Hydroxypropylmethylcellulose derivatives to stabilize polypeptides

Abstract

The present invention provides formulations comprising a polypeptide and a hydroxypropyl methyl cellulose acetate succinate (HPMCAS) derivative. The formulation is stable; , for example during high temperature processing and in possibly low pH environments. Furthermore, the HPMCAS derivative provides protection to a pH sensitive protein from acidic degradation products from aqueous hydrolysis of poly (lactic-co-glycolic acid) (PLGA) in a PLGA-based delivery system.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority to and the benefit of U.S. Provisional Patent Application No. 63 / 326,772, filed on April 1, 2022, which is incorporated herein by reference in its entirety. Technical Field

[0003] The present invention provides the use of hydroxypropylmethylcellulose acetate succinate (HPMCAS) derivatives as polymer excipients for stabilizing polypeptides; for example, during high temperature processing and in possible low pH environments. In addition, the HPMCAS derivatives provide protection for pH-sensitive proteins from acidic degradation products from aqueous hydrolysis of poly(lactic-co-glycolic acid) (PLGA) in PLGA-based delivery systems. Background Art

[0004] Protein-based therapeutic drugs are prone to physical and chemical degradation during manufacturing, storage and delivery (Le Basle, Y et al., J Pharm Sci, 2020.109(1): p.169-190). Although the understanding of how excipients stabilize liquid and solid proteins has enabled the successful development of many protein-based drugs, there is still a need to identify new excipients and design formulations to achieve 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 includes physicochemical phenomena unrelated to small molecule therapeutic drugs and conformational structural changes (Manning, MC et al., Pharm Res, 2010, 27(4): 544-75). In the case of polymer controlled release systems, protein drugs should be kept in the formulation at physiological temperature 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 when the rods stay at body temperature for a long time, the drug is exposed to a low pH microenvironment due to polymer hydrolysis. In the absence of stabilizing excipients, protein drugs may degrade inside the delivery system and when released in vivo (Rajagopal, K et al., J Pharm Sci, 2013, 102(8):2655-66).

[0005] Preparation of PLGA rods using hot melt extrusion involves mixing a spray-dried protein formulation with solid PLGA, microcompounding at 90°C, followed by extrusion, cooling the extrudate and cutting it into solid rods of the desired size (Rajagopal, K et al., J Pharm Sci, 2013, 102(8):2655-66). It has been previously demonstrated that the addition of hydrophilic small molecule excipients (such as trehalose or tetrahydropyrimidine) to the spray-dried formulation can stabilize the protein at high temperatures, allowing the hot melt extrusion process to be used to prepare 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). Although these excipients stabilize the protein in a solid state during the preparation and storage of PLGA rods during their shelf life, they do not provide stability to the low pH microenvironment created by hydrolytic polymer degradation after rod implantation in vivo (Chang, DP et al., J Pharm Sci, 2015, 104(10):3404-17). Since the hydrolysis of the polymer leads to the formation of lactic acid and glycolic acid (Pitt, CG et al., Biomaterials, 1981, 2(4):215-20), the accumulation of these acids in the PLGA delivery system 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, due to their small size and hydrophilicity, their release rate is faster than that of proteins. This situation leaves the protein unprotected within the rod and leads to aggregation, incomplete drug release and promotes low pH chemical degradation reactions (such as isomerization and fragmentation), which may lead to loss of activity (Rajagopal, K et al., Mol Pharm, 2019. 16(1):349-358; Liu, Y and SP Schwendeman, Mol Pharm, 2012, 9(5):1342-50; Fu, K et al., Pharm Res, 2000, 17(1):100-6; Lu, W and TG Park, PDA J Pharm Sci Technol, 1995, 49(1):13-9; Shao, PG. and LC Bailey, Pharm Dev Technol, 2000, 5(1):1-9).Studies by Zhu et al. have shown that large globular proteins such as BSA undergo extensive aggregation within PLGA systems during prolonged incubation at 37°C in PBS buffer (Zhu, G and SP Schwendeman, Pharm Res, 2000, 17(3):351-7). Therefore, excipients that neutralize the acidic microclimate and resist protein degradation are necessary for stable release of proteins from PLGA systems.

[0006] Various approaches have been explored to stabilize proteins in PLGA devices, such as the addition of the alkaline additive Mg(OH)2 to stabilize BSA (Zhu, G et al., Nat Biotechnol, 2000, 18(1):52-7), and the addition of divalent metal cation salts (such as CaCl2 or MnCl2) to stabilize octreotide (Sophocleous, AM et al., J Control Release, 2009. 137(3):179-84). However, the above methods rely on small inorganic ions as additives to neutralize degradation products or prevent the protein from interacting with degradation products from PLGA hydrolysis, rather than as excipients to provide protection for the protein.

[0007] The use of polymeric excipients is required to stabilize the protein in the spray-dried formulation at elevated temperatures and also to neutralize the low pH environment created within PLGA.

[0008] All references cited herein, including patent applications and publications, are hereby incorporated by reference in their entirety. Summary of the invention

[0009] In some aspects, the present invention provides a formulation comprising a polypeptide and hydroxypropylmethylcellulose acetate succinate (HPMCAS). In some embodiments, HPMCAS comprises about 8% to about 12% acetate and about 7% to about 15% succinate. In some embodiments, HPMCAS comprises about 8% acetate and about 15% succinate (HPMCAS-LF). In other embodiments, 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 concentration of the histidine buffer in the formulation is about 5mM to about 20mM. In some embodiments, the concentration of the histidine buffer in the formulation is about 10mM. In some embodiments, the formulation further comprises a polysorbate. In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the concentration of the polysorbate in the formulation is about 0.005% (w / v) to about 0.5% (w / v). In some embodiments, the concentration of the polysorbate in the formulation is 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.

[0010] In some embodiments of the present invention, the polypeptide in the preparation 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 (Fab')2 fragments. In some embodiments, the antibody fragment is a (Fab')2 fragment. In some embodiments, the concentration of the polypeptide in the preparation is about 5 mg / mL to about 100 mg / mL or about 10 mg / mL to about 80 mg / mL. In some embodiments, the concentration of the polypeptide in the preparation is about 10 mg / mL.

[0011] In some aspects, the present invention provides 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 1:1 (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), and wherein the pH of the formulation is about 5.5 to about 7.0. In some aspects, the present invention provides 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 1:1 (mg / mg); a histidine HCl buffer at a concentration of about 10 mM; polysorbate 20 at a concentration of about 0.01% (w / v), and wherein the pH of the formulation is about 6.5. In some aspects, the present invention provides 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 1:1 (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), and wherein the pH of the formulation is about 5.5 to about 7.0. In some aspects, the present invention provides 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 1:1 (mg / mg); a histidine HCl buffer at a concentration of about 10 mM; polysorbate 20 at a concentration of about 0.01% (w / v), and wherein the pH of the formulation is about 6.5.

[0012] In some embodiments of the invention, the formulation is spray dried to form a spray-dried formulation of a 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 at about 37°C for at least about 4 weeks. In some embodiments, the polypeptide in the formulation shows reduced aggregation and / or reduced chemical degradation. In some embodiments, the reduced chemical degradation includes the formation of reduced succinimide variants of the polypeptide and / or the formation of reduced pyroglutamic acid variants of the polypeptide. In some embodiments, the formulation is an amorphous glassy formulation.

[0013] In some embodiments of the invention, the spray-dried formulation of a polypeptide or antibody (e.g., an antibody) is encapsulated in a polymer system that produces 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 acid-co-glycolic acid) (PLGA). In some embodiments, the spray-dried formulation of a polypeptide is encapsulated in a PLGA rod.

[0014] In some aspects, the present invention provides a composition comprising any formulation described herein; for example, a composition comprising a spray-dried formulation of a polypeptide (e.g., an antibody) as described herein. In some embodiments, the present invention provides a composition comprising lactic acid / glycolic acid polymer particles comprising any formulation described herein. In some embodiments, the present invention provides a composition comprising lactic acid / glycolic acid polymer particles comprising any spray-dried formulation 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.

[0015] In some aspects, the present invention provides a method for preparing a spray-dried polypeptide, the method comprising preparing a formulation as described herein, and subjecting the formulation to spray drying. In some aspects, the present invention provides a method for preparing a spray-dried antibody, the method comprising preparing a formulation as described herein, and subjecting the formulation to spray drying. In some embodiments, spray drying is performed 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.

[0016] In some aspects, the present invention provides a method for preparing lactic acid / glycolic acid polymer particles comprising a polypeptide, the method comprising encapsulating a formulation as described herein in a lactic acid / glycolic acid polymer system. In some aspects, the present invention provides a method for preparing lactic acid / glycolic acid polymer particles comprising a polypeptide, the method comprising encapsulating a spray-dried formulation as described herein in a lactic acid / glycolic acid polymer system. In some embodiments, the lactic acid / glycolic acid polymer particles are PLGA particles.

[0017] In some aspects, the invention provides an article of manufacture comprising any formulation (eg, a spray-dried formulation) or composition as described herein. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1The structure of trehalose is shown along with the general structure of cellulose. The bottom subfigure shows the nature of the R groups defining Metolose, AS-LF, and AS-HF as well as the molecular weight and glass transition temperature of the polymer excipients and trehalose

[0019] Figure 2 The glass transition temperatures of the formulations as represented by the differential scanning calorimetry (DSC) thermograms of all five spray-dried Fab2 formulations are shown.

[0020] Figure 3 Schematic diagram showing the preparation of spray-dried AS-LF and AS-HF formulations. AS-HF or AS-LF polymer was dissolved in water, titrated to pH 6.5 with histidine base, and then mixed with Fab2 solution in pH 5.5 buffer. The turbid polymer and protein mixed solution was spray dried to obtain a solid formulation.

[0021] Figure 4A and 4B Characterization of the spray-dried formulations is shown. Figure 4A Shown is the particle size distribution of the spray-dried Fab2 formulation measured using laser diffraction. Figure 4B SEM images of spray-dried formulations are shown. The scale bar in each image corresponds to 10 μm.

[0022] Figure 5A and 5B Fab2 aggregation in the spray-dried formulation is shown. Fab2 monomer content in the spray-dried formulation as a function of time: After exposure to 90°C for 5 hours ( Figure 5A ); and after 4 weeks of exposure at 37°C ( Figure 5B ).

[0023] Figures 6A to 6D The chemical degradation of Fab2 in the spray-dried formulation is shown. Ion exchange chromatograms of Fab2 in different formulations: after 5 hours at 90°C ( Fig. 6A ); and after 4 weeks at 37°C ( Figure 6B ). Pyroglutamate formation (measured as the ratio of PyroE to the main peak area) over time: at 90 °C ( Figure 6C ); and at 37°C ( Fig.6D ).

[0024] Fig. 7A and 7B The buffer capacity of AS excipients is shown. HPMCAS excipients are able to resist changes in pH ( Fig. 7A ): After titration with strong base (0.5N NaOH) in the raw material form ( Figure 7B ); and during titration with strong acid (0.1 N HCl) in spray-dried Fab2 formulations.

[0025] Figure 8 Fab2 stability within PLGA rods is shown. For all four formulations, changes in the extracted Fab2 monomer content upon recovery after 3 weeks at 37°C within PLGA rods.

[0026] Fig. 9 Schematic diagram showing the distribution of proteins and polymers in spray-dried formulations. In the solid state, spray drying of protein-polymer mixtures produces two types of distributions. The electrostatic interaction between Fab2 and HPMCAS polymer ensures uniform blending in the solid state, while in Metolose formulations, physicochemical incompatibility leads to stratification and phase separation after spray drying.

[0027] Fig.10 Shown are the excipient glass transition temperatures represented by differential scanning calorimetry (DSC) thermograms for all four excipients used in our studies.

[0028] Fig.11 Aggregation of MAb1 in spray-dried formulations is shown. The change in monomer content of MAb1 in spray-dried formulations containing different excipient to protein weight ratios (E / P) after exposure to 90°C for different time periods (0 to 5 hours) is shown over time.

[0029] Fig.12 Aggregation of MAb1 in spray-dried formulations is shown. The change in monomer content of MAb1 in spray-dried formulations containing different excipient to protein weight ratios (E / P) after exposure to 37°C for different time periods (0 to 8 weeks) is shown over time.

[0030] Fig.13 Aggregation of MAb2 in spray-dried formulations is shown. The monomer content of MAb2 in spray-dried formulations containing different excipient to protein weight ratios (E / P) is shown over time after exposure to 90°C for different time periods (0 to 4 hours or 5 hours).

[0031] Fig.14 Aggregation of MAb2 in spray-dried formulations is shown. The monomer content of MAb2 in spray-dried formulations containing different excipient to protein weight ratios (E / P) is shown over time after exposure to 37°C for different time periods (0 to 8 weeks).

[0032] Fig.15 The buffer capacity of MAb1 formulations containing different excipients is shown. The pH change after adding dilute hydrochloric acid to a MAb1 formulation dissolved in water after spray drying is shown. The arrow indicates the increase in excipient content in the formulation containing the protein.

[0033] Fig.16The buffer capacity of MAb2 formulations containing different excipients is shown. The pH change after adding dilute hydrochloric acid to a MAb2 formulation dissolved in water after spray drying is shown. The arrow indicates the increase in excipient content in the formulation containing protein.

[0034] Fig.17 MAb1 stability in PLGA rods is shown. Shown is the change in the extracted MAb1 monomer content of the four formulations when recovered after 4 weeks at 37°C in PLGA rods.

[0035] Fig.18 MAb2 stability in PLGA rods is shown. Shown is the change in the extracted MAb2 monomer content of the four formulations when recovered after 4 weeks at 37°C in PLGA rods.

[0036] Fig.19 Aggregation of MAb3 in spray-dried formulations is shown. Shown is the change in monomer content of MAb3 in spray-dried formulations containing different excipient to protein weight ratios (E / P) after exposure to 90°C for different time periods (0 to 5 hours) over time.

[0037] Fig. 20 Aggregation of MAb3 in spray-dried formulations is shown. Shown is the change in monomer content of MAb3 in spray-dried formulations containing different excipient to protein weight ratios (E / P) after exposure to 37°C for different time periods (0 to 3 months) over time. DETAILED DESCRIPTION

[0038] In some aspects, the present invention provides a formulation comprising a polypeptide (e.g., an antibody) and a hydroxypropylmethylcellulose acetate succinate (HPMCAS) derivative. The use of HPMCAS provides stability for proteins during high temperature processing and in possible low pH environments of drug delivery platforms. In addition, in a delivery system based on poly (lactic acid-co-glycolic acid) (PLGA), HPMCAS provides protection for pH-sensitive polypeptides from the effects of acidic degradation products from aqueous hydrolysis of PLGA. In some embodiments, HPMCAS comprises about 8% acetate and about 15% succinate (HPMCAS-LF) or about 12% acetate and about 7% succinate (HPMCAS-HF).

[0039] In some aspects, 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, wherein the ratio of HPMCAS to polypeptide in the formulation is about 1:1 (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), and wherein the pH of the formulation is about 6.0 to about 7.0.

[0040] In some aspects, 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, wherein the ratio of HPMCAS to polypeptide in the formulation is about 1:1 (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), and wherein the pH of the formulation is about 6.0 to about 7.0, wherein the polypeptide in the formulation is stable at about 90° C. for at least about 5 hours and / or at about 37° C. for at least about 4 weeks.

[0041] Also provided are methods of preparing the formulations, spray-dried formulations, and PLGA-encapsulated polypeptides, and articles of manufacture comprising the formulations, spray-dried formulations, and PLGA-encapsulated polypeptides.

[0042] definition

[0043] "Spray drying" refers to a process in which a liquid or slurry containing a protein or monoclonal antibody is atomized and dried using a gas (usually air or nitrogen) at a temperature above ambient temperature to produce dry powder particles containing the protein or monoclonal antibody. During the process, the liquid evaporates and forms dry particles. In one embodiment, spray drying is performed using a spray dryer, for example, the spray dryer has 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 a variety of methods such as a cyclone separator, high pressure gas, static charge, etc. This definition of spray drying herein explicitly excludes freeze drying or crystallization of monoclonal antibodies.

[0044] "Dry" particles, proteins or monoclonal antibodies herein have been subjected to a drying process such that their water content has been significantly reduced. In one embodiment, the particles, proteins or monoclonal antibodies have a water content of less than about 10%, such as less than about 5%, for example, where the water content is measured by chemical titration (e.g., Karl Fischer) or weight loss (high temperature heating).

[0045] As used herein, "pre-spray drying preparation" refers to a preparation of a polypeptide (eg, antibody) and one or more excipients such as a stabilizer (eg, HPMCAS derivative) and optionally a buffer. In one embodiment, the preparation is in liquid form.

[0046] "Suspension formulations" are liquid formulations comprising solid particles (e.g., spray-dried antibody particles), which are dispersed in the entire liquid phase in which they are insoluble. In one embodiment, the solid particles in the suspension formulation have an average particle size (e.g., as analyzed by laser diffraction) of about 2 microns to about 30 microns (e.g., about 5 microns to about 10 microns). Optionally, the solid particles in the suspension formulation have a peak value (highest percentage) particle size (e.g., as analyzed by laser diffraction) less than about 30 microns and optionally less than about 10 microns. Suspension formulations can be prepared by merging spray-dried antibody particles with non-aqueous suspension vehicles. In one embodiment, the suspension formulation is adjusted to be suitable for or suitable for subcutaneous administration to a subject or patient.

[0047] The terms "polypeptide" or "protein" are used interchangeably herein and refer to polymers of amino acids of any length. The polymer may be linear or branched, it may contain modified amino acids, and it may be interspersed with non-amino acids. These terms also encompass amino acid polymers that have been modified naturally or by intervention; for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation or modification (such as conjugation with a labeling component). Also included within the definition are, for example, polypeptides containing one or more analogs of amino acids (including, for example, non-natural amino acids, etc.) and other modifications known in the art. The terms "polypeptide" and "protein" as used herein specifically encompass antibodies.

[0048] A "purified" polypeptide (e.g., an antibody or immunoadhesin) is one that has been improved in purity so that it exists in a purer form than it exists in its natural environment and / or when initially synthesized and / or amplified under laboratory conditions. Purity is a relative term and does not necessarily mean absolute purity.

[0049] A polypeptide that "binds" an antigen of interest (e.g., a tumor-associated polypeptide antigen target) is one that binds the antigen with sufficiently high affinity so that the polypeptide can be used as a diagnostic and / or therapeutic agent targeting cells or tissues expressing the antigen, and does not significantly cross-react with other polypeptides. In such embodiments, the extent of binding of the polypeptide to a "non-target" polypeptide will be less than about 10% of the extent of binding of the polypeptide to its specific target polypeptide as measured by fluorescence activated cell sorting (FACS) analysis or radioimmunoprecipitation (RIA).

[0050] With respect to the binding of a polypeptide to a target molecule, the term "specific binding" or "specifically binds to" or "specific for" a particular polypeptide or an epitope on a particular polypeptide target means binding that is distinct from non-specific interactions. Specific binding can be measured, for example, by measuring the binding of a molecule compared to the binding of a control molecule, which is typically a molecule with a similar structure but without binding activity. For example, specific binding can be determined by competition with a control molecule similar to the target (excess unlabeled target). In this case, if the binding of the labeled target to the probe is competitively inhibited by excess unlabeled target, specific binding is indicated.

[0051] 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 complete antibodies, and antibody fragments, so long as they exhibit the desired biological activity. The term "immunoglobulin" (Ig) is used interchangeably with antibody herein.

[0052] Antibodies are naturally occurring immunoglobulin molecules that have different structures, all based on the immunoglobulin fold. For example, an IgG antibody has two "heavy" chains and two "light" chains that undergo disulfide bonding to form a functional antibody. Each heavy and light chain itself contains a "constant" (C) and a "variable" (V) region. The V region determines the antigen-binding specificity of the antibody, while the C region provides structural support and function 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 an antibody to specifically bind to a specific antigen.

[0053] The antigen binding specificity of an antibody is determined by the structural features of the V region. Variability is not evenly distributed between the 110 amino acids of the variable domain. On the contrary, the V region is composed of relatively unchanged segments of a framework region (FR) consisting of 15 to 30 amino acids, which are separated by shorter regions that vary greatly, and these shorter regions are called "hypervariable regions", each of which is 9 to 12 amino acids long. The variable domains of natural heavy and light chains each include four FRs, which mainly use a β-folded structure, connected by three hypervariable regions ("HVRs"), which form a loop connecting the β-folded structure and form a part of the β-folded structure in some cases. The hypervariable regions in each chain are closely bound together by FR, and together with the hypervariable regions in another chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 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 have respective effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0054] Each V region generally contains three hypervariable regions, such as complementarity determining regions ("CDRs", each of which contains "hypervariable loops"), and four framework regions. Thus, an antibody binding site, i.e., the minimum structural unit required to bind a specific desired antigen with substantial affinity, will typically include three CDRs, and at least three, preferably four, framework regions interspersed therebetween to maintain and present the CDRs in the proper conformation. A classic four-chain antibody has a V H and V L The antigen binding site is defined by the V domains. Some antibodies, such as camelid and shark antibodies, lack light chains and rely solely on the binding site formed by the heavy chain. Single domain engineered immunoglobulins can be prepared in which the binding site is formed by either the heavy chain or the light chain alone, without the need for V H With V L cooperation between them.

