizumab (ACTEMRA)^TMChugai/Roche), IL-15 antibodies such as HuMax-Il-15 (Genmab/Ampen), chemokine receptor antibodies such as CCR2 antibodies (e.g., MLN 1202; Millienum), anti-complement antibodies such as C5 antibodies (e.g., eculizumab (eculizumab), 5G 1.1; Alexion), oral formulations of human immunoglobulins (e.g., IgPO; Protein Therapeutics), IL-12 antibodies such as ABT-874(CAT/Abbott), Teneliximab (Teneliximab) (BMS-224818; BMS), CD40 antibodies including S2C6 and humanized variants thereof (WO00/75348) and TNX 100(Chiron/Tanox), TNF- α antibodies including cA2 infliximab (infliximab)CDP571, MAK-195, adalimumab (HUMIRA)^TM) PEGylated TNF- α antibody fragments, such as CDP-870(Celltech), D2E7(Knoll), anti-TNF- α polyclonal antibodies (e.g., PassTNF; Verigen), CD22 antibodies, such as LL2 or epratuzumab (epratuzumab) ((R))(ii) a Immunomedics) including epratuzumab Y-90 and epratuzumab I-131, AbiogThe CD22 antibody from en (Abiogen, Italy), CMC 544(Wyeth/Celltech), combotox (UT soutwestern), BL22(NIH) and Lymposcan Tc99 (Immunodics).

Examples of CD20 antibodies include: "C2B 8", now known as "Rituximab"(U.S. Pat. No. 5,736,137); yttrium- [90 ]]Labeled 2B8 murine antibody, designated "Y2B 8" or "Ibritumomab Tiuxetan"Commercially available from IDEC Pharmaceuticals, inc (U.S. patent No. 5,736,137; 2B8 stored as ATCC accession No. HB11388 at 22.6.1993); murine IgG2a "B1", also known as "Tositumomab (Tositumomab)", optionally with¹³¹I tag, generated commercially available from Corixa "¹³¹I-B1' or "iodine I¹³¹Tositumomab "antibody (BEXXAR)^TM) (see also U.S. Pat. No. 5,595,721); murine monoclonal antibody "1F 5" (Press et al, Blood 69 (2): 584-591(1987)) and variants or humanizations thereof including "framework patches" 1F5(WO 2003/002607, Leung, S.; ATCC accession No. HB-96450); murine 2H7 and chimeric 2H7 antibody (U.S. patent No. 5,677,180); humanized 2H7(WO 2004/056312, Lowman et al); 2F2(HuMax-CD20), intact human high affinity antibodies that target the CD20 molecule in the cell membrane of B cells (Genmab, Denmark; see, e.g., Glennie and van de Winkel, Drug Discovery Today 8: 503-510(2003) and Cragg et al, Blood 101: 1045-1052 (2003); WO 2004/035607; US 2004/0167319); human monoclonal antibodies shown in WO 2004/035607 and US2004/0167319 (Teeling et al); antibodies with complex N-glycoside-linked sugar chains bound to the Fc region described in US 2004/0093621 (Shitara et al); monoclonal antibodies and antigen-binding fragments that bind CD20 (WO 2005/000901, Tedder et al), such as HB20-3, HB20-4, HB20-25 and MB 20-11; CD20 binding molecules, such as AME series antibodies, for example the AME 33 antibody shown in WO 2004/103404 and US2005/0025764 (Watkins et al, Eli)Lilly/Applied Molecular Evolution, AME); CD20 binding molecules, such as those described in US2005/0025764 (Watkins et al); an a20 antibody or variant thereof, such as a chimeric or humanized a20 antibody (cA 20, hA20, respectively) or IMMU-106(US 2003/0219433, immunology); CD 20-binding antibodies, including epitope-deleted Leu-16, 1H4 or 2B8, optionally conjugated with IL-2, such as US 2005/0069545a1 and WO 2005/16969(Carr et al); bispecific antibodies that bind to CD22 and CD20, e.g., hLL2xhA20(WO2005/14618, Chang et al); monoclonal antibodies L27, G28-2, 93-1B3, B-C1 or NU-B2(Valentine et al, in Leukocyte Typing III (McMichael, eds., page 440, Oxford university Press (1987)), 1H4(Haisma et al, Blood 92: 184(1998)), anti-CD 20auristatin conjugates (Seattle Genetics), anti-CD 20-IL2(EMD/Biovation/City of House), anti-CD 20 Mab therapy (EpiCyte), anti-CD 20 antibody TRU 015 (Trubion).

The term "antibody" herein includes monoclonal antibodies (including full length antibodies with immunoglobulin Fc regions), antibody compositions with polyepitopic specificity, multispecific antibodies (e.g., bispecific antibodies, diabodies, and single chain molecules), and antibody fragments (e.g., Fab, F (ab')₂And Fv). The term "immunoglobulin" (Ig) is used interchangeably herein with "antibody".

The basic 4-chain antibody unit is a heterotetrameric glycoprotein consisting of 2 identical light chains (L) and 2 identical heavy chains (H). IgM antibodies consist of 5 basic heterotetrameric units and an additional polypeptide called the J chain, and contain 10 antigen binding sites; IgA antibodies, in turn, comprise 2-5 basic 4-chain units that can multimerize in combination with the J chain to form a multivalent assembly. In the case of IgG, the 4 chain units are typically about 150,000 daltons. Each L chain is linked to an H chain by 1 covalent disulfide bond, while 2H chains are linked to each other by one or more (depending on the H chain isotype) disulfide bonds. Each H and L chain also has regularly spaced intrachain disulfide bridges. Each H chain has a variable domain at the N-terminus (V)_H) For α and gamma chain, V_HFollowed by 3 constant domains (C)_H) Whereas for μ and isoforms, V_HThe last 4C_HA domain. Each L chain has a variable domain at the N-terminus (V)_L) And a constant domain following its other end. V_LAnd V_HAre paired, and C_LTo the first constant domain (C) of the heavy chain_H1) And (6) pairing. Specific amino acid residues are believed to form interfaces between the light and heavy chain variable domains. Paired V_HAnd V_LTogether forming a single antigen binding site. For the structure and properties of different classes of antibodies see, e.g., basic Clinical Immunology, 8 th edition, Daniel p.sties, Abba i.terr and Tristram g.parsolw (eds.), Appleton&Lange, Norwalk, conn., 1994, page 71 and chapter 6.

Based on the amino acid sequence of its constant domain, L chains from any vertebrate species can be assigned to one of 2 well-defined different types, termed κ and λ. Depending on its heavy chain constant domain (C)_H) There are 5 classes of immunoglobulins, IgA, IgD, IgE, IgG and IgM, with heavy chains named α, gamma and mu, respectively, based on C_HRelatively subtle differences in sequence and function, the γ and α classes are further divided into subclasses, e.g., humans express the following subclasses IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2.

The term "variable" refers to the fact that the sequence of certain fragments of a variable domain differs significantly between antibodies. The V domain mediates antigen binding, defining the specificity of a particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domain. In contrast, the V regions consist of relatively invariant stretches of about 15-30 amino acid residues, called Framework Regions (FRs), separated by extremely variable, shorter regions of about 9-12 amino acid residues each in length, called "hypervariable regions" or sometimes called "complementarity determining regions" (CDRs). The variable domains of each native heavy and light chain comprise 4 FRs, largely adopting a β -sheet conformation, connected by 3 hypervariable regions, forming a circular junction, in some cases forming part of a β -sheet structure. The hypervariable regions in each chain are held together in close proximity by the FRs and together with the hypervariable regions from the other chains contribute to the formation of the antigen-binding site of the antibody (see Kabat et al, Sequences of proteins of Immunological Interest, 5 th edition, Public Health Service, national institutes of Health, Bethesda, Md. (1991). the constant domains are not directly involved in the binding of the antibody to the antigen, but exhibit a variety of effector functions, such as participation in antibody-dependent cellular cytotoxicity (ADCC).

The term "hypervariable region" (also referred to as "complementarity determining regions" or CDRs) refers herein to antibody amino acid residues (typically 3 or 4 short regions of high sequence variability) in the immunoglobulin V region domain that form the antigen-binding site and are the primary determinants of antigen specificity. There are at least two methods for identifying CDR residues: (1) methods based on cross-species sequence variability (i.e., Kabat et al, Sequences of Proteins of Immunological Interest (National Institute of Health, Bethesda, M S1991)); and (2) methods based on crystallographic studies of antigen-antibody complexes (Chothia, C. et al, J.mol.biol.196: 901-917 (1987)). However, both residue identification techniques define overlapping regions rather than identical regions, which can be combined to define hybrid CDRs.

As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous population of antibodies, i.e., a single antibody comprising the same population, except for mutations that may be naturally occurring and/or post-translational modifications (e.g., isomerization, amidation) that may be present in minor amounts. Monoclonal antibodies are highly specific, being directed against a single antigenic site. Furthermore, in contrast to conventional (polyclonal) antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to specificity, monoclonal antibodies are also advantageous in that they are synthesized by hybridoma cultures and are not contaminated with other immunoglobulins. The modifier "monoclonal" indicates that the characteristics of the antibody are obtained from a substantially homogeneous population of antibodies, and is not to be construed as requiring production of the antibody by any particular method. For example, the color may be determined by a color analysis performed by Kohler et al, Nature, 256: 495(1975) or the monoclonal antibody to be used according to the invention can be prepared by recombinant DNA methods (see, for example, U.S. Pat. No. 4,816,567). Also useful are, for example, Clackson et al, Nature, 352: 624-: 581-597(1991), the "monoclonal antibodies" are isolated from phage antibody libraries.

