HK1260077A1

HK1260077A1 - Formulations with reduced degradation of polysorbate

Info

Publication number: HK1260077A1
Application number: HK19119862.1A
Authority: HK
Inventors: Brian Connolly; Lydia HAMBURG; Emily HOLZ
Original assignee: 豪夫迈‧罗氏有限公司
Priority date: 2015-12-30
Filing date: 2016-12-28
Publication date: 2019-12-13

Description

Formulations for reducing polysorbate degradation

Cross Reference to Related Applications

This application claims the benefit of U.S. provisional application No. 62/272,965 filed on 30/12/2015, the contents of which are incorporated herein by reference in their entirety.

Technical Field

The present invention relates to aqueous pharmaceutical formulations comprising cyclodextrin and polysorbate and methods for reducing polysorbate degradation and for depolymerizing and solubilizing polysorbate degradation products.

Background

Pharmaceutical formulations typically contain polysorbates 20 and 80(PS20 and PS80), which are nonionic surfactants consisting of a hydrophilic polyoxyethylene head group and a hydrophobic fatty acid tail. Addition of surfactants to the formulations protects proteins from surface-induced denaturation and aggregation (Geisen, Diabetologia 27:212-218 (1984); Wang, int. J. pharm.289:1-30 (2005)). Protein aggregation can occur during processing, long term storage, transport and administration of Drug Substances (DS) and Drug Products (DP) (Cromwell et al, AAPS J.8: E572-E579 (2006)). It has been shown that the addition of a surfactant (e.g., PS20) minimizes the interaction of filtration (Maa et al, J.Pharm. Sci.87:808- "1998); Maa et al, Biotechnol. Bioeng.50: 319-" 328(1996)), agitation (Liu et al, J.Pharm. Sci.102:2460- "2470 (2013)), freeze-thaw (Kreilgaard et al, J.Pharm. Sci.87: 1597-" 1603 (1998); Hillgren et al, int.J.Pharm.237:57-69(2002)), lyophilization (Carpenter, Protein Sci.13:54-54 (2004); Carpenter et al, Pharm. Res.14:969- "975 (1998)), reconstitution (Webb et al, J.rm. Sci.91: 543: 558(2002)), administration (Kuru et al, Kuru. J.101: 3650)) with the storage interface.

To ensure stability of the Active Pharmaceutical Ingredient (API) during processing, long term storage and administration, it is important to prevent degradation of the polysorbate. However, PS20 is readily degraded by both hydrolytic and oxidative pathways (Kumru, et al, J.Pharm. Sci.101:3636-3650 (2012); Mahler et al, Abstr Pap Am Chem S.239 (2010)).

The oxidative degradation of polysorbates has been well characterized and widely studied (Kerwin, J.Pharm. Sci.97:2924-2935 (2008); Kishore et al, J.Pharm. Sci.100:721-731 (2011)). Oxidation typically occurs by two mechanisms, (1) autoxidation of the oxirane group and (2) free radical oxidation at the site of unsaturation (Kishore et al, J.pharm.Sci.100:721-731 (2011)). Although oxidative degradation of polysorbates has been observed, it has been shown that PS20 oxidation in protein formulations can be mitigated by co-formulation with antioxidants (e.g., methionine). Formulations containing tryptophan have also been developed to prevent oxidation of amino acid residues (US 2014/0322203; US 2014/0314). Oxidative and hydrolytic polysorbate degradation pathways can be distinguished by unique degradation product distributions. Hydrolytic polysorbate degradation produces primarily fatty acids and oxidative polysorbate degradation produces more diverse degradation products including peroxides, aldehydes, acids, ketones (keytone), n-alkanes, fatty acid esters, and other degradation products (Ravuri et al, pharm. Res.28:1194-1210 (2011)).

A stress model for oxidative polysorbate degradation using 2,2' -azobisisobutyramidine (AAPH) which degrades PS20 has been previously described (Borasov et al, J.Pharm. Sci.104:1005-1018 (2015)). Using similar methods, representative stress models can be used to develop formulations that reduce oxidative polysorbate degradation under relevant conditions.

A stress model for hydrolysis using purified esterases (e.g., pig liver esterase, etc.) and lipases (e.g., tween, etc.) has been previously described (Labrenz, j.pharm. Using similar methods, representative stress models can be used to develop formulations that reduce catalytic polysorbate degradation under relevant conditions.

Recently, enzymatic degradation of polysorbates in monoclonal antibody (mAb) formulations has been reported. For example, Labrenz attributed to a specific enzymatic mechanism for degradation of polysorbate 80(PS80) observed in CHO-derived mAb formulations, rather than the general biohydrolytic mechanism based on the PS20 degradation profile (Labrenz et al, J.Pharm. Sci.103:2268-2277 (2014)). Sequencing of the CHO cell genome identified various Host Cell Proteins (HCPs) such as lipases capable of degrading polysorbates (S.Hammond et al, Biotech.Bioeng.109: 1353-. Subsequently, Lee et al have shown that reducing the expression of a particular HCP significantly reduces the hydrolysis of PS80 relative to control samples. These recent findings confirm that lipases involved in the production of biologicals are expressed in upstream processes. Downstream purification processes (e.g., protein a) can remove HCPs; however, it has been shown That some HCPs can co-purify with API molecules with similar properties and thus leave traces in drug substances and drug products (k.lee, et al, a Chinese Hamster Ovary Cell Host Cell Protein thin images PS-80degradation. acc bio Conference (2015) it is speculated That lipases with high activity may even lead to significant polysorbate degradation at undetectable levels.

Polysorbate degradation has a number of consequences that may affect the stability and shelf life of protein pharmaceutical formulations. Polysorbate degradants include poorly soluble fatty acids, which can lead to the formation of visible and sub-visible particles in solution. Loss of PS20 may also reduce the protective effect of PS20 on protein formulations. Furthermore, a spiking study (spiking study) showed that some PS 20-related degradants may affect the stability of protein drugs; however, no effect was observed under pharmaceutically relevant conditions (Kishore et al, pharm. Res.28:1194-1210 (2011).

What is needed is a way to reduce polysorbate degradation so that the protective effect of the polysorbate on the formulation (e.g., polypeptide) is maintained over time. This will result in a more stable polypeptide formulation during processing, long term storage and administration, which in turn will extend the shelf life of the polypeptide formulation and reduce waste caused by degradation and overdue formulations.

All references, including patent applications and publications, cited herein are incorporated by reference in their entirety.

Disclosure of Invention

The present invention provides a method of reducing polysorbate degradation in an aqueous formulation comprising a polysorbate, the method comprising adding a cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5: 1. In some aspects, the invention provides a method of reducing polysorbate degradation in an aqueous formulation comprising a polysorbate, the method comprising adding cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5:1, wherein the formulation comprises about 0.005% -0.4% polysorbate. In some aspects, the invention provides a method of reducing polysorbate degradation in an aqueous formulation comprising a polysorbate, the method comprising adding cyclodextrin to the formulation to a concentration of about 0.01% -30%, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5:1, wherein the formulation comprises about 0.005% -0.4% polysorbate. In some aspects, the invention provides a method of reducing the amount of sub-visible particles (sub-visible particles) and visible particles (visible particles) in an aqueous formulation comprising a polysorbate, the method comprising adding a cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5:1, wherein the formulation comprises polysorbate and a polypeptide. In some aspects, the invention provides a method of depolymerizing and solubilizing a polysorbate degradation product in an aqueous formulation, the method comprising adding a cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5:1, wherein the formulation comprises a polysorbate and a polypeptide.

In some embodiments of the above aspects, the polysorbate is polysorbate 20 or polysorbate 80. In some embodiments, the cyclodextrin is HP-beta cyclodextrin, HP-gamma cyclodextrin, or sulfobutyl ether beta-cyclodextrin. In some embodiments, the polysorbate is present in the formulation at a concentration ranging from about 0.01% to 0.4%. In some embodiments, the polysorbate is present in the formulation at a concentration in the range of about 0.01% to 0.1%. In some embodiments, the polysorbate is at a concentration of about 0.02% in the formulation. In some embodiments, the formulation has a concentration of cyclodextrin in the range of about 0.5-30%. In some embodiments, the concentration of cyclodextrin in the formulation is about 15%.

In some embodiments of the above aspects and embodiments, polysorbate degradation is reduced by about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, less than about 1,000, about 750, about 500, about 250, about 150, about 100, about 50, or about 25 polysorbate particles greater than about 2 microns in diameter are formed per mL.

In some embodiments of the above aspects and embodiments, the agent comprises a polypeptide. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the concentration of the polypeptide in the formulation is about 1mg/mL to about 250 mg/mL.

In some embodiments of the above aspects and embodiments, the formulation is stable at about 2 ℃ to about 8 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, or at least about 24 months. In some embodiments, the formulation is stable at about 1 ℃ to about 10 ℃ for at least about 48 months. In some embodiments, the formulation is stable at about 2 ℃ to about 8 ℃ for at least about 48 months.

In some embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation has a pH of about 4.5 to about 6.0. In some embodiments, the formulation has a pH of about 6.0.

In some embodiments of the above aspects and embodiments, the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. In some embodiments, the formulation is a pharmaceutical formulation suitable for administration to a subject. In some embodiments, the formulation is a pharmaceutical formulation suitable for intravenous, subcutaneous, intramuscular, or intravitreal administration to a subject.

In some aspects, the invention provides an aqueous formulation comprising a polypeptide, a polysorbate, and a cyclodextrin, wherein the formulation has been stored at about 1 ℃ to about 10 ℃ for at least about 6 months, wherein the initial w/w ratio of cyclodextrin to polysorbate is at least about 37.5:1, and wherein the amount of polysorbate in the formulation is at least about 80% of the initial amount of polysorbate in the formulation. In some aspects, the invention provides an aqueous formulation comprising a polypeptide, a polysorbate, and a cyclodextrin, wherein the formulation has been stored at about 1 ℃ to about 10 ℃ for at least about 6 months, wherein the w/w ratio of cyclodextrin to polysorbate in the formulation is at least about 37.5:1, and wherein less than about 1% of the polysorbate has degraded.

In some embodiments of the above aspect, the cyclodextrin is HP-beta cyclodextrin, HP-gamma cyclodextrin, or sulfobutylether beta-cyclodextrin. In some embodiments, the polysorbate is present in the formulation at a concentration ranging from about 0.01% to 0.4%. In some embodiments, the polysorbate is present in the formulation at a concentration in the range of about 0.01% to 0.1%. In some embodiments, the polysorbate is at a concentration of about 0.02% in the formulation. In some embodiments, the formulation has a concentration of cyclodextrin in the range of about 0.5-30%. In some embodiments, the concentration of cyclodextrin in the formulation is about 15%.

In some embodiments of the above aspects and embodiments, the formulation is stable for at least about 6 months at about 2 ℃ to about 8 ℃. In some embodiments, the formulation is stable at about 1 ℃ to about 10 ℃ for at least about 48 months. In some embodiments, the formulation is stable at about 2 ℃ to about 8 ℃ for at least about 48 months.

In some embodiments of the above aspects and embodiments, the formulation further comprises a polypeptide. In some embodiments, the polypeptide is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the concentration of the polypeptide in the formulation is about 1mg/mL to about 250 mg/mL.

In some embodiments of the above aspects and embodiments, the formulation has a pH of about 4.5 to about 7.0. In some embodiments, the formulation has a pH of about 4.5 to about 6.0. In some embodiments, the formulation has a pH of about 6.0.

Drawings

Figure 1 shows the average (n-3) relative percentage determined by RP-ELSD at 40 ℃ by oxidation of samples with 5mM AAPH containing no excipient (control), 15% (w/v) sucrose and 15% (w/v) HP- β -CD for 24 hours.

Figure 2 shows the average (n-3) relative percentage of PS20 determined by RP-ELSD in protein-free samples containing 0.02% (w/v) PS20 with 0 and 15% (w/v) HP- β -CD using candida antarctica lipase B (black), lipoprotein lipase (grey) and rabbit liver esterase (white) digested samples.

FIGS. 3A-3D show the average (n ≧ 3) ≧ 2. mu.M (FIG. 3A),. gtoreq.5. mu.M (FIG. 3B),. gtoreq.10. mu.M (FIG. 3C) and. gtoreq.25. mu.M (FIG. 3D) particle counts/ml as determined by HIAC for samples digested with Candida antarctica lipase B (black), lipoprotein lipase (grey) and rabbit liver esterase (white) in protein-free samples containing 0.02% (w/v) PS20 and 0 and 15% (w/v) HP- β -CD.

Figure 4 shows the average (n-3) relative percentage of PS80 determined by RP-ELSD for a 5 hour protein-free sample containing 0.02% (w/v) PS80 digested with 15 μ g/mL PPL containing 0 and 15% (w/v) HP- β -CD at room temperature.

FIGS. 5A-5F show that for samples digested at room temperature for 5 hours with 15 μ g/mL PPL containing 0.02% (w/v) PS80 and 0 and 15% (w/v) HP- β -CD, the average (n ≧ 3) (FIG. 5A) ≧ 1.4 μ M, (FIG. 5B) ≧ 2 μ M, (FIG. 5C) ≧ 5 μ M, (FIG. 5D) ≧ 10 μ M, (FIG. 5E) ≧ 15 μ M, and (FIG. 5F) ≧ 25 μ M sub-visible particle counts/mL.

Figure 6 shows the average (n-3) relative percentage of PS20 determined by RP-ELSD as a function of time for samples digested with 15 μ g/mL PPL enzyme in protein-free samples containing 15% (w/v) sucrose (circles), HP- α -CD (diamonds), and HP- β -CD (triangles) at room temperature.

FIG. 7 shows the average (n 3) relative percentage of PS20 determined by RP-ELSD for samples digested with 15 μ g/mL PPL enzyme for 4.5 hours at room temperature in protein-free samples containing 0.02% (w/v) PS20 and no vehicle (control), 15% (w/v) sucrose, 1% (w/v) methionine, 15% (w/v) PEG 1500, 15% (w/v) PVP, 15% (w/v) HP- α -CD, 15% (w/v) HP- β -CD, 15% (w/v) SBE- β -CD, and 15% (w/v) HP- γ -CD.

FIGS. 8A-8D show that for samples digested with 15 μ g/mL PPL enzyme at room temperature for 4.5 hours in protein-free samples containing 0.02% (w/v) PS20 and no vehicle (control), containing 15% (w/v) sucrose, 1% (w/v) methionine, 15% (w/v) PEG 1500, 15% (w/v) PVP, 15% (w/v) HP- α -CD, 15% (w/v) HP- β -CD, 15% (w/v) SBE- β -CD and 15% (w/v) HP- γ -CD, the average (n 3) ≥ 2 μ M (FIG. 8A), ≥ 5 μ M (FIG. 8B), ≥ 10 μ M (FIG. 8C) and ≥ 25 μ M (FIG. 8D) particle count/ml as determined by RP-ELSD.

FIG. 9 shows the average (n-3) relative percentage of PS20 determined by RP-ELSD for samples digested with Candida antarctica lipase B in protein-free samples containing 0.02(w/v) PS20 and no vehicle (control), containing 15% (w/v) SBE- β -CD, 15% (w/v) HP- α -CD, 15% (w/v) HP- β -CD, 15% (w/v) HP- γ -CD, and 15% (w/v) sucrose.

FIGS. 10A and 10B show the average (n ≧ 3) (FIG. 10A) and (FIG. 10B) particle counts/ml determined by HIAC for samples after addition of various excipients (HP- α -CD, HP- β -CD, HP- γ -CD, SBE- β -CD, PVP, PEG 1500, sucrose and methionine) to evaluate the resolubilization of existing particles produced as a result of enzymatic PS-20 degradation.

FIGS. 11A and 11B show vials containing PS 20-related particles produced by enzymatic digestion with 15 μ g/mL PPL enzyme before (FIG. 11A) and after (FIG. 11B) addition of 15% (w/v) HP- β -CD at room temperature. After addition of 15% (w/v) HP- β -CD, no particles were visible.

FIGS. 12A-12F show the sub-visible particle counts/ml with HIAC for an average (n ≧ 3) (FIG. 12A) ≧ 1.4 μ M, (FIG. 12B) ≧ 2 μ M, (FIG. 12C) ≧ 5 μ M, (FIG. 12D) ≧ 10 μ M, (FIG. 12E) ≧ 15 μ M, and (FIG. 12F) ≧ 25 μ M in protein-free samples containing 0.02% (w/v) PS20 stored at 5 ℃ for 27 months.

Figure 13 shows the average (n-3) relative percentage of PS20 determined by RP-ELSD for samples digested with 15 μ g/mL PPL enzyme for 4.5 hours in protein-free samples containing 0.02% (w/v) PS20 and varying amounts of HP- β -CD at room temperature. Data were fitted using a sigmoidal model.

Fig. 14A-14D show the average (n-3) relative percentage of PS20 determined by RP-ELSD for samples containing (fig. 14A) 0.005%, (fig. 14B) 0.02%, (fig. 14C) 0.1%, and (fig. 14D) 0.4% PS20 digested with 15 μ g/mL PPL enzyme for 4.5 hours at room temperature in protein-free samples containing 0, 0.5, 5, and 15% (w/v) HP- β -CD without vehicle (control).

FIGS. 15A-15C show plate bar graphs (panel bar plot) showing the average (n ≧ 3) (FIG. 15A) ≥ 2 μ M, (FIG. 15B) ≥ 5 μ M, (FIG. 15C) ≥ 10 μ M particle count/mL for samples digested with 15 μ g/mL PPL enzyme for 4.5 hours at room temperature in protein-free samples containing 0.02% PS20 and 0, 0.1, 0.5, 5 and 15% (w/v) HP- β -CD.

Figure 16 shows the average (n-3) relative percentage of PS20 determined by RP-ELSD for samples digested with 15 μ g/mL PPL enzyme for 4.5 hours at room temperature in protein-free samples containing different HP- β -CD: PS20 ratios. Data were fitted using a sigmoidal model.

FIGS. 17A-17D show the relative percentage of PS20 determined by RP-ELSD for 4.5 hour (FIG. 17A) control, (FIG. 17B) monoclonal antibody (mAb), (FIG. 17C) bispecific antibody (BsAb), and (FIG. 17D) single Fab antibody (sFAb) samples digested with 15 μ g/mL PPL enzyme containing 0,5, and 15% (w/v) HP- β -CD at room temperature.

Detailed Description

The present invention relates to a method of reducing polysorbate degradation in an aqueous formulation comprising a polysorbate by adding cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5: 1. The invention also provides methods of reducing the amount of sub-visible particles and visible particles in an aqueous solution and methods of depolymerizing and solubilizing polysorbate degradation products comprising a polysorbate, the method comprising adding a cyclodextrin to the solution, wherein the ratio of cyclodextrin to polysorbate is greater than about 37.5: 1. The invention also provides a stable aqueous formulation comprising a polysorbate and a cyclodextrin, wherein the w/w ratio of cyclodextrin to polysorbate in the formulation is at least about 37.5: 1. In some embodiments, the formulation further comprises a polypeptide.

I. Definition of

The term "pharmaceutical formulation" refers to a formulation in a form such that the biological activity of the active ingredient is effective and free of additional components having unacceptable toxicity to the subject to which the formulation is to be administered. Such formulations are sterile.

"sterile" preparations are sterilized or free or substantially free of all living microorganisms and spores thereof.

A "stable" formulation is one in which the protein substantially retains its physical and/or chemical stability and/or biological activity after storage. Preferably, the formulation substantially retains its physical and chemical stability and its biological activity upon storage. The stable formulation also maintains its polysorbate content after storage. The shelf life is typically selected based on the expected shelf life of the formulation. Various analytical techniques for measuring Protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-. Stability may be measured at a selected exposure and/or temperature over a selected period of time. Stability can be assessed qualitatively and/or quantitatively in a variety of different ways, including assessment of aggregate formation (e.g., using size exclusion chromatography, by measuring turbidity and/or by visual inspection); evaluation of ROS formation (e.g., by using mild stress assay or 2,2' -azobis (2-amidinopropane) dihydrochloride (AAPH) stress assay); oxidation of specific amino acid residues of the protein (e.g., Trp residues and/or Met residues of monoclonal antibodies); charge heterogeneity is assessed by using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino-terminal or carboxy-terminal sequence analysis; mass spectrometry analysis; SDS-PAGE analysis to compare reduced and intact antibodies; peptide mapping (e.g., trypsin or LYS-C) analysis; evaluating the biological activity or target binding function of the protein (e.g., the antigen binding function of an antibody); and so on. Instability may involve any one or more of the following: aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation and/or Trp oxidation), isomerization (e.g., Asp isomerization), cleavage/hydrolysis/fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteines, N-terminal extension, C-terminal processing, glycosylation differences, and the like.

A protein "retains its physical stability" in a pharmaceutical formulation if it shows no or very little signs of aggregation, precipitation and/or denaturation as measured by visual inspection for color and/or clarity, or by UV light scattering or by size exclusion chromatography.

A protein "retains its chemical stability" in a pharmaceutical formulation if the chemical stability at a given time is such that the protein is considered to still retain its biological activity as defined below. Chemical stability can be assessed by detecting and quantifying chemical changes in the protein. Chemical changes may involve protein oxidation, and may be assessed, for example, using tryptic peptide mapping, reverse phase High Performance Liquid Chromatography (HPLC), and liquid chromatography-mass spectrometry (LC/MS). For example, other types of chemical changes include changes in the charge of a protein that can be assessed by ion exchange chromatography or icIEF.

A protein "retains its biological activity" in a pharmaceutical formulation if the biological activity of the protein 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 determined, for example, in an antigen binding assay for a monoclonal antibody.

As used herein, "biological activity" of a protein refers to the ability of the protein to bind to its target, e.g., the ability of a monoclonal antibody to bind to an antigen. It may further comprise a biological response that can be measured in vitro or in vivo. Such activity may be antagonistic or agonistic.

