Hydroxypropyl methylcellulose derivatives for stabilizing polypeptides

Abstract

The present invention provides a formulation comprising a polypeptide and a hydroxypropylmethylcellulose acetate succinate (HPMCAS) derivative. The formulation is stable, for example, during high temperature processing and potentially in low pH environments. Furthermore, the HPMCAS derivative provides protection of pH-sensitive proteins against acidic degradation products from aqueous hydrolysis of poly(lactic-co-glycolic acid) (PLGA) in PLGA-based delivery systems.

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims priority to and benefit of U.S. Provisional Patent Application No. 63 / 326,772, filed April 1, 2022, which is incorporated herein by reference in its entirety.

[0002] The present invention provides the use of hydroxypropylmethylcellulose acetate succinate (HPMCAS) derivatives as polymeric excipients to stabilize polypeptides (e.g., during high-temperature processing and potentially low-pH environments). Additionally, the HPMCAS derivatives provide protection for pH-sensitive proteins against acidic degradation products from aqueous hydrolysis of poly(lactic-co-glycolic acid) (PLGA) in PLGA-based delivery systems. [Background technology]

[0003] Protein-based therapeutics are susceptible to physical and chemical degradation reactions during manufacturing, storage, and delivery (Le Basle, Y et al., J Pharm Sci, 2020, 109(1): pp. 169-190). Understanding how excipients stabilize proteins in the liquid and solid states has enabled the successful development of many protein-based pharmaceuticals, but there remains a need to identify new excipients and design formulations that enable the development of polymer-based sustained release drugs (Fu, K et al., Nat Biotechnol, 2000, 18(1): 24-5; Putney, SD and PA Burke, Nat Biotechnol, 1998, 16(2): 153-7). Protein stability encompasses physicochemical phenomena and conformational changes not associated with small molecule therapeutics (Manning, MC et al., Pharm Res, 2010, 27(4): 544-75). In the case of polymer controlled release systems, protein drugs should remain within the formulation at physiological temperatures for several weeks to a year (Zhu, G et al., Nat Biotechnol, 2000, 18(1):52-7; Vaishya, R et al., Expert Opin Drug Deliv, 2015, 12(3):415-40. For example, in the preparation of drug-loaded PLGA rods, the drug is exposed to high temperatures during manufacturing and then to a low pH microenvironment due to polymer hydrolysis when the rods are at body temperature for extended periods of time. Without stabilizing excipients, protein drugs are subject to degradation, which can occur both within the delivery system and upon its release in vivo (Rajagopal, K et al., J Pharm Sci, 2013, 102(8):2655-66).

[0004] The preparation of PLGA rods using a hot melt extrusion process involves mixing a spray-dried protein formulation with solid PLGA and microcompounding at 90°C, followed by extrusion, cooling, and cutting the extrudate into solid rods of the desired size (Rajagopal, K et al., J Pharm Sci, 2013, 102(8):2655-66). It has previously been demonstrated that the inclusion of hydrophilic small molecule excipients such as trehalose or ectoine in spray-dried formulations stabilizes proteins at elevated temperatures, allowing the hot-melt extrusion process to be used for the preparation of protein-loaded PLGA rods (Rajagopal, K et al., Mol Pharm, 2019. 16(1):349-358; Nayak, PK et al., Molecular Pharmaceutics, 2020, 17(9):3291-3297). While these excipients stabilize proteins in the solid state during the preparation and shelf-life storage of PLGA rods, they do not provide stability to the low pH microenvironment that arises due to hydrolytic polymer degradation after the rods are implanted in the body (Chang, DP et al., J Pharm Sci, 2015, 104(10):3404-17). Because polymer hydrolysis leads to the formation of lactic acid and glycolic acid (Pitt, CG et al. al., Biomaterials, 1981, 2(4):215-20), and the accumulation of these acids within PLGA delivery systems creates an acidic microenvironment (Shenderova, A et al., Pharm Res, 1999. 16(2):241-8; Brunner, A et al., Pharm Res, 1999, 16(6):847-53. These excipients not only lack the buffering capacity to neutralize the acidic microenvironment, but also are released faster than proteins due to their small size and hydrophilicity.This scenario leaves the protein unprotected within the rod and promotes low-pH chemical degradation reactions such as isomerization and fragmentation, which contribute to aggregation, incomplete drug release, and potentially lead to loss of activity (Rajagopal, K et al., Mol Pharm, 2019, 16(1):349-358; Liu, Y and S.P. Schwendeman, Mol Pharm, 2012, 9(5):1342-50; Fu, K et al., Pharm Res, 2000, 17(1):100-6; Lu, W and T.G. Park, P.D.A. J Pharm Sci Technol, 1995, 49(1):13-9; Shao, P.G. and L.C. Bailey, Pharm Dev Technol, 2000, 5(1):1-9). A study by Zhu et al. showed that large globular proteins such as BSA undergo extensive aggregation in PLGA systems during prolonged incubation in PBS buffer at 37°C (Zhu, G and SP Schwendeman, Pharm Res, 2000, 17(3):351-7). Therefore, excipients that neutralize the acidic microclimate and resist proteolysis are necessary for stable protein release from PLGA systems.

[0005] Basic additives such as Mg(OH)2 to stabilize BSA (Zhu, G et al., Nat Biotechnol, 2000, 18(1):52-7), and CaCl2 or MnC to stabilize octreotide. l Various routes to protein stability in PLGA devices have been explored, including the addition of divalent metal cation salts such as 2 (Sophocleous, AM et al., J Control Release, 2009. 137(3):179-84). However, these approaches rely on small inorganic ions as additives to neutralize degradation products or prevent protein interaction with degradation species from PLGA hydrolysis without providing excipient protection for the protein.

[0006] What is needed is the use of polymeric excipients to stabilize proteins in spray-dried formulations at elevated temperatures and to neutralize the low pH environment created within PLGA.

[0007] All references cited herein, including patent applications and publications, are incorporated by reference in their entirety. Summary of the Invention

[0008] In some aspects, the present invention provides a formulation comprising a polypeptide and hydroxypropylmethylcellulose acetate succinate (HPMCAS). In some embodiments, the HPMCAS comprises about 8% to about 12% acetate and about 7% to about 15% succinate. In some embodiments, the HPMCAS comprises about 8% acetate and about 15% succinate (HPMCAS-LF). In other embodiments, the HPMCAS comprises about 12% acetate and about 7% succinate (HPMCAS-HF). In some embodiments, the ratio of HPMCAS to protein in the formulation is about 10:1 (mg / mg) to about 1:10 (mg / mg). In some embodiments, the ratio of HPMCAS to protein in the formulation is about 4:1 (mg / mg) to about 1:4 (mg / mg). In some embodiments, the ratio of HPMCAS to protein in the formulation is about 4:1 (mg / mg). In some embodiments, the ratio of HPMCAS to protein in the formulation is about 1:1 (mg / mg). In some embodiments, the ratio of HPMCAS to protein in the formulation is about 1:4 (mg / mg). In some embodiments, the ratio of HPMCAS to protein in the formulation is about 1:1 (mg / mg) to about 1:5 (mg / mg). In some embodiments, the formulation further comprises a histidine buffer. In some embodiments, the histidine buffer is a histidine HCl buffer. In some embodiments, the histidine buffer is present in the formulation at a concentration of about 5 mM to about 20 mM. In some embodiments, the histidine buffer is present in the formulation at a concentration of about 10 mM. In some embodiments, the formulation further comprises a polysorbate. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate in the formulation is at a concentration of about 0.005% (w / v) to about 0.5% (w / v). In some embodiments, the polysorbate is present in the formulation at a concentration of about 0.01% (w / v). In some embodiments, the pH of the formulation is about 5.5 to about 7.0. In some embodiments, the pH of the formulation is about 6.5.

[0009] In some embodiments of the invention, the polypeptide in the formulation is an antibody. In some embodiments, the polypeptide is a monoclonal antibody. In some embodiments, the polypeptide is a human antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, and a (Fab')2 fragment. In some embodiments, the antibody fragment is a (Fab')2 fragment. In some embodiments, the polypeptide is present in the formulation at a concentration of about 5 mg / mL to about 100 mg / mL or about 10 mg / mL to about 80 mg / mL. In some embodiments, the polypeptide is present in the formulation at a concentration of about 10 mg / mL.

[0010] In some embodiments, the present invention provides a formulation comprising an antibody at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS-LF in a ratio of HPMCAS-LF to antibody of about 1:1 (mg / mg), histidine-HCl buffer at a concentration of about 5 mM to about 20 mM, and polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), wherein the pH of the formulation is about 5.5 to about 7.0. In some embodiments, the present invention provides a formulation comprising an antibody at a concentration of about 10 mg / mL, HPMCAS-LF in a ratio of HPMCAS-LF to antibody of about 1:1 (mg / mg), histidine-HCl buffer at a concentration of about 10 mM, and polysorbate 20 at a concentration of about 0.01% (w / v), wherein the pH of the formulation is about 6.5. In some embodiments, the present invention provides a formulation comprising an antibody at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS-HF in a ratio of HPMCAS-HF to antibody of about 1:1 (mg / mg), histidine-HCl buffer at a concentration of about 5 mM to about 20 mM, and polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), wherein the pH of the formulation is about 5.5 to about 7.0. In some embodiments, the present invention provides a formulation comprising an antibody at a concentration of about 10 mg / mL, HPMCAS-HF in a ratio of HPMCAS-HF to antibody of about 1:1 (mg / mg), histidine-HCl buffer at a concentration of about 10 mM, and polysorbate 20 at a concentration of about 0.01% (w / v), wherein the pH of the formulation is about 6.5.

[0011] In some embodiments of the invention, the formulation is spray-dried to form a spray-dried formulation of the polypeptide or antibody. In some embodiments, the polypeptide (e.g., antibody) in the formulation is stable at about 90°C for at least about 5 hours and / or stable at about 37°C for at least about 4 weeks. In some embodiments, the polypeptide in the formulation exhibits reduced aggregation and / or reduced chemical degradation. In some embodiments, reduced chemical degradation includes reduced formation of succinimide variants of the polypeptide and / or reduced formation of pyroglutamate variants of the polypeptide. In some embodiments, the formulation is an amorphous glassy formulation.

[0012] In some embodiments of the present invention, a spray-dried formulation of a polypeptide (e.g., an antibody) is encapsulated in a polymer system that generates an acidic microclimate. In some embodiments, the spray-dried formulation of a polypeptide is encapsulated in a lactic acid / glycolic acid polymer system. In some embodiments, the spray-dried formulation of a polypeptide is encapsulated in poly(lactic-co-glycolic acid) (PLGA). In some embodiments, the spray-dried formulation of a polypeptide is encapsulated in a PLGA rod.

[0013] In some aspects, the present invention provides compositions comprising any of the formulations described herein; for example, compositions comprising a spray-dried formulation of a polypeptide (e.g., an antibody) described herein. In some embodiments, the present invention provides compositions comprising lactic acid / glycolic acid polymer particles comprising any of the formulations described herein. In some embodiments, the present invention provides compositions comprising lactic acid / glycolic acid polymer particles comprising any of the spray-dried formulations described herein. In some embodiments, the lactic acid / glycolic acid polymer particles are PLGA particles. In some embodiments, the lactic acid / glycolic acid polymer particles are PLGA rods.

[0014] In some aspects, the invention provides methods for preparing a spray-dried polypeptide, the method comprising preparing a formulation described herein and subjecting the formulation to spray drying. In some aspects, the invention provides methods for preparing a spray-dried antibody, the method comprising preparing a formulation described herein and subjecting the formulation to spray drying. In some embodiments, the spray drying is carried out using a spray dryer comprising an inlet and an outlet. In some embodiments, the inlet has a temperature of about 90°C to about 120°C, about 100°C, or about 110°C. In some embodiments, the outlet has a temperature of about 60°C.

[0015] In some aspects, the present invention provides methods for preparing lactic acid / glycolic acid polymer particles comprising a polypeptide, the methods comprising encapsulating a formulation described herein in a lactic acid / glycolic acid polymer system. In some aspects, the present invention provides methods for preparing lactic acid / glycolic acid polymer particles comprising a polypeptide, the methods comprising encapsulating a spray-dried formulation described herein in a lactic acid / glycolic acid polymer system. In some embodiments, the lactic acid / glycolic acid polymer particles are PLGA particles.

[0016] In some aspects, the present invention provides articles of manufacture comprising any of the formulations (e.g., spray-dried formulations) or compositions described herein. [Brief explanation of the drawings]

[0017] [Figure 1] Figure 1 shows the structure of trehalose and the general structure of cellulose. The bottom panel shows the nature of the R groups that define metolose, AS-LF, and AS-HF, as well as the molecular weights and glass transition temperatures of the polymer excipients, along with trehalose.

[0018] [Figure 2] FIG. 2 shows the glass transition temperatures of the formulations as revealed by differential scanning calorimetry (DSC) thermograms of all five spray-dried Fab2 formulations.

[0019] [Figure 3] Figure 3 is a schematic diagram showing the preparation of spray-dried AS-LF and AS-HF formulations. The AS-HF or AS-LF polymer was dissolved in water, titrated to pH 6.5 with histidine base, and then mixed with a solution of Fab2 in pH 5.5 buffer. The cloudy polymer-protein mixture was spray-dried to obtain a solid formulation.

[0020] [Figure 4]Figures 4A and 4B show the characterization of the spray-dried formulation. Figure 4A shows the size distribution of the spray-dried Fab2 formulation measured using laser diffraction. Figure 4B shows an SEM image of the spray-dried formulation. The scale bar in each image corresponds to 10 μm.

[0021] [Figure 5] Figures 5A and 5B show Fab2 aggregation in spray-dried formulations. Change in monomer content as a function of time for Fab2 in spray-dried formulations after exposure to 90°C for 5 hours (Figure 5A) and 37°C for 4 weeks (Figure 5B).

[0022] [Figure 6] Figures 6A-6D show the chemical degradation of Fab2 in spray-dried formulations. Ion-exchange chromatograms of Fab2 in different formulations after 5 hours at 90°C (Figure 6A) and after 4 weeks at 37°C (Figure 6B). Pyroglutamate formation, measured as the ratio of PyroE to main peak area, with time at 90°C (Figure 6C) and 37°C (Figure 6D).

[0023] [Figure 7] Figures 7A and 7B show the buffering capacity of the HPMCAS excipient, which resists changes in pH during titration with a strong base (0.5 N NaOH) in (Figure 7A) the raw form and a strong acid (0.1 N HCl) in (Figure 7B) the spray-dried Fab2 formulation.

[0024] [Figure 8] Figure 8 shows Fab2 stability within PLGA rods. Changes in extracted Fab2 monomer content at harvest after 3 weeks in PLGA rods at 37°C for all four formulations.

[0025] [Figure 9]Figure 9 is a schematic diagram showing the distribution of protein and polymer in spray-dried formulations. Spray drying of a protein-polymer mixture results in two types of distribution in the solid state. Electrostatic interactions between Fab2 and the HPMCAS polymer ensure a uniform blend in the solid state, whereas in the metolose formulation, physicochemical incompatibility leads to demixing and phase separation after spray drying.

[0026] [Figure 10] FIG. 10 shows the excipient glass transition temperatures as revealed by differential scanning calorimetry (DSC) thermograms for all four excipients used in our study.

[0027] [Figure 11] Figure 11 shows MAb1 aggregation in spray-dried formulations. The change in monomer content as a function of time is shown for MAb1 in spray-dried formulations containing various excipient-to-protein weight ratios (E / P) after exposure to 90°C for various periods (0-5 hours).

[0028] [Figure 12] Figure 12 shows MAb1 aggregation in spray-dried formulations. The change in monomer content as a function of time is shown for MAb1 in spray-dried formulations containing various excipient-to-protein weight ratios (E / P) after exposure to 37°C for various periods (0-8 weeks).

[0029] [Figure 13] Figure 13 shows MAb2 aggregation in spray-dried formulations. The change in monomer content as a function of time is shown for MAb2 in spray-dried formulations containing various excipient-to-protein weight ratios (E / P) after exposure to 90°C for various periods (0 to 4 or 5 hours).

[0030] [Figure 14]Figure 14 shows MAb2 aggregation in spray-dried formulations. The change in monomer content as a function of time is shown for MAb2 in spray-dried formulations containing various excipient-to-protein weight ratios (E / P) after exposure to 37°C for various periods (0 to 8 weeks).

[0031] [Figure 15] Figure 15 shows the buffering capacity of MAb1 formulations containing different excipients. The change in pH is shown when dilute hydrochloric acid is added to a spray-dried MAb1 formulation dissolved in water. The arrows indicate increasing excipient content in the protein-containing formulation.

[0032] [Figure 16] Figure 16 shows the buffering capacity of MAb2 formulations containing different excipients. The change in pH is shown when dilute hydrochloric acid is added to a spray-dried MAb2 formulation dissolved in water. The arrows indicate increasing excipient content in the protein-containing formulation.

[0033] [Figure 17] Figure 17 shows MAb1 stability in PLGA rods. The change in extracted MAb1 monomer content upon recovery after 4 weeks in PLGA rods at 37°C is shown for the four formulations.

[0034] [Figure 18] Figure 18 shows MAb2 stability in PLGA rods. The change in extracted MAb2 monomer content upon recovery after 4 weeks in PLGA rods at 37°C is shown for the four formulations.

[0035] [Figure 19] Figure 19 shows MAb3 aggregation in spray-dried formulations. The change in monomer content as a function of time is shown for MAb3 in spray-dried formulations containing various excipient-to-protein weight ratios (E / P) after exposure to 90°C for various periods (0-5 hours).

[0036] [Figure 20]Figure 20 shows MAb3 aggregation in spray-dried formulations. The change in monomer content as a function of time is shown for MAb3 in spray-dried formulations containing various excipient-to-protein weight ratios (E / P) after exposure to 37°C for various periods (0 to 3 months). DETAILED DESCRIPTION OF THE INVENTION

[0037] In some aspects, the present invention provides formulations comprising a polypeptide (e.g., an antibody) and a hydroxypropylmethylcellulose acetate succinate (HPMCAS) derivative. The use of HPMCAS provides stability to the protein during high-temperature processing and potentially low-pH environments of the drug delivery platform. Additionally, within the poly(lactic-co-glycolic acid) (PLGA)-based delivery system, HPMCAS provides protection for pH-sensitive polypeptides against acidic degradation products from aqueous hydrolysis of PLGA. In some embodiments, the HPMCAS contains about 8% acetate and about 15% succinate (HPMCAS-LF) or about 12% acetate and about 7% succinate (HPMCAS-HF).

[0038] In some embodiments, the present invention provides a formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS in a ratio of HPMCAS to polypeptide of about 1:1 (mg:mg) in the formulation, histidine-HCl buffer at a concentration of about 5 mM to about 20 mM, and polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), wherein the formulation has a pH of about 6.0 to about 7.0.

[0039] In some aspects, the invention provides a spray-dried formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS in a ratio of HPMCAS to polypeptide of about 1:1 (mg:mg) in the formulation, histidine-HCl buffer at a concentration of about 5 mM to about 20 mM, and polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), wherein the pH of the formulation is about 6.0 to about 7.0, and the polypeptide in the formulation is stable at about 90°C for at least about 5 hours and / or stable at about 37°C for at least about 4 weeks.

[0040] Formulations, spray-dried formulations, and methods for preparing the PLGA-encapsulated polypeptides, as well as articles of manufacture comprising the formulations, spray-dried formulations, and PLGA-encapsulated polypeptides, are also provided. definition

[0041] "Spray drying" refers to the process of atomizing and drying a liquid or slurry containing a protein or monoclonal antibody using a gas (usually air or nitrogen) at temperatures above ambient temperature to produce dry powder particles containing the protein or monoclonal antibody. During the process, the liquid evaporates and dry particles are formed. In one embodiment, spray drying is carried out using a spray dryer having, for example, an air inlet temperature of about 80°C to about 220°C and an air outlet temperature of about 50°C to about 100°C. The particles can be separated from the gas by various methods, such as cyclones, high-pressure gas, or electrostatic charging. This definition of spray drying herein specifically excludes lyophilization or crystallization of the monoclonal antibody.

[0042] A "dry" particle, protein, or monoclonal antibody herein has been subjected to a drying process to significantly reduce its moisture content. In one embodiment, the particle, protein, or monoclonal antibody has a moisture content of less than about 10%, e.g., less than about 5%, e.g., when the moisture content is measured by chemical titration (e.g., Karl Fischer) or weight loss (high temperature heating) methods.

[0043] As used herein, a "pre-spray dried preparation" refers to a preparation of a polypeptide (e.g., an antibody) and one or more excipients, such as a stabilizer (e.g., an HPMCAS derivative), and optionally a buffer. In one embodiment, the preparation is in liquid form.

[0044] A "suspension formulation" is a liquid formulation containing solid particles (e.g., spray-dried antibody particles) dispersed throughout a liquid phase in which they are not soluble. In one embodiment, the solid particles in the suspension formulation have an average particle size of about 2 to about 30 microns, e.g., about 5 to about 10 microns (e.g., as analyzed by laser diffraction). Optionally, the solid particles in the suspension formulation have a peak (highest percentage) particle size of less than about 30 microns, optionally less than about 10 microns (e.g., as analyzed by laser diffraction). A suspension formulation can be prepared by combining spray-dried antibody particles with a non-aqueous suspension vehicle. In one embodiment, the suspension formulation is adapted or suitable for subcutaneous administration to a subject or patient.

[0045] The terms "polypeptide" and "protein" are used interchangeably herein to refer to polymers of amino acids of any length. Polymers may be linear or branched, may comprise modified amino acids, and may be interrupted by non-amino acids. These terms also encompass amino acid polymers that are modified naturally or by intervention, e.g., disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification, such as conjugation with a labeling component. For example, polypeptides containing one or more analogs of an amino acid (including, e.g., unnatural amino acids), as well as other modifications known in the art, are also included within this definition. The terms "polypeptide" and "protein," as used herein, specifically encompass antibodies.

[0046] A "purified" polypeptide (e.g., an antibody or immunoadhesin) means that the polypeptide is present in a purer form than it is in its natural environment and / or that the purity of the polypeptide has been increased when it is initially synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily imply absolute purity.

[0047] A polypeptide that "binds to" an antigen of interest, e.g., a tumor-associated polypeptide antigen target, is one that binds the antigen with sufficient affinity such that the polypeptide is useful as a diagnostic and / or therapeutic agent in targeting cells or tissues that express the antigen and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the polypeptide to "non-target" polypeptides is less than about 10% of the binding of the polypeptide to its particular target polypeptide, as determined by fluorescence-activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0048] With respect to the binding of a polypeptide to a target molecule, the terms "specific binding" or "specifically binds to," or "specific for" a particular polypeptide or epitope on a particular polypeptide target, refer to binding that is distinct from nonspecific interactions. Specific binding can be measured, for example, by determining the binding of a molecule relative to the binding of a control molecule, which is generally a molecule of similar structure that does not have binding activity. For example, specific binding can be determined by competition for the target with a similar control molecule, e.g., excess unlabeled. In this case, specific binding is indicated if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target.

[0049] The term "antibody" herein is used in the broadest sense and specifically encompasses monoclonal antibodies, polyclonal antibodies, multispecific antibodies (e.g., bispecific antibodies) formed from at least two intact antibodies, and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0050] Antibodies are naturally occurring immunoglobulin molecules with various structures, all of which are based on the immunoglobulin fold. For example, IgG antibodies have two "heavy" chains and two "light" chains that are disulfide-bonded to form a functional antibody. Each heavy and light chain contains a "constant" (C) region and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and functions in non-antigen-specific interactions with immune effectors. The antigen-binding specificity of an antibody or an antigen-binding fragment of an antibody is the ability of the antibody to specifically bind to a particular antigen.