[0055] The term "variable" refers to the fact that some parts of the variable domains have very different sequences between antibodies and are used for the binding and specificity of each specific antibody to its specific antigen. However, variability is not evenly distributed in the variable domains of antibodies. It is concentrated in three segments called hypervariable regions in the light chain and heavy chain variable domains. The more conservative part of the variable domain is called the framework region (FR). The variable domains of natural heavy and light chains each contain four FRs, which mainly adopt a β-folded structure and are connected by three hypervariable regions, which form a loop connecting the β-folded structure and form a part of the β-folded structure in some cases. The hypervariable regions in each chain are tightly bound together by FR, and together with the hypervariable regions in the other chain, contribute to the formation of the antigen binding site of the antibody (see Kabat et al., Sequences of Proteins of Immunological Interest, 5th edition, 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 have respective effector functions, such as participation of the antibody in antibody-dependent cellular cytotoxicity (ADCC).

[0056] As used herein, the term "hypervariable region" refers to the amino acid residues in an antibody that are responsible for antigen binding. A hypervariable region may include amino acid residues from a "complementarity determining region" or "CDR" (e.g., V L Around residues 24-34 (L1), 50-56 (L2), and 89-97 (L3), and V H 31-35B (H1), 50-65 (H2), and 95-102 (H3) in the H1 (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., V L Residues 26-32 (L1), 50-52 (L2), and 91-96 (L3) in V H 26-32 (H1), 52A-55 (H2) and 96-101 (H3) (Chothia and Lesk J. Mol. Biol. 196:901-917 (1987)).

[0057] "Framework" or "FR" residues are those variable domain residues other than the hypervariable region residues as herein defined.

[0058] "Antibody fragments" include a portion of an intact antibody, preferably including its antigen binding region. Examples of antibody fragments include Fab, Fab', F(ab')2, 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-armed antibodies, single variable domain antibodies, minibodies, single-chain antibody molecules; multispecific antibodies formed from antibody fragments (e.g., including 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 engagers (BiTEs), and trifunctional antibodies.

[0059] 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, whose name reflects its ability to crystallize readily. Pepsin treatment produces the F(ab')2 fragment, which has two antigen-binding sites and is still capable of cross-linking antigen.

[0060] "Fv" is the smallest antibody fragment that contains a complete antigen recognition and antigen binding site. This region consists of a dimer of a heavy chain variable domain and a light chain variable domain in tight, non-covalent association. In this configuration, the three hypervariable regions of each variable domain interact to form a V H -V L The antigen binding site is defined on the surface of the dimer. The six hypervariable regions together 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 to an antigen, although its affinity is lower than that of the complete binding site.

[0061] The Fab fragment also contains the constant domain of the light chain and the first constant domain (CH1) of the heavy chain. The difference between the Fab' fragment and the Fab fragment is that the Fab' fragment adds some residues at the carboxyl terminus of the heavy chain CH1 domain, which residues include one or more cysteines from the antibody hinge region. Fab'-SH is the nomenclature herein for Fab' in which the cysteine ​​residues of the constant domain carry at least one free thiol group. F(ab')2 antibody fragments were originally produced as paired Fab' fragments with hinge cysteines between them. Other chemical couplings of antibody fragments are also known.

[0062] 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.

[0063] According to the amino acid sequence of its heavy chain constant domain, antibodies can be divided into different classes. There are the following five major classes of complete antibodies: IgA, IgD, IgE, IgG and IgM, and several of these classes can be further divided into "subclasses" (isotypes), such as IgG1, IgG2, IgG3, IgG4, IgA and IgA2. The heavy chain constant domains corresponding to different classes of antibodies are respectively called α, δ, ε, γ and μ. The subunit structure and three-dimensional configuration of different classes of immunoglobulins are well known.

[0064] "Single-chain Fv" or "scFv" antibody fragments comprise the V H and V L In some embodiments, the Fv polypeptide is further present in V H and V L A polypeptide linker is included between the domains so that the scFv can form the desired antigen binding structure. For a review of scFv, see Plückthun's The Pharmacology of Monoclonal Antibodies, Vol. 113, Rosenburg and Moore, ed., Springer-Verlag, New York, pp. 269-315 (1994).

[0065] The term "diabodies" refers to small antibody fragments with two antigen-binding sites, wherein the fragments are comprised of two antigen-binding sites on the same polypeptide chain (V H -V L ) in the light chain variable domain (V L ) connected to the heavy chain variable domain (V H ). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementary domains of another chain and create two antigen-binding sites. Diabodies are more fully described in, for example, EP 404,097; WO 93 / 11161; and Hollinger et al., Proc. Natl. Acad. Sci. USA, 90:6444-6448 (1993).

[0066] The term "multispecific antibody" is used in the broadest sense and specifically encompasses antibodies with multiple epitope specificities. Such multispecific antibodies include, but are not limited to, antibodies comprising a heavy chain variable domain (VH ) and the light chain variable domain (V L ) of the antibody, wherein V H V L Units have multiple epitope specificity, with two or more V L and V H Domain antibody, each V H V L Units bind to different epitopes, 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, covalently or non-covalently linked antibody fragments. "Multi-epitope specificity" refers to the ability to specifically bind to two or more different epitopes on the same or different targets. "Monospecificity" refers to the ability to bind only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5M to 0.001pM, 3M to 0.001pM, 1M to 0.001pM, 0.5M to 0.001pM or 0.1M to 0.001pM.

[0067] "Single domain antibodies" (sdAbs) or "single variable domain (SVD) antibodies" generally refer to antibodies in which a single variable domain (VH or VL) can confer antigen binding. In other words, the single variable domain does not need to interact with another variable domain to recognize the target antigen. Examples of single domain antibodies include antibodies derived from camelids (alpacas and camels) and cartilaginous fish (e.g., nurse sharks) and antibodies derived from recombinant methods of human and mouse antibodies (Nature (1989) 341:544-546; Dev Comp Immunol (2006) 30:43-56; Trend 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).

[0068] As used herein, the term "monoclonal antibody" refers to an antibody obtained from a substantially homogeneous antibody population, that is, the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants that may be produced during the production of the monoclonal antibody, which are generally present in small amounts. Contrary to polyclonal antibody preparations that generally include different antibodies for different determinants (epitopes), each monoclonal antibody is directed to a single determinant on an antigen. In addition to specificity, the advantage of monoclonal antibodies is that they are synthesized without being contaminated by other immunoglobulins. The modifier "monoclonal" indicates that the feature of an antibody is obtained from a substantially homogeneous antibody population, and should not be construed as requiring antibodies to be produced by any ad hoc method. For example, the monoclonal antibody used according to the method provided herein can be prepared by the hybridoma method first described by Kohler et al., Nature, 256:495 (1975), or can be prepared by a recombinant DNA method (see, for example, U.S. Patent No. 4,816,567). "Monoclonal antibodies" can 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).

[0069] The monoclonal antibodies herein specifically include "chimeric" antibodies (immunoglobulins) in which a portion of the heavy and / or light chains is identical or homologous to the corresponding sequence in an antibody from a particular species or belonging to a particular antibody class or subclass, and the remainder of one or more chains is identical or homologous to the corresponding sequence in an antibody from another species or belonging to another antibody class or subclass, as well as fragments of these antibodies, as 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)). The chimeric antibodies of interest herein include "primatization" antibodies, which comprise variable domain antigen-binding sequences derived from non-human primates (e.g., Old World monkeys, such as baboons, rhesus monkeys or cynomolgus monkeys) and human constant region sequences (U.S. Pat. No. 5,693,780).

[0070] "Humanized" forms of non-human (e.g., mouse) antibodies are chimeric antibodies containing minimal sequences derived from non-human immunoglobulins. In most cases, humanized antibodies are human immunoglobulins (receptor antibodies), wherein the residues in the hypervariable regions of the receptor are replaced by residues from the hypervariable regions of non-human species (donor antibodies), such as mice, rats, rabbits, or non-human primates, which have the desired specificity, affinity, and function. In some cases, the framework region (FR) residues of human immunoglobulins are replaced by corresponding non-human residues. In addition, humanized antibodies may include residues that are not present in receptor antibodies or donor antibodies. These modifications are intended to further improve antibody performance. In general, humanized antibodies will substantially include at least one variable domain in all, typically two variable domains, wherein all or substantially all hypervariable loops correspond to the hypervariable loops of non-human immunoglobulins, and all or substantially all FRs are FRs of human immunoglobulin sequences, except for FR substitutions as described above. Humanized antibodies also optionally include at least a portion of an immunoglobulin constant region, which is typically a human immunoglobulin. For more 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).

[0071] For the purposes of this article, a "complete antibody" is an antibody comprising a heavy variable domain and a light variable domain and an Fc region. The constant domain may be a native sequence constant domain (e.g., a human native sequence constant domain) or an amino acid sequence variant thereof. Preferably, the complete antibody has one or more effector functions.

[0072] "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 bonds varies between the heavy chains of different immunoglobulin isotypes. Each heavy and light chain also has regularly spaced intrachain disulfide bonds. Each heavy chain has a variable domain (V H ), followed by multiple constant domains. Each light chain has a variable domain (V L ), and a constant domain at the other end; the constant domain of the light chain is aligned with the first constant domain of the heavy chain, and the variable domain of the light chain is aligned with the variable domain of the heavy chain. It is believed that specific amino acid residues form an interface between the light chain and heavy chain variable domains.

[0073] A "naked antibody" is an antibody (as defined herein) that is not conjugated to a heterologous molecule, such as a cytotoxic moiety or a radiolabel.

[0074] In some embodiments, antibody "effector function" refers to the biological activity attributable to the Fc region (native sequence Fc region or amino acid sequence variant Fc region) of an antibody, and varies with 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; downregulation of cell surface receptors.

[0075] "Antibody-dependent cell-mediated cytotoxicity" and "ADCC" refer to a cell-mediated reaction in which nonspecific cytotoxic cells (e.g., natural killer (NK) cells, neutrophils, and macrophages) expressing Fc receptors (FcRs) recognize bound antibodies on target cells, subsequently causing target cell lysis. NK cells, the main cells that mediate ADCC, express only FcγRIII, while monocytes express FcγRI, FcγRII, and FcγRIII. FcR expression on hematopoietic cells is summarized in Table 3 on page 464 of Ravetch and Kinet, Annu. Rev. Immunol 9:457-92 (1991). To assess the ADCC activity of a target molecule, an in vitro ADCC assay, such as described in U.S. Pat. Nos. 5,500,362 or 5,821,337, may be performed. Useful effector cells for such assays include peripheral blood mononuclear cells (PBMCs) and natural killer (NK) cells. Alternatively or additionally, ADCC activity of the target molecule can be assessed in vivo, for example, in an animal model such as that disclosed in Clynes et al., Proc. Natl. Acad. Sci. (USA) 95:652-656 (1998).

[0076] "Human effector cells" are leukocytes that express one or more FcRs and perform effector functions. In some embodiments, these cells express at least FcγRIII and perform ADCC effector functions. Examples of human leukocytes that mediate ADCC include peripheral blood mononuclear cells (PBMCs), natural killer (NK) cells, monocytes, cytotoxic T cells, and neutrophils; PBMCs and NK cells are preferred.

[0077] The term "Fc receptor" or "FcR" is 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. In addition, a preferred FcR is one that binds to an IgG antibody (a gamma receptor), and includes receptors of the FcγRI, FcγRII, and FcγRIII subclasses, including allelic variants and alternatively spliced ​​forms of these receptors. FcγRII receptors include FcγRIIA ("activating receptor") and FcγRIIB ("inhibiting receptor"), both of which have similar amino acid sequences, differing primarily in their cytoplasmic domains. The activating receptor FcγRIIA contains an immunoreceptor tyrosine-based activation motif (ITAM) in its cytoplasmic domain. The inhibitory receptor FcγRIIB contains an immunoreceptor tyrosine-based inhibition motif (ITIM) in its cytoplasmic domain. (See Annu. Rev. Immunol. 15: 203-234 (1997)). For a review of FcRs, see: 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). The term "FcR" herein encompasses other FcRs, including those to be identified in the future. The term also includes the neonatal receptor FcRn, which is 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)).

[0078] Reference herein to "about" a value or parameter includes (and describes) variations involving the value or parameter itself. For example, description referring to "about X" includes description of "X".

[0079] As used herein and in the appended claims, the singular forms "a", "an", "or", and "the" include plural referents unless the context clearly dictates otherwise. It should be understood that the aspects and variations of the invention described herein include "consisting of" and / or "consisting essentially of" the aspects and variations.

[0080] preparation

[0081] In some aspects, the present invention provides a formulation comprising a polypeptide and hydroxypropylmethylcellulose acetate succinate (HPMCAS). In some embodiments, the HPMCAS in the formulation comprises an acetate group between about 1% to about 20%. In some embodiments, the HPMCAS in the formulation comprises an acetate group of 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%. In some embodiments, the HPMCAS in the formulation comprises a succinate group between about 1% to about 20%. In some embodiments, the HPMCAS in the formulation comprises 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 comprises between about 1% to about 20% acetate and between about 1% to about 20% succinate. In some embodiments, the HPMCAS in the formulation comprises about 8% acetate and about 15% succinate (HPMCAS-LF) or about 12% acetate and about 7% succinate (HPMCAS-HF).

[0082] 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), 1:1 (mg / mg) and 1:2 (mg / mg), 1:2 (mg / mg), 1:3 ... g) to 1:10 (mg / mg), 1:2 (mg / mg) to 1:9 (mg / mg), 1:2 (mg / mg) to 1:8 (mg / mg), 1:2 (mg / mg) to 1:7 (mg / mg), 1:2 (mg / mg) to 1:6 (mg / mg), 1:2 (mg / mg) to 1:5 (mg / mg), 1:2 (mg / mg) to 1:4 (mg / mg), 1:2 (mg / mg) to 1:3 (mg / mg), 1:3 (mg / mg) to 1:10 (mg / mg), 1:3 (mg / mg) to 1:9 (mg / mg), 1:3 (mg / mg) to 1:8 (mg / mg ), 1:3(mg / mg) to 1:7(mg / mg), 1:3(mg / mg) to 1:6(mg / mg), 1:3(mg / mg) to 1:5(mg / mg), 1:3(mg / mg) to 1:4(mg / mg), 1:4(mg / mg) to 1:10(mg / mg), 1:4( mg / mg) to 1:9 (mg / mg), 1:4 (mg / mg) to 1:8 (mg / mg), 1:4 (mg / mg) to 1:7 (mg / mg), 1:4 (mg / mg) to 1:6 (mg / mg), 1:4 (mg / mg) to 1:5 (mg / mg), 1:5 (mg / mg) to 1:10(mg / mg), 1:5(mg / mg) to 1:9(mg / mg), 1:5(mg / mg) to 1:8(mg / mg), 1:5(mg / mg) to 1:7(mg / mg), 1:5(mg / mg) to 1:6(mg / mg), 1:6(mg / mg) to 1:10(m g / mg), 1:6 (mg / mg) to 1:9 (mg / mg), 1:6 (mg / mg) to 1:8 (mg / mg), 1:6 (mg / mg) to 1:7 (mg / mg), 1:7 (mg / mg) to 1:10 (mg / mg), 1:7 (mg / mg) to 1:9 (mg / mg),In some embodiments, the ratio of HPMCAS to protein in the formulation is between 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) 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) to 1:1 (mg / mg). mg), 10:1 (mg / mg) to 2:1 (mg / mg), 9:1 (mg / mg) to 2:1 (mg / mg), 8:1 (mg / mg) to 2:1 (mg / mg), 7:1 (mg / mg) to 2:1 (mg / mg), 6:1 (mg / mg) to 2:1 (mg / mg), 5:1 (mg / mg) to 2:1 (mg / mg), 4:1 (mg / mg) to 2:1 (mg / mg), 3:1 (mg / mg) to 2:1 (mg / mg), 10:1 (mg / mg) to 3:1 (mg / mg), 9:1 (mg / mg) to 3:1 (mg / m g), 8:1 (mg / mg) to 3:1 (mg / mg), 7:1 (mg / mg) to 3:1 (mg / mg), 6:1 (mg / mg) to 3:1 (mg / mg), 5:1 (mg / mg) to 3:1 (mg / mg), 4:1 (mg / mg) to 3:1 (mg / mg ), 10:1 (mg / mg) to 4:1 (mg / mg), 9:1 (mg / mg) to 4:1 (mg / mg), 8:1 (mg / mg) to 4:1 (mg / mg), 7:1 (mg / mg) to 4:1 (mg / mg), 6:1 (mg / mg) to 4:1 (mg / mg) , 5:1 (mg / mg) to 4:1 (mg / mg), 10:1 (mg / mg) to 5:1 (mg / mg), 9:1 (mg / mg) to 5:1 (mg / mg), 8:1 (mg / mg) to 5:1 (mg / mg), 7:1 (mg / mg) to 5:1 (mg / mg) , 6:1 (mg / mg) to 5:1 (mg / mg), 10:1 (mg / mg) to 6:1 (mg / mg), 9:1 (mg / mg) to 6:1 (mg / mg), 8:1 (mg / mg) to 6:1 (mg / mg), 7:1 (mg / mg) to 6:1 (mg / mg),10:1 (mg / mg) to 7:1 (mg / mg), 9:1 (mg / mg) to 7:1 (mg / mg), 8:1 (mg / mg) to 7:1 (mg / mg), 10:1 (mg / mg) to 8:1 (mg / mg), 9:1 (mg / mg) to 8:1 (mg / mg), or 10:1 (mg / mg) to 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).

[0083] 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 concentration of the histidine buffer (e.g., histidine HCl buffer) in the formulation is about 1 mM to about 50 mM. In some embodiments, the concentration of 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 In some embodiments, the concentration of the histidine buffer in the formulation is about any of 1 mM, 5 mM, 10 mM, 15 mM, 20 mM, 25 mM, 30 mM, 40 mM, or 50 mM.

[0084] In some embodiments, the formulation further comprises a surfactant (i.e., the formulation comprises a polypeptide, HPMCAS, a 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 concentration of the surfactant (e.g., polysorbate) in the formulation is about 0.005% (w / v) to about 1.0% (w / v). In some embodiments, the concentration of the surfactant (e.g., a polysorbate, such as polysorbate 20) in the formulation is between about 0.005% (w / v) and 1.0% (w / v), 0.01% (w / v) and 1.0% (w / v), 0.05% (w / v) and 1.0% (w / v), 0.1% (w / v) and 1.0% (w / v), 0.5% (w / v) and 1.0%, (w / v), 0.005% (w / v) and 0.5% (w / v), 0.01% (w / v) and 0.5% (w / v). w / 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 0.005% (w / v) to 0.01% (w / v). In some embodiments, the concentration of the surfactant (e.g., a polysorbate such as polysorbate 20) in the formulation is 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).

[0085] 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 8.0, 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.

[0086] In some embodiments, the polypeptide of the preparation is an antibody. In some embodiments, the polypeptide of the preparation is a monoclonal antibody. In some embodiments, the polypeptide of the preparation is a human antibody, a chimeric antibody or a humanized antibody. In some embodiments, the polypeptide of the preparation is an antibody fragment selected from Fab, Fab'-SH, Fv, scFv and (Fab')2 fragments. In some embodiments, the concentration of the polypeptide in the preparation is about 5 mg / mL to about 100 mg / mL.