Monoclonal antibodies specifically include "chimeric" antibodies (immunoglobulins) and fragments of such antibodies (so long as the fragments exhibit the desired biological activity) in which a portion of the heavy and/or light chain is identical with or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody type or subclass, and the remainder of the chain is identical with or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody type or subclass (U.S. Pat. No. 4,816,567; Morrison et al, Proc. Natl. Acad. Sci. USA, 81: 6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies comprising variable domain antigen binding sequences derived from a non-human primate (e.g., old world monkey, ape, etc.) and human constant region sequences.

An "intact" antibody is one that comprises an antigen binding site and a CL, and at least a heavy chain domain C_H1、C_H2 and C_H3. The constant domain can be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the intact antibody has one or more effector functions.

Antibody fragments include a portion of an intact antibody, preferably the antigen binding and/or variable regions of an intact antibody. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; linear antibodies (see U.S. Pat. No. 5,641,870, example 2; Zapata et al, Protein Eng.8 (10): 1057-10)62[1995]) (ii) a Single chain antibody molecules formed from antibody fragments and multispecific antibodies.

Papain digestion of antibodies produces 2 identical antigen binding fragments, called "Fab" fragments, and a residual "Fc" fragment, the name reflecting the ability to crystallize readily. The Fab fragment consists of the entire variable domains (V) of the L chain and H chain_H) And a first constant domain of a heavy chain (C)_H1) And (4) forming. For antigen binding, each Fab fragment is monovalent, i.e., it has a single antigen binding site. Pepsin treatment of antibodies produced a single large F (ab')₂Fragment of, said F (ab')₂The fragments roughly correspond to 2 disulfide-linked Fab fragments with different antigen binding activity and are still capable of crosslinking the antigen. Fab' fragments differ from Fab fragments in C_H1 domain has several additional residues at the carboxy terminus, including one or more cysteines from the antibody hinge region. Fab '-SH is herein the name for Fab' in which the cysteine residues of the constant domains have a free thiol group. F (ab')₂Antibody fragments were initially produced as pairs of Fab' fragments with hinge cysteines in between. Other chemical couplings of antibody fragments are also known.

The Fc fragment contains the carboxy-terminal portions of the two H chains held together by a disulfide. The effector functions of antibodies are determined by the sequence of the Fc region, which is also recognized by Fc receptors (fcrs) found on certain cell types.

"Fv" is the smallest antibody fragment that contains a complete antigen recognition and binding site. The fragment consists of a dimer of one heavy and one light chain variable region domain in close, non-covalent association. The folding of these two domains gives rise to 6 hypervariable loops (3 loops each from the H and L chains), provides amino acid residues for antigen binding, and confers antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv comprising 3 CDRs specific for an antigen) has the ability to recognize and bind antigen, albeit with a lower affinity than the entire binding site.

"Single-chain Fv", also abbreviated as "sFv" or "scFv", is an antibody fragment comprising VH and VL antibody domains joined into a single polypeptide chain. Preferably, the sFv polypeptide is further comprised in V_HAnd V_LA polypeptide linker between the domains that enables the sFv to form the structure required for antigen binding. For an overview of sFv, see Pluckthun in the pharmaceutical of monoclonal antibodies, Vol.113, Rosenburg and Moore, Springer-Verlag, New York, p.269-315 (1994).

The term "diabodies" refers to diabodies that are constructed at V_HAnd V_LSmall antibody fragments prepared from sFv fragments (see previous section) with short linkers (about 5-10 residues) between the domains, such that inter-chain rather than intra-chain V domain pairing is formed, resulting in a bivalent fragment, i.e. a fragment with 2 antigen binding sites. Bispecific diabodies are heterodimers of two "crossover" sFv fragments, where the V of both antibodies_HAnd V_LThe domains are present on different polypeptide chains. Diabodies are described in detail in, for example, EP 404,097; WO 93/11161; hollinger et al, proc.natl.acad.sci.usa 90: 6444- > 6448 (1993).

An antibody that "specifically binds" or is "specific for" a particular polypeptide or an epitope on a particular polypeptide is an antibody that binds to the particular polypeptide or an epitope on the particular polypeptide, but does not substantially bind to any other polypeptide or polypeptide epitope.

The term "solid phase" describes a non-aqueous matrix to which the antibodies of the invention can be attached. Examples of solid phases encompassed herein include solid phases formed partially or completely from glass (e.g., controlled pore glass), polysaccharides (e.g., agarose), polyacrylamides, polystyrenes, polyvinyl alcohols, and polysiloxanes. In certain embodiments, depending on the context, the solid phase may comprise the wells of an assay plate; in other cases, it is a purification column (e.g., an affinity chromatography column). The term also includes discontinuous solid phases of dispersed particles, such as those described in U.S. Pat. No. 4,275,149.

"humanized" forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains or fragments thereof (e.g., Fv, Fab ', F (ab') which are mostly human in sequence₂Or other antigen-binding subsequences of antibodies) that contain minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region (also a CDR) of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody) (e.g., mouse, rat, or rabbit) having the desired specificity, affinity, and capacity. In some cases, Fv Framework Region (FR) residues of the human immunoglobulin are substituted for corresponding non-human residues. In addition, as used herein, a "humanized antibody" also comprises residues that are present in neither the recipient antibody nor the donor antibody. These modifications were made to further improve and optimize antibody performance. The humanized antibody optionally further comprises at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For further details, see Jones et al, Nature, 321: 522-525 (1986); reichmann et al, Nature, 332: 323-329 (1988); and Presta, curr.op.struct.biol., 2: 593-596(1992).

A "species-dependent antibody", e.g., a mammalian anti-human IgE antibody, is an antibody that binds with greater affinity to an antigen from a first mammalian species than to a homolog of the antigen from a second mammalian species. In general, a species-dependent antibody "specifically binds" to a human antigen (i.e., has no more than about 1x 10)^{- 7}M, alternatively no more than about 1x10^-8M, alternatively no more than about 1x10^-9M, but has a binding affinity (Kd) for an antigen homolog from a second non-human mammalian species that is at least about 50-fold, at least about 500-fold, or at least about 1000-fold weaker than its binding affinity for a non-human antigen. The species-dependent antibody may be any of the various antibody types described above, but is preferably a humanized or human antibodyAn antibody.

The "effector function" of an antibody refers to the biological activity attributed to the Fc region of an antibody (either the native sequence Fc region or the amino acid sequence variant Fc region), and varies with the antibody isotype. Examples of effector functions of antibodies include: c1q binding and complement-dependent cytotoxicity; fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; down-regulation of cell surface receptors (e.g., B cell receptors); and B cell activation.

"antibody-dependent cell-mediated cytotoxicity" or ADCC refers to a form of cytotoxicity in which secreted Ig binds to Fc receptors (fcrs) present on certain cytotoxic cells (e.g., natural killer cells (NK), neutrophils, and macrophages), enabling these cytotoxic effector cells to specifically bind to target cells bearing antigens, and then killing the target cells with cytotoxins. Antibodies "arm" cytotoxic cells and are necessary to kill target cells by this mechanism. The primary cell mediating ADCC, NK cells, expresses only Fc γ RIII, whereas monocytes express Fc γ RI, Fc γ RII and Fc γ RIII. Ravatch and Kinet, annu.rev.immunol.9: fc expression on hematopoietic cells is summarized in table 3 on page 464 of 457-92 (1991). To assess ADCC activity of a target molecule, an in vitro ACDD assay may be performed, as described in U.S. Pat. No. 5,500,362 or 5,821,337. Effector cells useful for such assays include Peripheral Blood Mononuclear Cells (PBMC) and natural killer cells (NK). Alternatively, or additionally, the ADCC activity of the target molecule may be assessed in vivo, for example, in a cell as described by Clynes et al, PNAS USA 95: 652-.

"Fc receptor" or "FeR" describes a receptor that binds to the Fc region of an antibody. A preferred FcR is a native sequence human FcR. Furthermore, preferred fcrs are those which bind IgG antibodies (gamma receptors) and include the fcrs of the Fc γ RI, Fc γ RII and Fc γ RIII subclasses, including allelic variants and alternatively cleaved forms of these receptors, Fc γ RII receptors including Fc γ RIIA ("activating receptor") and Fc γ RIIB ("inhibiting receptor"), having similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor Fc γ RIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor Fc γ RIIB contains an immunoreceptor tyrosine-based inhibitory motif (ITIM) in its cytoplasmic domain (see, M.Daeron, Annu.Rev.Immunol.15: 203-234 (1997)). Ravatch and Kinet, annu.rev.immunol.9: 457-92 (1991); capel et al, immunolmethods 4: 25-34 (1994); and deHaas et al, j.lab.clin.med.126: FcR was reviewed in 330-41 (1995). Other fcrs, including those to be identified in the future, are encompassed by the term "FcR" herein. The term also includes the neonatal receptor FcRn, which is responsible for transfer of maternal IgG into the fetus. Guyer et al, j.immunol.117: 587(1976) and Kim et al, j.immunol.24: 249(1994).