[0051] The antigen-binding specificity of antibodies is determined by structural features of the V regions. The variability is not evenly distributed across the 110 amino acid length of the variable domains. Instead, V regions consist of relatively invariant stretches of 15-30 amino acids called framework regions (FRs), separated by short regions of extreme variability called "hypervariable regions," each 9-12 amino acids long. Native heavy and light chain variable domains each contain four FRs that primarily adopt a β-sheet configuration, connected by three hypervariable regions ("HVRs"), which form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions within each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as contributing to antibody-dependent cellular cytotoxicity (ADCC).

[0052] Each V region typically contains three hypervariable regions, e.g., complementarity-determining regions ("CDRs"), each containing a "hypervariable loop," and four framework regions. Thus, an antibody combining site, which is the minimum structural unit required to bind with substantial affinity to a particular desired antigen, typically contains three CDRs and at least three, and preferably four, framework regions interspersed therebetween to hold and present the CDRs in the proper conformation. Classical four-chain antibodies contain a V H Domain and V L They have antigen-binding sites determined by the combined efforts of the V domains. Certain antibodies, such as camel and shark antibodies, lack light chains and rely on binding sites formed exclusively by heavy chains. Single-domain engineered immunoglobulins are characterized by V H and V LIn the absence of cooperation between the heavy and light chains, the binding site can be prepared to be formed by only the heavy or light chain.

[0053] The term "variable" refers to the fact that the sequences of certain portions of the variable domains vary widely among antibodies and are used in the binding and specificity of each particular antibody to its particular antigen. However, the variability is not evenly distributed throughout the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in both the light-chain and heavy-chain variable domains. The more highly conserved portions of the variable domains are called framework regions (FRs). Native heavy and light-chain variable domains each contain four FRs, which largely adopt a β-sheet configuration, connected by three hypervariable regions, which form loops that connect, and in some cases form part of, the β-sheet structure. The hypervariable regions within each chain are held together in close proximity by the FRs and, together with the hypervariable regions from the other chain, contribute to the formation of the antigen-binding site of antibodies (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, MD. (1991)). The constant domains are not involved directly in binding an antibody to an antigen, but exhibit various effector functions, such as contributing to antibody-dependent cellular cytotoxicity (ADCC).

[0054] The term "hypervariable region" as used herein refers to the amino acid residues of an antibody that are responsible for antigen binding. A hypervariable region comprises amino acid residues from the "complementarity determining regions" or "CDRs" (e.g., V L Approximately residues 24-34 (L1), 50-56 (L2), and 89-97 (L3) of V Habout residues 31-35B (H1), 50-65 (H2), and 95-102 (H3) of the VH1 protein (Kabat et al., Sequences of Proteins of Immunological Interest, 5th Ed. Public Health Service, National Institutes of Health, Bethesda, Md. (1991)) and / or those residues from the "hypervariable loops" (e.g., VH1, VH2, VH3, VH4, VH5, VH6, VH7, VH8, VH9, VL10, VL20, VL30, VL41, VL52, VL60, VL71, VL82, VL93, VL94, VL105, VL110, VL120, VL130, VL140, VL150, VL160, VL170, VL180, VL1 L Residues 26–32 (L1), 50–52 (L2), and 91–96 (L3), and V H 26-32 (H1), 52A-55 (H2), and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0055] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0056] "Antibody fragments" include portions of intact antibodies, preferably including their antigen-binding regions. Examples of antibody fragments include Fab, Fab', F(ab'), and Fv fragments, diabodies, tandem diabodies (taDb), triabodies, linear antibodies (e.g., U.S. Pat. No. 5,641,870, Example 2; Zapata et al., Protein Eng. 8(10):1057-1062(1995)); one-arm antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules, multispecific antibodies formed from antibody fragments (e.g., but not limited to, Db-Fc, taDb-Fc, taDb-CH3, (scFV)4-Fc, sc-Fv, di-scFv, bi-scFv, or tandem (di, tri)-scFv); bispecific T cell-inducing antibodies (BiTEs), and trifunctional antibodies.

[0057] Papain digestion of antibodies produces two identical antigen-binding fragments called "Fab" fragments, each with a single antigen-binding site, and a residual "Fc" fragment, named for its ability to crystallize readily. Pepsin treatment yields an F(ab')2 fragment that has two antigen-binding sites and is still capable of cross-linking antigen.

[0058] "Fv" is the minimum antibody fragment which contains a complete antigen-recognition and antigen-binding site. This region consists of a dimer of one heavy- and one light-chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to form the V H -V L The six hypervariable regions define an antigen-binding site on the surface of the dimer. Together, the six hypervariable regions confer antigen-binding specificity to the antibody. However, even a single variable domain (or half of an Fv containing only three hypervariable regions specific for an antigen) has the ability to recognize and bind antigen, although with lower affinity than the entire binding site.

[0059] Fab fragments also contain the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. Fab' fragments differ from Fab fragments by the addition of a few residues at the carboxy terminus of the heavy chain CH1 domain, including one or more cysteines from the antibody hinge region. Fab'-SH is the designation used herein for Fab' in which the cysteine ​​residue(s) of the constant domains bear at least one free thiol group. F(ab')2 antibody fragments were originally produced as pairs of Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0060] The "light chains" of antibodies (immunoglobulins) from any vertebrate species can be assigned to one of two clearly distinct types, called kappa (κ) and lambda (λ), based on the amino acid sequences of their constant domains.

[0061] Antibodies can be assigned to different classes depending on the amino acid sequence of the constant domain of their heavy chain. There are five major classes of intact antibodies: IgA, IgD, IgE, IgG, and IgM, and some of these may be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chain constant domains that correspond to the different classes of antibodies are called α, δ, ε, γ, and μ, respectively. The subunit structures and three-dimensional configurations of the different classes of immunoglobulins are well known.

[0062] "Single-chain Fv" or "scFv" antibody fragments are fragments of the V H and V L In some embodiments, the Fv polypeptide comprises a V domain, which enables the scFv to form the desired structure for antigen binding. H Domains and V L For a review of scFvs, see Pluckthun, The Pharmacology of Monoclonal Antibodies, vol. 113, Rosenburg and Moore eds., Springer-Verlag, New York, pp. 269-315 (1994).

[0063] The term "diabody" refers to small antibody fragments with two antigen-binding sites, which are composed of a light-chain variable domain (V) within the same polypeptide chain. L ) bound to the heavy chain variable domain (V H ) including (V H -V L(The term "diabodies" is used interchangeably with "diabodies" and is used interchangeably with "diabodies" in the context of "antigen binding sites" and "antigen binding sites ...

[0064] The term "multispecific antibody" is used in the broadest sense and specifically includes antibodies with polyepitopic specificity. Such multispecific antibodies include heavy chain variable domains (V H ) and the light chain variable domain (V L ), an antibody comprising V H V L Antibodies with polyepitopic specificity, where the unit is two or more V L Domain and V H Each V H V L Examples of multispecific antibodies include, but are not limited to, antibodies that bind to different epitopes, antibodies having two or more single variable domains, each of which binds to a different epitope, full-length antibodies, antibody fragments such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, and covalently or non-covalently linked antibody fragments. "Polyepitopic specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different target(s). "Monospecificity" refers to the ability to bind to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 M to 0.001 pM, 3 M to 0.001 pM, 1 M to 0.001 pM, 0.5 M to 0.001 pM, or 0.1 M to 0.001 pM.

[0065] The phrase "single domain antibody" (sdAb) or "single variable domain (SVD) antibody" generally refers to an antibody in which a single variable domain (VH or VL) is capable of conferring antigen binding. In other words, the single variable domain does not need to interact with other variable domains to recognize the target antigen. Examples of single domain antibodies include antibodies derived from camelids (llamas and camels) and cartilaginous fish (e.g., nurse sharks), as well as antibodies obtained by recombinant methods from human and murine antibodies (Nature (1989) 341:544-546, Dev Comp Immunol (2006) 30:43-56, Trends Biochem Sci (2001) 26:230-235, Trends Biotechnol (2003): 21:484-490, WO 2005 / 035572, WO 03 / 035694, FEBs Lett (1994) 339:285-290, WO 00 / 29004, WO 02 / 051870).

[0066] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, excluding possible variants that may arise during monoclonal antibody production, although such variants will generally be present in minor amounts. In contrast to polyclonal antibody preparations, which typically include different antibodies directed against different determinants (epitopes), each monoclonal antibody is directed against a single determinant on the antigen. In addition to their specificity, monoclonal antibodies are advantageous in that they are uncontaminated by other immunoglobulins. The modifier "monoclonal" indicates the character of the antibody as being obtained from a substantially homogeneous antibody population, and is not to be construed as requiring production of the antibody by any particular method. For example, the monoclonal antibodies to be used in accordance with the methods provided herein may be made by the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (see, e.g., U.S. Pat. No. 4,816,567). The "monoclonal antibodies" may also be isolated from phage antibody libraries using the techniques described in, for example, Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991).

[0067] The term "monoclonal antibodies" as used herein specifically includes "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chain is identical to 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 to 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 (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851-6855 (1984)). Chimeric antibodies of interest herein include "primatized" antibodies containing variable domain antigen-binding sequences derived from a non-human primate (e.g., an Old World monkey such as a baboon, rhesus monkey, or cynomolgus monkey) and human constant region sequences (U.S. Pat. No. 5,693,780).

[0068] "Humanized" forms of non-human (e.g., murine) antibodies are chimeric antibodies that contain minimal sequence derived from non-human immunoglobulin. Most often, humanized antibodies are human immunoglobulins (recipient antibody) in which residues from a hypervariable region of the recipient are replaced by residues from a hypervariable region of a non-human species (donor antibody), such as mouse, rat, rabbit, or non-human primate, possessing the desired specificity, affinity, and capacity. In some instances, framework region (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 are made to further refine antibody performance. Generally, humanized antibodies comprise substantially all of at least one, and typically two, variable domains, in which all or substantially all of the hypervariable loops correspond to those of a non-human immunoglobulin and all or substantially all of the FRs are those of a human immunoglobulin sequence, except for FR substitution(s) noted above. A humanized antibody optionally comprises at least a portion of an immunoglobulin constant region, typically that of a human immunoglobulin. For further details, see Jones 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).

[0069] As used herein, an "intact antibody" comprises heavy and light variable domains, as well as an Fc region. The constant domains may be native sequence constant domains (e.g., human native sequence constant domains) or amino acid sequence variants thereof. Preferably, the intact antibody possesses one or more effector functions.

[0070] "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, while 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 has at one end a variable domain (V H ) followed by a number of 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.

[0071] A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or radiolabel.

[0072] In some embodiments, antibody "effector functions" refer to the biological activities attributable to the Fc region (a native sequence Fc region or amino acid sequence variant Fc region) of an antibody, which vary depending on the antibody isotype. Examples of antibody effector functions include: C1q binding and complement-dependent cytotoxicity; Fc receptor binding; antibody-dependent cell-mediated cytotoxicity (ADCC); phagocytosis; and down-regulation of cell surface receptors.

[0073] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells expressing Fc receptors (FcR), such as natural killer (NK) cells, neutrophils, and macrophages, recognize bound antibodies on target cells and subsequently cause lysis of the target cells. NK cells, the primary cells for mediating ADCC, express FcγRIII only, whereas monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3, page 464, of Ravetch and Kinet, Annu. Rev. Immunol. 9:457-92 (1991). To assess ADCC activity of a molecule of interest, an in vitro ADCC assay, such as that described in U.S. Pat. No. 5,500,362 or U.S. Pat. No. 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMC) and natural killer (NK) cells. Alternatively, or additionally, ADCC activity of the molecule of interest may be assessed in vivo, e.g., in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0074] A "human effector cell" is a leukocyte that expresses one or more FcRs and performs effector function. In some embodiments, the cells express at least FcγRIII and perform ADCC effector function. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils, of which PBMCs and NK cells are preferred.

[0075] The terms "Fc receptor" or "FcR" are used to describe a receptor that binds to the Fc region of an antibody. In some embodiments, the FcR is a native-sequence human FcR. Furthermore, preferred FcRs are those that bind IgG antibodies (gamma receptors), including receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA (an "activating receptor") and FcγRIIB (an "inhibiting receptor"), which have similar amino acid sequences that differ primarily in their cytoplasmic domains. Activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. Inhibiting receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See M. Daeron, Annu. Rev. Immunol. 15:203-234 (1997)). FcRs are reviewed in Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991), Capel et al., Immunomethods 4:25-34 (1994), and de Haas et al., J. Lab. Clin. Med. 126:330-41 (1995). Other FcRs, including those identified in the future, are encompassed by the term "FcR" herein. This term also encompasses FcRn, the neonatal receptor responsible for the transfer of maternal IgG to the fetus (Guyer et al., J. Immunol. 117:587 (1976) and Kim et al., J. Immunol. 24:249 (1994)).

[0076] Reference herein to "about" a value or parameter includes (and describes) variations on the value or parameter itself. For example, a reference to "about X" includes the description of "X."

[0077] As used in this specification and the appended claims, the singular forms "a," "or," and "the" include plural referents unless the context clearly dictates otherwise. It is understood that aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" aspects and variations. formulation

[0078] In some aspects, the invention provides a formulation comprising a polypeptide and hydroxypropyl methylcellulose acetate succinate (HPMCAS). In some embodiments, the HPMCAS in the formulation comprises between about 1% acetate and about 20% acetate. In some embodiments, the HPMCAS in the formulation comprises between about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or greater than 20% acetate. In some embodiments, the HPMCAS in the formulation comprises between about 1% succinate and about 20% succinate. In some embodiments, the HPMCAS in the formulation contains about 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, 20%, or more than 20% succinate. In some embodiments, the HPMCAS in the formulation contains about 1% acetate to about 20% acetate and about 1% succinate to about 20% acetate. In some embodiments, the HPMCAS in the formulation contains about 8% acetate and about 15% succinate (HPMCAS-LF) or about 12% acetate and about 7% succinate (HPMCAS-HF).

[0079] In some embodiments, the ratio of HPMCAS to protein in the formulation is between about 10:1 (mg / mg) and about 1:10 (mg / mg). In some embodiments, the ratio of HPMCAS to protein in the formulation is between about 1:1 (mg / mg) and 1:10 (mg / mg), 1:1 (mg / mg) and 1:9 (mg / mg), 1:1 (mg / mg) and 1:8 (mg / mg), 1:1 (mg / mg) and 1:7 (mg / mg), 1:1 (mg / mg) and 1:6 (mg / mg), 1:1 (mg / mg) and 1:5 (mg / mg), 1:1 (mg / mg) and 1:4 (mg / mg), 1:1 (mg / mg) and 1:3 (mg / mg), or 1:1 (mg / mg) and 1:2 (mg / mg). ), 1:2(mg / mg)~1:10(mg / mg), 1:2(mg / mg)~1:9(mg / mg), 1:2(mg / mg)~1:8(mg / mg), 1:2(mg / mg)~1:7(mg / mg), 1:2(mg / mg)~1:6(mg / mg), 1: 2(mg / mg)~1:5(mg / mg), 1:2(mg / mg)~1:4(mg / mg), 1:2(mg / mg)~1:3(mg / mg), 1:3(mg / mg)~1:10(mg / mg), 1:3(mg / mg)~1:9(mg / mg), 1:3(mg) / mg)~1:8(mg / mg), 1:3(mg / mg)~1:7(mg / mg), 1:3(mg / mg)~1:6(mg / mg), 1:3(mg / mg)~1:5(mg / mg), 1:3(mg / mg)~1:4(mg / mg), 1:4(mg / mg)~ 1:10(mg / mg), 1:4(mg / mg)~1:9(mg / mg), 1:4(mg / mg)~1:8(mg / mg), 1:4(mg / mg)~1:7(mg / mg), 1:4(mg / mg)~1:6(mg / mg), 1:4(mg / mg)~1:5( mg / mg), 1:5(mg / mg)~1:10(mg / mg), 1:5(mg / mg)~1:9(mg / mg), 1:5(mg / mg)~1:8(mg / mg), 1:5(mg / mg)~1:7(mg / mg), 1:5(mg / mg)~1:6(mg / m) g), 1:6(mg / mg)~1:10(mg / mg), 1:6(mg / mg)~1:9(mg / mg), 1:6(mg / mg)~1:8(mg / mg), 1:6(mg / mg)~1:7(mg / mg), 1:7(mg / mg)~1:10(mg / mg),The ratio is between 1:7 (mg / mg) and 1:9 (mg / mg), 1:7 (mg / mg) and 1:8 (mg / mg), 1:8 (mg / mg) and 1:10 (mg / mg), 1:8 (mg / mg) and 1:9 (mg / mg), or 1:9 (mg / mg) and 1:10 (mg / mg). In some embodiments, the ratio of HPMCAS to protein in the formulation is about 10:1 (mg / mg) to 1:1 (mg / mg), 9:1 (mg / mg) to 1:1 (mg / mg), 8:1 (mg / mg) to 1:1 (mg / mg), 7:1 (mg / mg) to 1:1 (mg / mg), 6:1 (mg / mg) to 1:1 (mg / mg), 5:1 (mg / mg) to 1:1 (mg / mg), 4:1 (mg / mg) to 1:1 (mg / mg), 3:1 (mg / mg) to 1:1 (mg / mg), 2:1 (mg / mg), or g)~1:1(mg / mg), 10:1(mg / mg)~2:1(mg / mg), 9:1(mg / mg)~2:1(mg / mg), 8:1(mg / mg)~2:1(mg / mg), 7:1(mg / mg)~2:1(mg / mg), 6:1(mg) / mg)~2:1(mg / mg), 5:1(mg / mg)~2:1(mg / mg), 4:1(mg / mg)~2:1(mg / mg), 3:1(mg / mg)~2:1(mg / mg), 10:1(mg / mg)~3:1(mg / mg), 9:1(m g / mg)~3:1(mg / mg), 8:1(mg / mg)~3:1(mg / mg), 7:1(mg / mg)~3:1(mg / mg), 6:1(mg / mg)~3:1(mg / mg), 5:1(mg / mg)~3:1(mg / mg), 4:1( mg / mg)~3:1(mg / mg), 10:1(mg / mg)~4:1(mg / mg), 9:1(mg / mg)~4:1(mg / mg), 8:1(mg / mg)~4:1(mg / mg), 7:1(mg / mg)~4:1(mg / mg), 6:1 (mg / mg)~4:1(mg / mg), 5:1(mg / mg)~4:1(mg / mg), 10:1(mg / mg)~5:1(mg / mg), 9:1(mg / mg)~5:1(mg / mg), 8:1(mg / mg)~5:1(mg / mg), 7 :1(mg / mg)~5:1(mg / mg), 6:1(mg / mg)~5:1(mg / mg), 10:1(mg / mg)~6:1(mg / mg), 9:1(mg / mg)~6:1(mg / mg), 8:1(mg / mg)~6:1(mg / mg),The ratio is between 7:1 (mg / mg) and 6:1 (mg / mg), 10:1 (mg / mg) and 7:1 (mg / mg), 9:1 (mg / mg) and 7:1 (mg / mg), 8:1 (mg / mg) and 7:1 (mg / mg), 10:1 (mg / mg) and 8:1 (mg / mg), 9:1 (mg / mg) and 8:1 (mg / mg), or 10:1 (mg / mg) and 9:1 (mg / mg). In some embodiments, the ratio of HPMCAS to protein in the formulation is about any of 10:1 (mg / mg), 9:1 (mg / mg), 8:1 (mg / mg), 7:1 (mg / mg), 6:1 (mg / mg), 5:1 (mg / mg), 4:1 (mg / mg), 3:1 (mg / mg), 2:1 (mg / mg), 1:1 (mg / mg), 1:2 (mg / mg), 1:3 (mg / mg), 1:4 (mg / mg), 1:5 (mg / mg), 1:6 (mg / mg), 1:7 (mg / mg), 1:8 (mg / mg), 1:9 (mg / mg), or 1:10 (mg / mg).

[0080] In some embodiments, the formulation further comprises a histidine buffer (i.e., the formulation comprises a polypeptide, HPMCAS, and a histidine buffer). In some embodiments, the histidine buffer is a histidine HCl buffer. In some embodiments, the histidine buffer (e.g., histidine HCl buffer) is present in the formulation at a concentration of about 1 mM to about 50 mM. In some embodiments, the histidine buffer in the formulation is about 1 mM to 50 mM, 5 mM to 50 mM, 10 mM to 50 mM, 15 mM to 50 mM, 20 mM to 50 mM, 25 mM to 50 mM, 30 mM to 50 mM, 40 mM to 50 mM, 1 mM to 40 mM, 5 mM to 40 mM, 10 mM to 40 mM, 15 mM to 40 mM, 20 mM to 40 mM, 25 mM to 40 mM, 30 mM to 40 mM, 1 mM to 30 mM, 5 mM to 30 mM, 1 The concentration is between 0 mM to 30 mM, 15 mM to 30 mM, 20 mM to 30 mM, 25 mM to 30 mM, 1 mM to 25 mM, 5 mM to 25 mM, 10 mM to 25 mM, 15 mM to 25 mM, 20 mM to 25 mM, 1 mM to 20 mM, 5 mM to 20 mM, 10 mM to 20 mM, 15 mM to 20 mM, 1 mM to 15 mM, 5 mM to 15 mM, 10 mM to 15 mM, 1 mM to 10 mM, 5 mM to 10 mM, and 1 mM to 5 mM. In some embodiments, the histidine buffer is present in the formulation at a concentration of about any of 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM.

[0081] In some embodiments, the formulation further comprises a surfactant (i.e., the formulation comprises the polypeptide, HPMCAS, histidine buffer, and a surfactant). In some embodiments, the surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the surfactant (e.g., polysorbate) in the formulation is at a concentration of about 0.005% (w / v) to about 1.0% (w / v). In some embodiments, the surfactant (e.g., polysorbate such as polysorbate 20) in the formulation is about 0.005% (w / v) to 1.0% (w / v), 0.01% (w / v) to 1.0% (w / v), 0.05% (w / v) to 1.0% (w / v), 0.1% (w / v) to 1.0% (w / v), 0.5% (w / v) to 1.0% (w / v), 0.005% (w / v) to 0.5% (w / v), or 0.01% (w / v) to 0.5% (w / v). / v), 0.05% (w / v) to 0.5% (w / v), 0.1% (w / v) to 0.5% (w / v), 0.005% (w / v) to 0.1% (w / v), 0.01% (w / v) to 0.1% (w / v), 0.05% (w / v) to 0.1% (w / v), 0.005% (w / v) to 0.05% (w / v), 0.01% (w / v) to 0.05% (w / v), or any concentration between 0.005% (w / v) and 0.01% (w / v). In some embodiments, the surfactant (e.g., a polysorbate such as polysorbate 20) in the formulation is at a concentration of about any of 0.005% (w / v), 0.01% (w / v), 0.05% (w / v), 0.1% (w / v), 0.5% (w / v), or 1% (w / v).

[0082] In some embodiments, the pH of the formulation is about 4.0 to about 9.0. In some embodiments, the pH of the formulation is about 4.0 to 9.0, 5.0 to 9.0, 5.5 to 9.0, 6.0 to 9.0, 6.5 to 9.0, 7.0 to 9.0, 7.5 to 9.0, 8.0 to 9.0, 4.0 to 8.0, 5.0 to 8.0, 5.5 to 8.0, 6.0 to 8.0, 6.5 to 8.0, 7.0 to 8.0, 7.5 to 8.0, 4.0 to 7.5, 5.0 to 7.5, 5.5 to In some embodiments, the pH of the formulation is about 4.0, 5.0, 5.3, 5.5, 6.0, 6.5, 7.0, 7.5, 4.0, 7.0, 5.0, 5.5, 7.0, 4.0, 5.5, 5.0, 5.5, 6.0, 6.5, 4.0, 5.0, 5.5, 5.0, 5.5, 4.0, 5.0, 5.5, 5.0, 5.5, or 4.0, 5.0, In some embodiments, the pH of the formulation is about 4.0, 5.0, 5.3, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, or 9.0.