[0087] In some embodiments, the concentration of the polypeptide (e.g., antibody) in the formulation is between 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, 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, 100 mg / mL to 100 mg / mL, 15 ... g / mL to 90mg / mL, 10mg / mL to 90mg / mL, 15mg / mL to 90mg / mL, 20mg / mL to 90mg / mL, 25mg / mL to 90mg / mL, 30mg / mL to 90mg / mL, 40mg / mL to 90mg / mL, 50mg / mL to 90mg / mL, 60mg / mL to 90mg / mL, 70mg / mL to 90mg / mL, 75mg / mL to 90mg / mL, 80mg / mL to 90mg / mL, 5mg / mL to 80mg / mL, 10mg / mL to 80mg / mL, 15mg / mL to 80mg / mL, 20mg / mL to 90mg / mL, 25mg / mL to 80mg / mL, 30mg / mL to 80mg / mL, 40mg / mL to 80mg / mL, 50mg / mL to 80mg / mL, 60mg / mL to 80mg / mL, 70mg / mL to 80mg / mL, 75mg / mL to 80mg / mL, 5mg / mL to 75mg / mL, 10mg / mL to 75mg / mL, 15mg / mL to 75mg / mL, 20mg / mL to 75mg / mL, 25mg / mL to 75mg / mL, 30mg / mL to 75mg / mL, 40mg / mL to 75mg / mL, 50mg / mL to 75mg / mL, 60mg / mL to 80mg / mL, 70mg / mL to 80mg / mL, 75mg / mL to 80mg / mL, 5mg / mL to 75mg / mL, 10mg / mL to 75mg / mL, 15mg / mL to 75mg / mL, 20mg / mL to 75mg / mL, 25mg / mL to 75mg / mL, 30mg / mL to 75mg / mL, 40mg / mL to 75mg / mL, 50mg / mL to 75mg / mL, mL to 75mg / mL, 70mg / mL to 75mg / mL, 5mg / mL to 70mg / mL, 10mg / mL to 70mg / mL, 15mg / mL to 70mg / mL, 20mg / mL to 70mg / mL, 25mg / mL to 70mg / mL, 30mg / mL to 70mg / mL, 40mg / mL to 70mg / mL, 50mg / mL to 70mg / mL, 60mg / mL to 70mg / mL, 5mg / mL to 65mg / mL, 10mg / mL to 65mg / mL, 15mg / mL to 65mg / mL, 20mg / mL to 65mg / mL, 25mg / mL to 65mg / mL,30mg / mL to 65mg / mL, 40mg / mL to 65mg / mL, 50mg / mL to 65mg / mL, 60mg / mL to 65mg / mL, 5mg / mL to 60mg / mL, 10mg / mL to 60mg / mL, 15mg / mL to 60mg / mL, 20mg / mL to 60mg / mL, 25mg / mL to 60mg / mL, 30mg / mL to 60mg / mL, 40mg / mL to 60mg / mL, 50mg / mL to 60mg / mL, 5mg / mL to 50mg / mL, 10mg / mL to 50mg / mL, 15mg / mL to 50mg / mL, 20mg / mL to 50mg / mL, 25mg / mL to 50mg / mL, 30mg / mL to 50mg / mL, 40mg / mL to 50mg / mL, 5mg / mL to 40mg / mL, 10 Any between 10 mg / mL and 40 mg / mL, 15 mg / mL and 40 mg / mL, 20 mg / mL and 40 mg / mL, 25 mg / mL and 40 mg / mL, 30 mg / mL and 40 mg / mL, 5 mg / mL and 30 mg / mL, 10 mg / mL and 30 mg / mL, 15 mg / mL and 30 mg / mL, 20 mg / mL and 30 mg / mL, 25 mg / mL and 30 mg / mL, 5 mg / mL and 25 mg / mL, 10 mg / mL and 25 mg / mL, 15 mg / mL and 25 mg / mL, 20 mg / mL and 25 mg / mL, 5 mg / mL and 20 mg / mL, 10 mg / mL and 20 mg / mL, 15 mg / mL and 20 mg / mL, 5 mg / mL and 15 mg / mL, 10 mg / mL and 15 mg / mL, or 5 mg / mL to 10 mg / mL. In some embodiments, the concentration of the polypeptide (e.g., antibody) in the formulation is about 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 more than 100 mg / mL.

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

[0089] 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, wherein the ratio of HPMCAS-HF to the polypeptide in the formulation is between about 1:1 (mg / mg) and 1:5 (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), and wherein the pH of the formulation is about 6.0 to about 7.0. In some embodiments, the present invention provides a formulation comprising: a polypeptide (e.g., an antibody) at a concentration of about 10 mg / mL; HPMCAS-HF, wherein the ratio of HPMCAS-HF to the polypeptide in the formulation is about 1:1 (mg:mg); a histidine HCl buffer at a concentration of about 10 mM; polysorbate 20 at a concentration of about 0.01% (w / v), and wherein the pH of the formulation is about 6.5.

[0090] Spray dried preparations

[0091] In some aspects of the invention, the formulation of the invention is 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, wherein the ratio of HPMCAS-LF to polypeptide in the formulation is between about 1:1 (mg / mg) and 1:5 (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), and wherein the pH of the formulation is about 6.0 to about 7.0. In some embodiments, the present invention provides a spray-dried formulation comprising: a polypeptide (e.g., an antibody) at a concentration of about 10 mg / mL; HPMCAS-LF, wherein the ratio of HPMCAS-LF to the polypeptide in the formulation is about 1:1 (mg / mg); a histidine HCl buffer at a concentration of about 10 mM; polysorbate 20 at a concentration of about 0.01% (w / v), and wherein the pH of the formulation is about 6.5.

[0092] 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, wherein the ratio of HPMCAS-HF to the polypeptide in the formulation is between about 1:1 (mg / mg) and 1:5 (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), and wherein the pH of the formulation is about 6.0 to about 7.0. In some embodiments, the present invention provides a spray-dried formulation comprising: a polypeptide (e.g., an antibody) at a concentration of about 10 mg / mL; HPMCAS-HF, wherein the ratio of HPMCAS-HF to the polypeptide in the formulation is about 1:1 (mg / mg); a histidine HCl buffer at a concentration of about 10 mM; polysorbate 20 at a concentration of about 0.01% (w / v), and wherein the pH of the formulation is about 6.5.

[0093] In some embodiments, the polypeptide (e.g., antibody) of 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 of 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, 9 hours to 10 hours. In some embodiments, the polypeptide of 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.

[0094] In some embodiments, the polypeptide (e.g., antibody) of the spray-dried formulation is stable for at least about 1 week to more than 6 weeks at about 37° C. In some embodiments, the polypeptide of the spray-dried formulation is stable for about 1 week to 6 weeks, 2 weeks to 6 weeks, 3 weeks to 6 weeks, 4 weeks to 6 weeks, or 5 weeks to 6 weeks at about 37° C. In some embodiments, the polypeptide of the spray-dried formulation is stable for about 1 week, 2 weeks, 3 weeks, 4 weeks, 5 weeks, 6 weeks, or any one of more than 6 weeks at about 37° C.

[0095] 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 that does not include HPMCAS. In some embodiments, the reduced chemical degradation includes reduced formation of succinimide variants of the polypeptide and / or reduced formation of pyroglutamate variants of the polypeptide.

[0096] 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. Amorphous formulations are non-crystalline formulations that do not have long-range order. In some embodiments, glass is a rigid inert matrix that can be measured, for example, by a glass transition temperature.

[0097] Encapsulated Peptides

[0098] In some embodiments, the invention provides polypeptides in encapsulated form. In some embodiments, the spray-dried formulations of the invention are encapsulated in an acid polymer system. In some embodiments, the acid polymer system is a lactic acid / glycolic acid polymer system. In some embodiments, the acid polymer system is a poly(lactic acid-co-glycolic acid) (PLGA) system. In some embodiments, the acid polymer system generates 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 formed when the spray-dried polypeptide is encapsulated.

[0099] 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 PLGA rods.

[0100] Method for producing a preparation of a polypeptide

[0101] In some embodiments, the present invention provides a method for producing a formulation, the method comprising adding hydroxypropylmethylcellulose acetate succinate (HPMCAS) to a polypeptide formulation. In some embodiments, 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., histidine HCl buffer) is added to the formulation. In some embodiments, the histidine buffer is added to the formulation at a concentration of about 5mM to about 20mM. In some embodiments, the histidine buffer is added to the formulation at a concentration of about 10mM. In some embodiments, a polysorbate (e.g., polysorbate 20) is added to the formulation. In some embodiments, polysorbate is added to the formulation at a concentration of about 0.005% (w / v) to about 0.5% (w / v). In some embodiments, 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, a polypeptide (e.g., an 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, a polypeptide (e.g., an antibody) is added to the formulation at a concentration of about 10 mg / mL.

[0102] Additional aspects of formulations

[0103] In some embodiments, the polypeptide in the polypeptide preparation maintains functional activity.

[0104] Formulations to be used for in vivo administration must be sterile. This can be readily accomplished by filtration through sterile filtration membranes.

[0105] The preparations herein may also contain more than one active compound that is necessary for the specific indications treated, preferably active ingredients with complementary activities that do not adversely affect each other. For example, in addition to polypeptides, it may be necessary to include other polypeptides (e.g., antibodies) in a preparation. Alternatively or additionally, the composition may further include chemotherapeutic agents, cytotoxic agents, cytokines, growth inhibitors, antihormonal agents and / or cardioprotectants. Such molecules are suitably present in an effective amount combination for the intended purpose.

[0106] Spray drying

[0107] In some aspects, the present invention provides a spray-dried preparation of a polypeptide. In some embodiments, a preparation comprising a polypeptide and HPMCAS as described herein is spray-dried. Spray drying is a method of producing dry powder from a liquid or slurry by rapid drying with hot gas. In some embodiments, spray drying is a method of drying heat-sensitive materials such as drugs (e.g., polypeptides such as antibodies) and / or materials that may require extremely consistent fine particle sizes. Spray drying as used herein is different from freeze drying commonly used to prepare monoclonal antibody preparations because it is carried out at a temperature higher than ambient temperature. Spray drying temperatures are generally expressed as "air inlet" and "air outlet" temperatures. In one embodiment, spray drying is carried out 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. and about 220° C., about 80° C. and about 180° C., about 80° C. and about 160° C., about 80° C. and about 140° C., about 90° C. and about 220° C., about 90° C. and about 180° C., about 90° C. and about 160° C., about 90° C. and about 140° C., about 100° C. and about 220° C., about 100° C. and about 180° C., about 100° C. and about 160° C., about 100° C. and about 140° C., about 110° C. and about 220° C., about 110° C. and about 180° C., about 110° C. and about 160° C., or about 110° C. and about 140° C. In some embodiments, the air inlet temperature is any of 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 any of about 50° C. to about 100° C., 50° C. to about 90° C., 50° C. to about 80° C., 50° C. to about 70° C., 50° C. to about 60° C., about 60° C. to about 100° C., 60° C. to about 90° C., 60° C. to about 80° C., 60° C. to about 70° C., about 70° C. to about 100° C., 70° C. to about 90° C., 70° C. to about 80° C., about 80° C. to about 100° C., 80° C. to about 90° C., or about 90° C. to 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.

[0108] The spray drying process generally involves: atomization of a liquid feed; drying of the liquid droplets; and separation or recovery of the dried product.

[0109] In some embodiments, atomizers used in the invention described herein include, but are not limited to, rotary atomizers, pneumatic nozzle atomizers, ultrasonic nozzle atomizers, and sonic nozzles.

[0110] The contact between the liquid feed and the dry air can occur in two different modes. In the co-current system, the dry air and the particles (droplets) move in the same direction through the drying chamber. When the dry air and the droplets move in opposite directions, it is called the countercurrent mode. The particles produced in the countercurrent mode usually show a higher temperature than the exhaust air. The exhausted air itself can leave the system or can be recycled. By selecting and adjusting different process parameters (dry airflow, dry air temperature, etc.) from various spray dryer designs (size, atomizer, aseptic conditions, etc.), the final powder properties (such as particle size, shape and structure or even sterility) can be modified. If the resulting moisture of the recovered powder is not low enough, it may be necessary to carry out post-processing, for example in the form of secondary drying (for example, using a desktop freeze dryer), fluidized bed dryer and cooler, contact dryer or even microwave dryer.

[0111] As the liquid feed atomizes, its surface area to mass ratio increases, heat transfer between the air and the droplets accelerates, and the droplets can dry relatively quickly. Two convective processes may be involved: heat transfer of moisture (air to droplets) and mass transfer (droplets to air). In the latter, moisture permeates through the boundary layer around each droplet. The transfer rate may be affected by temperature, humidity, the transport properties of the surrounding air, the droplet diameter, and the relative velocity between the droplet and the air.

[0112] The final step of the spray drying process is usually to separate the powder from the air / gas and remove the dried product. In some embodiments, this step is as efficient as possible to obtain a high powder yield and prevent the powder from being discharged into the atmosphere and causing air pollution. To this end, a variety of methods can be used, such as cyclone separators, bag filters, electrostatic precipitators, high pressure gas, electrostatic charges, and combinations thereof.

[0113] The spray drying process produces particles comprising the polypeptide (eg, antibody).

[0114] In one embodiment, the characteristics of the spray-dried powder include any one or more of the following:

[0115] (a) average particle size: about 2 microns to about 30 microns; for example, about 2 microns to about 10 microns;

[0116] (b) Particle morphology: mainly spherical particles, collapsed spheres, some pits or holes in the particles, and “dried raisin” shapes;

[0117] (c) water content: less than about 10%, such as less than about 5%, for example, where the water content is measured by chemical titration (e.g., Karl Fischer) or weight loss (high temperature heating); and

[0118] (d) Stability: For example, assessed by suspending the particles in a vehicle and evaluating the physical stability and / or chemical stability and / or biological activity of the suspension preparation. In one embodiment, the monomer percentage of such preparations is 95% to 100%, for example, as assessed by size exclusion chromatography (SEC).

[0119] Controlled release formulations of peptides

[0120] The sustained release formulation according to the present invention generally comprises a polypeptide (e.g., antibody) prepared together with HPMCAS as described herein, a polymer carrier and a release modifier for changing the release rate of the polypeptide from the polymer carrier. By changing the manufacturing conditions of the delivery composition based on polymer, the release kinetics of the resulting composition can be adjusted. The polymer carrier generally comprises one or more biodegradable polymers or copolymers or a combination thereof. For example, the polymer carrier can be selected from polylactic acid (PLA), polyglycolic acid (PGA), polylactide-co-glycolide (PLGA), polyester, poly-(orthoester), poly-(phosphazene), poly-(phosphate), 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), polyvinyl pyrrolidone (PVP), poloxamer, sodium carboxymethyl cellulose (NaCMC), poly(acrylic acid), polyvinyl acetyl phthalate (PVAP), polysaccharides (e.g., sodium alginate, carrageenan, guar gum, and xanthan gum), poly(meth)acrylates, L. L, 100-55, L 30D-55, L 100, S. S100, FS, FS 30D, 934P, 940, 974P, 971 (Lubrizol) and AA-1. In some embodiments, the formulation comprises a polymer described in Maskova, E. et al., J. Controlled Release, 2020, 324: 695-727 (incorporated herein in its entirety).

[0121] In certain embodiments, the polymer carrier is PLGA. Release modifiers are generally long-chain fatty alcohols, preferably comprising 10 to 40 carbon atoms. Commonly used release modifiers include octanol, nonanol, decanol, lauryl alcohol, myristyl alcohol, cetyl alcohol, palmitoleyl alcohol, stearyl alcohol, isostearyl alcohol, elaidyl alcohol, oleyl alcohol, linoleyl alcohol, polyunsaturated 9E, 12E- 18diene -1- alcohol (polyunsaturated elaidolinoleyl alcohol), polyunsaturated linoleyl alcohol, 9E, 12E- 18diene -1- alcohol (elaidolinolenyl alcohol), polyunsaturated ricinoleyl alcohol, arachidyl alcohol, behenyl alcohol, docosanol, lignocerol, ceryl alcohol (ceryl alcohol), nonacosanol, octacosanol, myristyl alcohol, triacontanol and tetratriacontanol (geddyl alcohol).

[0122] In certain embodiments, a polypeptide (e.g., an antibody) formulated with HPMCAS is incorporated into a sustained-release composition based on microspheres. 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 changing the conditions for preparing the microspheres. Methods for producing such sustained-release formulations are described in US2005 / 0281861 and US2008 / 0107694.

[0123] In certain embodiments, the polypeptide formulated with HPMCAS is incorporated into a biodegradable implant such as a microneedle. Matrix implants such as microneedles are commonly used to treat ocular diseases that require a loading dose over a period of 1 day to 6 months, followed by a gradual reduction in the drug dose (Davis et al. (2004) Curr Opin Mol Therap 6, 195-205). They are most commonly made of copolymers of polylactic acid (PLA) and / or polylactic-glycolic acid (PLGA), which degrade into water and carbon dioxide. The rate and extent of drug release from the implant can be reduced by changing the relative concentrations of lactide (slow) and glycolide (fast), changing the polymer weight ratio, adding additional polymer coatings, or using hydrophobic insoluble drugs. The release of the drug generally follows first-order kinetics, with an initial burst of drug release followed by a rapid decline in drug content. Biodegradable implants do not need to be removed because they dissolve over time (Hsu (2007) Curr Opin Ophthalmol 18, 235-9). The biodegradable implant also allows for flexible adjustment of dosage and treatment duration (from short duration (weeks) to longer duration (months to a year)) based on the polymer PLA / PLGA ratio, which is another advantage in tailoring drug delivery based on disease progression, as dosage and treatment requirements may change over time.

[0124] Sustained release formulations of polypeptides were developed using polylactic-glycolic acid polymer (PLGA) because of its biocompatibility and broad biodegradability. The degradation products of PLGA (lactic acid and glycolic acid) are rapidly cleared from the body. In addition, the degradability of the 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 polymer", in: M. Chasin and R. Langer (eds.), Biodegradable Polymers as Drug Delivery Systems (Marcel Dekker: New York, 1990), pp. 1-41.

[0125] In some embodiments, the controlled release formulation of the polypeptide is produced by a hot melt extrusion process. For example, a 5 g or 7 cm 3 HAAKE barrel capacity TMMiniCTW micro conical twin screw compounder (Thermo Scientific, Karlsruhe, Germany) is used for hot melt extrusion. In certain embodiments, the extruder barrel is preheated to 90°C, and the screw speed is set to 40rpm. In certain embodiments, the front barrel opening of the extruder is kept closed in the recirculation mode. In certain embodiments, the pre-weighed solid polymer and spray-dried polypeptide are manually slowly fed into the extruder, and recirculated back to the barrel for micro-compounding. In certain embodiments, the polypeptide loading for implants is fixed to 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%, about 10% to about 15% (w / w) of polypeptide / PLGA spray-dried formulations. In some embodiments, the polypeptide loading for implants is fixed to a spray-dried formulation of any one of about 1%, 5%, 10%, 15%, 20% or 30% (w / w) polypeptide / PLGA. In some embodiments, the polypeptide loading for implants is fixed to a spray-dried formulation of about 15% (w / w) polypeptide / PLGA. In some embodiments, after complete feeding, melt phase blending continues for about 30 min in a recirculation mode and is then extruded through an about 0.5 mm circular die. In some embodiments, the extrudate is cooled to room temperature, cut into cylinders with a desired length, and stored at 2° C. to 8° C. until further use.

[0126] Hydroxypropyl Methylcellulose Acetate Succinate (HPMCAS)

[0127] In some aspects, the present invention provides formulations of polypeptides and HPMCAS. In some embodiments, HPMCAS provides protection for the polypeptide during the high temperature spray drying process and provides protection against low pH microenvironments during acidic polymer encapsulation.

[0128] The reaction of HMPC with acetic acid and succinic acid produces an HPMC derivative, which is called hydroxypropyl methylcellulose acetate succinate (abbreviated as HPMCAS), in which the hydroxyl groups in HPMC are modified to form the corresponding esters. The reaction with acetic acid converts the hydroxyl groups in HPMC into acetyl groups, while the reaction with succinic acid converts the hydroxyl groups in HPMC into 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). When used as a drug carrier, the acetyl and succinyl substitution content has a significant effect on the solubilization effect of HPMCAS. Since the acid dissociation constant (pKa) value of the succinate group of HPMCAS is about 5, the polymer is mainly unionized at pH values ​​below 4, and partially to completely ionized at pH values ​​of about 5 or higher. Therefore, the HPMC derivative is a polymer buffer containing succinic acid. In its non-ionized state (pH <5), HPMCAS is insoluble in water; while under neutral (pH 6.0 to 7.5) conditions, it forms a stable water-soluble colloidal assembly. It is a solubilizer for poorly soluble drugs (Friesen, DT et al., Mol Pharm, 2008.5(6):1003-19), insoluble in gastric juice, and rapidly dissolves in the small intestine (Curatolo, W et al., Pharm Res, 2009, 26(6):1419-31). Although HPMCAS-based dispersions are suitable for small molecule-based drug sustained release applications, their use in large protein therapeutics has not been explored previously. HPMCAS in spray-dried powder form is insoluble below pH 6, begins to dissolve at about pH 6 to pH 6.5, and completely dissolves at pH ≥ 6.5. Therefore, the advantage is that HPMCAS will provide protection by coating the protein in spray-dried form (thus remaining as SD powder) and prevent it from interacting with acidic counterions in the low pH microenvironment. Therefore, we have tried to take advantage of the low pH insolubility of HPMCAS and use it as a spray-dried drug carrier in the acidic microclimate of the PLGA system. However, almost all delivery systems using HPMCAS are currently for oral administration of small molecule drugs, and no studies have shown that they can be used to deliver large molecule proteins (Woodley, JF, Crit Rev Ther Drug Carrier Syst, 1994, 11 (2-3): 61-95). Specifically, the HPMCAS formulation described herein is a new strategy to stabilize proteins without significant effects on any specific delivery route. In addition, non-ionic HPMCAS with a high glass transition temperature (> 100 ° C) can be used as an excipient in spray-dried protein therapeutics and for high temperature manufacturing of drug delivery systems.

[0129] Maskova, E. et al. (J. Controlled Release, 2020, 324: 695-727) provide a review of HPMC as an excipient for oral and oral mucosal drug delivery.