A protein that is "susceptible to oxidation" is a protein that contains one or more residues that have been found to be susceptible to oxidation, such as, but not limited to, methionine (Met), cysteine (Cys), histidine (His), tryptophan (Trp), and tyrosine (Tyr). For example, tryptophan amino acids in the Fab portion of a monoclonal antibody or methionine amino acids in the Fc portion of a monoclonal antibody may be susceptible to oxidation.

By "isotonic" is meant that the formulation of interest has substantially the same osmotic pressure as human blood. Isotonic formulations typically have an osmotic pressure of about 250 to 350 mOsm. Isotonicity can be measured, for example, using a vapor pressure or ice freezing osmometer.

As used herein, "buffer" refers to a buffered solution that resists changes in pH by the action of its acid-base conjugate components. The buffer of the present invention preferably has a pH of about 4.5 to about 8.0. For example, histidine acetate is an example of a buffer that will control pH in this range.

"preservatives" are compounds that may optionally be included in a formulation to substantially reduce the bacterial effects therein, thus, for example, facilitating the preparation of a multi-purpose formulation. Examples of potential preservatives include octadecyl dimethyl benzyl ammonium chloride, hexamethonium chloride, benzalkonium chloride (a mixture of alkyl benzyl dimethyl ammonium chlorides wherein 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 paraben), catechol, resorcinol, cyclohexanol, 3-pentanol and m-cresol. In one embodiment, the preservative herein is benzyl alcohol.

As used herein, "surfactant" refers to a surface active agent, preferably a nonionic surfactant. Examples of surfactants herein include polysorbates (e.g., polysorbate 20 and polysorbate 80); poloxamers (e.g., poloxamer 188); triton; sodium Dodecyl Sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-or stearyl-sarcosine; linoleyl-, myristyl-or cetyl-betaine; lauramidopropyl, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-betaine (e.g. lauramidopropyl); myristamidopropyl-, palmitoamidopropyl-, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl taurate or disodium methyl oleyl taurate; and MONAQUAT^TMSeries (Mona Industries, inc., Paterson, n.j.); polyethylene glycol, polypropylene glycol, and copolymers of ethylene glycol and propylene glycol (e.g., Pluronics, PF68, etc.); etc. of the embodiments

As used herein, a "pharmaceutically acceptable" excipient or carrier includes pharmaceutically acceptable carriers, stabilizers, buffers, acids, bases, sugars, preservatives, surfactants, tonicity agents and The like as are well known in The art (Remington: The Science and Practice)of Pharmacy,22nd Ed., Pharmaceutical Press, 2012). Examples of pharmaceutically acceptable excipients include buffers such as phosphate, citrate, acetate and other organic acids; antioxidants including ascorbic acid, L-tryptophan, and methionine; low molecular weight (less than about 10 residues) polypeptides; proteins, such as serum albumin, gelatin, or immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone (PVP); amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides, and other carbohydrates including glucose, mannose, or dextrins; metal complexes, such as Zn-protein complexes; chelating agents, such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions, such as sodium; and/or nonionic surfactants such as polysorbates, poloxamers, polyethylene glycols (PEGs), and PLURONICS^TM. "pharmaceutically acceptable" excipients or carriers are those which are reasonably capable of being administered to a subject to provide an effective dose of the active ingredient employed and which are non-toxic to the subject at the dosages and concentrations employed.

The term "polysorbate" (also abbreviated as PS) as used herein refers to pegylated sorbitan esterified with fatty acids and includes polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate) and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

The term "cyclodextrin" refers to a family of compounds comprising glucose molecules bound in a ring-like structure with d-glucopyranose units linked to α - (1,4) glycosidic linkages. Exemplary cyclodextrins include 2-hydroxypropyl- β -cyclodextrin (HP- β -CD or HP- β -cyclodextrin), 2-hydroxypropyl- α -cyclodextrin (HP- α -CD or HP- α -cyclodextrin), 2-hydroxypropyl- γ -cyclodextrin (HP- γ -CD or HP- γ -cyclodextrin), β -cyclodextrin (β -CD or β -cyclodextrin), sulfobutylether β -cyclodextrin (SBE- β -CD or SBE- β -cyclodextrin), α -cyclodextrin (α -CD or α -cyclodextrin), and γ -cyclodextrin (γ -CD or γ -cyclodextrin). Synonyms for cyclodextrins include Cavitron, cyclic oligosaccharides, cycloamulose and cyclodextran.

The term "tonicity agent" refers to an agent used to adjust or maintain the relative concentration of a solution. Preferred tonicity agents include polyhydric sugar alcohols, preferably trihydric or higher sugar alcohols, such as glycerol, erythritol, arabitol, xylitol, sorbitol, and mannitol.

The term "stabilizing agent" refers to an agent that stabilizes a large number of charged biomolecules, such as proteins and antibodies. Tonicity agents may also be used as stabilizing agents when used with a wide variety of charged biomolecules.

By "reduced polysorbate degradation" is meant conditions under which more polysorbate remains in the sample after a period of time compared to a control under similar storage conditions. For example, a sample with 95% polysorbate after a period of time shows reduced degradation of polysorbate compared to a control sample with 50% polysorbate remaining after the same period of time.

The term "depolymerization" as used herein refers to a reduction in visible and/or sub-visible particles caused by degradation of polysorbate. For example, if the amount of visible and/or sub-visible particles is reduced when added to a solution containing polysorbate, the agent is effective in depolymerizing polysorbate degradation products.

The term "solubilization" refers to the dissolution of a solid in a liquid. For example, if a compound is more readily dissolved in the presence of the agent, the agent effectively dissolves the compound.

The term "w/w ratio" refers to the amount of one solute by mass divided by the amount of another solute by mass. For example, a solution containing 100mg cyclodextrin and 1mg polysorbate has a w/w ratio of cyclodextrin to polysorbate of 100: 1. According to one embodiment, the w/w ratio of cyclodextrin to polysorbate is greater than about 37.5: 1.

By "aqueous formulation" is meant a water-based liquid formulation suitable for administration. The formulation may contain a therapeutic agent, such as an antibody or small molecule, and is preferably sterile. The aqueous formulation may also contain buffers, stabilizers, tonicity agents and excipients.

The formulated protein is preferably substantially pure and desirably substantially homogeneous (e.g., free of contaminating proteins, etc.). By "substantially pure" protein is meant a protein comprising at least about 90 wt.% protein (e.g., monoclonal antibody), preferably at least about 95 wt.%, 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 (e.g., monoclonal antibody) based on the total weight of the composition.

The terms "protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of amino acids of any length. The polymer may be linear or branched, it may comprise modified amino acids, and it may be interrupted by non-amino acids. These terms also include amino acid polymers that have been modified, either naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other procedure or modification, such as conjugation to a labeling component. Also included in the definition are, for example, proteins containing one or more analogs of an amino acid (including, for example, unnatural amino acids, etc.), as well as other modifications known in the art. Examples of proteins 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; leptin; coagulation factors such as factor VIIIC, factor IX, tissue factor and von Willebrand factor; anti-coagulation factors, such as protein C; atrial natriuretic factor; a pulmonary surfactant; plasminogen activators, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; a hematopoietic growth factor; tumor necrosis factor-alpha and-beta; tumor necrosis factor receptors such as death receptor 5 and CD 120; TNF-related apoptosis-inducing ligand (TRAIL); b Cell Maturation Antigen (BCMA); b lymphocyte stimulating factor (BLyS); proliferation-inducing ligand (APRIL); enkephalin; RANTES (regulated on activation of normal T-cell expression and secretion); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; mullerian (Muellian) inhibiting substances; a relaxin a chain; a relaxin B chain; relaxin original; 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; platelet-derived endothelial cell growth factor (PD-ECGF); vascular endothelial growth factor family proteins (e.g., VEGF-A, VEGF-B, VEGF-C, VEGF-D and P1 GF); platelet Derived Growth Factor (PDGF) family proteins (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D and dimers thereof); fibroblast Growth Factor (FGF) families such as aFGF, bFGF, FGF4, and FGF 9; epidermal Growth Factor (EGF); receptors for hormones or growth factors, such as VEGF receptors (e.g., VEGFR1, VEGFR2, and VEGFR3), Epidermal Growth Factor (EGF) receptors (e.g., ErbB1, ErbB2, ErbB3, and ErbB4 receptors), platelet-derived growth factor (PDGF) receptors (e.g., PDGFR- α and PDGFR- β), and fibroblast growth factor receptors; TIE ligands (angiogenin, ANGPT1, ANGPT 2); angiogenin receptors such as TIE1 and TIE 2; 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-b; 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); chemokines, such as CXCL12 and CXCR 4; interferons such as interferon- α, - β, and- γ; colony Stimulating Factors (CSF), such as M-CSF, GM-CSF, and G-CSF; cytokines such as Interleukins (IL), e.g., IL-1 through IL-10; a midkine; superoxide dismutase; a T cell receptor; surface membrane proteins; an attenuation acceleration factor; viral antigens, such as 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; ephrin; bv 8; delta-like ligand 4(DLL 4); del-1; BMP 9; BMP 10; follistatin; hepatocyte Growth Factor (HGF)/Scatter Factor (SF); ALK 1; robo 4; ESM 1; leucosin (Perlecan); EGF-like domain, multiple 7(EGFL 7); CTGF and family members thereof; thrombospondin, such as thrombospondin 1 and thrombospondin 2; collagen, such as collagen IV and collagen XVIII; neurofibrillary proteins such as NRP1 and NRP 2; pleiotrophin (Pleiotrophin, PTN); a Progranulin; proliferation protein (Proliferin); notch proteins such as Notch1 and Notch 4; sema3A, Sema3C, and Sema 3F; tumor associated antigens, such as CA125 (ovarian cancer antigen); an immunoadhesin; as well as fragments and/or variants of any of the above-listed proteins and antibodies (including antibody fragments) that bind to one or more proteins (including, for example, any of the above-mentioned proteins).

The term "antibody" herein is used in the broadest sense and specifically covers monoclonal antibodies (including full length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

An "isolated" protein (e.g., an isolated antibody) is a protein that has been identified and isolated and/or recovered from a component of its natural environment. Contaminant components of their natural environment are substances that would interfere with the research, diagnostic or therapeutic uses of the protein, and may include enzymes, hormones, and other proteinaceous or nonproteinaceous solutes. Since at least one component of the protein's natural environment will not be present, the isolated protein comprises the protein in situ within the recombinant cell. Typically, however, the isolated protein will be prepared by at least one purification step.

A "natural antibody" is typically a heterotetrameric glycoprotein of about 150,000 daltons, consisting of two identical light chains (L) and two identical heavy chains (H). Each light chain is linked to a heavy chain by one covalent disulfide bond, while the number of disulfide bonds varies between heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain has a variable domain (VH) at one end followed by a plurality of constant domains. Each light chain has a variable domain (VL) at one end and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the light chain variable domain is aligned with the variable domain of the heavy chain. It is believed that particular amino acid residues form an interface between the light and heavy chain variable domains.

The term "constant domain" refers to a portion of an immunoglobulin molecule having a more conserved amino acid sequence relative to other portions of the immunoglobulin (the variable domains comprising the antigen binding site). The constant domains comprise the CH1, CH2, and CH3 domains of the heavy chain (collectively referred to as CH) and the CHL (or CL) domain of the light chain.

The "variable region" or "variable domain" of an antibody refers to the amino-terminal domain of the heavy or light chain of the antibody. The variable domain of the heavy chain may be referred to as "VH". The variable domain of the light chain may be referred to as "VL". These domains are usually the most variable parts of an antibody and contain an antigen binding site.

The term "variable" refers to the fact that certain portions of the variable domains differ widely in sequence among antibodies and are used for the binding and specificity of each particular antibody for its particular antigen. However, the variability is not evenly distributed throughout the variable domains of the antibody. It is concentrated in three segments called hypervariable regions (HVRs) or both the light and heavy chain variable domains. In some embodiments, the HVRs are Complementarity Determining Regions (CDRs).

The more highly conserved portions of the variable domains are called Framework Regions (FR). The variable domains of native heavy and light chains each comprise four FR regions, largely in a β -sheet configuration, connected by three HVRs, which form a loop junction, and in some cases form part of a β -sheet structure. The HVRs in each chain are held together by the FR region and, together with HVRs from the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, National Institute of Health, Bethesda, Md. (1991)). The constant domains are not directly involved in binding of the antibody to the antigen, but exhibit various effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity.

The "light chain" of an antibody (immunoglobulin) from any mammalian species can be classified into one of two distinctly different classes, termed kappa ("κ") and lambda ("λ"), based on the amino acid sequence of its constant domain.

The term IgG "isotype" or "subclass" as used herein refers to any subclass of immunoglobulin defined by the chemical and antigenic characteristics of its constant regions. Antibodies (immunoglobulins) can be assigned to different classes depending on the amino acid sequence of their heavy chain constant domains. There are five main types of immunoglobulins: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA 2. The heavy chain constant domains corresponding to different classes of immunoglobulins are referred to as α, β, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of different classes of immunoglobulins are well known and are generally described, for example, in Cellular and mol. The antibody may be part of a larger fusion molecule formed by covalent or non-covalent binding of the antibody to one or more other proteins or peptides.

The terms "full-length antibody," "intact antibody," and "full antibody" are used interchangeably herein to refer to an antibody in its substantially intact form, rather than an antibody fragment as defined below. These terms particularly refer to antibodies having a heavy chain comprising an Fc region.

An "antibody fragment" comprises a portion of an intact antibody, preferably comprising the antigen binding region thereof. Examples of antibody fragments include Fab, Fab ', F (ab')₂And Fv fragments; a diabody; a linear antibody; a single chain antibody molecule; and multispecific antibodies formed from antibody fragments.

Papain digestion of antibodies produces two identical antigen-binding fragments, called "Fab" fragments, each having a single antigen-binding site and a residual "Fc" fragment, the name reflecting its ability to crystallize readily. Pepsin treatment produces a peptide which has two antigen binding sites and is still capable of cross-linkingF (ab') of antigen₂And (3) fragment. Fab fragments contain both the heavy and light chain variable domains, and also the constant domain of the light chain and the first constant domain of the heavy chain (CH 1). Fab' fragments differ from Fab fragments by the addition of several residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab '-SH is the designation herein for Fab', where the cysteine residues of the constant domains carry a free thiol group. F (ab')₂Antibody fragments were originally produced as Fab' fragment pairs with hinge cysteines between them. Chemical coupling of other antibody fragments is also known.

"Fv" is the smallest antibody fragment that contains the entire antigen-binding site. In one embodiment, a two-chain Fv species consists of a dimer of one heavy and one light chain variable domain in tight, non-covalent association. In single chain Fv (scfv) species, one heavy chain and one light chain variable domain may be covalently linked by a flexible peptide linker, such that the light and heavy chains may associate in a "dimeric" structure, similar to those in two-chain Fv species. It is in this configuration that the three HVRs of each variable domain interact to define an antigen binding site on the surface of the VH-VL dimer. The six HVRs collectively confer antibody antigen binding specificity. However, even a single variable domain (or half of an Fv comprising only three HVRs specific for an antigen) has the ability to recognize and bind antigen, although with less affinity than the entire binding site.

"Single chain Fv" or "scFv" antibody fragments comprise the VH and VL domains of an antibody, wherein these domains are present in a single polypeptide chain. Typically, the scFv polypeptide further comprises a polypeptide linker between the VH and VL domains which enables the scFv to form the structure required for antigen binding. For reviews on scFv see, for example, Pluckth ü n, in The Pharmacology of Monoclonal Antibodies, vol.113, Rosenburg and Moore eds, Springer-Verlag, New York, pp.269-315 (1994).

The term "diabodies" refers to antibody fragments with two antigen-binding sites, which fragments comprise a heavy chain variable domain (VH) connected to a light chain variable domain (VL) in the same polypeptide chain (VH-VL). By using linkers that are too short to pair between two domains on the same strand, these domains are forced to pair with the complementary domains of the other strand and create two antigen binding sites. Diabodies may be bivalent or bispecific. Diabodies are described more fully in, for example, EP 404,097; WO 1993/01161; hudson et al, nat. Med.9: 129-; and Hollinger et al, Proc. Natl. Acad. Sci. USA 90: 6444-. Tri-and tetrabodies are also described in Hudson et al, nat. Med.9:129-134 (2003).

The term "monoclonal antibody" as used herein refers to an antibody obtained from a substantially homogeneous population of antibodies, e.g., the individual antibodies comprising the population are identical except for mutations that may be present in minor amounts (e.g., naturally occurring mutations). Thus, the modifier "monoclonal" indicates that the antibody is not characterized as a mixture of discrete antibodies. In certain embodiments, such monoclonal antibodies generally include antibodies comprising a polypeptide sequence that binds a target, wherein the target-binding polypeptide sequence is obtained by a process that includes selecting a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process may be to select a unique clone from a plurality of clones (such as a hybridoma clone bank, a phage clone bank, or a recombinant DNA clone bank). It will be appreciated that selected target binding sequences may be further altered, for example to improve affinity for the target, to humanize the target binding sequence, to improve its production in cell culture, to reduce its immunogenicity in vivo, to produce multispecific antibodies, etc., and that antibodies comprising altered target binding sequences are also monoclonal antibodies of the invention. In contrast to polyclonal antibody preparations, which typically contain different antibodies directed against different determinants (epitopes), each monoclonal antibody of a monoclonal antibody preparation is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibody preparations have the advantage that they are generally uncontaminated by other immunoglobulins.

The modifier "monoclonal" indicates the character of the antibody as being 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, Monoclonal Antibodies used according to the invention can be prepared by a variety of techniques, including, for example, the Hybridoma method (e.g., Kohler and Milstein, Nature,256:495-97 (1975); Hongo et al, Hybridoma,14(3):253-260(1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2 nd. 1988); Hammerling et al, in: Monoclonal Antibodies and T-Hybridomas pp.563-681 Elsevier, N.Y. (1981)), the recombinant DNA method (see, for example, U.S. Pat. No.4,816,567), the phage display technique (see, for example, Nature 2004, 352:624-628 (1991); Marks et al, J.mol. biol.222: Sil. 1247 (1992) and Level et al (99; Level et al, 2000-99; Level et al, Biostrain J.1247.), (III) and Level et al (132) (Level et al, Level) (Legend. 31; 35; Legend. J.32; Legend. 31; 35; Legend; U.32; Legend; E. J.32; Legend; 35; SEQ ID. 31; SEQ ID. 32; 35; USA; 35; SEQ ID. 31; 35; USA; Biostrain; 35; Biond; SEQ ID.),340; SEQ ID.),32; SEQ ID.),340; SEQ ID.),310), and techniques for producing human or human-like antibodies in animals having part or all of the animal's human immunoglobulin locus or genes encoding human immunoglobulin sequences (see, e.g., WO 1998/24893; WO 1996/34096; WO 1996/33735; WO 1991/10741; Jakobovits et al, Proc. Natl. Acad. Sci. USA 90:2551 (1993); Jakobovits et al, Nature 362:255-258 (1993); Bruggemann et al, Yeast in Immunol.7:33 (1993); No. 5,545,807; 5,545, 806; 5,569, 825; 5,625,126; 5,633,425; and 5,661,016; Marks et al, Bio/Technology 10:779-783 (1992); Lonberg et al, Nature 368: 856-; 859 (Imrrison, Nature: 812; Fisherd. H et al, Nature: H. J. H. 14: 1996; Nature technologies: 14: 1996; Nature: 14: 1996; Bioscher., 1996; Nature 78: 368; Biogemann; Australin., 1996; Australin. 93: 1996; Australin. Acad. 93: 5,661,016; Australin. 851; Australine., USA; U.851; U.7: 5,661,016; U.7: 82; USA.

Monoclonal antibodies herein specifically include "chimeric" antibodies in which a portion of the heavy and/or light chain is identical or homologous to corresponding sequences in antibodies derived from a particular species or belonging to a particular antibody class or subclass, while the remainder of the chain is identical or homologous to corresponding sequences in antibodies derived from another species or belonging to another antibody class or subclass, as well as fragments of such antibodies, so long as they exhibit the desired biological activity (see, e.g., U.S. Pat. No.4,816,567; and Morrison et al, Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). The chimeric antibody comprisesAn antibody, wherein the antigen binding region of the antibody is derived from an antibody produced by, for example, immunizing cynomolgus monkeys with an antigen of interest.

"humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequences derived from non-human immunoglobulins. In one embodiment, the humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced with residues from an HVR of a non-human species (donor antibody) such as mouse, rat, rabbit or non-human primate having the desired specificity, affinity, and/or capacity. In some cases, FR residues of the human immunoglobulin are substituted with corresponding non-human residues. In addition, humanized antibodies may comprise residues not found in the recipient antibody or the donor antibody. These modifications can be made to further improve antibody performance. Typically, the humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence. The humanized antibody optionally will also comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. For more details see, e.g., Jones et al, Nature 321:522-525 (1986); riechmann et al, Nature 332: 323-E329 (1988); and Presta, curr, Op, Structure, biol.2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. allergy, Asthma & Immunol.1: 105-; harris, biochem. Soc. transactions 23: 1035-; hurle and Gross, curr. Op. Biotech.5: 428-; and us patent nos. 6,982,321 and 7,087,409.

A "human antibody" is an antibody having an amino acid sequence corresponding to the amino acid sequence of an antibody produced by a human and/or made using any of the techniques disclosed herein for making human antibodies. This definition of human antibody specifically excludes humanized antibodies comprising non-human antigen binding residues. Human antibodies can be generated using a variety of techniques known in the art, including phage display libraries. Hoogenboom and Winter, J.mol.biol.,227:381 (1991); marks et al, J.mol.biol.,222:581 (1991).Also useful for the preparation of human Monoclonal Antibodies are those described in Cole et al, Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); boerner et al, J.Immunol.,147(1):86-95(1991), See also van Dijk and van de Winkel, Curr.Opin. Pharmacol.,5:368-74 (2001). Human antibodies are prepared by administering an antigen to a transgenic animal that has been modified to produce such antibodies in response to antigen challenge, but whose endogenous locus has failed, e.g., an immunized XENOMOUSE (see, e.g., for xenomice)^TMU.S. patent nos. 6,075,181 and 6,150,584 of the art). See also, e.g., Li et al Proc. Natl. Acad. Sci. USA,103:3557-3562(2006) for human antibody production by human B-cell hybridoma technology.