A "human effector cell" is a leukocyte that expresses one or more fcrs and performs effector functions. Preferably, the cell expresses at least FcRIII and exerts effector functions. Examples of human leukocytes that mediate ADCC include Peripheral Blood Mononuclear Cells (PBMCs), natural killer cells (NK), monocytes, cytotoxic T cells, and neutrophils, with PBMC and MNK cells being preferred. The effector cells may be isolated from a natural source (e.g., blood).

"complement-dependent cytotoxicity" or "CDC" refers to the lysis of a target cell in the presence of complement. Activation of the classical complement pathway is initiated by the binding of the first component of the complement system (C1q) to antibodies (of the appropriate subclass) that bind their cognate antigen. To assess complement activation, one can implement, for example, the methods described in Gazzano-Santoro et al, j.immunol.methods 202: 163 (1996).

When "isolated" is used to describe the various polypeptides and antibodies disclosed herein, it is meant that the polypeptide or antibody has been identified, isolated and/or recovered from a component of its production environment. Preferably, an isolated polypeptide is not associated with all other components from its production environment. Contaminating components of its production environment, such as those caused by recombinant transfected cells, are materials that generally interfere with diagnostic or therapeutic uses for the polypeptide, and may include enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In preferred embodiments, the polypeptide will be purified (1) to an extent sufficient to obtain at least 15 residues of the N-terminal or internal amino acid sequence by using a spinning cup sequencer, or (2) homogeneous (by SDS-PAGE under non-reducing or reducing conditions using Coomassie blue or, preferably, silver staining). Generally, however, an isolated polypeptide or antibody is prepared by at least one purification step.

An "isolated" nucleic acid molecule encoding a polypeptide and antibody herein is a nucleic acid molecule identified and isolated from at least one contaminating nucleic acid molecule with which it is ordinarily associated in its production environment. Preferably, an isolated nucleic acid is not associated with all components associated with the production environment. Isolated nucleic acid molecules encoding the polypeptides and antibodies herein are in a form or environment other than that in which they are naturally found. Thus, an isolated nucleic acid molecule is distinct from a nucleic acid encoding a polypeptide or antibody as found naturally in a cell herein.

The term "control sequences" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Suitable control sequences for prokaryotes include, for example, a promoter, optionally an operator sequence, and a ribosome binding site. Eukaryotic cells are known to utilize promoters, polyadenylation signals, and enhancers.

A nucleic acid is "operably linked" when it is placed into a functional relationship with another nucleic acid sequence. For example, DNA for a presequence or secretion leader is operably linked to DNA for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or if the ribosome binding site is placed so as to facilitate translation, it is operably linked to a coding sequence. In general, "operably linked" means that the linked DNA sequences are contiguous and, in the case of secretory leader sequences, contiguous and in reading phase. However, enhancers need not be contiguous. Ligation is achieved by ligation at convenient restriction sites. If such sites are not present, synthetic oligonucleotide adaptors or linkers are used according to conventional practice.

The term "epitopic" as used herein refers to a chimeric polypeptide comprising a polypeptide or antibody as described herein fused to a "tag polypeptide". The tag polypeptide has enough residues to provide an epitope against which an antibody is made, yet is short enough so as not to interfere with the activity of the polypeptide to which it is fused. Preferably, the tag polypeptide is sufficiently unique that the antibody does not substantially cross-react with other epitopes. Suitable tag polypeptides generally have at least 6 amino acid residues, and typically between about 8 and 50 amino acid residues (preferably between about 10 and 20 amino acid residues).

As used herein, the term "immunoadhesin" refers to antibody-like molecules that combine the binding specificity of a heterologous protein ("adhesin") with the effector functions of an immunoglobulin constant domain. Structurally, immunoadhesins comprise fusions of amino acid sequences having the desired binding specificity and immunoglobulin constant domain sequences that are distinct from the antigen recognition and binding sites of the antibody (i.e., "heterologous"). The adhesion moiety of an immunoadhesin molecule is typically a contiguous sequence of amino acids comprising at least a binding site for a receptor or ligand. The immunoglobulin constant domain sequence in the immunoadhesin may be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3 or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD or IgM. Ig fusions preferably include the replacement of at least one variable region within an Ig molecule with a domain of a polypeptide or antibody described herein. In particularly preferred embodiments, the immunoglobulin fusion comprises the hinge, CH2 and CH3, or hinge, CH1, CH2 and CH3 regions of an IgG1 molecule. For the production of immunoglobulin fusions see also U.S. Pat. No. 5,428,130, published 6, 7 mesh, 1995.

The term "pharmaceutical formulation" refers to a preparation in a form that allows the biological activity of the active ingredient to be effective and that does not contain other components of unacceptable toxicity to the subject to which the formulation is administered.

An antibody has "biological activity" in a pharmaceutical formulation if the biological activity of the antibody at a given time is within about 10% (within the error of the assay) of the biological activity exhibited when the pharmaceutical formulation was prepared, as first determined by the ability of the antibody to bind to an antigen in vitro or in vivo and the resulting measurable biological response.

A "stable" or "stabilized" formulation is one in which the protein substantially retains its physiological and/or chemical stability after storage. Stability can be measured at a selected temperature for a selected period of time. Preferably, the formulation is stable at room temperature (-30 ℃) or at 40 ℃ for at least 1 month, and/or at about 2-8 ℃ for at least 1 year, preferably at least 2 years. For example, the degree of aggregation during storage can be used as an indicator of protein stability. Thus, a "stable" formulation may be one in which less than about 10%, and preferably less than about 5%, of the protein is present in the formulation as aggregates. A variety of analytical techniques for measuring Protein stability are available in the art, reviewed, for example, in peptidean Protein Drug Delivery, 247-: 29-90 (1993).

Increasing the "stability" of a protein-containing formulation refers to reducing (as compared to an untreated protein-containing formulation) or preventing the formation of protein aggregates in the formulation.

The term "aqueous solution" refers to a solution in which water is the dissolution matrix or solvent. When a substance is dissolved in a liquid, the mixture is called a solution. The dissolved substance is the solute and the liquid in which the dissolution is carried out is the solvent (in this case water).

The term "stabilizing agent" or "stabilizer" is used herein to refer to chemicals or compounds added to solutions and mixtures or suspensions or compositions or therapeutic compositions to maintain them in a stable or unaltered state; or chemicals or compounds used because they produce changes involving atoms or molecules, resulting in a more stable or unchanged state.

The term "aggregate" or "aggregation" herein means to be brought together, or to be aggregated or integrated, e.g. as in the aggregation of peptides, polypeptides, antibodies or variants thereof. Aggregates may self-aggregate, or aggregate due to other factors, such as aggregating agents, precipitating agents, agitation, or other means and methods, resulting in the peptides, polypeptides, antibodies, or variants thereof, being brought together.

Agitation-induced aggregation is the formation of aggregates induced by agitation in a solution containing protein, where agitation is a motion induced by shaking or agitation.

An antibody that is "susceptible to aggregation" is one that is observed to aggregate with other antibody molecules, particularly under conditions of agitation.

By "inhibiting" agitation-induced aggregation is meant preventing, reducing, or reducing the amount of agitation-induced aggregation as measured by comparing the amount of aggregates present in a protein-containing solution that contains at least one agitation-induced aggregated inhibitor to the amount of aggregates present in a protein-containing solution that does not contain at least one agitation-induced aggregated inhibitor.

The amount of inhibition of agitation-induced aggregation is the amount of inhibition of agitation-induced aggregation.

Methods useful in the present invention for measuring agitation-induced aggregation include gel electrophoresis, isoelectric focusing, capillary electrophoresis, chromatography such as size exclusion chromatography, ion exchange chromatography and reverse-phase high performance liquid chromatography, peptide mapping, oligosaccharide mapping, mass spectrometry, ultraviolet absorption spectroscopy, fluorescence spectroscopy, circular dichroism spectroscopy, isothermal titration calorimetry, differential scanning calorimetry, analytical ultracentrifugation, dynamic light scattering, proteolysis and cross-linking, turbidity measurements, filtration retardation assays, immunoassays, fluorescent dye binding assays, protein staining assays, microscopy, and detection of aggregation by ELISA or other binding assays.

An "isotonic" formulation is one that has substantially the same osmotic pressure as human blood. Isotonic formulations generally have an osmotic pressure of from about 250 to 350 mOsm. The term "hypotonic" describes a formulation having an osmotic pressure that is lower than that of human blood. Accordingly, the term "hypertonic" is used to describe a formulation having an osmotic pressure higher than that of human blood. Isotonicity can be measured, for example, using a vapor pressure or ice-freezing type osmometer. The formulations of the present invention are hypertonic as a result of the addition of salts and/or buffers.