[0130] Peptides

[0131] Examples of polypeptides used 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, but are not limited to, 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; coagulation factors such as factor VIIIC, factor IX, tissue factor and von Willebrands factor; anticoagulant factors such as protein C; atrial natriuretic factor; pulmonary surfactant; plasminogen activators such as urokinase or human urine or tissue-type plasminogen activator (t-PA); bombesin; thrombin; hematopoietic growth factors; tumor necrosis factor-α and tumor necrosis factor-β; enkephalinase; RANTES (regulated expression and secretion of normal T-cell activation); human macrophage inflammatory protein (MIP-1-α); serum albumin such as human serum albumin; Muellerian-inhibiting substance (Muellerian-inhibiting substance substance); relaxin A chain; relaxin B chain; prorelaxin; mouse gonadotropin-related peptide; enzyme; microbial protein, such as β-lactamase; DNA enzyme; IgE; cytotoxic T lymphocyte-associated antigen (CTLA), for example CTLA-4; inhibin; activin; vascular endothelial growth factor (VEGF); receptor for hormone or growth factor; protein A or D; rheumatoid factor; neurotrophic factor, such as bone-derived neurotrophic factor (BDNF), neurotrophin-3, neurotrophin-4, neurotrophin-5 or neurotrophin-6 (NT-3, NT-4, NT-5 or NT-6), or nerve growth factor such as NGF-b; platelet-derived growth factor (PDGF); fibroblast growth factor such as aFGF, bFGF; epidermal growth factor ( EGF); transforming growth factor (TGF), such as TGF-α and TGF-β, including TGF-β1, TGF-β2, TGF-β3, TGF-β4 or TGF-β5; insulin-like growth factor-I and insulin-like growth factor-II (IGF-I and IGF-II); des(1-3)-IGF-I (brain IGF-I), insulin-like growth factor binding protein (IGFBP); cytokines; CD proteins, such as CD3, CD4, CD8, CD19 and CD20; erythropoietin; osteoinductive factors; immunotoxins; fusion polypeptides, i.e., polypeptides contained on two or more heterologous polypeptides or fragments thereof and encoded by recombinant nucleic acids; Fc-containing polypeptides, for example, fusion proteins comprising an immunoglobulin Fc region or a fragment thereof fused to a second polypeptide; immunoconjugates;Bone morphogenetic protein (BMP); interferons, such as interferon-α, interferon-β and interferon-γ; colony stimulating factors (CSF), such as M-CSF, GM-CSF and G-CSF; interleukins (IL), such as IL-1 to IL-10; superoxide dismutase; T cell receptors; surface membrane proteins; decay accelerating factor; viral antigens, such as part of the AIDS envelope; transporters; 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 above proteins, as well as antibodies (including antibody fragments) that bind to proteins (e.g., any of the above proteins). ;

[0132] Antibody

[0133] In some embodiments of any of the methods described herein, the polypeptide or agent described herein is an antibody.

[0134] The molecular targets of the 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; (iii) cell adhesion molecules, such as LFA-1, Mac1, p150,95, VLA-4, IC AM-1, VCAM and αv / β3 integrins, including their α or β subunits (e.g., anti-CD11a, anti-CD18 or anti-CD11b antibodies); (iv) growth factors, such as VEGF; IgE; blood group antigens; flk2 / flt3 receptors; obesity (OB) receptor; mpl receptor; CTLA-4; protein C, BR3, c-met, tissue factor, β7, etc.; (v) cell surface and transmembrane tumor-associated antigens (TAA), such as those described in U.S. Pat. No. 7,521,541.

[0135] Other exemplary antibodies include those selected from, but 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-Aβ antibodies, 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 -1BBL 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-CD14 antibody, anti-CD15 antibody, anti-CD19 antibody, anti-CD22 antibody, anti-CD23 antibody, anti-CD30 antibody, anti-CD31 antibody, anti-CD33 antibody, anti-CD34 antibody, anti-CD35 antibody, anti-CD38 antibody, anti-CD41 antibody, anti-L CA / CD45 antibody, 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-fibrillin antibody, anti-keratin antibody and anti-Tn antigen antibody.

[0136] Polyclonal antibodies

[0137] In some embodiments, the antibody is a polyclonal antibody. Polyclonal antibodies are preferably produced in animals by multiple subcutaneous (sc) or intraperitoneal (ip) injections of the relevant antigen and an adjuvant. Bifunctional or derivatizing agents, such as maleimidobenzoylsulfosuccinimide ester (conjugated through cysteine ​​residues), N-hydroxysuccinimide (conjugated through lysine residues), glutaraldehyde, succinic anhydride, SOCl2 or R 1 N=C=NR (where R and R 1 It may be useful to conjugate the antigen of interest to a polypeptide that is immunogenic in the species to be immunized (e.g., keyhole limpethemocyanin, serum albumin, bovine thyroglobulin, or soybean trypsin inhibitor).

[0138] Animals are immunized against antigens, immunogenic conjugates or derivatives by mixing, for example, 100 μg or 5 μg of 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 immunity of the animal is enhanced by subcutaneous injection at multiple sites with a dose of 1 / 5 to 1 / 10 of the original amount of peptide or conjugate in Freund's complete adjuvant. After 7 to 14 days, the animal is bled and the antibody titer of the serum is determined. The animal is boosted until the titer is stable. In some embodiments, the animal is boosted with the same antigen but conjugated to different polypeptides and / or conjugated by different cross-linking agents. Conjugates can also be prepared as polypeptide fusions in recombinant cell cultures. In addition, aggregating agents such as alum are suitable for enhancing immune responses.

[0139] Monoclonal antibodies

[0140] In some embodiments, the antibodies purified in the reusable chromatography material purified by the method of the present invention are monoclonal antibodies. Monoclonal antibodies are obtained from a substantially homogeneous antibody population, i.e., the individual antibodies comprising the population are identical and / or bind to the same epitope, except for possible variants generated during the production of the monoclonal antibodies, which variants are usually present in small amounts. Therefore, the modifier "monoclonal" indicates that the antibody is not characterized as a mixture of discrete or polyclonal antibodies.

[0141] For example, monoclonal antibodies can be made using the hybridoma method first described by Kohler et al., Nature 256:495 (1975), or may be made by recombinant DNA methods (US Pat. No. 4,816,567).

[0142] In the hybridoma method, mice or other appropriate host animals (such as hamsters) are immunized as described herein to elicit lymphocytes that produce or are capable of producing antibodies that will specifically bind to the polypeptide for immunization. Alternatively, lymphocytes can be immunized in vitro. Lymphocytes are then fused with tumor 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)).

[0143] The hybridoma cells thus 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 phosphoribosyl transferase (HGPRT or HPRT), the culture medium for the hybridomas will typically include hypoxanthine, aminopterin and thymidine (HAT medium), which prevent the growth of cells lacking HGPRT.

[0144] In certain embodiments, myeloma cells are those effectively fused, which support selected antibody-producing cells to stably produce antibodies at high levels, and are sensitive to culture medium such as HAT culture medium. Wherein, in certain embodiments, myeloma cell line is a mouse myeloma system, such as derived from MOPC-21 and MPC-11 mouse tumors obtainable from Salk Institute Cell Distribution Center, San Diego, California USA, and derived from SP-2 or X63-Ag8-653 cells obtainable from American Type Culture Collection, Rockville, Maryland USA. Human myeloma and mouse-human heteromyeloma cell lines (Kozbor, J.Immunol.133:3001 (1984) for producing human monoclonal antibodies are also described; Brodeur et al., Monoclonal Antibody Production Techniques and Applications, pp. 51-63 (Marcel Dekker, Inc, New York, 1987)).

[0145] Culture medium in which hybridoma cells are grown is analyzed 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).

[0146] The binding affinity of the monoclonal antibody can be determined by, for example, Scatchard analysis of Munson et al., Anal. Biochem. 107:220 (1980).

[0147] After identifying hybridoma cells that produce antibodies with the desired specificity, affinity and / or activity, the clones can be subcloned by limiting dilution and grown by standard methods (Goding, Monoclonal Antibodies: Principles and Practice 59-103 (Academic Press, 1986)). Suitable culture media for this purpose include, for example, D-MEM or RPMI-1640 medium. In addition, hybridoma cells can be grown in vivo as ascites tumors in animals.

[0148] 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.

[0149] DNA encoding monoclonal antibodies is readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes that specifically bind to genes encoding the heavy and light chains of murine antibodies). In some embodiments, hybridoma cells are used as a source of such DNA. Once isolated, the DNA can be placed in an expression vector, which is then transfected into a host cell that does not otherwise produce immunoglobulin polypeptides, such as an E. coli cell, ape COS cells, Chinese hamster ovary (CHO) cells, or a myeloma cell, to synthesize monoclonal antibodies in a recombinant host cell. Review papers on recombinant expression in bacteria of DNA encoding antibodies include Skerra et al., Curr. Opinion in Immunol. 5: 256-262 (1993); and Plückthun, Immunol. Revs, 130: 151-188 (1992).

[0150] In a further embodiment, antibodies or antibody fragments can be isolated from antibody phage libraries produced 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 use of phage libraries to separate mouse antibodies and human antibodies, respectively. Subsequent publications describe the production of high-affinity (nM range) human antibodies (Marks et al., Bio / Technology, 10: 779-783 (1992)) and combinatorial infection and in vivo recombination as strategies for constructing extremely large phage libraries (Waterhouse et al., Nuc. Acids. Res. 21: 2265-2266 (1993)). Therefore, these techniques are viable alternatives to traditional monoclonal antibody hybridoma techniques for isolating monoclonal antibodies.

[0151] The DNA also can be modified, for example, by replacing the homologous murine sequences with coding sequences for human heavy and light chain constant domains (U.S. Pat. No. 4,816,567; Morrison et al., Proc. Natl Acad. Sci. USA 81:6851 (1984)), or by covalently linking to the immunoglobulin coding sequence all or part of the coding sequence for a non-immunoglobulin polypeptide.

[0152] Typically, such non-immunoglobulin polypeptide sequences 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 specific for an antigen and another antigen-binding site specific for a different antigen.

[0153] 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.

[0154] Humanized Antibodies

[0155] 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 from a non-human source. These non-human amino acid residues are generally referred to as "import" residues, which are generally taken from the "import" variable domain. Humanization can be performed substantially according to the method of Winter and his colleagues (Jones et al., Nature, 321: 522-525 (1986); Riechmann et al., Nature, 332: 323-327 (1988); Verhoeyen et al., Science, 239: 1534-1536 (1988)), by replacing the corresponding sequence of a human antibody with a hypervariable region sequence. Therefore, such "humanized" antibodies are chimeric antibodies (U.S. Patent No. 4,816,567), in which substantially less than a complete human variable domain has been replaced by a 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.

[0156] The selection of light chain and heavy chain for preparing the human variable domain of humanized antibodies is very important for reducing antigenicity. According to the so-called "best fit" method, the sequence of the variable domain of rodent antibodies is screened for the entire library of known human variable domain sequences. The human sequence closest to rodents is then used as the human framework region (FR) of humanized antibodies (Sims et al., J.Immunol.151:2296 (1993); Chothia et al., J.Mol.Biol.196:901 (1987)). Another method uses the specific framework region of the consensus sequence of all human antibodies derived from a specific subgroup of light chain or heavy chain variable region. The same framework can be used for several different humanized antibodies (Carter et al., Proc.Natl.Acad.Sci.USA89:4285 (1992); Presta et al., J.Immunol.151:2623 (1993)).

[0157] It is further important that the antibody is humanized while retaining high affinity and other favorable biological properties to the antigen. To achieve this purpose, in some embodiments of the method, humanized antibodies are prepared by using the three-dimensional model of the parent and humanized sequence to analyze the parent sequence and various conceptual humanized products. The three-dimensional immunoglobulin model is usually available and is familiar to those skilled in the art. Computer programs can be used to illustrate and display the possible three-dimensional conformational structure of selected candidate immunoglobulin sequences. Inspection of these displays allows analysis of the possible effects of residues in the function of candidate immunoglobulin sequences, that is, analysis affects the residues of the ability of candidate immunoglobulin to bind its antigen. In this way, it is possible to select and combine FR residues from receptor and imported sequence, thereby obtain required antibody characteristics, such as increase in affinity to the target antigen. Generally speaking, hypervariable region residues directly and most substantially participate in affecting antigen binding.

[0158] Human Antibodies

[0159] In some embodiments, the antibodies are human antibodies. 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 able to produce a full spectrum of human antibodies after immunization without producing endogenous immunoglobulins. For example, the antibody heavy chain joining region (JCR) has been described in chimeric and germline mutant mice. H Homozygous deletion of the ) gene results in complete inhibition of endogenous antibody production. Transfer of the human germline immunoglobulin gene array in such germline mutant mice will result in the production of human antibodies after antigen challenge. See, for example, 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. 5591669; 5,589,369; and 5,545,807.

[0160] 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 technology, antibody V domain genes are cloned in-frame into the major or minor coat polypeptide genes of filamentous phages (such as M13 or fd) and displayed as functional antibody fragments on the surface of phage particles. Since filamentous particles contain single-stranded DNA copies of phage genomes, selection based on antibody functional properties also leads to selection of genes encoding antibodies that exhibit these properties. Therefore, phages mimic some characteristics of B cells. Phage display can be performed in a variety of forms; its relevant review see, for example, Johnson, Kevin S. and Chiswell, David J., Current Opinion in Structural Biology 3:564-571 (1993). V gene fragments from several sources can be used for phage display. Clackson et al., Nature 352:624-628 (1991) isolated a variety of anti-oxazolone antibodies from a small random combinatorial library of V genes derived from the spleen of immunized mice. V gene repertoires from unimmunized human donors can be constructed essentially according to the techniques described in Marks et al., J. Mol. Biol. 222:581-597 (1991) or Griffith et al., EMBO J. 12:725-734 (1993), and antibodies against different antigen arrays (including self-antigens) can be isolated. See also U.S. Patent Nos. 5,565,332 and 5,573,905.

[0161] Human antibodies can also be generated by in vitro activated B cells (see US Pat. Nos. 5,567,610 and 5,229,275).

[0162] Antibody fragments

[0163] In some embodiments, the antibody is an antibody fragment. Various techniques for producing antibody fragments have been developed. Traditionally, these fragments are obtained by proteolytic digestion of intact antibodies (see, for example, Morimoto et al., Journal of Biochemical and Biophysical Methods 24: 107-117 (1992) and Brennan et al., Science 229: 81 (1985)). However, these fragments can now be produced directly by recombinant host cells. For example, antibody fragments can be isolated from the above-mentioned antibody phage library. Alternatively, Fab'-SH fragments can be directly recovered from Escherichia coli (E. coli), and chemically coupled to form F (ab') 2 fragments (Carter et al., Bio / Technology 10: 163-167 (1992)). According to another method, F (ab') 2 fragments can be directly isolated from recombinant host cell cultures. Other techniques for producing antibody fragments will be apparent to those skilled in the art. In other embodiments, the selected antibody is a single-chain Fv fragment (scFv). See WO 93 / 16185; U.S. Patent No. 5,571,894; and U.S. Patent No. 5,587,458. For example, the antibody fragment may also be a "linear antibody," as described, for example, in U.S. Patent No. 5,641,870. Such linear antibody fragments may be monospecific or bispecific.

[0164] In some embodiments, fragments of 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: Fab fragment, Fab' fragment, F(ab')2 fragment, scFv, di-scFv, bi-scFv, tandem (di,tri)-scFv, Fv, sdAb, trifunctional antibody, BiTE, diabody and tribody.

[0165] Bispecific Antibodies

[0166] In some embodiments, the antibody is a bispecific antibody. Bispecific antibodies are antibodies with binding specificity to 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 (FcγR) of IgG, such as FcγRI (CD64), FcγRII (CD32) and FcγRIII (CD16), thereby focusing cellular defense mechanisms on cells. Bispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2 bispecific antibodies).

[0167] Methods for preparing bispecific antibodies are known in the art. The traditional production of full-length bispecific antibodies is based on the coexpression of two immunoglobulin heavy chain-light chain pairs, wherein the two chains have different specificities (Millstein et al., Nature, 305:537-539 (1983)). Due to the random diversity of immunoglobulin heavy chains and light chains, these hybridomas (quadromas) produce a potential mixture of 10 different antibody molecules, of which only one has the correct bispecific structure. The purification of the correct molecules usually completed by affinity chromatography steps is quite troublesome, and the product yield is low. Similar processes are disclosed in WO 93 / 08829 and Traunecker et al., EMBO J., 10:3655-3659 (1991).

[0168] According to different methods, the antibody variable domain (antibody-antigen binding site) with the desired binding specificity is fused with the immunoglobulin constant domain sequence. In certain embodiments, the fusion uses the immunoglobulin heavy chain constant domain, including at least a portion of the hinge, CH2 and CH3 regions. In certain 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. The DNA encoding the immunoglobulin heavy chain fusion and (if necessary) immunoglobulin light chain is inserted into a separate expression vector and cotransfected into a suitable host organism. When the unequal ratios of the three polypeptide chains used in the construction provide the best yield, this provides great flexibility in adjusting the mutual ratio of the three polypeptide fragments in an embodiment. However, when at least two polypeptide chains are expressed with equal ratios resulting in high yields or when the ratio has no particular significance, it is possible to insert the coding sequence of two or all three polypeptide chains in one expression vector.

[0169] In some embodiments of the method, 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 promotes the separation of the desired bispecific compound from the unwanted immunoglobulin chain combination, because the presence of immunoglobulin light chains in only one half of the bispecific molecule provides a simple separation method. The method is disclosed in WO 94 / 04690. For more details on the generation of bispecific antibodies, see, for example, Suresh et al., Methods in Enzymology, 121: 210 (1986).

[0170] According to another approach described in U.S. Pat. No. 5,731,168, the interface between a pair of antibody molecules can be engineered to maximize the percentage of heterodimers recovered from recombinant cell culture. In some embodiments, the interface comprises the C H At least a portion of the 3 domain. In this method, one or more small amino acid side chains from the interface of the first antibody molecule are replaced with larger side chains (e.g., tyrosine or tryptophan). Compensatory "cavities" with the same or similar size as the large side chains are created on the interface of the second antibody molecule by replacing the large amino acid side chains with smaller amino acid side chains (e.g., alanine or threonine). This provides a mechanism that can increase the yield of heterodimers rather than other unwanted end products (such as homodimers).

[0171] Bispecific antibodies include cross-linked or "heteroconjugate" antibodies. For example, one antibody in the heteroconjugate can be coupled to avidin and the other to biotin. For example, such antibodies have been proposed to target immune system cells to unwanted cells (U.S. Patent No. 4,676,980), and for the treatment of HIV infection (WO 91 / 00360, WO92 / 200373 and EP 03089). Heteroconjugate antibodies can be prepared using any convenient cross-linking method. Suitable cross-linking agents are well known in the art and are disclosed in U.S. Patent No. 4,676,980 together with many cross-linking techniques.

[0172] The technology of generating bispecific antibodies from antibody fragments has also been described in the literature. For example, chemical bonding can be used to prepare bispecific antibodies. Brennan et al., Science 229:81 (1985) described 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 the formation of intermolecular disulfides. The generated Fab' fragments are then converted into thionitrobenzoate (TNB) derivatives. Then one of the Fab'-TNB derivatives is converted into Fab'-thiol by reduction with mercaptoethylamine, and mixed with another Fab'-TNB derivative of equimolar amount to form a bispecific antibody. The resulting bispecific antibody can be used as a reagent for selective enzyme fixation.

[0173] Various techniques for preparing and isolating bispecific antibody fragments directly from recombinant cell culture have also been described. For example, bispecific antibodies have been produced using leucine zippers. Kostelny et al., J. Immunol. 148(5): 1547-1553 (1992). By gene fusion, leucine zipper peptides from Fos protein and Jun protein are connected to the Fab' portion of two different antibodies. Antibody homodimers are reduced to form monomers in the hinge region and then oxidized to form antibody heterodimers. This method can also be used for the production of antibody homodimers. The "diabody" technology described by Hollinger et al., Proc. Natl. Acad. Sci. USA 90: 6444-6448 (1993) has provided an alternative mechanism for preparing bispecific antibody fragments. These fragments contain a light chain variable domain (V H )'s heavy chain variable domain (V L ), the linker is too short to allow pairing between the two domains on the same chain. H and V L The domain is forced to mate with the complementary V L and V H The domains pair to form two antigen binding sites. Another strategy for preparing bispecific antibody fragments by using single-chain Fv (sFv) dimers has also been reported. See Gruber et al., J. Immunol. 152: 5368 (1994).

[0174] Antibodies with more than two valency states are contemplated. For example, trispecific antibodies can be prepared. Tutt et al., J. Immunol. 147:60 (1991).

[0175] Multivalent Antibodies

[0176] In some embodiments, the antibody is a multivalent antibody. By expressing cells of the antigen bound by the antibody, the multivalent antibody can be internalized (and / or decomposed) faster than a bivalent antibody. The antibody provided herein can be a multivalent antibody (which is not an antibody of the IgM class) (e.g., a tetravalent antibody) with three or more antigen binding sites, which can be easily produced by recombinantly expressing nucleic acids encoding the polypeptide chains of the antibody. The multivalent antibody may include a dimerization domain and three or more antigen binding sites. The preferred dimerization domain includes (or is composed of) an Fc region or a hinge region. In this case, the antibody will include an Fc region and three or more antigen binding sites located at the amino terminus of the Fc region. The preferred multivalent antibody herein includes three to about eight, but preferably four antigen binding sites (or is composed of). The multivalent antibody includes at least one polypeptide chain (and preferably two polypeptide chains), wherein the polypeptide chain includes two or more variable domains. For example, the polypeptide chain may comprise VD1-(X1)n-VD2-(X2)n-Fc, wherein VD1 is a first variable domain, VD2 is a second variable domain, Fc is a polypeptide chain of the Fc region, X1 and X2 represent amino acids or polypeptides, and n is 0 or 1. For example, the polypeptide chain may comprise: VH-CH1-flexible linker-VH-CH1-Fc region chain; or VH-CH1-VH-CH1-Fc region chain. The multivalent antibody herein preferably further comprises at least two (and preferably four) light chain variable domain polypeptides. The multivalent antibody herein may, for example, comprise 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.