As used herein, the term "hypervariable region", "HVR" or "HV" refers to the regions of a sequence that are hypervariable and/or form the antibody variable domains of structurally defined loops. Typically, an antibody comprises six HVRs; three of VH (H1, H2, H3), three of VL (L1, L2, L3). Among natural antibodies, H3 and L3 showed the most diversity of 6 HVRs, and H3 is believed to play a unique role, particularly in conferring good specificity against antibodies. See, e.g., Xu et al, Immunity 13:37-45 (2000); johnson and Wu, in Methods in Molecular Biology 248:1-25(Lo, ed., Human Press, Totowa, N.J., 2003). In fact, naturally occurring camelid (camelid) antibodies consisting of a heavy chain are functional and stable only in the absence of a light chain. See, e.g., Hamers-Casterman et al, Nature 363: 446-; sheriff et al, Nature struct.biol.3:733-736 (1996).

Many HVR descriptions are in use and are included herein. Kabat Complementarity Determining Regions (CDRs) are based on sequence variability and are the most commonly used (Kabat et al, Sequences of Proteins of Immunological Interest,5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). Chothia instead refers to the position of the structural loops (Chothia and Lesk J. mol. biol.196:901-917 (1987)). The AbM HVR represents a compromise between the Kabat HVR and Chothia structural loops and is used by Oxford Molecular's AbM antibody modeling software. The "contact" HVR is based on an analysis of the available complex crystal structure. The residues for each of these HVRs are described below.

The HVRs can comprise "extended HVRs" as follows: 24-36 or 24-34(L1), 46-56 or 50-56(L2) and 89-97 or 89-96(L3) in VL and 26-35(H1), 50-65 or 49-65(H2) and 93-102, 94-102 or 95-102(H3) in VH. For each of these definitions, the variable domain residues are numbered according to Kabat et al (supra).

"framework" or "FR" residues are those variable domain residues other than the HVR residues as defined herein.

The term "variable domain residue numbering as in Kabat" or "amino acid position numbering as in Kabat" and variations thereof refers to the numbering system used in Kabat et al (supra) to edit the heavy or light chain variable domain of an antibody. Using this numbering system, the actual linear amino acid sequence may contain fewer or additional amino acids corresponding to a shortening of, or insertion into, the FR or HVR of the variable domain. For example, a heavy chain variable domain may comprise a single amino acid insert (residue 52a according to Kabat) after residue 52 of H2 and inserted residues (e.g. residues 82a, 82b, and 82c, etc. according to Kabat) after heavy chain FR residue 82. Kabat numbering of residues of a given antibody can be determined by alignment at regions of homology of the antibody sequence to a "standard" Kabat-numbered sequence

When referring to residues in the variable domain (about residues 1-107 of the light chain and residues 1-113 of the heavy chain), the Kabat numbering system is typically used (e.g., Kabat et al, Sequences of Immunological interest.5th Ed. public Health Service, National Institutes of Health, Bethesda, Md. (1991)). When referring to residues in the constant region of an immunoglobulin heavy chain (e.g., the EU index as reported by Kabat et al, supra), the "EU numbering system" or "EU index" is typically used. The "EU index in Kabat" refers to the residue numbering of the human IgG1 EU antibody.

The expression "linear antibody" refers to the antibodies described in Zapata et al (Protein Eng.,8(10):1057-1062 (1995)). Briefly, these antibodies comprise a pair of Fc segments (VH-CH1-VH-CH1) in tandem, which together with a complementary light chain polypeptide form a pair of antigen binding regions. Linear antibodies can be bispecific or monospecific.

Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. It may be useful to conjugate the relevant antigen (particularly when synthetic peptides are used) to a protein that is immunogenic in the species to be immunized. For example, bifunctional or derivatizing agents are used, such as maleimidobenzoyl sulfosuccinimide esters (conjugated via cysteine residues), N-hydroxysuccinimide esters (conjugated via lysine residues), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N ═ C ═ NR wherein R and R¹Is a different alkyl group, and the antigen may be conjugated to Keyhole Limpet Hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

The term "multispecific antibody" is used in the broadest sense and specifically covers antibodies comprising antigen binding domains with polyepitopic specificity (i.e., capable of specifically binding to two or more different epitopes on one biomolecule, or capable of specifically binding to epitopes on two or more different biomolecules). In some embodiments, the antigen binding domain of a multispecific antibody (such as a bispecific antibody) comprises two VH/VL units, wherein a first VH/VL unit specifically binds a first epitope and a second VH/VL unit specifically binds a second epitope, wherein each VH/VL unit comprises a heavy chain variable domain (VH) and a light chain variable domain (VL). Such multispecific antibodies include, but are not limited to, full-length antibodies, antibodies with two or more VL and VH domains, antibody fragments (such as Fab, Fv, dsFv, scFv), diabodies, bispecific diabodies and triabodies, antibody fragments that have been covalently or non-covalently linked. A VH/VL unit that also comprises at least a portion of a heavy chain constant region and/or at least a portion of a light chain constant region may also be referred to as a "hemimer" or "half antibody". In some embodiments, the half-antibody comprises at least a portion of a single heavy chain variable region and at least a portion of a single light chain variable region. In some such embodiments, a bispecific antibody comprising two half-antibodies and binding to two antigens comprises a first half-antibody that binds to a first antigen or first epitope but not to a second antigen or second epitope and a second half-antibody that binds to a second antigen or second epitope but not to the first antigen or first epitope. According to some embodiments, the multispecific antibody is an IgG antibody that binds to each antigen or epitope with an affinity of 5M to 0.001pM, 3M to 0.001pM, 1M to 0.001pM, 0.5M to 0.001pM, or 0.1M to 0.001 pM. In some embodiments, the semimer comprises a sufficient portion of the heavy chain variable region to allow for the formation of an intramolecular disulfide bond with the second semimer. In some embodiments, the hemimer comprises a knob mutation (knob mutation) or a hole mutation (hole mutation), for example to allow heterodimerization with a second hemimer or hemiantibody comprising a complementary hole mutation or knob mutation. The knob and hole mutations will be discussed further below.

A "bispecific antibody" is a multispecific antibody comprising antigen-binding domains capable of specifically binding to two different epitopes on one biomolecule, or capable of specifically binding to epitopes on two different biomolecules. Bispecific antibodies may also be referred to herein as having "dual specificity" or being "dual specific". Unless otherwise indicated, the order in which the bispecific antibody binds the antigens listed in the bispecific antibody name is arbitrary. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and optionally at least a portion of a heavy chain constant region, and a single light chain variable region and optionally at least a portion of a light chain constant region. In certain embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region, and does not comprise more than one single heavy chain variable region and does not comprise more than one single light chain variable region. In some embodiments, the bispecific antibody comprises two half antibodies, wherein each half antibody comprises a single heavy chain variable region and a single light chain variable region, and wherein the first half antibody binds to the first antigen and does not bind to the second antigen, and the second half antibody binds to the second antigen and does not bind to the first antigen.

The term "knob-to-hole" or "KnH" techniques as used herein refers to techniques for pairing two polypeptides together in vitro or in vivo by introducing a knob (knob) into one polypeptide and a cavity (hole) into the other polypeptide at the interface where they interact. For example, KnH has been introduced into the Fc: Fc binding interface of an antibody, the CL: CH1 interface or the VH/VL interface of an antibody (see, e.g., US 2011/0287009, US2007/0178552, WO 96/027011, WO 98/050431, and Zhu et al, 1997, Protein Science 6: 781-788). In some embodiments, during the manufacture of the multispecific antibody, KnH drives the pairing of two different heavy chains. For example, a multispecific antibody having KnH in its Fc region may also comprise a single variable domain linked to each Fc region, or further comprise different heavy chain variable domains paired with similar or different light chain variable domains. The KnH technique can also be used to pair two different receptor extracellular domains or any other polypeptide sequences that contain different target recognition sequences (e.g., including affibodies, peptibodies, and other Fc fusions).

The term "knob mutation" as used herein refers to a mutation that introduces a protuberance (knob) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a hole mutation (see, e.g., US 5,731,168, US 5,807,706, US 5,821,333, US 7,695,936, US 8,216,805, each incorporated herein by reference in its entirety).

The term "socket mutation" as used herein refers to a mutation that introduces a cavity (socket) into a polypeptide at the interface where the polypeptide interacts with another polypeptide. In some embodiments, the other polypeptide has a knob mutation (see, e.g., US 5,731,168, US 5,807,706, US 5,821,333, US 7,695,936, US 8,216,805, each of which is incorporated herein by reference in its entirety).

The term "about" as used herein refers to an acceptable error range for the corresponding value as determined by one of ordinary skill in the art, which will depend in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" can mean within 1 standard deviation or more than 1 standard deviation, according to practice in the art. Reference herein to a "value or parameter of" about "includes and describes embodiments that are directed to that value or parameter per se. For example, a description referring to "about X" includes a description of "X".

As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to "a compound" optionally includes combinations of two or more such compounds, and the like.

Formulation and preparation

The present invention relates to a method of reducing polysorbate degradation in an aqueous formulation comprising a polysorbate, the method comprising adding a cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5: 1. In some embodiments, the present invention provides a method of reducing the amount of sub-visible and visible particles in an aqueous solution comprising a polysorbate, the method comprising adding cyclodextrin to the solution, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5: 1. In some embodiments, the present invention provides a method of depolymerizing and solubilizing polysorbate degradation products in an aqueous formulation, the method comprising adding cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than about 37.5: 1. In some embodiments, the cyclodextrin is HP-beta cyclodextrin, HP-gamma cyclodextrin, or sulfobutyl ether beta-cyclodextrin. In some embodiments, the cyclodextrin is HP-alpha cyclodextrin. In some embodiments, the formulation further comprises a polypeptide.

In some embodiments, the method comprises adding polyvinylpyrrolidone (PVP) to the formulation, wherein the resulting w/w ratio of PVP to polysorbate is greater than about 37.5: 1. In some embodiments, the present invention provides a method of reducing the amount of sub-visible and visible particles in an aqueous solution comprising a polysorbate, the method comprising adding PVP to the solution, wherein the resulting w/w ratio of PVP to polysorbate is greater than about 37.5: 1. In some embodiments, the present invention provides a method of depolymerizing and solubilizing polysorbate degradation products in an aqueous formulation, the method comprising adding PVP to the formulation, wherein the resulting w/w ratio of PVP to polysorbate is greater than about 37.5: 1.

In some embodiments, the present invention provides an aqueous formulation comprising a polysorbate and cyclodextrin, wherein less than 1% of the polysorbate is degraded after storage at about 1 ℃ to about 10 ℃ for at least about 6 months to at least about 48 months, wherein the w/w ratio of cyclodextrin to polysorbate in the formulation is at least about 37.5: 1. In some embodiments, the present invention provides an aqueous formulation comprising polysorbate and PVP, wherein less than 1% of the polysorbate is degraded after storage at about 1 ℃ to about 10 ℃ for at least about 6 months to at least about 48 months, wherein the w/w ratio of PVP to polysorbate in the formulation is at least about 37.5: 1. In some embodiments, the formulation is stable at about 2 ℃ to about 8 ℃ for at least about 6 months to at least about 48 months, at least about 12 months, at least about 18 months, at least about 24 months, or at least about 48 months. In some embodiments, the formulation comprises about 0.005% to 0.4% polysorbate. In some embodiments, the formulation comprises about 0.005% -0.4% polysorbate, and the cyclodextrin is added to the formulation to a concentration of about 0.01% -30%. In some embodiments, the cyclodextrin is HP-beta cyclodextrin, HP-gamma cyclodextrin, or sulfobutyl ether beta-cyclodextrin. In some embodiments, the cyclodextrin is HP-alpha cyclodextrin. In some embodiments, polysorbate degradation is reduced by about 50%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In further embodiments, less than about 1,000, about 750, about 500, about 250, about 150, about 100, about 50, or about 25 polysorbate particles greater than about 2 microns in diameter are formed per mL. In some embodiments, the formulation further comprises a polypeptide. In some embodiments, the protein concentration is about 1mg/mL to about 250 mg/mL. In some embodiments, the protein concentration is greater than about 250 mg/mL. In some embodiments, the formulation has a pH of about 4.5 to about 7.0, or about 4.5 to about 6.0, or about 6.0. In some embodiments, the formulation further comprises one or more of a stabilizer, a buffer, a surfactant, and a tonicity agent. In further embodiments, the formulation is suitable for intravenous, subcutaneous, intramuscular, or intravitreal administration to a subject. In some embodiments, the polypeptide is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment. In some embodiments, the formulation further comprises a small molecule, nucleic acid, lipid, and/or carbohydrate.

The proteins and antibodies in the formulations can be prepared using methods known in the art. Provided herein are non-limiting exemplary methods for making antibodies (e.g., full-length antibodies, antibody fragments, and multispecific antibodies). Antibodies in aqueous formulations are prepared using techniques available in the art for producing antibodies, exemplary methods of which are described in more detail in the following sections. One skilled in the art can adapt the methods herein for use in preparing formulations comprising other proteins, such as peptide-based inhibitors. For well-established and commonly used techniques and procedures commonly used for the production of therapeutic proteins, see Sam Sambrook et al, Molecular Cloning: A Laboratory Manual,4th ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2012); current Protocols in Molecular Biology, F.M. Ausubel, et al eds. (2003); short Protocols in Molecular Biology, Ausubel et al, eds., J.Wiley and Sons (2002); horspin et al, Current Protocols in Protein Science, (2006); antibodies, A Laboratory Manual, Harlow and Lane, eds. (1988); R.I.Freshney, Culture of Animal Cells A Manual of Basic Technique and Specialized Application,6th ed., J.Wiley and Sons (2010), which is incorporated herein by reference in its entirety.

A. Antibody preparation

The antibodies in the aqueous formulations provided herein are directed against an antigen of interest. Preferably, the antigen is a biologically important polypeptide, and administration of the antibody to a mammal having a disorder can result in a therapeutic benefit in the mammal. However, antibodies directed against non-polypeptide antigens are also contemplated.

When the antigen is a polypeptide, it may be a transmembrane molecule (e.g. receptor) or a ligand such as a growth factor. Exemplary antigens include molecules such as Vascular Endothelial Growth Factor (VEGF); CD 20; ox-LDL; ox-ApoB 100; 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 Willebrand factor; anti-coagulation factors, such as protein C; atrial natriuretic factor; a pulmonary surfactant; plasminogen activators, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; a hematopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalin; RANTES (modulation activates normal T cell expression and secretion); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; a mullerian tube inhibiting substance; a relaxin a chain; a relaxin B chain; (ii) 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; receptors for hormones or growth factors; protein A or D; rheumatoid factor; neurotrophic factors such as Bone Derived Neurotrophic Factor (BDNF), neurotrophic factor-3, 4, -5 or-6 (NT-3, NT4, NT-5 or NT-6) or nerve growth factors 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-1 (brain IGF-1), insulin-like growth factor binding protein; 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- α, - β, and- γ; 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; an attenuation acceleration factor; viral antigens, such as 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; tumor associated antigens such as HER2, HER3, or HER4 receptors; and fragments of any of the above-listed polypeptides.

(i) Antigen preparation

Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens for antibody production. For transmembrane molecules, such as receptors, fragments of these (e.g., the extracellular domain of a 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 that have been transformed by recombinant techniques to express a transmembrane molecule. Other antigens and forms thereof for making antibodies will be apparent to those skilled in the art.

(ii) Certain antibody-based methods

Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and adjuvant. Using bifunctional or derivatizing agents, e.g. maleimidobenzoyl sulphosuccinimide ester (conjugated via a cysteine residue), N-hydroxysuccinimide (conjugated via a lysine residue), glutaraldehyde, succinic anhydride, SOCl₂Or R¹N ═ C ═ NR wherein R and R¹Are different alkyl groups and can be used to conjugate the antigen of interest to a protein that is immunogenic in the species to be immunized, such as Keyhole Limpet Hemocyanin (KLH), serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

Animals are immunized against an antigen, immunogenic conjugate or derivative by combining, for example, 100 μ g or 5 μ g of protein or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, animals were boosted with 1/5 to 1/10 initial amounts of peptide or conjugate in freund's complete adjuvant by subcutaneous injection at multiple sites. After 7 to 14 days, the animals were bled and the serum was assayed for antibody titer. Animals were boosted until titer stabilized (titer planteau). Preferably, the animal is boosted with a conjugate of the same antigen but conjugated to a different protein and/or by a different cross-linking agent. Conjugates can also be prepared in recombinant cell culture as protein fusions. Furthermore, polymerization agents such as alum are suitable for enhancing immune responses.

Monoclonal Antibodies of the invention can be prepared using the Hybridoma method described for the first time by Kohler et al, Nature,256:495(1975), and further described, for example, in Hongo et al, Hybridoma,14(3):253-260(1995), Harlow et al, Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press,2nd ed.1988); hammerling et al, in Monoclonal Antibodies and T-Cell hybrids 563-681(Elsevier, N.Y.,1981), and Ni, Xiandai Mianyixue,26(4):265-268(2006) (for human-human Hybridomas). Additional methods include, for example, U.S. Pat. No. 7,189,826 directed to the production of monoclonal human native IgM antibodies from hybridoma cell lines. The human hybridoma technique (Trioma technique) is described in Vollmers and Brandlein, Histology and Histopathology,20(3): 927-.

For various other hybridoma techniques, see, e.g., US 2006/258841; US 2006/183887 (fully human antibodies), US 2006/059575; US 2005/287149; US 2005/100546; US 2005/026229; and us patent nos. 7,078,492 and 7,153,507. An exemplary protocol for producing monoclonal antibodies using the hybridoma method is described below. In one embodiment, a mouse or other suitable host animal, such as a hamster, is immunized to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the protein used for immunization. Antibodies are produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of a polypeptide of the invention or a fragment thereof, and an adjuvant such as monophosphoryl lipid a (mpl)/Trehalose Dimycolate (TDM) (Ribi immunochem. The polypeptides (e.g., antigens) or fragments thereof of the invention can be prepared using methods well known in the art, such as recombinant methods, some of which are further described herein. Sera from immunized animals are assayed for anti-antigen antibodies and optionally given a booster immunization. Lymphocytes from animals that produce anti-antigen antibodies are isolated. Alternatively, lymphocytes can be immunized in vitro.

The lymphocytes are then fused with myeloma cells using a suitable fusing agent (such as polyethylene glycol) to form hybridoma cells. See, for example, Goding, Monoclonal Antibodies: Principles and Practice, pp.59-103Academic Press, (1986). Efficient fusion of myeloma cells can be used, supporting stable high-level antibody production by the selected antibody-producing cells, and sensitive to media such as HAT media. Exemplary myeloma cells include, but are not limited to, murine myeloma Cell lines, such as MOPC-21 and MPC-11 mouse tumors from the Salk Institute Cell Distribution Center, San Diego, Calif. USA, and SP-2 or X63-Ag8-653 cells from the American Type Culture Collection, Rockville, Md. USA. Human myeloma and mouse-human heteromyeloma cell lines have also been described for the Production of human Monoclonal antibodies (Kozbor, J.Immunol.,133:3001 (1984); Brodeur et al, Monoclonal Antibody Production Techniques and Applications, pp.51-63Marcel Dekker, Inc., New York (1987).

The hybridoma cells so prepared are seeded and grown in a suitable medium, such as one containing one or more substances that inhibit the growth or survival of the unfused parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas typically includes hypoxanthine, aminopterin, and thymidine (HAT medium), which substances prevent the growth of HGPRT-deficient cells. Preferably, serum-free hybridoma cell culture methods are used to reduce the use of animal-derived sera, such as fetal bovine serum, described, for example, in Even et al, Trends in Biotechnology,24(3),105-108 (2006).

Oligopeptides that are useful as tools for improving productivity of hybridoma cell cultures are described in Franek, Trends in Monoclonal Antibody Research,111-122 (2005). In particular, standard media are rich in certain amino acids (alanine, serine, asparagine, proline) or protein hydrolysate fractions, and apoptosis can be significantly inhibited by synthetic oligopeptides consisting of 3-6 amino acid residues. The peptide is present at a millimolar concentration or greater.

The medium in which the hybridoma cells are grown can be assayed to produce monoclonal antibodies that bind to the antibodies of the invention. The binding specificity of monoclonal antibodies produced by hybridoma cells can be determined by immunoprecipitation or by an in vitro binding assay such as Radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA). For example, the binding affinity of monoclonal antibodies can be determined by a Scatchard analysis. See, e.g., Munson et al, anal. biochem.,107:220 (1980).

After hybridoma cells producing antibodies with the desired specificity, affinity, and/or activity are identified, the clones can be subcloned by limiting dilution procedures and grown by standard methods. See, e.g., Goding (supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can grow in the animal as ascites tumors. Monoclonal antibodies secreted by the subclones are suitably isolated from the culture medium, ascites fluid or serum by conventional immunoglobulin purification procedures, such as protein a-sepharose, hydroxyapatite chromatography, gel electrophoresis, dialysis or affinity chromatography. One procedure for isolating proteins from hybridoma cells is described in US 2005/176122 and US patent No. 6,919,436. The method comprises using a minimum amount of a salt such as a lyotropic salt during binding and preferably also a small amount of an organic solvent during elution.

(iii) Certain library screening methods

The antibodies of the invention can be screened for antibodies with desired activity by using combinatorial libraries. For example, various methods are known in the art for generating phage display libraries and screening libraries for antibodies with desired binding characteristics. This method is generally described in Hoogenboom et al in Methods in Molecular Biology 178:1-37, O' Brien et al ed., Human Press, Totowa, N.J. (2001). For example, one method of producing an antibody of interest is by using a phage display library as described in Lee et al, J.mol.biol.340(5):1073-93 (2004).