A "reconstituted" formulation is a formulation prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration (e.g., parenteral administration) to a patient to be treated with the protein of interest, and in certain embodiments of the invention, may be a reconstituted formulation suitable for subcutaneous administration.

"surfactants" are surface active agents that can act on the surface of solid-solid, solid-liquid, liquid-liquid, and liquid-gas because their chemical composition contains hydrophilic and hydrophobic groups. These materials reduce the concentration of proteins in dilute solutions at air-water and/or water-solid interfaces where proteins can adsorb and possibly aggregate. The surfactant may bind to a hydrophobic interface in the protein formulation. Due to unfolding and subsequent aggregation of the protein monolayer, the proteins on the water surface will aggregate, especially when stirred.

"surfactants" may denature proteins, but may also stabilize them against surface denaturation. In general, ionic surfactants can denature proteins. However, even at relatively high concentrations (1% w/v), nonionic surfactants do not generally denature proteins. Most of the parent acceptable nonionic surfactantsFrom polysorbates or polyethers. Polysorbates 20 and 80 are contemporary surfactant stabilizers in commercially available protein formulations. However, other surfactants used in protein formulations include Pluronic F-68 and members of the "Brij" class. The nonionic surfactant may be sugar-based. The sugar-based surfactant may be an alkyl glycoside. The general structure of the alkyl glycoside is R₁-O-(CH₂) x-R, wherein R is independently CH₃Or cyclohexyl (C)₆H₁₁)，R₁Independently glucose or maltose. Exemplary alkylglycosides include those alkylglycosides wherein R is₁Is glucose, R is CH₃And x is 5 (n-hexyl- β -D-glucopyranoside), x is 6 (n-heptyl- β -D-glucopyranoside), x is 7 (n-octyl- β -D-glucopyranoside), x is 8 (n-nonyl- β -D-glucopyranoside), x is 9 (n-decyl- β -D-glucopyranoside), and x is 11 (n-dodecyl- β -D-glucopyranoside)₁Is maltose, R is CH₃And x is 5 (n-hexyl- β -D-maltopyranoside), x is 7 (n-octyl- β -D-maltopyranoside), x is 8 (n-nonyl- β -D-maltopyranoside), x is 9 (n-decyl- β -D-maltopyranoside), x is 10 (n-undecyl- β -D-maltopyranoside), x is 11 (n-dodecyl- β -D-maltopyranoside), x is 12 (n-tridecyl- β -D-maltopyranoside), x is 13 (n-tetradecyl- β -D-maltopyranoside), and x is 15 (n-hexadecyl- β -D-maltopyranoside)₁Is glucose, x is 3, and R is cyclohexyl (3-cyclohexyl-1-propyl- β -D-glucoside), wherein R is₁Is maltose, x is 4, and R is cyclohexyl (4-cyclohexyl-1-butyl- β -D-maltoside).

"pharmaceutically acceptable acids" include inorganic and organic acids that are non-toxic in the concentrations and manner of formulation. For example, suitable inorganic acids include hydrochloric acid, perchloric acid, hydrobromic acid, hydroiodic acid, nitric acid, sulfuric acid, sulfonic acid, sulfinic acid, sulfanilic acid, phosphoric acid, carbonic acid, and the like. Suitable organic acids include straight and branched chain alkanes, aromatic hydrocarbons, cyclic hydrocarbons, alicyclic, araliphatic, heterocyclic, saturated, unsaturated, mono-, di-and tricarboxylic acids, including, for example, formic acid, acetic acid, 2-glycolic acid, trifluoroacetic acid, phenylacetic acid, trimethylacetic acid, t-butylacetic acid, aminobenzoic acid, propionic acid, 2-hydroxypropionic acid, 2-oxopropionic acid, malonic acid, cyclopentylpropionic acid, 3-phenylpropionic acid, butyric acid, succinic acid, benzoic acid, 3- (4-hydroxybenzoyl) benzoic acid, 2-acetoxy-benzoic acid, ascorbic acid, cinnamic acid, dodecylsulfuric acid, stearic acid, muconic acid, mandelic acid, succinic acid, embonic acid (embonic acid), fumaric acid, malic acid, maleic acid, hydroxymaleic acid, malic acid, cinnamic acid, malonic acid, lactic acid, citric acid, tartaric acid, glycolic acid, gluconic acid, pyruvic acid, glyoxylic acid, oxalic acid, methanesulfonic acid, succinic acid, salicylic acid, phthalic acid, palmoic acid, palmeic acid, thiocyanic acid, methanesulfonic acid, ethanesulfonic acid, 1, 2-diethylsulfonic acid, 2-hydroxyethanesulfonic acid, benzenesulfonic acid, 4-chlorobenzenesulfonic acid, naphthalene-2-sulfonic acid, p-methylbenzenesulfonic acid, camphorsulfonic acid, 4-methylbicyclo [2.2.2] -oct-2-ene-1-carboxyl, glucoheptonic acid, 4' -methylenebis-3- (hydroxy-2-ene-1-carboxylic acid), hydroxynaphthoic acid.

"pharmaceutically acceptable bases" include inorganic and organic bases that are non-toxic in the concentrations and manner of formulation. For example, suitable bases include bases formed from metals that form inorganic bases, such as lithium, sodium, potassium, magnesium, calcium, ammonium, iron, zinc, copper, manganese, aluminum, N-methylglucamine, morpholine, piperidine, and organic non-toxic bases including primary, secondary and tertiary amines, substituted amines, cyclic amines and basic ion exchange resins (e.g., N (R')₄ ⁺[ wherein R' is independently H or C_1-4Alkyl groups, e.g., ammonium, Tris), e.g., isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, ethanolamine, 2-diethylaminoethanol, trimethamine, dicyclohexylamine, lysine, arginine, histidine, caffeine, procaine, hydrabamine, choline, betaine, ethylenediamine, glucosamine, methylglucamine, theobromine, and the likePurines, piperazine, piperidine, N-ethylpiperidine, polyamine resins, and the like. Particularly preferred organic non-toxic bases are isopropylamine, diethylamine, ethanolamine, trimethamine, dicyclohexylamine, choline and caffeine.

Other pharmaceutically acceptable acids and bases that may be used in the present invention include acids and bases derived from amino acids such as histidine, glycine, phenylalanine, aspartic acid, glutamic acid, lysine and asparagine.

"pharmaceutically acceptable" buffers and salts include those derived from acid and base addition salts of the above acids and bases. Specific buffers and/or salts include histidine, succinate and acetate.

A "lyoprotectant" is a molecule that, when combined with a protein of interest, substantially prevents or reduces the physicochemical instability of the protein upon lyophilization and subsequent storage. Exemplary lyoprotectants include sugars and their corresponding sugar alcohols; amino acids, such as monosodium glutamate or histidine; methylamines, such as betaine; lyotropic salts, such as magnesium sulfate; polyols, such as trihydric or higher molecular weight sugar alcohols, such as glycerol, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol;(ii) a And combinations thereof. Other exemplary lyoprotectants include glycerol and gelatin, as well as the saccharides melibiose, melezitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyols. Preferred sugar alcohols are monoglycosides, especially compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The side chain group of the glycoside may be a glucoside or a galactoside. Other examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred lyoprotectants are non-reducing sugarsTrehalose or sucrose.

The lyoprotectant is added to the pre-lyophilized formulation in a "lyoprotecting amount," which means that the protein substantially retains its physicochemical stability upon lyophilization and storage after lyophilization in the presence of the lyoprotecting amount of the lyoprotectant.

A "pharmaceutically acceptable sugar" is a molecule that, when combined with a protein of interest, substantially prevents or reduces the physicochemical instability of the protein in storage. When the formulation is intended to be lyophilized and then reconstituted, the "pharmaceutically acceptable sugar" is also referred to as a "lyoprotectant". Exemplary sugars and their corresponding sugar alcohols include: amino acids, such as sodium glutamate or histidine; methylamines, such as betaine; lyotropic salts, such as magnesium sulfate; polyols, such as trihydric or higher molecular weight sugar alcohols, such as glycerol, dextran, erythritol, glycerol, arabitol, xylitol, sorbitol and mannitol; propylene glycol; polyethylene glycol;(ii) a And combinations thereof. Other exemplary lyoprotectants include glycerol and gelatin, as well as the saccharides melibiose, melezitose, raffinose, mannotriose, and stachyose. Examples of reducing sugars include glucose, maltose, lactose, maltulose, isomaltulose and lactulose. Examples of non-reducing sugars include non-reducing glycosides of polyhydroxy compounds selected from sugar alcohols and other linear polyols. Preferred sugar alcohols are monoglycosides, especially compounds obtained by reduction of disaccharides such as lactose, maltose, lactulose and maltulose. The side chain group of the glycoside may be a glucoside or a galactoside. Other examples of sugar alcohols are glucitol, maltitol, lactitol and isomaltulose. Preferred lyoprotectants are the non-reducing sugars trehalose or sucrose.

The addition of a pharmaceutically acceptable sugar to a (e.g., pre-lyophilized) formulation in a "protective amount" means that the protein substantially retains its physicochemical stability during storage (e.g., after reconstitution and storage).