[0177] 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 ) of the antibody, wherein V H V L Units have multiple epitope specificity, with two or more V L and V H Domain antibody, each V H V LUnits bind to different epitopes, 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, covalently or non-covalently linked antibody fragments. In some embodiments, the antibody has multi-epitope specificity; for example, the ability to specifically bind to two or more different epitopes on the same or different targets. In some embodiments, the antibody is monospecific; for example, an antibody that binds only one epitope. According to one embodiment, the multispecific antibody is an IgG antibody that binds to each epitope with an affinity of 5ΓM to 0.001pM, 3ΓM to 0.001pM, 1ΓM to 0.001pM, 0.5ΓM to 0.001pM, or 0.1ΓM to 0.001pM.

[0178] Other Antibody Modifications

[0179] It may be desirable to modify the antibodies provided herein in terms of effector functions, 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 in the Fc region of the antibody. Alternatively or additionally, cysteine ​​residues can be introduced in the Fc region, thereby allowing the formation of interchain disulfide bonds in the region. The homodimeric antibodies thus produced may have improved internalization capacity 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, antibodies can be engineered to have dual Fc regions, which can have enhanced complement-mediated lysis and ADCC capabilities. See Stevenson et al., Anti-Cancer Drug Design 3: 219-230 (1989).

[0180] To increase the serum half-life of an antibody, amino acid changes may be made in the antibody as described in US 2006 / 0067930, which is incorporated herein by reference in its entirety.

[0181] Peptide variants and modifications

[0182] One or more amino acid sequence modifications of the polypeptides (including antibodies) described herein can be used in reusable chromatography materials that are purified by the methods described herein.

[0183] Variant polypeptide

[0184] "Polypeptide variant" refers to a polypeptide having at least about 80% amino acid sequence identity with the full-length native sequence of a polypeptide, a polypeptide sequence lacking a signal peptide, an extracellular domain of a polypeptide (with or without a signal peptide) as defined herein, preferably an active polypeptide. Such polypeptide variants include, for example, polypeptides, wherein one or more amino acid residues are added or missing at the N-terminus or C-terminus of the full-length native amino acid sequence. Typically, TAT polypeptide variants and full-length native sequence polypeptide sequences, polypeptide sequences lacking a signal peptide, the extracellular domain of a polypeptide (with or without a signal peptide) will have at least about 80% amino acid sequence identity, or at least about 85%, 90%, 95%, 96%, 97%, 98% or 99% amino acid sequence identity of any one. Optionally, compared to a native polypeptide sequence, a variant polypeptide will have no more than one conservative amino acid substitution, or compared to a native polypeptide sequence, including no more than about 2, 3, 4, 5, 6, 7, 8, 9 or 10 conservative amino acid substitutions.

[0185] For example, compared to the full-length native polypeptide, the variant polypeptide may be truncated at the N-terminus or C-terminus, or may lack internal residues. Some variant polypeptides may lack amino acid residues that are not essential for the desired biological activity. These variant polypeptides with truncations, deletions and insertions can be prepared by any of many conventional techniques. The desired variant polypeptide can be chemically synthesized. Another suitable technique includes separating and amplifying nucleic acid fragments encoding the desired variant polypeptides by polymerase chain reaction (PCR). Oligonucleotides defining the desired ends of the nucleic acid fragments are used at the 5' and 3' primers in PCR. Preferably, the variant polypeptide shares at least one biological and / or immunological activity with the native polypeptide disclosed herein.

[0186] Amino acid sequence insertions include amino and / or carboxyl terminal fusions ranging in length from one residue to polypeptides containing one hundred or more residues, and intrasequence insertions of one or more amino acid residues. Examples of terminal insertions include antibodies with an N-terminal methionyl residue or antibodies fused to a cytotoxic polypeptide. Other insertion variants of the antibody molecule include fusions to the N-terminus or C-terminus of an antibody that increases the serum half-life of the antibody.

[0187] For example, it may be desirable to improve the binding affinity and / or other biological properties of a polypeptide. Amino acid sequence variants of a polypeptide are prepared by introducing appropriate nucleotide changes into antibody nucleic acids or by peptide synthesis. Such modifications include, for example, deletions and / or insertions and / or substitutions of residues within the polypeptide amino acid sequence. Any combination of deletions, insertions and substitutions is performed to obtain the final construct, provided that the final construct has the desired properties. Amino acid changes can also change the post-translational processes of a polypeptide (e.g., an antibody), such as changing the number or position of glycosylation sites.

[0188] Guidance in determining which amino acid residues may be inserted, substituted or deleted without adversely affecting the desired activity can be found by comparing the polypeptide sequence to the sequences of homologous known polypeptide molecules and minimizing the number of amino acid sequence changes in regions of high homology.

[0189] The method that can be used to identify certain residues or regions of polypeptides (e.g., antibodies) (which are preferred positions for mutagenesis) is called "alanine scanning mutagenesis", as described in Cunningham and Wells, Science 244: 1081-1085 (1989). Here, a residue or a group of target 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 interaction of amino acids with antigens. Then, by introducing further or other variants at or for the substitution site, those amino acid positions that show functional sensitivity to substitution are refined. Therefore, although the site for introducing amino acid sequence variation is predetermined, it is not necessary to predetermine the nature of the mutation itself. For example, in order to analyze the performance of the mutation at a given site, ala scanning or random mutagenesis is performed at the target codon or region, and the desired activity of the expressed antibody variant is screened.

[0190] Another type of variant is an amino acid substitution variant. These variants have at least one amino acid residue replaced by a different residue in the antibody molecule. The most interesting sites for substitution mutagenesis include hypervariable regions, but FR changes are also envisioned. Conservative substitutions are shown under the heading "preferred substitutions" in Table A below. If this type of substitution causes a change in biological activity, then a more substantial change named "exemplary substitutions" or further described as below with reference to amino acid classes may be introduced into Table A, and the product may be screened.

[0191] Table A. Exemplary amino acid substitutions

[0192] Original residue Exemplary substitutions Preferred substitution Ala(A) Val; Leu; Ile Val Arg(R) Lys; Gln; Asn Lys Asn(N) Gln; His; Asp; Lys; Arg Gln Asp(D) Glu; Asn Glu Cys(C) Ser; Ala Ser Gln(Q) Asn;Glu Asn Glu(E) Asp; Gln Asp Gly(G) Ala Ala His(H) Asn; Gln; Lys; Arg Arg Ile(I) Leu; Val; Met; Ala; Phe; Norleucine Leu Leu(L) Norleucine; Ile; Val; Met; Ala; Phe Ile Lys(K) Arg; Gln; Asn Arg Met(M) Leu; Phe; Ile Leu Phe(F) Trp; Leu; Val; Ile; Ala; Tyr Tyr Pro(P) Ala Ala Ser(S) Thr Thr Thr(T) Val; Ser Ser Trp(W) Tyr; Phe Tyr Tyr(Y) Trp; Phe; Thr; Ser Phe Val(V) Ile; Leu; Met; Phe; Ala; norleucine Leu

[0193] Substantial modification of the biological properties of a polypeptide is accomplished by selecting substitutions that have a significantly different effect on maintaining (a) the structure of the polypeptide backbone in the area of ​​the substitution, e.g., as a 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 (in ALLehninger, Biochemistry 2nd Edition, Worth Publishers, New York (1975)):

[0194] (1) Non-polar: Ala (A), Val (V), Leu (L), Ile (I), Pro (P), Phe (F), Trp (W), Met (M)

[0195] (2) Non-electric polarity: Gly(G), Ser(S), Thr(T), Cys(C), Tyr(Y), Asn(N), Gln(Q)

[0196] (3) Acidic: Asp (D), Glu (E)

[0197] (4) Basicity: Lys (K), Arg (R), His (H)

[0198] Alternatively, naturally occurring residues can be divided into the following classes based on common side-chain properties:

[0199] (1) Hydrophobicity: norleucine, Met, Ala, Val, Leu, Ile;

[0200] (2) Neutral hydrophilicity: Cys, Ser, Thr, Asn, Gln;

[0201] (3) Acidic: Asp, Glu;

[0202] (4) Basic: His, Lys, Arg;

[0203] (5) Residues that affect chain orientation: Gly, Pro;

[0204] (6) Aromatic: Trp, Tyr, Phe.

[0205] Non-conservative substitutions will entail exchanging a member of one of these classes for another class.

[0206] Any cysteine ​​residues not involved in maintaining the proper conformation of the antibody may also be substituted with serine to improve the oxidative stability of the molecule and prevent abnormal cross-linking. Conversely, one or more cysteine ​​bonds may be added to a polypeptide to improve its stability (particularly when the antibody is an antibody fragment such as an Fv fragment).

[0207] A particularly preferred type of substitution variant involves replacing one or more hypervariable region residues of a parent antibody (e.g., a humanized antibody). In general, the resulting variants selected for further development will have improved biological properties relative to the parent antibody that produces them. A convenient method for generating such substitution variants involves affinity maturation using phage display. In short, several hypervariable region sites (e.g., 6 to 7 sites) are mutated to produce all possible amino substitutions at each site. The antibody variants thus generated are displayed as fusions of the gene III product of M13 packaged in each particle from filamentous phage particles in a monovalent manner. The biological activity (e.g., binding affinity) of the variants displayed by phage is then screened, as disclosed herein. In order 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 the crystal structure of the antigen-antibody complex to identify the contact points between the antibody and the target. According to the technology described herein, such contact residues and adjacent residues are candidates for substitution. Once such variants are generated, the panel of variants is screened as described herein, and antibodies with superior properties in one or more relevant assays can be selected for further development.

[0208] Another type of polypeptide amino acid variant changes the original glycosylation pattern of the antibody. The polypeptide may contain non-amino acid moieties. For example, the 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 caused by human intervention. The alteration refers to the deletion of one or more carbohydrate moieties found in the polypeptide, and / or the addition of one or more glycosylation sites not present in the polypeptide.

[0209] Glycosylation of polypeptides is usually 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 other than proline) are recognition sequences for enzymatic attachment of the carbohydrate moiety to the asparagine side chain. Thus, the presence of any 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, but 5-hydroxyproline or 5-hydroxylysine may also be used.

[0210] By altering the amino acid sequence to contain one or more of the above-described tripeptide sequences (for N-linked glycosylation sites), it is convenient to add glycosylation sites to the polypeptide. The alteration may also be accomplished by adding or substituting one or more serine or threonine residues in the sequence of the original antibody (for O-linked glycosylation sites).

[0211] 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 are targets for glycosylation. Enzymatic cleavage of carbohydrate moieties on a polypeptide can be achieved by the use of a variety of endo- and exo-glycosidases.

[0212] 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 groups 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.

[0213] Chimeric polypeptide

[0214] The polypeptides described herein can be modified in a manner to form a chimeric molecule comprising a polypeptide fused to another heterologous polypeptide or amino acid sequence. In some embodiments, the chimeric molecule comprises a fusion of a polypeptide and a tag polypeptide, and the tag polypeptide provides an epitope to which an anti-tag antibody can selectively bind. The epitope tag is usually placed at the amino or carboxyl terminus of the polypeptide. Antibodies to the tag polypeptide can be used to detect the presence of the polypeptide in this epitope-tagged form. In addition, the provision of the epitope tag enables the polypeptide to be easily purified by affinity purification using an anti-tag antibody or another type of affinity matrix that binds to the epitope tag.

[0215] In alternative embodiments, the chimeric molecule may comprise a fusion of a polypeptide with an immunoglobulin or a specific region of an immunoglobulin. The bivalent form of the chimeric molecule is referred to as an "immunoadhesin."

[0216] As used herein, the term "immunoadhesin" refers to an antibody-like molecule that combines the binding specificity of a heterologous polypeptide with the effector function of an immunoglobulin constant domain. Structurally, an immunoadhesin comprises a fusion of an amino acid sequence with a desired binding specificity that is different from the antigen recognition and binding site binding specificity of an antibody (i.e., "heterologous"). The adhesin portion of an immunoadhesin molecule is typically a contiguous amino acid sequence that comprises at least the binding site of a receptor or ligand. The immunoglobulin constant domain sequence in an immunoadhesin can be obtained from any immunoglobulin, such as IgG-1, IgG-2, IgG-3, or IgG-4 subtypes, IgA (including IgA-1 and IgA-2), IgE, IgD, or IgM.

[0217] The Ig fusion preferably comprises a polypeptide in a soluble (transmembrane domain deleted or inactivated) form replacing at least one variable region position in an Ig molecule. In a particularly preferred embodiment, the immunoglobulin fusion comprises the hinge region CH2 and CH3, or the hinge region CH1, CH2 and CH3 of an IgG1 molecule.

[0218] Peptide conjugates

[0219] Polypeptides for use in polypeptide formulations may be conjugated to cytotoxic agents (e.g., chemotherapeutic agents), growth inhibitory agents, toxins (e.g., enzymatically active toxins of bacterial, fungal, plant or animal origin or fragments thereof), or radioactive isotopes (i.e., radioconjugates).

[0220] Chemotherapeutic agents that can be used to produce such conjugates can be used. In addition, enzymatically active toxins and fragments thereof that can be used include diphtheria A chain, non-binding active fragments of diphtheria toxin, exotoxin A chain (from Pseudomonas aeruginosa), ricin A chain, abrin A chain, modeccin A chain, α-sarcin, Aleurites fordii protein, dianthus protein, pokeweed antiviral proteins (PAPI, PAPII and PAP-S), momordica charantia inhibitor, curcumin, crotonin, saponin inhibitor, gelatin, mitocin, restrictocin, phenomycin, enomycin and trichothecenes. A variety of radionuclides can be used to produce radioconjugated polypeptides. Examples include 212 Bi, 131 I. 131 In, 90 Y and 186 Re. Conjugates of polypeptides and cytotoxic agents are prepared using a variety of bifunctional protein coupling agents such as N-succinimidyl-3-(2-pyridyldithiol) propionate (SPDP), iminothiolane (IT), bifunctional derivatives of iminoesters (such as dimethyl adipate HCL), active esters (such as disuccinimidyl suberate), aldehydes (such as glutaraldehyde), bis-azido compounds (such as bis(p-azidobenzoyl)hexanediamine), bis-diazonium derivatives (such as bis-(p-diazoniumbenzoyl)-ethylenediamine), diisocyanates (such as toluene 2,6-diisocyanate), and bis-active fluorine compounds (such as 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.

[0221] Also contemplated herein are conjugates of the polypeptides with one or more small molecule toxins, such as calicheamicin, maytansinoids, trichothene, and CC1065, as well as derivatives of these toxins that have toxin activity.

[0222] Maytansinoids are mitotic inhibitors that work by inhibiting tubulin polymerization. Maytansine was originally isolated from the East African shrub Maytansinol (Maytenus serrata). Subsequently, it was found that some microorganisms also produced maytansinoids, such as maytansinol (maytansinol) and C-3 maytansinol esters. Synthetic maytansinol and its derivatives and analogs are also envisioned. Many linking groups for preparing polypeptide-maytansinoid conjugates are known in the art, including those disclosed in, for example, U.S. Patent No. 5,208,020. Linking groups include disulfide groups, thioether groups, acid-labile groups, light-labile groups, peptidase-labile groups or esterase-labile groups, as disclosed in the above-mentioned patents, preferably disulfide and thioether groups.

[0223] The joint can be connected to the maytansinol molecule at different positions, depending on the type of connection. For example, conventional coupling techniques can be used to form an ester bond by reacting with a hydroxyl group. The reaction can be carried out at the C-3 position with a hydroxyl group, the C-14 position modified by hydroxyl group, the C-15 position modified by hydroxyl group, and the C-20 position with a hydroxyl group. In a preferred embodiment, bonding is formed at the C-3 position of maytansinol or maytansinol analogs.

[0224] Another conjugate of interest comprises a polypeptide conjugated to one or more calicheamicin molecules. The calicheamicin antibiotic family is capable of producing double-stranded DNA breaks at sub-picomolar concentrations. For the preparation of conjugates of the calicheamicin family, see, e.g., U.S. Pat. No. 5,712,374. Structural analogs of calicheamicin that may be used include, but are not limited to, γ1 I , α2 I , α3 I , N-acetyl-γ1 I , PSAG and θ1 I Another anti-tumor drug to which the antibody can be conjugated is QFA, which is an antifolate. Both calicheamicin and QFA have intracellular sites of action and do not readily cross the plasma membrane. Therefore, cellular uptake of these agents by polypeptide (e.g., antibody)-mediated internalization greatly enhances their cytotoxic effects.

[0225] Other anti-tumor agents that can be conjugated to the polypeptides described herein include BCNU, streptozotocin, vincristine, and 5-fluorouracil (a family of agents collectively referred to as the LL-E33288 complex), and esperamicin.

[0226] In some embodiments, the polypeptide can be a conjugate between the polypeptide and a compound having nucleolytic activity (eg, a ribonuclease or a DNA endonuclease, such as a deoxyribonuclease; DNase).

[0227] In yet another embodiment, a polypeptide (e.g., an antibody) can be conjugated to a "receptor" (such as streptavidin) for tumor pretargeting, wherein the polypeptide receptor conjugate is administered to a patient, followed by removal of unbound conjugate from the circulation using a clearing agent, and then administration of a "ligand" (e.g., avidin) conjugated to a cytotoxic agent (e.g., a radionucleotide).

[0228] In some embodiments, the polypeptide can be conjugated to a prodrug activating enzyme that converts a prodrug (e.g., a peptide-based chemotherapeutic agent) into an active anticancer drug. The enzyme component of the immunoconjugate includes any enzyme that can act on a prodrug in this manner to convert it into its more active cytotoxic form.

[0229] Useful enzymes include, but are not limited to, alkaline phosphatase, which can be used to convert phosphate-containing prodrugs to free drugs; arylsulfatase, which can be used to convert sulfate-containing prodrugs to free drugs; cytosine deaminase, which can be used to convert non-toxic 5-fluorocytosine to the anticancer drug 5-fluorouracil; proteases, such as serrassin, thermolysin, subtilisin, carboxypeptidases, and cathepsins (such as cathepsins B and L), which can be used to convert peptide-containing prodrugs to free drugs; D-alanyl carboxypeptidases, which can be used to convert prodrugs containing D-amino acid substituents; carbohydrate cleavage enzymes, such as β-galactosidase and neuraminidase, which can be used to convert glycosylated prodrugs to free drugs; β-lactamase, which can be used to convert drugs derivatized with β-lactams to free drugs; and penicillin amidases, such as penicillin V amidase or penicillin G amidase, which can be used to convert drugs derivatized at their amine nitrogens to have phenoxyacetyl or phenylacetyl groups, respectively, to free drugs. Alternatively, antibodies with enzymatic activity, also known in the art as "abzymes," can be used to convert prodrugs into free active drugs.

[0230] other

[0231] Another type of covalent modification of polypeptides includes linking the polypeptide to one of a variety of non-protein polymers, such as polyethylene glycol, polypropylene glycol, polyoxyalkylene or copolymers of polyethylene glycol and polypropylene glycol. The polypeptide can also be embedded in microcapsules (e.g., hydroxymethylcellulose or gelatin microcapsules and poly(methyl methacrylate) microcapsules, respectively) prepared, for example, by coacervation techniques or by interfacial polymerization; in colloidal drug delivery systems (e.g., liposomes, albumin microspheres, microemulsions, nanoparticles and nanocapsules); or in coarse emulsions. Such techniques are disclosed in Remington's Pharmaceutical Sciences, 18th edition, Gennaro, AR, ed., (1990).

[0232] Obtaining polypeptides for use in formulations

[0233] Polypeptides to be purified using reusable chromatography materials purified by the methods described herein can be obtained using methods well known in the art, including recombinant methods. The following section provides guidance on these methods.

[0234] Polynucleotide

[0235]

[0046] "Polynucleotide" or "nucleic acid," used interchangeably herein, refer to polymers of nucleotides of any length and include DNA and RNA.

[0236] Polynucleotides encoding polypeptides can be obtained from any source, including but not limited to cDNA libraries prepared from tissues believed to have polypeptide mRNA and express the polypeptide at detectable levels. Therefore, polynucleotides encoding polypeptides can be conveniently obtained from cDNA libraries prepared from human tissues. Polypeptide encoding genes can also be obtained from genomic libraries or by known synthetic procedures (e.g., automatic nucleic acid synthesis).

[0237] For example, a polynucleotide may encode an entire immunoglobulin molecule chain, such as a light chain or a heavy chain. A complete heavy chain includes not only the heavy chain variable region (V H ), and includes the heavy chain constant region (C H ), the heavy chain constant region usually includes 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.

[0238] Other polypeptides that may be encoded by a polynucleotide include antigen-binding antibody fragments such as single domain antibodies ("dAbs"), Fv, scFv, Fab' and F(ab')2, and "minibodies". Minibodies are (usually) H 1 and C K or C LThe term "antibody" refers to a bivalent antibody fragment of a polypeptide having a domain. Since miniantibodies are smaller than conventional antibodies, they should achieve better tissue penetration in clinical / diagnostic use, but as bivalent antibodies, they should maintain a higher binding affinity than monovalent antibody fragments (such as dAbs). Therefore, unless the context otherwise indicates, the term "antibody" as used herein includes not only whole antibody molecules, but also antigen-binding antibody fragments of the above types. Preferably, each framework region present in the encoded polypeptide will include at least one amino acid substitution relative to the corresponding human receptor framework. Therefore, for example, relative to the receptor framework region, the framework region includes three, four, five, six, seven, eight, nine, ten, eleven, twelve, thirteen, fourteen or fifteen amino acid substitutions in total.