In principle, synthetic antibody clones are selected by screening phage libraries containing phage displaying various fragments of antibody variable regions (Fv) fused to phage coat proteins. Such phage libraries are panned by affinity chromatography against the desired antigen. Clones expressing Fv fragments capable of binding to the desired antigen are adsorbed to the antigen and thereby separated from the non-binding clones in the library. The bound clones are then eluted from the antigen and may be further enriched by additional antigen adsorption/elution cycles. Any antibody of the invention can be obtained as follows: suitable antigen screening programs were designed to select phage clones of Interest, followed by the construction of full-length antibody clones using Fv Sequences and suitable constant region (Fc) Sequences from phage clones of Interest, as described in Kabat et al, Sequences of Proteins of Immunological Interest, Fifth Edition, NIH Publication 1-3:91-3242, Bethesda Md. (1991).

In certain embodiments, the antigen-binding domain of an antibody is formed from two variable (V) regions of about 110 amino acids, each from a light chain (VL) and a heavy chain (VH), both of which present three hypervariable loops (HVRs) or Complementarity Determining Regions (CDRs). The variable domains can be functionally displayed on phage as single chain fv (scFv) fragments, in which VH and VL are covalently linked by a short flexible peptide, or as Fab fragments, in which they are each fused to a constant domain and interact non-covalently, as described in Winter et al, Ann.Rev.Immunol.,12:433-455 (1994). As used herein, scFv-encoding phage clones and Fab-encoding phage clones are collectively referred to as "Fv phage clones" or "Fv clones".

The repertoire of VH and VL genes (reteire) can be cloned separately by Polymerase Chain Reaction (PCR) and recombined randomly in a phage library, which can then be searched for antigen binding clones as described by Winter et al, Ann. Rev. Immunol.,12:433-455 (1994). Libraries from immune sources provide high affinity antibodies to the immunogen without the need to construct hybridomas. Alternatively, as described by Griffiths et al, EMBO J,12: 725-. Finally, an untargeted library can also be prepared synthetically as follows: unrearranged V gene fragments from stem cells were cloned and rearranged in vitro using PCR primers containing random sequences to encode the highly variable CDR3 regions, as described in Hoogenboom and Winter, J.Mol.biol.,227:381-388 (1992).

In certain embodiments, filamentous phages are used to display antibody fragments by fusion to the minor coat protein pIII. Antibody fragments may be displayed as single chain Fv fragments in which the VH and VL domains are linked to the same polypeptide chain by a flexible polypeptide spacer, for example as described by Marks et al, J.mol.biol.,222: 581-.

Typically, the nucleic acid encoding the antibody gene fragment is obtained from immune cells harvested from a human or animal. If a library favoring anti-antigen clones is desired, the subject is immunized with the antigen to generate an antibody response, and spleen cells and/or circulating B cells or other Peripheral Blood Lymphocytes (PBLs) are recovered for library construction. In one embodiment, a library of human antibody gene fragments biased for anti-antigen cloning is obtained by generating an anti-antigen antibody response in transgenic mice carrying a functional human immunoglobulin gene array (and lacking a functional endogenous antibody production system), such that antigen-immunized B cells produce human antibodies against the antigen. Transgenic mice that produce human antibodies are described below.

Additional enrichment of the anti-antigen reactive cell population can be obtained as follows: b cells expressing antigen-specific membrane-bound antibodies are isolated using a suitable screening procedure, for example by cell separation using antigen affinity chromatography or adsorption of cells to fluorochrome-labeled antigens, followed by Flow Activated Cell Sorting (FACS).

Alternatively, the use of spleen cells and/or B cells or other PBLs from non-immunized donors provides better representativeness of a possible repertoire of antibodies, and also allows the construction of antibody libraries using any animal (human or non-human) species, where the antigen is not antigenic. For libraries constructed for incorporation of in vitro antibody genes, stem cells are harvested from a subject to provide nucleic acids encoding unrearranged antibody gene segments. Immune cells of interest can be obtained from a variety of animal species, such as human, mouse, rat, lagomorpha, wolf (lupine), canine (Canine), feline (feline), porcine (porcine), bovine (bovine), equine (equine), and avian (avian) species, among others.

Nucleic acids encoding antibody variable gene segments (including VH and VL segments) are recovered from cells of interest and amplified. In the case of rearranged VH and VL gene libraries, the desired DNA may be obtained as follows: genomic DNA or mRNA is isolated from lymphocytes and different V genome libraries are prepared for expression by Polymerase Chain Reaction (PCR) using primers that match the 5 'and 3' ends of the rearranged VH and VL genes, as described by Orlandi et al, Proc. Natl. Acad. Sci. (USA),86: 3833-. The V gene can be amplified from cDNA and genomic DNA with a back primer at the 5' end of the exon encoding the mature V domain, and a forward primer based within the J segment, as described in Orlandi et al (1989) and Ward et al, Nature,341:544-546 (1989). However, for amplification from cDNA, reverse primers may also be based on leader exons as described in Jones et al, Biotechnol.,9:88-89(1991), and forward primers within the constant region as described in Sastry et al, Proc. Natl. Acad. Sci. (USA),86: 5728-. To maximize complementarity, degeneracy can be introduced into the primers, as described in Orlandi et al (1989) or Sasty et al (1989). In certain embodiments, library diversity is maximized by: PCR primers targeting each V gene family are used to amplify all available VH and VL permutations present in immune cell Nucleic acid samples, for example as described in Marks et al, J.mol.biol.,222:581-597(1991) or as described in Orum et al, Nucleic Acids Res.,21:4491-4498 (1993). For cloning of the amplified DNA into an expression vector, a rare restriction site may be introduced as a tag at one end within the PCR primers, as described by Orlandi et al (1989), or by further PCR amplification with tagged primers, as described by Clackson et al, Nature,352: 624-.

A repertoire of synthetically rearranged V genes can be obtained in vitro from V gene segments. Most human VH-gene segments have been cloned and sequenced (reported in Tomlinson et al, J.mol.biol.,227:776-798 (1992)) and mapped (reported in Matsuda et al, Nature Genet.,3:88-94 (1993)); segments of these clones (including all major conformations of the loops H1 and H2) can be used to generate different VH genomic libraries in which PCR primers encode H3 loops of different sequences and lengths, as described in Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992). VH repertoires can also be prepared with full sequence diversity centered in a single length of long H3 loop, as described by Barbas et al, Proc. Natl. Acad. Sci. USA,89:4457-4461 (1992). Human V κ and V λ segments (reported in Williams and Winter, Eur.J.Immunol.,23: 1456-. Based on a series of VH and VL folds and L3 and H3 lengths, synthetic V genome libraries will encode antibodies with considerable structural diversity. After amplification of the DNA encoding the V gene, germline V gene segments can be rearranged in vitro according to the method of Hoogenboom and Winter, J.mol.biol.,227:381-388 (1992).

Repertoires of antibody fragments can be constructed by combining VH and VL genomic libraries together in several ways. Various libraries can be generated in different vectors and the vectors recombined in vitro, for example as described in Hogrefe et al, Gene,128:119-126(1993), or by combinatorial infection in vivo, for example the loxP system described in Waterhouse et al, Nucl. acids Res.,21:2265-2266 (1993). The in vivo recombination method exploits the double-stranded nature of the Fab fragment to overcome the library size limitation imposed by E.coli transformation efficiency. The non-challenged VH and VL repertoires were cloned separately, one into a phagemid and the other into a phage vector. The two libraries are then combined by phage infection of phagemid-containing bacteria such that each cell contains a different combination and the library size is limited only by the number of cells present (about 10)¹²Individual clones). Both vectors contain in vivo recombination signals, such that the VH and VL genes are recombined onto a single replicon and co-packaged into phage virions. These huge libraries provide a large number of antibodies (K) of different affinities_d ^-1About 10^-8M)。

Alternatively, all libraries may be cloned sequentially into the same vector, e.g., as described in Barbas et al, Proc. Natl. Acad. Sci. USA,88: 7978-. PCR assembly can also be used to link VH and VL DNA with DNA encoding flexible peptide spacers to form single chain fv (scfv) repertoires. In another technique, "cellular PCR assembly" is used to combine VH and VL genes in lymphocytes by PCR, and then clone a repertoire of linked genes, as described in Embleton et al, Nucl. acids Res.,20:3831-3837 (1992).

Antibodies produced by an untapped library (natural or synthetic) may have intermediate affinity (K)_d ^-1Is about 10⁶To 10⁷M^-1) However, affinity maturation can also be simulated in vitro by construction and re-selection from a second library, as described in Winter et al (1994) (supra). For example, mutations can be randomly introduced in vitro by using the error-prone polymerase (reported in Leung et al, Technique 1:11-15 (1989)) in the method of Hawkins et al, J.mol.biol.,226:889-896(1992) or in the method of Gram et al, Proc.Natl.Acad.Sci USA,89:3576-3580 (1992). Furthermore, affinity maturation can be performed as follows: one or more CDRs are randomly mutated, e.g., in selected individual Fv clones using PCR with primers generated with random sequences carrying the CDRs of interest, and higher affinity clones are screened. WO 9607754 (published 3/14 1996) describes a method for inducing mutagenesis in complementarity determining regions of immunoglobulin light chains to generate a light chain gene library. Another useful method is to recombine VH or VL domains selected by phage display with a repertoire of naturally occurring V domain variants obtained from naive donors and screen for higher affinity in several rounds of chain transformations (chain reshuffling), as described in Marks et al, Biotechnol.,10: 779-. This technique allows to generate an affinity of about 10^-9M or lower, and antibody fragments.

Screening of the library can be accomplished by a variety of techniques known in the art. For example, the wells of an adsorption plate can be coated with an antigen, expressed on host cells attached to the adsorption plate or used for cell sorting, or bound to biotin for capture with streptavidin-coated beads, or any other method for panning a phage display library.

Contacting a phage library sample with the immobilized antigen under conditions suitable for binding at least a portion of the phage particles with the adsorbent. Typically, conditions including pH, ionic strength, temperature, etc., are selected to mimic physiological conditions. The solid phase bound phage is washed and then eluted with an acid (e.g., as described in Barbas et al, Proc. Natl. Acad. Sci USA,88: 7978-. Phages can be enriched 20-1,000-fold in a single round of selection. Moreover, enriched phage can be grown in bacterial culture and subjected to more rounds of selection.

The efficiency of selection depends on many factors, including the kinetics of dissociation during washing, and whether multiple antibody fragments on a single phage can bind to the antigen simultaneously. Antibodies with fast dissociation kinetics (and weak binding affinity) can be retained by using short washes, multivalent phage display, and high antigen coating density in the solid phase. The high density not only stabilizes the phage through multivalent interactions, but also facilitates the reassociation of dissociated phage. Selection of antibodies with slow dissociation kinetics (and good binding affinity) can be facilitated by the use of long washes and monovalent phage display (as described in Bass et al, Proteins,8: 309-.

It is possible to select between phage antibodies of different affinities, even if the affinities are slightly different, for the antigen. However, random mutagenesis of selected antibodies (e.g., as performed in some affinity maturation techniques) may result in many mutants, most binding to antigen, and a few with higher affinity. In the case of restricted antigens, rare high affinity phages may be eliminated. To retain all higher affinity mutants, the phage may be incubated with an excess of biotinylated antigen, but with biotinylated antigen at a lower molarity than the target molarity affinity constant for the antigen. High affinity binding phage can then be captured by streptavidin-coated paramagnetic beads. This "equilibrium capture" allows the selection of antibodies based on their binding affinity, with a sensitivity that allows the isolation of mutant clones from a large excess of phage with as little as two times as high affinity. The conditions used to wash phage bound to the solid phase can also be manipulated to differentiate based on dissociation kinetics.

Antigenic clones can be selected for activity. In certain embodiments, the invention provides anti-antigen antibodies that bind to live cells that naturally express an antigen or bind to a free floating antigen or an antigen linked to other cellular structures. Fv clones corresponding to such anti-antigen antibodies can be selected as follows: (1) isolating anti-antigen clones from the phage library as described above, and optionally amplifying the population of isolated phage clones by culturing the population in a suitable bacterial host; (2) the antigen and the second protein need to be selected for blocking and non-blocking activity, respectively; (3) adsorbing the anti-antigen phage clones to the immobilized antigen; (4) eluting any unwanted clones that recognize antigen binding determinants that overlap or share with binding determinants of the second protein with an excess of the second protein; and (5) the adsorbed clones remain after the elution step (4). Optionally, clones with the desired blocking/non-blocking properties can be further enriched by repeating the selection procedure described herein one or more times.

DNA encoding the hybridoma-derived monoclonal antibodies or phage-display Fv clones of the invention can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide primers designed to specifically amplify the heavy and light chain coding regions of interest from a hybridoma or phage DNA template). Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells that do not otherwise produce immunoglobulin, such as E.coli cells, simian COS cells, Chinese Hamster Ovary (CHO) cells, or myeloma cells, to obtain synthesis of the desired monoclonal antibody in the recombinant host cell. Review articles on recombinant expression of DNA encoding antibodies in bacteria include Skerra et al, curr. opinion in Immunol.,5:256(1993) and Pluckthun, Immunol. Revs,130:151 (1992).

The DNA encoding the Fv clones of the invention can be combined with known DNA sequences encoding the heavy and/or light chain constant regions (e.g., suitable DNA sequences are available from Kabat et al, supra) to form clones encoding full or partial length heavy and/or light chains. It will be appreciated that constant regions of any isotype may be used for this purpose, including IgG, IgM, IgA, IgD and IgE constant regions, and that such constant regions may be obtained from any human or animal species. Fv clones derived from variable domain DNA of one animal species (such as human) and then fused with constant region DNA of another animal species to form coding sequences for "hybrid" full-length heavy and/or light chains are included in the definition of "chimeric" and "hybrid" antibodies as used herein. In certain embodiments, Fv clones derived from human variable DNA are fused to human constant region DNA to form coding sequences for full or partial length human heavy and/or light chains.

The DNA encoding anti-antigen antibodies derived from the hybridomas of the present invention may also be modified, for example, by substituting the coding sequences for human heavy and light chain constant domains for homologous murine sequences derived from hybridoma clones (e.g., as in the method of Morrison et al, Proc. Natl. Acad. Sci. USA,81:6851-6855 (1984)). The DNA encoding the antibody or fragment derived from the hybridoma or Fv clone may be further modified by covalent linkage to an immunoglobulin coding sequence, the coding sequence for all or part of a non-immunoglobulin polypeptide. In this way, "chimeric" or "hybrid" antibodies are prepared that have the binding specificity of the antibodies derived from Fv clones or hybridoma clones of the invention.

(iv) Humanized antibody and human antibody

Various methods for humanizing non-human antibodies are known in the art. For example, a humanized antibody has one or more amino acid residues introduced from a non-human source. These non-human amino acid residues are often referred to as "import" residues, which are typically taken from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and co-workers (Jones et al, Nature,321:522-525 (1986); Riechmann et al, Nature,332:323-327 (1988); Verhoeyen et al, Science,239:1534-1536(1988)) by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody. Thus, such "humanized" antibodies are chimeric antibodies (U.S. Pat. No.4,816,567) in which substantially less than an intact human variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some CDR residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

The choice of light and heavy chain human variable domains for making humanized antibodies is very important to reduce antigenicity. The sequence of the variable domain of a rodent antibody is screened against a complete library of known human variable domain sequences according to the so-called "best-fit" method. The human Framework (FR) that received the closest human sequence to the rodent as the humanized antibody (Sims et al, J.Immunol.,151:2296 (1993); Chothia et al, J.mol.biol.,196:901 (1987)). Another approach uses specific frameworks derived from consensus sequences of all human antibodies of a specific subgroup of light or heavy chains. The same framework can be used for several different humanized antibodies (Carter et al, Proc. Natl. Acad. Sci. USA,89:4285 (1992); Presta et al, J. Immunol.,151:2623 (1993)).

It is further important that antibodies be humanized and retain high affinity for the antigen and other favorable biological properties. To achieve this goal, according to one embodiment of the method, humanized antibodies are prepared by a process of analyzing the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are generally available and familiar to those skilled in the art. Computer programs can be used to illustrate and display possible three-dimensional conformational structures of selected candidate immunoglobulin sequences. Examination of these displays allows analysis of the likely role of the residues in the function of the candidate immunoglobulin sequence, i.e., analysis of residues that affect the ability of the candidate immunoglobulin to bind its antigen. In this manner, FR residues can be selected and combined from the recipient and import sequences to achieve a desired antibody characteristic, such as increased affinity for the target antigen(s). In general, hypervariable region residues are directly and most substantially involved in affecting antigen binding.

As described above, the human antibodies of the invention can be constructed by combining Fv clone variable domain sequences selected from human-derived phage display libraries with known human constant domain sequences. Alternatively, the human monoclonal antibodies of the invention can be prepared by the hybridoma method. Human myeloma and mouse-human heteromyeloma cell lines for the production of human monoclonal antibodies have been described, for example, by Kozbor j.immunol.,133:3001 (1984); brodeur et al, Monoclonal Antibody Production Techniques and Applications, Marcel Dekker, Inc., New York, pp.51-63 (1987); and Boerner et al, J.Immunol.,147:86 (1991).

It is possible to generate transgenic animals (e.g., mice) that, when immunized, are capable of producing a complete repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, antibody heavy chain joining regions (J) have been described in chimeric and germline mutant mice_H) Homozygous deletion of the gene results in complete inhibition of endogenous antibody production. Transfer of human germline immunoglobulin gene arrays in such germline mutant mice will result in the production of human antibodies upon antigen challenge. See, e.g., Jakobovits et al, Proc.Natl.Acad.Sci.USA,90:2551 (1993); jakobovits et al, Nature,362:255-258 (1993); bruggermann et al, Yeast in Immuno, 7:33 (1993); and Duchosal et al Nature 355:258 (1992).

Gene shuffling (Gene shuffling) can also be used to derive human antibodies from non-human (e.g., rodent) antibodies, where the human antibodies have similar affinity and specificity for the starting non-human antibody. According to this method, also known as "epitope imprinting", the heavy or light chain variable regions of non-human antibody fragments obtained by phage display techniques described herein are replaced with a repertoire of human V domain genes, resulting in a population of non-human chain/human chain scFv or Fab chimeras. Selection with antigen results in the isolation of a non-human chain/human chain chimeric scFv or Fab, where the human chain restores the antigen binding site that was destroyed after removal of the corresponding non-human chain in the primary phage display clone, i.e. the epitope dominates (imprints) the selection of the human chain counterpart. When this process is repeated to replace the remaining non-human chains, human antibodies are obtained (see PCT WO 93/06213 published on 4/1 1993). Unlike humanization of traditional non-human antibodies by CDR grafting, this technique provides fully human antibodies, which do not have FR or CDR residues of non-human origin.

(v) Antibody fragments

Antibody fragments may be produced by conventional means such as enzymatic digestion or by recombinant techniques. In some cases, it may be advantageous to use antibody fragments rather than whole antibodies. The smaller size of the fragments allows for rapid clearance and may result in improved access to solid tumors. For a review of some antibody fragments, see Hudson et al nat. Med.9: 129-.

Various techniques have been developed for the production of antibody fragments. Traditionally, these fragments have been obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al, Journal of Biochemical and Biophysical Methods 24:107-117 (1992); and Brennan et al, Science,229:81 (1985)). However, these fragments can now be produced directly by recombinant host cells. Fab, Fv and ScFv antibody fragments can all be expressed in and secreted from E.coli, so that large amounts of these fragments can be readily produced. Antibody fragments can be isolated from the antibody phage libraries discussed above. Alternatively, Fab '-SH fragments can be recovered directly from E.coli and chemically coupled to form F (ab')₂Fragments (Carter et al, Bio/Technology 10: 163-. According to another method, F (ab')₂And (3) fragment. Fab and F (ab') with increased in vivo half-life comprising salvage receptor binding epitope residues₂Fragments are described in U.S. Pat. No. 5,869,046. Other techniques for producing antibody fragments will be apparent to the skilled artisan. In certain embodiments, the antibody is a single chain Fv fragment (scFv). See WO 93/16185; U.S. patent nos. 5,571,894 and 5,587,458. Fv and scFv are the only species that have an intact binding site without constant regions; thus, they may be suitable for reduced non-specific binding during in vivo use. scFv fusion proteins can be constructed to produce fusion of the effector protein at the amino or carboxy terminus of the scFv. See, Antibody Engineering, ed.borrebaeck (supra). The antibody fragment may alsoIs a "linear antibody," for example, as described in U.S. patent No. 5,641,870. Such linear antibodies may be monospecific or bispecific.

(vi) Multispecific antibodies

Multispecific antibodies have binding specificities for at least two different epitopes, where the epitopes are typically from different antigens. While such molecules typically bind only two different epitopes (i.e., bispecific antibodies, BsAbs), antibodies with additional specificity, such as trispecific antibodies, are included in this expression when used herein. Bispecific antibodies can be prepared as full length antibodies or antibody fragments (e.g., F (ab')₂Bispecific antibodies).

Methods of making bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the co-expression of two immunoglobulin heavy-light chain pairs, where the two chains have different specificities (Millstein et al, Nature,305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (cell hybridomas) produce a potential mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. The purification of the correct molecule, usually by an affinity chromatography step, is very cumbersome and the product yield is low. Similar procedures are described in WO 93/08829 and Trauecker et al, EMBO J.,10:3655-3659 (1991).

According to different methods, antibody variable domains (antibody-antigen binding sites) with the desired binding specificity are fused to immunoglobulin constant domain sequences. The fusion is preferably with an immunoglobulin heavy chain constant domain comprising at least part of the hinge, CH2 and CH3 regions. Typically, the first heavy chain constant region (CH1) contains the sites necessary for light chain binding present in at least one of the fusions. The DNA encoding the immunoglobulin heavy chain fusion and, if necessary, the immunoglobulin light chain is inserted into separate expression vectors and co-transfected into a suitable host organism. This provides great flexibility in embodiments for adjusting the mutual proportions of the three polypeptide fragments, while the unequal ratios of the three polypeptide chains used in the construction provide the best yield. However, when at least two polypeptide chains are expressed at the same ratio to produce high yields or when the ratio is not of particular significance, the coding sequences for two or all three polypeptide chains can be inserted into one expression vector.