The "diluent" of interest herein is pharmaceutically acceptable (safe and non-toxic for administration to humans) and is useful for preparing liquid formulations (e.g., formulations that are reconstituted after lyophilization). Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose [ solution. In alternative embodiments, the diluent may comprise an aqueous solution of a salt and/or a buffer.

A "preservative" is a compound that can be added to the formulations herein to reduce bacterial activity. For example, the addition of a preservative may facilitate the production of a multiple use (multi-dose) formulation. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides, where the alkyl group is a long chain compound), and benzethonium chloride. Other types of preservatives include aromatic alcohols such as phenol, butanol and benzyl alcohol, alkyl parabens such as methyl or propyl parabens, catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. The most preferred preservative herein is benzyl alcohol.

"treatment" refers to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already suffering from the disease, as well as those in need of prevention of the condition.

"mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, domesticated and farm animals, and zoo, sports, or pet animals, such as dogs, horses, rabbits, cattle, pigs, hamsters, gerbils, mice, ferrets, rats, cats, and the like. Preferably, the mammal is a human.

A "disorder" is any condition that benefits from treatment with a protein. Including chronic and acute disorders or diseases, including those pathological conditions that induce the disorder in question in a mammal. Non-limiting examples of conditions treated herein include cancer and inflammation.

A "therapeutically effective amount" is the minimum concentration required to produce a measurable improvement in, or prevention of, a particular condition. A therapeutically effective amount of a protein is known to be generally known in the art, and a therapeutically effective amount of a protein as hereinafter discovered can be determined by standard techniques generally known to those skilled in the art (e.g., a general physician).

Methods for the preparation of antibodies (including toxin-conjugated antibodies) and other proteins formulated as described herein are generally known in the art and are described in detail, for example, in WO 2007/001851.

Antibodies and other proteins may be formulated in accordance with the present invention in aqueous or lyophilized form, which is capable of reconstitution into an aqueous form.

The formulations described herein may be prepared as reconstituted lyophilized formulations. The proteins or antibodies described herein are lyophilized and then reconstituted to produce the liquid formulations of the invention. In this particular embodiment, after the protein of interest is prepared as described above, a "pre-lyophilized" formulation is produced. The amount of protein present in the pre-lyophilized formulation is determined taking into account the desired dose volume, mode of administration, etc. For example, the starting concentration of intact antibody may be from about 2mg/ml to about 50mg/ml, preferably from about 5mg/ml to about 40mg/ml and most preferably from about 20-30 mg/ml.

The protein to be formulated is generally present in solution. For example, in the liquid formulations of the present invention, the protein may be present in a pH buffered solution at a pH of from about 4 to 8, and preferably about 5 to 7. The buffer concentration may be from about 1mM to about 20mM, alternatively from about 3mM to about 15mM, depending on, for example, the desired degree of tonicity (tonicity) of the buffer and formulation (e.g., of the reconstituted formulation). Exemplary buffers and/or salts are pharmaceutically acceptable and may be produced from suitable acids, bases and salts thereof, such as those defined under "pharmaceutically acceptable" acids, bases or buffers.

In one embodiment, a lyoprotectant is added to the pre-lyophilized formulation. The amount of lyoprotectant in the pre-lyophilized formulation is generally such that the resulting formulation upon reconstitution is isotonic. However, hypertonic reconstituted formulations may also be suitable. Furthermore, the amount of lyoprotectant cannot be too low, so that unacceptable amounts of protein degradation/aggregation occur upon lyophilization. However, exemplary lyoprotectant concentrations in the pre-lyophilized formulation are from about 10mM to about 400mM, alternatively from about 30mM to about 300mM, alternatively from about 50mM to about 100 mM. Exemplary lyoprotectants include sugars and sugar alcohols, such as sucrose, mannose, trehalose, glucose, sorbitol, mannitol. However, certain lyoprotectants may also have an effect on increasing the viscosity of the formulation under certain circumstances. Therefore, care needs to be taken in selecting a particular lyoprotectant that minimizes or neutralizes this effect. Other lyoprotectants are described in the definition of "lyoprotectant" above, which is also referred to herein as a "pharmaceutically acceptable sugar".

The ratio of protein to lyoprotectant may vary for each particular protein or combination of antibody and lyoprotectant. In the case where an antibody is selected as the protein and a sugar (e.g., sucrose or trehalose) as the lyoprotectant for generating an isotonic reconstituted formulation with a high protein concentration, the molar ratio of lyoprotectant to antibody can be from about 100 to about 1500 moles lyoprotectant to 1 mole antibody, and preferably from about 200 to about 1000 moles lyoprotectant to 1 mole antibody, e.g., from about 200 to about 600 moles lyoprotectant to 1 mole antibody.

A mixture of lyoprotectants (e.g., sucrose or trehalose) and bulking agents (e.g., mannitol or glycerol) can be used to prepare the pre-lyophilized formulation. The bulking agent may allow the production of a homogeneous lyophilized cake without excessive voids or the like therein. Other pharmaceutically acceptable carriers, excipients or stabilizers, such as those described in Remington's Pharmaceutical Sciences 16 th edition, Osol, a. eds (1980), may also be included in the pre-lyophilized formulation (and/or lyophilized and/or reconstituted) so long as they do not adversely affect the desired characteristics of the formulation. Acceptable carriers, excipients, or stabilizers are non-toxic to recipients at the dosages and concentrations employed, and include: other buffering agents; a preservative; a co-solvent; antioxidants, including ascorbic acid and methionine; chelating agents such as EDTA; metal complexes (e.g., Zn-protein complexes); biodegradable polymers such as polyesters; and/or salt-forming counterions, such as sodium.

The formulations herein may also contain more than one protein necessary for a particular therapeutic indication, preferably a protein having complementary activity that does not adversely affect other proteins. For example, it may be desirable to provide two or more antibodies that bind a desired target (e.g., receptor or antigen) in a single formulation. Such proteins are suitably present in combination in an amount effective for the intended purpose.

The formulations used for in vivo administration must be sterile. This may conveniently be achieved by filtration through sterile filtration membranes before or after lyophilization and reconstitution. Alternatively, sterilization of the entire mixture may be achieved by, for example, autoclaving the ingredients other than the protein at about 120 ℃ for about 30 minutes.

After mixing the protein, optional lyoprotectant, and other optional components together, the formulation is lyophilized. There are a number of different freeze dryers available for this purpose, for example Hull50^TM(Hull, USA) or GT20^TM(Leybold-Heraeus, Germany) lyophilizer. Lyophilization is accomplished by freezing the formulation and subsequently subliming ice from the frozen contents at a temperature suitable for primary drying. Under this condition, the product temperature is below the eutectic point or collapse temperature of the formulation. Typically, the storage temperature (shelf temperature) for primary drying ranges from about-30 to 25 deg.C (provided that the product remains frozen during primary drying) at a suitable pressure (typically from about 50-250 mTorr). The formulation, size and type of container (e.g., glass bottle) holding the sample, and volume of liquid will primarily determine the time required for drying, which can range from several hours to several days (e.g., 40-60 hrs). Optionally, depending on the desired residual moisture level in the product, a secondary drying stage may also be implemented. The temperature for performing the secondary drying is about 0 to 40 deg.CThe range of C depends primarily on the type and size of the container, and the type of protein used. For example, the storage temperature throughout the water removal stage of lyophilization can be from about 15-30 ℃ (e.g., about 20 ℃). The time and pressure required for secondary drying is the time and pressure to produce a suitable lyophilized cake, depending on, for example, temperature and other parameters. The time for secondary drying is determined by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g., 10-15 hours). The pressure may be the same as the pressure used during the primary drying step. The freeze-drying conditions may vary depending on the formulation and vial size.

Reconstituting the lyophilized formulation with a pharmaceutically acceptable diluent prior to administration to a patient such that the protein concentration in the reconstituted formulation is at least about 50mg/ml, for example from about 50mg/ml to about 400mg/ml, alternatively from about 80mg/ml to about 300mg/ml, alternatively from about 90mg/ml to about 150 mg/ml. Such high protein concentrations in reconstituted formulations are considered particularly useful when subcutaneous delivery of the reconstituted formulation is desired. However, for other routes of administration, such as intravenous administration, lower protein concentrations in the reconstituted formulation may be desirable (e.g., from about 5-50mg/ml, or from about 10-40mg/ml protein in the reconstituted formulation). In certain embodiments, the protein concentration in the reconstituted formulation is significantly higher than the protein concentration in the pre-lyophilized formulation. For example, the protein concentration in the reconstituted formulation may be about 2-40 times, alternatively 3-10 times, alternatively 3-6 times (e.g., at least 3 times or at least 4 times) the concentration in the pre-lyophilized formulation.

Reconstitution generally occurs at a temperature of about 25 ℃ to ensure adequate hydration, although other temperatures may be used if desired. The time required for reconstitution depends on, for example, the type of diluent, excipients and the amount of protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWF), pH buffered solutions (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives As noted above, preferred preservatives are aromatic alcohols such as benzyl alcohol or phenol alcohol. The amount of preservative used is determined by evaluating the compatibility of different preservative concentrations with the protein and preservative efficacy tests. For example, if the preservative is an aromatic alcohol (e.g., benzyl alcohol), it may be present in an amount of from about 0.1-2.0%, and preferably from about 0.5-1.5%, but most preferably from about 1.0-1.2%.