[0239] Suitably, the polynucleotides described herein may be isolated and / or purified. In some embodiments, the polynucleotide is an isolated polynucleotide.

[0240] The term "isolated polynucleotide" is intended to indicate that the molecule is removed or separated from its normal or natural environment, or is produced in a way that it does not exist 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.

[0241] Suitably, the polynucleotides are substantially purified such that the polynucleotide of interest constitutes the predominant (ie, most abundant) polynucleotide present in the composition.

[0242] Expression of polynucleotides

[0243] The following description mainly relates to the production of polypeptides by culturing cells transformed or transfected with a vector containing a polynucleotide encoding a polypeptide. Of course, it is contemplated that alternative methods well known in the art can be used to prepare polypeptides. For example, solid phase techniques can be used to produce appropriate amino acid sequences or portions thereof by direct peptide synthesis (see, for example, Stewart et al., Solid-Phase Peptide Synthesis WH Freeman Co, San Francisco, Calif. (1969); Merrifield, J. Am. Chem. Soc. 85: 2149-2154 (1963)). In vitro protein synthesis can be performed using manual techniques or by automation. For example, Applied Biosystems peptide synthesizer (Foster City, Calif.) can be used to complete automatic synthesis according to the manufacturer's instructions. The various parts of the polypeptide can be chemically synthesized separately and combined using chemical or enzymatic methods to produce the desired polypeptide.

[0244] The polynucleotides as described herein are inserted into expression vectors for producing polypeptides. The term "control sequence" refers to DNA sequences necessary for expressing 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.

[0245] A polypeptide is "operably linked" when it is in a functional relationship with another polynucleotide sequence. For example, a nucleic acid of a presequence or secretory leader is operably linked to a 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 that affects the transcription of a sequence; or a ribosome binding site is operably linked to a coding sequence that is positioned to facilitate translation. In general, "operably linked" means that the nucleic acid sequences connected are continuous, and for a secretory leader sequence, continuous and at the reading stage. However, enhancers do not have to be continuous. The connection is accomplished by connecting at a convenient restriction site. If such a site does not exist, a synthetic oligonucleotide adapter or joint is used according to conventional practice.

[0246] For antibodies, the light chain and heavy chain can be cloned into the same or different expression vectors. The nucleic acid fragment encoding the immunoglobulin chain can be operably linked to one or more control sequences in the expression vector to ensure the expression of the immunoglobulin polypeptide.

[0247] The vector containing the polynucleotide sequence (e.g., variable heavy chain and / or variable light chain encoding sequence and optional expression control sequence) can be transferred to the host cell by well-known methods, which depends on the type of cell host. For example, calcium chloride transfection is commonly used for prokaryotic cells, while calcium phosphate treatment, electroporation, lipofection, gene gun or virus-based transfection can be used for other cell hosts. (Generally see Sambrook et al., Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Press, 2nd edition, 1989)). Other methods for transforming mammalian cells include the use of polybrene, protoplast fusion, liposomes, electroporation and microinjection. For the production of transgenic animals, the transgene can be microinjected into fertilized oocytes, or it can be incorporated into the genome of embryonic stem cells, and the nucleus of such cells is transferred to enucleated oocytes.

[0248] Carrier

[0249] The term "vector" includes expression vectors and transformation vectors as well as shuttle vectors.

[0250] The term "expression vector" means a construct capable of in vivo or in vitro expression.

[0251] The term "transformation vector" means a construct that can be transferred from one entity to another, which entities may belong to the same species or may belong to different species. If the construct can be transferred from one species to another, such as from an Escherichia coli plasmid to a bacterium, such as a bacterium of the genus Bacillus, the transformation vector is sometimes referred to as a "shuttle vector". It can even be a construct that can be transferred from an E. coli plasmid to an Agrobacterium and then to a plant.

[0252] The vector can be transformed into an appropriate host cell to provide expression of the polypeptide as described below. Various vectors are publicly available. The vector can be in the form of, for example, a plasmid, a cosmid, a viral particle or a bacteriophage. The appropriate nucleotide sequence can be inserted into the vector by various procedures. In general, DNA is inserted into an appropriate restriction endonuclease site using techniques known in the art. The construction of a suitable vector containing one or more of these components adopts standard ligation techniques known to those skilled in the art.

[0253] The vector may be, for example, a plasmid, a virus or a phage vector provided with an origin of replication, optionally a promoter for expressing the polynucleotide and optionally a regulator of the promoter. The vector may contain one or more selectable marker genes well known in the art.

[0254] These expression vectors are typically replicable in the host organism either as an episome or as an integral part of the host chromosomal DNA.

[0255] Host cells

[0256] The host cell can be, for example, a bacterial, yeast or other fungal cell, an insect cell, a plant cell, or a mammalian cell.

[0257] A genetically manipulated transgenic multicellular host organism may be used to produce the polypeptide. The organism may be, for example, a transgenic mammalian organism (eg, a transgenic goat or mouse strain).

[0258] Suitable prokaryotes include, but are not limited to, true bacteria, such as Gram-negative or Gram-positive organisms, e.g., Enterobacteriaceae, such as E. coli. Various E. coli strains are publicly available, such as E. coli K12 strain MM294 (ATCC 31,446); E. coli X1776 (ATCC 31,537); E. coli W3110 (ATCC 27,325) and K5 772 (ATCC 53,635). Other suitable prokaryotic host cells include Enterobacteriaceae, such as Escherichia, e.g., E. coli; Enterobacter; Erwinia; Klebsiella; Proteus; Salmonella, e.g., Salmonella typhimurium; typhimurium); and Serratia, such as Serratia marcescans; and Shigella; and Bacilli, such as B. subtilis and B. licheniformis (e.g., B. licheniformis 41P); Pseudomonas, such as Pseudomonas aeruginosa; and Streptomyces. These examples are illustrative and non-limiting. Strain W3110 is a particularly preferred host or parent host because it is a commonly used host strain for fermentation of recombinant polynucleotide products. Preferably, the host cell secretes very small amounts of proteolytic enzymes.For example, strain W3110 can be modified to generate a gene mutation in a gene encoding a polypeptide endogenous to the host, examples of such hosts include Escherichia coli (E. coli) W3110 strain 1A2, which has a complete genotype tonA; Escherichia coli (E. coli) W3110 strain 9E4, which has a complete genotype tonA ptr3; Escherichia coli (E. coli) W3110 strain 27C7 (ATCC 55,244), which has a complete genotype tonA ptr3 phoA E15 (argF-lac) 169degP ompT kan'; Escherichia coli (E. coli) W3110 strain 37D6, which has a complete genotype tonA ptr3 phoA E15 (argF-lac) 169degP ompT rbs7 ilvG kan'; E. coli W3110 strain 40B4, which is strain 37D6 with a non-kanamycin resistance degP deletion mutation; and an E. coli strain with a mutant periplasmic protease. Alternatively, in vitro cloning methods (such as PCR or other nucleic acid polymerase reactions) are suitable.

[0259] In these prokaryotic hosts, expression vectors can be prepared, which will generally contain expression control sequences (e.g., origin of replication) compatible with the host cell. In addition, there will be any number of various well-known promoters, such as a lactose promoter system, a tryptophan (trp) promoter system, a β-lactamase promoter system, or a promoter system from bacteriophage λ. The promoter will generally control expression, optionally together with an operator sequence, and have a ribosomal binding site sequence, etc., for initiating and completing transcription and translation.

[0260] 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. Yeast (Saccharomyces cerevisiae) is a commonly used lower eukaryotic host microorganism. Others include Schizosaccharomyces pombe; Kluyveromyces hosts, such as K. lactis (MW98-8C, CBS683, CBS4574), K. fragilis (ATCC 12,424), K. bulgaricus (ATCC 16,045), K. wickeramii (ATCC 24,178), K. waltii (ATCC 56,500), K. drosophilarum (ATCC 36,906), K. thermotolerans, and K. marxianus; Yarrowia (EP 402,226); Pichia pastoris (ATCC 16,045); Pastoris;Candida;Trichodermareesia;Neurospora crassa;Schwanniomyces, such as Schwanniomyces occidentalis;And filamentous fungi, such as Neurospora, Penicillium, Tolypocladium;And Aspergillus hosts, such as A. nidulans and A. niger. Methylotrophic yeasts are suitable herein and include, but are not limited to, yeasts capable of growing on methanol, selected from Hansenula, Candida, Kloeckera, Pichia, Saccharomyces, Torulopsis, and Rhodotorula. Saccharomyces is a preferred yeast host, which has a suitable vector with the control sequences (e.g., promoters), replication origins, termination sequences, etc. required for expression. Typical promoters include 3-phosphoglycerate kinase and other glycolytic enzymes. Inducible yeast promoters include those from alcohol dehydrogenase, isocytochrome C, and enzymes responsible for maltose and galactose utilization, etc.

[0261] In addition to microorganisms, mammalian tissue cell cultures can also be used to express and produce polypeptides as described herein, and are preferred in some cases (see Winnacker, From Genes to Clones VCH Publishers, NY, NY (1987)). For some embodiments, eukaryotic cells may be preferred because many suitable host cell lines capable of secreting heterologous polypeptides (e.g., complete immunoglobulins) have been developed in the art, and include 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 cell is a CHO cell.

[0262] In some embodiments, the host cell is a vertebrate host cell. Examples of mammalian host cell lines that can be used are: monkey kidney CV1 cell line transformed by SV40 (COS-7, ATCC CRL 1651); human embryonic kidney line (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 cell lines); mouse testicular support cells; monkey kidney cells (CV1ATCC CCL 70); African green monkey kidney cells (VERO-76, ATCC CRL-1587); human cervical cancer cells (HELA, ATCC CCL 2); canine kidney cells (MDCK, ATCC CCL 34); buffalo rat liver cells (BRL 3A, ATCC CRL 1442); human lung cells (W138, ATCC CCL75); human liver cells (Hep G2, HB 8065); mouse mammary tumor (MMT 060562, ATCC CCL51); TRI cells; MRC 5 cells; FS4 cells; and human hepatoma cell line (Hep G2).

[0263] Products

[0264] The polypeptide formulations described herein may be included in an article of manufacture. The article of manufacture may include a container containing a polypeptide (e.g., a preparation of a polypeptide, a spray-dried preparation of a polypeptide, or a polypeptide in a controlled release form). Preferably, the article of manufacture includes: (a) a container containing a composition comprising a polypeptide and / or a polypeptide formulation described herein; and (b) a package insert containing instructions for administering the formulation to a subject.

[0265] The article includes a container and a label or package insert on or associated with the container. Suitable containers include, for example, bottles, vials, syringes, etc. The container can be formed of a variety of materials such as glass or plastic. The container holds or contains the preparation and may have a sterile access port (for example, the container may be an intravenous solution bag or a vial with a stopper that can be pierced by a hypodermic needle). At least one active agent in the composition is a polypeptide. The label or package insert indicates the use of the composition in the subject and is accompanied by specific instructions for the dosage and interval of the polypeptide and any other drugs provided. The article may further include other substances required from a commercial and user perspective, including other buffers, diluents, filters, needles, and syringes. In some embodiments, the container is a syringe. In some embodiments, the syringe is further contained in an injection device. In some embodiments, the injection device is an automatic syringe.

[0266] "Package insert" is used to refer to instructions customarily included in commercial packages of therapeutic products, that contain information about the indications, usage, dosage, administration, contraindications, other therapeutic products for use in combination with the product in the package and / or warnings concerning the use of such therapeutic products.

[0267] Exemplary Embodiments

[0268] The present invention provides the following exemplary embodiments.

[0269] 1. A preparation comprising a polypeptide and hydroxypropylmethylcellulose acetate succinate (HPMCAS).

[0270] 2. The formulation of embodiment 1, wherein the HPMCAS comprises about 8% to about 12% acetate and about 7% to about 15% succinate.

[0271] 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).

[0272] 4. The formulation of any one of embodiments 1 to 3, wherein the ratio of HPMCAS to protein in the formulation is about 4:1 (mg / mg) to about 1:4 (mg / mg), optionally wherein the ratio is about 4:1 (mg / mg), about 1:1 (mg / mg) or about 1:4 (mg / mg).

[0273] 5. The formulation of any one of embodiments 1 to 4, wherein the formulation further comprises a histidine buffer.

[0274] 6. The formulation of embodiment 5, wherein the histidine buffer is histidine HCl buffer.

[0275] 7. The formulation of embodiment 5 or 6, wherein the concentration of the histidine buffer in the formulation is about 5 mM to about 20 mM, optionally wherein the concentration of the histidine buffer in the formulation is about 10 mM.

[0276] 8. The formulation of any one of embodiments 1 to 7, wherein the formulation further comprises polysorbate.

[0277] 9. The formulation of embodiment 8, wherein the polysorbate is polysorbate 20.

[0278] 10. The formulation of embodiment 8 or 9, wherein the concentration of polysorbate in the formulation is about 0.005% (w / v) to about 0.5% (w / v).

[0279] 11. The formulation of any one of embodiments 8 to 10, wherein the concentration of polysorbate in the formulation is about 0.01% (w / v).

[0280] 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.

[0281] 13. The formulation of any one of embodiments 1 to 12, wherein the pH of the formulation is about 6.5.

[0282] 14. The formulation of any one of embodiments 1 to 13, wherein the polypeptide is an antibody.

[0283] 15. The formulation of any one of embodiments 1 to 14, wherein the polypeptide is a monoclonal antibody.

[0284] 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.

[0285] 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.

[0286] 18. The formulation of any one of embodiments 1 to 17, wherein the concentration of the polypeptide in the formulation is about 5 mg / mL to about 100 mg / mL.

[0287] 19. The formulation of any one of embodiments 1 to 18, wherein the concentration of the polypeptide in the formulation is about 10 mg / mL.

[0288] 20. A preparation comprising:

[0289] The antibody at a concentration of about 5 mg / mL to about 50 mg / mL,

[0290] HPMCAS-LF, wherein the ratio of HPMCAS-LF to the antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg),

[0291] Histidine HCl buffer at a concentration of about 5 mM to about 20 mM,

[0292] Polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), and wherein the pH of the formulation is about 5.5 to about 7.0.

[0293] 21. A preparation comprising:

[0294] The antibody concentration is about 10 mg / mL,

[0295] HPMCAS-LF, wherein the ratio of HPMCAS-LF to the antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg),

[0296] Histidine HCl buffer at a concentration of about 10 mM,

[0297] Polysorbate 20 at a concentration of about 0.01% (w / v), and

[0298] The pH of the formulation is about 6.5.

[0299] 22. A preparation comprising:

[0300] The antibody at a concentration of about 5 mg / mL to about 50 mg / mL,

[0301] HPMCAS-HF, wherein the ratio of HPMCAS-HF to the antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg),

[0302] Histidine HCl buffer at a concentration of about 5 mM to about 20 mM,

[0303] Polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), and wherein the pH of the formulation is about 5.5 to about 7.0.

[0304] 23. A preparation comprising:

[0305] The antibody concentration is about 10 mg / mL,

[0306] HPMCAS-HF, wherein the ratio of HPMCAS-HF to the antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg),

[0307] Histidine HCl buffer at a concentration of about 10 mM,

[0308] Polysorbate 20 at a concentration of about 0.01% (w / v), and

[0309] The pH of the formulation is about 6.5.

[0310] 24. The formulation of any one of embodiments 20 to 23, wherein the antibody is a monoclonal antibody.

[0311] 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.

[0312] 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, optionally wherein the antibody fragment is a (Fab')2 fragment.

[0313] 27. The formulation of any one of embodiments 1 to 26, wherein the formulation is spray dried to form a spray-dried formulation of the polypeptide or antibody.

[0314] 28. The spray-dried formulation of the 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 at about 37°C for at least about 4 weeks.

[0315] 29. The 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.

[0316] 30. The spray-dried formulation of the polypeptide or antibody of 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.

[0317] 31. The 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.

[0318] 32. The spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 31, wherein the spray-dried formulation of a polypeptide or antibody is encapsulated in a polymer system that generates an acidic microclimate.

[0319] 33. The spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 32, wherein the spray-dried formulation of a polypeptide or antibody is encapsulated in a lactic acid / glycolic acid polymer system.

[0320] 34. The spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 33, wherein the spray-dried formulation of a polypeptide or antibody is encapsulated in poly(lactic-co-glycolic acid) (PLGA).

[0321] 35. The spray-dried formulation of a polypeptide or antibody according to any one of embodiments 27 to 33, wherein the spray-dried formulation of a polypeptide or antibody is encapsulated in a PLGA rod.

[0322] 36. A composition comprising the formulation of any one of embodiments 1 to 27.

[0323] 37. A composition comprising a spray-dried formulation of the polypeptide or antibody of any one of embodiments 27 to 35.

[0324] 38. A composition comprising lactic acid / glycolic acid polymer particles comprising the formulation of any one of embodiments 1 to 27.

[0325] 39. A composition comprising lactic acid / glycolic acid polymer particles comprising the spray-dried formulation of any one of embodiments 27 to 35.

[0326] 40. The composition of embodiment 38 or 39, wherein the lactic acid / glycolic acid polymer particles are PLGA particles.

[0327] 41. The composition of any one of embodiments 38 to 40, wherein the lactic acid / glycolic acid polymer particles are PLGA rods.

[0328] 42. A method of preparing a spray-dried polypeptide, the method comprising preparing the formulation of any one of embodiments 1 to 19, and subjecting the formulation to spray drying.

[0329] 43. A method of preparing a spray-dried antibody, the method comprising preparing the formulation of any one of embodiments 20 to 26, and subjecting the formulation to spray drying.

[0330] 44. The method of embodiment 42 or 43, wherein spray drying is performed using a spray dryer comprising an inlet and an outlet.

[0331] 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.

[0332] 46. ​​The method of embodiment 44, wherein the outlet has a temperature of about 60°C.

[0333] 47. A method of preparing polypeptide-containing lactic acid / glycolic acid polymer particles, the method comprising encapsulating the formulation of any one of embodiments 1 to 26 in a lactic acid / glycolic acid polymer system.

[0334] 48. A method of preparing lactic acid / glycolic acid polymer particles comprising a polypeptide, the method comprising encapsulating the spray-dried formulation of any one of embodiments 27 to 30 in a lactic acid / glycolic acid polymer system.

[0335] 49. The method of embodiment 47 or 48, wherein the lactic acid / glycolic acid polymer particles are PLGA particles.

[0336] 50. An article of manufacture comprising the formulation of any one of embodiments 1 to 27, the spray-dried formulation of any one of embodiments 28 to 35, or the composition of any one of embodiments 36 to 41.

[0337] 51. An article of manufacture comprising a spray-dried formulation prepared by the method of any one of embodiments 42 to 46.

[0338] 52. An article comprising a polypeptide encapsulated by lactic acid / glycolic acid polymer particles prepared by the method of any one of embodiments 47 to 49.

[0339] All features disclosed in this specification may be combined in any combination. Each feature disclosed in this specification may be replaced by an alternative feature for the same, equivalent or similar purpose. Therefore, unless otherwise expressly stated, each feature disclosed is only an example of a general series of equivalent or similar features.

[0340] Further details of the present invention are illustrated by the following non-limiting examples.The disclosures of all references in this specification are expressly incorporated herein by reference.

[0341] Examples

[0342] Abbreviations: AS-HF (HPMCAS containing about 12% acetate and about 7% succinate); AS-LF (HPMCAS containing about 8% acetate and about 15% succinate); E / P (excipient / protein ratio); HPMC (hydroxypropyl methylcellulose, also known as hypromellose); HPMCAS (hydroxypropyl methylcellulose acetate succinate); IEC (ion exchange chromatography); PLGA (poly(lactic-co-glycolic acid)); and SEC (size exclusion chromatography).

[0343] The following examples are intended only as illustrations of the present invention and therefore should not be considered to limit the present invention in any way.The following examples and detailed description are provided by way of illustration only and not limitation.

[0344] Example 1. Hydroxypropyl methylcellulose derivatives stabilize antibody fragments against pH changes

[0345] The acetate and succinate derivatives of hydroxypropyl methylcellulose (HPMC or hypromellose) were evaluated for their utility in stabilizing a model protein (labeled as Fab2) in the context of PLGA rods. Two different HPMC derivatives were investigated; one containing about 8% acetate and about 15% succinate (referred to as AS-LF) and the other containing about 12% acetate and 7% succinate (referred to as AS-HF).

[0346] The performance of HPMCAS excipients in spray-dried formulations containing Fab2 was compared with trehalose, Metolose, and excipient-free formulations at 90°C for several hours and at 37°C for 4 weeks. The role of trehalose in spray-dried formulations and in stabilizing Fab2 at high temperatures has been previously studied (Rajagopal, K et al., Mol Pharm, 2019.16(1):349-358). A temperature of 90°C was selected to guide the compatibility of spray-dried formulations for high temperature processing (such as hot melt extrusion used in the preparation of PLGA polymer rods for drug delivery). A 37°C stability study was performed to provide a meaningful relative stability between formulations that can be applied to 2°C to 8°C. In addition, 37°C also simulates body temperature and records the effects of physiological temperatures experienced by protein drugs exposed to PLGA rods during long-term residence in a sustained delivery system. Fab2 was encapsulated in a PLGA cylindrical rod as a spray-dried powder by hot melt extrusion. The stability of Fab2 within PLGA rods when formulated in the presence of HPMCAS excipient, Metolose, and Trehalose was then studied under physiological conditions at 37°C for 3 weeks. To characterize the stability of the protein encapsulated in PLGA rods, the aggregation or chemical degradation of Fab2 extracted from the rods was investigated. Although unlike Trehalose, which is hydrolyzable in the human body, HPMCAS is not biodegradable, thus limiting its use in parenteral delivery.

