Composition and methods for stabilizing liquid protein formulations

Abstract

To provide liquid pharmaceutical formulations comprising polypeptides and surfactants.SOLUTION: The present invention provides a liquid formulation comprising a polypeptide and a surfactant, wherein at least about 70% (wt.%) of the surfactant are isosorbide polyoxyethylene (POE) fatty acid esters.SELECTED DRAWING: Figure 5

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of priority to U.S. Provisional Patent Application No. 62 / 868,615, filed June 28, 2019, the disclosure of which is incorporated herein by reference in its entirety. [Technical field]

[0002] The present disclosure relates generally to stable liquid pharmaceutical formulations comprising a polypeptide and a surfactant and methods of making the same. [Background technology]

[0003] Polysorbates (PS), a commonly used surfactant in biopharmaceutical protein formulations, have been shown to be susceptible to various degradation pathways, including chemical hydrolysis, oxidation, and enzymatic hydrolysis. Degradation of PS can lead to the generation of various peroxides, which subsequently oxidize amino acid residues (e.g., methionine) in proteins during long-term storage. Levine et al. Proc. Natl. Acad. Sci. USA (1996) 93, 15036-15040. Oxidation of these amino acid residues potentially adversely affects the biological activity of proteins, thereby limiting the protective effect of PS in protein formulations. Furthermore, because polysorbates are heterogeneous mixtures, the pattern of degradation may differ significantly among the various pathways. Thus, there remains a need for more efficient excipients for surfactants in the development of pharmaceutical protein formulations. Summary of the Invention

[0004] The present disclosure discloses a liquid formulation comprising a polypeptide and a surfactant, wherein at least about 70% (by weight) of the surfactant is an isosorbide polyoxyethylene (POE) fatty acid ester. In some embodiments, the isosorbide POE fatty acid ester comprises about 5-30 POE units. In some embodiments, the isosorbide POE fatty acid ester comprises about 20 POE units. In some embodiments, the isosorbide POE fatty acid ester comprises an optionally substituted C 4-28 Alkyl and optionally substituted C 4-28 alkenyl. In some embodiments, the isosorbide POE fatty acid ester is a monoester, a diester, or a mixture thereof. In some embodiments, the isosorbide POE fatty acid ester is selected from the group consisting of isosorbide POE monolaurate, isosorbide POE monomyristate, isosorbide POE monopalmitate, isosorbide POE monostearate, and isosorbide POE monooleate.

[0005] In some embodiments, the isosorbide POE fatty acid ester has the formula (I): TIFF2025023952000002.tif23170[in formula: a and b are independently an integer from 2 to 28, provided that the sum of a and b is an integer from 5 to 30; R 1 and R 2 are independently hydrogen and -C(O)R" (wherein R" is an optionally substituted C 3-27 Alkyl or optionally substituted C 3-27 alkenyl); R 3 and R 4 are independently prime] It is a compound of the formula:

[0006] In some embodiments, the sum of a and b is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the sum of a and b is 9. In some embodiments, the sum of a and b is 20. In some embodiments, R 1 is H and R 2 is -C(O)R''. In some embodiments, R 2 is H and R 1 is -C(O)R''. In some embodiments, R 1 and R 2 In some embodiments, R″ is an unsubstituted C 3-27 In some embodiments, R″ is an unsubstituted C 11 In some embodiments, R″ is an unsubstituted C 3-27 In some embodiments, R″ is an unsubstituted C 17 It is alkenyl.

[0007] In some embodiments, the surfactant further comprises a POE fatty acid ester. In some embodiments, at least about 80% (wt%) of the surfactant is an isosorbide POE fatty acid ester and a POE fatty acid ester. In some embodiments, at least about 85%, at least about 90%, or at least about 95% (wt%) of the surfactant is an isosorbide POE fatty acid ester and a POE fatty acid ester. In some embodiments, at least about 90% (wt%) of the surfactant is an isosorbide POE fatty acid ester and a POE fatty acid ester. In some embodiments, the POE fatty acid ester is an optionally substituted C 4-28 Alkyl and optionally substituted C 4-28alkenyl. In some embodiments, the POE fatty acid ester is selected from the group consisting of POE monolaurate, POE monomyristate, POE monopalmitate, POE monostearate, and POE monooleate. In some embodiments, less than about 20% of the surfactant is a POE fatty acid ester. In some embodiments, less than about 10% of the surfactant is a POE fatty acid ester. In some embodiments, the surfactant is about 0.0005%-0.2% (w:v) in the liquid formulation. In some embodiments, the surfactant comprises an isosorbide POE fatty acid ester in an amount greater than the POE fatty acid ester. In some embodiments, the surfactant further comprises a sorbitan POE fatty acid ester. In some embodiments, less than about 10%, less than about 8%, less than about 5%, less than about 3%, or less than about 1% of the surfactant is a sorbitan POE fatty acid ester.

[0008] In some embodiments, the polypeptide is a protein. In some embodiments, the protein is an antibody selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, and an antibody fragment. In some embodiments, the antibody fragment is selected from the group consisting of a Fab, a Fab', a F(ab')2, and an Fv fragment. In some embodiments, the antibody has a concentration of about 0.001 mg / mL to about 300 mg / mL. In some embodiments, the liquid formulation is a reconstituted lyophilized formulation. In some embodiments, the liquid formulation is further diluted with infusion fluid to a concentration of about 0.001 mg / mL to about 100 mg / mL. In some embodiments, the liquid formulation is substantially free of aggregates. In some embodiments, the liquid formulation has low free fatty acid particle formation.

[0009] Also provided herein is an article of manufacture comprising a container enclosing any of the liquid formulations described herein. In some embodiments, the container is an IV bag. In some embodiments, the IV bag comprises an injection device. In some embodiments, the IV bag comprises an infusion fluid. Also provided herein is a lyophilized formulation comprising a polypeptide and a surfactant, wherein at least about 70% (wt%) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester. In some embodiments, the lyophilized formulation is prepared by lyophilizing any of the liquid formulations disclosed herein.

[0010] Also provided herein is a method of making a liquid formulation comprising adding a polypeptide and a surfactant to an aqueous solution, wherein at least 70% (by weight) of the surfactant is an isosorbide POE fatty acid ester. In some embodiments, the isosorbide POE fatty acid ester comprises about 5-30 POE units. In some embodiments, the isosorbide POE fatty acid ester comprises about 20 POE units. In some embodiments, the isosorbide POE fatty acid ester comprises an optionally substituted C 4ー28 Alkyl and optionally substituted C 4-28 alkenyl. In some embodiments, the isosorbide POE fatty acid ester is a monoester, a diester, or a mixture thereof. In some embodiments, the isosorbide POE fatty acid ester is selected from the group consisting of isosorbide POE monolaurate, isosorbide POE monomyristate, isosorbide POE monopalmitate, isosorbide POE monostearate, and isosorbide POE monooleate.

[0011] In some embodiments, the isosorbide POE fatty acid ester has the formula (I): TIFF2025023952000003.tif25170[in formula: a and b are independently an integer from 2 to 28, provided that the sum of a and b is an integer from 5 to 30; R 1 and R 2are independently hydrogen and -C(O)R" (wherein R" is an optionally substituted C 3-27 Alkyl or optionally substituted C 3-27 alkenyl); R 3 and R 4 are independently hydrogen. It is a compound of the formula:

[0012] In some embodiments, the sum of a and b is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, or 25. In some embodiments, the sum of a and b is 9. In some embodiments, the sum of a and b is 20. In some embodiments, R 1 is H and R 2 is -C(O)R''. In some embodiments, R 2 is H and R 1 is -C(O)R''. In some embodiments, R 1 and R 2 In some embodiments, R″ is an unsubstituted C 3-27 In some embodiments, R″ is an unsubstituted C 11 In some embodiments, R″ is an unsubstituted C 3-27 In some embodiments, R″ is an unsubstituted C 17 It is alkenyl.

[0013] In some embodiments, the surfactant further comprises a POE fatty acid ester. In some embodiments, at least about 80% (wt%) of the surfactant is an isosorbide POE fatty acid ester and a POE fatty acid ester. In some embodiments, at least about 85%, at least about 90%, or at least about 95% (wt%) of the surfactant is an isosorbide POE fatty acid ester and a POE fatty acid ester. In some embodiments, the method further comprises adding a POE fatty acid ester. In some embodiments, the POE fatty acid ester is an optionally substituted C4-28 Alkyl and optionally substituted C 4-28 alkenyl. In some embodiments, the POE fatty acid ester is selected from the group consisting of POE monolaurate, POE monomyristate, POE monopalmitate, POE monostearate, and POE monooleate. In some embodiments, less than about 20% of the surfactant is a POE fatty acid ester. In some embodiments, less than about 10% of the surfactant is a POE fatty acid ester. In some embodiments, the surfactant is about 0.0005%-0.2% (w:v) in the liquid formulation. In some embodiments, the surfactant comprises an isosorbide POE fatty acid ester in an amount greater than the POE fatty acid ester. In some embodiments, the surfactant further comprises a sorbitan POE fatty acid ester. In some embodiments, less than about 10%, less than about 8%, less than about 5%, less than about 3%, or less than about 1% of the surfactant is a sorbitan POE fatty acid ester.

[0014] In some embodiments, the polypeptide is a protein. In some embodiments, the protein is an antibody selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, and an antibody fragment. In some embodiments, the antibody fragment is selected from the group consisting of a Fab, a Fab', a F(ab')2, and an Fv fragment. In some embodiments, the antibody has a concentration of about 0.1 mg / mL to about 300 mg / mL. In some embodiments, the liquid formulation is a reconstituted lyophilized formulation. In some embodiments, the liquid formulation is further diluted with infusion fluid to a concentration of about 0.1 mg / mL to about 2 mg / mL. In some embodiments, the liquid formulation is further lyophilized to prepare a lyophilized formulation. In some embodiments, the liquid formulation is substantially free of aggregates. In some embodiments, the liquid formulation has low free fatty acid particle formation.

[0015] It should be understood that one, some, or all of the features of the various embodiments described herein may be combined to form other embodiments of the present invention. These and other aspects of the present invention will be apparent to those skilled in the art. These and other embodiments of the present invention are further described in the detailed description that follows. [Brief description of the drawings]

[0016] [Figure 1] 1A and 1B show UPLC analysis of the purity and identity of the polysorbate 20 fraction (FIG. 1A) and the polysorbate 80 fraction (FIG. 1B).

[0017] [Diagram 2] 2A and 2B show the critical micelle concentration (CMC) of the polysorbate 20 fraction (FIG. 2A) and the polysorbate 80 fraction (FIG. 2B) using the fluorescent dye N-phenylnaphthalen-1-amine (NPN).

[0018] [Diagram 3] 3A and 3B show the surface tension over time for the polysorbate 20 fraction (FIG. 3A) and the polysorbate 80 fraction (FIG. 3B).

[0019] [Figure 4] 4A and 4B show the micelle size of the polysorbate 20 fraction (FIG. 4A) and the polysorbate 80 fraction (FIG. 4B), each fraction having a concentration of 1% by weight.

[0020] [Diagram 5] FIG. 5 shows images of antibody formulations containing various concentrations of polysorbate 20 fractions.

[0021] [Figure 6] Figures 6A and 6B show images of antibody formulations containing various concentrations of polysorbate 20 fractions.

[0022] [Figure 7]7A and 7B show the HIAC results for antibody formulations of mAbB and mABC containing various concentrations of the polysorbate 20 fraction.

[0023] [Figure 8] 8A and 8B show the HIAC results for antibody formulations of mAbB and mAbC containing various concentrations of the polysorbate 20 fraction. [Figure 9] 9A and 9B show the HIAC results for antibody formulations of mAbB and mAbC containing various concentrations of the polysorbate 20 fraction.

[0024] [Figure 10] Figures 10A and 10B show images of antibody formulations containing various concentrations of polysorbate 20 fractions stored at 40°C for various times.

[0025] [Figure 11] Figures 11A and 11B show HIAC results for antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 40°C as indicated.

[0026] [Figure 12] 12A and 12B show the results of SEC-HPLC of antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 40° C. [Figure 13] 13A and 13B show the results of SEC-HPLC of antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 40° C.

[0027] [Figure 14] FIG. 14 shows the results of IEC for antibody formulations of mAbB containing polysorbate 20 fraction stored at 40° C.

[0028] [Figure 15]Figures 15A and 15B show images of antibody formulations containing various concentrations of polysorbate 20 fractions stored at 25°C for various times.

[0029] [Figure 16] Figures 16A and 16B show HIAC results for antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 25°C.

[0030] [Figure 17] Figures 17A and 17B show the results of SEC-HPLC of antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 25°C. [Figure 18] Figures 18A and 18B show the results of SEC-HPLC of antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 25°C.

[0031] [Figure 19] FIG. 19 shows the results of IEC for antibody formulations of mAbB containing polysorbate 20 fraction stored at 25° C.

[0032] [Figure 20] Figures 20A and 20B show images of antibody formulations containing various concentrations of polysorbate 20 fractions stored at 5°C for various times.

[0033] [Figure 21] Figures 21A and 21B show HIAC results for antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 5°C.

[0034] [Figure 22] Figures 22A and 22B show the results of SEC-HPLC of antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 5°C. [Figure 23]Figures 23A and 23B show the results of SEC-HPLC of antibody formulations of mAbB and mAbC containing various concentrations of polysorbate 20 fractions stored at 5°C.

[0035] [Figure 24] FIG. 24 shows the results of IEC for antibody formulations of mAbB containing polysorbate 20 fraction stored at 5° C.

[0036] [Diagram 25] FIG. 25 shows the HIAC results from forced degradation of PS20 and F2a (each in formulation buffer at pH=6.0) by 2.5 U / mL of lipase enzyme from Pseudomonas cepacia (PCL). DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0037] The present disclosure is based on the discovery that certain fractions of polysorbate provide strong protective effect in pharmaceutical protein formulations.Specifically, they allow the use of less surfactant for the same protective effect, thereby minimizing the adverse effect on protein biological activity resulting from polysorbate degradation.In one aspect, the present disclosure provides protein formulations that include one or more such fractions of polysorbate.The protein formulations described herein have demonstrated increased protein stability in formulations.The present disclosure also provides kits and methods for making protein formulations.

[0038] I. Definition Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by those skilled in the art to which this disclosure belongs. All patents, applications, published applications and other publications mentioned herein are incorporated by reference in their entirety. If the definitions set forth in this paragraph are contrary to or inconsistent with the definitions set forth in the patents, applications or other publications incorporated herein by reference, the definitions set forth in this paragraph shall take precedence over the definitions incorporated herein by reference.

[0039] It is understood that certain features of the disclosure that are for clarity described in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various features of the disclosure that are for brevity described in the context of a single embodiment may also be provided separately or in any suitable subcombination. All combinations of the embodiments with respect to specific method steps, reagents, or conditions are specifically embraced by the disclosure and are disclosed herein just as if each and every combination was individually and expressly disclosed.

[0040] As used in this specification and the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0041] Reference herein to "about" a value or parameter includes (and describes) variations that are directed to the value or parameter itself. For example, a statement referring to "about X" includes the statement of "X."

[0042] The term "pharmaceutical formulation" refers to a preparation in which the biological activity of the active ingredient is effective and which does not contain additional ingredients that are unacceptably toxic to the subject to which the formulation is administered. In some embodiments, the formulation is sterile.

[0043] A "reconstituted" formulation is one prepared by dissolving a lyophilized protein or antibody formulation in a diluent such that the protein is dispersed in the reconstituted formulation. The reconstituted formulation is suitable for administration (e.g., parenteral administration) to a patient treated with the protein of interest, and in certain embodiments of the invention may be suitable for subcutaneous administration.