Preferably, the reconstituted formulation has less than 6000 particles ≧ 10 μm in size per vial.

Therapeutic formulations for storage are prepared by mixing the active ingredient in the desired purity with optional pharmaceutically acceptable carriers, excipients or stabilizers (Remington's Pharmaceutical Sciences, 18 th edition, mack publishing co., Easton, pa.18042[1990 ]). Acceptable carriers, excipients, or stabilizers are nontoxic to recipients at the dosages and concentrations employed, and include buffers; antioxidants, including ascorbic acid, methionine, vitamin E, sodium metabisulfite, preservatives, isotonicifiers, stabilizers, metal complexes (e.g., Zn-protein complexes), and/or chelating agents, such as EDTA.

When the therapeutic agent is an antibody fragment, a minimal fragment that specifically binds to the binding domain of the target protein is preferred. For example, based on the variable region sequences of the antibody, antibody fragments, or even peptide molecules, can be designed that retain the ability to bind to the target protein sequence. Such peptides may be chemically synthesized and/or produced by recombinant DNA techniques (see, e.g., Marasco et al, Proc. Natl. Acad. Sci. USA 90: 7889-.

Buffers are used to control pH within a range that optimizes therapeutic efficacy, especially where stability is pH dependent. The buffer is preferably present at a concentration of from about 50mM to about 250 mM. Suitable buffering agents for use in the present invention include organic and inorganic acids, and salts thereof. For example, citric acid, phosphoric acid, succinic acid, tartaric acid, fumaric acid, gluconic acid, oxalic acid, lactic acid, acetic acid. In addition, the buffer may consist of histidine and a trimethylamine salt such as Tris.

Preservatives are added to retard microbial growth, typically present in the range of from 0.2% to 1.0% (w/v). Suitable preservatives for use in the present invention include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium halides (e.g., chloride, bromide, iodide), benzethonium chloride, thimerosal, phenol, butanol or benzyl alcohol; alkyl parabens such as methyl or propyl paraben; catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol.

Tonicity agents, sometimes referred to as "stabilizers," are present to adjust or maintain the osmotic pressure of the liquid composition. When used with large charged biomolecules (e.g., proteins and antibodies), they are often referred to as "stabilizers" because they can interact with the charged groups of the amino acid side chains, thereby reducing the likelihood of intermolecular and intramolecular interactions. The tonicity agent may be present in any amount between 0.1% to 25% by weight, preferably 1 to 5% by weight, taking into account the relative amounts of the other ingredients. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

Other excipients include active agents that may act as one or more of the following: (1) an extender, (2) a solubility enhancer, (3) a stabilizer, and (4) an active agent that prevents denaturation or adsorption to the walls of the container. Such excipients include: polyhydric sugar alcohols (enumerated above); amino acids such as alanine, glycine, glutamine, asparagine, histidine, arginine, lysine, ornithine, leucine, 2-phenylalanine, glutamic acid, threonine, and the like; organic sugars or sugar alcohols, such as sucrose, lactose, lactitol, trehalose, stachyose, mannose, sorbose, xylose, ribose, ribitol, myoisitose, myoisitol, galactose, galactitol, glycerol, cyclitols (e.g., inositol) polyethylene glycol; sulfur-containing reducing agents such as urea, glutathione, lipoic acid, sodium thioglycolate, thioglycerol, α -monothioglycerol and sodium thiosulfate; low molecular weight proteins, such as human serum albumin, bovine serum albumin, gelatin, or other immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone; monosaccharides (e.g., xylose, mannose, fructose, glucose; disaccharides (e.g., lactose, maltose, sucrose), trisaccharides such as raffinose, and polysaccharides such as dextrin or dextran.

For the formulation to be administered in vitro, it must be sterile. The preparation may be rendered sterile by filtration through sterile filtration membranes. The therapeutic compositions herein are typically placed in a container having a sterile access port, such as an intravenous bag or a vial having a stopper pierceable by a hypodermic injection needle.

The route of administration is according to known and acceptable methods, e.g. by single or multiple bolus injection or infusion over a longer period of time in a suitable manner, e.g. by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes of injection or infusion, topical administration, inhalation, sustained release or sustained release.

The formulations herein may also contain more than one active compound necessary to treat a particular indication, preferably those compounds having complementary activities that do not adversely affect each other. Alternatively or additionally, the composition may comprise a cytotoxic agent, cytokine or growth inhibitory agent. Such molecules are suitably present in combination in amounts effective for the intended purpose.

The active ingredients may also be entrapped in microcapsules prepared, for example, by coacervation techniques or interfacial polymerization, such as hydroxymethylcellulose or gelatin microcapsules and poly (methylmethacylate) microcapsules, in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules) or macroemulsions, respectively. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18 th edition, supra.

Sustained release articles can be prepared. Suitable examples of sustained release articles include semipermeable matrices of solid hydrophobic polymers containing the antibody, which matrices are in the form of shaped articles, e.g. films or microcapsules. Examples of sustained release matrices include polyesters, hydrogels (examples)E.g., poly (2-hydroxyethyl-methacrylic acid), or poly (vinyl alcohol)), polylactic acid (U.S. Pat. No. 3,773,919), copolymers of L-glutamic acid and γ -ethyl-L-glutamate, non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolic acid copolymers, e.g., LUPRON DEPOT^TM(injectable microspheres composed of lactic acid-glycolic acid copolymer and leuprolyl acetate) and poly-D- (-) -3-hydroxybutyric acid. Microencapsulation of recombinant proteins for sustained release has been successfully performed with human growth hormone (rhGH), interferon- (rhIFN-), interleukin-2, and MN rpg 120. Johnson et al, nat.med.2: 795-799 (1996); yasuda et al, biomed. ther.27: 1221-1223 (1993); hora et al, Bio/Technology 8: 755, 758 (1990); cleland, "Design and production of Single Immunization Vaccines Using polylactic polyol microspheres systems," in Vaccine Design: the Subunit and Adjuvant Approach, edited by Powell and Newman (Plenum Press: New York, 1995), page 439-462; WO 97/03692; WO 96/40072; WO 96/07399; and U.S. patent No. 5,654,010.

Due to their biocompatibility and broad biodegradable properties, polylactic-co-glycolic acid (PLGA) polymers can be used to develop sustained release formulations of these proteins. The degradation products of PLGA, lactic acid and glycolic acid, can be rapidly eliminated in the human body. In addition, the degradability of the polymer can be adjusted from months to years, depending on its molecular weight and composition. Lewis, "Controlled release of biologically active agents from active/polysaccharide Polymers", in Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker; New York, 1990), M.Chasin and R.Langer (eds.) pages 1-41.

Polymers (such as ethylene-vinyl acetate and lactic-glycolic acid) allow the release of molecules over a period of 100 days, while some hydrogels release proteins over a shorter period of time. When encapsulated antibodies remain in the body for extended periods of time, they may denature or aggregate due to exposure to moisture at 37 ℃, resulting in loss of biological activity and possibly altered immunogenicity. Rational countermeasures can be designed for stabilization according to the mechanism involved. For example, if the aggregation mechanism is found to be intermolecular S — S bond formation through disulfide bond exchange, stabilization can be achieved by modifying sulfhydryl residues, lyophilizing from acidic solutions, controlling moisture content, using appropriate additives, and developing specific polymer matrix compositions.

Liposomes or proteinaceous compositions can also be used to formulate the proteins or antibodies disclosed herein. See U.S. Pat. nos. 4,925,673 and 5,013,556.

The stability of the proteins and antibodies described herein can be enhanced by the use of non-toxic "water-soluble multivalent metal salts". Examples include Ca²⁺、Mg²⁺、Zn²⁺、Fe²⁺、Fe³⁺、Cu²⁺、Sn²⁺、Sn³⁺、Al²⁺And Al³⁺. Examples of the anion which can form a water-soluble salt with the above polyvalent metal cation include anions formed from inorganic acids and/or organic acids. Such water soluble salts have a solubility in water (at 20 ℃) of at least about 20mg/ml, alternatively at least about 100mg/ml, alternatively at least about 200 mg/ml.

Suitable inorganic acids that can be used to form the "water-soluble polyvalent metal salt" include hydrochloric acid, acetic acid, sulfuric acid, nitric acid, thiocyanic acid, and phosphoric acid. Suitable organic acids that may be used include aliphatic carboxylic acids and aromatic acids. Aliphatic acids within this definition may be defined as saturated or unsaturated C_2-9Carboxylic acids (e.g., aliphatic monocarboxylic acids, dicarboxylic acids, and tricarboxylic acids). For example, monocarboxylic acids within this definition include saturated C_2-9Monocarboxylic acids acetic acid, propionic acid, butyric acid, valeric acid, caproic acid, enanthic acid, caprylic acid, pelargonic acid and capryonic acid, and unsaturated C_2-9Monocarboxylic acids acrylic acid, preprolic methacrylic acid, crotonic acid and isocrotonic acid. Exemplary dicarboxylic acids include saturated C_2-9Dicarboxylic acids malonic, succinic, glutaric, adipic and pimelic acid, and unsaturated C_2-9Dicarboxylic acids include maleic acid, fumaric acid, citraconic acid and methyl fumaric acid. Exemplary tricarboxylic acids include saturated C_2-9The tricarboxylic acid tricarballylic acid and 1,2, 3-butanetricarboxylic acid. In addition, the carboxylic acids of this definition may also contain one or two hydroxyl groups to form hydroxycarboxylic acids. Exemplary hydroxycarboxylic acids include glycolic acid, lactic acid, glyceric acid, tartronic acid, malic acid, tartaric acid, and citric acid. Aromatic acids within this definition include benzoic acid and salicylic acid.