[0347] Materials and Methods

[0348] Materials. Fab2 was obtained from Genentech (South San Francisco, CA). α-α Trehalose dihydrate was obtained from Ferro Pfanstiehl Laboratories (Cleveland, OH); hydroxypropyl methylcellulose acetate succinate (HPMCAS) was obtained from Shin-Etsu Chemical Co, Ltd. (Tokyo, Japan) in two different grades, HPMCAS-HF (12% acetyl, 7% succinyl, dissolution pH ≥ 6.5) and HPMC AS-LF (8% acetyl, 15% succinyl, dissolution pH ≥ 5.5). Hydroxypropyl methylcellulose (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 Burnswick, NJ). PLGA RG755S was purchased from Evonik Corporation (Piscataway, NJ).

[0349] Spray drying of Fab2. 40 mg / mL of Fab2 (MWCO = 10,000 Da) was dialyzed against 10 mM histidine / histidine HCl buffer (pH 5.5) containing 0.01% (w / v) polysorbate 20. The dialysis buffer was changed four times over 16 hours. 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). HPMC AS-LF and HPMC AS-HF grades were initially insoluble in water when prepared at 20 mg / mL and the final pH of the mixture was approximately 4, as specified by the manufacturer. 20mg / mL HPMC AS-LF and HPMC AS-HF working solutions were prepared in water containing 10mg / mL histidine base and 0.01% polysorbate 20, with a final pH of 6.5. The required amount of trehalose dihydrate, HPMC AS-LF, HPMCAS-HF and Metolose were added to Fab2 to obtain a solution with the same excipient / protein mass ratio of 1.0 (see Table 1-1). Then, the aqueous formulation was spray-dried using a Buchi B290 micro-spray dryer (New Castle, NJ) at inlet and outlet temperatures of 110°C ± 2°C and 60°C ± 2°C, respectively. The pump power was 8%, and the aspirator was operated at 100% capacity. The liquid feed rate was 3.4mL / min, and the compressed air flow rate was 600L / h. The collected spray-dried powder was transferred to a clean, dry 15cc Lyo glass vial and stored in a vacuum chamber under nitrogen until further use. All spray-dried powders were further secondary dried in a lyophilizer (Advantage Pro Lyophilizer, SP Scientific, UK).

[0350] Scanning electron microscopy (SEM). The morphology of the spray-dried particles was imaged using a Quanta 3D (Hillsboro, OR) FEGSEM. The samples were mounted on aluminum stubs with a carbon adhesive and sputtered with gold / palladium (Cressington sputter coater, TED Pella, Inc.) to improve their conductivity. SEM images were collected at low voltage to minimize any potential sample damage or surface charging.

[0351] Particle size characterization was performed 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 the spray-dried powder was dispersed in 1 ml of isopropanol, and the dispersion was added dropwise to 50 ml of isopropanol until the target light resistance level was reached. The particle size distribution was calculated using a particle size classification program using the refractive index of isopropanol (1.3776).

[0352] High temperature heat treatment of Fab2. Weigh the spray-dried powder (2mg to 7mg, equivalent to 1mg to 1.5mg Fab2) from each preparation and place it in a 7mL glass vial. The vial lid is opened and placed in a Binder forced air convection oven (Bohemia, NY), preheated and balanced to the desired temperature of 90 ± 5°C. Take out the sample at 1, 2, 3, 4 and 5h, cover it immediately, and cool it to room temperature. The vial contents are dissolved in purified water so that the final protein concentration is about 1mg / mL. Observe the clarity of the reconstituted water sample, and the obviously clarified sample is used for SEC and IEC analysis.

[0353] Size exclusion chromatography (SEC). For Fab2, size exclusion chromatography (SEC) HPLC analysis was performed using an Agilent 1200 series HPLC system (Santa Clara, CA) equipped with a TOSOH TSKgel G2000SWXL (7.8 × 300mm internal diameter, 5 μm particle size) column. Samples were analyzed at 25 ° C in isocratic mode using 0.20M K3PO4 and 0.25M KCl (pH 6.2) as mobile phases (flow rate of 0.7mL / min). 20 μL samples were injected at a concentration of 1 mg / mL, and the total run time was 20min. Detection was performed using the absorbance at 280nm. Percent peak area was calculated by dividing the peak area of ​​each group at each time point by the total peak area.

[0354] Ion exchange chromatography (IEC).Ion exchange chromatography (IEC) HPLC analysis was performed using an Agilent 1200 series HPLC system (Santa Clara, CA) equipped with two Dionex (Sunnyvale, CA) ProPac SAX-10 (2 × 250mm) strong anion exchange columns and a diode array detector (DAD) connected in series. Mobile phase A (solvent A) was 20mM Tris buffer, pH 8.2, and mobile phase B (solvent B) was 250mM sodium chloride dissolved in solvent A. Before analysis, the sample was diluted to about 1mg / mL with solvent A, and 20 μL of sample was injected. Using a linear gradient of 20% solvent A from 100% solvent A at 0min to 20% solvent A at 45min, Fab2 charge variants were separated in a total running time of about 60min. Detection was performed using the absorbance at 280nm. IEC peaks were separated into main peaks, acidic peaks and basic peaks.

[0355] Separation of Fab2 from AS-LF and AS-HF spray-dried formulations. The stability of Fab2 in HPMCAS powder formulations was measured by SEC and IEC by redissolving the powder in water and separating Fab2 from HPMCAS. The redissolved Fab2 spray-dried powder containing AS-LF and AS-HF was titrated with 0.1N HCl to reduce the pH of the solution to pH 3 and precipitate the HPMCAS excipient. The precipitated sample was centrifuged at 4000 g, and the supernatant was diluted in the mobile phase corresponding to the assay for Fab2 analysis purposes.

[0356] Differential Scanning Calorimetry (DSC). DSC analysis was performed under modulated conditions on a TA Instruments DSC Q200 (New Castle DE) calorimeter. Approximately 2 ± 1 mg of sample was weighed and placed in an aluminum pan and hermetically sealed. The sample was equilibrated at 5°C for 10 min, then heated at 2°C / min and modulated to 120°C at ± 1.00°C / min, and cooled back to 5°C at 2°C / min. After the sample was equilibrated again at 5°C, the same heating and modulation slope was repeated a second time until 180°C was reached. The first heating slope was completed to eliminate any thermal history associated with the sample due to storage or handling conditions. For reporting purposes, the second heating slope of the sample was used, which did not contain any thermal history. The glass transition temperature is reported as the half-height midpoint of the second heating cycle ( Figure 2 ).

[0357] Base and acid titration.Base titration experiments were performed on separate solutions of HPMC AS-LF and HPMC AS-HF excipients in water using a Mettler Toledo pH meter (Mettler Toledo, Columbus, OH) with a Micro-Pro-ISM pH probe. Mixtures of HPMC AS-LF and HPMC AS-HF in water were prepared at 5 wt % and 7 wt %, respectively. The initial pH values ​​of the two mixtures were measured to be about 4, and most HPMCAS excipients were insoluble in water. The solution was titrated gradually with 0.5N NaOH, and vortexed continuously until fully miscible. After fully miscible, the pH of HPMC AS-LF and HPMC AS-HF in water was measured to exceed pH 6. Similar acid titration experiments were performed on spray-dried powder samples of HPMCAS, Metolose, trehalose and excipient-free formulations in water, in this case, by titrating to pH 3 with 0.1N HCl.

[0358] Hot melt extrusion (HME) of spray-dried Fab2 and PLGA cylindrical rods. Use HAAKETMMiniCTW micro-conical twin-screw compounder (Thermo Scientific, Karlsruhe, Germany) with barrel capacity of 5g or 7cm3 for hot melt extrusion. The extruder barrel is preheated to 90°C, and the screw speed is set to 40rpm. The front barrel opening of the extruder remains closed in recirculation mode. The pre-weighed solid polymer and spray-dried Fab2 are manually slowly fed into the extruder and recycled back to the barrel for micro-compounding. The Fab2 loading of implants using different spray-dried formulations is fixed to 15% (w / w) Fab2 / PLGA. After complete feeding, melt phase blending is continued for 30min in recirculation mode, and then extruded through a 0.5mm round die. The extrudate is cooled to room temperature, cut into cylinders of desired length and stored at 2 to 8°C until further used for release studies.

[0359] 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 rods and 1 mL of PBSTN (1xPBS, 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 rods were cut into five 1 mg segments and placed in individually labeled glass vials. The exact weight of the rods 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 removed and 1.5 mL was collected. Eppendorf tube (Eppendorf, CT, USA), and replaced with 1mL fresh PBSTN. As previously described, SEC method was used to measure the concentration of Fab2 in the release buffer. After release for 3 weeks, the buffer was thoroughly sucked out from the rod in the vial, and then the rod was rinsed twice with 1mL water, and air-dried for 3 hours to 4 hours in a chemical / biological safety cabinet until there was no visible liquid inside the vial. The rod was then placed in a vacuum desiccator for at least one week to dry out all moisture completely.

[0360] Recovery of Fab2 from PLGA rod-shaped object. After the rod-shaped object is dried, the same rod-shaped object is used to analyze the remaining Fab2 in the rod-shaped object. Utilize the extraction scheme to remove Fab2 from the polymer and separate it. First, the dry rod-shaped object is transferred to a conical bottom glass bottle (Worldwide Glass Resource, 4mL), and then the rod-shaped object is dissolved by adding 4mL tetrahydrofuran (THF) and stirring the bottle until the rod-shaped object is visibly dissolved. PLGA is soluble in THF, and protein is insoluble in THF. The bottle containing the dissolved PLGA rod-shaped object is centrifuged to form a Fab2 precipitation at the bottom of the bottle. Protein precipitation is easily disturbed, so about 3.5mL THF is removed from the supernatant. Then the bottle is rinsed once more with THF, and centrifuged. The Fab2 precipitation at the bottom of the tube is rinsed twice with THF and centrifuged to repeat the process again. The purpose of this operation is to remove any remaining polymer residues after the first rinse with THF. Therefore, a precipitation rich in Fab2 in the form of wet powder is left. Evaporate the remaining THF, leaving dry Fab2 powder. The final dried Fab2 powder was reconstituted with 1 ml PBS buffer and used for analytical testing.

[0361] result

[0362] Spray-dried formulations of Fab2. Five Fab2 formulations at a concentration of 10 mg / mL were prepared and the stability after spray drying was evaluated (Table 1-1). All five formulations were formulated in 10 mM His-HCl / His buffer and 0.01% w / v polysorbate 20. One of the formulations had only 10 mM His-HCl / His buffer and 0.01% w / v polysorbate 20, without any sugar or HPMCAS, and was used as a control. Two formulations containing HPMCAS excipients 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 schematically shown 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 molar ratio of excipient to protein varied due to the molecular weight of the excipients and ranged from 0.3 to 137, depending on the formulation. As shown in Table 1-1, the pH of all formulations was measured before liquid spray drying and after spray drying by reconstitution in water. The two pH measurements were identical, indicating that no change in ionization state occurred due to spray drying.

[0363] Properties of spray-dried Fab2. All five formulations after spray drying yielded micron-sized particles ( Figure 4A ), the particles have similar particle morphology, ranging from spherical to collapsed spherical ( Figure 4B ). In different excipient formulations, the diameter of the particles ranged from 3 microns to 10 microns. All spray-dried powders were redissolved 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 scheme highlighted in the methods section was followed before analyzing Fab2. After spray drying, the monomer content obtained by SEC in all formulations was similar to the main charge variant peak obtained by IEC and was comparable to Fab2 before spray drying (Table 1-1).

[0364] Table 1-1. Details of the spray-dried Fab2 formulations evaluated for stability studies

[0365]

[0366] 1 The pH after spray drying was measured after dissolving 1% solids in water.

[0367] Effect of HPMCAS on 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 both temperatures, the greatest level of aggregation was observed in the case of the formulation without excipients, followed by the formulation containing Metolose. In the case of the formulation without excipients, the maximum monomer loss after 5 hours at 90°C was 6.2% ( Figure 5A ), and the maximum monomer loss after four weeks at 37°C was 1.5% ( Figure 5B ). The second was the Metolose formulation, which had a monomer loss of 5% at 90°C and a monomer loss of 0.9% at 37°C. In formulations containing HPMCAS excipients and trehalose, monomer loss was comparable and significantly reduced regardless of the nature of the excipients. After five hours of incubation at 90°C, trehalose, AS-LF, and AS-HF formulations showed the least monomer loss (0.7% to 0.9%). After four weeks of incubation at 37°C, trehalose, AS-LF, and AS-HF formulations again showed the least monomer loss (≤0.2%). Shorter incubation periods at 90°C caused more aggregation compared to longer incubation periods at 37°C. Comparing the excipient-dependent physical aggregation of Fab2 after storage at the two temperatures, the polymer excipients AS-LF and AS-HF provided the same anti-aggregation protection as trehalose.

[0368] The IEC method relies on anion exchange columns to separate different Fab2 related charge variant substances into basic or acidic variants. Basic peaks are observed on the left side of the Fab2 main peak, and acidic charge variants are displayed on the right side of the Fab2 main peak. The detailed characterization and types of peaks on IEC and the chemical degradation mechanism of Fab2 have been previously reported (Rajagopal, K et al., Mol Pharm, 2019.16 (1): 349-358). Studies have shown that the peak immediately adjacent to the left side of the Fab2 main peak is formed by the pyroglutamic acid peak formed by the intramolecular cyclization of N-terminal glutamic acid in Fab2. The peaks at about 19 minutes and 21 minutes correspond to the succinimide intermediates of two and one aspartic acid residues in the Fab2 sequence, respectively. The formation of such pyroglutamic acid and succinimide intermediates leads to the removal of net negative charge, and produces basic peaks on anionic IECs. However, the formation of deamidated substances by asparagine side chains leads to an increase in net negative charge and Fab2 isoelectric point (pI), thereby causing acidic variant peaks on anionic IECs. No major Fab2 deamidation peak was observed during the assay.

[0369] In the case of Fab2, different pathways of chemical degradation were observed after exposure to 90°C and 37°C ( Fig. 6A and 6BThe succinimide product formed by cyclization of one of the aspartic acid side chains in Fab2 and the pyroglutamate product (PyroE) formed by cyclization of the N-terminal glutamate gave basic peaks at about 21 minutes and 24.2 minutes, respectively, on the IEC. For all formulations, increases in both degradation reactions were observed after exposure of Fab2 to 90°C ( Fig. 6A At 37°C, all preparations showed a new peak at about 19 minutes after stress ( Figure 6B ), and the peak disappeared at about 21 minutes, indicating that a new succinimide product was formed from the second day aspartic acid after exposure to 37°C.

[0370] The presence of excipients in the formulation did not affect succinimide formation at both 90°C and 37°C. However, the nature of the excipients showed an effect on the magnitude of pyroglutamate formation. The IEC chromatograms have been normalized to the main peak. Pyroglutamate formation was quantified by measuring the ratio of the main peak to the pyroglutamate peak area ( Figure 6C and 6D ). Compared with the formulation without excipients, the presence of HPMCAS excipient had no effect on the formation of pyroglutamate in Fab2. In contrast, trehalose inhibited the formation of the pyroglutamate peak. This is consistent with our previous results that trehalose inhibited the formation of the pyroglutamate peak 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 under both 90°C and 37°C stress conditions.

[0371] Buffering capacity of HPMCAS excipients. Alkaline titration experiments with HPMCAS raw materials revealed the resistance of both excipients to pH changes after the addition of sodium hydroxide ( Fig. 7A This type of base titration simulates the aqueous environment formed by HPMCAS chains during the preparation of the HPMCAS stock solution for spray drying ( Figure 3 ). AS-LF showed resistance to pH changes of about pH 5.2 upon subsequent base addition, and AS-HF showed resistance to pH changes of about pH 5.8, further confirming the fact that both excipients provide a buffering capacity range as specified by the manufacturers. During the base titration experiments, AS-LF was more resistant to pH changes within its observed buffering range of about pH 5.2, i.e., almost twice as resistant as AS-HF, as determined by the number of millimoles of hydroxyl groups introduced per unit mass of each excipient ( Fig. 7A To investigate the neutralizing effect of the HPMCAS excipient within the acidic microclimate of the PLGA device, the pH change was monitored during acid titration of the HPMCAS spray-dried powder formulation with strong hydrochloric acid ( Figure 7B). The presence of HPMCAS excipient in the spray-dried Fab2 powder resists pH changes caused by acid addition at conditions of about pH 5. In the case of trehalose and excipient-free formulations, the pH dropped significantly (from pH 5.5 to 2.5) with the addition of only 1 mM acid compared to spray-dried protein powders without HPMCAS excipient. Similarly, in the case of Metolose, which is structurally identical to HPMCAS but does not contain acetate and succinate functional groups, no buffering capacity was observed. Importantly, it was interesting to find that the amount of acid required to produce a similar pH change in the spray-dried protein formulation containing HPMCAS was almost 6 times as high (about 6 mM) compared to the formulation without HPMCAS.

[0372] Stability of Fab2 encapsulated in PLGA implants during in vitro release. To investigate the effect of excipients on protein stability in PLGA implanted rods, spray-dried formulations of Fab2 containing trehalose, AS-LF, AS-HF and no excipients were encapsulated in 1 mg PLGA cylindrical rods to study drug release and PLGA implant degradation. After incubation in PBSTN at 37°C for 3 weeks, Fab2 was recovered from PLGA rods loaded with 15% drug and analyzed for aggregate stability ( Figure 8 ). This operation was performed in triplicate for PLGA rods loaded with different Fab2s. In the absence of any excipients, Fab2 aggregation was higher and the monomer content was reduced by 9.5%. The rod-shaped formulation containing trehalose showed a similar loss of monomer content (7%) after 3 weeks. For all HPMCAS excipient formulations, the least amount of Fab2 aggregates was observed, with a 4% (AS-LF) and 3.2% (AS-HF) reduction in monomer content.

[0373] discuss

[0374] Spray drying provides a physically stable amorphous drug form, allowing the spray dried product to be processed into poorly soluble polymer drug delivery systems. Amorphous drugs can be molecularly dispersed in a stable micron form for sustained delivery drug release systems, such as millimeter-scale PLGA cylindrical rods. Thermogravimetric analysis of spray dried powders ( Figure 2) and glass transition temperature (Tg) measurements (Table 1-1) can provide information about identifying the hot melt extrusion processing temperature window for preparing PLGA rods. In addition, spray drying can produce solid drug forms of uniform size and morphology that can be manufactured by reproducible, controllable and scalable processes. Physical characterization of spray-dried powders showed that the properties of the excipients did not affect the overall particle size or morphology of the spray-dried powders, as shown in SEM images (Figure 4). The collapsed shape of the particles has been reported previously in similar drying experiments, which varies with formulation composition and spray drying parameters such as temperature, gas flow rate and feed rate (Rajagopal, K et al., Mol Pharm, 2019.16(1):349-358).

[0375] Controlling the spray drying conditions is very important for maintaining protein stability and further processing during the entire operation. The stability of our spray-dried Fab2 does not seem to be affected by the drying conditions and the nature or absence of the excipients. In addition, since drying is a rapid solvent evaporation and droplet formation method, it is essential to have an appropriate amount of excipients for a homogeneous protein-excipient solid phase matrix with a single Tg value. Because the dispersion properties 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, the powder T g T g Corresponding ( Figure 1 ), and the excipient's T g The larger the T of the overall excipient-protein formulation, the g Therefore, the T g (79°C) is higher than that of AS-HF (67°C) and AS-LF formulations (59°C). Since all spray-dried formulations were processed under similar RH conditions, the effect of moisture should be minimal.

[0376] Previous studies have shown 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, Fab2 has been confirmed to be stable in powder form when exposed to 90°C and 37°C, providing similar anti-aggregation protection as trehalose ( Figure 5A and 5B However, the presence of HPMCAS did not inhibit the formation of pyroglutamic acid in the solid preparation ( Figures 6A to 6D). Since the properties of the excipient will determine the appropriate excipient loading required to protect the antibody (Costantino, HR et al., JPharm Sci, 1998, 87(11): 1412-20), it is necessary to further optimize the excipient loading to investigate the maximum protection provided by HPMCAS. However, HPMCAS as a polymer excipient may be superior to the small molecule excipient trehalose in preferentially protecting antibodies when PLGA rods are stored under high humidity conditions and during drug release because it has a slower overall flowability (Mensink, MA et al., Eur J Pharm Biopharm, 2017, 114: 288-295). Current studies have confirmed that when the mass ratio of excipient to protein is 1.0, HPMCAS excipient provides excellent protection for Fab2 in PLGA rods under release conditions.