[0044] The terms "protein," "polypeptide," and "peptide" are used herein to refer to polymers of amino acids of any length. The polymers may be linear or branched, may contain modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified, either naturally or by intervention, such as disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. Typically, a protein as used herein has a molecular weight of at least about 5-20 kD, alternatively at least about 15-20 kD, or at least about 20 kD. 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, are also included in this definition. In certain embodiments, examples of polypeptides encompassed by the definition herein include mammalian polypeptides such as, for example, renin; growth hormones, e.g., human growth hormone and bovine growth hormone; growth hormone releasing factor; parathyroid hormone; thyroid stimulating hormone; lipoproteins; alpha-1-antitrypsin; insulin A chain; insulin B chain; proinsulin; follicle stimulating hormone; calcitonin; luteinizing hormone; glucagon; leptin; clotting factors, e.g., factor VIIIC, factor IX, tissue factor, and von Willebrand factor; anticoagulants, e.g., protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators, e.g., urokinase or human urinary or fetal bovine growth hormone; or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and -beta; tumor necrosis factor receptors, e.g., death receptor 5 and CD120; TNF-related apoptosis-inducing ligand (TRAIL); B-cell maturation antigen (BCMA); B-lymphocyte stimulatory factor (BLyS); proliferation-inducing ligand (APRIL); enkephalinase; RANTES (regulated upon activation and normally expressed and secreted by T cells); human macrophage inflammatory protein (MIP-1-alpha); serum albumin, e.g., human serum albumin; Müllerian inhibitory substance; relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide;Microbial proteins, e.g., beta-lactamase; DNase; IgE; cytotoxic T-lymphocyte-associated antigen (CTLA), e.g., CTLA-4; inhibin; activin; platelet-derived endothelial cell growth factor (PD-ECGF); vascular endothelial growth factor family proteins (e.g., VEGF-A, VEGF-B, VEGF-C, VEGFD, and PlGF); platelet-derived growth factor (PDGF) family proteins (e.g., PDGF-A, PDGF-B, PDGF-C, PDGF-D, and dimers thereof); fibroblast growth factor (FGF) family, e.g., aFGF, bFGF, FGF4, and FGF9; epidermal growth factor (EGF); hormone or growth factor receptors, e.g., VEGF receptor(s) (e.g., VEGFR1, VEGFR2, and VEGFR3), epidermal growth factor (EGF) receptor(s) (e.g., ErbB1, ErbB2, ErbB3, and ErbB4) receptor), platelet-derived growth factor (PDGF) receptor(s) (e.g., PDGFR-α and PDGFR-β), and fibroblast growth factor receptor(s); TIE ligands (angiopoietin, ANGPT1, ANGPT2), angiopoietin receptors, e.g., TIE1 and TIE2; protein A or D; rheumatoid factor; neurotrophic factors, e.g., bone-derived neurotrophic factor (BDNF), neurotrophin-3, -4, -5, or -6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factor, e.g., NGF-b; transforming growth factor (TGF), e.g., TGF-β1, TGF-β2, TGF-β3, TGF-β4, TGF-β5, TGF-β6, TGF-β7, TGF-β8, TGF-β9, TGF-β10, TGF-β11, TGF-β12, TGF-β13, TGF-β14, TGF-β15, TGF-β16, TGF-β17, TGF-β18, TGF-β19, TGF-β20, TGF-β210, TGF-β220, TGF-β23, TGF-β24, TGF-β25, TGF-β26, TGF-β27, TGF-β28, TGF-β29 ... TGF-alpha and TGF-beta, including TGF-β4, or TGF-β5; insulin-like growth factors I and II (IGF-I and IGF-II); des(l-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (IGFBPs); CD proteins, such as CD3, CD4, CDS, CD19, and CD20; erythropoietin; bone morphogenetic factors; immunotoxins; bone morphogenetic proteins (BMPs); chemokines, such as CXCL12 and CXCR4; interferons, such as interferon-alpha, -beta, and -gamma; colony stimulating factors (CSFs), such as M-CSF, GM-CSP, and G-CSF;Cytokines, e.g., interleukins (IL), e.g., IL-1 to IL-10; midkine; superoxide dismutase; T cell receptors; surface membrane proteins; decay-accelerating factors; viral antigens, e.g., parts of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, e.g., CDlla, CDllb, CDllc, CD18, ICAM, VLA-4, and VCAM; ephrins; Bv8; delta-like ligand 4 (DLL4); Del-1; BMP9; BMPlO; follistatin; hepatocyte growth factor (HGF) / scatter factor (SF); Alkl; Robo4; ESMl; perlecan; EGF-like domain multiple 7 (EGFL7); CTGF and members of its family; Thrombospondins, such as thrombospondin 1 and thrombospondin 2; collagens, such as collagen IV and collagen XVIII; neuropilins, such as NRP1 and NRP2; pleiotrophin (PTN); progranulin; proliferin; Notch proteins, such as Notch1 and Notch4; semaphorins, such as Sema3A, Sema3C, and Sema3F; tumor associated antigens, such as CA125 (ovarian cancer antigen), or receptors for HER2, HER3, or HER4; immunoadhesins; and fragments and / or variants of any of the above polypeptides, as well as antibodies, including antibody fragments, that bind to one or more proteins, including, for example, any of the above proteins;

[0045] The term "antibody" herein is used in the broadest sense and specifically includes monoclonal antibodies (such as full-length monoclonal antibodies), polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody fragments so long as they exhibit the desired biological activity.

[0046] An "isolated" antibody is one that has been identified and separated and / or recovered from a component of its natural environment. Contaminant components of its natural environment are substances that would interfere with the antibody's experimental, diagnostic, or therapeutic uses, such as enzymes, hormones, and other proteinaceous or non-proteinaceous solutes. In some embodiments, the antibody is purified (1) to greater than 95% by weight, and in some embodiments, greater than 99% by weight, e.g., as determined by the Lowry method; (2) to a degree sufficient to obtain at least 15 residues of N-terminal or internal amino acid sequence, e.g., using a spinning cup sequenator; or (3) to homogeneity by SDS-PAGE under reducing or non-reducing conditions, e.g., using Coomassie blue or silver staining. An isolated antibody includes the antibody in situ within recombinant cells, since at least one component of the antibody's natural environment will not be present. Ordinarily, however, an isolated antibody will be prepared by at least one purification step.

[0047] "Native antibodies" are usually heterotetrameric glycoproteins of about 150,000 daltons, composed of two identical light (L) chains and two identical heavy (H) chains. Each light chain is linked to a heavy chain by one covalent disulfide bond; the number of disulfide linkages varies among the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bridges. Each heavy chain contains a variable domain (V H ) followed by multiple constant domains. Each light chain has a variable domain (V L ) at one end and a constant domain at the other end. The light chain constant domain 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. Particular amino acid residues are believed to form an interface between the light chain variable domain and the heavy chain variable domain.

[0048] The terms "full length antibody," "intact antibody," and "whole antibody" are used interchangeably herein and refer to an antibody in its substantially intact form, as described below, and not to antibody fragments. These terms specifically refer to antibodies that comprise a heavy chain that contains an Fe region.

[0049] An "antibody fragment" includes a portion of an intact antibody, optionally including its antigen-binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, and Fv fragments; diabodies; linear antibodies; single-chain antibody molecules; and multispecific antibodies formed from antibody fragments.

[0050] The term "monoclonal antibody" as used herein refers to an antibody obtained from a population of substantially homogenous antibodies, e.g., the individual antibodies that make up the population are identical except for possible mutations that may be present in minor amounts, e.g., naturally occurring mutations. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of separate antibodies. In certain embodiments, such monoclonal antibodies typically include antibodies that include a polypeptide sequence that binds to a target, the target-binding polypeptide sequence being obtained by a process that includes the selection of a single target-binding polypeptide sequence from a plurality of polypeptide sequences. For example, the selection process can be the selection of a unique clone from a plurality of clones, such as a pool of hybridoma clones, phage clones, or recombinant DNA clones. It is understood that the selected target-binding sequence may be further modified, e.g., to improve affinity to the target, humanize the target-binding sequence, improve its production in cell culture, reduce its immunogenicity in vivo, create a multispecific antibody, etc., and an antibody that includes a modified target-binding sequence is also a monoclonal antibody of the present invention. In contrast to polyclonal antibody preparations, which typically include 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 are advantageous in that they are typically 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.

[0051] 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(s) 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)). Chimeric antibodies include PRIMATTZED® antibodies, whose antigen-binding regions are derived from, for example, antibodies produced by immunizing macaque monkeys with the antigen of interest.

[0052] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence from non-human immunoglobulin. In one embodiment, a humanized antibody is a human immunoglobulin (recipient antibody) in which residues from an HVR of the recipient are replaced by 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 instances, FR residues of the human immunoglobulin are replaced by corresponding non-human residues. Furthermore, humanized antibodies may comprise residues that are not found in the recipient or donor antibody. These modifications can be made to further refine antibody performance. In general, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, with all or substantially all of the hypervariable loops corresponding to those of a non-human immunoglobulin and all or substantially all of the FRs being those of a human immunoglobulin sequence. A humanized antibody optionally also comprises at least a portion of an immunoglobulin constant region (Fe), typically that of a human immunoglobulin. For further details, see, e.g., Ones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988); and Presta, Curr. Op. Struct. Biol. 2:593-596 (1992). See also, e.g., Vaswani and Hamilton, Ann. Allergy, Asthma & Immunol. 1:105-115 (1998); Harris, Biochem. Soc. Transactions 23:1035-1038 (1995); Hurle and Gross, Curr. Op. Biotech. 5:428-433 (1994); and U.S. Patent Nos. 6,982,321 and 7,087,409.

[0053] A "human antibody" is an antibody that has an amino acid sequence that corresponds to that of an antibody produced by a human and / or that has been made using any of the techniques disclosed herein for making human antibodies. This definition of a human antibody specifically excludes humanized antibodies that contain non-human antigen-binding residues. Human antibodies can be produced using a variety of techniques known in the art, such as phage display libraries. The methods described in Hoogenboom and Winter, J.Mal.Biol., 227:381 (1991); Marks et al., J.Mal.Biol., 222:581 (1991). Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R.Liss, p.77 (1985); Boerner et al., J.Immunol., 147(1):86-95 (1991) can also be used to prepare human monoclonal antibodies. See also van Dijk and van de Winkel, Curr. Opin. Pharmacol., 5:368-74 (2001). Human antibodies can be prepared by administering antigen to transgenic animals, e.g., immunized xenomouse, that have been engineered to produce such antibodies in response to antigen challenge, but whose endogenous loci have been disabled (see, e.g., U.S. Patent Nos. 6,075,181 and 6,150,584 for XENOMOUSE™ technology). Also, for human antibodies produced by human B cell hybridoma technology, see, e.g., Li et al., Proc. Natl. Acad. Sci. USA, 103:3557-3562 (2006).

[0054] A "stable" formulation is one in which the protein therein essentially retains its physical and / or chemical stability and / or biological activity upon storage. In some embodiments, the formulation essentially retains its physical and chemical stability and its biological activity upon storage. The storage period is generally selected based on the intended shelf life of the formulation. A variety of analytical techniques for measuring protein stability are available in the art and are reviewed, for example, in Peptide and Protein Drug Delivery, 247-301, Vincent Lee Ed., Marcel Dekker, Inc., New York, NY, Pubs. (1991) and Jones, A. Adv. Drug Delivery Rev. 10:29-90 (1993). Stability can be measured over a selected period of time at a selected exposure and / or temperature. Stability can be qualitatively and / or quantitatively assessed 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); assessment of ROS formation (e.g., by using a light stress assay or a 2,2'-azobis(2-amidinopropane) dihydrochloride (AAPH) stress assay); oxidation of specific amino acid residues of the protein (e.g., Trp and / or Met residues of a monoclonal antibody); by assessing charge heterogeneity using cation exchange chromatography, image capillary isoelectric focusing (icIEF) or capillary zone electrophoresis; amino- or carboxy-terminal sequence analysis; mass spectrometry; SDS-PAGE analysis to compare reduced and intact antibodies; peptide map (e.g., trypsin or LYS-C) analysis; assessment of the biological activity or target binding function of the protein (e.g., antigen binding function of an antibody); etc.Instability may include any one or more of aggregation, deamidation (e.g., Asn deamidation), oxidation (e.g., Met oxidation and / or Trp oxidation), isomerization (e.g., Asp isomerization), clipping / hydrolysis / fragmentation (e.g., hinge region fragmentation), succinimide formation, unpaired cysteine(s), N-terminal extension, C-terminal processing, differential glycosylation, excipient degradation, formation of particulates (e.g., free fatty acid particles), and the like.

[0055] A protein "retains its physical stability" in a pharmaceutical formulation if it shows little or no signs of aggregation, precipitation, and / or denaturation upon visual inspection of color and / or clarity, or as measured by UV light scattering or size exclusion chromatography.

[0056] 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 chemically modified forms of the protein. Chemical modifications can include protein oxidation, which can be assessed, for example, using tryptic peptide mapping, reversed-phase high performance liquid chromatography (HPLC), and liquid chromatography-mass spectrometry (LC / MS). Other types of chemical modifications include protein charge modifications, which can be assessed, for example, by ion exchange chromatography or icIEF.

[0057] 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 at the time the pharmaceutical formulation was prepared, as determined, for example, by an antigen binding assay of a monoclonal antibody. As used herein, the "biological activity" of a protein refers to the ability of the protein to bind to a target, e.g., the ability of a monoclonal antibody to bind to an antigen. This may further include biological responses that may be measured in vitro or in vivo. Such activity may be an antagonistic or agonistic activity.

[0058] As used herein, the terms "polyethylene glycol," "PEG," "polyethylene oxide," "PEO," "polyoxyethylene," and "POE" may be used interchangeably and refer to a polyether compound composed of two or more ethylene oxide subunits. "Polyethylene glycol" may be composed of ethylene oxide oligomers (e.g., having 2 to 9 ethylene oxide monomer subunits) or ethylene oxide polymers (e.g., having 10 or more than 9 ethylene oxide monomer subunits).

[0059] "Fatty acids" are carboxylic acids with a long-chain hydrocarbon side group. They are composed of organic monobasic acids derived from hydrocarbons by oxidation of the methyl group to an alcohol, an aldehyde, and then an acid equivalent. Fatty acids can be either saturated or unsaturated. In the case of unsaturated fatty acids, they can have the cis (Z) or trans (E) configuration, or a combination of both.

[0060] "Alkyl" includes straight and branched carbon chains having the specified number of carbon atoms, e.g., 1 to 20 carbon atoms, or 1 to 8 carbon atoms, or 1 to 6 carbon atoms. For example, C 1-6Alkyl includes both straight and branched chain alkyls of 1 to 6 carbon atoms. When an alkyl residue having a specific number of carbons is recited, all branched and straight chain versions having that number of carbons are intended to be included, thus, for example, "propyl" includes n-propyl and isopropyl, and "butyl" includes n-butyl, sec-butyl, isobutyl and t-butyl. Examples of alkyl groups include, but are not limited to, methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2-pentyl, 3-pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl and 3-methylpentyl. When a range of values ​​is given (e.g., C 1-6 alkyl), each value within that range and all intervening ranges are included. For example, "C 1-6 Alkyl" is C1, C2, C3, C4, C5, C6, C 1-6 , C 2-6 , C 3-6 , C 4-6 , C 5-6 , C 1-5 , C 2-5 , C 3-5 , C 4-5 , C 1-4 , C 2-4 , C 3-4 , C 1-3 , C 2-3 , and C 1-2 Examples of such alkyl groups include alkyl.

[0061] "Alkenyl" refers to an unsaturated branched or straight chain alkyl group having a specified number of carbon atoms (e.g., 2 to 8 or 2 to 6 carbon atoms) and at least one carbon-carbon double bond. The group may be in either the cis or trans configuration (Z or E configuration) about the double bond(s). Alkenyl groups include, but are not limited to, ethenyl, propenyl (e.g., prop-1-en-1-yl, prop-1-en-2-yl, prop-2-en-1-yl (allyl), prop-2-en-2-yl), and butenyl (e.g., but-1-en-1-yl, but-1-en-2-yl, 2-methyl-prop-1-en-1-yl, but-2-en-1-yl, but-2-en-1-yl, but-2-en-2-yl, buta-1,3-dien-1-yl, buta-1,3-dien-2-yl).

[0062] The term "substituted" means that the particular group or moiety bears one or more substituents, including, but not limited to, alkoxy, acyl, acyloxy, alkoxycarbonyl, carbonylalkoxy, acylamino, amino, aminoacyl, aminocarbonylamino, aminocarbonyloxy, cycloalkyl, cycloalkenyl, aryl, heteroaryl, aryloxy, cyano, azido, halo, hydroxyl, nitro, carboxyl, thiol, thioalkyl, alkyl, alkenyl, alkynyl, heterocyclyl, aralkyl, aminosulfonyl, sulfonylamino, sulfonyl, oxo, and the like. The term "unsubstituted" means that the specified group does not bear any substituents. When the term "substituted" is used to describe a structural system, it is meant that the substitution occurs at any valence-allowed position on the system. When a group or moiety bears two or more substituents, it is understood that the substituents may be the same or different from one another. In some embodiments, a substituent or moiety bears 1-5 substituents. In some embodiments, a substituent or moiety bears one substituent. In some embodiments, a substituent or moiety bears two substituents. In some embodiments, the substituent or moiety has 3 substituents. In some embodiments, the substituent or moiety has 4 substituents. In some embodiments, the substituent or moiety has 5 substituents.

[0063] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description is meant to include the case where the event or circumstance occurs and the case where it does not occur. For example, "optionally substituted alkyl" encompasses both "alkyl" and "substituted alkyl" as defined herein. With respect to any group containing one or more substituents, it will be understood by those skilled in the art that such groups are not intended to introduce any substitution or substitution pattern that is sterically impractical, synthetically unfeasible, and / or inherently unstable. When a group or moiety is optionally substituted, it will also be understood that the present disclosure includes both embodiments in which the group or moiety is substituted and embodiments in which the group or moiety is unsubstituted.

[0064] A "sterile" preparation is aseptic or free or essentially free of all viable microorganisms and their spores.