In one embodiment of the method, the bispecific antibody consists of a hybrid immunoglobulin heavy chain with a first binding specificity in one arm and a hybrid immunoglobulin heavy chain-light chain pair (providing a second binding specificity) in the other arm. It was found that this asymmetric structure helps to separate the desired bispecific compound from the undesired immunoglobulin chain combinations, since the presence of the immunoglobulin light chain in only half of the bispecific molecule provides a convenient way of separation. This process is disclosed in WO 94/04690. For more details on the generation of bispecific antibodies see, e.g., Suresh et al, Methods in Enzymology,121:210 (1986).

According to another approach described in WO96/27011, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. C with one interface comprising antibody constant domains_H3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). By replacing large amino acid side chains with smaller ones (e.g., alanine or threonine), complementary "cavities" of the same or similar size as the large side chains are created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers relative to other undesired end products such as dimers.

Bispecific antibodies include cross-linked or "heteroconjugated" antibodies. For example, one antibody in the heterologous conjugate may be coupled to avidin and the other to biotin. For example, antibodies have been proposed which target immune system cells to unwanted cells (U.S. Pat. No.4,676,980) and are useful in the treatment of HIV infection (WO 91/00360, WO 92/200373 and EP 03089). Heteroconjugated antibodies can be prepared using any convenient crosslinking method. Suitable crosslinking agents are well known in the art and are disclosed in U.S. Pat. No.4,676,980, along with some crosslinking techniques.

Techniques for generating bispecific antibodies from antibody fragments are also described in the literature. For example, bispecific antibodies can be prepared using chemical ligation. Brennan et al, Science,229:81(1985) describe a procedure in which intact antibodies are proteolytically cleaved to yield F (ab')₂And (3) fragment. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize the vicinal dithiols and prevent intermolecular disulfide formation. The resulting Fab' fragments are then converted to Thionitrobenzoate (TNB) derivatives. One of the Fab ' -TNB derivatives is then reconverted to the Fab ' -thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab ' -TNB derivative to form the bispecific antibody. The bispecific antibodies produced can be used as selective immobilization agents for enzymes.

Recent advances have facilitated the recovery of Fab' -SH fragments directly from E.coli, which can be chemically coupled to form bispecific antibodies. A fully humanized bispecific antibody F (ab')₂The generation of molecules. Each Fab' fragment was separately secreted from E.coli and subjected to directed chemical coupling in vitro to form bispecific antibodies.

Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell cultures are also described. For example, bispecific antibodies are produced using leucine zippers. Kostelny et al, J.Immunol.,148(5):1547-1553 (1992). Leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. Antibody homodimers were reduced at the hinge region to form monomers and then re-oxidized to form antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technique described by Hollinger et al, Proc. Natl. Acad. Sci. USA,90: 6444-. A fragment comprises a heavy chain variable domain (VH) linked to a light chain variable domain (VL) by a linker that is too short to pair between the two domains on the same chain. Thus, V of a segment_HAnd V_LThe domains being forced into another segmentComplementary V_LAnd V_HThe domains pair, thereby forming two antigen binding sites. Another strategy for making bispecific antibody fragments using single chain fv (sFv) dimers has also been reported. See Gruber et al, J.Immunol,152:5368 (1994).

Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tuft et al, J.Immunol.147:60 (1991).

(vii) Single domain antibodies

In some embodiments, the antibodies of the invention are single domain antibodies. A single domain antibody is a single polypeptide chain comprising all or part of a heavy chain variable domain or all or part of a light chain variable domain of an antibody. In certain embodiments, the single domain antibody is a human single domain antibody (Domantis, Inc., Waltham, Mass.; see, e.g., U.S. Pat. No. 6,248,516). In one embodiment, a single domain antibody consists of all or part of the heavy chain variable domain of an antibody.

(viii) Antibody variants

In some embodiments, amino acid sequence modifications of the antibodies described herein are contemplated. For example, it may be desirable to improve the binding affinity and/or other biological properties of an antibody. Amino acid sequence variants of an antibody can be prepared by introducing appropriate changes into the nucleotide sequence encoding the antibody or by peptide synthesis. Such modifications include, for example, deletions from and/or insertions into and/or substitutions of residues within the amino acid sequence of the antibody. Any combination of deletions, insertions, and substitutions may be made to arrive at the final construct, so long as the final construct possesses the desired characteristics. Amino acid changes can be introduced into the subject antibody amino acid sequences at the time the sequences are prepared.

(ix) Antibody derivatives

The antibodies of the invention may be further modified to contain other non-proteinaceous moieties known in the art and readily available. In certain embodiments, the moiety suitable for derivatizing the antibody is a water-soluble polymer. Non-limiting examples of water-soluble polymers include, but are not limited to, polyethylene glycol (PEG), copolymers of ethylene glycol/propylene glycol, carboxymethylcellulose, dextran, polyvinyl alcohol, polyvinylpyrrolidone, poly-1, 3-dioxolane, poly-1, 3, 6-trioxane, ethylene/maleic anhydride copolymers, polyaminoacids (homopolymers or random copolymers) and dextran or poly (n-vinyl pyrrolidone) polyethylene glycol, propylene glycol homopolymers, propylene oxide/ethylene oxide copolymers, polyoxyethylated polyols (e.g., glycerol), polyvinyl alcohol, and mixtures thereof. Polyethylene glycol propionaldehyde may have advantages in manufacturing due to its stability in water. The polymer may have any molecular weight and may be branched or unbranched. The number of polymers attached to the antibody can vary, and if more than one polymer is attached, they can be the same or different molecules. In general, the amount and/or type of polymer used for derivatization may be determined based on considerations including, but not limited to, the particular properties or functions of the antibody to be improved, whether the antibody derivative is to be used in a therapy under defined conditions, and the like.

(x) Vectors, host cells, recombinant methods

Antibodies can also be produced using recombinant methods. For recombinant production of anti-antigen antibodies, nucleic acids encoding the antibodies are isolated and inserted into replicable vectors for further cloning (amplification of the DNA) or for expression. DNA encoding the antibody can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that are capable of binding specifically to genes encoding the heavy and light chains of the antibody). Many vectors are available. Carrier components typically include, but are not limited to, one or more of the following: a signal sequence, an origin of replication, one or more marker genes, an enhancer element, a promoter and a transcription termination sequence.

(a) Component of a Signal sequence

The antibodies of the invention can be produced recombinantly not only directly, but also with fusion polypeptides having a heterologous polypeptide, preferably a signal sequence or other polypeptide having a specific cleavage site at the N-terminus of the mature protein or polypeptide. Preferably, the heterologous signal sequence of choice is one that is recognized and processed (e.g., cleaved by a signal peptidase) by the host cell. For prokaryotic host cells that do not recognize and process the native antibody signal sequence, the signal sequence is replaced by a prokaryotic signal sequence selected, for example, from the group consisting of the alkaline phosphatase, penicillinase, lpp, or heat-stable enterotoxin II leaders. For yeast secretion, the native signal sequence may be replaced by, for example, a yeast invertase leader, a factor leader (including Saccharomyces (Saccharomyces) and Kluyveromyces (Kluyveromyces) alpha-factor leader), or an acid phosphatase leader, a Candida albicans (C.albicans) glucoamylase leader, or a signal sequence as described in WO 90/13646. In mammalian cell expression, mammalian signal sequences are available as well as viral secretory leaders, such as the herpes simplex gD signal sequence.

(b) Origin of replication

Both expression and cloning vectors contain nucleic acid sequences that enable the vector to replicate in one or more selected host cells. Typically, in cloning vectors, the sequence is one that enables the vector to replicate independently of the host chromosomal DNA, and includes an origin of replication or an autonomously replicating sequence. Such sequences are well known for a variety of bacteria, yeasts and viruses. The origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the 2. mu. plasmid origin is suitable for yeast, and various viral origins (SV40, polyoma, adenovirus, VSV or BPV) can be used for cloning vectors in mammalian cells. Typically, mammalian expression vectors do not require an origin of replication component (the SV40 origin is typically used only because it contains an early promoter).

(c) Selection of Gene Components

Expression and cloning vectors may contain a selection gene, also known as a selectable marker. Typical selection genes encode proteins that: (a) conferring resistance to antibiotics or other toxins (e.g., ampicillin, neomycin, methotrexate, or tetracycline), (b) supplementing auxotrophic deficiencies, or (c) providing key nutrients not available from complex media, such as the gene encoding bacillus (bacillus) D-alanine racemase.

One example of a selection scheme utilizes drugs to prevent growth of the host cell. Those cells successfully transformed with the heterologous gene produce a protein conferring drug resistance and thus survive the selection protocol. Examples of such dominant selection use the drugs neomycin, mycophenolic acid and hygromycin.

Another example of a suitable selection marker for mammalian cells is a cell capable of identifying a nucleic acid encoding an antibody competent to take up, such as DHFR, Glutamine Synthetase (GS), thymidine kinase, metallothionein-I and-II, preferably primate metallothionein genes, adenosine deaminase, ornithine decarboxylase, etc.

For example, cells transformed with the DHFR gene are identified by culturing the transformants in a medium containing the competitive antagonist methotrexate (Mtx) for DHFR. Under these conditions, the DHFR gene is amplified along with any other co-transformed nucleic acids. A Chinese Hamster Ovary (CHO) cell line deficient in endogenous DHFR activity (e.g., ATCC CRL-9096) can be used.

Alternatively, cells transformed with the GS gene are identified by culturing the transformants in a medium containing the GS inhibitor L-methionine sulfoximine (Msx). Under these conditions, the GS gene is amplified along with any other co-transformed nucleic acids. The GS selection/amplification system can be used in combination with the DHFR selection/amplification system described above.

Alternatively, host cells transformed or co-transformed with a DNA sequence encoding an antibody of interest, a wild-type DHFR gene and another selectable marker such as aminoglycoside 3' -phosphotransferase (APH), particularly wild-type hosts containing endogenous DHFR, can be selected by cell growth in medium containing a selection agent for the selectable marker, such as an aminoglycoside antibiotic, e.g., kanamycin, neomycin or G418. See U.S. patent No.4,965,199.

A suitable selection gene for yeast is the trp1 gene present in the yeast plasmid YRp7 (Stinchcomb et al, Nature,282:39 (1979)). the trp1 gene provides a selection marker for mutants of yeast lacking the ability to grow in tryptophan, such as ATCC No.44076 or PEP 4-1. Jones, Genetics,85:12 (1977). The presence of trp1 lesions in the genome of the yeast host cell provides an effective environment for detecting transformation in the absence of tryptophan. Similarly, Leu2 deficient yeast strains (ATCC 20,622 or 38,626) were complemented by known plasmids carrying the Leu2 gene.

In addition, a vector derived from the 1.6 μm circular plasmid pKD1 can be used for the transformation of Kluyveromyces (Kluyveromyces yeast). Alternatively, expression systems for large-scale production of recombinant calf chymosin from kluyveromyces lactis (k.lactis) have been reported. Van den Berg, Bio/Technology,8:135 (1990). Also disclosed are stable multicopy expression vectors for secreting mature recombinant human serum albumin using an industrial strain of kluyveromyces. Fleer et al, Bio/Technology,9: 968-.

(d) Promoter component

Expression and cloning vectors typically contain a promoter that is recognized by the host organism and is operably linked to nucleic acid encoding an antibody. Promoters suitable for use in prokaryotic hosts include the phoA promoter, the beta-lactamase and lactose promoter systems, the alkaline phosphatase promoter, the tryptophan (trp) promoter system, and hybrid promoters such as the tac promoter. However, other known bacterial promoters are suitable. Promoters for use in bacterial systems will also comprise Shine-Dalgarno (s.d.) sequences operably linked to DNA encoding the antibody.

Promoter sequences are known for eukaryotes. Virtually all eukaryotic genes have an AT-rich region located approximately 25 to 30 bases upstream of the transcription start site. Another sequence found 70 to 80 bases upstream of the start of transcription of many genes is the CNCAAT region, where N can be any nucleotide. At the 3 'end of most eukaryotic genes is an AATAAA sequence, which may be a signal for adding a polyA tail at the 3' end of the coding sequence. All these sequences are suitable for insertion into eukaryotic expression vectors.

Examples of promoter sequences suitable for use in a yeast host include promoters for 3-phosphoglycerate kinase or other glycolytic enzymes such as enolase, glyceraldehyde-3-phosphate dehydrogenase, hexokinase, pyruvate decarboxylase, phosphofructokinase, glucose-6-phosphate isomerase, 3-phosphoglycerate mutase, pyruvate kinase, triosephosphate isomerase, phosphoglucose isomerase, and glucokinase.

Other yeast promoters, which are inducible promoters with the additional advantage of transcription controlled by growth conditions, are the promoter regions for the following enzymes: alcohol dehydrogenase 2, isocytochrome C, acid phosphatase, degradative enzymes associated with nitrogen metabolism, metallothionein, glyceraldehyde-3-phosphate dehydrogenase, and enzymes responsible for maltose and galactose utilization. Suitable vectors and promoters for yeast expression are further described in EP 73,657. Yeast enhancers are also advantageously used with yeast promoters.

For example, antibody transcription of a vector in a mammalian host cell can be controlled by a promoter obtained from the genome of a virus, such as polyoma virus, fowlpox virus, adenovirus (such as adenovirus 2), bovine papilloma virus, avian sarcoma virus, cytomegalovirus, a retrovirus, hepatitis b virus, simian virus 40(SV40), or from a heterologous mammalian promoter (e.g., an actin promoter or an immunoglobulin promoter from a heat shock promoter, provided such promoter is compatible with the host cell system).

The early and late promoters of the SV40 virus are conveniently obtained as an SV40 restriction fragment that also contains the SV40 viral origin of replication. The immediate early promoter of human cytomegalovirus is conveniently obtained as a HindIII E restriction fragment. A system for expressing DNA in a mammalian host using bovine papilloma virus as a vector is disclosed in U.S. patent No.4,419,446. An improvement of this system is described in U.S. patent No.4,601, 978. For the expression of human interferon-beta cDNA in mouse cells under the control of the thymidine kinase promoter from herpes simplex virus, see also Reyes et al, Nature297: 598-. Alternatively, Rous sarcoma virus long terminal repeat can be used as a promoter.

(e) Enhancer element component

Transcription of DNA encoding the antibodies of the invention by higher eukaryotes is often increased by inserting enhancer sequences into the vector. Many enhancer sequences are now known from mammalian genes (globin, elastase, albumin, alpha-fetoprotein, and insulin). However, typically an enhancer from a eukaryotic cell virus will be used. Examples include the SV40 enhancer on the late side of the replication origin (bp100-270), the cytomegalovirus early promoter enhancer, the polyoma enhancer on the late side of the replication origin, and adenovirus enhancers. For elements that promote eukaryotic promoter activation, see Yaniv, Nature297: 17-18 (1982). Enhancers may be spliced into the vector at positions 5' or 3' to the antibody coding sequence, but are preferably located at sites 5' to the promoter.

(f) Transcription terminator component

Expression vectors used in eukaryotic host cells (yeast, fungi, insect, plant, animal, human, or nucleated cells from other multicellular organisms) will also contain sequences necessary for the termination of transcription and for stabilizing the mRNA. These sequences are typically available from the 5 'and occasionally 3' untranslated regions of eukaryotic or viral DNA or cDNA. These regions contain nucleotide segments transcribed as polyadenylated fragments in the untranslated portion of the mRNA encoding the antibody. One useful transcription termination component is the bovine growth hormone polyadenylation region. See WO94/11026 and expression vectors disclosed therein.

(g) Selection and transformation of host cells

Suitable host cells for cloning or expressing the DNA in the vectors herein are the prokaryotes, yeast or higher eukaryote cells described above. Suitable prokaryotes for this purpose include eubacteria, such as gram-negative or gram-positive organisms, for example Enterobacteriaceae (Enterobacteriaceae) such as Escherichia (Escherichia) such as Escherichia coli, Enterobacter (Enterobacter), Erwinia (Erwinia), Klebsiella (Klebsiella), Proteus (Proteus), Salmonella (Salmonella) such as Salmonella typhimurium (Salmonella typhimurium), Serratia (Serratia) such as Serratia marcescens (Serratia marcescens) and Shigella (Shigella), and bacillus (bacillus) such as bacillus subtilis (b. subtilis) and bacillus (b. liciformis) (e.g. bacillus licheniformis (bacillus licheniformis) (disclosed in DD 266,710, 4.12.1989), Pseudomonas (Pseudomonas) such as Pseudomonas aeruginosa (Streptomyces). A preferred E.coli cloning host is E.coli 294(ATCC 31,446), although other strains such as E.coli B, E.coli X1776(ATCC 31,537), and E.coli W3110(ATCC 27,325) are also suitable. These examples are illustrative and not limiting.

Full-length antibodies, antibody fusion proteins, and antibody fragments can be produced in bacteria, which themselves exhibit effectiveness for tumor cell destruction, particularly when glycosylation and Fc effector function are not required, such as when a therapeutic antibody is conjugated to a cytotoxic agent (e.g., a toxin). Full-length antibodies have a greater half-life in circulation. The production speed of the Escherichia coli is higher, and the cost benefit is higher. For expression of antibody fragments and polypeptides in bacteria, see, e.g., U.S. Pat. No. 5,648,237 (Carter et al), U.S. Pat. No. 5,789,199 (Joly et al), U.S. Pat. No. 5,840,523 (Simmons et al), which describe a Translation Initiation Region (TIR) and signal sequences for optimized expression and secretion. See also Charlton, Methods in Molecular Biology, vol.248, b.k.c.lo, ed., Humana Press, Totowa, n.j., pp.245-254(2003), which describes the expression of antibody fragments in e. After expression, the antibody can be isolated from a soluble fraction of E.coli cell paste (cell paste) and can be purified, for example, by protein A or G column depending on the isotype. The final purification can be carried out analogously to the purification of antibodies expressed, for example, in CHO cells.

In addition to prokaryotes, eukaryotic microorganisms such as filamentous fungi or yeast are suitable cloning or expression hosts for vectors encoding antibodies. Saccharomyces cerevisiae or commonly used baker's yeast is the most commonly used lower eukaryotic host microorganism. However, many other genera, species and strains are generally available and useful in the present invention, such as Schizosaccharomyces pombe (Schizosaccharomyces pombe); kluyveromyces hosts such as, for example, Kluyveromyces lactis (k.lactis), Kluyveromyces fragilis (k.fragilis) (ATCC 12,424), Kluyveromyces bulgaricus (k.bulgaricus) (ATCC 16,045), Kluyveromyces williamsii (k.winkeramii) (ATCC 24,178), k.wallidii (ATCC 56,500), Kluyveromyces drosophilus (k.drosophilarium) (ATCC 36,906), Kluyveromyces thermotolerans (k.thermotolerans), and Kluyveromyces marxianus (k.marxianus); yarrowia (EP 402,226); pichia pastoris (Pichia pastoris) (EP 183,070); candida genus (Candida); trichoderma reesei (Trichoderma reesei) (EP 244,234); neurospora crassa (Neurospora crassa); schwanniomyces (Schwanniomyces), such as Schwanniomyces occidentalis; and filamentous fungi such as, for example, Neurospora (Neurospora), Penicillium (Penicillium), torticollis (Tolypocladium), and Aspergillus (Aspergillus) hosts such as Aspergillus nidulans (a. nidulans) and Aspergillus niger (a. niger). For a review on the discussion of the use of yeast and filamentous fungi for the production of therapeutic proteins, see, e.g., Gerngross, nat. Biotech.22:1409-1414 (2004).

Certain fungal and yeast strains may be selected in which the glycosylation pathway has been "humanized" resulting in the production of antibodies with partially or fully human glycosylation patterns. See, for example, Li et al, nat. Biotech.24:210-215(2006) (humanization of the glycosylation pathway in Pichia pastoris is described); and Gerngross et al (supra).

Host cells suitable for expression of glycosylated antibodies are also derived from multicellular organisms (invertebrates and vertebrates). Examples of invertebrate cells include plant and insect cells. A number of baculovirus strains and variants and corresponding permissive insect host cells have been identified which are derived from hosts such as Spodoptera frugiperda (caterpillars), Aedes aegypti (mosquitoes), Aedes albopictus (mosquitoes), Drosophila melanogaster (Drosophila melanogaster) and Bombyx mori (Bombyx mori). A variety of viral strains are publicly available for transfection, such as the L-1 variant of Autographa californica (Autographa californica) NPV and the Bm-5 strain of Bombyx mori (Bombyx mori) NPV, and such viruses may be used as viruses herein in accordance with the present invention, particularly for transfecting Spodoptera frugiperda cells.

Plant cell cultures of cotton, corn, potato, soybean, petunia, tomato, lemna (Leninaceae), alfalfa (m.truncatula) and tobacco may also be used as hosts. See, e.g., U.S. Pat. Nos. 5,959,177, 6,040,498, 6,420,548, 7,125,978, and 6,417,429 (describing PLANTIBODIIES for antibody production in transgenic plants^TMA technique).

Vertebrate cells can be used as hosts, and propagation of vertebrate cells in culture (tissue culture) has become a routine procedure.Examples of useful mammalian host cell lines include, but are not limited to, monkey kidney CV1 cell line transformed with SV40 (COS-7, ATCC CRL 1651); human embryonic kidney cell lines (293 or 293 cells subcloned for growth in suspension culture, Graham et al, J.Gen Virol.36:59 (1977)); baby hamster kidney cells (BHK, ATCC CCL 10); mouse Sertoli (sertoli) cells (TM4, Mather, biol. reprod.23:243-251 (1980)); monkey kidney cells (CV1, ATCC CCL 70); vero kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); bovine murine (buffalo rat) hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL 51); TRI cells (Mather et al, Annals N.Y.Acad.Sci.383:44-68 (1982)); MRC5 cells; FS4 cells; and a human hepatoma cell line (Hep G2). Other useful mammalian host cell lines include Chinese Hamster Ovary (CHO) cells, including DHFR^-CHO cells (Urlaub et al, Proc. Natl. Acad. Sci. USA 77:4216 (1980)); and myeloma cell lines such as NS0 and Sp 2/0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki and Wu, Methods in Molecular Biology,248:255-268 (2003).