Common water-soluble multivalent metal salts that may be used to help stabilize the encapsulated polypeptides of the invention include, for example: (1) inorganic acid metal salt halides (e.g., zinc chloride, calcium chloride), sulfates, nitrates, phosphates, and thiocyanates; (2) metal salts of aliphatic carboxylic acids (e.g., calcium acetate, zinc acetate, calcium propionate, zinc lactate, and zinc tartrate); and (3) aromatic carboxylic acid metal salts of benzoic acid salts (e.g., zinc benzoate) and salicylic acid salts.

For the prevention or treatment of a disease, the appropriate dosage of the active agent depends on the type of disease to be treated, the severity and course of the disease, whether the active agent is administered for prophylactic or therapeutic purposes, previous therapy, the patient's clinical history and response to the active agent, as defined above, and the discretion of the attending physician. The active agent is suitably administered to the patient at one time or in a series of treatments.

The methods of the invention may be combined with known methods of treating a condition, as a combined or additional therapeutic step, or as an additional component of a therapeutic formulation.

The dosage of the pharmaceutical composition of the invention and the desired drug concentration may vary depending on the particular use contemplated. Determination of the appropriate dosage or route of administration is known to those of ordinary skill in the art. Animal experiments provide reliable guidance for determining effective doses for human therapy. The interspecies analogy of effective doses (interspecies Scaling) may be carried out following rules set forth In Mordenti, J. and Chappell, W. "The Use of intersections Scaling In Autocortics," In Autocortics and New drug development, Yacobi et al, Pergamon Press, New York 1989, pages 42-46.

When the polypeptides or antibodies described herein are administered in vivo, a typical dose may be from about 10ng/kg of mammal body weight up to about 100mg/kg of mammal body weight or more per day, preferably from about 1 mg/kg/day to 10 mg/kg/day, depending on the route of administration. Guidance in specific dosages and delivery methods is provided in the literature; see, e.g., U.S. patent nos. 4,657,760; 5,206,344 or 5,225,212. It is also within the scope of the invention that different formulations are effective for different treatments and different conditions, and that administration intended to treat a particular organ or tissue may need to be delivered in a manner different from administration to another organ or tissue. Furthermore, the dose may be administered by one or more separate administrations or by continuous perfusion. For repeated administrations over several days or longer, depending on the condition, treatment is continued until the desired suppression of disease symptoms occurs. However, other dosage regimens may also be useful. The progress of the therapy can be conveniently monitored by conventional techniques and assays.

The formulations of the present invention (including but not limited to reconstituted formulations) are administered to a mammal (preferably a human) in need of treatment with a protein according to known methods, such as intravenously as a bolus or by continuous infusion over a period of time, by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intraarterial, intrasynovial, intrathecal, oral, topical or inhalation routes.

In a preferred embodiment, the formulation is administered to the mammal subcutaneously (i.e., subcutaneously). For such purposes, the formulation may be injected using a syringe. However, other devices for administering the formulation are also available, such as injection devices (e.g., Inject-ase)^TMAnd Genject^TMA device); injection pen (e.g. GenPen)^TM) (ii) a Automatic injection device, needleless device (e.g. mediJector)^TMAnd BioJector^TM) (ii) a And a subcutaneous patch (patch) delivery system.

In a specific embodiment, the invention relates to a kit for single dose administration of units. Such kits comprise a container, including a single or multi-chambered pre-filled syringe, of an aqueous formulation of a therapeutic protein or antibody. An exemplary pre-filled syringe is available from Vetter GmbH, Ravensburg, germany.

The appropriate dosage of the protein (the "therapeutically effective amount") depends, for example, on the condition to be treated, the severity and course of the condition, whether the protein is administered for prophylactic or therapeutic purposes, previous therapy, the clinical history and responsiveness of the patient to the protein, the type of protein used, and the discretion of the attending physician. The protein is suitably administered to the patient in one or over a series of treatments, and may be administered to the patient at any time after diagnosis. The protein may be administered as the sole therapy or in combination with other drugs or therapies useful for treating the condition of interest.

When the protein of choice is an antibody, the initial candidate dose administered to the patient is from about 0.1-20mg/kg, e.g., by one or more divided administrations. However, other dosage regimens may also be useful. The progress of the therapy can be conveniently monitored by conventional techniques.

In another embodiment of the invention, an article of manufacture containing a formulation, and preferably instructions for its use, is provided. The article comprises a container. Suitable containers include, for example, bottles, vials (e.g., dual chamber vials), syringes (e.g., single chamber or dual chamber syringes), and test tubes. The container may be formed from a variety of materials, such as glass or plastic. A label on or associated with the container containing the formulation may indicate instructions for reconstitution or use. The label may also indicate that the formulation is for subcutaneous administration or is intended for subcutaneous administration. The container holding the formulation may be a multi-use vial that allows repeated administration (e.g., 2-6 administrations) of the reconstituted formulation. The article of manufacture may also comprise a second container comprising a suitable diluent (e.g., BWFI). After mixing the diluent and the lyophilized formulation, the final concentration of protein in the reconstituted formulation is typically at least 50 mg/ml. The article of manufacture may also contain other materials as needed from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

The present invention will be more fully understood from the following examples. But should not be construed as limiting the scope of the invention. All references throughout this disclosure are expressly incorporated herein by reference.

Example 1: investigation of the Effect of fatty acids, Polysorbate and POE sorbitan on protein aggregation

This example illustrates how polysorbates, fatty acids, and POE sorbitan affect protein aggregation in aqueous solutions.

The protective effect of POE sorbitan against agitation-induced aggregation of anti-IL 13 monoclonal antibodies in solution was evaluated using an agitation-induced protein aggregation assay. Specifically, in this study, a buffer solution (20mM His-OAc, pH 5.7) containing 1mg/ml of anti-IL 13 monoclonal antibody was prepared in combination with the following potential stabilizing additives:

(i) no additive, control;

(ii) lauric acid (29 ppm);

(iii) lauric acid (29ppm) and polysorbate 20(24 ppm);

(iv) POE sorbitan 20 "(a + b + c + d ═ 20)" (150 ppm);

(v) POE sorbitan 20 "(a + b + c + d ═ 20)" (150ppm) and polysorbate 20(24 ppm);

(vi) POE sorbitan 20 "(a + b + c + d ═ 20)" (150ppm) and lauric acid (29 ppm);

(vii) POE sorbitan 20 "(a + b + c + d ═ 20)" (150ppm), lauric acid (29ppm) and polysorbate 20(24 ppm);

(viii) polysorbate 20(24 ppm).

9ml of the monoclonal antibody containing formulation was placed into separate 15ml Forma Virium vials (in triplicate), the vials were sealed, and then allowed to stir on a bench top shaker at room temperature (70rpm) for 0, 4, or 24 hours. Upon completion, the contents of each vial were immediately subjected to UV spectrometry analysis (340-360nm) to measure the turbidity of the solution. Turbidity was obtained at 25 ℃ using a 1cm path length chamber and an Agilent 8453UV spectrophotometer. Absorbance values at 340, 345, 350, 355 and 360nm where no protein formulation chromophore absorbs light are averaged and scattering of insoluble protein aggregates can be determined. The average absorbance at the above wavelengths represents the turbidity of the sample. In this regard, it is generally known in the art that the turbidity of a protein-containing solution is directly and quantitatively related to the amount of protein aggregation in the solution (see, e.g., Dani et al, J.Pharm.Sci., 96 (6): 1504-1517 (2007)). The results of these analyses are shown in fig. 1.

The data shown in figure 1 indicate that certain polysorbate degradants (including fatty acids, lauric acid) have an adverse effect on protein stability in aqueous solution and cause protein aggregation upon stirring. In contrast, the addition of POE sorbitan to the aqueous solution containing the antibody prevented the formation of protein aggregates upon stirring. Thus, these data indicate that POE sorbitan has the effect of preventing protein aggregation upon stirring, thus enhancing the stability of the therapeutic protein in solution.

Example 2: investigation of the Effect of POE sorbitan and PEG on protein aggregation

This example illustrates the use of POE sorbitan and PEG as stabilizers to prevent or reduce protein aggregation.

Using the agitation-induced protein aggregation assay, the protective effect of various POE sorbitans and PEGs against aggregation of two monoclonal antibodies (anti-IL 13 and anti-IgE antibodies) induced by agitation in solution was evaluated.