[0065] II. Polypeptide Formulations Provided herein is a formulation comprising a polypeptide and a surfactant, wherein the surfactant comprises one or more components of polysorbate. In some embodiments, the formulation is a liquid formulation. In some embodiments, the formulation is a lyophilized formulation. Surfactants

[0066] Biopharmaceutical formulations are commonly formulated with surfactants to protect the active pharmaceutical ingredient from interfacial stresses. Interactions with interfaces, especially air-water, have been shown to cause aggregation of therapeutic proteins during agitation or long-term storage. Polysorbates are commonly used surfactants to prevent these types of interactions for biopharmaceuticals. They are generally selected for their high surface activity, low critical micelle concentration (CMC) and low toxicity. However, these surfactants are heterogeneous mixtures of related compounds that, in combination, endow them with the unique properties required for biopharmaceutical formulations.

[0067] Polysorbates (PS) are a class of emulsifiers typically described as ethoxylated sorbitan esterified with fatty acids. PS are a mixture of related compounds with multiple layers of heterogeneity. The first layer is the tail length of the fatty acid esters. USP and EP monographs indicate that the distribution of these esters for PS20 is up to 1% caproic (C6) esters, 10% caprylic (C8) esters, 10% capric (C9) esters, 10% capric (C10) esters, 10% capric (C11) esters, 10% capric (C12) esters, 10% capric (C13) esters, 10% capric (C14) esters, 10% capric (C15) esters, 10% capric (C16) esters, 10% capric (C17) esters, 10% capric (C18) esters, 10% capric (C19) esters, 10% capric (C20) esters, 10% capric (C21) esters, 10% capric (C22) esters, 10% capric (C23) esters, 10% capric (C24) esters, 10% capric (C25) esters, 10% capric (C26) esters, 10% capric (C27) esters, 10% capric (C28) esters, 10% capric (C29) esters, 10% capric (C21) esters, 10% capric (C22) esters, 10% capric (C23) esters, 10% capric (C24) esters, 10% capric (C25) esters, 10% capric 10 ) ester, 7% stearic acid (C 18 ) ester, 11% monounsaturated C 18 Esters, and 3% diunsaturated C 18 Contains esters, 40-60% lauric acid (C 12 ) ester, 14-25% myristic acid (C 14 ) esters, and 7-15% palmitic acid (C 16 ) esters. Further heterogeneity comes from the length of the POE chain and the presence of di- and tri-esters. Furthermore, isosorbide is also formed during the synthesis of sorbitan from sorbitol, which can generate its own series of PS20-like compounds with two POE arms instead of four. See also Table 1 below. Various components have been found to have significantly different solution and interface properties. In particular, isosorbide POE fatty acid ester components have greater protective properties for polypeptides. Thus, using a higher concentration of isosorbide POE fatty acid ester (e.g., at least 70% (wt%)) than polysorbates (e.g., as described in EP or USP monographs) allows the use of smaller amounts of surfactant while providing greater protection to the polypeptide in the liquid formulation.

[0068] Provided herein is a biopharmaceutical formulation comprising a surfactant with certain components of polysorbate that are effective for protecting polypeptides in the formulation.In some embodiments, these components allow the use of less surfactant, but provide greater stability.In some embodiments, the less amount of surfactant used results in less formation of free fatty acid particles.In some embodiments, these components provide greater protection of polypeptides against free fatty acid particles.

[0069] In one embodiment, the surfactant comprises an isosorbide POE fatty acid ester. In another embodiment, the surfactant comprises an isosorbide POE fatty acid ester and a POE fatty acid ester.

[0070] In some embodiments, each isosorbide POE fatty acid ester and each POE fatty acid ester independently has about 5 to 10 POE units, about 10 to 15 POE units, about 15 to 20 POE units, about 20 to 25 POE units, about 25 to 30 POE units, or about 15 to 30 POE units. In some embodiments, each isosorbide POE fatty acid ester and each POE fatty acid ester independently has 5POE units, 6POE units, 7POE units, 8POE units, 9POE units, 10POE units, 11POE units, 12POE units, 13POE units, 14POE units, 15POE units, 16POE units, 17POE units, 18POE units, 19POE units, 20POE units, 21POE units, 22POE units, 23POE units, 24POE units, 25POE units, 26POE units, 27POE units, 28POE units, 29POE units, 30POE units, or a combination thereof. In some embodiments, the isosorbide POE fatty acid esters independently have about 5-10 POE units, about 10-15 POE units, about 15-20 POE units, about 20-25 POE units, about 25-30 POE units, about 15-30 POE units. In some embodiments, the isosorbide POE fatty acid esters independently have about 5 POE units, 6 POE units, 7 POE units, 8 POE units, 9 POE units, 10 POE units, 11 POE units, 12 POE units, 13 POE units, 14 POE units, 15 POE units, 16 POE units, 17 POE units, 18 POE units, 19 POE units, 20 POE units, 21 POE units, 22 POE units, 23 POE units, 24 POE units, 25 POE units, 26 POE units, 27 POE units, 28 POE units, 29 POE units, 30 POE units, or combinations thereof. In some embodiments, the isosorbide POE fatty acid ester has about 20 POE units. In some embodiments, both the isosorbide POE fatty acid ester and the POE fatty acid ester have about 5-30 POE units. In some embodiments, both the isosorbide POE fatty acid ester and the POE fatty acid ester have about 20 POE units.

[0071] In some embodiments, each isosorbide POE fatty acid ester and each POE fatty acid ester independently has a fatty acid chain independently selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl. In some embodiments, the alkyl is linear. In some embodiments, the fatty acid chain is an unsubstituted C 4-28 In some embodiments, the fatty acid chain is a C substituted with a substituent selected from the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl, or heterocyclyl. 4-28 In some embodiments, the fatty acid chain is an unsubstituted C alkyl. 4-28 In some embodiments, the alkenyl is linear or branched. In some embodiments, the compounds described herein have one or more double bonds. In some embodiments, each double bond has a cis configuration. In some embodiments, each double bond has a trans configuration. In some embodiments, the fatty acid chain is a C substituted with a substituent selected from the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl, or heterocyclyl. 4-28 It is alkenyl.

[0072] In some embodiments, each isosorbide POE fatty acid ester independently has 1, 2, 3, or 4 POE arms. In some embodiments, each isosorbide POE fatty acid ester has 2 POE arms.

[0073] In some embodiments, the isosorbide POE fatty acid ester has the following formula (I): TIFF2025023952000004.tif24170[in formula: a and b are independently an integer of 2 to 28, provided that the sum of a and b is an integer of 5 to 30; R1 and R 2 are independently hydrogen and -C(O)R" (wherein R" is an optionally substituted C 3-27 Alkyl or optionally substituted C 3-27 alkenyl); R 3 and R 4 are independently hydrogen. It has the structure:

[0074] In some embodiments of Formula (I), the sum of a and b is about 5-10, about 10-15, about 15-20, about 20-25, about 25-30, or about 15-30. In some aspects of Formula (I), the sum of a and b is 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments, the sum of a and b is 9. In some embodiments, the sum of a and b is 20. In some aspects of Formula (I), R 1 and R 2 At least one of R is not hydrogen. 1 and R 2 In some embodiments of formula (I), R″ is an optionally substituted C(O)R″. 3-27 In some embodiments of formula (I), R″ is an unsubstituted C 3-27 In some embodiments of formula (I), C is alkyl. 3-27 The alkyl is linear. In some embodiments of formula (I), C 3-27 In some embodiments of formula (I), R″ is an unsubstituted C5 alkyl, C7 alkyl, C9 alkyl, C 11 Alkyl, C 13 Alkyl, C 15 Alkyl, C 17 Alkyl, C 19 Alkyl, C 21 Alkyl or C 23 In some embodiments of formula (I), R″ is an unsubstituted linear C alkyl. 11In some embodiments of formula (I), R″ is an optionally substituted C 3-27 In some embodiments of formula (I), R″ is an unsubstituted C 3-27 In some embodiments of formula (I), C is alkenyl. 3-27 In some embodiments of formula (I), R″ is an unsubstituted C5 alkenyl, C7 alkenyl, C9 alkenyl, C 11 Alkenyl, C 13 Alkenyl, C 15 Alkenyl, C 17 Alkenyl, C 19 Alkenyl, C 21 Alkenyl, or C 23 alkenyl. In some aspects of Formula (I), R" has two or more double bonds. In some embodiments of Formula (I), two or more double bonds have a cis configuration. In some embodiments of Formula (I), two or more double bonds have a trans configuration. In some embodiments of Formula (I), R" has one double bond having a cis configuration. In some embodiments of Formula (I), R" has one double bond having a trans configuration. In some aspects of Formula (I), R" is -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)9CH=CH(CH2)5CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, and -(CH2) 11 In some embodiments of formula (I), R″ is selected from the group consisting of: In some aspects of formula (I), R″ is —(CH2)7CH═CH(CH2)7CH3, and both double bonds have a cis configuration. In some embodiments of formula (I), R″ is: -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and both double bonds have a trans configuration.

[0075] In some embodiments, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% (wt%) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester. In some embodiments, at least 70% (wt%) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester. In some embodiments, at least 80% (wt%) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester. In some embodiments, at least 90% (wt%) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester. In some embodiments, at least 95% (wt%) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester.

[0076] In some embodiments, the isosorbide POE fatty acid ester is selected from monoesters, diesters, triesters, tetraesters, and combinations thereof. In some embodiments, the isosorbide POE fatty acid ester is a monoester. In some embodiments, the isosorbide POE fatty acid monoester is selected from the group consisting of isosorbide POE monocaproate, isosorbide POE monocaprylate, isosorbide POE monocaprate, isosorbide POE monolaurate, isosorbide POE monomyristate, isosorbide POE monopalmitate, isosorbide POE monopalmitoleate, isosorbide POE monostearate, isosorbide POE monooleate, isosorbide POE monolinoleate, isosorbide POE monolinolenate, and combinations thereof. In some embodiments, the isosorbide POE fatty acid ester is a diester. In some embodiments, the isosorbide POE fatty acid diester is selected from the group consisting of isosorbide POE dicaproate, isosorbide POE dicaprylate, isosorbide POE dicaprate, isosorbide POE dilaurate, isosorbide POE dimyristate, isosorbide POE dipalmitate, isosorbide POE dipalmitoleate, isosorbide POE distearate, isosorbide POE dileate, isosorbide POE dilinoleate, isosorbide POE dilinolenate, and combinations thereof. In some embodiments, the isosorbide POE fatty acid ester is a compound of formula (I).

[0077] In some embodiments, the isosorbide POE fatty acid ester and the POE fatty acid ester have the same fatty acid chain. In some embodiments, the isosorbide POE fatty acid ester and the POE fatty acid ester have different fatty acid chains. In some embodiments, the POE fatty acid ester is selected from the group consisting of POE monocaproate, POE monocaprylate, POE monocaprate, POE monolaurate, POE monomyristate, POE monopalmitate, POE monopalmitoleate, POE monostearate, POE monooleate, POE monolinoleate, POE monolinolenate, POE dicaproate, POE dicaprylate, POE dicaprate, POE dilaurate, POE dimyristate, POE dipalmitate, POE dipalmitoleate, POE distearate, POE dioleate, POE dilinoleate, POE dilinolenate, and combinations thereof. In some embodiments, the surfactant comprises isosorbide POE monolaurate and POE monolaurate. In some embodiments, the surfactant comprises isosorbide POE monopalmitate and POE monopalmitate. In some embodiments, the surfactant comprises isosorbide POE monomyristate and POE monomyristate. In some embodiments, the surfactant comprises isosorbide POE monooleate and POE monooleate. In some embodiments, the surfactant comprises isosorbide POE monolinoleate and POE monolinoleate. In some embodiments, the surfactant comprises a greater amount of isosorbide POE fatty acid ester than POE fatty acid ester. In some embodiments, less than about 40%, less than about 35%, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 7.5%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, less than about 1%, less than about 0.75%, less than about 0.5%, or less than about 0.1% (wt %) of the surfactant is POE fatty acid ester.

[0078] In another aspect, the surfactant further comprises a sorbitan POE fatty acid ester. In some embodiments, each sorbitan POE fatty acid ester independently has about 5-10 POE units, about 10-15 units, about 15-20 POE units, about 20-25 POE units, about 25-30 POE units, or about 15-30 POE units. In some embodiments, each sorbitan POE fatty acid ester independently has 5POE units, 6POE units, 7POE units, 8POE units, 9POE units, 10POE units, 11POE units, 12POE units, 13POE units, 14POE units, 15POE units, 16POE units, 17POE units, 18POE units, 19POE units, 20POE units, 21POE units, 22POE units, 23POE units, 24POE units, 25POE units, 26POE units, 27POE units, 28POE units, 29POE units, 30POE units, or a combination thereof.

[0079] In some embodiments, each sorbitan POE fatty acid ester has a fatty acid chain independently selected from the group consisting of optionally substituted alkyl, optionally substituted alkenyl, and optionally substituted alkynyl. In some embodiments, the alkyl is linear. In some embodiments, the fatty acid chain is an unsubstituted C 4-28 In some embodiments, the fatty acid chain is a C substituted with a substituent selected from the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl, or heterocyclyl. 4-28 In some embodiments, the fatty acid chain is an unsubstituted C alkyl. 4-28In some embodiments, the alkenyl is linear or branched. In some embodiments, the compounds described herein have one or more double bonds. In some embodiments, each double bond has a cis configuration. In some embodiments, each double bond has a trans configuration. In some embodiments, the fatty acid chain is a C substituted with a substituent selected from the group consisting of acyl, hydroxyl, cycloalkyl, alkoxy, acyloxy, amino, aminoacyl, nitro, halo, thiol, thioalkyl, alkyl, alkenyl, alkynyl, or heterocyclyl. 4-28 It is alkenyl.

[0080] In some embodiments, each sorbitan POE fatty acid ester independently has 2, 3, or 4 POE arms. In some embodiments, each sorbitan POE fatty acid ester has 4 POE arms.

[0081] In some embodiments, the sorbitan POE fatty acid ester has the following formula (II): TIFF2025023952000005.tif25170[in formula: w, z, y, and x are independently an integer from 2 to 24, provided that the sum of w, z, y, and x is an integer from 15 to 30; R 5 , R 6 , R 7 and R 8 are independently selected from the group consisting of hydrogen and -C(O)R', where R' is an optionally substituted C 3-27 Alkyl or optionally substituted C 3-27 alkenyl; R 9 is hydrogen] It has the structure:

[0082] In some embodiments of Formula (II), the sum of w, z, y, and x is about 15 to 20, about 20 to 25, about 25 to 30, or about 15 to 30. In some embodiments of Formula (II), the sum of w, z, y, and x is 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30. In some embodiments of Formula (II), R 5 , R 6 , R 7 and R 8 At least one of R is not hydrogen. 5 , R 6 , R 7 and R 8 At least two of R are not hydrogen. 5 , R 6 , R 7 and R 8 is -C(O)R'. In some embodiments, R 5 , R 6 and R 7 is H and R 8 is -C(O)R'. In some embodiments, R 5 and R 6 is H and R 3 and R 4 is -C(O)R'. In some embodiments, R 6 is H and R 5 , R 7 and R 8 In some embodiments of formula (II), R' is -C(O)R'. In some embodiments of formula (II), R' is an optionally substituted C 3-27 In some embodiments of formula (II), R' is an unsubstituted C 3-27 In some embodiments of formula (II), C 3-27 The alkyl is linear. In some embodiments of formula (II), C 3-27 In some embodiments of formula (II), R' is an unsubstituted C5 alkyl, C7 alkyl, C9 alkyl, C 11 Alkyl, C 13 Alkyl, C 15 Alkyl, C 17Alkyl, C 19 Alkyl, C 21 Alkyl or C 23 In some embodiments of formula (II), R' is an unsubstituted linear C alkyl. 11 In some embodiments of formula (II), R' is an optionally substituted C alkyl. 3-27 In some embodiments of formula (II), R' is an unsubstituted C 3-27 In some embodiments of formula (II), C is alkenyl. 3-27 In some embodiments of formula (II), R' is an unsubstituted C5 alkenyl, C7 alkenyl, C9 alkenyl, C 11 Alkenyl, C 13 Alkenyl, C 15 Alkenyl, C 17 Alkenyl, C 19 Alkenyl, C 21 Alkenyl, or C 23 alkenyl. In some aspects of formula (II), R' has two or more double bonds. In some embodiments of formula (II), two or more double bonds have a cis configuration. In some embodiments of formula (II), two or more double bonds have a trans configuration. In some embodiments of formula (II), R' has one double bond having a cis configuration. In some embodiments of formula (II), R' has one double bond having a trans configuration. In some embodiments of formula (II), R' is -(CH2)7CH=CH(CH2)3CH3, -(CH2)7CH=CH(CH2)5CH3, -(CH2)4CH=CH(CH2)8CH3, -(CH2)7CH=CH(CH2)7CH3, -(CH2)9CH=CH(CH2)5CH3, -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, -(CH2)7CH=CHCH2CH=CHCH2CH=CHCH2CH3, -(CH2)3CH=CHCH2CH=CHCH2CH=CHCH2CH=CH(CH2)4CH3, and -(CH2) 11In some embodiments of formula (II), R' is selected from the group consisting of: In some aspects of formula (II), R' is -(CH2)7CH=CH(CH2)7CH3, and both double bonds have a cis configuration. In some embodiments of formula (II), R' is -(CH2)7CH=CHCH2CH=CH(CH2)4CH3, and both double bonds have a trans configuration.