[0083] In some embodiments, the polypeptide of the formulation is an antibody. In some embodiments, the polypeptide of the formulation is a monoclonal antibody. In some embodiments, the polypeptide of the formulation is a human antibody, a chimeric antibody, or a humanized antibody. In some embodiments, the polypeptide of the formulation is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments. In some embodiments, the polypeptide is present in the formulation at a concentration of about 5 mg / mL to about 100 mg / mL.

[0084] In some embodiments, the polypeptide (e.g., an antibody) is present in the formulation at about 5 mg / mL to 100 mg / mL, 10 mg / mL to 100 mg / mL, 15 mg / mL to 100 mg / mL, 20 mg / mL to 100 mg / mL, 25 mg / mL to 100 mg / mL, 30 mg / mL to 100 mg / mL, 40 mg / mL to 100 mg / mL, 50 mg / mL to 100 mg / mL, 60 mg / mL to 100 mg / mL, 70 mg / mL to 100 mg / mL, 75 mg / mL to 100 mg / mL, 80 mg / mL to 100 mg / mL, 90 mg / mL to 100 mg / mL, 5 mg / mL to 90 mg / mL, 10 mg / mL to 90 mg / mL, 15 mg / mL to 90 mg / mL, 20 mg / mL to 90 mg / mL, 25 mg / mL to 90 mg / mL, 30 mg / mL to 90 mg / mL, 40 mg / mL to 90 mg / mL, 50 mg / mL to 90 mg / mL, 60 mg / mL to 90 mg / mL, 70 mg / mL to 90 mg / mL, 75 mg / mL to 90 mg / mL, 80 mg / mL to 90 mg / mL, 5 mg / mL to 80 mg / mL, 10 mg / mL to 80 mg / mL, 15 mg / mL to 80 mg / mL, 20 mg / mL to 90 mg / mL, 25 mg / mL to 80 mg / mL, 30 mg / mL to 80 mg / mL, 40 mg / mL to 80 mg / mL, 50 mg / mL to 80 mg / mL, 60 mg / mL to 80 mg / mL, 70 mg / mL to 80 mg / mL, 75 mg / mL to 80 mg / mL, 5 mg / mL to 75 mg / mL, 10 mg / mL to 75 mg / mL, 15 mg / mL to 75 mg / mL, 20 mg / mL to 75 mg / mL, 25 mg / mL to 75 mg / mL, 30 mg / mL to 75 mg / mL, 40 mg / mL to 75 mg / mL, 50 mg / mL to 75 mg / mL, 60 mg / mL to 75 mg / mL, 70 mg / mL to 75 mg / mL, 5 mg / mL to 70 mg / mL, 10 mg / mL to 70 mg / mL, 15 mg / mL to 70 mg / mL, 20 mg / mL to 70 mg / mL, 25 mg / mL to 70 mg / mL, 30 mg / mL to 70 mg / mL, 40 mg / mL to 70 mg / mL, 50 mg / mL to 70 mg / mL, 60 mg / mL to 70 mg / mL, 5 mg / mL to 65 mg / mL, 10 mg / mL to 65 mg / mL, 15 mg / mL to 65 mg / mL, 20 mg / mL to 65 mg / mL, 25 mg / mL to 65 mg / mL,30mg / mL~65mg / mL, 40mg / mL~65mg / mL, 50mg / mL~65mg / mL, 60mg / mL~65mg / mL, 5mg / mL~60mg / mL, 10 mg / mL~60mg / mL, 15mg / mL~60mg / mL, 20mg / mL~60mg / mL, 25mg / mL~60mg / mL, 30mg / mL~60mg / mL, 40m g / mL~60mg / mL, 50mg / mL~60mg / mL, 5mg / mL~50mg / mL, 10mg / mL~50mg / mL, 15mg / mL~50mg / mL, 20mg / mL mL~50mg / mL, 25mg / mL~50mg / mL, 30mg / mL~50mg / mL, 40mg / mL~50mg / mL, 5mg / mL~40mg / mL, 10mg / mL

[0042] The present invention is present in a concentration of any of the following: 15mg / mL to 40mg / mL, 20mg / mL to 40mg / mL, 25mg / mL to 40mg / mL, 30mg / mL to 40mg / mL, 5mg / mL to 30mg / mL, 10mg / mL to 30mg / mL, 15mg / mL to 30mg / mL, 20mg / mL to 30mg / mL, 25mg / mL to 30mg / mL, 5mg / mL to 25mg / mL, 10mg / mL to 25mg / mL, 15mg / mL to 25mg / mL, 20mg / mL to 25mg / mL, 5mg / mL to 20mg / mL, 10mg / mL to 20mg / mL, 15mg / mL to 20mg / mL, 5mg / mL to 15mg / mL, 10mg / mL to 15mg / mL, or 5mg / mL to 10mg / mL. In some embodiments, the polypeptide (e.g., antibody) is present in the formulation at a concentration of about any of 5 mg / mL, 10 mg / mL, 15 mg / mL, 20 mg / mL, 25 mg / mL, 30 mg / mL, 35 mg / mL, 40 mg / mL, 45 mg / mL, 50 mg / mL, 55 mg / mL, 60 mg / mL, 65 mg / mL, 70 mg / mL, 75 mg / mL, 80 mg / mL, 85 mg / mL, 90 mg / mL, 95 mg / mL, 100 mg / mL, or greater than 100 mg / mL.

[0085] In some embodiments, the present invention provides a formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS-LF in a ratio of HPMCAS-LF to polypeptide of about 1:1 (mg / mg) to 1:5 (mg / mg) in the formulation, histidine-HCl buffer at a concentration of about 5 mM to about 20 mM, and polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), wherein the formulation has a pH of about 6.0 to about 7.0. In some embodiments, the invention provides a formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 10 mg / mL, HPMCAS-LF in the formulation at a ratio of about 1:1 (mg / mg) to HPMCAS-LF and polypeptide, histidine-HCl buffer at a concentration of about 10 mM, and polysorbate 20 at a concentration of about 0.01% (w / v), wherein the pH of the formulation is about 6.5.

[0086] In some embodiments, the present invention provides a formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS-HF in a ratio of HPMCAS-HF to polypeptide of about 1:1 (mg / mg) to 1:5 (mg / mg) in the formulation, histidine-HCl buffer at a concentration of about 5 mM to about 20 mM, and polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), wherein the formulation has a pH of about 6.0 to about 7.0. In some embodiments, the invention provides a formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 10 mg / mL, HPMCAS-HF in the formulation having a ratio of HPMCAS-HF to polypeptide of about 1:1 (mg / mg), histidine-HCl buffer at a concentration of about 10 mM, and polysorbate 20 at a concentration of about 0.01% (w / v), wherein the pH of the formulation is about 6.5. Spray-dried formulation

[0087] In some aspects of the invention, the formulations of the invention are spray-dried. In some embodiments, the invention provides a spray-dried formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS-LF in a ratio of HPMCAS-LF to polypeptide of about 1:1 (mg / mg) to 1:5 (mg / mg) in the formulation, histidine-HCl buffer at a concentration of about 5 mM to about 20 mM, and polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), wherein the pH of the formulation is about 6.0 to about 7.0. In some embodiments, the invention provides a spray-dried formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 10 mg / mL, HPMCAS-LF in the formulation at a ratio of HPMCAS-LF to polypeptide of about 1:1 (mg / mg), histidine-HCl buffer at a concentration of about 10 mM, and polysorbate 20 at a concentration of about 0.01% (w / v), wherein the pH of the formulation is about 6.5.

[0088] In some embodiments, the present invention provides a spray-dried formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS-HF in a ratio of HPMCAS-HF to polypeptide of about 1:1 (mg / mg) to 1:5 (mg / mg) in the formulation, histidine-HCl buffer at a concentration of about 5 mM to about 20 mM, and polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), wherein the spray-dried formulation has a pH of about 6.0 to about 7.0. In some embodiments, the invention provides a spray-dried formulation comprising a polypeptide (e.g., an antibody) at a concentration of about 10 mg / mL, HPMCAS-HF in the formulation having a ratio of HPMCAS-HF to polypeptide of about 1:1 (mg / mg), histidine-HCl buffer at a concentration of about 10 mM, and polysorbate 20 at a concentration of about 0.01% (w / v), wherein the pH of the formulation is about 6.5.

[0089] In some embodiments, the polypeptide (e.g., an antibody) in the spray-dried formulation is stable at about 90° C. for at least about 15 minutes to at least about 10 hours. In some embodiments, the polypeptide in the spray-dried formulation is stable at about 90° C. for about 15 minutes to 10 hours, 30 minutes to 10 hours, 45 minutes to 10 hours, 1 hour to 10 hours, 2 hours to 10 hours, 3 hours to 10 hours, 4 hours to 10 hours, 5 hours to 10 hours, 6 hours to 10 hours, 7 hours to 10 hours, 8 hours to 10 hours, or 9 hours to 10 hours. In some embodiments, the polypeptide in the spray-dried formulation is stable at about 90° C. for at least about 15 minutes, 30 minutes, 45 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 6 hours, 7 hours, 8 hours, 9 hours, or 10 hours.

[0090] In some embodiments, the polypeptide (e.g., antibody) in the spray-dried formulation is stable for at least about 1 to 6 weeks at about 37° C. In some embodiments, the polypeptide in the spray-dried formulation is stable at about 37° C. for any of about 1 to 6 weeks, 2 to 6 weeks, 3 to 6 weeks, 4 to 6 weeks, or 5 to 6 weeks. In some embodiments, the polypeptide in the spray-dried formulation is stable at about 37° C. for any of about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or more than 6 weeks.

[0091] In some embodiments of the invention, the polypeptide (e.g., antibody) in the formulation exhibits reduced aggregation and / or reduced chemical degradation. In some embodiments of the invention, the polypeptide in the formulation exhibits reduced aggregation and / or reduced chemical degradation compared to a similar formulation without HPMCAS. In some embodiments, reduced chemical degradation includes reduced formation of succinimide variants of the polypeptide and / or reduced formation of pyroglutamate variants of the polypeptide.

[0092] In some embodiments, the spray-dried formulation is a powder. In some embodiments, the spray-dried formulation provides an amorphous formulation of the polypeptide. In some embodiments, the spray-dried formulation provides an amorphous glassy formulation of the polypeptide. An amorphous formulation is a non-crystalline formulation that does not have long-range order. In some embodiments, the glass is a rigid inert matrix that can be measured, for example, by glass transition temperature. Encapsulated forms of polypeptides

[0093] In some embodiments, the present invention provides encapsulated forms of polypeptides. In some embodiments, the spray-dried formulations of the present invention are encapsulated in an acidic polymer system. In some embodiments, the acidic polymer system is a lactic / glycolic acid polymer system. In some embodiments, the acidic polymer system is a poly(lactic-co-glycolic acid) (PLGA) system. In some embodiments, the acidic polymer system creates a low pH microenvironment for the spray-dried polypeptide. In some embodiments, the low pH microenvironment is about pH 3 to about pH 5. In some embodiments, the low pH microenvironment is less than about pH 5. In some embodiments, the low pH microenvironment is less than about pH 6. In some embodiments, the low pH microenvironment is less than about pH 7. Without being bound by theory, the presence of HPMCAS in the spray-dried formulation stabilizes the polypeptide in the low pH microenvironment created when the spray-dried polypeptide is encapsulated.

[0094] In some embodiments, the spray-dried formulation of the polypeptide is encapsulated in PLGA. In some embodiments, the spray-dried formulation of the polypeptide is encapsulated in a PLGA rod. Method for producing a polypeptide formulation

[0095] In some embodiments, the present invention provides a method for preparing a polypeptide formulation, comprising adding hydroxypropylmethylcellulose acetate succinate (HPMCAS) to the formulation. In some embodiments, the HPMCAS comprises about 8% acetate and about 15% succinate (HPMCAS-LF) or about 12% acetate and about 7% succinate (HPMCAS-HF). In some embodiments, the ratio of HPMCAS to protein in the formulation is about 1:1 (mg / mg) to about 1:5 (mg / mg). In some embodiments, a histidine buffer (e.g., a histidine HCl buffer) is added to the formulation. In some embodiments, the histidine buffer is added to the formulation at a concentration of about 5 mM to about 20 mM. In some embodiments, the histidine buffer is added to the formulation at a concentration of about 10 mM. In some embodiments, a polysorbate (e.g., polysorbate 20) is added to the formulation. In some embodiments, the polysorbate is added to the formulation at a concentration of about 0.005% (w / v) to about 0.5% (w / v). In some embodiments, the polysorbate is added to the formulation at a concentration of about 0.01% (w / v). In some embodiments, the pH of the formulation is adjusted to about 6.0 to about 7.0. In some embodiments, the pH of the formulation is adjusted to about 6.5. In some embodiments, the polypeptide (e.g., antibody) is added to the formulation at a concentration of about 5 mg / mL to about 100 mg / mL or about 10 mg / mL to about 80 mg / mL. In some embodiments, the polypeptide (e.g., antibody) is added to the formulation at a concentration of about 10 mg / mL. Further aspects of the formulation

[0096] In some embodiments, the polypeptides in the polypeptide formulation maintain functional activity.

[0097] Formulations to be used for in vivo administration must be sterile, which is readily accomplished by filtration through sterile filtration membranes.

[0098] The formulations herein may contain two or more active compounds as needed for the particular indication being treated, preferably active compounds with complementary activities that do not adversely affect each other. For example, in addition to a polypeptide, it may be desirable to include an additional polypeptide (e.g., an antibody) in a single formulation. Alternatively, or in addition, the composition may further comprise a chemotherapeutic agent, a cytotoxic agent, a cytokine, a growth inhibitor, an antihormonal agent, and / or a cardioprotectant. Such molecules are suitably present in combination in amounts effective for the intended purpose. spray drying

[0099] In some aspects, the present invention provides spray-dried formulations of polypeptides. In some embodiments, formulations comprising a polypeptide and HPMCAS described herein are spray-dried. Spray drying is a process for producing a dry powder from a liquid or slurry by rapid drying with hot gas. In some embodiments, spray drying is a method for drying heat-sensitive materials, such as pharmaceuticals (e.g., polypeptides such as antibodies) and / or materials that may require very consistent and fine particle size. Spray drying, as used herein, differs from freeze-drying, which is commonly used to prepare monoclonal antibody formulations, because it is performed at temperatures above ambient temperature. Spray-drying temperatures are generally expressed as "air inlet" and "air outlet" temperatures. In one embodiment, spray drying is performed at an air inlet temperature of about 100°C to about 220°C (e.g., about 120°C to about 160°C) and an air outlet temperature of about 50°C to about 100°C (e.g., about 60°C to about 80°C, about 100°C, or about 120°C). In some embodiments, the air inlet temperature is about 110°C and the air outlet temperature is about 60°C. In some embodiments, the air inlet temperature is about 80° C. to about 220° C. and the air outlet temperature is about 50° C. to about 100° C. In some embodiments, the air inlet temperature is between about 80° C. to about 220° C., about 80° C. to about 180° C., about 80° C. to about 160° C., about 80° C. to about 140° C., about 90° C. to about 220° C., about 90° C. to about 180° C., about 90° C. to about 160° C., about 90° C. to about 140° C., about 100° C. to about 220° C., about 100° C. to about 180° C., about 100° C. to about 160° C., about 100° C. to about 140° C., about 110° C. to about 220° C., about 110° C. to about 180° C., about 110° C. to about 160° C., or about 110° C. to about 140° C. In some embodiments, the air inlet temperature is about 80°C, 90°C, 100°C, 110°C, 120°C, 130°C, 140°C, 150°C, 160°C, 170°C, 180°C, 190°C, 200°C, 210°C, or 220°C.In some embodiments, the air outlet temperature is between about 50°C and about 100°C, 50°C and about 90°C, 50°C and about 80°C, 50°C and about 70°C, 50°C and about 60°C, about 60°C and about 100°C, 60°C and about 90°C, 60°C and about 80°C, 60°C and about 70°C, about 70°C and about 100°C, 70°C and about 90°C, 70°C and about 80°C, about 80°C and about 100°C, 80°C and about 90°C, or about 90°C and about 100°C. In some embodiments, the air outlet temperature is about 50°C, about 60°C, about 70°C, about 80°C, about 90°C, or about 100°C.

[0100] The spray drying process generally involves: atomization of a liquid feed; drying of the droplets; and separation or recovery of the dried product.

[0101] In some embodiments, atomizers used in the invention herein include, but are not limited to, rotary atomizers, pneumatic nozzle atomizers, ultrasonic nozzle atomizers, and sonic nozzles.

[0102] Contact between the liquid feed and the drying air can occur in two different modes. In a co-current system, the drying air and particles (droplets) move in the same direction through the drying chamber. When the drying air and droplets move in opposite directions, this is called counter-current mode. Particles produced in counter-current mode typically exhibit a higher temperature than the discharge air. The discharged air itself can exit the system or be recycled. By choosing from various spray dryer designs (size, atomizer, aseptic conditions, etc.) and adjusting different process parameters (drying air flow, drying air temperature, etc.), the final powder properties, such as particle size, shape and structure, or sterility, can be modified. If the resulting moisture content of the recovered powder is not sufficiently low, post-processing may be required, for example, in the form of secondary drying (e.g., using a benchtop freeze dryer), a fluidized bed dryer and cooler, a contact dryer, or even a microwave dryer.

[0103] When a liquid feed is atomized, its surface area-to-mass ratio increases, promoting heat transfer between the air and the droplets, allowing the droplets to dry relatively quickly. Two convective processes can be involved: heat transfer (from the air to the droplets) and moisture mass transfer (from the droplets to the air). In the latter, moisture penetrates through the boundary layer surrounding each droplet. The rate of transfer can be affected by temperature, humidity, transport properties of the surrounding air, droplet diameter, and the relative velocity between the droplets and the air.

[0104] The final step in the spray drying process is typically the separation of the powder from the air / gas and removal of the dried product. In some embodiments, this step is as effective as possible to obtain a high powder yield and prevent air pollution due to the release of the powder into the atmosphere. For this purpose, various methods are available, such as cyclones, bag filters, electrostatic precipitators, high-pressure gas, electrostatic charges, and combinations thereof.

[0105] The spray drying process produces particles that include a polypeptide (eg, an antibody).

[0106] In one embodiment, the properties of the spray-dried powder include any one or more of the following: (a) average particle size: about 2 microns to about 30 microns; for example, about 2 microns to about 10 microns; (b) Particle morphology: mainly spherical particles, collapsed spheres, some depressions or holes within the particle, "dried raisin" shape; (c) water content: for example, less than about 10%, e.g., less than about 5%, when the water content is measured by chemical titration (e.g., Karl Fischer) or weight loss (high temperature heating) methods; and (d) Stability: For example, by suspending particles in a vehicle and assessing the physical and / or chemical stability and / or biological activity of the suspension preparation. In one embodiment, the percent monomer in such a preparation is 95%-100%, as assessed, for example, by size exclusion chromatography (SEC). Controlled release formulations of polypeptides

[0107] Sustained-release formulations according to the present invention typically include a polypeptide (e.g., an antibody) formulated with HPMCAS as described herein, a polymeric carrier, and a release-modifying agent for modifying the release rate of the polypeptide from the polymeric carrier. The release kinetics of the resulting composition can be adjusted by varying the manufacturing conditions of the polymer-based delivery composition. The polymeric carrier typically includes one or more biodegradable polymers or copolymers, or a combination thereof. For example, the polymeric carrier can be selected from polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polyester, poly(orthoester), poly(phosphazine), poly(phosphate ester), polyethylene glycol (PEG), polycaprolactone, or a combination thereof. Other polymers for use in the formulations of the present invention include, but are not limited to, poly(meth)acrylic acid derivatives, insoluble cellulose derivatives, polyvinyl acetate, hydroxypropyl cellulose (HPC), polyethylene oxide (PEO), polyvinyl alcohol (PVA), polyvinylpyrrolidone (PVP), poloxamers, sodium carboxymethylcellulose (NaCMC), poly(acrylic acid), polyvinylacetylphthalate (PVAP), polysaccharides (e.g., sodium alginate, carrageenan, guar, and xanthan gum), poly(meth)acrylates, Eudragit (登録商標) L, Eudragit (登録商標) L, 100-55, Eudragit (登録商標) L 30D-55, Eudragit (登録商標) L 100, Eudragit (登録商標) S, Eudragit (登録商標) S100, Eudragit (登録商標) FS, Eudragit (登録商標) FS 30D, Carbopol (登録商標) , Carbopol (登録商標) 934P, Carbopol (登録商標) 940, Carbopol (登録商標) 974P, Carbopol (登録商標) 971 (Lubrizol), and Noveon (登録商標)In some embodiments, the formulation comprises a polymer described in Maskova, E. et al., J. Controlled Release, 2020, 324:695-727, the entire contents of which are incorporated herein.

[0108] In certain embodiments, the polymer carrier is PLGA. Release-modifying agents are typically long-chain fatty alcohols, preferably containing 10 to 40 carbon atoms. Commonly used release-modifying agents include caprylic alcohol, pelargonic alcohol, capric alcohol, lauryl alcohol, myristyl alcohol, cetyl alcohol, palmitoleic alcohol, stearyl alcohol, isostearyl alcohol, elaidyl alcohol, oleyl alcohol, linoleyl alcohol, polyunsaturated elaidolinoleyl alcohol, polyunsaturated linolenyl alcohol, elaidolinolenyl alcohol, polyunsaturated ricinoleyl alcohol, arachidyl alcohol, behenyl alcohol, erucyl alcohol, lignoceryl alcohol, ceryl alcohol, montanyl alcohol, crityl alcohol, myricyl alcohol, melissyl alcohol, and geddyl alcohol.

[0109] In certain embodiments, the polypeptide (e.g., antibody) formulated with HPMCAS is incorporated into a microsphere-based sustained-release composition. In certain embodiments, the microspheres are prepared from PLGA. The amount of polypeptide incorporated into the microspheres and the release rate of the polypeptide can be controlled by varying the conditions used to prepare the microspheres. Processes for producing such sustained-release formulations are described in U.S. Patent Application Publication Nos. 2005 / 0281861 and 2008 / 0107694.

[0110] In certain embodiments, the polypeptide formulated with HPMCAS is incorporated into a biodegradable implant (e.g., a microneedle). Matrix implants (e.g., microneedles) are typically used to treat ocular diseases requiring a loading dose followed by tapering doses of the drug over a period of one day to six months (Davis et al. (2004) Curr Opin Mol Therap 6, 195-205). They are most commonly made from copolymers polylactic acid (PLA) and / or polylactic-glycolic acid (PLGA), which decompose into water and carbon dioxide. The rate and extent of drug release from the implant can be reduced by altering the relative concentrations of lactide (slow) and glycolide (fast), changing the polymer weight ratio, adding an additional coat of polymer, or using a hydrophobic, insoluble drug. Drug release generally follows first-order kinetics, with an initial burst of drug release followed by a rapid decline in drug levels. Biodegradable implants dissolve over time and do not require removal (Hsu (2007) Curr Opin Ophthalmol 18, 235-9). Biodegradable implants also allow for dosage and treatment flexibility, from short-term (weeks) to long-term (months to a year), depending on the polymer PLA / PLGA ratio, another advantage in tailoring drug delivery to disease progression, as dosage and treatment requirements can change over time.

[0111] A sustained-release formulation of polypeptides was developed using polylactic-coglycolic acid (PLGA) polymer due to its biocompatibility and wide range of biodegradability. The degradation products of PLGA, lactic acid, and glycolic acid, can be rapidly eliminated by the human body. Furthermore, the degradability of this polymer can be adjusted from several months to several years depending on its molecular weight and composition. Lewis, "Controlled release of bioactive agents from lactide / glycolide polymers," in M. Chasin and R. Langer (Eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.