Host cells are transformed with the above-described expression or cloning vectors for antibody production and cultured in conventional nutrient media with appropriate induction of promoters, selection of transformants, or amplification of genes encoding the desired sequences.

(h) Culturing host cells

Host cells for producing the antibodies of the invention can be cultured in a variety of media. Commercially available media such as Ham F10(Sigma), minimal essential medium (MEM, Sigma), RPMI-1640(Sigma), and Dulbecco's modified Eagle's medium (DMEM, Sigma) are suitable for culturing the host cells. In addition, any of the media described in the following documents can be used as the medium for the host cells: ham et al, meth.Enz.58:44 (1979); barnes et al, anal. biochem.102:255 (1980); 4,767,704 th; 4,657,866, respectively; 4,927,762, respectively; 4,560,655; or U.S. Pat. No. 5,122,469; WO 90/03430; WO 87/00195; or us patent review 30,985. Any of these media may be supplemented with hormones and/or as neededOther growth factors (such as insulin, transferrin or epidermal growth factor), salts (such as sodium chloride, calcium, magnesium and phosphate), buffers (such as HEPES), nucleotides (such as adenosine and thymidine), antibiotics (such as GENTAMYCIN)^TMDrugs), trace elements (defined as inorganic compounds usually present in final concentrations in the micromolar range), and glucose or an equivalent energy source. Any other necessary supplements known to those skilled in the art may also be included at suitable concentrations. It will be apparent to the skilled person that culture conditions, such as temperature, pH, etc., are previously selected for the host cell for expression.

(xi) Purification of antibodies

When using recombinant techniques, the antibody may be produced intracellularly, in the periplasmic space, or secreted directly into the culture medium. If the antibody is produced intracellularly, as a first step, particulate debris (host cells or lysed fragments) is removed, for example by centrifugation or ultrafiltration. Carter et al, Bio/Technology 10:163-167(1992) describe methods for isolating antibodies secreted into the periplasmic space of E.coli. Briefly, the cell paste was thawed in the presence of sodium acetate (pH 3.5), EDTA, and phenylmethylsulfonyl fluoride (PMSF) within about 30 minutes. Cell debris can be removed by centrifugation. In the case of secretion of the antibody into the culture medium, the supernatant from such an expression system is usually first concentrated using a commercially available protein concentration filter, such as an Amicon or Millipore Pellicon ultrafiltration unit. Protease inhibitors such as PMSF may be included in any of the foregoing steps to inhibit proteolysis, and antibiotics may be included to prevent the growth of adventitious contaminants.

Antibody compositions prepared from cells can be purified using, for example, hydroxyapatite chromatography, hydrophobic interaction chromatography, gel electrophoresis, dialysis, and affinity chromatography, with affinity chromatography being one of the typically preferred purification steps. The suitability of protein a as an affinity ligand depends on the type and isotype of any immunoglobulin Fc domain present in the antibody. Protein A can be used to purify antibodies based on human gamma 1, gamma 2 or gamma 4 heavy chains (Lindmark et al, J.Immunol. meth.62:1-13 (1983)). Protein G is recommended for all mouse isoforms and human gamma 3(Guss et al, EMBO J.5: 1567)1575(1986)). The matrix to which the affinity ligand is attached is typically agarose, but other matrices are also useful. Mechanically stable matrices such as controlled pore glass or poly (styrene divinyl) benzene allow faster flow rates and shorter processing times than achieved with agarose. When the antibody comprises C_H3 domain, Bakerbond ABX^TMResins (j.t.baker, phillips burg, n.j.) can be used for purification. Other techniques for protein purification such as fractional distillation on ion exchange columns, ethanol precipitation, reverse phase HPLC, silica chromatography, heparin Sepharose on anion or cation exchange resins such as polyaspartic acid columns^TMThe chromatographic analysis, chromatofocusing, SDS-PAGE and ammonium sulphate precipitation also depend on the antibody to be recovered.

In general, various methodologies for preparing antibodies for research, testing, and clinical use are well known in the art, consistent with the above methodology, and/or as deemed appropriate by one of skill in the art for a particular antibody of interest.

B. Selection of biologically active antibodies

Antibodies produced as described above may be subjected to one or more "bioactivity" assays to select antibodies with beneficial properties from a therapeutic standpoint. Antibodies can be screened for their ability to bind to the antigen against which they are produced. For example, for an anti-DR 5 antibody (e.g., drozitumab), the antigen binding properties of the antibody can be evaluated in an assay that detects the ability to bind to death receptor 5(DR 5).

In another embodiment, the affinity of the antibody can be determined by, for example, saturation binding; ELISA; and/or a competition assay (e.g., RIA).

In addition, the antibodies can be assayed for other biological activities, for example, to evaluate their effectiveness as therapeutics. Such assays are known in the art and depend on the intended use of the benzo antigen and antibody.

To screen for Antibodies that bind to a particular epitope on an antigen of interest, a conventional cross-blocking assay, such as described in Antibodies, a Laboratory Manual, Cold Spring Harbor Laboratory, Ed Harlow and David Lane (1988), can be performed. Alternatively, epitope mapping, e.g., as in Champe et al, J.biol.chem.270:1388-1394(1995), is performed to determine whether an antibody binds to an epitope of interest.

C. Preparation of the formulations

Provided herein are formulations comprising a polysorbate and a cyclodextrin having reduced degradation of the polysorbate. In some embodiments, the cyclodextrin is 2-hydroxypropyl- β -cyclodextrin (HP- β -CD). In some embodiments, the cyclodextrin is 2-hydroxypropyl-a-cyclodextrin (HP-a-CD), 2-hydroxypropyl- γ -cyclodextrin (HP- γ -CD). In some embodiments, the cyclodextrin is beta-cyclodextrin (beta-CD). In some embodiments, the cyclodextrin is sulfobutyl ether β -cyclodextrin (SBE- β -CD). In some embodiments, the cyclodextrin is alpha-cyclodextrin (alpha-CD). In some embodiments, the cyclodextrin is gamma-cyclodextrin (gamma-CD). In some embodiments, the formulation comprises polysorbate and polyvinylpyrrolidone (PVP) and has reduced polysorbate degradation. In some embodiments, the formulation further comprises a polypeptide. In some embodiments, the polysorbate is in the range of about 0.001% to about 15% or any range between these values. In certain embodiments, the polysorbate is in a range of about 0.001% to about 0.4%, 0.01% to about 0.4%, about 0.01% to about 0.3%, about 0.01% to about 0.2%, about 0.01% to about 0.1%. In some embodiments, the formulation comprises about 0.001%, about 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.4%, about 1%, about 5%, or about 15% polysorbate. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 40. In some embodiments, the polysorbate is polysorbate 60. In some embodiments, the polysorbate is polysorbate 80.

In some embodiments, the polyalkylpyrrolidone is a class of polymer molecules made from the monomer N-vinylpyrrolidone. In some embodiments, the polyvinylpyrrolidone (PVP) is povidone (soluble PVP). In some embodiments, the PVP is povidone K12 (approximate MW: 2.5 kDa). In some embodiments, the PVP is povidone K15 (approximate MW: 8 kDa). In some embodiments, the PVP is povidone K17 (approximate MW: 10 kDa). In some embodiments, the PVP is povidone K25 (approximate MW: 30 kDa). In some embodiments, the PVP is povidone K30 (approximate MW: 50 kDa). In some embodiments, the PVP is povidone K60 (approximate MW: 400 kDa). In some embodiments, the PVP is povidone K90 (approximate MW: 1,000 kDa). In some embodiments, the PVP is povidone K120(3,000 kDa). In some embodiments, the PVP is crospovidone (insoluble PVP). In some embodiments, the PVP is copovidone.

In some embodiments, the cyclodextrin is in the range of about 0.5% to about 30%. In some embodiments, the cyclodextrin ranges from about 1% to about 25%, or from about 5% to about 20%, or from about 10% to about 15%. In further embodiments, the concentration of the cyclodextrin is about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%. In some embodiments, the PVP is in the range of about 0.5% to about 30%.

In some embodiments, the w/w ratio of cyclodextrin to polysorbate in the formulation is greater than about 37.5: 1. In some embodiments, the w/w ratio of cyclodextrin to polysorbate in the formulation is greater than about 50:1, greater than about 100:1, greater than about 150:1, greater than about 250:1, greater than about 750:1, greater than about 1000:1 or greater than about 3000: 1. In some embodiments, the w/w ratio of cyclodextrin to polysorbate is not between 67:1 and 1000: 1. In some embodiments, the w/w ratio of PVP to polysorbate in the formulation is greater than about 37.5: 1.

In some embodiments, the aqueous formulation comprises a polypeptide at a concentration ranging from about 10mg/mL to about 250mg/mL or any range therebetween. In some embodiments, the concentration of the polypeptide is greater than about 250 mg/mL. In some embodiments, the concentration of the polypeptide ranges from about 10mg/mL to 250mg/mL, 50mg/mL to 250mg/mL, 100mg/mL to 250mg/mL, 150mg/mL to 250mg/mL, 200mg/mL to 250mg/mL, 10mg/mL to 200mg/mL, 50mg/mL to 200mg/mL, 100mg/mL to 200mg/mL, 150mg/mL to 200mg/mL, 10mg/mL to 150mg/mL, 50mg/mL to 150mg/mL, 100mg/mL to 150mg/mL, 10mg/mL to 100mg/mL, 50mg/mL to 100mg/mL, 10mg/mL to 50mg/mL, or any range therebetween.

In some embodiments, the aqueous formulation comprises an antibody. In some embodiments, the antibody is directed to Vascular Endothelial Growth Factor (VEGF); CD 20; ox-LDL; ox-ApoB 100; renin; growth hormones, including human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; a 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 Willebrand factor; anti-coagulation factors, such as protein C; atrial natriuretic factor; a pulmonary surfactant; plasminogen activators, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; a hematopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalin; RANTES (modulation activates normal T cell expression and secretion); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; a muller tube inhibiting substance; a relaxin a chain; a relaxin B chain; (ii) 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; receptors for hormones or growth factors; protein A or D; rheumatoid factor; neurotrophic factors such as Bone Derived Neurotrophic Factor (BDNF), neurotrophic factor-3, 4, -5 or-6 (NT-3, NT4, NT-5 or NT-6) or nerve growth factors 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-1 (brain IGF-1), insulin-like growth factor binding protein; 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- α, - β, and- γ; 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; an attenuation acceleration factor; viral antigens, such as 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; tumor associated antigens such as HER2, HER3, or HER4 receptors; and fragments of any of the above-listed polypeptides. In some embodiments, the antibody is not an anti-CD 20 antibody. In some embodiments, the formulation does not comprise an anti-CD 20 antibody and 0.2% polysorbate (e.g., polysorbate 80). In some embodiments, the formulation does not comprise 10% HP-gamma cyclodextrin and 0.03% polysorbate 20. In some embodiments, the formulation does not comprise an anti-CD 20 antibody, 10% HP-gamma cyclodextrin, and 0.03% polysorbate 20.

In some embodiments, the aqueous formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. The aqueous formulations of the present invention may be prepared in a pH buffered solution. The buffer of the present invention has a pH in the range of about pH 4.5 to about 9.0. In certain embodiments, the pH is in the range of about 4.5 to about 7.0, in the range of about 4.5 to about 6.5, in the range of about 4.5 to about 6.0, in the range of about pH 4.5 to about 5.5, in the range of about pH 4.5 to about 5.0, in the range of about pH 5.0 to about 7.0, in the range of about pH 5.5 to about 7.0, in the range of about pH 5.7 to about 6.8, in the range of about pH 5.8 to about 6.5, in the range of about pH 5.9 to about 6.5, in the range of about pH 6.0 to about 6.5 or in the range of about pH 6.2 to about 6.5. In certain embodiments, the pH of the liquid formulation is in the range of about 4.7 to about 5.2, in the range of about 5.0 to 6.0, or in the range of about 5.2 to about 5.8. In certain embodiments of the invention, the liquid formulation has a pH of 6.2 or about 6.2. In certain embodiments of the invention, the liquid formulation has a pH of 6.0 or about 6.0.

Examples of buffering agents to control the pH within this range include organic and inorganic acids and salts thereof. For example, acetates (e.g., histidine acetate, arginine acetate, sodium acetate), succinates (e.g., histidine succinate, arginine succinate, sodium succinate), gluconates, phosphates, fumarates, oxalates, lactates, citrates and combinations thereof. The buffer concentration may be from about 1mM to about 600mM, depending, for example, on the buffer and the desired isotonicity of the formulation.

Additional surfactants may optionally be added to the aqueous formulation. Exemplary surfactants include nonionic surfactants, such as poloxamers (e.g., poloxamer 188 and the like). The amount of surfactant added is such that it reduces aggregation of the formulated antibody and/or minimizes particle formation in the formulation and/or reduces adsorption. For example, the surfactant may be present in the formulation in an amount of about 0.001% to about 0.5%, about 0.005% to about 0.2%, about 0.01% to about 0.1%, or about 0.02% to about 0.06%, or about 0.03% to about 0.05%. In certain embodiments, the surfactant is present in the formulation in an amount of 0.04% or about 0.04%. In certain embodiments, the surfactant is present in the formulation in an amount of 0.02% or about 0.02%. In one embodiment, the formulation does not comprise a surfactant.

Tonicity agents, sometimes referred to as "stabilizers," are present to adjust or maintain the tonicity of the liquid in the composition. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with charged amino acid side chain groups, thereby reducing the likelihood of intermolecular and intramolecular interactions. Tonicity agents may be present in any amount from 0.1% to 25%, or more preferably from 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.

In some embodiments, the formulation is for in vivo administration. In some embodiments, the formulation is sterile. The formulation may be rendered sterile by filtration through sterile filtration membranes. The therapeutic formulations herein are typically placed in a container having a sterile access port, such as an intravenous solution bag or vial having a stopper pierceable by a hypodermic injection needle. The route of administration is according to known and acceptable methods, such as by single or multiple bolus injections or prolonged infusion in a suitable manner, e.g., by injection or infusion by subcutaneous, intravenous, intraperitoneal, intramuscular, intraarterial, intralesional or intraarticular routes, topical administration, inhalation or by sustained or slow release means.

The aqueous formulations provided by the present invention comprise a polypeptide, a polysorbate, and a cyclodextrin and exhibit enhanced polysorbate stability after storage for a period of time. In one embodiment, polysorbate stability is expressed as the relative percentage of polysorbate remaining in the formulation after a period of storage. For example, if a formulation initially contains 0.1% polysorbate and contains 0.09% polysorbate after a period of storage, 10% of the polysorbate has been degraded. In another embodiment, the amount of polysorbate in solution is determined by reverse phase ultra high performance liquid chromatography using evaporative light scattering detection (RP-ELSD) (Kim, J & Qiu, J.2014, Analytica Chimica Acta 806: 144-151). In some embodiments, the concentration of polysorbate in the sample is determined by comparing the sample results to a standard curve generated using different polysorbate concentrations.

In some embodiments, less than 5% of the polysorbate has degraded after storage of the formulation at about 1 ℃ to about 10 ℃ for about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, less than 5% of the polysorbate is degraded after the formulation is stored at about 2 ℃ to about 8 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, less than 5% of the polysorbate has degraded after storage of the formulation at about 4 ℃ to about 6 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months.

In some embodiments, less than 1% of the polysorbate has degraded after storage of the formulation at about 1 ℃ to about 10 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, less than 1% of the polysorbate is degraded after the formulation is stored at about 2 ℃ to about 8 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, less than 1% of the polysorbate has degraded after storage of the formulation at about 4 ℃ to about 6 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months.

In some embodiments, less than 0.1% of the polysorbate has degraded after storage of the formulation at about 1 ℃ to about 10 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, less than 0.1% of the polysorbate has degraded after storage of the formulation at about 2 ℃ to about 8 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, less than 0.1% of the polysorbate has degraded after storage of the formulation at about 4 ℃ to about 6 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months.

In some embodiments, less than 5% of the polysorbate has degraded after storage of the formulation at about 22 ℃ to about 28 ℃ for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months. In some embodiments, less than 1% of the polysorbate has degraded after storage of the formulation at about 22 ℃ to about 28 ℃ for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months. In some embodiments, less than 0.1% of the polysorbate has degraded after storage of the formulation at about 22 ℃ to about 28 ℃ for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.

In some embodiments, less than 5% of the polysorbate has degraded after storage of the formulation at about-15 ℃ to about-25 ℃ for at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, at least about 48 months, at least about 54 months, at least about 60 months, at least about 66 months, or at least about 72 months. In some embodiments, less than 1% of the polysorbate is degraded after the formulation is stored at about-15 ℃ to about-25 ℃ for at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, at least about 48 months, at least about 54 months, at least about 60 months, at least about 66 months, or at least about 72 months. In some embodiments, less than 0.1% of the polysorbate is degraded after storage of the formulation at about-15 ℃ to about-25 ℃ for at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, at least about 48 months, at least about 54 months, at least about 60 months, at least about 66 months, or at least about 72 months.

In some embodiments, the formulation is stored at about-8 ℃ to about-80 ℃. In some embodiments, the formulation is stored at about-20 ℃, -40 ℃, -70 ℃, or-80 ℃.

The formulations provided by the present invention are effective in reducing polysorbate degradation products such as visible and sub-visible particles. In one embodiment, the visible particles are observed by placing the sample in a glass vial and rotating the sample in the presence of tyndall light. In one embodiment, sub-visible particles are analyzed using a high precision (HIAC) particle counter. In some embodiments, a HIAC 9703 particle counter equipped with an HRDL-150 detector and a 1mL syringe may be used. In some embodiments, the performance of the instrument can be verified at 3000 counts/mL using NIST traceable 2 μm polystyrene bead standards prior to each measurement session control (session). In some embodiments, the HIAC instrument may be configured for a flow rate of 10mL/min, a tare volume of 0.1mL, and a sample volume of 0.4 mL. In certain embodiments, samples may be analyzed using 4 runs of 0.4mL small (sip), and the first run of each sample is discarded to prevent measurement errors due to sample carryover (carryover). 2. Filter sizes of 5, 10, 15 and 25 μm were available for analysis.

In some embodiments, the formulation has less than about 10,000, about 5,000, about 1,000, about 500, about 250, about 150, about 100, about 50, or about 25 particles greater than 1.4 μ in diameter per mL. In some embodiments, the formulation has less than about 10,000, about 5,000, about 1,000, about 500, about 250, about 150, about 100, about 50, or about 25 particles greater than 2 μ in diameter per mL. In some embodiments, the formulation has less than about 1250, about 150, about 100, about 50, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 particles greater than 5 μ in diameter per mL. In some embodiments, the formulation has less than about 250, about 150, about 100, about 50, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 particle greater than 10 μ in diameter per mL. In some embodiments, the formulation has less than about 250, about 150, about 100, about 50, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 particles greater than 15 μ in diameter per mL. In some embodiments, the formulation has less than about 250, about 150, about 100, about 50, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 particles greater than 25 μ in diameter per mL.

Administration of the formulations

The aqueous formulation is administered to a mammal, preferably a human, in need of treatment with a protein (e.g., an antibody) by intramuscular, intraperitoneal, intracerobrospinal, subcutaneous, intravitreal, intraarticular, intrasynovial, intrathecal, ocular, oral, topical, or inhalation routes according to known methods, such as a bolus injection or by intravenous administration by continuous infusion over a period of time. In one embodiment, the aqueous formulation is administered to the mammal by intravenous administration. For this purpose, the formulation can be injected, for example, using a syringe or through an IV line. In one embodiment, the liquid formulation is administered to the mammal by subcutaneous administration.

For example, the appropriate dosage of the protein ("therapeutically effective amount") will depend on, for example, 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 patient's clinical history and response to the protein, the type of protein used, and the judgment of the attending physician. The protein is suitably administered to the patient at one time or over a series of treatments, and may be administered to the patient at any time from the start of diagnosis. The protein may be administered as the sole treatment or in combination with other drugs or therapies used to treat the condition. The term "treatment" as used herein refers to both therapeutic treatment and prophylactic or preventative measures. Patients in need of treatment include patients already suffering from the condition as well as patients in need of prevention of the condition. As used herein, a "disorder" is any disorder that would benefit from treatment, including but not limited to chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder.

In a pharmacological sense, in the context of the present invention, a "therapeutically effective amount" of a protein (e.g. an antibody) refers to an amount effective to prevent or treat a disorder for which the antibody is effective. As a general proposition, whether by one or more administrations, the therapeutically effective amount of protein administered will be in the range of about 0.1 to about 50mg/kg of patient body weight, with typical ranges for protein employed being, for example, about 0.3 to about 20mg/kg, preferably about 0.3 to about 15mg/kg, per daily administration. However, other dosage regimens may be useful. For example, the protein may be administered at a dose of about 100 or 400mg every 1, 2,3 or 4 weeks, or at a dose of about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, 10.0, 15.0 or 20.0mg/kg every 1, 2,3 or 4 weeks. The dose may be administered as a single dose or as multiple doses (e.g., 2 or 3 doses), such as infusion. The progress of the therapy is readily monitored by conventional techniques.

Methods of reducing polysorbate degradation

Provided herein are methods of reducing polysorbate degradation in an aqueous formulation containing a polysorbate, the method comprising adding a cyclodextrin to the formulation. Also provided herein are methods of reducing the amount of visible and sub-visible particles in an aqueous solution containing a polysorbate, the method comprising adding a cyclodextrin to the formulation. The invention also includes a method of depolymerizing and solubilizing polysorbate degradation products in an aqueous solution, the method comprising adding cyclodextrin to the formulation. In some embodiments, the formulation further comprises a polypeptide, a nucleic acid, a lipid, and/or a carbohydrate.