[0083] In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (wt%) of the surfactant is a sorbitan POE fatty acid ester. In some embodiments, at least 1% (wt%) of the surfactant is a sorbitan POE fatty acid ester. In some embodiments, at least 5% (wt%) of the surfactant is a sorbitan POE fatty acid ester. In some embodiments, at least 10% (wt%) of the surfactant is a sorbitan POE fatty acid ester. In some embodiments, at least 15% (wt%) of the surfactant is a sorbitan POE fatty acid ester.

[0084] In some embodiments, each sorbitan POE fatty acid ester is independently a monoester, a diester, a triester, or a tetraester. In some embodiments, each sorbitan POE fatty acid ester is sorbitan POE monocaproate, sorbitan POE monocaprylate, sorbitan POE monocaprate, sorbitan POE monolaurate, sorbitan POE monomyristate, sorbitan POE monopalmitate, sorbitan POE monopalmitoleate, sorbitan POE monostearate, sorbitan POE monooleate, sorbitan POE monolinoleate, sorbitan POE monolinolenate, sorbitan POE dicaproate, sorbitan POE dicaprylate, sorbitan POE dicaprate, sorbitan POE dilaurate, sorbitan POE dimyristate ...laurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorbitan POE dilaurate, sorb In some embodiments, the sorbitan POE fatty acid ester is independently selected from the group consisting of sorbitan POE dipalmitate, sorbitan POE dipalmitoleate, sorbitan POE distearate, sorbitan POE dioleate, sorbitan POE dilinoleate, sorbitan POE dilinolenate, sorbitan POE tricaproate, sorbitan POE tricaprylate, sorbitan POE tricaprate, sorbitan POE trilaurate, sorbitan POE trimyristate, sorbitan POE tripalmitate, sorbitan POE tripalmitate, sorbitan POE tristearate, sorbitan POE trioelate, sorbitan POE trilinoleate, and sorbitan POE trilinolenate. In some embodiments, the sorbitan POE fatty acid ester is a compound of formula (II).

[0085] In another aspect, the surfactants disclosed herein have a critical micelle concentration (CMC) of greater than about 0.001%, about 0.002%, about 0.003%, about 0.004%, about 0.005%, about 0.006%, about 0.007%, about 0.008%, about 0.009%, 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%, or about 0.1% (w:v). In another embodiment, the surfactant has a surface tension of less than about 20 mN / m, about 25 mN / m, about 30 mN / m, about 35 mN / m, about 40 mN / m, about 45 mN / m, about 50 mN / m, about 55 mN / m, or about 60 mN / m. Polypeptides

[0086] The present disclosure relates to liquid formulations comprising a polypeptide and a surfactant. In some embodiments, the polypeptide in the liquid formulations described herein is essentially pure. In some embodiments, the polypeptide in the liquid formulations described herein is essentially homogenous (i.e., free of contaminating proteins). An "essentially pure" polypeptide refers to a composition comprising at least about 90% by weight of the polypeptide, based on the total weight of the composition. In some embodiments, the polypeptide is at least 95% by weight, based on the total weight of the composition. An "essentially homogenous" polypeptide refers to a composition comprising at least about 99% by weight of the polypeptide, based on the total weight of the composition.

[0087] In some embodiments, the polypeptide is an antibody. The antibodies herein are directed against an "antigen" of interest. In some embodiments, the antigen is a biologically important protein, and administration of the antibody to a mammal suffering from a disease or disorder may provide a therapeutic benefit to the mammal. In some embodiments, antibodies directed against non-protein antigens (such as tumor-associated glycolipid antigens; see, e.g., U.S. Pat. No. 5,091,178) are also contemplated. When the antigen is a protein, it may be a ligand, such as a transmembrane molecule (e.g., a receptor) or a growth factor. Exemplary antigens include any of the proteins described herein. In some embodiments, molecular targets of antibodies encompassed by the present disclosure include CD polypeptides such as CD3, CD4, CD8, CD19, CD20 and CD34; members of the HER receptor family, such as EGF receptor (HER1), HER2, HER3 or HER4 receptor; interleukins (IL), such as IL-1 to IL-10; cell adhesion molecules such as LFA-1, Macl, p150, 95, VLA-4, ICAM-1, VCAM and av / b3 integrin (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); growth factors, such as VEGF; IgE; blood group antigens; flk2 / flt3 receptor; obesity (OB) receptor; mpl receptor; CTLA-4; programmed cell death protein 1 (PD-1); programmed death ligand 1 (PD-L1); polypeptide C, etc. Soluble antigens or fragments thereof, optionally conjugated to other molecules, can be used as immunogens to generate antibodies. For transmembrane molecules such as receptors, fragments of these (e.g., the extracellular domain of a receptor) can be used as the immunogen. Alternatively, cells expressing the transmembrane molecule can be used as the immunogen. Such cells can be from natural sources (e.g., cancer cell lines) or can be cells transformed by recombinant techniques to express the transmembrane molecule.

[0088] In some embodiments, antibodies include, but are not limited to, polyclonal antibodies, monoclonal antibodies, humanized antibodies, human antibodies, bispecific antibodies, polyspecific antibodies, chimeric antibodies, and heteroconjugate antibodies. In some embodiments, antibodies include antibody fragments and whole antibodies. In some embodiments, the antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, and Fv fragments.

[0089] The polypeptide in the formulation can be prepared using methods known in the art, for example, by culturing cells transformed or transfected with a vector containing a nucleic acid encoding the polypeptide, or by synthetic techniques (e.g., recombinant techniques and peptide synthesis or a combination of these techniques), or can be isolated from an endogenous source of the polypeptide.

[0090] A. Protein Preparation Preparation of the proteins formulated by the disclosed methods by recombinant means can be accomplished by transfecting or transforming suitable host cells with an expression or cloning vector and culturing in conventional nutrient media modified appropriately for inducing promoters, selecting transformants, or amplifying genes encoding the desired sequences. Culture conditions, such as medium, temperature, and pH, can be selected by one of skill in the art without undue experimentation. In general, principles, protocols, and practical techniques for maximizing cell culture productivity can be found in Mammalian Cell Biotechnology: A Practical Approach, M. Butler, Ed. (IRL Press, 1991) and Sambrook et al., Molecular Cloning: A Laboratory Manual, New York: Cold Spring Harbor Press. Methods of transfection are known to those of skill in the art and include, for example, CaPO4 and CaCl2 transfection, electroporation, microinjection, and the like. Suitable techniques are also described in Sambrook et al., supra. Further transfection techniques are described in Shaw et al., Gene 23:315 (1983); WO 89 / 05859; Graham et al., Virology 52:456-457 (1978), and U.S. Pat. No. 4,399,216.

[0091] Nucleic acids encoding the desired proteins for formulation by the present methods can be inserted into replicable vectors for cloning or expression. Suitable vectors are publicly available and can take the form of a plasmid, cosmid, viral particle or phage. Appropriate nucleic acid sequences can be inserted into vectors by a variety of procedures. Generally, DNA is inserted into an appropriate restriction endonuclease site(s) using techniques known in the art. Vector components generally include, but are not limited to, one or more of a signal sequence, an origin of replication, one or more marker genes and enhancer elements, a promoter, and a transcription termination sequence. Construction of suitable vectors containing one or more of these components employs standard ligation techniques known to those skilled in the art.

[0092] The formulated form of the protein can be recovered from culture medium or host cell lysate. If membrane-bound, it can be released from the membrane using a suitable detergent or by enzymatic cleavage. Cells used for expression can also be disrupted by various physical or chemical means, such as freeze-thaw cycling, sonication, mechanical disruption, or cell lysing agents.

[0093] Purification of the formulated protein may be carried out by any suitable technique known in the art, such as fractionation on ion exchange columns, ethanol precipitation, reverse phase HPLC, chromatography on silica or cation exchange resins (e.g., DEAE), chromatofocusing, SDS-PAGE, ammonium sulfate precipitation, gel filtration using Protein A Sepharose columns (e.g., Sephadex® G-75) to remove contaminants such as IgG, and metal chelate columns to bind epitope-tagged forms.

[0094] B. Antibody preparation In certain embodiments of the invention, the protein of choice is an antibody. Techniques for the production of antibodies, including polyclonal, monoclonal, humanized, bispecific and heteroconjugate antibodies, are provided below.

[0095] 1. Polyclonal antibodies Polyclonal antibodies are generally raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. It may be useful to conjugate the relevant antigen to a protein that is immunogenic in the species being immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor. Examples of adjuvants that can be used include Freund's complete adjuvant, and MPL-TDM adjuvant (monophosphoryl lipid A, synthetic trehalose dicorynomycolate). The immunization protocol can be selected by one skilled in the art without undue experimentation.

[0096] One month later, the animals are boosted with 1 / 5 to 1 / 10 the original amount of peptide or conjugate in Freund's complete adjuvant by subcutaneous injection at multiple sites. 7 to 14 days later, the animals are bled and the serum is assayed for antibody titer. Animals are boosted until the titer plateaus. In some embodiments, animals are boosted with conjugates of the same antigen, but conjugated to a different protein and / or through a different cross-linking reagent. Conjugates can also be made in recombinant cell culture as protein fusions. Aggregating agents such as alum are also suitably used to enhance the immune response.

[0097] 2. Monoclonal antibodies. A monoclonal antibody is obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical except for possible naturally occurring mutations that may be present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of discrete antibodies.

[0098] For example, the monoclonal antibodies may be made using the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or may be made by recombinant DNA methods (U.S. Pat. No. 4,816,567).

[0099] In the hybridoma method, a mouse, or other suitable host animal, such as a hamster, is immunized as described herein above to elicit lymphocytes that produce, or are capable of producing, antibodies that will specifically bind to the protein used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused to myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0100] The immunizing agent typically includes the formulated protein. Generally, peripheral blood lymphocytes ("PBLs") are used if cells of human origin are desired, or spleen cells or lymph node cells are used if non-human mammalian sources are desired. The lymphocytes are then fused with an immortalized cell line using a suitable fusing agent, such as polyethylene glycol, to form a hybridoma cell. Goding, Monoclonal antibodies: Principles and Practice, Academic Press (1986), pp. 59-103.

[0101] Immortalized cell lines are usually transformed mammalian cells, especially myeloma cells of rodent, bovine and human origin. Usually, rat or mouse myeloma cell lines are used. Thus, hybridoma cells are seeded and cultured in a suitable medium, optionally containing one or more substances that inhibit the growth or survival of unfused parental myeloma cells. For example, if parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the medium for hybridoma typically contains hypoxanthine, aminopterin and thymidine (HAT medium), which prevents the growth of HGPRT-deficient cells.

[0102] Suitable immortalized myeloma cells are those that fuse efficiently, support stable high-level production of antibodies by selected antibody-producing cells, and are sensitive to media such as HAT medium. In some embodiments, the myeloma cell line is a mouse myeloma line, such as those derived from MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, Calif., USA, and SP-2 cells (and derivatives thereof, such as X63-Ag8-653) available from the American Type Culture Collection, Manassas, Va., 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.51-63 (Marcel Dekker, Inc., New York, 1987)).

[0103] The culture medium in which the hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of the monoclonal antibodies produced by the hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0104] The culture medium in which the hybridoma cells are cultured can again be assayed for the presence of monoclonal antibodies against the desired antigen. In some embodiments, the binding affinity and binding specificity of the monoclonal antibody can be determined by immunoprecipitation or by in vitro binding assays, such as radioimmunoassay (RIA) or enzyme-linked assay (ELISA). Such techniques and assays are known in the art. The binding affinity of the monoclonal antibody can be determined, for example, by the Scatchard analysis of Munson et al., Anal. Biochem., 107:220 (1980).

[0105] After hybridoma cells that produce 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 (Goding, supra). Suitable media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.

[0106] The monoclonal antibodies secreted by the subclones are suitably separated from the culture medium, ascites fluid, or serum by conventional immunoglobulin purification procedures such as, for example, protein A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0107] Monoclonal antibodies can also be produced by recombinant DNA methods, such as those described in U.S. Pat. No. 4,816,567 and described above. DNA encoding the monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of mouse antibodies). In some embodiments, hybridoma cells serve as a source of such DNA. Once isolated, the DNA may be placed into an expression vector, which is then transfected into host cells, such as E. coli cells, monkey COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells, that do not produce immunoglobulin proteins, to synthesize the monoclonal antibodies in such recombinant host cells. Review articles on recombinant expression in bacteria of DNA encoding the antibody include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Pliickthun, Immunol. Revs. 130:151-188 (1992).

[0108] In further embodiments, antibodies can be isolated from antibody phage libraries generated using techniques described in McCafferty et al., Nature, 348:552-554 (1990). Clackson et al., Nature, 352:624-628 (1991) and Marks et al., J. Mal. Biol., 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications describe the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / I'echnology, 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing very large phage libraries (Waterhouse et al., Nucl. Acids Res., 21:2265-2266 (1993)). Thus, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0109] The DNA can also be modified, for example, by substituting the coding sequence for human heavy and light chain constant domains for the homologous murine sequences (U.S. Pat. No. 4,816,567; Morrison, et al., Proc. Natl Acad. Sci. USA, 81:6851 (1984)), or by covalently linking all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence. Typically, such non-immunoglobulin polypeptides are substituted for the constant domains of an antibody, or they are substituted for the variable domains of one antigen-binding site of an antibody to create a chimeric bivalent antibody containing one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0110] The monoclonal antibodies described herein may be monovalent, and their preparation is well known in the art. For example, one method involves recombinant expression of an immunoglobulin light chain and a modified heavy chain. The heavy chain is generally truncated at any point in the Fc region to prevent heavy chain cross-linking. Alternatively, the relevant cysteine ​​residue may be replaced with another amino acid residue or deleted to prevent cross-linking. In vitro methods are also suitable for preparing monovalent antibodies. Digestion of antibodies to produce fragments thereof, particularly Fab fragments, can be accomplished using routine techniques known in the art.

[0111] Chimeric or hybrid antibodies can also be prepared in vitro using known methods in synthetic protein chemistry involving crosslinking agents. For example, immunotoxins can be constructed using a disulfide exchange reaction or by forming a thioether bond. Examples of suitable reagents for this purpose include iminothiolate and methyl-4-mercaptobutyrimidate.

[0112] 3. Humanized antibodies. The antibodies of the invention may further include humanized or human antibodies. Humanized forms of non-human (e.g., murine) antibodies are chimeric immunoglobulins, immunoglobulin chains, or fragments thereof (such as Fv, Fab, Fab', F(ab')2, or other antigen-binding subsequences of antibodies) that contain minimal sequence derived from the non-human immunoglobulin. Humanized antibodies include human immunoglobulins (recipient antibody) in which residues from the complementarity determining regions (CDRs) of the recipient are replaced by residues from the CDRs of a non-human species (donor antibody) such as mouse, rat, or rabbit having the desired specificity, affinity, and capacity. In some instances, Fv framework residues of the human immunoglobulin are replaced by corresponding non-human residues. Humanized antibodies may also include residues that are found neither in the recipient antibody nor in the imported CDR or framework sequences. Generally, a humanized antibody will comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the CDR regions correspond to those of a non-human immunoglobulin and all or substantially all of the FR regions are of a human immunoglobulin consensus sequence. The humanized antibody will also most advantageously comprise at least a portion of an immunoglobulin constant region (Fc), typically that of a human immunoglobulin. Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-329 (1988) and Presta, Curr. Opin. Struct. Biol. 2:593-596 (1992).

[0113] Methods for humanizing non-human antibodies are well known in the art. In general, a humanized antibody has one or more amino acid residues introduced into it from a source that is non-human. Such non-human amino acid residues are commonly referred to as "import" residues, typically coming from an "import" variable domain. Humanization is essentially performed by substituting rodent CDRs or CDR sequences for the corresponding sequences of a human antibody according to the method of Winter and colleagues (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)). Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact human variable domain has been substituted with the corresponding sequences of 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.

[0114] The choice of human variable domains (both heavy and light chains) used to generate humanized antibodies is very important to reduce antigenicity. According to the so-called "best-fit" method, the sequence of the variable domain of a rodent antibody is screened against the entire library of known human variable domain sequences. The human sequence that is closest to the rodent sequence is then accepted as the human framework for the humanized antibody. Sims et al., J. Immunol., 151:2296 (1993); Chothia et al., J. Mal. Biol., 196:901 (1987). Another method uses a particular framework derived from the consensus sequence of all human antibodies of a particular 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).

[0115] It is further important that antibodies be humanized with retention of high affinity for the antigen and other favorable biological properties. To achieve this goal, in some embodiments, humanized antibodies are prepared through a process of analysis of the parental sequences and various conceptual humanized products using three-dimensional models of the parental and humanized sequences. Three-dimensional immunoglobulin models are publicly available and are familiar to those skilled in the art. Computer programs are available which illustrate and display predicted three-dimensional conformational structures of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the likely role of the residues in the functioning of the candidate immunoglobulin sequence, i.e., analysis of residues that influence the ability of the candidate immunoglobulin to bind to its antigen. In this way, FR residues can be selected and combined from the recipient and import sequences to result in the desired antibody characteristic, such as improved affinity for the target antigen(s). In general, the CDR residues are directly and most substantially involved in influencing antigen binding.