[0112] In some embodiments, the controlled release formulation of the polypeptide is manufactured by a hot melt extrusion process. For example, hot melt extrusion can be performed on a 5 g or 7 cm 3Compounding can be performed using a HAAKE™ MiniCTW Micro-Conical Twin Screw Compounder (Thermo Scientific, Karlsruhe, Germany) with a barrel capacity of 1000 rpm. In some embodiments, the extruder barrel is preheated to 90°C and the screw speed is set to 40 rpm. In some embodiments, the front barrel opening of the extruder remains closed in recirculation mode. In some embodiments, pre-weighed solid polymer and spray-dried polypeptide are slowly fed manually into the extruder and recirculated back into the barrel for microcompounding. In some embodiments, the polypeptide loading for the implant is about 1% to about 30%, about 1% to about 20%, about 1% to about 15%, about 5% to about 30%, about 5% to about 20%, about 5% to about 15%, about 10% to about 30%, about 10% to about 20%, or about 10% to about 15% (w / w) polypeptide / PLGA-immobilized spray-dried formulations are used. In some embodiments, the polypeptide load for the implant is a spray-dried formulation fixed at about 1%, 5%, 10%, 15%, 20%, or 30% (w / w) polypeptide / PLGA. In some embodiments, the polypeptide load for the implant is a spray-dried formulation fixed at about 15% (w / w) polypeptide / PLGA. In some embodiments, the melt-phase blending is continued in a recirculation mode for about 30 minutes after complete feeding, and then extruded through a circular die of about 0.5 mm. In some embodiments, the extrudate is cooled to room temperature, cut into cylinders of the desired length, and stored at 2-8°C until further use. Hydroxypropyl methylcellulose acetate succinate (HPMCAS)

[0113] In some aspects, the invention provides formulations of a polypeptide and HPMCAS. In some embodiments, the HPMCAS provides protection for the polypeptide during the high temperature spray drying process, as well as protection against the low pH microenvironment during acidic polymer encapsulation.

[0114] Reaction of HMPC with acetic acid and succinic acid produces an HPMC derivative known as hydroxypropylmethylcellulose acetate succinate (referred to as HPMCAS), in which the hydroxyl groups in HPMC are modified to form the respective esters. Reaction with acetic acid converts the hydroxyl groups in HPMC to acetyl groups, while reaction with succinic acid converts the hydroxyl groups in HPMC to succinyl groups. In some embodiments, HMPC is an HPMC derivative. In some embodiments, HPMC contains 8% acetic acid (referred to as HPMCAS-LF). In some embodiments, HPMC contains 7% succinic acid (referred to as HPMCAS-HF). The acetyl and succinoyl substitution levels significantly affect the solubility-enhancing effect of HPMCAS when used as a drug carrier. Due to the acid dissociation constant (pKa) value of approximately 5 for the succinate group of HPMCAS, the polymer is primarily non-ionized at pH values ​​below 4 and partially to fully ionized at pH values ​​above approximately 5. Thus, HPMC derivatives are polymeric buffers containing succinic acid. In its unionized state (pH < 5), HPMCAS is insoluble in water and forms stable colloidal aggregates that are water-soluble near neutral conditions (pH 6.0–7.5). It is a solubility enhancer for poorly soluble drugs (Friesen, DT et al., Mol Pharm, 2008, 5(6):1003–19), is insoluble in gastric juice, and dissolves rapidly in the small intestine (Curatolo, W et al., Pharm Res, 2009, 26(6):1419–31). HPMCAS-based dispersions are suitable for small molecule-based sustained drug release applications, but their use with large protein therapeutics has not been explored. Spray-dried powder form of HPMCAS is insoluble below pH 6, begins to become soluble near pH 6–6.5, and is completely soluble at pH > 6.5. Thus, the advantage is that HPMCAS provides protection by coating the protein in spray-dried form (thus still remaining as an SD powder), preventing it from interacting with acidic counterions in the low pH microenvironment. Consequently, we sought to exploit the low pH insolubility of HPMCAS for use as a spray-dried drug carrier within the acidic microclimate of a PLGA system.However, most of the current delivery systems using HPMCAS are orally administered for small molecule drugs, and no studies have demonstrated their potential use in the delivery of large proteins (Woodley, JF, Crit Rev Ther Drug Carrier Syst, 1994, 11(2-3):61-95). Specifically, the HPMCAS formulations described herein are a novel strategy for stabilizing proteins and have no apparent implications for any particular delivery route. Furthermore, HPMCAS in its non-ionized state, with a high glass transition (>100°C), may be suitable for use in spray-dried protein therapeutics as well as an excipient for high-temperature manufacturing of drug delivery systems.

[0115] A review of HPMC as an excipient for oral and buccal drug delivery is provided by Maskova, E. et al., J. Controlled Release, 2020, 324:695-727. Polypeptides

[0116] Examples of polypeptides for use in the formulations described herein include, but are not limited to, immunoglobulins, immunoadhesins, antibodies, enzymes, hormones, fusion proteins, Fc-containing proteins, immunoconjugates, cytokines, and interleukins. Examples of polypeptides include mammalian proteins, such as renin, hormones, growth hormones including human growth hormone and bovine growth hormone, growth hormone releasing factor; parathyroid hormone, thyroid stimulating hormone, lipoproteins, alpha-1-antitrypsin, insulin A chain, insulin B chain, proinsulin, follicle stimulating hormone, calcitonin, luteinizing hormone, glucagon, clotting factors such as factor VIIIC, factor IX, tissue factor, and von Willebrand factor, anticoagulants such as protein C, atrial natriuretic factor; pulmonary surfactant, plasminogen activators such as urokinase or human urinary or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-alpha and -beta; enkephalinase; RANTES (regulated upon activation and normally expressed and secreted by T cells), human macrophage inflammatory protein (MIP-1-alpha), serum albumin, e.g. for example, human serum albumin, Mullerian inhibitory substance, relaxin A chain; relaxin B chain, prorelaxin, mouse gonadotropin-related peptide, enzymes, microbial proteins, for example, beta-lactamase; DNase; IgE; cytotoxic T lymphocyte-associated antigen (CTLA), for example, CTLA-4; inhibin, activin, vascular endothelial growth factor (VEGF); hormone or growth factor receptors, protein A or D; rheumatoid factor; neurotrophic factors, for example, bone-derived neurotrophic factor (BDNF), neurotrophic factor (NF-κB ... neurotrophin-3, neurotrophin-4, neurotrophin-5, or neurotrophin-6 (NT-3, NT-4, NT-5, or NT-6), or nerve growth factors, such as NGF-β; platelet-derived growth factor (PDGF); fibroblast growth factors, such as aFGF and bFGF; epidermal growth factor (EGF); transforming growth factors (TGFs), such as TGF-alpha and TGF-beta, including TGF-β1, TGF-β2, TGF-β3, TGF-β4, or TGF-β5;Insulin-like growth factors I and II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding proteins (IGFBPs), cytokines, CD proteins such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors; immunotoxins, fusion polypeptides, i.e., polypeptides composed of two or more heterologous polypeptides or fragments thereof, Fc-containing polypeptides, e.g., fusion proteins comprising an immunoglobulin Fc region fused to a second polypeptide or a fragment thereof, immunoconjugates, bone morphogenetic proteins (BMPs), interferons, e.g., interferon-alpha, interferon-beta, and interferon-gamma; colony-stimulating factors (CSFs), e.g., Examples include, but are not limited to, M-CSF, GM-CSF, and G-CSF; interleukins (IL), such as IL-1 through IL-10; superoxide dismutase; T-cell receptors; surface membrane proteins; decay-accelerating factors; viral antigens, such as portions of the AIDS envelope; transport proteins; homing receptors; addressins; regulatory proteins; integrins, such as CD11a, CD11b, CD11c, CD18, ICAM, VLA-4, and VCAM; tumor-associated antigens, such as CA125 (ovarian cancer antigen), or HER2, HER3, or HER4 receptors, immunoadhesins, and fragments and / or variants of any of the foregoing proteins, and proteins, such as antibodies, e.g., antibody fragments, that bind to any of the above proteins; antibody

[0117] In some embodiments of any of the methods described herein, the polypeptide or preparation described herein is an antibody.

[0118] Molecular targets of antibodies include CD proteins and their ligands, such as, but not limited to, (i) CD3, CD4, CD8, CD19, CD11a, CD20, CD22, CD34, CD40, CD79α (CD79a), and CD79β (CD79b), (ii) members of the ErbB receptor family, such as EGF receptor, HER2, HER3, or HER4 receptor, and (iii) cell adhesion molecules, such as LFA-1, Mac1, p150,95, VLA-4, ICAM-1, VCAM, and α v / β3 integrins, such as either their alpha or beta subunits (e.g., anti-CD11a, anti-CD18, or anti-CD11b antibodies), (iv) growth factors, such as VEGF, IgE, blood group antigens, flk2 / flt3 receptor, obesity (OB) receptor, mpl receptor, CTLA-4, protein C, BR3, c-met, tissue factor, β7, and the like; and (v) cell surface and transmembrane tumor-associated antigens (TAAs), such as those described in U.S. Pat. No. 7,521,541.

[0119] Other exemplary antibodies include, but are not limited to, anti-CD20 antibodies, anti-CD40 antibodies, anti-HER2 antibodies, anti-IL6 antibodies, anti-IgE antibodies, anti-IL13 antibodies, anti-Flu A antibodies, anti-TIGIT antibodies, anti-PD-L1 antibodies, anti-VEGF-A antibodies, anti-VEGF-A / ANG2 antibodies, anti-CD79b antibodies, anti-ST2 antibodies, anti-factor D antibodies, anti-factor IX antibodies, anti-factor X antibodies, anti-Abeta, anti-tau antibodies, anti-CEA antibodies, anti-CEA / CD3 antibodies, anti-CD20 / CD3 antibodies, anti-FcRH5 / CD3 antibodies, anti-Her2 / CD3 antibodies, anti-FGFR1 / KLB antibodies, FAP-4-1 BBL fusion protein, FAP-IL2v fusion protein, anti-estrogen receptor antibody, anti-progesterone receptor antibody, anti-p53 antibody, anti-EGFR antibody, anti-cathepsin D antibody, anti-Bcl-2 antibody, anti-E-cadherin antibody, anti-CA125 antibody, anti-CA15-3 antibody, anti-CA19-9 antibody, anti-c-erbB-2 antibody, anti-P-glycoprotein antibody, anti-CEA antibody, anti-retinoblastoma protein antibody, anti-ras oncoprotein antibody, anti-Lewis X antibody, anti-Ki-67 antibody, anti-PCNA antibody, anti-CD3 antibody, anti-CD4 antibody, anti-CD5 antibody, anti-CD7 antibody, anti-CD8 antibody, anti-CD9 / p24 antibody, anti-CD10 antibody, anti-CD11a antibody, anti-CD11c antibody, anti-CD13 antibody, anti-CD 14 antibodies, anti-CD15 antibodies, anti-CD19 antibodies, anti-CD22 antibodies, anti-CD23 antibodies, anti-CD30 antibodies, anti-CD31 antibodies, anti-CD33 antibodies, anti-CD34 antibodies, anti-CD35 antibodies, anti-CD38 antibodies, anti-CD41 antibodies, anti-LCA / CD45 antibodies, Examples of antibodies include anti-CD45RO antibody, anti-CD45RA antibody, anti-CD39 antibody, anti-CD100 antibody, anti-CD95 / Fas antibody, anti-CD99 antibody, anti-CD106 antibody, anti-ubiquitin antibody, anti-CD71 antibody, anti-c-myc antibody, anti-cytokeratin antibody, anti-vimentin antibody, anti-HPV protein antibody, anti-κ light chain antibody, anti-λ light chain antibody, anti-melanosome antibody, anti-prostate specific antigen antibody, anti-S-100 antibody, anti-tau antigen antibody, anti-fibrin antibody, anti-keratin antibody and anti-Tn-antigen antibody. Polyclonal antibodies

[0120] In some embodiments, the antibody is a polyclonal antibody. Polyclonal antibodies are preferably raised in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents, such as maleimidobenzoyl sulfosuccinimide ester (conjugates via cysteine ​​residues), N-hydroxysuccinimide (via lysine residues), glutaraldehyde, succinic anhydride, SOCl, or R 1 N=C=NR, where R and R 1 are different alkyl groups) to conjugate the relevant antigen to a polypeptide that is immunogenic in the species to be immunized, such as keyhole limpet hemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor.

[0121] For example, animals are immunized against the antigen, immunogenic conjugate, or derivative by combining 100 μg or 5 μg of the polypeptide or conjugate (for rabbits or mice, respectively) with 3 volumes of Freund's complete adjuvant and injecting the solution intradermally at multiple sites. One month later, 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. Seven 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 a conjugate of the same antigen but conjugated to a different polypeptide and / or through a different cross-linking reagent. Conjugates can also be produced in recombinant cell culture as polypeptide fusions. Aggregating agents such as alum are also suitably used to enhance the immune response. Monoclonal antibodies

[0122] In some embodiments, antibodies purified on reusable chromatographic materials washed by the methods of the invention are monoclonal antibodies. Monoclonal antibodies are obtained from a population of substantially homogeneous antibodies, i.e., the individual antibodies within the population are identical and / or bind to the same epitope, excluding possible variants that arise during monoclonal antibody production, although such variants are generally present in minor amounts. Thus, the modifier "monoclonal" indicates the character of the antibody as not being a mixture of individual or polyclonal antibodies.

[0123] For example, 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. Patent No. 4,816,567).

[0124] In the hybridoma method, a mouse or other suitable host animal, such as a hamster, is immunized as described herein to elicit lymphocytes that produce, or are capable of producing, antibodies that specifically bind to the polypeptide used for immunization. Alternatively, lymphocytes may be immunized in vitro. The lymphocytes are then fused with myeloma cells using a suitable fusing agent, such as polyethylene glycol, to form hybridoma cells (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)).

[0125] The hybridoma cells so prepared are seeded and grown in a suitable culture medium that preferably contains one or more substances that inhibit the growth or survival of the unfused, parental myeloma cells. For example, if the parental myeloma cells lack the enzyme hypoxanthine guanine phosphoribosyltransferase (HGPRT or HPRT), the culture medium for the hybridomas typically contains hypoxanthine, aminopterin, and thymidine (HAT medium), which substances inhibit the growth of HGPRT-deficient cells.

[0126] In some embodiments, the myeloma cells are those that fuse efficiently, support stable, high-level antibody production by selected antibody-producing cells, and are sensitive to a medium such as HAT medium. In particular, in some embodiments, the myeloma cell line is derived from a mouse myeloma line, such as MOPC-21 and MPC-11 mouse tumors available from the Salk Institute Cell Distribution Center, San Diego, California, USA, and SP-2 or X63-Ag8-653 cells available from the American Type Culture Collection, Rockville, Maryland, 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)).

[0127] Culture medium in which hybridoma cells are growing is assayed for production of monoclonal antibodies directed against the antigen. In some embodiments, the binding specificity of monoclonal antibodies produced by hybridoma cells is determined by immunoprecipitation or by an in vitro binding assay, such as radioimmunoassay (RIA) or enzyme-linked immunosorbent assay (ELISA).

[0128] The binding affinity of the monoclonal antibody can, for example, be determined by the Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

[0129] After hybridoma cells producing antibodies with the desired specificity, affinity, and / or activity are identified, the clones may be subcloned by limiting dilution procedures and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice, pp. 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM medium or RPMI-1640 medium. Additionally, hybridoma cells may be grown in vivo as ascites tumors in animals.

[0130] 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, polypeptide A-Sepharose, hydroxylapatite chromatography, gel electrophoresis, dialysis, or affinity chromatography.

[0131] DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of binding specifically to genes encoding the heavy and light chains of murine antibodies). In some embodiments, hybridoma cells serve as a source of such DNA. Once isolated, the DNA can be placed into an expression vector and then transfected into host cells, such as E. coli cells, simian COS cells, Chinese hamster ovary (CHO) cells, or myeloma cells that do not otherwise produce immunoglobulin proteins, to obtain the synthesis of monoclonal antibodies in the recombinant host cells. Review articles on recombinant expression of antibody-encoding DNA in bacteria include Skerra et al., Curr. Opinion in Immunol. 5:256-262 (1993) and Pluckthun, Immunol. Revs., 130:151-188 (1992).

[0132] In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries generated using the techniques described in McCafferty et al., Nature 348:552-554 (1990). Clackson et al., Nature 352:624-628 (1991) and Marks et al., J. Mol. Biol. 222:581-597 (1991) describe the isolation of murine and human antibodies, respectively, using phage libraries. Subsequent publications described the production of high affinity (nM range) human antibodies by chain shuffling (Marks et al., Bio / Technology 10:779-783 (1992)), as well as combinatorial infection and in vivo recombination as strategies for constructing extremely large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21:2265-2266 (1993)). These techniques are therefore viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0133] The DNA may also be modified, for example, by substituting human heavy and light chain constant domains for the homologous murine sequences in the coding sequence (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl. Acad. Sci. USA 81:6851 (1984)), or by covalently joining all or part of the coding sequence for a non-immunoglobulin polypeptide to the immunoglobulin coding sequence.

[0134] 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 comprising one antigen-binding site with specificity for an antigen and another antigen-binding site with specificity for a different antigen.

[0135] In some embodiments of any of the methods described herein, the antibody is IgA, IgD, IgE, IgG, or IgM. In some embodiments, the antibody is an IgG monoclonal antibody. humanized antibodies

[0136] In some embodiments, the antibody is a humanized antibody. Methods for humanizing non-human antibodies have been described in the art. In some embodiments, a humanized antibody has one or more amino acid residues introduced into it from a non-human source. These non-human amino acid residues are often referred to as "import" residues, typically from an "import" variable domain. Humanization can be performed essentially according to the method of Winter and coworkers (Jones et al., Nature 321:522-525 (1986); Riechmann et al., Nature 332:323-327 (1988); Verhoeyen et al., Science 239:1534-1536 (1988)), by substituting hypervariable region sequences for the corresponding sequences of a human antibody. Such "humanized" antibodies are thus chimeric antibodies (U.S. Pat. No. 4,816,567) in which substantially less than an intact variable domain has been substituted by the corresponding sequence from a non-human species. In practice, humanized antibodies are typically human antibodies in which some hypervariable region residues and possibly some FR residues are substituted by residues from analogous sites in rodent antibodies.

[0137] The selection of human variable domains (both heavy and light chains) used to generate humanized antibodies is crucial to reducing 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 region (FR) of the humanized antibody (Sims et al., J. Immunol. 151:2296 (1993); Chothia et al., J. Mol. Biol. 196:901 (1987)). Another method uses a specific framework region derived from the consensus sequence of all human antibodies of a particular subgroup of light or heavy chain variable regions. The same framework may 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)).

[0138] 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 of the present methods, humanized antibodies are prepared by 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 commonly available and are familiar to those skilled in the art. Computer programs are available that illustrate and display probable three-dimensional conformations of selected candidate immunoglobulin sequences. Inspection of these displays permits 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, hypervariable region residues are directly and most substantially involved in influencing antigen binding. Human antibodies

[0139] In some embodiments, the antibody is a human antibody. As an alternative to humanization, human antibodies can be generated. 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 antibody heavy chain joining region (J) in chimeric and germ-line mutant mice can be expressed in a variety of ways. H It has been described that homozygous deletion of the .gamma.) gene results in complete inhibition of endogenous antibody production. Transfer 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. 7:33 (1993); and U.S. Patent Nos. 5,591,669; 5,589,369; and 5,545,807.

[0140] Alternatively, phage display technology (McCafferty et al., Nature 348:552-553 (1990)) can be used to produce human antibodies and antibody fragments in vitro from immunoglobulin variable (V) domain gene repertoires from unimmunized donors. According to this technique, antibody V domain genes are cloned in-frame into either a major or minor coat polypeptide 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 result in selection of the gene encoding the antibody exhibiting those properties. Thus, the phage mimics some of the properties of B cells. Phage display can be performed in a variety of formats, for reviews of which see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). Several 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. V gene repertoires may be constructed from unimmunized human donors, and antibodies to a diverse array of antigens (including self-antigens) may 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.

[0141] Human antibodies may also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275). antibody fragment

[0142] In some embodiments, the antibody is an antibody fragment. Various techniques have been developed to produce antibody fragments. Traditionally, these fragments were obtained by proteolytic digestion of intact antibodies (see, e.g., Morimoto et al., Journal of Biochemical and Biophysical Methods 24:107-117 (1992) and Brennan et al., Science, 229:81 (1985)). However, these fragments can now be produced directly from recombinant host cells. For example, antibody fragments can be isolated from the antibody phage libraries discussed 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. Other techniques for producing antibody fragments will be apparent to those skilled in the art. 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 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.

[0143] In some embodiments, fragments of the antibodies described herein are provided. In some embodiments, the antibody fragment is an antigen-binding fragment. In some embodiments, the antigen-binding fragment is selected from the group consisting of a Fab fragment, a Fab' fragment, a F(ab')2 fragment, a scFv, a di-scFv, a bi-scFv, a tandem (di, tri)-scFv, an Fv, an sdAb, a trifunctional antibody, a BiTE, a diabody, and a triabody. bispecific antibody

[0144] In some embodiments, the antibody is a bispecific antibody. A bispecific antibody is an antibody that has binding specificities for at least two different epitopes. Exemplary bispecific antibodies can bind to two different epitopes. Alternatively, the bispecific antibody binding arm can be combined with an arm that binds to a triggering molecule on a leukocyte, such as a T cell receptor molecule (e.g., CD2 or CD3), or an Fc receptor for IgG (FcγR), such as FcγRI (CD64), FcγRII (CD32), and FcγRIII (CD16), to target cellular defense mechanisms against the cell. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0145] Methods for producing bispecific antibodies are known in the art. Traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy-light chain pairs, with these two chains having different specificities (Millstein et al., Nature 305:537-539 (1983)). Due to the random assortment of immunoglobulin heavy and light chains, these hybridomas (quadromas) may produce a mixture of 10 different antibody molecules, only one of which has the correct bispecific structure. Purification of the correct molecule, usually performed by affinity chromatography steps, is somewhat cumbersome, and product yields are low. Similar procedures are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0146] According to a different approach, antibody variable domains with the desired binding specificities (antibody-antigen combining sites) are fused to immunoglobulin constant domain sequences. In some embodiments, the fusions are with an immunoglobulin heavy-chain constant domain, comprising at least part of the hinge, CH2, and CH3 regions. In some embodiments, the first heavy-chain constant region (CH1), containing the site necessary for light-chain binding, is present in at least one of the fusions. DNAs encoding the immunoglobulin heavy chain fusions and, if desired, 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 relative proportions of the three polypeptide fragments in embodiments where unequal ratios of the three polypeptide chains used in the construction provide optimal yields. However, if expression of at least two polypeptide chains in equal ratios results in high yields, or if the ratios are not particularly critical, it is possible to insert the coding sequences for two or all three polypeptide chains into a single expression vector.

[0147] In some embodiments of this approach, the bispecific antibody is 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 this asymmetric structure facilitates separation of the desired bispecific compound from undesired immunoglobulin chain combinations, as the presence of an immunoglobulin light chain in only one half of the bispecific molecule provides a facile separation method. This approach is disclosed in WO 94 / 04690. For further details on the generation of bispecific antibodies, see, e.g., Suresh et al., Methods in Enzymology 121:210 (1986).

[0148] According to another approach described in U.S. Patent No. 5,731,168, the interface between a pair of antibody molecules may be engineered to maximize the percentage of heterodimers which are recovered from recombinant cell culture. In some embodiments, the interface is located between the C and C regions of the antibody constant domains.H The antibody comprises at least a portion of the three domains. In this method, one or more small amino acid side chains from the interface of a first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). By replacing the large amino acid side chains with smaller ones (e.g., alanine or threonine), compensatory "cavities" of identical or similar size to the large side chain(s) are created on the interface of the second antibody molecule. This provides a mechanism for increasing the yield of heterodimers over other unwanted end-products such as homodimers.