In this set of studies, buffer solutions containing 1mg/ml of anti-IL 13 antibody (20mM His-OAc, pH 5.7) or anti-IgE antibody (His-HisCl, pH 6.0) were prepared with the following additions:

(i) no additive, control;

(ii) POE sorbitan 20 "(a + b + c + d ═ 20)" at a concentration of 200 ppm;

(iii) POE sorbitan 20 "(a + b + c + d ═ 20)" at a concentration of 1000 ppm;

(iv) POE sorbitan 20 "(a + b + c + d ═ 20)" at a concentration of 5000 ppm;

(v) PEG 1000 at a concentration of 200 ppm;

(vi) PEG 1000 at a concentration of 1000 ppm;

(vii) PEG 1000 at a concentration of 5000 ppm;

(viii) PEG 6000 at a concentration of 200 ppm;

(ix) PEG 6000 at a concentration of 1000 ppm;

(x) PEG 6000 at a concentration of 5000 ppm.

9ml of each formulation containing the monoclonal antibody was stored in separate 15ml Forma Vitrium vials (in triplicate), the vials were sealed, and then allowed to stir on a bench top shaker at room temperature (70rpm) for 0 hours, 4 hours, or 24 hours. Upon completion, the contents of each vial were immediately subjected to (a) a UV spectrometry analysis to measure the protein concentration after filtration, (b) a UV spectrometry analysis (340-360nm) to measure the turbidity of the solution, and (c) a light absorbance analysis for determining the protein particle size and distribution.

A.UVSpectrometry methodMeasurement of protein concentration

Immediately after shaking as described above, the solution containing the protein was filtered to remove protein aggregates, and then the protein concentration in the filtrate was determined by UV spectrometry. Protein concentration data was obtained using a 0.5 or 1cm pathlength chamber and Agilent 8453UV Spectrophotometer at 25 ℃. The extinction coefficient E used at 278nm was 1.45 and 1.60mLmg^-1cm^-1To determine the concentration of the antibody after filtration through a 0.2 μm syringe filter. The scattering effect was calculated by subtracting the absorbance at 320nm from the absorbance at 278 nm. In this regard, it is generally known in the art that the concentration of protein in a solution containing the protein can be quantitatively measured using UV absorption analysis (see, e.g., Liu et al, J.Pharm.Sci., 94 (9): 1928-. The results of the data obtained for the anti-IL 13 antibody and the anti-IgE antibody are shown in figures 2 and 3, respectively.

The data in fig. 2 and 3 show that stirring the untreated control antibody formulation induced measurable and significant aggregation and protein loss upon filtration. In contrast, the addition of POE sorbitan or PEG at various concentrations all tested prevented agitation-induced protein aggregate formation and thus protein loss upon filtration. These data indicate that POE sorbitan and polyethylene glycol function as effective protein stabilizers in aqueous solutions by preventing or reducing protein aggregate formation in solution.

B.UVSpectrometry methodMeasurement of solution turbidity

As described above, UV spectrometry at 340-360nm provides an effective means to quantitatively determine the amount of protein aggregates present in a solution, where turbidity is directly related to the amount of aggregated protein present. The results obtained from the turbidity analysis of the anti-IL 13 antibody and anti-IgE antibody are shown in fig. 4 and 5, respectively.

The data in fig. 4 and 5 show that stirring the untreated control antibody formulation induced measurable and significant antibody aggregation therein. In contrast, addition of POE sorbitan or PEG at various concentrations all tested prevented and/or reduced agitation-induced protein aggregate formation. These data indicate that POE sorbitan and polyethylene glycol function as effective protein stabilizers in aqueous solutions by preventing or reducing protein aggregate formation in solution.

C. Determination of particle size distribution

The above aqueous solution containing the antibody is also analyzed to determine the particle size distribution of the protein contained therein. Specifically, the number and size of insoluble particles between 2 and 50 μm were measured at room temperature using a HIAC/Royco9703 liquid particle counter connected to a HIAC/Royco 3000A liquid syringe sampler, HRLD-150 sensor, and analyzed using PacificSpec version 2.0 software. The upper limit of detection is 18000 particles/ml and samples above this threshold are measured after appropriate dilution. Each sample was measured 4 times at a volume of 1.0mL per injection. The 1 st injection was discarded and the average was obtained from the last 3 injections. Between each sample analysis, the system was washed with water for injection until the instrument had a2 μm particle count < 10. Sub-visible particles of ≧ 2, 5, 10, 15, 25, 35 and 50 μm are expressed as cumulative counts per ml.

The results obtained from these analyses are shown in FIGS. 6-11, respectively. The data in fig. 6-11 show that stirring the untreated control antibody formulation induced measurable and significant antibody aggregation therein. In contrast, addition of POE sorbitan or PEG at various concentrations all tested prevented and/or reduced agitation-induced protein aggregate formation. These data indicate that POE sorbitan and polyethylene glycol function as effective protein stabilizers in aqueous solutions by preventing or reducing protein aggregate formation in solution.

Claims (40)

1. A composition of matter comprising a protein and POE sorbitan.

2. The composition of claim 1, wherein the protein is an antibody.

3. The composition of matter of claim 1, wherein the POE sorbitan is present at a concentration of from about 20ppm to about 100,000 ppm.

4. The composition of matter of claim 1, wherein the POE sorbitan is selected from the group consisting of POE sorbitan 10, POE sorbitan 20, POE sorbitan 40, and POE sorbitan 80.

5. The composition of matter of claim 1, which is surfactant free.

6. The composition of matter of claim 5, wherein the surfactant is a polysorbate.

7. The composition of matter of claim 1, which is in an aqueous form.

8. The composition of matter of claim 1, in lyophilized form.

9. The composition of matter of claim 1, further comprising polyethylene glycol.

10. An article of manufacture comprising a container containing the composition of matter of claim 1.

11. A composition of matter comprising a protein and polyethylene glycol, wherein the polyethylene glycol is present at a concentration of 10,000ppm or less.

12. The composition of matter of claim 11, wherein the protein is an antibody.

13. The composition of matter of claim 11, wherein the polyethylene glycol is present at a concentration of from 20ppm to 10,000 ppm.

14. The composition of matter of claim 11, wherein the polyethylene glycol is selected from the group consisting of PEG 1000 and PEG 6000.

15. The composition of matter of claim 11, being surfactant free.

16. The composition of matter of claim 15, wherein the surfactant is a polysorbate.

17. The composition of matter of claim 11, which is in an aqueous form.

18. The composition of matter of claim 11, in lyophilized form.

19. The composition of matter of claim 11, further comprising POE sorbitan.

20. An article of manufacture comprising a container containing the composition of matter of claim 11.

21. A method of preparing a stable protein-containing formulation, the method comprising mixing a protein with POE sorbitan, thereby providing the stable protein-containing formulation.

22. The method of claim 21, wherein the protein is an antibody.

23. The method of claim 21, wherein the POE sorbitan is selected from the group consisting of POE sorbitan 10, POE sorbitan 20, POE sorbitan 40, and POE sorbitan 80.

24. The method of claim 21, further comprising the step of lyophilizing the stable protein-containing formulation.

25. A method of preparing a stable protein-containing formulation, the method comprising mixing a protein with polyethylene glycol, wherein the polyethylene glycol is present at a concentration of 10,000ppm or less, thereby providing the stable protein-containing formulation.

26. The method of claim 25, wherein the protein is an antibody.

27. The method of claim 25, wherein the polyethylene glycol is selected from the group consisting of PEG 1000 and PEG 6000.

28. The method of claim 25, further comprising the step of lyophilizing the stable protein-containing formulation.

29. A method of increasing the stability of a protein in an aqueous solution, the method comprising mixing the protein with a POE sorbitan, wherein the POE sorbitan increases the stability of the protein in an aqueous solution.

30. The method of claim 29, wherein the protein is an antibody.

31. The method of claim 29, wherein the POE sorbitan is selected from the group consisting of POE sorbitan 10, POE sorbitan 20, POE sorbitan 40, and POE sorbitan 80.

32. A method of increasing the stability of a protein in an aqueous solution, the method comprising mixing the protein with polyethylene glycol, wherein the polyethylene glycol is present at a concentration of 10,000ppm or less and increasing the stability of the protein in the aqueous solution.

33. The method of claim 32, wherein the protein is an antibody.

34. The method of claim 32, wherein the polyethylene glycol is selected from the group consisting of PEG 1000 and PEG 6000.

35. A method of preventing or reducing aggregation of a protein in an aqueous solution, the method comprising mixing the protein with POE sorbitan, wherein the POE sorbitan prevents or reduces aggregation of the protein in an aqueous solution.

36. The method of claim 35, wherein the protein is an antibody.

37. The method of claim 35, wherein the POE sorbitan is selected from the group consisting of POE sorbitan 10, POE sorbitan 20, POE sorbitan 40, and POE sorbitan 80.

38. A method of preventing or reducing aggregation of a protein in an aqueous solution, the method comprising mixing the protein with polyethylene glycol, wherein the polyethylene glycol is present at a concentration of 10,000ppm or less, and preventing or reducing aggregation of the protein in an aqueous solution.

39. The method of claim 38, wherein the protein is an antibody.

40. The method of claim 38, wherein the polyethylene glycol is selected from the group consisting of PEG 1000 and PEG 6000.