[0116] Various forms of humanized antibodies are contemplated. For example, the humanized antibody may be an antibody fragment, such as a Fab, which is optionally conjugated with one or more cytotoxic agent(s) to generate an immunoconjugate. Alternatively, the humanized antibody may be an intact antibody, such as an intact IgG1 antibody.

[0117] 4. Human antibodies As an alternative to humanization, human antibodies can be produced. For example, it is now possible to produce transgenic animals (e.g., mice) that are capable, upon immunization, of producing a full repertoire of human antibodies in the absence of endogenous immunoglobulin production. For example, the heavy chain joining region (J) of the antibody can be expressed in chimeric and germ-line mutant mice. HIt has been described that homozygous deletion of the .) gene completely inhibits endogenous antibody production. Introduction of the human germ-line immunoglobulin gene array in such germ-line mutant mice results 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., Year in Immuno., 1:33 (1993); U.S. Patent No. 5,591,669 and WO 97 / 17852.

[0118] Alternatively, phage display technology can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from non-immunized donors. McCafferty et al., Nature 348:552-553 (1990); Hoogenboom and Winter, J. Mal. Biol. 227:381 (1991). According to this technology, antibody V domain genes are cloned in frame into either a major or minor coat protein gene of a filamentous bacteriophage, such as M13 or fd, and displayed as functional antibody fragments on the surface of the phage particle. Because the filamentous particle contains a single-stranded DNA copy of the phage genome, selections based on the functional properties of the antibody also lead to selection of the gene encoding the antibody exhibiting those properties. In this way, the phage mimics some of the properties of the B cell. Phage display can be carried out in a variety of formats, as reviewed, for example, in Johnson, Kevin S. and Chiswell, David J., Curr. Opin Struct. Biol. 3:564-571 (1993). Multiple sources of V gene segments can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a diverse array of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleens of immunized mice. Repertoires of V genes from unimmunized human donors can be constructed, and antibodies against a diverse array of antigens, including self-antigens, can be isolated essentially according to the techniques described by Marks et al., J. Mol. Biol. 222:581-597 (1991), or Griffith et al., EMBO J. 12:725-734 (1993). See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0119] The techniques of Cole et al. and Boerner et al. can also be used to prepare human monoclonal antibodies (Cole et al., Monoclonal Antibodies and Cancer Therapy, Alan R. Liss, p. 77 (1985) and Boerner et al., J. Immunol. 147(1):86-95 (1991)). Similarly, human antibodies can be made by introducing human immunoglobulin loci into transgenic animals, e.g., mice in which the endogenous immunoglobulin genes have been partially or completely inactivated. Upon antigenic challenge, human antibody production is observed, which closely resembles that seen in humans in all respects, including gene rearrangement, assembly, and antibody repertoire. This approach is described, for example, in U.S. Pat. Nos. 5,545,807, 5,545,806, 5,569,825, 5,625,126, 5,633,425, and 5,661,016, as well as in the following scientific publications: Marks et al., Bio / I'echnology 10:779-783 (1992); Lenberg et al., Nature 368:856-859 (1994); Morrison, Nature 368:812-13 (1994), Fishwild et al., Nature Biotechnology 14:845-51 (1996), Neuberger, Nature Biotechnology 14:826 (1996), and Lenberg and Huszar, Intern. Rev. Immunol. 13:65-93 (1995).

[0120] Finally, human antibodies can also be generated in vitro by activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0121] 5. Antibody fragment In certain circumstances, there are advantages to using antibody fragments rather than whole antibodies: the smaller fragment size allows for rapid clearance and may result in improved access to solid tumors.

[0122] Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were derived via proteolytic digestion of intact antibodies (e.g., Morimoto et al., J Biochem Biophys. Method. 24:107-117 (1992); and Brennan et al., Science 229:81 (1985)). However, such 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, allowing the facile production of large amounts of these fragments. Antibody fragments can be isolated from the antibody phage libraries described above. Alternatively, Fab'-SH fragments can be directly recovered from E. coli and chemically coupled to form F(ab')2 fragments (Carter et al., Bio / Technology 10:163-167 (1992)). According to another approach, F(ab')2 fragments can be directly isolated from recombinant host cell culture. Fab and F(ab')2 fragments with increased in vivo half-lives are described in U.S. Pat. No. 5,869,046. In other embodiments, the antibody of choice is a single chain Fv fragment (scFv). See WO 93 / 16185, U.S. Pat. No. 5,571,894, and U.S. Pat. No. 5,587,458. The antibody fragment may also be a "linear antibody", e.g., as described in U.S. Pat. No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0123] 6. Bispecific and multispecific antibodies. Bispecific antibodies (BsAbs) are antibodies that have binding specificities for at least two different epitopes, including on the same or different proteins. Alternatively, one arm can be equipped to bind to a target antigen and another arm can be combined with an arm that binds to a trigger molecule on a white blood cell, such as a T cell receptor molecule (e.g., CD3) or an Fc receptor for IgG (FcγR), such as FcγRl (CD64), FcγRII (CD32), and FcγRIII (CD16), in order to focus and localize cellular defense mechanisms to the target antigen-expressing cells. Such antibodies can be derived from full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0124] Bispecific antibodies can also be used to localize cytotoxic agents to cells expressing the target antigen. Such antibodies have one arm that binds to the desired antigen and another arm that binds to the cytotoxic agent (e.g., saporin, anti-interferon-a, vinca alkoloid, ricin A chain, methotrexate, or radioisotope hapten). Examples of known bispecific antibodies include anti-ErbB2 / anti-FcgRIII (WO 96 / 16673), anti-ErbB2 / anti-FcgRI (U.S. Pat. No. 5,837,234), and anti-ErbB2 / anti-CD3 (U.S. Pat. No. 5,821,337).

[0125] Methods for making bispecific antibodies are known in the art. 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). Because the immunoglobulin heavy and light chains are randomly assorted, these hybridomas (quadromas) can produce a mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. Purification of the correct molecule is usually done by affinity chromatography steps, which are quite laborious and give low product yields. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0126] According to a different approach, antibody variable domains (antibody-antigen binding sites) with the desired binding specificity are fused to immunoglobulin constant domain sequences. In some embodiments, the fusion is between an immunoglobulin heavy chain constant domain, including at least a portion of the hinge region, CH2 region, and CH3 region. In some embodiments, the first heavy chain constant region (CH1), which contains the site required for light chain binding, is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, optionally, the immunoglobulin light chain are inserted into separate expression vectors and co-transfected into a suitable host organism. This allows for great flexibility in adjusting the mutual proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields. However, in cases where expression of at least two polypeptide chains in equal ratios provides high yields, or where the ratios are not critical, it is possible to insert the coding sequences for two or all three polypeptide chains into one expression vector.

[0127] In some embodiments, bispecific antibodies are composed 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 has been found that such an asymmetric structure facilitates separation of the desired bispecific compound from unwanted immunoglobulin chain combinations, since the presence of an immunoglobulin light chain in only one half of the bispecific molecule facilitates separation. This approach is disclosed in WO 94 / 04690. For further details on bispecific antibody generation, see, e.g., Suresh et al., Methods in Enzymology 121:210 (1986).

[0128] According to another approach described in WO 96 / 27011 or US Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. In some embodiments, the interface comprises at least a portion of the CH3 region of the antibody constant domain. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with a larger side chain (e.g., tyrosine or tryptophan). By replacing the large amino acid side chain with a smaller one (e.g., alanine or threonine), a compensatory "cavity" of the same or similar size as the large side chain(s) is created at the interface of the second antibody molecule. This provides a mechanism to increase the yield of heterodimers over other unwanted end-products such as homodimers.

[0129] Techniques for generating bispecific antibodies from antibody fragments have been described in the literature. For example, bispecific antibodies can be prepared using chemical linkage. Brennan et al., Science 229:81 (1985) describe a procedure in which intact antibodies are proteolytically cleaved to generate F(ab')2 fragments. These fragments are reduced in the presence of the dithiol complexing agent sodium arsenite to stabilize vicinal dithiols and prevent intermolecular disulfide formation. The generated Fab' fragments are then converted to thionitrobenzoate (TNB) derivatives. One of the Fab'-TNB derivatives is then reconverted to a Fab'-TNB derivative to form the bispecific antibody. The generated bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0130] Fab' fragments may be directly recovered from E. coli and chemically coupled to form bispecific antibodies. Shalaby et al., J. Exp. Med. 175:217-225 (1992) described the production of fully humanized bispecific antibody F(ab')2 molecules. Each Fab' fragment was secreted separately from E. coli and subjected to directed chemical coupling in vitro to form the bispecific antibody. The bispecific antibody thus formed was capable of binding to cells overexpressing the ErbB2 receptor and normal human T cells, and was also capable of inducing the lytic activity of human cytotoxic lymphocytes against human breast tumor targets.

[0131] Various techniques have also been described for producing and isolating bivalent antibody fragments directly from recombinant cell culture. For example, bivalent heterodimers have been produced using leucine zippers. Kostelny et al., J. Immunol., 148(5):1547-1553 (1992). The leucine zipper peptides from the Fos and Jun proteins were linked to the Fab' portions of two different antibodies by gene fusion. The antibody homodimers were reduced at the hinge region to form monomers and then reoxidized to form the antibody heterodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993) provided an alternative mechanism for making bispecific / bivalent antibody fragments. The fragments consist of a light chain variable domain (V) connected to a light chain variable domain (V) by a linker that is too short to allow pairing between the two domains on the same chain. L ) connected to a heavy chain variable domain (V H ) in a given fragment. H and V L The domain is a complementary V L and V H The Fv domains are forced to pair together to form two antigen-binding sites. Another strategy for making bispecific / bivalent antibody fragments by the use of single-chain Fv (scFv) dimers has also been reported. See Gruber et al., J. Immunol., 152:5368 (1994).

[0132] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0133] Exemplary bispecific antibodies may bind to two different epitopes on a given molecule. Alternatively, an anti-protein arm may be combined with an arm that binds to a trigger molecule on a white blood cell, such as a T cell receptor molecule (e.g., CD2, CD3, CD28, or B7), or an Fc receptor (FcγR) for IgG, such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to focus cellular defense mechanisms on cells expressing a particular protein. Bispecific antibodies may also be used to localize cytotoxic agents to cells expressing a particular protein. Such antibodies have a protein-binding arm and an arm that binds a cytotoxic agent or a radionuclide chelator, such as EOTUBE, DPTA, DOTA, or TETA. Another bispecific antibody of interest binds to a protein of interest and also binds tissue factor (TF).

[0134] formulation Provided herein is a liquid formulation comprising a polypeptide and a surfactant. Any surfactant described herein can be used in the liquid formulation. In some embodiments, the surfactant in the liquid formulation is about 0.0005%-0.2%, about 0.0005%-0.001%, about 0.001%-0.002%, about 0.002%-0.003%, about 0.003%-0.004%, about 0.004%-0.005%, about 0.005%-0.006%, about 0.006%-0.007%, about 0.007%-0.008%, about 0.008%-0.009%, about 0.009%-0.01%, about 0.01% or about 0.02% of the liquid formulation. %~0.015%, approx. 0.015%~0.02%, approx. 0.02%~0.025%, approx. 0.025%~0.03%, approx. 0.03%~0.035%, approx. 0.035%~0.04%, approx. 0.04%~0.045%, approx. 0.045%~0.05%, approx. 0.05%~0.055%, approx. 0.055%~0.06%, approx. 0.06%~0.065%, approx. 0.065%~0.07%, approx. 0.07%~0.075%, approx. 0.075%~0.08%, approx. 0.08%~0 .085%, approx. 0.085%-0.09%, approx. 0.09%-0.095%, approx. 0.095%-0.1%, approx. 0.1%-0.11%, approx. 0.11%-0.12%, approx. 0.12%-0.13%, approx. 0.13%-0.14%, approx. 0.14%-0.15%, approx. 0.15%-0.16%, approx. 0.16%-0.17%, approx. 0.17%-0.18%, approx. 0.18%-0.19%, approx. 0.19%-0.2%, approx. 0.0005%-0.01%, approx. 0.01%-0. 0.02%, about 0.02% to 0.03%, about 0.03% to 0.04%, about 0.04% to 0.05%, about 0.05% to 0.06%, about 0.06% to 0.07%, about 0.07% to 0.08%, about 0.08% to 0.09%, about 0.09% to 0.1%, about 0.1% to 0.12%, about 0.12% to 0.14%, about 0.14% to 0.16%, about 0.16% to 0.18%, about 0.18% to 0.2%, or about 0.0005% to 0.02% (w:v).

[0135] In some embodiments, the isosorbide POE fatty acid ester in the liquid formulation is about 0.00035%-0.2%, about 0.00035%-0.0005%, about 0.0005%-0.001%, about 0.001%-0.002%, about 0.002%-0.003%, about 0.003%-0.004%, about 0.004%-0.005%, about 0.005%-0.006%, about 0.006%-0.007%, about 0.007%-0.008%, about 0.008%-0.00 9%, approx. 0.009%~0.01%, approx. 0.01%~0.015%, approx. 0.015%~0.02%, approx. 0.02%~0.025%, approx. 0.025%~0.03%, approx. 0.03%~0.035%, approx. 0.035%~0.04%, approx. 0.04%~0.045%, approx. 0.045%~0.05%, approx. 0.05%~0.055%, approx. 0.055%~0.06%, approx. 0.06%~0.065%, approx. 0.065%~0.07%, approx. 0.07%~0.075%, approx. 0.075%~ 0.08%, approx. 0.08%~0.085%, approx. 0.085%~0.09%, approx. 0.09%~0.095%, approx. 0.095%~0.1%, approx. 0.1%~0.11%, approx. 0.11%~0.12%, approx. 0.12%~0.13%, approx. 0.13%~0.14%, approx. 0.14%~0.15%, approx. 0.15%~0.16%, approx. 0.16%~0.17%, approx. 0.17%~0.18%, approx. 0.18%~0.19%, approx. 0.19%~0.2%, approx. 0.00035%~0.01%, approx. 0 having a concentration of about 0.01% to 0.02%, about 0.02% to 0.03%, about 0.03% to 0.04%, about 0.04% to 0.05%, about 0.05% to 0.06%, about 0.06% to 0.07%, about 0.07% to 0.08%, about 0.08% to 0.09%, about 0.09% to 0.1%, about 0.1% to 0.12%, about 0.12% to 0.14%, about 0.14% to 0.16%, about 0.16% to 0.18%, about 0.18% to 0.2%, or about 0.00035% to 0.14% (w:v).

[0136] The concentration of the polypeptide in a liquid formulation can vary based on the storage configuration and the desired route of administration (eg, subcutaneous, intramuscular or intravitreal administration, intravenous injection or infusion, etc.). In some embodiments, the polypeptide in the liquid formulation is from about 0.1 mg / mL to 300 mg / mL, from about 0.1 mg / mL to 0.5 mg / mL, from about 0.5 mg / mL to 1 mg / mL, from about 1 mg / mL to 1.5 mg / mL, from about 1.5 mg / mL to 2 mg / mL, from about 2 mg / mL to 2.5 mg / mL, from about 2.5 mg / mL to 3 mg / mL, from about 3 mg / mL to 3.5 mg / mL, from about 3.5 mg / mL to 4 mg / mL, from about 4 mg / mL to 4.5 mg / mL, from about 4.5 mg / mL to 5 mg / mL, from about 0.1 mg / mL to 1 mg / mL, from about 1 mg / mL to 2 mg / mL, from about 2 mg / mL to 3 mg / mL, from about 3 mg / mL to 4 mg / mL, from about 4 mg / mL to 5 mg / mL , about 5 mg / mL to 10 mg / mL, about 10 mg / mL to 15 mg / mL, about 15 mg / mL to 20 mg / mL, about 20 mg / mL to 30 mg / mL, about 30 mg / mL to 40 mg / mL, about 40 mg / mL to 50 mg / mL, about 50 mg / mL to 100 mg / mL, about 100 mg / mL to 150 mg / mL, about 150 mg / mL to 200 mg / mL, about 200 mg / mL to 250 mg / mL, about 250 mg / mL to 300 mg / mL, about 0.1 mg / mL to 2 mg / mL, about 0.5 mg / mL to 2 mg / mL, about 50 mg / mL to 150 mg / mL, about 150 mg / mL to 200 mg / mL, or 200 mg / mL to 300 mg / mL. In some embodiments, the concentration of the polypeptide in the liquid formulation is about 0.5 mg / mL. In some embodiments, the polypeptide in the liquid formulation has a concentration of greater than about 50 mg / mL, greater than about 150 mg / mL, greater than about 200 mg / mL, greater than about 250 mg / mL, or greater than about 300 mg / mL.In some embodiments, the liquid formulation may be diluted to reduce the concentration of the polypeptide by about 1-5 times, about 5-10 times, about 10-15 times, about 15-20 times, about 20-30 times, about 30-40 times, about 40-50 times, about 50-100 times, about 100-150 times, about 150-200 times, about 200-300 times, about 300-400 times, about 400-500 times, about 500-600 times, about 600-700 times, about 700-800 times, about 800-900 times, about 900-1000 times, about 1000-1500 times, about 1500-2000 times, about 2000-2500 times, about 2500-3000 times, or about 3000-5000 times.