[0149] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one of the antibodies in the heteroconjugate can be conjugated to avidin and the other to biotin. Such antibodies have been proposed, for example, to target immune system cells to unwanted cells (U.S. Pat. No. 4,676,980) and to treat HIV infection (WO 91 / 00360, WO 92 / 200373, and EP 03089). Heteroconjugate antibodies may be made using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Pat. No. 4,676,980, along with several cross-linking techniques.

[0150] Techniques for generating bispecific antibodies from antibody fragments are also 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 the Fab'-thiol by reduction with mercaptoethylamine and mixed with an equimolar amount of the other Fab'-TNB derivative to form the bispecific antibody. The resulting bispecific antibody can be used as an agent for the selective immobilization of enzymes.

[0151] Various techniques for making and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies were produced using leucine zippers. Kostelny et al., J. Immunol. 148(5):1547-1553 (1992). Leucine zipper peptides from Fos and Jun proteins were attached 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 re-oxidized to form the antibody heterodimers. This method can also be used to produce antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90:6444-6448 (1993) provides an alternative mechanism for making bispecific antibody fragments. These fragments comprise a light-chain variable domain (V) separated by a linker that is too short to allow pairing between the two domains on the same chain. L ) connected to the heavy chain variable domain (V H ) is included. Therefore, the V H Domain and V L The domain is complementary to the V of another fragment. L Domain and VH The antibody fragments are then paired with the single-chain Fv (sFv) dimers, thereby forming two antigen-binding sites. Another method for making bispecific antibody fragments by the use of single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152:5368 (1994).

[0152] Antibodies with more than two valencies are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991). Multivalent antibodies

[0153] In some embodiments, the antibody is a multivalent antibody. Multivalent antibodies may be internalized (and / or catabolized) more quickly than bivalent antibodies by cells expressing the antigen to which the antibody binds. The antibodies provided herein may be multivalent antibodies (other than IgM class) having three or more antigen-binding sites (e.g., tetravalent antibodies), which can be easily produced by recombinant expression of nucleic acids encoding antibody polypeptide chains. The multivalent antibody may comprise a dimerization domain and three or more antigen-binding sites. A preferred dimerization domain comprises (or consists of) an Fc region or hinge region. In this scenario, the antibody comprises an Fc region and three or more antigen-binding sites amino-terminal to the Fc region. Preferred multivalent antibodies herein comprise (or consist of) three to about eight, but preferably four, antigen-binding sites. A multivalent antibody comprises at least one polypeptide chain (and preferably two polypeptide chains), and the polypeptide chain(s) comprise two or more variable domains. For example, the polypeptide chain(s) may comprise VD1-(X1)n-VD2-(X2)n-Fc, where VD1 is a first variable domain, VD2 is a second variable domain, Fc is one polypeptide chain of an Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain(s) may comprise a VH-CH1-flexible linker-VH-CH1-Fc region chain or a VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (preferably four) light chain variable domain polypeptides. The multivalent antibody herein may comprise, for example, about two to about eight light chain variable domain polypeptides. The light chain variable domain polypeptide contemplated herein comprises a light chain variable domain and optionally further comprises a CL domain.

[0154] In some embodiments, the antibody is a multispecific antibody. Examples of multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (V H ) and the light chain variable domain (V L ) containing antibodies (V H V Lunits have polyepitope specificity), two or more V L Domain and V H Antibodies with domains (each V H V L Examples of multispecific antibodies include antibodies with two or more single variable domains (each single variable domain binds to a different epitope), full-length antibodies, antibody fragments, such as Fab, Fv, dsFv, scFv, diabodies, bispecific diabodies, triabodies, trifunctional antibodies, and covalently or non-covalently linked antibody fragments. In some embodiments, the antibody has polyepitopic specificity, e.g., the ability to specifically bind to two or more different epitopes on the same or different target(s). In some embodiments, the antibody is monospecific, e.g., an antibody that binds to only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5 ΓM to 0.001 pM, 3 ΓM to 0.001 pM, 1 ΓM to 0.001 pM, 0.5 ΓM to 0.001 pM, or 0.1 ΓM to 0.001 pM. Other antibody modifications

[0155] It may be desirable to modify the antibodies provided herein with respect to effector function, for example, to enhance the antigen-dependent cell-mediated cytotoxicity (ADCC) and / or complement-dependent cytotoxicity (CDC) of the antibody. This can be achieved by introducing one or more amino acid substitutions into the Fc region of the antibody. Alternatively, or additionally, cysteine ​​residue(s) may be introduced into the Fc region, thereby allowing interchain disulfide bond formation in this region. The homodimeric antibody thus generated may have improved internalization capability and / or increased complement-mediated cell killing and antibody-dependent cellular cytotoxicity (ADCC). See Caron et al., J. Exp Med. 176:1191-1195 (1992) and Shopes, BJ, Immunol. 148:2918-2922 (1992). Homodimeric antibodies with enhanced anti-tumor activity can also be prepared using heterobifunctional cross-linkers as described in Wolff et al., Cancer Research 53:2560-2565 (1993). Alternatively, an antibody can be engineered which has dual Fc regions and may thereby have enhanced complement lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3:219-230 (1989).

[0156] To increase the serum half-life of the antibody, amino acid changes can be made in the antibody as described in US Patent Application Publication No. 2006 / 0067930, which is incorporated herein by reference in its entirety. Polypeptide Variants and Modifications

[0157] The amino acid sequence modification(s) of polypeptides, including antibodies, described herein may be used in reusable chromatographic materials that are cleaned by the methods described herein. Variant Polypeptides

[0158] By "polypeptide variant" is meant a polypeptide, preferably an active polypeptide, as defined herein having at least about 80% amino acid sequence identity with the full-length native sequence of the polypeptide, the polypeptide sequence lacking the signal peptide, or the extracellular domain of the polypeptide with or without the signal peptide. Such polypeptide variants include, for example, polypeptides in which one or more amino acid residues have been added or deleted at the N- or C-terminus of the full-length native amino acid sequence. Typically, a TAT polypeptide variant has at least about 80% amino acid sequence identity, or at least any of about 85%, about 90%, about 95%, about 96%, about 97%, about 98%, or about 99% amino acid sequence identity with the full-length native sequence polypeptide sequence, the polypeptide sequence lacking the signal peptide, or the extracellular domain of the polypeptide with or without the signal peptide. Optionally, the variant polypeptide has no more than one conservative amino acid substitution compared to the native polypeptide sequence, or has no more than about any of 2, 3, 4, 5, 6, 7, 8, 9, or 10 conservative amino acid substitutions compared to the native polypeptide sequence.

[0159] Variant polypeptides may be truncated at the N- or C-terminus, or may lack internal residues, for example, compared to the full-length native polypeptide. Certain variant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These variant polypeptides with truncations, deletions, and insertions may be prepared by any of several conventional techniques. The desired variant polypeptide may also be chemically synthesized. Another suitable technique involves isolating and amplifying a nucleic acid fragment encoding the desired variant polypeptide by polymerase chain reaction (PCR). Oligonucleotides that define the desired termini of the nucleic acid fragment are used at the 5' and 3' primers in the PCR. Preferably, the variant polypeptide shares at least one biological and / or immunological activity with the native polypeptides disclosed herein.

[0160] Amino acid sequence insertions include amino- and / or carboxyl-terminal fusions ranging in length from one residue to polypeptides containing 100 or more residues, as well as intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include an antibody with an N-terminal methionyl residue or an antibody fused to a cytotoxic polypeptide. Other insertional variants of antibody molecules include the fusion to the N- or C-terminus of the antibody to an enzyme or a polypeptide which increases the serum half-life of the antibody.

[0161] For example, it may be desirable to improve the binding affinity and / or other biological properties of the polypeptide. Amino acid sequence variants of the polypeptide are prepared by introducing appropriate nucleotide changes into the antibody nucleic acid or by peptide synthesis. Such modifications include, for example, deletions from, and / or insertions into, and / or substitutions of residues within the amino acid sequence of the polypeptide. Any combination of deletions, insertions, and substitutions can be made to arrive at the final construct, provided that the final construct possesses the desired characteristics. Amino acid changes can also alter post-translational processing of the polypeptide (e.g., antibody), for example, changing the number or position of glycosylation sites.

[0162] Guidance in determining which amino acid residues can be inserted, substituted, or deleted without adversely affecting a desired activity can be found by comparing the sequence of a polypeptide to the sequences of known polypeptide molecules of the same type, and minimizing the number of amino acid sequence changes made in areas of high homology.

[0163] A useful method for identifying particular residues or regions of a polypeptide (e.g., an antibody) that are preferred sites for mutagenesis is called "alanine scanning mutagenesis," described by Cunningham and Wells, Science 244:1081-1085 (1989). In this method, a residue or target group of residues (e.g., charged residues such as Arg, Asp, His, Lys, and Glu) is identified and replaced with neutral or negatively charged amino acids (most preferably alanine or polyalanine) to affect the amino acid's interaction with the antigen. Those amino acid locations demonstrating functional sensitivity to the substitution are then refined by introducing further or other variants at or for those substitution sites. Thus, while the site for introducing an amino acid sequence variation is predetermined, the nature of the mutation need not itself be predetermined. For example, to analyze the performance of a mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the expressed antibody variants are screened for the desired activity.

[0164] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue in the antibody molecule replaced with a different residue. The most interesting sites for substitutional mutagenesis include the hypervariable regions, although FR alterations are also contemplated. Conservative substitutions are shown in Table 1 below under the heading of "preferred substitutions." If such substitutions result in a change in biological activity, more substantial changes, designated "exemplary substitutions" in Table A or further described below with reference to amino acid classes, can be introduced and the products screened. [Table 1]

[0165] Substantial modification of the biological properties of a polypeptide is achieved by selecting substitutions that differ significantly in their effect on maintaining (a) the structure of the polypeptide backbone in the region of substitution, e.g., sheet or helical conformation, (b) the charge or hydrophobicity of the molecule at the target site, or (c) the bulk of the side chain. Amino acids can be grouped according to the similarity of their side chain properties (A.L. Lehninger, Biochemistry second ed. Worth Publishers, New York (1975)): (1) Non-polar: Ala(A), Val(V), Leu(L), Ile(I), Pro(P), Phe(F), Trp(W), Met(M) (2) Uncharged polar: Gly (G), Ser (S), Thr (T), Cys (C), Tyr (Y), Asn (N), Gln (Q) (3) Acidic: Asp(D), Glu(E) (4) Basic: Lys(K), Arg(R), His(H)

[0166] Alternatively, naturally occurring residues can be divided into groups based on common side chain properties. (1) Hydrophobic: Norleucine, Met, Ala, Val, Leu, Ile; (2) Neutral hydrophilic: Cys, Ser, Thr, Asn, Gln; (3) Acidic: Asp, Glu; (4) basic: His, Lys, Arg; (5) Residues affecting chain orientation: Gly, Pro; (6) Aromatic: Trp, Tyr, Phe.

[0167] Non-conservative substitutions involve exchanging a member of one of these classes for another class.

[0168] Any cysteine ​​residue not involved in maintaining the proper conformation of the antibody may also generally be substituted with serine to improve the oxidative stability of the molecule and prevent aberrant crosslinking. Conversely, cysteine ​​bond(s) may be added to a polypeptide to improve its stability (particularly where the antibody is an antibody fragment such as an Fv fragment).

[0169] A particularly preferred type of substitutional variant involves substituting one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). Generally, the resulting variant(s) selected for further development will have improved biological properties relative to the parent antibody from which they are generated. A convenient method for generating such substitutional variants involves affinity maturation using phage display. Briefly, several hypervariable region sites (e.g., 6-7 sites) are mutated to generate all possible amino acid substitutions at each site. The antibody variants thus generated are displayed in a monovalent manner from filamentous phage particles as fusions to the gene III product of M13 packaged within each particle. The phage-displayed variants are then screened for their biological activity (e.g., binding affinity) as disclosed herein. To identify candidate hypervariable region sites for modification, alanine scanning mutagenesis can be performed to identify hypervariable region residues that contribute significantly to antigen binding. Alternatively, or additionally, it may be beneficial to analyze a crystal structure of the antigen-antibody complex to identify contact points between the antibody and target. Such contact and adjacent residues are candidates for substitution according to the techniques detailed herein. Once such variants are generated, the panel of variants can be subjected to screening as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0170] Another type of amino acid variant of a polypeptide alters the original glycosylation pattern of the antibody. A polypeptide may contain non-amino acid moieties. For example, a polypeptide may be glycosylated. Such glycosylation may occur naturally during expression of the polypeptide in a host cell or host organism, or may be a deliberate modification resulting from human intervention. By alteration is meant deleting one or more carbohydrate moieties found in the polypeptide and / or adding one or more glycosylation sites that are not present in the polypeptide.

[0171] Glycosylation of polypeptides is typically either N-linked or O-linked. N-linked refers to the attachment of the carbohydrate moiety to the side chain of an asparagine residue. The tripeptide sequences asparagine-X-serine and asparagine-X-threonine, where X is any amino acid except proline, are the recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of either of these tripeptide sequences in a polypeptide creates a potential glycosylation site. O-linked glycosylation refers to the attachment of one of the sugars N-acetylgalactosamine, galactose, or xylose to a hydroxyamino acid, most commonly serine or threonine, although 5-hydroxyproline or 5-hydroxylysine may also be used.

[0172] Addition of glycosylation sites to the polypeptide is conveniently accomplished by altering the amino acid sequence such that it contains one or more of the above-described tripeptide sequences (for N-linked glycosylation sites). The alteration may also be made by the addition of, or substitution by, one or more serine or threonine residues to the sequence of the original antibody (for O-linked glycosylation sites).

[0173] Removal of carbohydrate moieties present on a polypeptide can be accomplished chemically or enzymatically, or by mutational substitution of codons encoding amino acid residues that serve as targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on polypeptides can be achieved by the use of a variety of endo- and exo-glycosidases.

[0174] Other modifications include deamidation of glutaminyl and asparaginyl residues to the corresponding glutamyl and aspartyl residues, respectively, hydroxylation of proline and lysine, phosphorylation of the hydroxyl group of seryl or threonyl residues, methylation of the α-amino groups of lysine, arginine, and histidine side chains, acetylation of the N-terminal amine, and amidation of any C-terminal carboxyl group. Chimeric Polypeptides

[0175] The polypeptides described herein can be modified in ways to form chimeric molecules comprising the polypeptide fused to another, heterologous polypeptide or amino acid sequence. In some embodiments, a chimeric molecule comprises a fusion of the polypeptide with a tag polypeptide that provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is generally located at the amino- or carboxyl-terminus of the polypeptide. The presence of such an epitope-tagged form of the polypeptide can be detected using an antibody against the tag polypeptide. Providing an epitope tag also allows the polypeptide to be readily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

[0176] In an alternative embodiment, the chimeric molecule may comprise a fusion of the polypeptide with an immunoglobulin or a particular region of an immunoglobulin. The bivalent form of the chimeric molecule is referred to as an "immunoadhesin."

[0177] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide with the effector functions of immunoglobulin constant domains. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence with the desired binding specificity, which is other than the antigen recognition and binding site of an antibody (i.e., "heterologous"), with an immunoglobulin constant domain sequence. The adhesin portion of the immunoadhesin molecule is typically a contiguous amino acid sequence that includes at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin may be derived from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

[0178] Ig fusions preferably include the substitution of a soluble (transmembrane domain deleted or inactivated) form of a polypeptide in place of at least one variable region within an Ig molecule. In certain preferred embodiments, the immunoglobulin fusion includes the hinge, CH2, and CH3, or the hinge, CH1, CH2, and CH3 regions of an IgG1 molecule. Polypeptide Conjugates

[0179] Polypeptides for use in polypeptide formulations may be conjugated to a cytotoxic agent, such as a chemotherapeutic agent, a growth inhibitory agent, a toxin (e.g., an enzymatically active toxin of bacterial, fungal, plant, or animal origin, or fragments thereof), or a radioactive isotope (i.e., a radioconjugate).

[0180] Chemotherapeutic agents useful in the production of such conjugates can be used. Additionally, enzymatically active toxins and their fragments that can be used include diphtheria A chain, nonbinding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, alpha-sarcin, Aleurites fordii proteins, diansin proteins, Phytolaca americana proteins (PAPI, PAPII, and PAP-S), momordica charantia inhibitor, curcin, crotin, sapaonaria officinalis inhibitor, gelonin, mitogenin, restrictocin, phenomycin, enomycin, and the trichothecenes. A variety of radioisotopes are available for the production of radioactive conjugated polypeptides. Examples include: 212 Bi, 131 I, 131 In, 90 Y, and 186 Conjugates of polypeptides and cytotoxic agents, including Re, have been prepared using a variety of bifunctional protein coupling agents, such as N-succinimidyl-3-(2-pyridyldithiol)propionate (SPDP), iminothiolane (IT), bifunctional derivatives of imidoesters (e.g., dimethyl adipimidate HCl), active esters (e.g., disuccinimidyl suberate), aldehydes (e.g., glutaraldehyde), bis-azido compounds (e.g., bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (e.g., bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (e.g., tolyene 2,6-diisocyanate), and bis-active fluorine compounds (e.g., 1,5-difluoro-2,4-dinitrobenzene). For example, ricin immunotoxins can be prepared as described in Vitetta et al., Science 238:1098 (1987). Carbon-14-labeled 1-isothiocyanatobenzyl-3-methyldiethylenetriaminepentaacetic acid (MX-DTPA) is an exemplary chelating agent for conjugating radionucleotides to polypeptides.

[0181] Conjugates of a polypeptide and one or more small molecule toxins, such as a calicheamicin, a maytansinoid, a trichothecene, and CC1065, and derivatives of these toxins that have toxin activity, are also contemplated herein.

[0182] Maytansinoids are mitotic inhibitors that act by inhibiting tubulin polymerization. Maytansine was first isolated from the East African shrub Maytenus serrata. It was subsequently discovered that certain microorganisms also produce maytansinoids, such as maytansinol and C-3 maytansinol esters. Synthetic maytansinol and its derivatives and analogs are also contemplated. Numerous linking groups are known in the art for preparing polypeptide-maytansinoid conjugates, including those disclosed in U.S. Pat. No. 5,208,020. Linking groups include disulfide, thioether, acid-labile, photolabile, peptidase-labile, or esterase-labile groups, as disclosed in the aforementioned patents; disulfide and thioether groups are preferred.

[0183] Depending on the type of bond, the linker can be attached to the maytansinoid molecule at various positions. For example, an ester bond can be formed by reaction with a hydroxyl group using conventional coupling techniques. Reactions can occur at the C-3 position with a hydroxyl group, the C-14 position with a hydroxymethyl modification, the C-15 position with a hydroxyl group, and the C-20 position with a hydroxyl group. In a preferred embodiment, the bond is formed at the C-3 position of maytansinol or a maytansinol analog.

[0184] Another conjugate of interest comprises a polypeptide conjugated to one or more calicheamicin molecules. The calicheamicin family of antibiotics can produce double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of calicheamicin family conjugates, see, e.g., U.S. Patent No. 5,712,374. Structural analogs of calicheamicin that can be used include γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG, and θ1 I Another antitumor drug to which the antibody can be conjugated is the antifolate drug QFA. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents through polypeptide (e.g., antibody)-mediated internalization greatly enhances their cytotoxic effects.

[0185] Other anti-tumor drugs that can be conjugated to the polypeptides described herein include BCNU, streptosycin, vincristine, and 5-fluorouracil, a family of drugs collectively known as LL-E33288 complexes, and esperamicin.

[0186] In some embodiments, the polypeptide can be a conjugate between a polypeptide and a compound with nucleolytic activity (eg, a ribonuclease, or a DNA endonuclease, such as a deoxyribonuclease, DNase).

[0187] In yet another embodiment, a polypeptide (e.g., an antibody) may be conjugated to a "receptor" (such as streptavidin) for use in pre-tumor targeting, in which case the polypeptide-receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a clearing agent, followed by administration of a "ligand" (e.g., avidin) that has been conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0188] In some embodiments, the polypeptide may be conjugated to a prodrug-activating enzyme that converts a prodrug (e.g., a peptidyl chemotherapeutic agent) into an active anticancer drug. The enzyme component of the immunoconjugate includes any enzyme that can act on a prodrug in such a way as to convert it into its more active, cytotoxic form.

[0189] Useful enzymes include alkaline phosphatases, which are useful for converting phosphate-containing prodrugs to free drugs; arylsulfatases, which are useful for converting sulfate-containing prodrugs to free drugs; cytosine deaminases, which are useful for converting the non-toxic 5-fluorocytosine to the anti-cancer drug 5-fluorouracil; proteases such as Serratia protease, thermolysin, subtilisin, carboxypeptidase, and cathepsins (e.g., cathepsins B and L), which are useful for converting peptide-containing prodrugs to free drugs; and enzymes containing D-amino acid substituents. carbohydrate-cleaving enzymes such as β-galactosidase and neuraminidase, which are useful for converting glycosylated prodrugs to free drugs, β-lactamases, which are useful for converting β-lactam-derivatized drugs to free drugs, and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which are useful for converting drugs derivatized with a phenoxyacetyl or phenylacetyl group, respectively, at the amine nitrogen, to free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert prodrugs to free active drugs. others

[0190] Another type of covalent modification of polypeptides involves linking the polypeptide to one of a variety of non-proteinaceous polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylenes, or copolymers of polyethylene glycol and polypropylene glycol. Polypeptides can also be encapsulated in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles, and nanocapsules), or in microemulsions, for example, by coacervation techniques or microcapsules prepared by interfacial polymerization (e.g., hydroxymethylcellulose or gelatin microcapsules and poly-(methylmethacylate) microcapsules, respectively). Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, AR, Ed., (1990). Obtaining Polypeptides for Use in Formulations

[0191] Polypeptides purified using reusable chromatography materials washed in the manner described herein can be obtained using methods well known in the art, including recombinant methods. The following sections provide guidance regarding these methods. Polynucleotides

[0192] "Polynucleotide" or "nucleic acid," used interchangeably herein, refer to polymers of nucleotides of any length, and include DNA and RNA.

[0193] Polynucleotides encoding polypeptides can be obtained from any source, including, but not limited to, cDNA libraries prepared from tissues believed to harbor polypeptide mRNA and express it at detectable levels. Thus, polynucleotides encoding polypeptides can be conveniently obtained from cDNA libraries prepared from human tissues. Genes encoding polypeptides can also be obtained from genomic libraries or by known synthetic procedures (e.g., automated nucleic acid synthesis).

[0194] For example, a polynucleotide can encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes a heavy chain variable region (V H ), as well as the heavy chain constant region (C H ), which typically contains three constant domains: C H 1. C H 2 and C H 3, and a "hinge" region. In some cases, the presence of a constant region is desirable.

[0195] Other polypeptides that may be encoded by the polynucleotides include antigen-binding antibody fragments, such as single domain antibodies ("dAbs"), Fvs, scFvs, Fab's, and F(ab')2s, and "minibodies." Minibodies are (typically) C H 1 and C K or C LMinibodies are bivalent antibody fragments in which domains have been truncated. Because minibodies are smaller than conventional antibodies, they should achieve good tissue penetration for clinical / diagnostic applications, but they are bivalent and must retain higher binding affinity than monovalent antibody fragments such as dAbs. Thus, unless the context dictates otherwise, the term "antibody" as used herein encompasses not only all antibody molecules but also antigen-binding antibody fragments of the types discussed above. Preferably, each framework region present in the encoded polypeptide contains at least one amino acid substitution relative to the corresponding human receptor framework. Thus, for example, the framework region may contain a total of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15 amino acid substitutions relative to the receptor framework region.

[0196] Suitably, the polynucleotides described herein may be isolated and / or purified. In some embodiments, the polynucleotides are isolated polynucleotides.