[0377] The solid-state stabilization of proteins by good excipients can be explained based on the following factors: its glass transition, water displacement trend and its chemical stability (Colaco, CJ et al., Science, 1995, 268 (5212): 788). In a dry state, the selected excipient should be able to prevent protein denaturation by preferentially excluding water molecules. One such excipient is trehalose, which provides the maximum hydrogen bond formation protection for proteins in a dry state (Allison, SD et al., ArchBiochem Biophys, 1999, 365 (2): 289-98). Therefore, the displacement of water molecules and the direct protein-excipient interaction formed by hydrogen bonds are the benchmarks for keeping proteins in a folded state in solid formulations. In our previous study, it was determined that when the excipient to protein mass ratio was 1.0, the trehalose saturation concentration provided maximum protection for Fab2 (Rajagopal, K et al., Mol Pharm, 2019.16 (1): 349-358). At the current excipient to protein mass ratio of 1.0, the molar number of HPMCAS excipient is much lower than the molar number of trehalose. It is reported that the hydrogen bonding of polymer sugars to proteins is different from that of disaccharides (Allison, SD et al., Arch Biochem Biophys, 1999, 365 (2): 289-98). Although the strength of the hydrogen bond formation interaction between Fab2 and HPMCAS is beyond the current scope, HPMCAS seems to be similar to trehalose in providing equivalent protection to prevent Fab2 aggregation at 37°C and 90°C ( Figure 5A and 5B Further studies are necessary to determine the concentration dependence of Fab2 stability on HPMCAS.

[0378] The data presented herein support the use of HPMCAS as an amorphous solid dispersion carrier for protein drugs at high temperatures. At 90°C, the physical stability of the HPMCAS formulation was comparable to that of trehalose, which may be a function of the type and strength of hydrogen bonds formed with the protein in the glassy matrix. The hydroxyl groups of HPMCAS can form hydrogen bonds with proteins and improve the stability of the spray-dried solids. This should limit any molecular mobility, phase separation ( Fig. 9 ). Al-Obaidi et al. have reported that HPMCAS, as a strong hydrogen bond donor, improves the amorphous phase stability of hydrophobic drugs via hydrogen bond forming interactions with the drug carbonyl groups (Al-Obaidi, H. and G. Buckton, AAPS PharmSciTech, 2009, 10(4): 1172-7). In addition, the presence of the succinate moiety promotes additional intermolecular hydrogen bond forming interactions between HPMCAS and proteins. The free carboxylic acid group in the succinate group can simultaneously serve as a hydrogen bond donor and acceptor for the amide group of the protein. At the same time, Metolose, which has methoxy and hydroxypropoxy groups similar to HPMCAS but does not contain additional succinate groups, will lack hydrogen bond donor and acceptor sites for proteins. Therefore, in the case of Metolose formulations, the protein excipient interaction will be weaker. As evidenced by the Tg (150°C) value of the Metolose spray-dried formulation, it may exist as a separate protein and excipient phase in the dry state ( Fig. 9 ), driven by protein-polymer demixing. Therefore, spray-dried Metolose has the same Tg value as pure Metolose ( Figure 1 and Fig.10 ), which is not affected by any protein interaction. In contrast, the spray-dried molecular dispersion of protein and HPMCAS produced a single amorphous phase with a glass transition that was different from that of pure HPMCAS (Table 1-1 and Fig.10 ).

[0379] Interestingly, trehalose, but not HPMCAS, was able to prevent chemical degradation of solid-state Fab2 ( Figure 6C and 6D All spray-dried formulations had increased basic variants after stress at 90°C, with the least amount of basic variants formed in the formulation containing trehalose ( Fig. 6A and 6B). This result has been previously reported in the case of Fab2, as it is prone to heat-dependent dehydration / isomerization, resulting in reduced chemical stability (Rajagopal, K et al., Mol Pharm, 2019. 16(1): 349-358). It is noteworthy that more succinimide and pyroglutamate were formed in the HPMCAS formulation compared to the formulation containing trehalose. Pyroglutamate is the product of the cyclization of N-terminal glutamate on proteins. As hypothesized in our previous study, the hydrogen bond formation interaction between the donor hydroxyl group of trehalose and the acceptor N-terminal amino group of Fab2 is able to inhibit pyroglutamate formation (Rajagopal, K et al., Mol Pharm, 2019. 16(1): 349-358). Unexpectedly, similar amine-hydroxyl interactions do not appear to exist in the case of HPMCAS, resulting in an increase in pyroglutamate species during the duration of stress at 90°C and 37°C. Additionally, the glass transition temperatures of the AS-LF and AS-HF spray-dried formulations containing protein were slightly lower than that of the trehalose spray-dried protein. Thus, the mobility in the HPMCAS formulation may be slightly higher, limiting the hydrogen bonding interactions of the OH groups with the N-terminal amine Fab groups. Without being bound by theory, there may be a large excess of HPMCAS excipient (at a 1:1 w / w excipient to Fab loading ratio) over trehalose, so in the case of the spray-dried HPMCAS powder, the excess excipient that is not bound to the protein may lead to instability and may result in a lower glass transition temperature.

[0380] Current data indicate that HPMCAS is able to resist pH changes of around pH 5 when added to aqueous media ( Fig. 7A ). In the neutral state (when pH = 7), it is soluble due to the deprotonated state of its succinate group (pKa of about 5), forming a stable polymer colloid. Since the succinate anion is formed at neutral to alkaline pH, the net negative charge on HPMCAS prevents the polymer chains from interacting with each other. As the HPMCAS concentration increases, the polymer chains begin to aggregate after reaching the maximum capacity for colloid formation. As the succinate groups transition to a state dominated by non-ionized protonation, the pH of the system gradually decreases (pH ≤ 4), and eventually precipitates out. This pH-driven deprotonation and protonation phenomenon of the HPMCAS chains is reversible. In addition, when the pH is around the pKa of the succinate group, the HPMCAS / water mixture acts as a buffer, absorbing any excess protons introduced into the aqueous mixture. This buffering capacity was demonstrated in titration studies using pure raw materials and HPMCAS spray-dried protein powder ( Fig. 7A and 7B). Using this unique physicochemical property of HPMCAS, stable drug-polymer colloids (when ionized) and spray-dried drug dispersions (when not ionized) have been formulated (Friesen, DT et al., Mol Pharm, 2008, 5(6): 1003-19). During spray drying with the addition of a pH 5.5 stock solution of Fab2, the HPMCAS / water mixture (pH about 6.5) showed no pH change. This suggests that the hydrochloric acid protons from the Fab2 solution are neutralized by the succinate anions present in the excipient, thereby acting as a buffer.

[0381] Many publications have pointed out that HPMCAS is able to maintain the supersaturation of the drug in the solution state due to the advantage of the polymer as a precipitation inhibitor, forming a stable drug / polymer colloidal solution. Friesen et al. have reported that spray-dried drug dispersions made of HPMCAS appear in a colloidal state after dissolution in water, which dissolves after 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 and is very important for maintaining the solubility of the drug under physiological conditions. The present invention includes forming a similar colloidal solution of HPMCAS and Fab2 after exposing HPMCAS spray-dried Fab2 to water and during the release of the drug from the PLGA rod ( Figure 3 ). The pH of the rapidly formed colloid should be the same as the pH of the stock solution before spray drying, which is itself in a high-energy colloidal state due to the mixing of the HPMCAS stock solution (pH 6.5) with the Fab2 stock solution (pH 5.5). This should be higher than the acid dissociation constant of the succinate group, resulting in ionization of the HPMCAS chains. Such a system consisting of ionized HPMCAS chains with Fab2 acts as an electrostatic assembly, should be able to neutralize added protons, and act as a buffer against pH changes. To demonstrate such properties of HPMCAS, the examples of the present invention show stable protein released when exposed to acidic protons from the hydrolysis of PLGA rod implants.

[0382] When preparing polymer rods loaded with proteins, an appropriate combination of excipients is necessary to withstand the high temperature processing conditions of hot melt extrusion and the acidic degradation products produced by 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 effectively maintain solid-state stability and resistance to pH changes also promote the stability of Fab2 during in vitro release. Importantly, the current results show that the HPMCAS excipient provides better protection against aggregation of Fab2 within PLGA implant rods than formulations without any excipients or containing trehalose. Similar results showing the role of inorganic alkaline additives in protecting proteins delivered by 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 the non-covalent protein aggregation caused by the acidic microclimate present in the PLGA hydrolysate.

[0383] In other words, polymer excipients with chemical components that can neutralize acidic degradation products in PLGA systems (such as acetate and succinate) help stabilize drug release. The possible interaction between acidic hydrolysis products and Fab2 seemed to weaken within 3 weeks, but the impact of long-term storage in the rod environment in the presence of HPMCAS needs to be studied.

[0384] Example 2. Hydroxypropyl methylcellulose derivatives stabilize solid monoclonal antibodies against pH changes in aqueous phase

[0385] The acetate and succinate derivatives of hydroxypropyl methylcellulose (HPMC or hypromellose) were evaluated for their effectiveness in stabilizing three full-length monoclonal antibodies (MAbs) in the context of PLGA rods. Two different HPMC derivatives were investigated; one containing about 8% acetate and about 15% succinate (referred to as AS-LF) and the other containing about 12% acetate and 7% succinate (referred to as AS-HF). The materials and methods used 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.

[0386] Materials and Methods

[0387] Materials. MAb1, MAb2, and MAb3 were obtained from Genentech (South San Francisco, CA). MAb1 is a recombinant humanized IgG1κ antibody that reacts with HER2. MAb2 is a recombinant humanized IgG1κ antibody that reacts with VEGF-A. MAb3 is a recombinant humanized IgG1κ antibody that reacts with IgE.

[0388] Size Exclusion Chromatography (SEC). For MAb1, MAb2 and MAb3, size exclusion chromatography (SEC) HPLC analysis was performed using an Agilent 1200 series HPLC system (Santa Clara, CA) equipped with a TOSOH TSKgel G3000SWXL (7.8×300mm inner diameter, 5μm particle size) column. Samples were analyzed at 25°C in isocratic mode using 0.20M K3PO4 and 0.25M KCl (pH6.2) as mobile phases (flow rate of 0.5mL / min). 20μL samples were injected at a concentration of 1mg / mL, and the total run time was 30min. Detection was performed using the absorbance at 280nm. Percent peak area was calculated by dividing the peak area of ​​each group at each time point by the total peak area.

[0389] Ion exchange chromatography (IEC). For MAb1, an Agilent 1200 series HPLC system (Santa Clara, CA) equipped with a ThermoScientific / Dionex (Santa Clara, CA) ProPac WCX-10 analytical (4×250 mm) weak cation exchange column and a diode array detector (DAD) was used for ion exchange chromatography (IEC) HPLC analysis. 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. Before analysis, the sample was diluted to about 1 mg / mL with solvent A, and 20 μL of sample was injected at a flow rate of 0.5 mL / min. A linear gradient from 85% solvent A at 0 min to 0% solvent A at 45 min was used to separate MAb1 charge variants in a total running time of about 60 min. Detection was performed using the absorbance at 214 nm. The IEC peak was separated into a main peak, an acidic peak, and a basic peak.

[0390] For MAb2, an Agilent 1200 series HPLC system (Santa Clara, CA) equipped with a ThermoScientific / Dionex (Santa Clara, CA) ProPac WCX-10 analytical (4×250mm) weak cation exchange column and a diode array detector (DAD) was used to perform ion exchange chromatography (IEC) HPLC analysis. Mobile phase A (solvent A) was 20mMACES buffer, pH 6.5, and mobile phase B (solvent B) was 200mM sodium chloride dissolved in solvent A. Before analysis, the sample was diluted to about 1mg / mL with solvent A, and 20 μL of sample was injected at a flow rate of 0.5mL / min. A linear gradient of 70% solvent A from 0min to 0% solvent A at 50min was used to separate MAb2 charge variants in a total of about 90 minutes of running time. Detection was performed using the absorbance at 280nm. IEC peaks were separated into main peaks, acidic peaks, and alkaline peaks.

[0391] For MAb3, an Agilent 1200 series HPLC system (Santa Clara, CA) equipped with a ThermoScientific / Dionex (Santa Clara, CA) ProPac WCX-10 analytical (4×250 mm) weak cation exchange column and a diode array detector (DAD) was used for ion exchange chromatography (IEC) HPLC analysis. 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, the sample was diluted to about 1 mg / mL with solvent A, and 20 μL of sample was injected at a flow rate of 0.8 mL / min. A linear gradient from 50% solvent A at 0 min to 0% solvent A at 60 min was used to separate MAb3 charge variants in a total run time of about 90 minutes. Detection was performed using the absorbance at 280 nm. The IEC peak was separated into a main peak, an acidic peak, and a basic peak.

[0392] The performance of HPMCAS excipients in spray-dried formulations containing three full-length MAbs was compared with trehalose, metolose, and excipient-free formulations at 90°C for several hours and at 37°C for several weeks. The temperature of 90°C was selected to guide the compatibility of spray-dried formulations for high temperature processing (such as hot melt extrusion used in the preparation of PLGA polymer rods for drug delivery). The stability at 37°C was evaluated to provide a meaningful relative stability between formulations suitable for storage at 2°C to 8°C. In addition, 37°C also simulates body temperature and records the effects of physiological temperatures experienced by protein drugs during long-term residence in sustained delivery systems containing PLGA rods. Two of the three MAbs (MAb1 and MAb2) were encapsulated in PLGA cylindrical rods by hot melt extrusion as spray-dried powders with an E / P mass ratio of 1 / 1. The stability of the MAbs within PLGA rods when formulated in the presence of HPMCAS excipients and trehalose was subsequently studied under physiological conditions at 37°C for several weeks. To characterize the stability of proteins encapsulated in PLGA rods, MAbs extracted from the rods were investigated for aggregation or chemical degradation. Although unlike trehalose, which is hydrolyzable in the human body, HPMCAS is not biodegradable, thus limiting its application in parenteral delivery.

[0393] result

[0394] Spray-dried formulations of MAbs. Thirteen formulations containing either 5 mg / mL or 10 mg / mL of each mAb 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 of the formulations had only 10 mM His-HCl / His buffer and 0.01% w / v polysorbate 20 and did not contain any sugar or HPMCAS (control formulation). Formulations containing HPMCAS excipient were formulated in 10 mM His-HCl / His buffer at pH 6.0±0.5 and 0.01% w / v polysorbate 20. Spray-dried AS-LF and AS-HF formulations containing MAbs were prepared as for Fab2. Figure 3Schematically shown. The mass ratio of trehalose, metolose, AS-LF and AS-HF excipients to protein in each formulation is 1: 1, 1: 4 and 4: 1 (w / w). However, the molar ratio of excipient to protein varies due to the molecular weight of the excipient and ranges from 0.3 to 1752.9, depending on the formulation. As shown in Table 2-1 for MAb1 and Table 2-2 for MAb2, the pH of all formulations was measured before liquid spray drying and after spray drying by redissolving in water. The pH measurements were similar, indicating that there was almost no change in the ionization state caused by spray drying.

[0395] Characteristics of spray-dried MAbs. All 13 formulations after spray drying obtained micron-sized particles with similar particle morphology ranging from spherical to collapsed spherical. In different excipient formulations, the diameter range of particles containing MAb1 was 3.8 microns to 10.0 microns. In different excipient formulations, the diameter range of particles containing MAb2 was 4.4 microns to 11.2 microns. All spray-dried powders were redissolved in water, and aggregation was analyzed using size exclusion chromatography (SEC), and chemical degradation was analyzed using ion exchange chromatography (IEC). For formulations comprising AS-LF and AS-HF, separation protocols were followed before analyzing MAbs. The SEC monomer content of all formulations after spray drying was similar, and comparable to the MAb formulations before spray drying (Table 2-1 and Table 2-2).

[0396] 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 Fig.11 , Fig.13 and Fig.19 ) and tested at 37°C for several weeks ( Fig.12 , Fig.14 and Fig. 20 The greatest level of aggregation was observed with the MAb formulation without excipients (control), followed by the MAb formulation containing Metolose when stored at 90°C, and MAb3 stored at 37°C.

[0397] Table 2-1. Details of the spray-dried MAb1 formulations evaluated for stability studies

[0398]

[0399]

[0400] 1 The E / P=1 / 1, E / P=1 / 4, and E / P=4 / 1 conditions were analyzed using one sample of the MAb1 formulation containing no excipients (ie, the control).

[0401] 2 The pH after spray drying was measured after dissolving 1% solids in water.

[0402] Table 2-2. Details of the spray-dried MAb2 formulations evaluated for stability studies

[0403]

[0404]

[0405] 1 One sample of MAb2 formulation containing no excipients (ie, control) was used in SEC and IEC analysis for E / P=1 / 1 and E / P=1 / 4 conditions, and a separate sample was used in SEC and IEC analysis for E / P=4 / 1 condition.

[0406] 2 The pH after spray drying was measured after dissolving 1% solids in water.

[0407] When stored at 90° C., AS excipient and trehalose provided similar levels of aggregation protection for MAb1, with the highest level of protection observed in the E / P = 4 formulation. In contrast, Metolose, which does not contain acetate and succinate groups, did not protect MAb1 from aggregation.

[0408] Furthermore, when stored at 90°C, AS excipient and trehalose provided similar levels of aggregation protection for MAb2, with the highest level of protection observed in the E / P = 4 formulation. In contrast, Metolose, which does not contain acetate and succinate groups, provided less aggregation protection for MAb2.

[0409] Furthermore, when stored at 90° C., AS excipient and trehalose provided similar levels of aggregation protection for MAb3, with the highest level of protection observed in the E / P=1 / 4 formulation. In contrast, Metolose, which does not contain acetate and succinate groups, provided little protection for MAb3 from aggregation.

[0410] Buffering capacity of HPMCAS excipients. To investigate the neutralization effect of the HPMCAS excipients within the acidic microclimate of the PLGA device, the pH changes were monitored during acid titration of the HPMCAS spray-dried powder formulation with strong hydrochloric acid ( Fig.15 and Fig.16). The presence of HPMCAS excipient in the spray-dried MAb powder resists pH changes caused by acid addition at about pH 5 to pH 6. In the case of trehalose and a control formulation lacking an excipient, the pH dropped significantly (from pH 5.5 to 2.5) upon addition of just 1 mM acid compared to spray-dried protein powders without the HPMCAS excipient. Similarly, no buffering capacity was observed in the case of Metolose, which is structurally identical to HPMCAS but does not contain acetate and succinate functional groups. Importantly, it was interesting to find that the equivalent amount of acid required to produce a similar pH change in a spray-dried protein formulation containing HPMCAS was much greater than that required in a formulation without HPMCAS in a formulation with a high E / P ratio.

[0411] Stability of MAbs encapsulated in PLGA implants during in vitro release. To investigate the effect of excipients on protein stability within PLGA implants, spray-dried formulations of MAbs containing trehalose, AS-LF, AS-HF, and no excipients were encapsulated in 1 mg PLGA cylindrical rods for studying drug release and PLGA implant degradation. The monomer stability of MAbs recovered from drug-loaded PLGA rods stored in PBSTN at 37°C for several weeks was analyzed ( Fig.17 and Fig.18 ). PLGA rods containing trehalose had significantly reduced monomer content after 4 weeks. Much fewer aggregates of MAb1 and MAb2 were observed for both HPMCAS excipient formulations.

Claims

1. A preparation comprising a polypeptide and hydroxypropylmethylcellulose acetate succinate (HPMCAS).

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

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

5. The formulation according to any one of claims 1 to 4, wherein the formulation further comprises a histidine buffer.

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

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

8. The formulation according to any one of claims 1 to 7, wherein the formulation further comprises polysorbate.

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

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

11. The formulation of any one of claims 8 to 10, wherein the concentration of the polysorbate in the formulation is 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 according to any one of claims 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 claims 1 to 17, wherein the concentration of the polypeptide in the formulation is about 5 mg / mL to about 100 mg / mL.

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

20. A preparation comprising: The antibody at a concentration of about 5 mg / mL to about 50 mg / mL, HPMCAS-LF, wherein the ratio of the HPMCAS-LF to the 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 5 mM to about 20 mM, Polysorbate 20 at a concentration of about 0.005% (w / v) to about 0.5% (w / v), and wherein the pH of the formulation is from about 5.5 to about 7.

0.

21. A preparation comprising: The antibody concentration is about 10 mg / mL, HPMCAS-LF, wherein the ratio of the HPMCAS-LF to the 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), and wherein the pH of the formulation is about 6.

5.

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

0.

23. A preparation comprising: The antibody concentration is about 10 mg / mL, HPMCAS-HF, wherein the ratio of the HPMCAS-HF to the antibody in the formulation is about 4:1 (mg / mg), about 1:1 (mg / mg), or about 1:4 (mg / mg), and the concentration is about 10 mM histidine HCl buffer, Polysorbate 20 at a concentration of about 0.01% (w / v), and 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 Fab, Fab'-SH, Fv, scFv and (Fab')2 fragments, 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 a polypeptide or antibody.

28. The 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 at about 37°C for at least about 4 weeks.

29. The 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. The 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. The 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. The spray-dried formulation of a polypeptide or antibody according to any one of claims 27 to 31, wherein the spray-dried formulation of a polypeptide or antibody is encapsulated in a polymer system that generates an acidic microclimate.

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

49. The method of claim 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 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 comprising a spray-dried formulation prepared by the method of any one of claims 42 to 46.

52. An article comprising polypeptide-encapsulated lactic acid / glycolic acid polymer particles prepared by the method of any one of claims 47 to 49.

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