Provided herein are methods of reducing polysorbate in an aqueous formulation comprising polyvinylpyrrolidone (PVP) and polysorbate. Also provided herein are methods of reducing the amount of visible and sub-visible particles in an aqueous solution containing polysorbate, the method comprising adding PVP to the formulation. The invention also includes a method of depolymerizing and solubilizing polysorbate degradation products in an aqueous solution, the method comprising adding PVP to the formulation. In some embodiments, the formulation further comprises a polypeptide, a nucleic acid, a lipid, and/or a carbohydrate.

In some embodiments, the polysorbate is in the range of about 0.001% to about 0.4% or any range between these values. In certain embodiments, the polysorbate is in the range of about 0.001% to about 0.4%, about 0.01% to about 0.3%, about 0.01% to about 0.2%, or about 0.01% to about 0.1%. In some embodiments, the formulation comprises about 0.001%, about 0.005%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, or about 0.4% polysorbate. In some embodiments, it is the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 40. In some embodiments, the polysorbate is polysorbate 60. In some embodiments, the polysorbate is polysorbate 80.

In some embodiments, the cyclodextrin is added to a concentration of about 0.5% to about 30%. In some embodiments, the cyclodextrin ranges from about 1% to about 25%, from about 5% to about 20%, or from about 10% to about 15%. In further embodiments, the cyclodextrin is added to a concentration of about 0.5%, about 1%, about 5%, about 10%, about 15%, about 20%, about 25%, or about 30%. In some embodiments, PVP is added to a concentration of about 0.5% to about 30%.

In some embodiments, the formulation does not comprise 10% HP-gamma cyclodextrin and 0.03% polysorbate 20. In some embodiments, the formulation does not comprise an anti-CD 20 antibody, 10% HP-gamma cyclodextrin, and 0.03% polysorbate 20.

In some embodiments, the resulting w/w ratio of cyclodextrin to polysorbate in the formulation is greater than about 37.5: 1. In some embodiments, the resulting w/w ratio of cyclodextrin to polysorbate in the formulation is greater than about 50:1, greater than about 100:1, greater than about 150:1, greater than about 250:1, greater than about 750:1, greater than about 1000:1 or greater than about 3000: 1. In some embodiments, the w/w ratio of cyclodextrin to polysorbate in the formulation is not between 67:1 and 1000: 1. In some embodiments, the resulting w/w ratio of PVP to polysorbate in the formulation is greater than about 37.5: 1. In some embodiments, the resulting PVP to polysorbate ratio in the formulation is 250: 1.

In some embodiments, the cyclodextrin is 2-hydroxypropyl- β -cyclodextrin (HP- β -CD). In some embodiments, the cyclodextrin is 2-hydroxypropyl-a-cyclodextrin (HP-a-CD), 2-hydroxypropyl- γ -cyclodextrin (HP- γ -CD). In some embodiments, the cyclodextrin is sulfobutyl ether β -cyclodextrin (SBE- β -CD). In some embodiments, the cyclodextrin is beta-cyclodextrin (beta-CD). In some embodiments, the cyclodextrin is alpha-cyclodextrin (alpha-CD). In some embodiments, the cyclodextrin is gamma-cyclodextrin (gamma-CD).

In some embodiments, the aqueous formulation comprises a polypeptide at a concentration in the range of 10mg/mL to 250 mg/mL. In some embodiments, the concentration of the polypeptide is greater than 250 mg/mL. In some embodiments, the concentration of the polypeptide ranges from 30mg/mL to 150mg/mL, from 50mg/mL to 150mg/mL, or from 100 to 150 mg/mL.

In some embodiments, the aqueous formulation comprises an antibody. In some embodiments, the antibody is directed to Vascular Endothelial Growth Factor (VEGF); CD 20; ox-LDL; ox-ApoB 100; 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 Willebrand factor; anti-coagulation factors, such as protein C; atrial natriuretic factor; a pulmonary surfactant; plasminogen activators, such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; a hematopoietic growth factor; tumor necrosis factor-alpha and-beta; enkephalin; RANTES (modulation activates normal T cell expression and secretion); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, such as human serum albumin; a muller tube inhibiting substance; a relaxin a chain; a relaxin B chain; (ii) 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; receptors for hormones or growth factors; protein A or D; rheumatoid factor; neurotrophic factors such as Bone Derived Neurotrophic Factor (BDNF), neurotrophic factor-3, 4, -5 or-6 (NT-3, NT4, NT-5 or NT-6) or nerve growth factors 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-1 (brain IGF-1), insulin-like growth factor binding protein; 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- α, - β, and- γ; 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; an attenuation acceleration factor; viral antigens, such as 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; tumor associated antigens such as HER2, HER3, or HER4 receptors; and fragments of any of the above-listed polypeptides. In some embodiments, the antibody is not an anti-CD 20 antibody.

In some embodiments, the aqueous formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents. The process of the invention may be carried out in a pH buffered solution. The buffers of the present invention have a pH in the range of about pH 4.5 to about 9.0. In certain embodiments, the pH is in the range of about 4.5 to about 7.0, in the range of about 4.5 to about 6.6, in the range of about 4.5 to about 6.0, in the range of about pH 4.5 to about 5.5, in the range of about pH 4.5 to about 5.0, in the range of about pH 5.0 to about 7.0, in the range of about pH 5.5 to about 7.0, in the range of about pH 5.7 to about 6.8, in the range of about pH 5.8 to about 6.5, in the range of about pH 5.9 to about 6.5, in the range of about pH 6.0 to about 6.5 or in the range of about pH 6.2 to about 6.5. In certain embodiments, the pH of the liquid formulation is in the range of about 4.7 to about 5.2, in the range of about 5.0 to 6.0, or in the range of about 5.2 to about 5.8. In certain embodiments of the invention, the liquid formulation has a pH of 6.2 or about 6.2. In certain embodiments of the invention, the liquid formulation has a pH of 6.0 or about 6.0.

The methods may involve the use of tonicity agents, sometimes referred to as "stabilizers," to adjust or maintain the tonicity of the aqueous formulation. When used with large charged biomolecules such as proteins and antibodies, they are often referred to as "stabilizers" because they can interact with charged amino acid side chain groups, thereby reducing the likelihood of intermolecular and intramolecular interactions. Tonicity agents may be present in any amount from 0.1% to 25%, or more preferably from 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.

In some embodiments, the method results in less than 5% degradation of polysorbate after storage of the formulation at about 1 ℃ to about 10 ℃ for about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, the method results in less than 5% degradation of polysorbate after storage of the formulation at about 2 ℃ to about 8 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, the method results in less than 5% degradation of polysorbate after storage of the formulation at about 4 ℃ to about 6 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months.

In some embodiments, the method results in less than 1% degradation of polysorbate after storage of the formulation at about 1 ℃ to about 10 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, the method results in less than 1% degradation of polysorbate after storage of the formulation at about 2 ℃ to about 8 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, the method results in less than 1% degradation of polysorbate after storage of the formulation at about 4 ℃ to about 6 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months.

In some embodiments, the method results in less than 0.1% degradation of polysorbate after storage of the formulation at about 1 ℃ to about 10 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, the method results in less than 0.1% degradation of polysorbate after storage of the formulation at about 2 ℃ to about 8 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months. In some embodiments, the method results in less than 0.1% degradation of polysorbate after storage of the formulation at about 4 ℃ to about 6 ℃ for at least about 6 months, at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, or at least about 48 months.

In some embodiments, the method results in less than 5% polysorbate degradation after storage of the formulation at about 22 ℃ to about 28 ℃ for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months. In some embodiments, the method results in less than 1% degradation of polysorbate after storage of the formulation at about 22 ℃ to about 28 ℃ for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months. In some embodiments, the method results in less than 0.1% degradation of polysorbate after storage of the formulation at about 22 ℃ to about 28 ℃ for at least about 1 month, at least about 2 months, at least about 3 months, at least about 4 months, at least about 5 months, at least about 6 months, at least about 7 months, at least about 8 months, at least about 9 months, at least about 10 months, at least about 11 months, or at least about 12 months.

In some embodiments, the method results in less than 5% degradation of polysorbate after storage of the formulation at about-15 ℃ to about-25 ℃ for at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, at least about 48 months, at least about 54 months, at least about 60 months, at least about 66 months, or at least about 72 months. In some embodiments, the method results in less than 1% degradation of polysorbate after storage of the formulation at about-15 ℃ to about-25 ℃ for at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, at least about 48 months, at least about 54 months, at least about 60 months, at least about 66 months, or at least about 72 months. In some embodiments, the method results in less than 0.1% degradation of polysorbate after storage of the formulation at about-15 ℃ to about-25 ℃ for at least about 12 months, at least about 18 months, at least about 24 months, at least about 30 months, at least about 36 months, at least about 42 months, at least about 48 months, at least about 54 months, at least about 60 months, at least about 66 months, or at least about 72 months.

The method provided by the present invention is effective in reducing the number of particles, such as visible and sub-visible particles. In some embodiments, less than about 10,000, about 5,000, about 1,000, about 500, about 250, about 150, about 100, about 50, or about 25 particles greater than 1.4 μ in diameter are formed per mL. In some embodiments, less than about 10,000, about 5,000, about 1,000, about 500, about 250, about 150, about 100, about 50, or about 25 particles greater than 2 μ in diameter are formed per mL. In some embodiments, less than about 1250, about 150, about 100, about 50, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 particle greater than 5 μ in diameter is formed per mL. In some embodiments, less than about 250, about 150, about 100, about 50, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 particle greater than 10 μ in diameter is formed per mL. In some embodiments, less than about 250, about 150, about 100, about 50, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 particle greater than 15 μ in diameter is formed per mL. In some embodiments, less than about 250, about 150, about 100, about 50, about 25, about 20, about 15, about 10, about 5, about 4, about 3, about 2, or about 1 particles greater than 25 μ in diameter are formed per mL.

Also provided herein are methods of redissolving polysorbate degradation products in a formulation. In some embodiments, the number of particles greater than 1.4 μ present in the formulation is reduced by 100, 1000, 2000, 5000, or 10000 fold upon addition of polysorbate. In some embodiments, the number of particles greater than 2 μ in diameter present in the formulation is reduced by 100, 1000, 2000, 5000, or 10000 fold upon addition of polysorbate. In some embodiments, the number of particles greater than 5 μ in diameter present in the formulation is reduced by 100, 1000, 2000, 5000, or 10000 fold upon addition of polysorbate. In some embodiments, the number of particles greater than 10 μ in diameter present in the formulation is reduced by 100, 1000, 2000, 5000, or 10000 fold upon addition of polysorbate. In some embodiments, the number of particles greater than 15 μ in diameter present in the formulation is reduced by a factor of 100, 1000, 2000, 5000, or 10000 upon addition of polysorbate. In some embodiments, the number of particles greater than 25 μ in diameter in the formulation is reduced by a factor of 100, 1000, 2000, 5000, or 10000 upon addition of polysorbate.

V. product

In another embodiment of the invention, an article of manufacture is provided that includes a container holding a liquid formulation of the invention and optionally provides instructions for its use. Suitable containers include, for example, bottles, vials, and syringes. The container may be formed from a variety of materials, such as glass or plastic. An exemplary container is a 3-20cc single use glass vial. Alternatively, for multi-dose formulations, the container may be a 3-100cc glass vial. The container contains the formulation and a label on or associated with the container may indicate instructions for use. The article may also contain other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.

VI. kit

In another embodiment of the invention, a kit for reducing polysorbate degradation is provided. In some embodiments, the invention provides kits for reducing polysorbate degradation by the methods described herein. In some embodiments, kits are provided comprising any of the formulations provided herein. In one embodiment, such kits comprise a container of an aqueous formulation of a therapeutic peptide or antibody and a cyclodextrin solution that can be added to the aqueous formulation, wherein the ratio of cyclodextrin to polysorbate is greater than 37.5: 1. In one embodiment, such kits comprise a container of an aqueous preparation of a therapeutic peptide or antibody and a solution of polyvinylpyrrolidone (PVP) that can be added to the aqueous preparation, wherein the ratio of PVP to polysorbate is greater than 37.5: 1.

The description is to be construed as sufficient to enable those skilled in the art to practice the invention. Various modifications of the invention, in addition to those shown and described herein, will become apparent to those skilled in the art from the foregoing description and fall within the scope of the appended claims. All publications, patents, and patent applications cited herein are hereby incorporated by reference in their entirety for all purposes.

Examples

The invention will be more fully understood by reference to the following examples. However, they should not be construed as limiting the scope of the invention. It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and scope of the appended claims.

Materials and methods

Material

Hydroxypropyl-beta-cyclodextrin (HP-beta-CD) was obtained as Cavitron W7HP5Pharma from Ashland Inc (Ashland, Kentucky). Sulfobutyl ether- β -cyclodextrin (SBE- β -CD) was obtained as Captisol from Ligand Pharmaceuticals (La Jolla, California). Hydroxypropyl- α -cyclodextrin (HP- α -CD), hydroxypropyl- γ -cyclodextrin (HP- γ -CD), polyethylene glycol (PEG 1500), and methionine were obtained from Sigma Aldrich (st.louis, Missouri). Polyvinylpyrrolidone (PVP) was obtained as KollidonPovidone K-157 from Spectrum Chemical (Gardena, Calif.). Polysorbate 20(PS20) was obtained from Croda Inc (New Castle, Delaware). Porcine Pancreatic Lipase (PPL), lipoprotein lipase from Burkholderia sp (LPL), candida antarctica lipase b (candida antarctica lipase b) (calb), Rabbit Liver Esterase (RLE), and 2,2' -azobis (2-methylpropionamidine) dihydrochloride (AAPH) were obtained from Sigma Aldrich Inc.

Determination of sub-visible particle count by HIAC

Sub-visible particles were measured using a HIAC 9703 particle counter equipped with an HRDL-150 detector and a 1mL syringe. The performance of the instrument was verified at 3000 counts/mL using NIST traceable 2 μm polystyrene bead standards prior to each measurement session control (session). The HIAC instrument was configured for a flow rate of 10mL/min, a tare volume of 0.1mL, and a sample volume of 0.4 mL. Samples were analyzed using 4 runs of 0.4mL small (sip) and the first run of each sample was discarded to prevent measurement errors due to sample carryover (carryover). Results are reported as the average of 1.4, 2, 5, 10, 15, 25 and 50 μm analytical filter sizes.

Determination of polysorbate concentration

Polysorbate concentration was determined by reverse phase ultra performance liquid chromatography using evaporative light scattering detection (RP-ELSD). Samples were analyzed using an Agilent 1100 series high performance liquid chromatography system (HPLC) equipped with a Waters Oasis MAX column (20x 2.1mm,30 μm particle size). The HPLC system was equipped with an on-off valve to pass the column to waste or a Varian 380-LC evaporative light scattering detector set at 100 ℃. The mobile phase consisted of 2% aqueous formic acid (pump a) and 2% formic acid/isopropanol (pump B). The pump gradient was 10% pump B, linear to 20% pump B for 1 minute, isocratic at 20% pump B for 2.4 minutes, linear to 100% pump B for 0.1 minute, isocratic at 100% pump B for 1.1 minute, linear to 10% pump B for 0.1 minute, and finally isocratic at 10% pump B for 1.9 minutes during equilibration. The on-off valve directs column flow to waste at the beginning of each injection, then after 2.4 minutes fluid is directed to the detector until the end of the gradient. To quantify the PS20 concentration, a standard curve was generated by injecting 20. mu.L of a solution containing between 0% w/v and 0.4% w/v PS 20. For excipients that affect migration time of PS20 through the column, PS20 standard curve solutions containing relevant excipients were included in the analysis to facilitate accurate quantification.

Visible particle imaging by Seidenader

The sample holder was tilted to 60 degrees using a Seideneader V90-T visual inspection apparatus (Markt Schwaben, Germany) to inspect the vials for visible particles. Visual particle inspection of the samples was performed by placing the glass vials in a holder and rotating with tyndall light passing through the bottom of the vial. First a pre-rotation (i.e. a fast rotation) is performed to agitate the liquid, suspending and circulating the particles. After rotation and illumination, the particles move and the light causes reflections of the particles that make them visible (i.e., the tyndall effect). The visible particles were then observed using a magnifying glass. Video and photographs of visible particles were obtained using a Samsung (Seoul, South Korea) Galaxy apparatus.

Determination of turbidity

Turbidity was determined using UV spectroscopy. The UV absorbance of each sample was measured by recording the absorbance at 279nm and 320nm on an Agilent 8453 spectrophotometer using Chemstation software (Agilent Technologies, Santa Clara, CA) in a quartz cuvette with a 1cm path length.

Determination of protein concentration

Protein concentration was measured as follows. The UV absorbance of each sample was measured by recording the average absorbance between 340nm and 360nm on an Agilent 8453 spectrophotometer using Chemstation software (Agilent Technologies, Santa Clara, CA) in a quartz cuvette with a 1cm path length. UV concentration determinations were calculated by using experimentally determined absorbance of each protein. The measurement is blanked with an appropriate buffer.

Example 1: oxidative degradation of polysorbates

The ability of cyclodextrins to inhibit oxidative PS20 degradation was evaluated using 2,2' -azobisisobutyramidine (AAPH) (Borrisov et al, J.Pharm.Sci.104:1005-1018(2015)) which had previously been shown to degrade PS 20. To evaluate the ability of cyclodextrins to inhibit the oxidation of PS20, samples containing 15% (w/v) HP- β -CD or 15% (w/v) sucrose were compared to control samples (without any additional excipients). Polysorbate 20 degradation was determined by RP-ELSD for samples containing 15% (w/v) sucrose and 15% (w/v) HP- β -cyclodextrin oxidized with 5mM AAPH for 24 hours at 40 ℃ without vehicle (control).

As shown in fig. 1, the data indicate that both HP- β -CD and sucrose reduced the amount of PS20 degradation. A relative decrease of 17.9% in PS20 degradation was observed in the control samples after incubation with AAPH. In contrast, a 9.8% and 3.6% reduction in degradation of PS20 was observed for samples containing 15% (w/v) sucrose and HP- β -CD, respectively.

Example 2: inhibition of enzymatic degradation of polysorbate 20 by HP-beta-CD

The effect of 15% HP- β -CD on the enzymatic degradation of PS20 was measured. Samples of 0.02% PS20 containing 15% sucrose or 15% HP-beta-cyclodextrin in pH 5.5 buffer without additional excipients were digested at room temperature with each of Porcine Pancreatic Lipase (PPL), lipoprotein lipase (LPL), Candida Antarctica Lipase (CALB), and Rabbit Liver Esterase (RLE). PPL samples were digested with 15. mu.g/mL enzyme for 4.5 hours. LPL samples were digested with 70. mu.g/mL enzyme for 5 hours. CALB samples were digested with 0.1mg/mL of immobilized enzyme for 1 hour. RLE samples were digested with 15. mu.g/mL of enzyme for 5 hours. All digestions were performed at room temperature.

The PPL digestion was terminated by heat inactivation in a water bath at 85 ℃ for 30 minutes. LPL and RLE digestion are not stopped by heat inactivation, so samples are analyzed immediately for PS20 content. CALB digestion was stopped by filtering out the immobilized enzyme beads. To allow the particles to form, the CALB samples were left at 5 ℃ overnight. RLE and LPL samples were frozen overnight at-20 ℃ to prevent enzyme activity and then thawed on ice immediately prior to particle analysis.

All samples were analyzed for PS20 content by high performance liquid chromatography (Agilent 1100 series) with an online evaporative light scattering detector (Varian 380-LC series) as described above. Visual particle inspection was performed on a Seidenader visual inspection instrument. Sub-visible particle analysis was performed on a HIAC 9703 particle counter.

Samples treated with CALB, RLE, and LPL showed 59.2% -68.3% reduction in PS20 (fig. 2). In contrast, samples containing 15% HP- β -CD showed significant inhibition of PS20 degradation. Specifically, the PS20 concentration was reduced by 14.3% to 37.4% for these samples (fig. 2 and table 1).

TABLE 1 enzymatic degradation of polysorbate 20 by CALB, LPL and RLE

In addition, the HIAC data showed that 15% (w/v) HP- β -CD reduced the formation of sub-visible particles (SVP). After enzymatic degradation using a variety of enzymes (CALB, LPL and RLE), very little SVP/mL was observed for all particle size classes (. gtoreq.2,. gtoreq.5,. gtoreq.10 and. gtoreq.25 μm particles) when 15% (w/v) HP- β -CD was contained in the sample. LPL and RLE achieve similar results; however, the very small amount of ≧ 10 and >. gtoreq.25 μm particles excluded interpretation of ≧ 10 and ≧ 25 μm particle count measurements (FIGS. 3A-3D).

These findings indicate that HP- β -CD is a representative cyclodextrin complex capable of inhibiting enzymatic degradation of PS20 by a variety of enzymes. Without being bound by theory, this may suggest that the primary mechanism by which the cyclodextrin molecule protects PS20 involves a direct interaction between cyclodextrin and the polysorbate molecule.

Example 3: inhibition of enzymatic degradation of polysorbate 80 by HP-beta-CD

The effect of 15% (w/v) HP- β -CD on the enzymatic degradation of PS80 was measured. Samples of 0.02% (w/v) PS80 containing 0% and 15% (w/v) HP- β -CD were digested with 15 μ g/mL Porcine Pancreatic Lipase (PPL) in pH 5.5 buffer for 5 hours at room temperature. PPL digestion was stopped by heat inactivation in a water bath at 85 ℃ for 30 minutes.

After digestion with PPL, RP-ELSD showed a relative decrease in PS80 of about 19% (fig. 4). In contrast, a reduction of about 4% in degradation of PS80 was observed in samples containing 15% (w/v) HP- β -CD.

In addition, the HIAC data showed that 15% (w/v) HP- β -CD reduced the average (n-3) amount of sub-visible particles (SVP). After enzymatic degradation using PPL, very little SVP/mL was observed for all particle size classifications (≧ 1.4, ≧ 2, ≧ 5, ≧ 10 and ≧ 25 microns) when 15% (w/v) HP- β -CD was contained in the sample (FIGS. 5A-5F).