[0197] The term "isolated polynucleotide" is intended to indicate that the molecule has been removed or separated from its normal or natural environment, or has been produced in such a way that it is not present in its normal or natural environment. In some embodiments, the polynucleotide is a purified polynucleotide. The term purified is intended to indicate that at least some contaminating molecules or substances have been removed.

[0198] Suitably, the polynucleotides are substantially purified such that the related polynucleotide constitutes the predominant (ie, most abundant) polynucleotide present in the composition. Polynucleotide Expression

[0199] The following description primarily relates to producing polypeptides by culturing cells transformed or transfected with a vector containing a polynucleotide encoding the polypeptide. It is, of course, contemplated that alternative methods known in the art can be used to prepare polypeptides. For example, the appropriate amino acid sequence, or portions thereof, can be produced by direct peptide synthesis using solid-phase techniques (see, e.g., Stewart et al., Solid-Phase Peptide Synthesis, W.H. Freeman Co., San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc. 85:2149-2154 (1963)). In vitro protein synthesis can be carried out by manual techniques or automation. Automated synthesis can be achieved, for example, using an Applied Biosystems Peptide Synthesizer (Foster City, Calif.) using the manufacturer's instructions. Various portions of a polypeptide can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired polypeptide.

[0200] The polynucleotides described herein are inserted into an expression vector(s) for the production of a polypeptide. The term "regulatory sequence" refers to DNA sequences necessary for the expression of an operably linked coding sequence in a particular host organism. Control sequences include, but are not limited to, promoters (e.g., naturally associated or heterologous promoters), signal sequences, enhancer elements, and transcription termination sequences.

[0201] A polynucleotide is "operably linked" when it is placed into a functional relationship with another polynucleotide sequence. For example, nucleic acid for a presequence or secretory leader is operably linked to nucleic acid for a polypeptide if it is expressed as a preprotein that participates in the secretion of the polypeptide; a promoter or enhancer is operably linked to a coding sequence if it affects the transcription of the sequence; or a ribosome binding site is operably linked to a coding sequence if it is positioned so as to facilitate translation. Generally, "operably linked" means that linked nucleic acid sequences are contiguous, and, in the case of a secretory leader, contiguous and in reading phase. Enhancers, however, need not be contiguous. Linking is accomplished by ligation at convenient restriction sites. If such sites do not exist, synthetic oligonucleotide adaptors or linkers are used in accordance with conventional practice.

[0202] In the case of antibodies, the light and heavy chains can be cloned into the same or different expression vectors. The nucleic acid segments encoding immunoglobulin chains are operably linked to regulatory sequences in the expression vector(s) that ensure the expression of immunoglobulin polypeptides.

[0203] Vectors containing polynucleotide sequences (e.g., variable heavy and / or variable light chain coding sequences and any expression control sequences) can be transferred into host cells by well-known methods that vary depending on the type of cellular host. For example, calcium chloride transfection is commonly utilized for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, biolistic or viral-based transfection may be used for other cellular hosts. (See generally Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd ed., 1989).) Other methods used to transform mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation, and microinjection. To generate transgenic animals, transgenes can be microinjected into fertilized oocytes or integrated into the genome of embryonic stem cells, the nuclei of which are then transferred into enucleated oocytes. vector

[0204] The term "vector" includes expression vectors and transformation vectors as well as shuttle vectors.

[0205] The term "expression vector" means a construct capable of expression in vivo or in vitro.

[0206] The term "transformation vector" refers to a construct that can be transferred from one entity to another, which can be of the same species or a different species. If the construct can be transferred from one species to another, for example, from an E. coli plasmid to a bacterium such as Bacillus, the transformation vector is sometimes called a "shuttle vector." It can even be a construct that can be transferred from an E. coli plasmid to Agrobacterium to plants.

[0207] To provide expression of the polypeptide, the vector can be transformed into a suitable host cell as described below. Various vectors are publicly available. The vector can be, for example, in the form of a plasmid, cosmid, virus particle, or phage. The appropriate nucleic acid sequence can be inserted into the vector by various procedures. Generally, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. Construction of an appropriate vector containing one or more of these components uses standard ligation techniques known to those skilled in the art.

[0208] The vectors may be, for example, plasmid, virus or phage vectors provided with an origin of replication, optionally a promoter for the expression of the said polynucleotide and optionally a regulator of the promoter. The vectors may contain one or more selectable marker genes well known in the art.

[0209] These expression vectors are typically replicable in the host organisms either as episomes or as an integral part of the host chromosomal DNA. host cell

[0210] The host cell can be, for example, a bacterium, a yeast or other fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0211] Genetically engineered transgenic multicellular host organisms can be used to produce polypeptides. The organisms can be, for example, transgenic mammalian organisms (e.g., transgenic goat or mouse strains).

[0212] Suitable prokaryotes can include, but are not limited to, eubacteria, such as Gram-negative or Gram-positive organisms, for example, Enterobacteriaceae, such as E. coli. Various strains of E. coli are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446), E. coli X1776 (ATCC 31,537), E. coli strain W3110 (ATCC 27,325), and E. coli K5772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae such as Escherichia, e.g., Escherichia coli, Enterobacter, Erwinia, Klebsiella, Proteus, Salmonella, e.g., Salmonella typhimurium, Serratia, e.g., Serratia marcescens, and Shigella, as well as Bacillus, e.g., Bacillus subtilis and Bacillus licheniformis (e.g., Bacillus licheniformis 41P), Pseudomonas, e.g., Pseudomonas aeruginosa, and Streptomyces. These examples are illustrative and not limiting. Strain W3110 is a common host strain for recombinant polynucleotide product fermentation, and is therefore one particularly preferred host or parent host. Preferably, the host cell secretes minimal amounts of proteolytic enzymes. For example, strain W3110 may be modified to introduce a genetic mutation in a gene encoding a polypeptide endogenous to the host; examples of such hosts include E. coli W3110 strain 1A2, which has the intact tonA genotype; E. coli W3110 strain 9E4, which has the intact tonA ptr3 genotype; E. coli W3110 strain 27C7 (ATCC 55,244), which has the intact genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT kan'; E. coli W3110 strain 37D6, which has the intact genotype tonA ptr3 phoA E15(argF-lac)169 degP ompT rbs7 ilvG kan'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin-resistant degP deletion mutation; and E. coli strains with mutant periplasmic proteases. Alternatively, in vitro methods of cloning, such as PCR or other nucleic acid polymerase reactions, are suitable.

[0213] In these prokaryotic hosts, expression vectors can be made, which typically contain expression control sequences compatible with the host cell (e.g., an origin of replication). In addition, there are any number of well-known promoters available, such as the lactose promoter system, the tryptophan (trp) promoter system, the beta-lactamase promoter system, or a promoter system derived from phage lambda. The promoters typically optionally control expression with an operator sequence and contain ribosome binding site sequences, etc., for initiating and completing transcription and translation.

[0214] Eukaryotic microorganisms can be used for expression. Eukaryotic microorganisms, such as filamentous fungi or yeast, are suitable cloning or expression hosts for polypeptide-encoding vectors. Saccharomyces cerevisiae is a commonly used lower eukaryotic host microorganism. Other examples include Schizosaccharomyces pombe; Kluyveromyces spp., such as K. lactis (MW98-8C, CBS683, CBS4574), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. wartsii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotrysarans, and K. marcianus; Yarrowia arvensis (EP 402,226); Pichia pastoris; Candida, Trichoderma Hosts suitable for use include: Neurospora crassa; Schwanniomyces species, such as Schwanniomyces occidentalis; and filamentous fungi, such as Neurospora crassa, Penicillium, Tolypocladium, and Aspergillus, including A. nidulans and A. niger. Methylotrophic yeasts are suitable herein, including, but not limited to, yeasts capable of growing on methanol selected from the genera Hansenula, Candida, Krokella, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. Saccharomyces is a preferred yeast host, with suitable vectors containing expression control sequences (e.g., promoters), origins of replication, termination sequences, and the like, as needed. Typical promoters include those for 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include promoters from alcohol dehydrogenase, isocytochrome C, and enzymes involved in maltose and galactose utilization, among others.

[0215] In addition to microorganisms, mammalian tissue cell culture can also be used, and in some cases is preferred, to express and produce the polypeptides described herein (see Winnacker, From Genes to Clones VCH Publishers, NY, NY (1987)). In some embodiments, eukaryotic cells may be preferred, as numerous suitable host cell lines capable of secreting heterologous polypeptides (e.g., intact immunoglobulins) have been developed in the art, including CHO cell lines, various Cos cell lines, HeLa cells, preferably myeloma cell lines, or transformed B cells or hybridomas. In some embodiments, the mammalian host cells are CHO cells.

[0216] In some embodiments, the host cell is a vertebrate host cell. Examples of useful mammalian host cell lines include SV40-transformed monkey kidney CV1 cells (COS-7, ATCC CRL 1651), human embryonic kidney cells (293 or 293 cells subcloned for growth in suspension culture), baby hamster kidney cells (BHK, ATCC CCL 10), Chinese hamster ovary cells / DHFR (CHO or CHO-DP-12 lines), mouse Sertoli cells, monkey kidney cells (CV1 ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical carcinoma cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat hepatocytes (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL 75); human hepatocytes (Hep G2, HB 8065); and mouse mammary tumor (MMT) cells. 060562, ATCC CCL51); TRI cells, MRC5 cells, FS4 cells, and a human hepatoma line (Hep G2). manufactured goods

[0217] The polypeptide formulations described herein can be included in an article of manufacture. The article of manufacture can include a container containing the polypeptide (e.g., a formulation of the polypeptide, a spray-dried formulation of the polypeptide, or a controlled-release form of the polypeptide). Preferably, the article of manufacture includes (a) a container containing a composition comprising a polypeptide and / or polypeptide formulation described herein within the container, and (b) a package insert with instructions for administering the formulation to a subject.

[0218] The article of manufacture includes a container and a label or package insert inserted in or associated with the container. Suitable containers include, for example, bottles, vials, syringes, and the like. The container may be formed from a variety of materials, such as glass or plastic. The container holds or contains the formulation and may have a sterile access port (e.g., the container may be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). At least one active agent in the composition is a polypeptide. The label or insert indicates that the composition is to be used in a subject, with specific directions for the dosage amount and interval of the polypeptide and any other drugs provided. The article of manufacture may further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes. In some embodiments, the container is a syringe. In some embodiments, the syringe is further contained within an injection device. In some embodiments, the injection device is an autoinjector.

[0219] "Package insert" is used to refer to instructions customarily included in commercial packaging of therapeutic products, which contain information about the indications, uses, dosages, administration, contraindications of such therapeutic product, other therapeutic products with which the packaged product is combined, and / or warnings regarding their use. Illustrative Embodiments The present invention provides the following exemplary embodiments. 1. A formulation comprising a polypeptide and hydroxypropyl methylcellulose acetate succinate (HPMCAS). 2. The formulation of embodiment 1, wherein the HPMCAS comprises about 8% to about 12% acetate and about 7% to about 15% succinate. 3. The formulation of embodiment 2, wherein the HPMCAS comprises about 8% acetate and about 15% succinate (HPMCAS-LF), or about 12% acetate and about 7% succinate (HPMCAS-HF). 4. The formulation of any one of embodiments 1-3, wherein the ratio of HPMCAS to protein in the formulation is from about 4:1 (mg / mg) to about 1:4 (mg / mg), optionally, the ratio is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg). 5. The formulation of any one of embodiments 1-4, further comprising a histidine buffer. 6. The formulation of embodiment 5, wherein the histidine buffer is a histidine HCl buffer. 7. The formulation of embodiment 5 or 6, wherein the histidine buffer is present in the formulation at a concentration of about 5 mM to about 20 mM, and optionally, the histidine buffer is present in the formulation at a concentration of about 10 mM. 8. The formulation of any one of embodiments 1-7, further comprising a polysorbate. 9. The formulation of embodiment 8, wherein the polysorbate is polysorbate 20. 10. The formulation of embodiment 8 or 9, wherein the polysorbate is at a concentration of about 0.005% (w / v) to about 0.5% (w / v) in the formulation. 11. A formulation according to any one of embodiments 8 to 10, wherein the polysorbate is present in the formulation at a concentration of about 0.01% (w / v). 12. The formulation of any one of embodiments 1 to 11, wherein the pH of the formulation is from about 5.5 to about 7.0. 13. The formulation of any one of embodiments 1-12, wherein the pH of the formulation is about 6.5. 14. The formulation of any one of embodiments 1 to 13, wherein the polypeptide is an antibody. 15. The formulation of any one of embodiments 1 to 14, wherein the polypeptide is a monoclonal antibody. 16. The formulation of any one of embodiments 1 to 15, wherein the polypeptide is a human antibody, a chimeric antibody, or a humanized antibody. 17. The formulation of any one of embodiments 14 to 16, wherein the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments, optionally wherein the antibody fragment is a (Fab')2 fragment. 18. The formulation of any one of embodiments 1-17, wherein the polypeptide is present in the formulation at a concentration of from about 5 mg / mL to about 100 mg / mL. 19. The formulation of any one of embodiments 1-18, wherein the polypeptide is present in the formulation at a concentration of about 10 mg / mL. 20. A formulation comprising: an antibody at a concentration of about 5 mg / mL to about 50 mg / mL; HPMCAS-LF in a formulation having a ratio of HPMCAS-LF to antibody of about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg); a histidine-HCl buffer solution having a concentration of about 5 mM to about 20 mM; Polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v) Including, The formulation has a pH of about 5.5 to about 7.0. 21. A formulation comprising: an antibody at a concentration of about 10 mg / mL; HPMCAS-LF in a formulation having a ratio of HPMCAS-LF to antibody of about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg); histidine-HCl buffer at a concentration of about 10 mM; Polysorbate 20 at a concentration of approximately 0.01% (w / v) Including, A formulation wherein the pH of the formulation is about 6.5. 22. A formulation comprising: an antibody at a concentration of about 5 mg / mL to about 50 mg / mL; HPMCAS-HF in a formulation having a ratio of HPMCAS-HF to antibody of about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg); a histidine-HCl buffer solution having a concentration of about 5 mM to about 20 mM; Polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v) Including, The formulation has a pH of about 5.5 to about 7.0. 23. A formulation comprising: an antibody at a concentration of about 10 mg / mL; HPMCAS-HF in a formulation having a ratio of HPMCAS-HF to antibody of about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg); histidine-HCl buffer at a concentration of about 10 mM; Polysorbate 20 at a concentration of approximately 0.01% (w / v) Including, A formulation wherein the pH of the formulation is about 6.5. 24. The formulation of any one of embodiments 20-23, wherein the antibody is a monoclonal antibody. 25. The formulation of any one of embodiments 20 to 24, wherein the antibody is a human antibody, a chimeric antibody, or a humanized antibody. 26. The formulation of any one of embodiments 20 to 25, wherein the antibody is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv, and (Fab')2 fragments, and optionally, the antibody fragment is a (Fab')2 fragment. 27. The formulation of any one of embodiments 1-26, wherein the formulation is spray dried to form a spray-dried formulation of the polypeptide or antibody. 28. The spray-dried formulation of a polypeptide or antibody according to embodiment 27, wherein the polypeptide or antibody in the formulation is stable at about 90°C for at least about 5 hours and / or stable at about 37°C for at least about 4 weeks. 29. A spray-dried formulation of a polypeptide or antibody according to embodiment 27 or 28, wherein the polypeptide or antibody in the formulation exhibits reduced aggregation and / or reduced chemical degradation. 30. The spray-dried formulation of a polypeptide or antibody according to embodiment 29, wherein the reduced chemical degradation comprises reduced formation of succinimide variants of the polypeptide and / or reduced formation of pyroglutamate variants of the polypeptide. 31. A spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 30, wherein the formulation is an amorphous glassy formulation. 32. A spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 31, wherein the spray-dried formulation of the polypeptide or antibody is encapsulated in a polymer system that creates an acidic microclimate. 33. A spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 32, wherein the spray-dried formulation of the polypeptide or antibody is encapsulated in a lactic acid / glycolic acid polymer system. 34. A spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 33, wherein the spray-dried formulation of the polypeptide or antibody is encapsulated in poly(lactic-co-glycolic acid) (PLGA). 35. A spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 33, wherein the spray-dried formulation of the polypeptide or antibody is encapsulated in a PLGA rod. 36. A composition comprising a formulation according to any one of embodiments 1 to 27. 37. A composition comprising a spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 35. 38. A composition comprising lactic acid / glycolic acid polymer particles comprising a formulation according to any one of embodiments 1 to 27. 39. A composition comprising lactic acid / glycolic acid polymer particles comprising the spray-dried formulation of any one of embodiments 27-35. 40. The composition of embodiment 38 or 39, wherein the lactic acid / glycolic acid polymer particles are PLGA particles. 41. The composition of any one of embodiments 38 to 40, wherein the lactic acid / glycolic acid polymer particles are PLGA rods. 42. A method for preparing a spray-dried polypeptide, comprising preparing a formulation according to any one of embodiments 1 to 19, and subjecting the formulation to spray drying. 43. A method for preparing a spray-dried antibody, comprising preparing a formulation according to any one of embodiments 20 to 26, and subjecting the formulation to spray drying. 44. The method of embodiment 42 or 43, wherein the spray drying is carried out using a spray dryer comprising an inlet and an outlet. 45. The method of embodiment 44, wherein the inlet has a temperature of about 90°C to about 120°C, about 100°C, or about 110°C. 46. ​​The method of embodiment 44, wherein the outlet has a temperature of about 60°C. 47. A method for preparing lactic acid / glycolic acid polymer particles comprising a polypeptide, the method comprising encapsulating a formulation according to any one of embodiments 1 to 26 in a lactic acid / glycolic acid polymer system. 48. A method for preparing lactic acid / glycolic acid polymer particles comprising a polypeptide, the method comprising encapsulating a spray-dried formulation according to any one of embodiments 27 to 30 in a lactic acid / glycolic acid polymer system. 49. The method of embodiment 47 or 48, wherein the lactic acid / glycolic acid polymer particles are PLGA particles. 50. An article of manufacture comprising a formulation according to any one of embodiments 1 to 27, a spray-dried formulation according to any one of embodiments 28 to 35, or a composition according to any one of embodiments 36 to 41. 51. An article of manufacture comprising a spray-dried formulation prepared by the method of any one of embodiments 42-46. 52. An article of manufacture comprising a lactic acid / glycolic acid polymer particle-encapsulated polypeptide prepared by the method of any one of embodiments 47-49.

[0220] All features disclosed herein may be combined in any combination. Each feature disclosed herein may be replaced by an alternative feature serving the same, equivalent, or similar purpose. Thus, unless expressly stated otherwise, each feature disclosed is only an example of a generic series of equivalent or similar features.

[0221] Further details of the present invention are illustrated by the following non-limiting examples, the disclosures of all references herein being expressly incorporated herein by reference. [Example]

[0222] Abbreviations: AS-HF (HPMCAS containing approximately 12% acetate and approximately 7% succinate); AS-LF (HPMCAS containing approximately 8% acetate and approximately 15% succinate); E / P (excipient / protein ratio); HPMC (hydroxypropyl methylcellulose, also known as hypromellose); HPMCAS (hypromellose acetate succinate); IEC (ion exchange chromatography); PLGA (poly(lactic-co-glycolic acid)); SEC (size exclusion chromatography).

[0223] The following examples are intended to be merely illustrative of the present invention and therefore should not be construed as limiting the present invention in any way. The following examples and detailed description are offered by way of illustration and not by way of limitation. Example 1. Hydroxypropyl methylcellulose derivatives stabilize antibody fragments and resist pH changes

[0224] The usefulness of acetate and succinate derivatives of hydroxypropyl methylcellulose (HPMC or hypromellose) in stabilizing a model protein (labeled Fab2) in the context of PLGA rods was evaluated. Two different HPMC derivatives were investigated: one containing approximately 8% acetate and approximately 15% succinate (termed AS-LF), and the other containing approximately 12% acetate and 7% succinate (termed AS-HF).

[0225] The performance of the HPMCAS excipient in spray-dried formulations containing Fab2 was compared with that of trehalose, metolose, and excipient-free formulations over a period of several hours at 90°C and 4 weeks at 37°C. Trehalose has previously been studied for stabilization of Fab2 in spray-dried formulations and at elevated temperatures (Rajagopal, K et al., Mol Pharm, 2019.16(1):349-358). The 90°C temperature was chosen to guide the suitability of spray-dried formulations for high-temperature processing, such as hot-melt extrusion, used to prepare PLGA polymer rods for drug delivery. Stability at 37°C was performed to provide significant relative stability between formulations that may be applicable at temperatures between 2 and 8°C. Furthermore, 37°C also mimics body temperature and captures the effect of exposing a protein drug to physiological temperatures experienced during prolonged residence within a PLGA rod sustained delivery system. Fab2 as a spray-dried powder was encapsulated within PLGA cylindrical rods by hot-melt extrusion. The stability of Fab2 within PLGA rods was then studied under physiological conditions when formulated in the presence of HPMCAS excipients, metolose, and trehalose at 37°C for 3 weeks. To characterize protein stability when encapsulated in PLGA rods, Fab2 extracted from the rods was examined for aggregation or chemical degradation. Unlike trehalose, which is hydrolyzable in humans, HPMCAS is nonbiodegradable, limiting its use in parenteral delivery. Materials and Methods

[0226] Materials. Fab2 was obtained from Genentech (South San Francisco, CA). α-α trehalose dihydrate was obtained from Ferro Pfantiehl Laboratories (Cleveland, OH). Hypromellose acetate succinate (HPMCAS) was obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan) in two different grades: HPMC AS-HF (12% acetyl, 7% succinoyl, dissolution pH ≥ 6.5) and HPMC AS-LF (8% acetyl, 15% succinoyl, dissolution pH ≥ 5.5). Hypromellose (Metolose) was also obtained from Shin-Etsu Chemical Co., Ltd. (Tokyo, Japan). Histidine and histidine-HCl were obtained from Sigma-Aldrich (St. Louis, MO), and polysorbate 20 (PS20) was obtained from Spectrum Chemical (New Bernswick, NJ). PLGA RESOMER® RG755S was purchased from Evonik Corporation (Piscataway, NJ).

[0227] Spray drying of Fab2. 40 mg / mL of Fab2 was dialyzed against 10 mM histidine / histidine-HCl buffer (pH 5.5) containing 0.01% (w / v) polysorbate 20 (MWCO = 10,000 Da). The dialysis buffer was changed four times over 16 h. Fab2 was diluted to 20 mg / mL with 10 mM histidine / histidine-HCl buffer (pH 5.5) containing 0.01% polysorbate 20. Fab2 concentration was measured using UV spectroscopy (ε = 1.39 mL cm-1 mg-1 at 280 nm). As specified by the manufacturer, HPMC AS-LF and HPMC AS-HF grades were initially insoluble in water when prepared at 20 mg / mL with a final mixture pH of approximately 4. Working solutions of 20 mg / mL HPMC AS-LF and HPMC AS-HF were prepared in water containing 10 mg / mL histidine base and 0.01% polysorbate 20 at a final pH of 6.5. The required amounts of trehalose dihydrate, HPMC AS-LF, HPMC AS-HF, and metolose were added to Fab2 to obtain solutions with the same excipient / protein mass ratio of 1.0 (see Table 1). The aqueous formulations were then spray-dried using a Buchi Model B290 Mini Spray Dryer (New Castle, NJ) at inlet and outlet temperatures of 110 ± 2 °C and 60 ± 2 °C, respectively. The pump power was 8%, and the aspirator was operated at 100% capacity. The delivery rate was 3.4 mL / min, and the compressed air flow rate was 600 L / h. The collected spray-dried powders were transferred to clean, dry 15 cc Lyo glass vials and stored in a vacuum chamber under nitrogen until further use. All spray-dried powders were further dried in a freeze dryer (Advantage Pro Lyophilizer, SP Scientific, UK).