[0137] In some embodiments, the liquid formulation is diluted with an infusion fluid. In some embodiments, the infusion fluid includes, but is not limited to, a dextrose-containing solution, lactated Ringer's solution, normal saline, semi-normal saline, or buffered saline. In some embodiments, the saline is saline (about 0.9% (w:v)). In some embodiments, the saline is an isotonic saline. In some embodiments, the saline is a buffered saline, including, but not limited to, phosphate buffered saline or Krebs-Ringer's solution. In some embodiments, the saline is isotonic or nearly isotonic with blood from the subject. The saline includes a salt, such as sodium chloride, potassium chloride, magnesium chloride, or calcium chloride. In some embodiments, the saline includes one or more buffers, such as a phosphate buffer (such as sodium phosphate or potassium phosphate), sodium carbonate, or HEPES. When using a buffered saline, the pH is maintained in a range that optimizes the therapeutic effectiveness of the polypeptide, especially if its stability is pH-dependent.

[0138] A. Lyophilized formulations In some embodiments, the liquid formulations described herein may be prepared as reconstituted lyophilized formulations. The polypeptides described herein may be lyophilized and then reconstituted to produce liquid formulations. In some embodiments, after preparation of the protein of interest as described above, a "pre-lyophilized formulation" is made. In some embodiments, the polypeptide concentration in the reconstituted formulation is higher than in the pre-lyophilized formulation. In some embodiments, the polypeptide concentration in the reconstituted formulation is lower than in the pre-lyophilized formulation. The concentration of the polypeptide present in the pre-lyophilized and reconstituted liquid formulations is determined by considering the desired dose volume, mode of administration, etc.

[0139] 1. Preparation of lyophilized formulations. When preparing a lyophilized formulation, the protein to be formulated is generally present in a solution. For example, in the high ionic strength, low viscosity formulations of the present invention, the protein may be present in a pH buffer at a pH of about 4 to 8, preferably about 5 to 7. The concentration of the buffer may be, for example, about 1 mM to about 20 mM, alternatively about 3 mM to about 15 mM, depending on the buffer and the desired tonicity of the formulation (e.g., of the reconstituted formulation). Exemplary buffers and / or salts are those that are pharma- ceutically acceptable and may be made from suitable acids, bases and their salts, such as those defined under "pharmaceutically acceptable" acids, bases or buffers.

[0140] In some embodiments, a cryoprotectant is added to the pre-lyophilized formulation. The amount of lyoprotectant in the pre-lyophilized formulation is generally such that upon reconstitution the resulting formulation is isotonic. However, hypertonic reconstituted formulations may also be suitable. Furthermore, the amount of lyoprotectant should not be so low that an unacceptable amount of protein degradation / aggregation occurs upon lyophilization. However, exemplary lyoprotectant concentrations in the pre-lyophilized formulation are from about 10 mM to about 400 mM, alternatively from about 30 mM to about 300 mM, alternatively from about 50 mM to about 100 mM. Exemplary lyoprotectants include sugars and sugar alcohols, such as sucrose, mannose, trehalose, glucose, sorbitol, and mannitol. However, under certain circumstances, certain lyoprotectants may also contribute to an increase in the viscosity of the formulation. Thus, care should be taken to select a particular lyoprotectant that minimizes or neutralizes this effect. Additional lyoprotectants are described above under the definition of "lyoprotectant", also referred to herein as "pharmacologically acceptable sugars".

[0141] The ratio of protein to lyoprotectant may vary for each particular protein or antibody-lyoprotectant combination. In the case of an antibody as the selected protein and a sugar (e.g., sucrose or trehalose) as the lyoprotectant to generate an isotonic reconstituted formulation with high protein concentration, the molar ratio of lyoprotectant to antibody may be about 100 to about 1500 moles of lyoprotectant per mole of antibody, or about 200 to about 1000 moles of lyoprotectant per mole of antibody, for example, about 200 to about 600 moles of lyoprotectant per mole of antibody.

[0142] The formulations herein may optionally contain more than one protein for the particular indication being treated, e.g., those with complementary activities that do not adversely affect other proteins. For example, it may be desirable to provide two or more antibodies that bind to a desired target (e.g., a receptor or antigen) in a single formulation. Such proteins are suitably present in combination in amounts effective for the intended purpose.

[0143] Formulations to be used for in vivo administration must be sterile. This is readily accomplished by filtration through sterile filtration membranes, prior to or after lyophilization and reconstitution. Alternatively, sterility of the entire mixture can be achieved, for example, by autoclaving the components except protein at about 120° C. for about 30 minutes.

[0144] After the protein, optional cryoprotectant and other optional ingredients are mixed together, the formulation is lyophilized. Many different lyophilizers are available for this purpose, such as the Hull50™ (Hull, USA) or GT20™ (Leybold-Heraeus, Germany) lyophilizers. Lyophilization is achieved by freezing the formulation and then sublimating the ice from the freeze at a temperature suitable for primary drying. Under these conditions, the temperature of the product is below the eutectic point or the collapse temperature of the formulation. Typically, storage temperatures for primary drying range from about -30 to 25°C at suitable pressures, typically in the range of about 50 to 250 mTorr (provided that the product remains frozen during primary drying). The formulation, size and type of container (e.g., glass vial) holding the sample, as well as the volume of liquid, largely determine the time required for drying, which can range from a few hours to several days (e.g., 40 to 60 hours). Optionally, a secondary drying step can also be performed, depending on the desired residual moisture level in the product. The temperature at which the secondary drying is carried out ranges from about 0 to 40°C, depending mainly on the type and size of the container and the type of protein used. For example, the storage temperature throughout the water removal stage of lyophilization can be about 15 to 30°C (e.g., about 20°C). The time and pressure required for secondary drying are those that will produce a suitable lyophilized cake, depending, for example, on temperature and other parameters. The time of secondary drying is determined by the desired residual moisture level in the product and typically takes at least about 5 hours (e.g., 10 to 15 hours). The pressure may be the same as that used during the primary drying step. Lyophilization conditions can vary depending on the formulation and vial size.

[0145] 2. Reconstitution of lyophilized formulations. Prior to administration to a patient, the lyophilized formulation is reconstituted with a pharma- ceutically acceptable diluent such that the protein concentration in the reconstituted formulation is at least about 50 mg / mL, e.g., about 50 mg / mL to about 400 mg / mL, alternatively about 80 mg / mL to about 300 mg / mL, alternatively about 90 mg / mL to about 150 mg / mL. Such high protein concentrations in the reconstituted formulation are believed to be particularly useful when subcutaneous delivery of the reconstituted formulation is intended. However, for other routes of administration, such as intravenous administration, lower concentrations of protein in the reconstituted formulation may be desirable (e.g., about 0.1 to 2 mg / mL, about 2 to 10 mg / mL, or about 10 to 50 mg / mL of protein in the reconstituted formulation). In certain embodiments, the protein concentration in the reconstituted formulation is significantly higher than the protein concentration in the pre-lyophilized formulation. In certain embodiments, the protein concentration in the reconstituted formulation is significantly lower than the protein concentration in the pre-lyophilized formulation.

[0146] Reconstitution is generally performed at a temperature of about 25° C. to ensure complete hydration, although other temperatures may be used as needed. The time required for reconstitution depends, for example, on the type of diluent, the excipient(s), and the amount of protein. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), a pH buffer (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. The diluent optionally contains a preservative. Exemplary preservatives are described above, with aromatic alcohols, such as benzyl or phenol alcohol, being preferred preservatives. The amount of preservative used is determined by evaluating different preservative concentrations for compatibility with the protein and preservative efficacy testing. For example, if the preservative is an aromatic alcohol (such as benzyl alcohol), it can be present in an amount of about 0.1-2.0%, or about 0.5-1.5%, or about 1.0-1.2%.

[0147] In some embodiments, the liquid formulations, including but not limited to, reconstituted liquid formulations, are substantially free of aggregates. In some embodiments, the liquid formulations, including but not limited to, reconstituted liquid formulations, have low free fatty acid particle formation.

[0148] III. Methods for making liquid formulations Also provided herein is a method of making a liquid formulation comprising adding a polypeptide and a surfactant to an aqueous solution, where the surfactant comprises one or more components of a polysorbate.

[0149] In some embodiments, the surfactant comprises an isosorbide POE fatty acid ester. In some embodiments, the surfactant further comprises a POE fatty acid ester. In some embodiments, the isosorbide POE fatty acid ester and the POE fatty acid ester independently have about 5 to 30 POE units. In some embodiments, the isosorbide POE fatty acid ester and the POE fatty acid ester independently have about 5 to 30 POE units. In some embodiments, the isosorbide POE fatty acid ester and the POE fatty acid ester independently have an optionally substituted C 4-28 Alkyl and optionally substituted C 4-28alkenyl. In some embodiments, the isosorbide POE fatty acid ester is a compound of formula (I). In some embodiments, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% (wt%) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester. In some embodiments, the isosorbide POE fatty acid ester is selected from the group consisting of isosorbide POE monocaproate, isosorbide POE monocaprylate, isosorbide POE monocaprate, isosorbide POE monolaurate, isosorbide POE monomyristate, isosorbide POE monopalmitate, isosorbide POE monopalmitoleate, isosorbide POE monostearate, isosorbide POE monooleate, isosorbide POE monolinoleate, isosorbide POE monolinolenate, and combinations thereof. In some embodiments, the POE fatty acid ester is selected from the group consisting of POE monocaproate, POE monocaprylate, POE monocaprate, POE monolaurate, POE monomyristate, POE monopalmitate, POE monopalmitoleate, POE monostearate, POE monooleate, POE monolinolenate, and combinations thereof. In some embodiments, the surfactant comprises isosorbide POE monolaurate and POE monolaurate. In some embodiments, the surfactant comprises isosorbide POE monopalmitate and POE monopalmitate. In some embodiments, the surfactant comprises isosorbide POE monomyristate and POE monomyristate. In some embodiments, the surfactant comprises isosorbide POE monooleate and POE monooleate. In some embodiments, the surfactant comprises isosorbide POE monolinoleate and POE monolinoleate.

[0150] In some embodiments, the surfactant further comprises a sorbitan POE fatty acid ester. In some embodiments, the sorbitan POE fatty acid ester is independently optionally substituted C 4-28 Alkyl and optionally substituted C 4-28 alkenyl. In some embodiments, the sorbitan POE fatty acid ester is a compound of formula (II). In some embodiments, less than about 30%, less than about 25%, less than about 20%, less than about 15%, less than about 10%, less than about 9%, less than about 8%, less than about 7%, less than about 6%, less than about 5%, less than about 4%, less than about 3%, less than about 2%, or less than about 1% (wt%) of the surfactant is a sorbitan POE fatty acid ester. In some embodiments, at least 1% (wt%) of the surfactant is a sorbitan POE fatty acid ester. In some embodiments, at least 5% (wt%) of the surfactant is a sorbitan POE fatty acid ester. In some embodiments, at least 10% (wt%) of the surfactant is a sorbitan POE fatty acid ester. In some embodiments, at least 15% (wt%) of the surfactant is a sorbitan POE fatty acid ester.

[0151] In some embodiments, each sorbitan POE fatty acid ester is sorbitan POE monocaproate, sorbitan POE monocaprylate, sorbitan POE monocaprate, sorbitan POE monolaurate, sorbitan POE monomyristate, sorbitan POE monopalmitate, sorbitan POE monopalmitoleate, sorbitan POE monostearate, sorbitan POE monooleate, sorbitan POE monolinoleate, sorbitan POE monolinolenate, sorbitan POE dicaproate, sorbitan POE dicaprylate, sorbitan POE dicaprate, sorbitan POE dilaurate, sorbitan POE dimyristate, sorbitan POE monostearate, sorbitan POE monooleate, sorbitan POE monolinoleate, sorbitan POE dicaproate, sorbitan POE dicaprate, sorbitan POE dilaurate, sorbitan POE dimyristate, sorbitan POE monostearate ...stearate, sorbitan POE monostearate, sorbitan POE monostearate, sorbitan POE monostearate, sorbitan POE monostearate, sorbitan POE monostearate, sorbitan POE monostearate, sorbitan POE monostearate, sorbitan POE monostearate, In one embodiment, the sorbitan POE trilaurate, sorbitan POE trimyristate, sorbitan POE tripalmitate, sorbitan POE tripalmitate, sorbitan POE tristearate, sorbitan POE dilinoleate, sorbitan POE dilinolenate, sorbitan POE tricaproate, sorbitan POE tricaprylate, sorbitan POE tricaprate, sorbitan POE trilaurate, sorbitan POE trimyristate, sorbitan POE tripalmitate, sorbitan POE tripalmitate, sorbitan POE tristearate, sorbitan POE trieolate, sorbitan POE trilinoleate, and sorbitan POE trilinolenate.

[0152] Any of the surfactants described herein can be used in the method of making the liquid formulation.

[0153] In some embodiments, the surfactant in the liquid formulation is about 0.0005%-0.2%, about 0.0005%-0.001%, about 0.001%-0.002%, about 0.002%-0.003%, about 0.003%-0.004%, about 0.004%-0.005%, about 0.005%-0.006%, about 0.006%-0.007%, about 0.007%-0.008%, about 0.008%-0.009%, about 0.009%-0.01%, about 0.01% %~0.015%, approx. 0.015%~0.02%, approx. 0.02%~0.025%, approx. 0.025%~0.03%, approx. 0.03%~0.035%, approx. 0.035%~0.04%, approx. 0.04%~0.045%, approx. 0.045%~0.05%, approx. 0.05%~0.055%, approx. 0.055%~0.06%, approx. 0.06%~0.065%, approx. 0.065%~0.07%, approx. 0.07%~0.075%, approx. 0.075%~0.08%, approx. 0.08%~0 .085%, approx. 0.085%-0.09%, approx. 0.09%-0.095%, approx. 0.095%-0.1%, approx. 0.1%-0.11%, approx. 0.11%-0.12%, approx. 0.12%-0.13%, approx. 0.13%-0.14%, approx. 0.14%-0.15%, approx. 0.15%-0.16%, approx. 0.16%-0.17%, approx. 0.17%-0.18%, approx. 0.18%-0.19%, approx. 0.19%-0.2%, approx. 0.0005%-0.01%, approx. 0.01%-0. 0.02%, about 0.02% to 0.03%, about 0.03% to 0.04%, about 0.04% to 0.05%, about 0.05% to 0.06%, about 0.06% to 0.07%, about 0.07% to 0.08%, about 0.08% to 0.09%, about 0.09% to 0.1%, about 0.1% to 0.12%, about 0.12% to 0.14%, about 0.14% to 0.16%, about 0.16% to 0.18%, about 0.18% to 0.2%, or about 0.0005% to 0.02% (w:v).

[0154] The concentration of the polypeptide in a liquid formulation can vary based on the storage configuration and the desired route of administration (eg, subcutaneous, intramuscular or intravitreal administration, intravenous injection or infusion, etc.). In some embodiments, the polypeptide in the liquid formulation is from about 0.1 mg / mL to 300 mg / mL, from about 0.1 mg / mL to 0.5 mg / mL, from about 0.5 mg / mL to 1 mg / mL, from about 1 mg / mL to 1.5 mg / mL, from about 1.5 mg / mL to 2 mg / mL, from about 2 mg / mL to 2.5 mg / mL, from about 2.5 mg / mL to 3 mg / mL, from about 3 mg / mL to 3.5 mg / mL, from about 3.5 mg / mL to 4 mg / mL, from about 4 mg / mL to 4.5 mg / mL, from about 4.5 mg / mL to 5 mg / mL, from about 0.1 mg / mL to 1 mg / mL, from about 1 mg / mL to 2 mg / mL, from about 2 mg / mL to 3 mg / mL, from about 3 mg / mL to 4 mg / mL, from about 4 mg / mL to 5 mg / mL L, about 5 mg / mL to 10 mg / mL, about 10 mg / mL to 15 mg / mL, about 15 mg / mL to 20 mg / mL, about 20 mg / mL to 30 mg / mL, about 30 mg / mL to 40 mg / mL, about 40 mg / mL to 50 mg / mL, about 50 mg / mL to 100 mg / mL, about 100 mg / mL to 150 mg / mL, about 150 mg / mL to 200 mg / mL, about 200 mg / mL to 250 mg / mL, about 250 mg / mL to 300 mg / mL, about 0.1 mg / mL to 2 mg / mL, about 0.5 mg / mL to 2 mg / mL, about 50 mg / mL to 150 mg, about 150 mg / mL to 200 mg / mL, or 200 mg / mL to 300 mg / mL. In some embodiments, the concentration of the polypeptide in the liquid formulation is about 0.5 mg / mL. In some embodiments, the polypeptide in the liquid formulation has a concentration of greater than about 50 mg / mL, greater than about 150 mg / mL, greater than about 200 mg / mL, greater than about 250 mg / mL, or greater than about 300 mg / mL.