These findings indicate that HP- β -CD is a representative cyclodextrin complex capable of inhibiting the enzymatic degradation of PS 80. These results indicate that the ability of cyclodextrins to reduce polysorbate degradation, reduce particle formation, and solubilize existing particles is generally applicable to a variety of polysorbate molecules (e.g., PS20, PS40, PS60, PS80, etc.). Without being bound by theory, this may indicate that the primary mechanism by which cyclodextrin molecules protect polysorbates involves direct interactions between cyclodextrin and conserved chemical structural subunits (e.g., fatty acids) that comprise all polysorbate molecules.

Example 4: kinetics of enzymatic degradation of Polysorbate 20

To evaluate the kinetics of enzymatic PS20 degradation, samples were consumed at room temperature with 15. mu.g/mL PPL, and protein-free samples containing 0.02% (w/v) PS20 and 15% sucrose, 15% HP- β -CD, or 15% HP- α -CD were consumed with PPL incubated for 180 hours at about 25 ℃. All samples were analyzed for PS20 content by high performance liquid chromatography (Agilent 1100 series) with an online evaporative light scattering detector (Varian 380-LC series) as described above. Sub-visible particle analysis was performed on a HIAC 9703 particle counter.

The 15% sucrose curve in fig. 6 shows that PS20 degradation can be described by monophasic exponential decay. The half-life was 32.91 hours, and the smoothness was 44% (FIG. 6). These degradation kinetics support the use of a 4.5 hour digestion model (4.5 hours at 25 ℃ using PPL) for subsequent studies.

Example 5: inhibition of enzymatic degradation of polysorbates by cyclodextrins and other excipients

Several excipients were tested for their ability to protect PS20 from enzymatic hydrolysis, including HP- α -CD, HP- β -CD, HP- γ -CD, SBE- β -CD, PVP, PEG 1500, sucrose and methionine. After addition of the enzyme, a solution containing each excipient at a final concentration of 0.02% PS20 was prepared in pH 5.5 buffer. The concentration of HP-alpha-CD, HP-beta-CD, HP-gamma-CD and SBE-beta-CD in the excipient solution was 106 mM. Due to solubility limitations, the methionine samples contained 10mg/mL methionine. The remaining excipients (PVP, PEG 1500, sucrose) were added to 15% w/v to match the% w/v of HP- β -CD. Samples were digested with 15. mu.g/mL PPL enzyme for 4.5 hours at room temperature followed by heat inactivation for 30 minutes at 85 ℃. The PS20 concentration of each sample was analyzed using high performance liquid chromatography with an online evaporative light scattering detector. The samples were then left at 5 ℃ overnight to allow particles to form and the visible and sub-visible particles were analyzed as previously described.

The RP-ELSD results indicate that the excipient class is important for determining the extent of enzymatic PS20 degradation (fig. 7). After enzymatic digestion, PS20 degradation was reduced by 58% for the control sample (i.e., no vehicle). An equivalent reduction of 58% in PS20 was observed for the sample containing 15% (w/v) sucrose. These findings indicate that sucrose as an acyclic disaccharide (i.e. sucrose) has no inhibitory effect on enzymatic PS20 degradation. The inhibitory effect of cyclodextrins cannot be attributed solely to the mass dilution effect (mass dilution effect) since the results indicate that equal amounts of other excipients (e.g. sucrose) do not mitigate the catalytic polysorbate degradation.

Similarly, the methionine-containing samples had no inhibitory effect on enzymatic PS20 degradation or SVP formation (FIGS. 7 and 8A-8D). It is speculated that methionine prevents oxidative PS20 degradation, but not enzymatic PS20 degradation. Without being bound by theory, the PS20 degradation mechanism reproduced in this experiment was likely to be hydrolytic and independent of oxidative PS20 degradation.

The results show that PEG has a small inhibitory effect on PS20 degradation relative to the control sample. A51% reduction in PS20 was observed for the sample containing 5% (w/v) PEG 1500.

The cyclodextrin molecules evaluated (HP- α -CD, HP- β -CD and SBE- β -CD) had significant inhibitory effects on both enzymatic PS20 degradation and SVP formation (FIGS. 7 and 8A-8D). Interestingly, the number of sugar subunits may be important in determining the degree of cyclodextrin inhibition. For example, 1% and 7% reductions in PS20 were observed for HP- α -CD and HP- β -CD, respectively.

Further studies were conducted to evaluate the importance of HP- α -CD, HP- β -CD, and HP- γ -CD to further understand the importance of cyclodextrin ring size. The data shown in figure 9 indicate that no significant degradation of PS20 was observed for samples containing HP- α -CD and HP- β -CD; in contrast, about 82% degradation of PS20 was observed in the sample containing 15% HP-gamma-CD. Similarly, a significant decrease in PS20 concentration was observed in the control sample (i.e., no excipient and 15% sucrose) (fig. 9). These findings indicate that the smaller cyclodextrin HP- α -CD (cavity diameter:volume of the cavity:) And HP- β -CD (cavity diameter:volume of the cavity:) Is the specific ratio HP- γ -CD (cavity diameter:volume of the cavity:) More effective polysorbate 20 degradation inhibitor.

The size and volume of the cavities of each cyclodextrin may determine their effectiveness as molecular inhibitors of enzymatic PS20 degradation, rather than the physicochemical properties of each cyclodextrin. Without being bound by theory, this finding suggests that the protection mechanism may involve host-guest complexation between cyclodextrin and polysorbate 20 reaction sites.

Example 6: solubilization of visible and sub-visible particles associated with enzymatic polysorbate 20 degradation by cyclodextrins and other excipients

Several excipients were tested for their ability to solubilize the particles produced as a result of enzymatic PS20 degradation. Concentrated solutions of HP- α -CD, HP- β -CD, HP- γ -CD, SBE- β -CD, PVP, PEG 1500, sucrose and methionine were prepared in triplicate in pH 5.5 buffer. Particles from enzymatic PS20 degradation were prepared. Three solutions of 0.05% PS20 in pH 5.5 buffer were enzymatically degraded with 37.5. mu.g/mL PPL for 4.5 hours at room temperature, followed by heat inactivation at 85 ℃ for 30 minutes. The degraded PS20 solution was placed at 5 ℃ to allow the particles to crystallize.

After formation of particles in the degraded PS20 solution, the remaining sample preparation was performed in a cold room at 5 ℃ to prevent dissolution of PS 20-derived particles. Each degraded PS20 solution was divided into 11 portions and the concentrated excipient was incorporated into each aliquot. The final concentrations of excipients in each sample were as follows: 5% sucrose, 10mg/mL methionine, 5% PVP, 5% PEG 1500, 15% HP- β -CD, 5% HP- β -CD, 0.5% HP- β -CD, 35.5mM HP- α -CD, 35.5mM HP- γ -CD and 35.5mM SBE- β -CD. After addition of various excipients, the samples were left overnight at 5 ℃. The following day, the vials were inspected for visible particles under a seideneader visual detector. The sub-visible particle count was measured using a HIAC 9703 particle counter.

The results indicate that cyclodextrins (HP- α -CD, HP- β -CD, and HP- γ -CD) were able to significantly reduce the amount of SVP relative to the control and other excipient samples (FIGS. 10A and 10B). These results demonstrate that cyclodextrin can solubilize PS20 degradants resulting from enzymatic digestion of polysorbate 20, in addition to preventing enzymatic polysorbate degradation.

In addition, photographs were taken immediately before and after the addition of 15% (w/v) HP- β -CD. The photographs depict the solubilization of PS 20-related visible particles before (fig. 11A) and after (fig. 11B) the addition of 15% (w/v) HP- β -CD. The immediate solubilization of visible particles shown in the photographs provides convincing evidence that cyclodextrins can increase the solubility of visible and sub-visible particles associated with the degradation of PS 20.

Example 7: solubilization of visible and sub-visible particles associated with cyclodextrin oxidative polysorbate 20 degradation

HP- β -CD was tested at various concentrations for its ability to solubilize particles resulting from oxidative PS20 degradation. Concentrated HP- β -CD solutions were prepared in triplicate in pH 5.5 buffer. Storage of a protein-free sample containing 0.02% (w/v) PS20 at 5 ℃ for 27 months resulted in oxidative PS20 degradation and formation of visible and invisible particles associated with PS20 degradation products. Each degraded PS20 solution was divided into 3 portions and the concentrated vehicle was incorporated into each aliquot. The final concentrations of excipients in each sample were as follows: 0% (w/v) HP- β -CD (control), 5% (w/v) HP- β -CD and 15% (w/v) HP- β -CD. After adjustment of the HP-beta-CD concentration, the samples were left overnight at 5 ℃. The next day, sub-visible particle counts were measured using a HIAC 9703 particle counter.

HP- β -CD was tested at concentrations of 0%, 5% and 15% (w/v) for its effect on SVP re-solubilization. The results demonstrate a significant reduction in SVP in samples containing HP- β -CD relative to the control sample (0% HP- β -CD). As shown in fig. 12A-12F, 15% HP- β -CD effectively resolubilizes particles greater than or equal to 1.4 microns, while 5% HP- β -CD effectively resolubilizes particles greater than or equal to 2 microns.

These results demonstrate that cyclodextrin can solubilize PS20 degradation products resulting from oxidative digestion of polysorbate 20 in addition to preventing enzymatic polysorbate degradation and solubilizing PS20 degradants resulting from enzymatic digestion of polysorbate 20.

Example 8: HP-beta-CD concentration and effect of HP-beta-CD PS20 on degradation of PS20

To determine the effect of HP- β -CD concentration on PS20 degradation, samples containing 0.001, 0.01, 0.1, 1,5, or 15% PS20 were digested with 15 μ g/mL PPL for 4.5 hours. As shown in fig. 13, increasing the amount of HP- β -CD decreased PS20 degradation.

The effect of HP- β -CD concentration on PS20 enzymatic degradation at different PS20 concentrations was evaluated to identify the optimal concentration to inhibit enzymatic PS20 degradation. To evaluate the dependence of PS20 degradation on HP- β -CD concentration, RP-ELSD was used to determine the PS20 content of triplicate samples containing different HP- β -CD concentrations at various PS20 concentrations. Samples containing 0.005% (FIG. 14A), 0.02% (FIG. 14B), 0.1% (FIG. 14C) and 0.4% PS20 (FIG. 14D) were digested in protein-free samples containing 0, 0.5, 5 and 15% (w/v) HP- β -CD without vehicle (control) using 15 μ g/mL PPL enzyme at room temperature for 4.5 hours. PPL was added to each treatment solution at a ratio of 75mg PPL/mg PS20, and an equal volume of buffer was added to the control solution to determine the effect of HP- β -CD to polysorbate ratio on enzymatic degradation. Digestion was stopped by heat inactivation for 30 min in a water bath at 85 ℃. The PS20 concentration of each sample was analyzed using high performance liquid chromatography with an online evaporative light scattering detector as described above. The sample was then placed at 5 ℃ overnight to allow particles to form during analysis of visible and sub-visible particles as described above and placed on ice.

TABLE 2 ratio of HP-beta-CD to PS20

The amount of cyclodextrin required to completely inhibit enzymatic PS20 degradation was dependent on the concentration of PS20 (fig. 13 and 14A-D). At lower PS20 concentrations (e.g., 0.005% PS20 (fig. 14A)), only 0.5% HP- β -CD was required to completely inhibit PS20 degradation, while 15% HP- β -CD was required for the sample containing 0.1% PS20 (fig. 14C). Similarly, the formation of sub-visible particles larger than 2, 5 or 10 microns in diameter depends on the ratio of cyclodextrin to polysorbate. The sample containing 0.02% PS20 required 0.5% HP- β -CD to partially inhibit the formation of sub-visible particles and 15% HP- β -CD to completely inhibit the formation of sub-visible particles (FIGS. 15A-15C).

These results can be explained in the context of the HP- β -CD to PS20 ratio (w/w) (FIG. 16 and Table 2). The PS20 data indicate that sufficient HP- β -CD: PS20 (. gtoreq.37.5 w/w) was required to inhibit enzymatic PS20 degradation (Table 2). The results show that the ratio of HP- β -CD to PS20 is important in determining PS20 degradation across a wide concentration range of PS20 and HP- β -CD.

The results also demonstrate a possible mechanism for inhibition of PS20 degradation by HP- β -CD. In this case, the amount of PS20 degradation varied sigmoidally with increasing concentration of inhibitor (i.e., HP- β -CD) at the concentration of immobilized substrate (i.e., PS20) (fig. 13). Thus, without being bound by theory, increasing the cyclodextrin concentration can effectively reduce the free substrate concentration in solution, which reduces the rate of degradation of PS 20. Alternatively, the rate of PS20 degradation may be inhibited by the substrate-inhibitor complex.

Example 9: polysorbate degradation under antibody storage conditions

The effect of various classes of protein molecules (e.g., monoclonal antibodies (mabs), single Fab antibodies (sfabs), and bispecific antibodies (bsabs)) on the ability of cyclodextrins to reduce enzymatic PS20 degradation was evaluated. mAb, sFAb and BsAb drug samples were provided as their natural preparations. The samples were dialyzed and conditioned to a final protein concentration of 20mg/mL in the target formulation of 20mM histidine acetate with 0.02% PS20 at pH 5.5. Control samples were prepared to contain 20mM histidine acetate, pH 5.5, 0.02% PS 20. Each mAb, sFAb, BsAb, and control sample was aliquoted and conditioned with conditioning buffer to contain varying amounts (0%, 5%, and 15%) of HP β CD. Samples were digested with 15. mu.g/mL PPL enzyme for 4.5 hours at room temperature, followed by heat inactivation at 85 ℃ for 30 minutes. The PS20 concentration of each sample was analyzed using high performance liquid chromatography with an online evaporative light scattering detector. The samples were then left at 5 ℃ overnight to allow particles to form and the visible and sub-visible particles were analyzed as described above.

The results demonstrate that each sample containing 0% HP β CD has a different amount of PS20 degradation. The results show that the 0% HP β CD sample containing protein (fig. 17B-D) has a higher amount of PS20 degradation relative to the control sample (fig. 17A). Because proteins are expressed in Chinese Hamster Ovary (CHO) or e.coli cells, protein samples may contain other impurities (i.e., lipases, etc.) that contribute to the overall amount of PS20 degradation.

Although samples containing 0% HP β CD protein were observed to have higher amounts of PS20 degradation, the results indicate that comparable amounts of PS20 degradation were observed in samples containing 5% and 15% HP β CD. These results indicate that, although HP β CD may have varying amounts of impurities that catalyze degradation of PS20 (fig. 17A-D), it is equally effective in mitigating catalytic PS20 degradation for all molecular forms evaluated. This indicates that the presence of the protein molecule and its native hetereogenetic profile does not significantly affect the cyclodextrin to protein ratio necessary to mitigate degradation of PS 20. Without being bound by theory, this finding suggests that the mechanism of catalytic inhibition involves a cyclodextrin molecule that interacts directly with PS20, rather than an enzyme that degrades polysorbates. Otherwise, protein-containing samples containing additional enzymes that degrade polysorbates required more HP β CD to mitigate PS20 degradation compared to controls. Thus, the ratio of cyclodextrin to PS20 described herein should be broadly applicable to a variety of formulations comprising different protein molecules and a spectrum of impurities.

Conclusion

The studies performed showed the ability of cyclodextrins to inhibit enzymatic and oxidative degradation of polysorbates (PS20 and PS 80). The results indicate that PVP and cyclodextrin (i.e., HP- α -CD, HP- β -CD, HP- γ -CD, SBE- β -CD) are capable of preventing enzymatic degradation of PS 20. Further experiments demonstrated that HP- β -CD has a protective effect on polysorbates in the presence of multiple enzymes (i.e., CALB, RLE, LPL, and PLL). Without being bound by theory, the inhibition mechanism may involve an interaction between the inhibitor (i.e., cyclodextrin) and the substrate (i.e., polysorbate). The formation of the inclusion complex can either reduce the concentration of free substrate or directly sterically inhibit the interaction with the active site and substrate. Furthermore, concentration studies determined the optimal range that provided the HP- β -CD to PS20 ratio (. gtoreq.37.5 w/w) necessary to completely inhibit enzymatic PS20 degradation.

In addition to preventing enzymatic PS20 degradation, the results indicated that cyclodextrins were effective in reducing the amount of sub-visible and visible particles. In this way, the cyclodextrin depolymerizes and solubilizes the sub-visible and visible particles in solution. In addition to being effective in preventing particle formation, the results indicate that cyclodextrins can also effectively solubilize existing particles associated with polysorbate degradation. It is speculated that cyclodextrin also increases the solubility of free fatty acids as degradation products of polysorbates.

The findings of this study have broad practical significance. The results provide comprehensive evidence that cyclodextrin-containing formulations can be used to prevent enzymatic polysorbate degradation. In addition, cyclodextrins can be used to solubilize free fatty acids associated with polysorbate degradation from both oxidative and enzymatic degradation. Thus, cyclodextrins can also be used as diluents or reconstitution buffers for pharmaceutical products to solubilize degradants and particles associated with polysorbate degradation.

Claims

1. A method of using cyclodextrin to reduce polysorbate degradation in an aqueous formulation comprising a polysorbate, the method comprising adding cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than 37.5:1, wherein the formulation further comprises a polypeptide.

2. A method of using cyclodextrin to reduce polysorbate degradation in an aqueous formulation comprising a polysorbate, the method comprising adding cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than 37.5:1, wherein the formulation comprises 0.005% -0.4% polysorbate, wherein the formulation further comprises a polypeptide.

3. A method of using cyclodextrin to reduce polysorbate degradation in an aqueous formulation comprising a polysorbate, the method comprising adding cyclodextrin to the formulation to a concentration of 0.01% -30%, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than 37.5:1, wherein the formulation comprises 0.005% to 0.4% polysorbate, wherein the formulation further comprises a polypeptide.

4. A method of using cyclodextrin to reduce the amount of sub-visible and visible particles in an aqueous formulation comprising a polysorbate, the method comprising adding cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than 37.5:1, wherein the formulation further comprises a polypeptide.

5. A method of using cyclodextrin to depolymerize and solubilize a polysorbate degradation product in an aqueous formulation comprising a polysorbate, the method comprising adding cyclodextrin to the formulation, wherein the resulting w/w ratio of cyclodextrin to polysorbate is greater than 37.5:1, wherein the formulation further comprises a polypeptide.

6. The method of any one of claims 1-5, wherein the polysorbate is polysorbate 20 or polysorbate 80.

7. The method of any one of claims 1-5, wherein the cyclodextrin is HP-beta cyclodextrin, HP-gamma cyclodextrin, or sulfobutyl ether beta-cyclodextrin.

8. The method of any one of claims 1-5, wherein the concentration of polysorbate in the formulation is in the range of 0.01% to 0.4%.

9. The method of any one of claims 1-5, wherein the concentration of polysorbate in the formulation is in the range of 0.01% to 0.1%.

10. The method of any one of claims 1-5, wherein the concentration of polysorbate in the formulation is 0.02%.

11. The method of any one of claims 1-5, wherein the concentration of cyclodextrin in the formulation is in the range of 0.5 to 30%.

12. The method of any one of claims 1-5, wherein the concentration of cyclodextrin in the formulation is 15%.

13. The method of any one of claims 1-5, wherein polysorbate degradation is reduced by 50%, 75%, 80%, 85%, 90%, 95%, or 99%.

14. The method of any one of claims 1-4, wherein less than 1,000 polysorbate particles greater than 2 microns in diameter are formed per mL.

15. The method of any one of claims 1-4, wherein less than 750 polysorbate particles greater than 2 microns in diameter are formed per mL.

16. The method of any one of claims 1-4, wherein less than 500 polysorbate particles greater than 2 microns in diameter are formed per mL.

17. The method of any one of claims 1-4, wherein less than 250 polysorbate particles greater than 2 microns in diameter are formed per mL.

18. The method of any one of claims 1-4, wherein less than 150 polysorbate particles greater than 2 microns in diameter are formed per mL.

19. The method of any one of claims 1-4, wherein less than 100 polysorbate particles greater than 2 microns in diameter are formed per mL.

20. The method of any one of claims 1-4, wherein less than 50 polysorbate particles greater than 2 microns in diameter are formed per mL.

21. The method of any one of claims 1-4, wherein less than 25 polysorbate particles greater than 2 microns in diameter are formed per mL.

22. The method of any one of claims 1-5, wherein the formulation is stable at 2 ℃ to 8 ℃ for at least 6 months.

23. The method of any one of claims 1-5, wherein the formulation is stable at 2 ℃ to 8 ℃ for at least 12 months.

24. The method of any one of claims 1-5, wherein the formulation is stable at 2 ℃ to 8 ℃ for at least 18 months.

25. The method of any one of claims 1-5, wherein the formulation is stable at 2 ℃ to 8 ℃ for at least 24 months.

26. The method of any one of claims 1-5, wherein the formulation is stable at 1 ℃ to 10 ℃ for at least 48 months.

27. The method of any one of claims 1-5, wherein the formulation is stable at 2 ℃ to 8 ℃ for at least 48 months.

28. The method of claim 1, wherein the polypeptide is an antibody.

29. The method of claim 28, wherein the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, a multispecific antibody, or an antibody fragment.

30. The method of any one of claims 1-5, wherein the concentration of the polypeptide in the formulation is 1mg/mL to 250 mg/mL.

31. The method of any one of claims 1-5, wherein the formulation has a pH of 4.5 to 7.0.

32. The method of any one of claims 1-5, wherein the formulation has a pH of 4.5 to 6.0.

33. The method of any one of claims 1-5, wherein the formulation has a pH of 6.0.

34. The method of any one of claims 1-5, wherein the formulation further comprises one or more excipients selected from the group consisting of stabilizers, buffers, surfactants, and tonicity agents.

35. The method of any one of claims 1-5, wherein the formulation is a pharmaceutical formulation suitable for administration to a subject.

36. The method of any one of claims 1-5, wherein the formulation is a pharmaceutical formulation suitable for intravenous, subcutaneous, intramuscular, or intravitreal administration to a subject.