[0228] Scanning Electron Microscopy (SEM). The morphology of the spray-dried particles was imaged with a Quanta 3D (Hillsboro, Oregon) FEG SEM. Samples were mounted on aluminum stubs with carbon adhesive tape and sputter-coated with gold / palladium (Cressington Sputter Coater, TED Pella, Inc.) to improve electrical conductivity. SEM images were collected at low voltage to minimize any potential sample damage or surface charging.

[0229] Particle size characterization by laser diffraction. The particle size distribution of the spray-dried Fab2 powder was measured using a Partica LA-950V2 Laser Diffraction Particle Size Distribution Analyzer (Horiba Ltd., Kyoto, Japan). Approximately 1 mg of spray-dried powder was dispersed in 1 ml of isopropanol, and this dispersion was added dropwise to 50 ml of isopropanol until the target light obscuration level was achieved. The refractive index of isopropanol (1.3776) was used to calculate the particle size distribution using a particle size program.

[0230] High-Temperature Heat Treatment of Fab2. Spray-dried powder from each formulation (2–7 mg, equivalent to 1–1.5 mg of Fab2) was weighed into a 7 mL glass vial. The vial was uncapped and placed in a Binder forced-air convection oven (Bohemia, NY), preheated, and equilibrated to the desired temperature at 90 ± 5 °C. Samples were removed at 1, 2, 3, 4, and 5 hours, immediately capped, and cooled to room temperature. The vial contents were dissolved in purified water to a final protein concentration of approximately 1 mg / mL. Reconstituted aqueous samples were observed for clarity, and visually clear samples were used for SEC and IEC analysis.

[0231] Size-exclusion chromatography (SEC). For Fab2 size-exclusion chromatography (SEC), HPLC was performed using an Agilent 1200 Series HPLC system (Santa Clara, CA) equipped with a TOSOH TSKgel G2000SWXL (7.8 × 300 mm id, 5 μm particle size) column. Samples were analyzed at 25 °C in isocratic mode using 0.20 M K3PO4 and 0.25 M KCl, pH 6.2, as the mobile phase at a flow rate of 0.7 mL / min. 20 μL of sample at 1 mg / mL concentration was injected, and the total run time was 20 min. Absorbance at 280 nm was used for detection. The percent peak area was calculated by dividing the peak area of ​​each group at each time point by the total peak area.

[0232] Ion exchange chromatography (IEC). IEC HPLC was performed using an Agilent 1200 Series HPLC system (Santa Clara, CA) with two Dionex (Sunnyvale, CA) ProPac SAX-10 (2 x 250 mm) strong anion exchange columns connected in series with a diode array detector (DAD). Mobile phase A (solvent A) was 20 mM Tris buffer, pH 8.2, and mobile phase B (solvent B) was 250 mM sodium chloride dissolved in solvent A. Prior to analysis, samples were diluted to approximately 1 mg / mL in solvent A, and 20 μL of sample was injected. Fab2 charge variants were separated using a linear gradient starting from 100% solvent A in 0 min to 20% solvent A in 45 min, with a total run time of approximately 60 min. Absorbance at 280 nm was used for detection. The IEC peak was resolved into a main peak, an acidic peak, and a basic peak.

[0233] Separation of Fab2 from AS-LF and AS-HF Spray-Dried Formulations. The stability of Fab2 in HPMCAS powder formulations was analytically determined via SEC and IEC by reconstituting the powder in water and separating Fab2 from HPMCAS. Reconstituted Fab2 spray-dried powders containing AS-LF and AS-HF were titrated with 0.1 N HCl to lower the pH of the solution to pH 3, precipitating the HPMCAS excipient. The precipitated samples were centrifuged at 4000 g, and the supernatant was used for Fab2 analysis by diluting with the appropriate mobile phase for the assay.

[0234] Differential Scanning Calorimetry (DSC). DSC was performed on a TA Instruments DSC Q200 (New Castle, DE) calorimeter under modulated conditions. Approximately 2 ± 1 mg of sample was weighed into an aluminum pan and sealed. The sample was equilibrated at 5°C for 10 minutes, then heated to 120°C at 2°C / min with a modulation of ± 1.00°C / min and cooled to 5°C at 2°C / min. After re-equilibrating the sample at 5°C, the same heating and modulation ramp was repeated twice to 180°C. The first heating ramp was performed to eliminate any thermal history associated with the sample due to storage or handling conditions. For reporting purposes, the second heating ramp was used for samples without thermal history. The glass transition temperature was reported as the half-height midpoint of the second heating cycle (Figure 2).

[0235] Base and Acid Titrations. Base titration experiments were performed on separate aqueous solutions of HPMC AS-LF and HPMC AS-HF excipients using a Mettler Toledo pH meter (Mettler Toledo, Columbus, OH) equipped with a MicroPro-ISM pH probe. Mixtures of HPMC AS-LF and HPMC AS-HF in water were prepared at 5% and 7% by weight, respectively. The initial pH of both mixtures was measured to be near 4, indicating that the majority of the HPMCAS excipient was insoluble in water. The solutions were gradually titrated with 0.5 N NaOH and continuously swirled until complete mixing. The pH of both HPMC AS-LF and HPMC AS-HF in water was measured to be above pH 6 upon complete mixing. Similar acid titration experiments were performed in water on spray-dried powder samples of HPMCAS, metolose, trehalose, and excipient-free formulations, in this case by titration to pH 3 with 0.1 N HCl.

[0236] Hot-melt extrusion (HME) of spray-dried Fab2 and PLGA cylindrical rods. Hot-melt extrusion was performed using a HAAKE™ MiniCTW Micro-Conical Twin Screw Compounder (Thermo Scientific, Karlsruhe, Germany) with a barrel capacity of 5 g or 7 cm3. The extruder barrel was preheated to 90 °C, and the screw speed was set at 40 rpm. The front barrel opening of the extruder was kept closed in recirculation mode. Pre-weighed solid polymer and spray-dried Fab2 were manually slowly fed into the extruder and recirculated back into the barrel for microcompounding. The Fab2 loading of implants using different spray-dried formulations was fixed at 15% (w / w) Fab2 / PLGA. Melt-phase blending continued for 30 min in recirculation mode after complete feeding and then extruded through a 0.5 mm circular die. The extrudates were cooled to room temperature, cut into cylinders of the desired length, and stored at 2–8 °C until further use in release studies.

[0237] In vitro stability study setup. The in vitro stability setup for studying Fab2 from PLGA rods was performed in glass vials (VWR borosilicate glass vials, 7.4 mL) containing 5 mg of polymer rod and 1 mL of PBSTN (1x PBS containing 0.01% (w / v) PS20 and 0.02% (w / v) sodium azide). The study temperature was maintained at 37 °C in a humidity-controlled incubator at 80% RH to minimize evaporation. For sample preparation, the polymer rod was cut into five 1 mg segments and placed in individually labeled glass vials. The exact weight of the rod was recorded using a microbalance (Mettler Toledo XPE56, Columbus, OH). Release studies were performed in triplicate, and the release buffer was sampled on day 1 and then every 7 days. At each time point, the release buffer (PBSTN) was completely pipetted into a 1.5 mL LoBind® Eppendorf tube (Eppendorf, CT, USA) and replaced with 1 mL of fresh PBSTN. The Fab2 concentration in the release buffer was measured using the SEC method described previously. After 3 weeks of release, the buffer was completely aspirated from the rod in the vial. The rod was then rinsed twice with 1 mL of water and air-dried in a chemical / biosafety cabinet for 3-4 hours until no visible liquid remained in the vial. The rod was then placed in a vacuum desiccator for at least 1 week to allow all water to completely dry.

[0238] Recovery of Fab2 from PLGA rods. After drying, the same rods were used to analyze the Fab2 remaining within the rods. Fab2 was removed and isolated from the polymer using an extraction protocol. First, the dried rods were transferred to tapered-bottom glass vials (Worldwide Glass Resource, 4 mL). The rods were then dissolved by adding 4 mL of tetrahydrofuran (THF) and vortexing the vial until the rods were visibly dissolved. PLGA is soluble in THF, whereas proteins are not. The vials containing dissolved PLGA rods were centrifuged to produce a Fab2 pellet at the bottom of the vial. Because the protein pellet is easily disturbed, approximately 3.5 mL of THF was removed from the supernatant. The vials were then rinsed with additional THF and centrifuged. The Fab2 pellet at the bottom of the tube was rinsed a second time with THF, centrifuged, and the process repeated. This was done to remove any polymer residue remaining after the initial THF rinse, thus leaving the Fab2-rich pellet in the form of a wet powder. The remaining THF was evaporated to leave a dry Fab2 powder, which was finally reconstituted in 1 ml of PBS buffer and used for analytical testing. result

[0239] Spray-dried formulations of Fab2. Five formulations of 10 mg / mL Fab2 were prepared and evaluated for stability after spray drying (Table 1-1). All five formulations were formulated in 10 mM His-HCl / His buffer and 0.01% w / v polysorbate 20. One formulation contained only 10 mM His-HCl / His buffer and 0.01% w / v polysorbate 20, without any sugar or HPMCAS, and served as a control. Two formulations containing the HPMCAS excipient were formulated in pH 6.5, 10 mM His-HCl / His buffer, and 0.01% w / v polysorbate 20. The preparation of spray-dried AS-LF and AS-HF formulations containing Fab2 is shown schematically in Figure 3. The mass ratio of trehalose, metolose, AS-LF, and AS-HF excipients to protein in each formulation was 1:1. However, the excipient to protein molar ratio varied due to differences in excipient molecular weight, ranging from 0.3 to 137 depending on the formulation. The pH of all formulations was measured in the liquid state before spray drying and by reconstitution in water after spray drying, as shown in Table 1. Both pH measurements were identical, indicating no change in ionization state due to spray drying.

[0240] Characterization of Spray-Dried Fab2. All five formulations after spray drying yielded micron-sized particles (Figure 4A) with similar particle morphology, ranging from spherical to collapsed spheres (Figure 4B). Particles ranged from 3 to 10 microns in diameter across the different excipient formulations. All spray-dried powders were reconstituted in water and analyzed for aggregation using size exclusion chromatography (SEC) and chemical degradation using ion exchange chromatography (IEC). For formulations containing AS-LF and AS-HF, the separation protocol was followed as highlighted in the methods section before analyzing Fab2. The monomer content by SEC and the major charge variant peak by IEC were similar for all formulations after spray drying and comparable to Fab2 before spray drying (Table 1). [Table 2]

[0241] Effect of HPMCAS on the solid-state stability of Fab2. The stability of Fab2 in spray-dried formulations was tested at 90°C for 5 hours and at 37°C for 4 weeks. At these temperatures, the greatest levels of aggregation were observed for the excipient-free formulation, followed by the metolose-containing formulation. For the excipient-free formulation, the maximum monomer loss was 6.2% after 5 hours at 90°C (Figure 5A), and 1.5% after 4 weeks at 37°C (Figure 5B). This was followed by the metolose formulation, which had 5% monomer loss at 90°C and 0.9% monomer loss at 37°C. For formulations containing HPMCAS excipient and trehalose, monomer loss was comparable and significantly reduced regardless of the nature of the excipient. After 5 hours of incubation at 90°C, the trehalose, AS-LF, and AS-HF formulations showed the least monomer loss (0.7%-0.9%). Upon 4 weeks of incubation at 37°C, the trehalose, AS-LF, and AS-HF formulations again showed minimal monomer loss (≦0.2%). Shorter incubation periods at 90°C caused more aggregation than longer incubation periods at 37°C. Compared to the excipient-dependent physical aggregation of Fab2 upon storage at both temperatures, the polymer excipients AS-LF and AS-HF offered equivalent protection against aggregation as trehalose.

[0242] The IEC method relied on an anion exchange column to separate different Fab2-associated charge variant species into either basic or acidic variants. A basic peak was observed to the left of the main Fab2 peak, and an acidic charge variant appeared to the right of the main Fab2 peak. Detailed characterization and identification of the peaks in Fab2 IEC and the chemical degradation mechanism have been previously reported (Rajagopal, K et al., Mol Pharm, 2019.16(1):349-358). The peak immediately to the left of the main Fab2 peak has been studied as a pyroglutamate peak formed via intramolecular cyclization of the N-terminal glutamic acid in Fab2. The peaks around 19 and 21 minutes correspond to the succinimide intermediate products of two and one aspartic acid residue in the Fab2 sequence, respectively. The formation of such pyroglutamate and succinimide intermediate products results in the removal of a net negative charge, resulting in a basic peak in anionic IEC. On the other hand, the formation of deamidated species from the asparagine side chain results in a net negative charge and an increase in the Fab2 isoelectric point (pI), thereby resulting in an acidic variant peak on the anionic IEC. No major Fab2 deamidated peak was observed during the assay.

[0243] In the case of Fab2, chemical degradation was observed following exposure to 90°C and 37°C via various pathways (Figures 6A and 6B). The succinimide product formed via cyclization of one aspartic acid side chain in Fab2 and the pyroglutamate product (PyroE) formed via cyclization of the N-terminal glutamic acid gave rise to basic peaks at approximately 21 and 24.2 minutes in IEC, respectively. An increase in both of these degradation reactions was observed upon exposure of Fab2 to 90°C for all formulations (Figure 6A). In contrast, at 37°C, a new peak appeared around 19 minutes in all formulations after stress (Figure 6B), and the peak around 21 minutes disappeared, suggesting the formation of a new succinimide product from the second aspartic acid following exposure to 37°C.

[0244] At both 90°C and 37°C, the presence of excipients in the formulations did not affect succinimide formation. However, the nature of the excipients was shown to affect the magnitude of pyroglutamate formation. IEC chromatograms were normalized to the main peak. Pyroglutamate formation was quantified by measuring the ratio of the area of ​​the main peak to the pyroglutamate peak (Figures 6C and 6D). The presence of the HPMCAS excipient did not affect Fab2 pyroglutamate formation, in contrast to trehalose, which suppressed pyroglutamate peak formation compared to excipient-free formulations. This is consistent with our previous results, which showed that trehalose suppressed pyroglutamate peak formation in Fab2 under similar conditions (Rajagopal, K et al., Mol Pharm, 2019.16(1):349-358). At the same time, the presence of HPMCAS excipient did not improve the chemical stability of Fab2 during stress conditions at both 90°C and 37°C.

[0245] Buffering Capacity of HPMCAS Excipients. Base titration experiments of the HPMCAS raw material revealed the resistance of both excipients to pH changes upon the addition of sodium hydroxide (Figure 7A). Such base titrations mimicked a similar aqueous ambient environment experienced by the HPMCAS chain during the preparation of the HPMCAS stock solution for spray drying (Figure 3). AS-LF exhibited resistance to pH changes around pH 5.2 and AS-HF around pH 5.8 upon subsequent base addition, further confirming the fact that these two excipients provided the range of buffering capacities specified by the manufacturer. During the base titration experiments, AS-LF was more resistant to pH changes within its observed buffering range around pH 5.2, i.e., nearly twice as resistant as AS-HF, as determined from the mmol of hydroxyl groups introduced per mass of each excipient (Figure 7A). To investigate the neutralizing effect of the HPMCAS excipient within the acidic microclimate of the PLGA device, we monitored the pH change upon acid titration of the HPMCAS spray-dried powder formulation with strong hydrochloric acid (Figure 7B). The presence of the HPMCAS excipient in the spray-dried Fab2 powder resisted pH changes induced by the addition of acid near pH 5. Compared to spray-dried protein powders without the HPMCAS excipient, the pH drop for trehalose and excipient-free formulations was significant (pH 5.5-2.5) with the addition of only 1 mM acid. Similarly, no buffering capacity was observed for metolose, which is structurally identical to HPMCAS except for the acetate and succinate functional groups. Importantly, we found that spray-dried protein formulations containing HPMCAS required nearly six-fold higher (approximately 6 mM) equivalent amounts of acid to induce a similar pH change compared to formulations without HPMCAS.

[0246] Stabilization of Fab2 Encapsulated in PLGA Implants During In Vitro Release. To investigate the effect of excipients on protein stability within PLGA implant rods, spray-dried Fab2 formulations containing trehalose, AS-LF, and AS-HF, but no excipients, were encapsulated into 1 mg PLGA cylindrical rods to study drug release and PLGA implant degradation. After 3 weeks of incubation in PBSTN at 37°C, Fab2 recovered from 15% drug-loaded PLGA rods was analyzed for aggregate stability (Figure 8). This was performed in triplicate for different Fab2-loaded PLGA rods. In the absence of excipients, Fab2 aggregation was higher, with a 9.5% decrease in monomer content. Trehalose-containing rod formulations showed a similar loss of 7% monomer content after 3 weeks. Minimal Fab2 aggregates were observed for all HPMCAS excipient formulations, with a 4% (AS-LF) and 3.2% (AS-HF) decrease in monomer content. Consideration

[0247] Spray drying provides a physically stable amorphous drug form that allows the spray-dried product to be processed into poorly soluble polymeric drug delivery systems. Amorphous drugs can be molecularly dispersed in a stable micron morphology for sustained-release drug release systems, such as millimeter-scale PLGA cylindrical rods. Thermogravimetric analysis (Figure 2) and glass transition temperature (Tg) measurements (Table 1) of the spray-dried powder can provide information to identify the hot-melt extrusion processing temperature range for preparing PLGA rods. Furthermore, spray drying produces solid drug forms of uniform size and morphology that can be manufactured by a reproducible, controllable, and scalable process. Physical characterization of the spray-dried powder suggests that the nature of the excipients does not affect the overall size or morphology of the spray-dried powder, as evidenced by SEM images (Figure 4). The collapsed shape of our particles has been previously reported in similar drying experiments as a function of formulation composition and spray drying parameters such as temperature, air flow rate and feed rate (Rajagopal, K et al., Mol Pharm, 2019.16(1):349-358).

[0248] Controlling spray-drying conditions is important for maintaining protein stability throughout the entire operation and for further processing. The stability of our spray-dried Fab2 did not appear to be affected by the drying conditions and the nature or absence of excipients. Furthermore, because drying is a rapid process of solvent evaporation and droplet formation, having an appropriate amount of excipient is important for a homogeneous protein-excipient solid-phase matrix with a single Tg value. Therefore, the properties of the dispersion are related to the Tg of the pure excipient and the final relative humidity (RH) of the product (Jang, JW et al., PDA J Pharm Sci Technol, 1995, 49(4):166-74). The type of excipient affects the Tg of the spray-dried protein powder. g At the same time, powder T g is the T of the pure excipient g (Figure 1) and the T g The higher the T of the entire excipient-protein formulation, the g Therefore, the T of the trehalose preparation was g (79°C) is higher than the AS-HF (67°C) and AS-LF (59°C) formulations. Since all spray-dried formulations were processed under similar RH conditions, the effect of moisture should be of minimal importance.

[0249] Previous studies have demonstrated that Fab2 is known to undergo aggregation and chemical degradation when stressed at 90°C in the absence of excipients (Rajagopal, K et al., Mol Pharm, 2019, 16(1):349-358). In the presence of AS-LF or AS-HF, we confirmed that Fab2 was stable in powder form when exposed to 90°C and 37°C, providing protection against aggregation similar to trehalose (Figures 5A and 5B). However, the presence of HPMCAS did not suppress pyroglutamate formation in solid formulations (Figures 6A-6D). Because the nature of the excipient determines the appropriate amount of excipient loading required to protect the antibody (Costantino, HR et al., J Pharm Sci, 1998, 87(11):1412-20), further optimization of excipient loading is required to determine the maximum protection provided by HPMCAS. However, HPMCAS as a polymeric excipient may be superior to the small molecule excipient trehalose in preferentially protecting antibodies during storage under high humidity conditions in PLGA rods and during drug release due to its slower overall mobility (Mensink, MA et al., Eur J Pharm Biopharm, 2017, 114:288-295). This study confirmed that at an excipient-to-protein mass ratio of 1.0, HPMCAS excipient provided superior protection for Fab2 inside PLGA rods under release conditions.

[0250] The solid-state stabilization of proteins by good excipients can be explained based on their glass transition, water displacement tendency, and chemical stability (Colaco, CJ et al., Science, 1995, 268(5212):788). In the dry state, the selected excipient should be able to prevent protein denaturation by preferentially excluding water molecules. One such excipient of choice is trehalose, which provides maximum hydrogen-bond protection to proteins in the dry state (Allison, SD et al., Arch Biochem Biophys, 1999, 365(2):289-98). Therefore, direct protein-excipient interactions through water displacement and hydrogen bonding are the benchmark for maintaining a folded protein in solid formulations. In our previous study, the saturating concentration of trehalose to provide maximum protection for Fab2 was determined at an excipient-to-protein mass ratio of 1.0 (Rajagopal, K et al., Mol Pharm, 2019, 16(1):349-358). At the current excipient-to-protein mass ratio of 1.0, the molar ratio of HPMCAS excipient is much lower than that of trehalose. Polymeric sugars have been reported to hydrogen bond with proteins in a manner different from that of disaccharides (Allison, SD et al., Arch Biochem Biophys, 1999, 365(2):289-98). While it is beyond our current scope to examine the strength of the hydrogen-bonding interaction between Fab2 and HPMCAS, HPMCAS appears to provide Fab2 with comparable protection against aggregation at 37°C and 90°C as trehalose (Figures 5A and 5B). Further studies will be required to determine the concentration dependence of Fab2 stability toward HPMCAS.

[0251] The data presented here support the use of HPMCAS as an amorphous solid dispersion carrier for protein drugs at elevated temperatures. At 90 °C, the physical stability of HPMCAS formulations is comparable to that of trehalose, which may be due to the type and strength of hydrogen bonds with proteins in the glassy matrix. The hydroxyl groups of HPMCAS can form hydrogen bonds with proteins, extending the stability of spray-dried solids. This should limit any molecular mobility and phase separation that would otherwise occur in a suitable protein-polymer mixed phase, forming tighter protein-excipient interactions (Figure 9). Al-Obaidi et al. reported HPMCAS as a strong hydrogen-bond donor, improving the amorphous phase stability of hydrophobic drugs through hydrogen-bonding interactions with drug carbonyl groups (Al-Obaidi, H. and G. Buckton, AAPS PharmSciTech, 2009, 10(4):1172-7). Furthermore, the presence of succinate moieties promotes additional intermolecular hydrogen-bonding interactions between HPMCAS and proteins. The free carboxylic acid group in succinate can act as both a hydrogen bond donor and acceptor for protein amide groups. At the same time, Metolose, which has similar methoxy and hydroxypropoxy groups as HPMCAS but lacks additional succinate groups, lacks hydrogen bond donor and acceptor sites for proteins. Therefore, protein-excipient interactions are likely weaker in Metolose formulations. As evidenced by the Tg (150 °C) values ​​of Metolose spray-dried formulations, distinct protein and excipient phases likely exist in the dried state, driven by protein-polymer demixing (Figure 9). Therefore, spray-dried Metolose has the same Tg value as pure Metolose (Figures 1 and 10) and is not affected by any protein interactions. In contrast, spray-dried molecular dispersions of protein and HPMCAS produced a single amorphous phase with a glass transition different from that of pure HPMCAS (Table 1 and Figure 10).