[0155] In some embodiments, the method further comprises diluting the liquid formulation to reduce the concentration of the polypeptide by about 1-5 times, about 5-10 times, about 10-15 times, about 15-20 times, about 20-30 times, about 30-40 times, about 40-50 times, about 50-100 times, about 100-150 times, about 150-200 times, about 200-300 times, about 300-400 times, about 400-500 times, about 500-600 times, about 600-700 times, about 700-800 times, about 800-900 times, about 900-1000 times, about 1000-1500 times, about 1500-2000 times, about 2000-2500 times, about 2500-3000 times, or about 3000-5000 times. In some embodiments, the method further comprises diluting the liquid formulation with an infusion fluid. In some embodiments, the infusion fluid includes, but is not limited to, a dextrose-containing solution, lactated Ringer's solution, normal saline, or buffered saline. In some embodiments, the saline is saline (about 0.9% (w:v)). In some embodiments, the saline is an isotonic saline. In some embodiments, the saline is a buffered saline, including, but not limited to, phosphate buffered saline or Krebs-Ringer's solution. In some embodiments, the saline is isotonic or nearly isotonic with blood from the subject. The saline includes a salt, such as sodium chloride, potassium chloride, magnesium chloride, or calcium chloride. In some embodiments, the saline includes one or more buffers, such as a phosphate buffer (such as sodium phosphate or potassium phosphate), sodium carbonate, or HEPES. When using a buffered saline, the pH is maintained in a range that optimizes the therapeutic effectiveness of the polypeptide, especially if its stability is pH-dependent. In some embodiments, the liquid formulation is diluted prior to administration to the subject.

[0156] When a liquid formulation is prepared by reconstituting a lyophilized formulation, reconstitution is generally performed at a temperature of about 25° C. to ensure complete hydration, although other temperatures may be used as desired. The time required for reconstitution depends, for example, on the type of diluent, the excipient(s) and the amount of polypeptide. Exemplary diluents include sterile water, bacteriostatic water for injection (BWFI), pH buffer (e.g., phosphate buffered saline), sterile saline solution, Ringer's solution, or dextrose solution. The diluent optionally contains a preservative.

[0157] In some embodiments, the polypeptide used in the above methods is an antibody. In some embodiments, the antibody is a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a bispecific antibody, a polyspecific antibody, a chimeric antibody, or a heteroconjugate antibody. In some embodiments, the antibody includes antibody fragments and whole antibodies. In some embodiments, the antibody fragment is selected from the group consisting of Fab, Fab', F(ab')2, and Fv fragments.

[0158] In another aspect, the above method can be used to make any of the liquid formulations described herein.

[0159] IV. Manufactured products Provided herein is an article of manufacture that includes a container enclosing a liquid formulation, the liquid formulation comprising a polypeptide and a surfactant, the surfactant comprising one or more components of a polysorbate.

[0160] The article of manufacture includes a container. Suitable containers include, but are not limited to, bottles, vials (e.g., dual chamber vials), syringes (e.g., single or dual chamber syringes), test tubes, and intravenous therapy (IV) bags. In some embodiments, the IV bag contains an infusion fluid. In some embodiments, the infusion fluid includes, but is not limited to, a dextrose-containing solution, lactated Ringer's solution, saline, or buffered saline. The container may be formed from a variety of materials, such as glass, metal alloys (e.g., stainless steel), or plastic. A label on or associated with the container holding the liquid formulation may indicate instructions for reconstitution and / or use. In some embodiments, the label may further indicate that the formulation is useful or intended for subcutaneous administration. In some embodiments, the label may further indicate that the formulation is useful or intended for intravenous administration (e.g., by intramuscular, intraperitoneal, intraspinal, subcutaneous, intra-articular, intrasynovial, or intrathecal administration, e.g., as a bolus or by continuous infusion over a period of time). In some embodiments, the label may further indicate that the formulation is useful or intended for intravitreal administration. The container holding the formulation may be a multi-use vial that allows for repeated administration (e.g., 2-6 administrations) of the liquid formulation. In some embodiments, the article of manufacture comprises a polypeptide at a concentration of about 0.1 mg / mL to about 300 mg / mL. The article of manufacture may further comprise a second container that comprises a suitable diluent (e.g., water, formulation buffer, surfactant solution, and infusion fluid, such as dextrose-containing solutions, saline, lactated Ringer's solution, and BWFI). Upon mixing the diluent with the liquid formulation, the final protein concentration in the diluted formulation is generally at least 0.001 mg / mL. In some embodiments, the diluent is mixed with the lyophilized formulation to form a reconstituted liquid formulation. In some embodiments, the final protein concentration in the liquid formulation, including the reconstituted liquid formulation, is about 0.5 mg / mL to about 2 mg / mL. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, syringes, and package inserts with instructions for use.In some embodiments, the article of manufacture contains a syringe or device for intravenous administration and / or a sterile buffer for preparing the lyophilized composition for administration.

[0161] In another aspect, the article of manufacture may contain any of the liquid formulations described herein. In another aspect, the article of manufacture may contain a liquid formulation prepared according to any of the methods of making described herein.

[0162] V. Medications and Methods of Treatment Using Liquid Formulations Also provided herein is a method of treating a disease or disorder in a subject, comprising administering an effective amount of a liquid formulation described herein to a subject in need thereof. Also provided herein is the use of a liquid formulation described herein in the preparation of a medicament for treating a patient in need of treatment with a polypeptide in a liquid formulation. Also provided is a liquid formulation described herein for treating a disease or disorder in a subject in need of treatment with a polypeptide in a liquid formulation. Also provided is a liquid formulation described herein for treating a patient, comprising administering an effective amount of the liquid formulation to the patient.

[0163] When the antibody in the formulation binds to HER2, the suspension formulation can be used to treat cancer.Cancer generally comprises HER2-expressing cells, so that the HER2 antibody herein can bind to cancer cells.Therefore, the present invention in this embodiment relates to a method for treating HER2-expressing cancer in a subject, comprising administering to the subject a HER2 antibody pharmaceutical formulation in an amount effective for treating cancer.The exemplary cancers that are treated herein with HER2 antibody (e.g., trastuzumab or pertuzumab) are HER2-positive breast cancer or gastric cancer.

[0164] If the antibody in the formulation binds to a B cell surface marker such as CD20, the formulation may be used to treat a B cell malignancy such as NHL or CLL, or an autoimmune disease (e.g., rheumatoid arthritis or vasculitis).

[0165] Where the antibody in the formulation binds to VEGF (e.g., bevacizumab), the formulation may be used to inhibit angiogenesis, to treat cancer (e.g., colorectal, non-small cell lung (NSCL), glioblastoma, breast cancer, and renal cell carcinoma), or to treat age-related macular degeneration (AMD) or macular edema.

[0166] When the indication is cancer, the patient may be treated with a combination of the suspension formulation and a chemotherapeutic agent. The combined administration includes co-administration or simultaneous administration using separate formulations or a single pharmaceutical formulation, and sequential administration in any order, where there is a period during which both (or all) active agents simultaneously exert their biological activity. Thus, the chemotherapeutic agent may be administered prior to or subsequent to administration of the composition. In this embodiment, the timing between at least one administration of the chemotherapeutic agent and at least one administration of the formulation is about one month or less, or about two weeks or less. Alternatively, the chemotherapeutic agent and the formulation are administered to the patient simultaneously in a single formulation or separate formulations.

[0167] Treatment with the suspension formulation will result in amelioration of signs or symptoms of the disease or disorder. Additionally, treatment with a combination of a chemotherapeutic agent and an antibody formulation may provide synergistic or additive therapeutic benefit to the patient.

[0168] In some embodiments, the liquid formulations described herein are administered intravenously. In some embodiments, the liquid formulations described herein are administered by intravenous injection at a controlled administration rate such that administration occurs over at least about 30 minutes or more. The administration schedule and actual dosage administered may vary depending on factors such as the nature and severity of the infection, the age, weight and general health of the patient, and the disease or disorder being treated, and are ascertainable by a medical professional. The liquid formulations are suitably administered to the subject at one time or over a series of treatments. Depending on the type and severity of the disease, about 1 μg / kg to 50 mg / kg (e.g., 0.1 to 20 mg / kg) of antibody is an initial candidate dosage for administration to the patient, whether by one or more separate administrations, for example. The dosage of the antibody is generally about 0.05 mg / kg to about 10 mg / kg. Thus, when a chemotherapeutic agent is administered, it is usually administered at a known dosage or is optionally reduced due to the combined effects of the drugs or negative side effects resulting from the administration of the chemotherapeutic agent. EXAMPLES

[0169] The following examples are offered to illustrate, but not to limit, the invention. Those of skill in the art will recognize that the procedures set forth below can be modified using methods known to those of skill in the art.

[0170] material: Monoclonal antibodies A-D were manufactured by Genentech, Inc. (South San Francisco, CA, USA). Polysorbate 20, containing approximately 99% lauric acid ester, was synthesized by BASF SE (Ludwigshafen, Germany). Polysorbate 80 HX2 ultrapure grade, containing approximately 98% oleic acid ester, was synthesized by NOF America Corp. (Irvine, CA). All polysorbate samples were stored with nitrogen overlay at 2-8 °C when not in use. Acetonitrile and methanol (HPLC grade) were purchased from Avantor Performance Materials, Inc. (Pierre-Burgh, NJ).

[0171] Example 1 Polysorbate Fractionation and Analysis Polysorbate Fraction Fractionation of polysorbates (PS) was performed using a Gilson PLC-2050 Prep HPLC system. For all PS samples, 2 grams of polysorbate 20 (PS20) or polysorbate 80 (PS80) was dissolved in 8 mL of >18 MΩ cm water and injected onto a Waters X-Bridge BEH C4 column (30 × 100 mm). Samples were then fractionated using a gradient of >18 MΩ cm water (mobile phase A) and acetonitrile (mobile phase B). Gradient conditions were as follows: 0.0–3.0 min, 5% B; 3.0–20.0 min, 5–100% B; 20.0–27.0 min, 100% B; 27–27.1 min, 100–5% B; 27.1–30.0 min, 5% B. The flow rate was 40 mL / min, and the total run time was 30 min. Fractions of 25 mL were collected during the run. These fractions were then analyzed using a reversed-phase ultra-high performance liquid chromatography method with charged aerosol detection (RP-UHPLC-CAD) described in the following paragraphs. After analysis, the fractions were pooled and dried using a rotary evaporator.

[0172] Polysorbate fraction purity analysis The fractionated PS samples were pooled and assessed for purity using a Waters Acquity UPLC H-Class system equipped with a Thermo Corona Ultra CAD detector. The column used was a Waters Acquity BEH C8 column (2.1 × 50 mm, 120 Å, and 2.5 μm particle size). The gradient was as follows: 0.0–0.5 min, 5% B; 0.5–5.0 min, 5–100% B; 5.0–6.0 min, 100% B; 6.0–6.1 min, 100–5% B; 6.1–8.0 min, 5% B. CAD was performed with data collection at 10 Hz and nebulizer temperature at 30 °C.

[0173] result The fractions of PS20 and PS80 were purified and dried on a rotary evaporator. Their relative purity as determined by RP-UHPLC-CAD is shown in Table 1 below. Three fractions were purified and analyzed from each PS type. In the case of PS20, these fractions were: POE-sorbitan monolaurate ("Fraction 1 or F1a"), POE-isosorbide monolaurate and POE monolaurate (N approx. 10) ("Fraction 2 or F2a"), and POE-sorbitan dilaurate ("Fraction 3 or F3a"). In the case of PS80, the fractions were: POE-sorbitan monooleate ("Fraction 1 or F1b"), POE-isosorbide monooleate and POE monooleate (N approx. 10) ("Fraction 2 or F2b"), and POE-sorbitan dioleate ("Fraction 3 or F3b"). The identity of each fraction was confirmed by LC-MS analysis. Reverse-phase UHPLC-CAD analysis confirmed that the most hydrophobic polysorbate ester fraction was F3, followed by F2, and then F1, which was the least hydrophobic (Figures 1A and 1B). [Table 1]

[0174] Example 2 Critical Micelle Concentration (CMC) Determination Purified PS fractions were assessed for their critical micelle concentration (CMC) using the fluorescent dye N-phenylnaphthalen-1-amine (NPN). The assay was performed by performing two-fold serial dilutions at pH 8.0 into a diluent consisting of 0.15 M sodium chloride, 0.05 M TRIS, 5% ACN, 5 μM N-phenyl-1-naphthylamine, and 15 ppm Brij35. Samples were immediately analyzed on a Molecular Devices Spectramax M5 fluorescent plate reader with excitation at 350 nm and emission at 420 nm.

[0175] For PS20, the order of increasing CMC was F3a>F2a>F1a, consistent with the order of hydrophobicity. The CMCs were widely separated, approximately 0.1 wt% for F1a, approximately 0.015 wt% for F2a, and approximately 0.001 wt% for F3a, which corresponds to a change of roughly 500 fluorescence units from baseline (Figure 2A). In contrast, the PS80 ester CMC had a similar order, but a much narrower range of values, spanning only 0.001-0.003 wt% (Figure 2B).

[0176] Example 3 Surface activity of fractionated PS samples The surface activity of the purified fractions of PS was determined using a Kruess (Hamburg, Germany) K100 Force Tensiometer using roughened platinum Wilhemy plates. Samples were measured at room temperature (20–25 °C) with a relative humidity above 70% to prevent evaporation. Prior to testing, the purified fractions of PS20 were each dissolved in purified water to 0.2 mg / mL. Three milliliters of sample were placed in the sample holder, taking special care not to introduce air bubbles. The change in surface tension of each sample was measured once every second until equilibrium was reached in 60 min. The same procedure was followed for the PS80 analysis.

[0177] The surface tension results showed that at equilibrium, at the same concentration of 0.02 wt%, PS20 F3a and F2a fractions were significantly more effective at lowering surface tension than the less hydrophobic F1a fraction (Figure 3A). A similar trend is observed for PS80 F1b-F3b fractions (Figure 3B). Interestingly, the surface tension of PS20 fraction F3a is almost the same as F2a, and that of PS80 fraction F3b is also similar to F2b. Furthermore, unfractionated PS20, which contains all the ester fractions, lowered surface tension even more than F3a or F2a fractions (Figure 3A). In contrast, unfractionated PS80 showed a surface tension between its F1b and F3b fractions (Figure 3B).

[0178] Example 4 Dynamic Light Scattering of Micelle Size Dynamic light scattering (DLS) was performed on a Malvern (Westborough, MA) Zetasizer Nano instrument. Measurements were performed by diluting each fraction sample in water to a final concentration of 10 mg / mL, above the CMC of each fraction.

[0179] Using dynamic light scattering (DLS) and a fraction concentration of 1 wt%, well above the CMC of all ester fractions studied, the micelle radius or hydrodynamic radius (R h For PS 20, the R h The R of F1a monoester was approximately 4 nm, and the F2a and F3a fractions were approximately 3.9 nm. h is rather small, at around 3.5 nm (Figure 4A). In the case of PS80, the micelle diameter is R h The unfractionated PS80 sample (R h The R of F1b was also slightly increased by about 4.8 nm. h is smallest in the PS80 sample set, but R h It is not so large at about 4.3 nm (Figure 4B).

[0180] Example 5. Stir protection studies The purified fractions were tested for their ability to prevent surface-induced aggregation using agitation studies. mAbA was diluted to 0.5mg / mL in 0.9% saline, which contained various amounts of each of the PS20 fractions (F1a, F2a and F3a 0.0001%-0.01%, w:v) in 10mL PETG vials. These vials were agitated for 2 hours at ambient temperature at 180RPM on an orbital shaker. After agitation, samples were observed using a Bosch APK system at 10x magnification and rotation.

[0181] During agitation studies, it was observed that F2a required the lowest concentration to protect against visible particle formation upon agitation, with F1a requiring the most. F3a and all-laurate PS20 yielded similar results (Figure 5). F2a provided the most protection for the mAb against agitation stress and particle formation, although it had a higher CMC and a comparable equilibrium surface tension effect to F3a and all-laurate PS20.

[0182] Example 6 IV Bag Agitation Model Study The purified fractions were tested for their ability to prevent surface-induced aggregation using agitation studies. mAbB and mAbC were diluted to 0.5 mg / mL in 0.9% saline buffer containing various amounts of each of the PS20 fractions (F1a, F2a and F3a 0.0001%-0.01%, w / v) in 5 mL PETG vials. These samples were agitated for 2 hours at ambient temperature at 180 RPM on an orbital shaker. After agitation, the samples were observed using a Bosch APK system at 10x magnification and rotation. The samples were subjected to HIAC (High Precision Fluid Particle Counting) to quantify SVPs (Sub-Visible Particles) in the formulation and SEC-HPLC (Size Exclusion High Performance Liquid Chromatography) to quantify the concentration of HMWFs (High Molecular Weight Fractions) and active antibody in the formulation. SEC and IEC were performed on an Agilent 1260 HPLC equipped with a binary pump and a diode array detector. Subvisible particle measurements were performed on a Royco HIAC 9703+ Pharmaceutical Particle Counter.

[0183] The observations suggested that all fractions tested protected mAbB (Figure 6A) and mAbC (Figure 6B). HIAC results for mAb B (Figure 7A) and mAbC (Figure 7B) showed that all fractions were able to reduce the number of subvisible particles, showing lower levels of aggregation. F2a is as good or better than HP or all-laurate PS20 in reducing aggregation levels. SEC-HPLC assay results showed that all fractions reduced %HMWF during stirring (Figures 8A and 8B) and better preserved the soluble fractions (Figures 9A and 9B) of both mAbB (Figures 8A and 9A) and mAbC (Figures 8B and 9B) antibody formulations. F2a is particularly effective at lower detergent concentrations (Figures 8A, 8B, 9A, and 9B). All detergents were successful in mitigating protein degradation.

[0184] Example 7 Stability Studies Long-term formulation stability was tested for formulations of PS20 and F2a (each at 0.02% w / v) containing proteins mAbC and mAbD (each at 30 mg / mL). Formulations were stored at 5°C, 25°C, and 40°C and subjected to visual inspection (APK), HIAC, and SEC-HPLC at various intervals. Formulations of PS20 and F2a were also subjected to IEC at 5°C, 25°C, and 40°C.

[0185] Visual inspection of formulations stored at 40°C indicated that both surfactants were effective in limiting particle formation in both mAb C (Figure 10A) and mAb D (Figure 10B) formulations at 40°C for up to 1 month.

[0186] HIAC results for formulations stored at 40°C showed that both surfactants were effective in limiting the formation of subvisible particles in both mAbC (Figure 11A) and mAbD (Figure 11B) formulations at 40°C, even when stored for up to one month.

[0187] SEC-HPLC results of formulations stored at 40° C. showed that both surfactants were effective in limiting aggregation in both mAbC (FIG. 12A) and mAbD (FIG. 12B) formulations. SEC results also showed that both surfactants were effective in maintaining the concentration of active antibody in both mAbC (FIG. 13A) and mAbD (FIG. 13B) formulations for up to 1 month at 40° C.

[0188] IEC data for formulations stored at 40° C. showed that both surfactants limited degradation of mAbC, exhibiting similar efficacy at 40° C. for 1 month (FIG. 14).

[0189] Visual inspection data for formulations stored at 25° C. showed that both surfactants were effective in limiting aggregation in formulations of mAbC (FIG. 15A) and mAbD (FIG. 15B) for up to 3 months.

[0190] HIAC results for formulations stored at 25°C showed that both surfactants were effective in limiting the formation of subvisible particles in both mAbC (Figure 16A) and mAbD (Figure 16B) formulations at 25°C, even when stored for up to 3 months.

[0191] SEC-HPLC results of formulations stored at 25° C. showed that both surfactants were effective in limiting aggregation in both mAbC (FIG. 17A) and mAbD (FIG. 17B) formulations. SEC results also showed that both surfactants were effective in maintaining the concentration of active antibody in both mAbC (FIG. 18A) and mAbD (FIG. 18B) formulations for up to 3 months at 25° C.

[0192] IEC data for formulations stored at 25° C. showed that both surfactants limited degradation of mAbC with similar effectiveness for 3 months at 25° C. (FIG. 19).

[0193] Visual inspection data for formulations stored at 5° C. showed that both surfactants were effective in preventing aggregation in formulations of mAbC (FIG. 20A) and mAbD (FIG. 20B) for up to 3 months.

[0194] HIAC results for formulations stored at 5°C showed that both surfactants were effective in limiting the formation of subvisible particles in both mAbC (Figure 21A) and mAbD (Figure 21B) formulations at 5°C, even when stored for up to 3 months.

[0195] SEC-HPLC results of formulations stored at 5° C. showed that both surfactants were effective in limiting aggregation in both mAbC (FIG. 22A) and mAbD (FIG. 22B) formulations. SEC results also showed that both surfactants were effective in maintaining the concentration of active antibody in both mAbC (FIG. 23A) and mAbD (FIG. 23B) formulations for up to 3 months at 5° C.

[0196] IEC data for formulations stored at 5° C. showed that both surfactants limited the degradation of mAbC with similar effectiveness for 3 months at 5° C. (FIG. 24).

[0197] Example 8 Forced Degradation Studies PS20 and F2a (each in formulation buffer at pH=6.0) were subjected to forced degradation by lipase enzyme from Pseudomonas cepacia (PCL) at a concentration of 2.5 U / mL. The resulting mixture was subjected to HIAC to quantify the percentage of sub-visible particles and intact surfactant. The results showed that F2a produced less SVPs despite more degradation by the enzyme (Figure 25).

[0198] overview F2a has been shown to be more protective for biopharmaceuticals when compared to the commonly used surfactant excipient PS20. This surfactant has attractive properties that make it a superior alternative to traditional polysorbates such as PS20 and PS80.

[0199] In an agitation stress study, F2a was found to be more protective from particle formation for mAb A when tested with APK. In a separate study, F2a showed similar protection from agitation stress between F2a and HP PS20 against mAbs B and C based on particle testing by APK, subvisible particle counts by HIAC, %HMWF by SEC-HPLC, and soluble antibody concentration tested by SEC-HPLC.

[0200] In stability studies, F2a was observed to be as effective as HP PS20 in preventing particle formation by APK and HIAC for two mAbs, mAbC and mAbD. F2a also had similar protection as HP PS20 in preventing HMWF by SEC-HPLC for both mAbC and mAbD upon storage at 5°C, 25°C and 40°C.

[0201] Forced degradation studies have shown that F2a can be enzymatically degraded prior to the formation of subvisible and visible fatty acid particles more easily than HP PS20, providing an advantage to F2a as a surfactant that is less susceptible to this type of fatty acid-related particle formation.

Claims

1. A liquid formulation comprising a polypeptide and a surfactant, wherein at least about 70% (by weight) of the surfactant is an isosorbide polyoxyethylene (POE) fatty acid ester.

2. 2. The liquid formulation of claim 1, wherein the isosorbide POE fatty acid ester comprises about 5 to 30 POE units.

3. 3. The liquid formulation of claim 1 or 2, wherein the isosorbide POE fatty acid ester contains about 20 POE units.

4. The isosorbide POE fatty acid ester is optionally substituted C ４－２８ Alkyl and optionally substituted C ４－２８ 4. The liquid formulation of claim 1, further comprising a fatty acid chain selected from the group consisting of alkenyl.

5. The liquid formulation according to claim 1 , wherein the isosorbide POE fatty acid ester is a monoester.

6. 6. The liquid formulation of claim 5, wherein the monoester is selected from the group consisting of isosorbide POE monolaurate, isosorbide POE monomyristate, isosorbide POE monopalmitate, isosorbide POE monostearate and isosorbide POE monooleate.

7. 5. The liquid formulation of claim 1, wherein the isosorbide POE fatty acid ester is a monoester, a diester, or a mixture thereof.

8. The isosorbide POE fatty acid ester has the formula (I): [During the ceremony a and b are independently an integer from 2 to 28, provided that the sum of a and b is an integer from 5 to 30; R １ and R ２ are independently hydrogen and —C(O)R″ (wherein R″ is an optionally substituted C ３－２７ Alkyl or optionally substituted C ３－２７ alkenyl; R ３ and R ４ are independently hydrogen. The liquid formulation of claim 1, wherein the compound is

9. 9. The liquid formulation of claim 8, wherein the sum of a and b is 9.

10. R １ is H, and R ２ The liquid formulation according to claim 8 or 9, wherein is -C(O)R''.

11. R １ is -C(O)R'', and R ２ The liquid formulation according to claim 8 or 9, wherein is H.

12. R １ and R ２ The liquid formulation according to claim 8 or 9, wherein both of the following are -C(O)R''.

13. R″ is unsubstituted C ３－２７ 13. The liquid formulation of any one of claims 8 to 12, wherein the alkyl group is alkyl.

14. R″ is unsubstituted C １１ The liquid formulation of claim 13, wherein the alkyl group is an alkyl group.

15. R″ is unsubstituted C ３－２７ 13. The liquid formulation of claim 8, wherein the alkyl group is alkenyl.

16. R″ is unsubstituted C １７ The liquid formulation of claim 15, wherein the alkyl group is alkenyl.

17. 17. The liquid formulation of any one of claims 1 to 16, wherein at least about 80% (by weight) of the surfactant is an isosorbide POE fatty acid ester.

18. 18. The liquid formulation of claim 1, wherein the surfactant further comprises a POE fatty acid ester.

19. 20. The liquid formulation of claim 18, wherein the surfactant comprises an isosorbide POE fatty acid ester in an amount greater than the POE fatty acid ester.

20. The POE fatty acid ester is an optionally substituted C ４－２８ Alkyl and optionally substituted C ４－２８ 20. The liquid formulation of claim 18 or 19, comprising a fatty acid chain selected from the group consisting of alkenyl.

21. 21. The liquid formulation of claim 20, wherein the POE fatty acid ester is selected from the group consisting of POE monolaurate, POE monomyristate, POE monopalmitate, POE monostearate and POE monooleate.

22. 22. The liquid formulation of any one of claims 18 to 21, wherein less than about 20% (wt%) of the surfactant is a POE fatty acid ester.

23. 23. The liquid formulation of any one of claims 1 to 22, wherein the surfactant is about 0.0005% to 0.2% (w:v) of the liquid formulation.

24. 24. The liquid formulation of any one of claims 1 to 23, wherein the surfactant further comprises a sorbitan POE fatty acid ester.

25. 25. The liquid formulation of any one of claims 1 to 24, wherein the polypeptide is a protein.

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

27. 27. The liquid formulation of claim 26, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, and an antibody fragment.

28. The antibody fragment is Fab, Fab', F(ab') ２ 28. The liquid formulation of claim 27, wherein the antibody is selected from the group consisting of:

29. 29. The liquid formulation of any one of claims 26 to 28, wherein the concentration of the antibody is from about 0.1 mg / mL to about 300 mg / mL.

30. 30. The liquid formulation of claim 29, wherein the concentration of the antibody is from about 100 mg / mL to about 300 mg / mL.

31. 31. The liquid formulation of any one of claims 1 to 30, wherein the liquid formulation is a reconstituted lyophilized formulation.

32. 31. The liquid formulation of claim 29 or 30, wherein the liquid formulation is further diluted with infusion fluid to a polypeptide concentration of about 0.001 mg / mL to about 0.5 mg / mL.

33. 33. The liquid formulation of any one of claims 1-32, wherein the liquid formulation is substantially free of aggregates.

34. 33. The liquid formulation of any one of claims 1 to 32, wherein the liquid formulation has low free fatty acid particle formation.

35. 35. The liquid formulation of any one of claims 18 to 34, wherein at least about 80% (by weight) of the surfactants are isosorbide polyoxyethylene (POE) fatty acid esters and POE fatty acid esters.

36. 36. The liquid formulation of any one of claims 18 to 35, wherein at least about 90% (by weight) of the surfactants are isosorbide polyoxyethylene (POE) fatty acid esters and POE fatty acid esters.

37. A lyophilized formulation comprising a polypeptide and a surfactant, wherein at least about 70% (by weight) of the surfactant is an isosorbide polyoxyethylene (POE) fatty acid ester and a POE fatty acid ester, the lyophilized formulation being prepared by lyophilizing a liquid formulation described in any one of claims 18 to 36.

38. 37. An article of manufacture comprising a container enclosing the liquid formulation of any one of claims 1 to 36.

39. 40. The article of manufacture of claim 38, wherein the container is an IV bag.

40. 40. The article of manufacture of claim 39, wherein the IV bag comprises an injection device.

41. 41. The article of manufacture of claim 39 or 40, wherein the IV bag contains an infusion solution.

42. 39. The article of manufacture of claim 38, wherein the container is a vial or a prefilled syringe.

43. 40. The article of manufacture of claim 38, further comprising a second container enclosing a diluent.

44. 38. An article of manufacture comprising a container enclosing the lyophilized formulation of claim 37.

45. 45. The article of manufacture of claim 44, further comprising a second container enclosing a diluent.

46. A method for making a liquid formulation comprising adding a polypeptide and a surfactant to an aqueous solution, wherein at least 70% (by weight) of the surfactant is an isosorbide POE fatty acid ester.

47. 47. The method of claim 46, wherein the isosorbide POE fatty acid ester comprises about 5 to 30 POE units.

48. 48. The method of claim 47, wherein the isosorbide POE fatty acid ester contains about 20 POE units.

49. The isosorbide POE fatty acid ester is optionally substituted C ４－２８ Alkyl and optionally substituted C ４－２８ 49. The method of any one of claims 46 to 48, comprising a fatty acid chain selected from the group consisting of alkenyl.

50. 49. The method of any one of claims 46 to 48, wherein the isosorbide POE fatty acid ester is a monoester.

51. 51. The method of claim 50, wherein the monoester is selected from the group consisting of isosorbide POE monolaurate, isosorbide POE monomyristate, isosorbide POE monopalmitate, isosorbide POE monostearate, and isosorbide POE monooleate.

52. 50. The method of any one of claims 46 to 49, wherein the isosorbide POE fatty acid ester is a monoester, a diester, or a mixture thereof.

53. The isosorbide POE fatty acid ester has the formula (I): [During the ceremony a and b are independently an integer from 2 to 28, provided that the sum of a and b is an integer from 5 to 30; R １ and R ２ are independently hydrogen and —C(O)R″ (wherein R″ is an optionally substituted C ３－２７ Alkyl or optionally substituted C ３－２７ alkenyl; R ３ and R ４ are independently hydrogen. The method of claim 46, wherein the compound is

54. 54. The method of claim 53, wherein the sum of a and b is 9.

55. R １ is H, and R ２ The method of claim 53 or 54, wherein is -C(O)R''.

56. R ２ is -C(O)R'', and R １ The method of claim 53 or 54, wherein is H.

57. R １ and R ２ The method of claim 53 or 54, wherein both of are --C(O)R''.

58. R″ is unsubstituted C ３－２７ 58. The method of any one of claims 53 to 57, wherein said alkyl is aryl.

59. R″ is unsubstituted C １１ 57. The method of claim 56, wherein said alkyl is alkyl.

60. R″ is unsubstituted C ３－２７ 58. The method of any one of claims 53 to 57, wherein said alkyl group is alkenyl.

61. R″ is unsubstituted C １７ The method of claim 60, wherein the alkyl group is alkenyl.

62. 62. The method of any one of claims 46 to 61, wherein the surfactant further comprises a POE fatty acid ester.

63. 63. The method of claim 62, wherein at least about 80% (by weight) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester.

64. 64. The method of claim 62 or 63, wherein at least about 90% (by weight) of the surfactant is isosorbide POE fatty acid ester and POE fatty acid ester.

65. 65. The method of any one of claims 62 to 64, wherein the surfactant comprises an amount of isosorbide POE fatty acid ester greater than the amount of POE fatty acid ester.

66. The POE fatty acid ester is an optionally substituted C ４－２８ Alkyl and optionally substituted C ４－２８ 66. The method of any one of claims 62 to 65, comprising a fatty acid chain selected from the group consisting of alkenyl.

67. 67. The method of claim 66, wherein the POE fatty acid ester is selected from the group consisting of POE monolaurate, POE monomyristate, POE monopalmitate, POE monostearate and POE monooleate.

68. 68. The method of any one of claims 62 to 67, wherein less than about 20% (by weight) of the surfactant is a POE fatty acid ester.

69. 69. The method of any one of claims 46 to 68, wherein the surfactant further comprises a sorbitan POE fatty acid ester.

70. 70. The method of any one of claims 46 to 69, wherein the surfactant is about 0.0005% to 0.2% (w:v) of the liquid formulation.

71. 71. The method of any one of claims 46 to 70, wherein the polypeptide is a protein.

72. 72. The method of claim 71, wherein the protein is an antibody.

73. 73. The method of claim 72, wherein the antibody is selected from the group consisting of a polyclonal antibody, a monoclonal antibody, a humanized antibody, a human antibody, a chimeric antibody, and an antibody fragment.

74. The antibody fragment is Fab, Fab', F(ab') ２ and an Fv fragment.

75. 75. The method of any one of claims 72 to 74, wherein the concentration of the antibody is from about 0.1 mg / mL to about 300 mg / mL.

76. 76. The method of claim 75, wherein the concentration of the antibody is from about 100 mg / mL to about 300 mg / mL.

77. 77. The method of claim 75 or 76, wherein the liquid formulation is further diluted with infusion fluid to a concentration of about 0.1 mg / mL to about 2 mg / mL.

78. 78. The method of any one of claims 46-77, further comprising lyophilizing the liquid formulation to produce a lyophilized formulation.

79. 79. The method of any one of claims 46-78, wherein the liquid formulation is substantially free of aggregates.

80. 79. The method of any one of claims 46 to 78, wherein the liquid formulation has low free fatty acid particle formation.

81. 5. The liquid formulation according to claim 1, wherein the isosorbide POE fatty acid ester is isosorbide POE monolaurate.

82. 49. The method of any one of claims 46 to 48, wherein the isosorbide POE fatty acid ester is isosorbide POE monolaurate.

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