[0252] Interestingly, trehalose, but not HPMCAS, prevented chemical degradation of Fab2 in the solid state (Figures 6C and 6D). All spray-dried formulations had an increase in basic variants upon stress at 90 °C, with the least amount of basic variants formed in the trehalose-containing formulation (Figures 6A and 6B). This has previously been reported for Fab2, due to its susceptibility to heat-dependent dehydration / isomerization, which contributes to reduced chemical stability (Rajagopal, K et al., Mol Pharm, 2019.16(1):349-358). It is noteworthy that succinimide and pyroglutamate formation was greater in the HPMCAS formulation than in the trehalose-containing formulation. Pyroglutamate is the product of cyclization of N-terminal glutamic acid on proteins. As hypothesized in our previous study, hydrogen-bonding interactions between the donor hydroxyl groups of trehalose and the acceptor N-terminal amine group of Fab2 could suppress pyroglutamate formation (Rajagopal, K et al., Mol Pharm, 2019.16(1):349-358). Surprisingly, in the case of HPMCAS, similar amine-hydroxyl interactions do not appear to exist, resulting in an increase in pyroglutamate species with stress durations at 90°C and 37°C. Furthermore, the glass transition temperatures of AS-LF and AS-HF spray-dried formulations containing proteins are slightly lower than those of trehalose-spray-dried proteins. Therefore, mobility may be slightly higher in HPMCAS formulations, which limit hydrogen-bonding interactions between OH groups and the N-terminal amine Fab group. Without being bound by theory, it is possible that there is a significant excess of HPMCAS excipient over trehalose (1:1 w / w excipient to Fab loading), and therefore excess excipient not bound to the protein may drive instability and decrease the glass transition temperature in the case of spray dried HPMCAS powders.

[0253] The present data show that HPMCAS can tolerate pH changes around 5 when added to an aqueous medium (Figure 7A). At neutral pH (pH = 7), it is soluble due to the deprotonated state of its succinate groups (pKa ~ 5), forming stable polymer colloids. The net negative charge on HPMCAS, resulting from the formation of succinate anions at neutral to basic pH, prevents the polymer chains from interacting with each other. As the HPMCAS concentration increases and reaches its maximum capacity for colloid formation, the polymer chains begin to aggregate. The transition to a predominantly unionized protonated state of the succinate groups gradually lowers the pH of the system (pH ≤ 4), eventually resulting in precipitation. This pH-driven phenomenon of deprotonation and protonation of HPMCAS chains is reversible. Furthermore, when the pH is near the pKa of succinate, the HPMCAS / water mixture acts as a buffer, absorbing any excess protons introduced into the aqueous mixture. This buffering capacity was demonstrated in our titration studies using pure raw material and HPMCAS spray-dried protein powder (Figures 7A and 7B). Taking advantage of the unique physicochemical properties of HPMCAS, stable drug-polymer colloids (when ionized) and spray-dried drug dispersions (when non-ionized) have been formulated (Friesen, DT et al., Mol Pharm, 2008, 5(6):1003-19). During the addition of Fab2 stock at pH 5.5 for spray drying, the HPMCAS / water mixture (pH 6.5) showed no pH change. As suggested, hydrochloric acid protons from the Fab2 solution are neutralized by succinate anions present in the excipient, thus acting as a buffer.

[0254] Numerous publications have shown that HPMCAS can maintain supersaturation with the drug in solution due to the polymer's superiority as a precipitation inhibitor, forming stable drug / polymer colloidal solutions. Friesen et al. reported the emergence of a colloidal state during aqueous dissolution of spray-dried drug dispersions made from HPMCAS, which dissolves upon dilution to form free drug and polymer chains (Friesen, DT et al., Mol Pharm, 2008, 5(6):1003-19). This colloidal state is essentially amorphous, which is important for maintaining drug solubility under physiological conditions. The present invention involves the formation of a similar colloidal solution of HPMCAS and Fab2 upon exposure of HPMCAS spray-dried Fab2 to water and during drug release from PLGA rods (Figure 3). The pH of the rapidly forming colloid should be the same as the pH of the pre-spray-dried stock solution, which was itself in a high-energy colloidal state due to the mixing of the HPMCAS stock (pH 6.5) with the Fab2 stock (pH 5.5). This should exceed the acid dissociation constant of the succinate group, ionizing the HPMCAS chain. Such a system consisting of ionized HPMCAS chains with Fab2 acting as an electrostatic assembly should be able to neutralize exogenously added protons and act as a buffer to withstand pH changes. To demonstrate this property of HPMCAS, this example shows a stable protein released upon exposure to acidic protons, likely from aqueous hydrolysis of a PLGA rod implant.

[0255] In preparing protein-loaded polymer rods, an appropriate combination of excipients is required to withstand the high-temperature processing conditions of hot-melt extrusion and acidic degradation products from PLGA hydrolysis (Rajagopal, K et al., J Pharm Sci, 2013, 102(8):2655-66; Zhu, G and SP Schwendeman, Pharm Res, 2000, 17(3):351-7). In general, the same excipients that were effective in maintaining solid-state stability and resisting pH changes also promoted Fab2 stability during in vitro release. Importantly, the present results demonstrate that the HPMCAS excipient provides better protection against Fab2 aggregation within PLGA implant rods compared to the absence of excipients or trehalose-containing formulations. Similar results demonstrating the effectiveness of inorganic basic additives in protecting proteins delivered from PLGA implants have been reported previously (Zhu, G and SP Schwendeman, Pharm Res, 2000, 17(3):351-7). This further demonstrates that the HPMCAS excipient can limit non-covalent protein aggregation caused by the acidic microclimate resulting from PLGA hydrolysis products.

[0256] In other words, having polymer excipients with chemical properties such as acetate and succinate that can neutralize acidic degradation products in PLGA systems aided in stable drug release. While the potential interaction between acidic hydrolysis products and Fab2 appears to decrease over a 3-week period, the effects of long-term storage within the rod environment need to be studied in the presence of HPMCAS. Example 2. Hydroxypropyl methylcellulose derivatives stabilize monoclonal antibodies in the solid state and resist pH changes in the aqueous phase

[0257] The usefulness of acetate and succinate derivatives of hydroxypropyl methylcellulose (HPMC or hypromellose) in stabilizing three full-length monoclonal antibodies (MAbs) in the context of PLGA rods was evaluated. Two different HPMC derivatives were investigated: one containing approximately 8% acetate and approximately 15% succinate (referred to as AS-LF), and the other containing approximately 12% acetate and 7% succinate (referred to as AS-HF). The materials and methods utilized to test the MAb formulations were as described in Example 1 for testing the Fab2 formulations, except for the materials and methods specific to the MAb formulations described below. Materials and Methods

[0258] Materials. MAb1, MAb2, and MAb3 were obtained from Genentech (South San Francisco, CA). MAb1 is a recombinant humanized IgG1κ antibody reactive with HER2. MAb2 is a recombinant humanized IgG1κ antibody reactive with VEGF-A. MAb3 is a recombinant humanized IgG1κ antibody reactive with IgE.

[0259] Size-exclusion chromatography (SEC). For MAb1, MAb2, and MAb3 size-exclusion chromatography (SEC), HPLC was performed using an Agilent 1200 Series HPLC system (Santa Clara, CA) equipped with a TOSOH TSKgel G3000SWXL (7.8 × 300 mm id, 5 μm particle size) column. Samples were analyzed at 25 °C in isocratic mode using 0.20 M K3PO4 and 0.25 M KCl, pH 6.2, as the mobile phase at a flow rate of 0.5 mL / min. 20 μL of sample at 1 mg / mL concentration was injected, and the total run time was 30 min. Absorbance at 280 nm was used for detection. The percent peak area was calculated by dividing the peak area of ​​each group at each time point by the total peak area.

[0260] Ion Exchange Chromatography (IEC). For MAb1, ion exchange chromatography (IEC) HPLC was performed using an Agilent 1200 Series HPLC system (Santa Clara, CA) equipped with a Thermo Scientific / Dionex (Sunnyvale, CA) ProPac WCX-10 analytical (4 x 250 mm) weak cation exchange column and a diode array detector (DAD). Mobile phase A (solvent A) was 10 mM sodium phosphate buffer, pH 7.5, and mobile phase B (solvent B) was 100 mM sodium chloride dissolved in solvent A. Prior to analysis, samples were diluted to approximately 1 mg / mL in solvent A, and 20 μL of sample was injected at a flow rate of 0.5 mL / min. A linear gradient starting from 85% solvent A at 0 min to 0% solvent A at 45 min was used to separate MAb1 charge variants for a total run time of approximately 60 min. Absorbance at 214 nm was used for detection. The IEC peak was separated into a main peak, an acidic peak, and a basic peak.

[0261] For MAb2, ion exchange chromatography (IEC) HPLC was performed using an Agilent 1200 Series HPLC system (Santa Clara, CA) equipped with a Thermo Scientific / Dionex (Sunnyvale, CA) ProPac WCX-10 analytical (4 x 250 mm) weak cation exchange column and a diode array detector (DAD). Mobile phase A (solvent A) was 20 mM ACES buffer, pH 6.5, and mobile phase B (solvent B) was 200 mM sodium chloride dissolved in solvent A. Prior to analysis, samples were diluted to approximately 1 mg / mL in solvent A, and 20 μL of sample was injected at a flow rate of 0.5 mL / min. A linear gradient starting from 70% solvent A at 0 min to 0% solvent A at 50 min was used to separate MAb2 charge variants over a total run time of approximately 90 min. Absorbance at 280 nm was used for detection. The IEC peak was resolved into a main peak, an acidic peak, and a basic peak.

[0262] For MAb3, ion exchange chromatography (IEC) HPLC was performed using an Agilent 1200 Series HPLC system (Santa Clara, CA) equipped with a Thermo Scientific / Dionex (Sunnyvale, CA) ProPac WCX-10 analytical (4 x 250 mm) weak cation exchange column and a diode array detector (DAD). Mobile phase A (solvent A) was 20 mM MES buffer, pH 6.1, and mobile phase B (solvent B) was 100 mM sodium chloride dissolved in solvent A. Prior to analysis, samples were diluted to approximately 1 mg / mL in solvent A, and 20 μL of sample was injected at a flow rate of 0.8 mL / min. A linear gradient starting from 50% solvent A at 0 min to 0% solvent A at 60 min was used to separate MAb3 charge variants over a total run time of approximately 90 min. Absorbance at 280 nm was used for detection. The IEC peak was resolved into a main peak, an acidic peak, and a basic peak.

[0263] The performance of the HPMCAS excipient in spray-dried formulations containing three full-length MAbs was compared with that of formulations without trehalose, metolose, and excipients at 90°C for several hours and 37°C for several weeks. The 90°C temperature was selected to guide the suitability of the spray-dried formulations for high-temperature processing, such as hot-melt extrusion, used to prepare PLGA polymer rods for drug delivery. Stability at 37°C was evaluated to provide significant relative stability among formulations that may be applicable for storage at 2-8°C. Furthermore, 37°C also mimics body temperature and captures the effects of exposing protein drugs to physiological temperatures experienced during prolonged residence within sustained delivery systems containing PLGA rods. Two of the three MAbs (MAb1 and MAb2) were encapsulated into PLGA cylindrical rods by hot-melt extrusion as spray-dried powders at a 1 / 1 E / P mass ratio. MAb stability within PLGA rods was then studied at 37°C for several weeks when formulated in the presence of HPMCAS excipients and trehalose under physiological conditions. To characterize protein stability when encapsulated in PLGA rods, MAbs extracted from the rods were examined for aggregation or chemical degradation. Unlike trehalose, which is hydrolyzable in humans, HPMCAS is nonbiodegradable, limiting its application in parenteral delivery. result

[0264] Spray-dried formulations of MAbs. Thirteen formulations of each mAb, either 5 mg / mL or 10 mg / mL, were prepared and evaluated for stability after spray drying. All 13 formulations were formulated in 10 mM His-HCl / His buffer and 0.01% w / v polysorbate 20. One formulation contained only 10 mM His-HCl / His buffer and 0.01% w / v polysorbate 20, without any sugar or HPMCAS (control formulation). A formulation containing the HPMCAS excipient was formulated at pH 6.0 ± 0.5, 10 mM His-HCl / His buffer, and 0.01% w / v polysorbate 20. The preparation of spray-dried AS-LF and AS-HF formulations containing MAbs is shown schematically in Figure 3 for Fab2. The mass ratios of trehalose, metolose, AS-LF, and AS-HF excipients to protein in each formulation were 1:1, 1:4, and 4:1 (w / w). However, the molar ratios of excipients to protein varied due to differences in excipient molecular weight and ranged from 0.3 to 1752.9 depending on the formulation. The pH of all formulations was measured in the liquid state before spray drying and by reconstitution in water after spray drying, as shown in Table 2-1 for MAb1 and Table 2-2 for MAb2. The pH measurements were similar, indicating little change in ionization state due to spray drying.

[0265] Characterization of Spray-Dried MAbs. All 13 formulations after spray drying yielded micron-sized particles with similar particle morphology, ranging from spherical to collapsed spheres. Particles containing MAb1 ranged in diameter from 3.8 to 10.0 microns across the different excipient formulations. Particles containing MAb2 ranged in diameter from 4.4 to 11.2 microns across the different excipient formulations. All spray-dried powders were reconstituted in water and analyzed for aggregation using size exclusion chromatography (SEC) and chemical degradation using ion exchange chromatography (IEC). For formulations containing AS-LF and AS-HF, a separation protocol was followed before analyzing the MAbs. Monomer content by SEC was similar for all formulations after spray drying and comparable to the MAb formulations before spray drying (Tables 2-1 and 2-2).

[0266] Effect of HPMCAS on Solid-State Stability of MAbs. The stability of MAbs in spray-dried formulations was tested at 90°C for several hours (see Figures 11, 13, and 19) and at 37°C for several weeks (see Figures 12, 14, and 20). The greatest levels of aggregation were observed for the excipient-free MAb formulation (control) when stored at 90°C, followed by the MAb formulation containing metolose, and for MAb3 when stored at 37°C. [Table 3] [Table 4]

[0267] The AS excipient and trehalose provided similar levels of protection against aggregation to MAb1 when stored at 90°C, with the greatest level of protection observed in the E / P = 4 formulation. In contrast, metolose, which lacks acetate and succinate groups, did not protect MAb1 against aggregation.

[0268] In addition, the AS excipient and trehalose provided similar levels of protection against aggregation to MAb2 when stored at 90°C, with the greatest level of protection observed in the E / P = 4 formulation. In contrast, metolose, which lacks acetate and succinate groups, provided less protection to MAb2 against aggregation.

[0269] Furthermore, the AS excipient and trehalose provided similar levels of protection against aggregation to MAb3 when stored at 90°C, with the greatest level of protection observed in the E / P = 1 / 4 formulation. In contrast, metolose, which lacks acetate and succinate groups, provided little or no protection of MAb3 against aggregation.

[0270] Buffering Capacity of HPMCAS Excipient. To investigate the neutralizing effect of HPMCAS excipient in the acidic microclimate of PLGA devices, we monitored the pH change during acid titration of HPMCAS spray-dried powder formulations with strong hydrochloric acid (Figures 15 and 16). The presence of HPMCAS excipient in spray-dried MAb powders resisted pH changes due to the addition of acid around pH 5 to pH 6. Compared to spray-dried protein powders without HPMCAS excipient, the pH drop in the control formulation lacking trehalose and excipients was significant (pH 5.5-2.5) with the addition of only 1 mM acid. Similarly, no buffering capacity was observed with metolose, which is structurally identical to HPMCAS except for the acetate and succinate functional groups. Importantly, we found that spray-dried protein formulations containing HPMCAS required a much greater equivalent amount of acid to undergo similar pH changes compared to formulations without HPMCAS in formulations with high E / P ratios.

[0271] Stabilization of MAb encapsulated in PLGA implants during in vitro release. To examine the effect of excipients on protein stability within PLGA implants, spray-dried MAb formulations containing trehalose, AS-LF, or AS-HF but no excipients were encapsulated into 1 mg PLGA cylindrical rods to study drug release and PLGA implant degradation. MAb recovered from drug-loaded PLGA rods stored in PBSTN at 37°C for several weeks was analyzed for monomer stability (Figures 17 and 18). Trehalose-containing PLGA rods showed substantial loss of monomer content after 4 weeks. Much less MAb1 and MAb2 aggregates were observed for both HPMCAS excipient formulations.

Claims

1. A formulation comprising a polypeptide and hydroxypropyl methylcellulose acetate succinate (HPMCAS).

2. 10. The formulation of claim 1, wherein the HPMCAS comprises from about 8% to about 12% acetate and from about 7% to about 15% succinate.

3. 3. The formulation of claim 2, wherein the HPMCAS comprises about 8% acetate and about 15% succinate (HPMCAS-LF) or about 12% acetate and about 7% succinate (HPMCAS-HF).

4. 4. The formulation of any one of claims 1 to 3, wherein the ratio of HPMCAS to protein in the formulation is from about 4:1 (mg / mg) to about 1:4 (mg / mg), optionally the ratio is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg).

5. The formulation of any one of claims 1 to 4, further comprising a histidine buffer.

6. 6. The formulation of claim 5, wherein the histidine buffer is a histidine HCl buffer.

7. 7. The formulation of claim 5 or 6, wherein the histidine buffer is present in the formulation at a concentration of about 5 mM to about 20 mM, optionally wherein the histidine buffer is present in the formulation at a concentration of about 10 mM.

8. The formulation of any one of claims 1 to 7, further comprising a polysorbate.

9. 9. The formulation of claim 8, wherein the polysorbate is polysorbate 20.

10. 10. The formulation of claim 8 or 9, wherein the polysorbate is present in the formulation at a concentration of about 0.005% (w / v) to about 0.5% (w / v).

11. 11. The formulation of any one of claims 8 to 10, wherein the polysorbate is present in the formulation at a concentration of about 0.01% (w / v).

12. The formulation of any one of claims 1 to 11, wherein the pH of the formulation is from about 5.5 to about 7.

0.

13. The formulation of any one of claims 1 to 12, wherein the pH of the formulation is about 6.

5.

14. The formulation of any one of claims 1 to 13, wherein the polypeptide is an antibody.

15. The formulation of any one of claims 1 to 14, wherein the polypeptide is a monoclonal antibody.

16. The formulation of any one of claims 1 to 15, wherein the polypeptide is a human antibody, a chimeric antibody or a humanized antibody.

17. The formulation of any one of claims 14 to 16, wherein the antibody is an antibody fragment selected from a Fab, a Fab'-SH, an Fv, a scFv, and a (Fab')2 fragment, optionally wherein the antibody fragment is a (Fab')2 fragment.

18. 18. The formulation of any one of claims 1 to 17, wherein the polypeptide is present in the formulation at a concentration of about 5 mg / mL to about 100 mg / mL.

19. The formulation of any one of claims 1 to 18, wherein the polypeptide is present in the formulation at a concentration of about 10 mg / mL.

20. A formulation comprising: an antibody at a concentration of about 5 mg / mL to about 50 mg / mL; HPMCAS-LF, wherein the ratio of HPMCAS-LF to antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg); a histidine-HCl buffer at a concentration of about 5 mM to about 20 mM; Polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v); Including, The formulation has a pH of about 5.5 to about 7.

0.

21. A formulation comprising: an antibody at a concentration of about 10 mg / mL; HPMCAS-LF, wherein the ratio of HPMCAS-LF to antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg); Histidine-HCl buffer at a concentration of about 10 mM; Polysorbate 20 at a concentration of about 0.01% (w / v) Including, A formulation, wherein the pH of the formulation is about 6.

5.

22. A formulation comprising: an antibody at a concentration of about 5 mg / mL to about 50 mg / mL; HPMCAS-HF, wherein the ratio of HPMCAS-HF to antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg); a histidine-HCl buffer at a concentration of about 5 mM to about 20 mM; Polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v); Including, The formulation has a pH of about 5.5 to about 7.

0.

23. A formulation comprising: an antibody at a concentration of about 10 mg / mL; HPMCAS-HF, wherein the ratio of HPMCAS-HF to antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg); Histidine-HCl buffer at a concentration of about 10 mM; Polysorbate 20 at a concentration of about 0.01% (w / v) Including, A formulation, wherein the pH of the formulation is about 6.

5.

24. The formulation of any one of claims 20 to 23, wherein the antibody is a monoclonal antibody.

25. The formulation of any one of claims 20 to 24, wherein the antibody is a human antibody, a chimeric antibody or a humanized antibody.

26. The formulation of any one of claims 20 to 25, wherein the antibody is an antibody fragment selected from a Fab, a Fab'-SH, an Fv, a scFv, and a (Fab')2 fragment, optionally wherein the antibody fragment is a (Fab')2 fragment.

27. The formulation of any one of claims 1 to 26, wherein the formulation is spray dried to form a spray dried formulation of the polypeptide or antibody.

28. 28. A spray-dried formulation of a polypeptide or antibody according to claim 27, wherein the polypeptide or antibody in the formulation is stable at about 90°C for at least about 5 hours and / or stable at about 37°C for at least about 4 weeks.

29. 29. A spray-dried formulation of a polypeptide or antibody according to claim 27 or 28, wherein the polypeptide or antibody in the formulation exhibits reduced aggregation and / or reduced chemical degradation.

30. 30. A spray dried formulation of a polypeptide or antibody according to claim 29, wherein the reduced chemical degradation comprises reduced formation of succinimide variants of the polypeptide and / or reduced formation of pyroglutamate variants of the polypeptide.

31. A spray dried formulation of a polypeptide or antibody according to any one of claims 27 to 30, wherein the formulation is an amorphous glassy formulation.

32. A spray-dried formulation of a polypeptide or antibody according to any one of claims 27 to 31, wherein the spray-dried formulation of the polypeptide or antibody is encapsulated in a polymer system which creates an acidic microclimate.

33. A spray-dried formulation of a polypeptide or antibody according to any one of claims 27 to 32, wherein the spray-dried formulation of the polypeptide or antibody is encapsulated in a lactic / glycolic acid polymer system.

34. A spray-dried formulation of a polypeptide or antibody according to any one of claims 27 to 33, wherein the spray-dried formulation of the polypeptide or antibody is encapsulated in poly(lactic-co-glycolic acid) (PLGA).

35. A spray-dried formulation of a polypeptide or antibody according to any one of claims 27 to 33, wherein the spray-dried formulation of the polypeptide or antibody is encapsulated in a PLGA rod.

36. A composition comprising a formulation according to any one of claims 1 to 27.

37. A composition comprising a spray dried formulation of a polypeptide or antibody according to any one of claims 27 to 35.

38. A composition comprising lactic / glycolic acid polymer particles comprising the formulation according to any one of claims 1 to 27.

39. A composition comprising lactic / glycolic acid polymer particles comprising the spray-dried formulation of any one of claims 27 to 35.

40. 40. The composition of claim 38 or 39, wherein the lactic / glycolic acid polymer particles are PLGA particles.

41. The composition of any one of claims 38 to 40, wherein the lactic / glycolic acid polymer particles are PLGA rods.

42. 20. A method for preparing a spray-dried polypeptide, the method comprising preparing a formulation according to any one of claims 1 to 19 and subjecting said formulation to spray drying.

43. 27. A method of preparing a spray-dried antibody, comprising preparing a formulation according to any one of claims 20 to 26 and subjecting said formulation to spray drying.

44. 44. The method of claim 42 or 43, wherein the spray drying is carried out using a spray dryer comprising an inlet and an outlet.

45. 45. The method of claim 44, wherein the inlet has a temperature of about 90°C to about 120°C, about 100°C, or about 110°C.

46. 45. The method of claim 44, wherein the outlet has a temperature of about 60°C.

47. A method for preparing a lactic / glycolic acid polymer particle comprising a polypeptide, the method comprising encapsulating a formulation according to any one of claims 1 to 26 in a lactic / glycolic acid polymer system.

48. A method for preparing lactic / glycolic acid polymer particles comprising a polypeptide, the method comprising encapsulating a spray-dried formulation according to any one of claims 27 to 30 in a lactic / glycolic acid polymer system.

49. 49. The method of claim 47 or 48, wherein the lactic / glycolic acid polymer particles are PLGA particles.

50. 42. An article of manufacture comprising a formulation according to any one of claims 1 to 27, a spray dried formulation according to any one of claims 28 to 35, or a composition according to any one of claims 36 to 41.

51. An article of manufacture comprising a spray dried formulation prepared by the method of any one of claims 42 to 46.

52. 50. An article of manufacture comprising a lactic / glycolic acid polymer particle encapsulating a polypeptide prepared by the method of any one of claims 47-49.

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