Highly concentrated protein formulations containing polysorbate excipients and methods for producing same

Abstract

The present disclosure relates to formulations comprising proteins (e.g., antibodies) that include an Fc region and surfactants such as polysorbates (e.g., polysorbate 20), as well as methods of purifying such proteins that improve the stability of surfactants such as polysorbates in such formulations.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 489,132, filed March 8, 2023, which is incorporated herein by reference in its entirety.

[0002] Incorporation by reference of sequence listing The Sequence Listing XML associated with this application has been provided electronically in XML file format and is incorporated herein by reference. The filename of the XML file containing the Sequence Listing XML is "MUNO-012_00US_SeqList_ST26". The XML file is 44,393 bytes, was created on February 28, 2023, and is being submitted electronically hereby. [Technical Field]

[0003] The present disclosure relates to formulations comprising a protein having an Fc region and one or more excipients including a surfactant such as polysorbate, and methods of purifying a protein having an Fc region that improve the stability of such surfactants in such formulations. [Background technology]

[0004] Surfactants (e.g., polysorbate 20, polysorbate 80) are typically added to drug substance or drug-stage liquid protein formulations, especially those with high protein concentrations (>50 g / L or >100 g / L), to stabilize the product under various thermal, mechanical, and interfacial shear conditions (see Khan et al., Eur. J. of Pharm. and Biopharmaceutics 97:60, 2015; Wang et al., Antibody Therapeutics 4:262–273, 2021). This stabilizing effect of surfactants protects antibody or protein formulations from thermal, mechanical, and interfacial shear stresses as the material undergoes drug product fabrication (fill / finish), packaging, storage, handling, and distribution. Degradation of surfactants results in the loss of this protective effect against stress conditions, potentially resulting in undesirable consequences such as particle formation, impact on purity, and other quality attributes of the drug substance or drug product (see Liu, Lu, et al., J Pharm Sci 102:2460, 2013). Therefore, the stability of surfactants (e.g., polysorbate 20 (PS20) or polysorbate 80 (PS80)) needs to be improved at the drug substance stage to protect molecules from freeze-thaw cycles and the shear stresses of drug product filling, packaging, shipping, and storage. Summary of the Invention

[0005] In one aspect, provided herein is a formulation comprising (i) a protein having an Fc region at a concentration greater than 50 g / L and (ii) one or more excipients including a polysorbate, wherein the concentration of the polysorbate remains within 30% of the starting concentration of the polysorbate after storage for at least one day, and / or the presence of polysorbate degradation products does not increase by more than two-fold the starting concentration of the degradation products after storage for at least one day. In some embodiments, a concentration greater than 50 mg / ml is equal to or greater than 75 g / L or 100 g / L. In some embodiments, a concentration greater than 100 mg / ml is equal to or greater than 150 g / L, 170 g / L, or 200 g / L.

[0006] In some embodiments, the protein having an Fc region is an antibody. In some embodiments, the antibody is a humanized or fully human monoclonal antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an anti-FcRn antibody that specifically binds to FcRn. In some embodiments, the anti-FcRn antibody is a fully human monoclonal IgG1 antibody. In some embodiments, the anti-FcRn antibody is (a) (i) the three heavy chain CDR amino acid sequences of SEQ ID NO: 27 (HCDR1), SEQ ID NO: 28 (HCDR2), and SEQ ID NO: 29 (HCDR3); and (ii) the three light chain CDR amino acid sequences of SEQ ID NO: 30 (LCDR1), SEQ ID NO: 31 (LCDR2), and SEQ ID NO: 32 (LCDR3); or (b) (i) three heavy chain CDR amino acid sequences of SEQ ID NO: 49 (HCDR1), SEQ ID NO: 22 (HCDR2), SEQ ID NO: 23 (HCDR3); and (ii) three light chain CDR amino acid sequences of SEQ ID NO: 50 (LCDR1), SEQ ID NO: 25 (LCDR2), SEQ ID NO: 26 (LCDR3). In some embodiments, the anti-FcRn antibody comprises (a) a heavy chain variable region amino acid sequence of SEQ ID NO: 6 and a light chain variable region amino acid sequence of SEQ ID NO: 16; or (b) a heavy chain amino acid sequence of SEQ ID NO: 51; and a light chain amino acid sequence of SEQ ID NO: 52.

[0007] In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the polysorbate is at a concentration of about 0.002% to 0.2%. In some embodiments, the polysorbate is at a concentration of 0.01 to 0.05%. In some embodiments, the pharmaceutical formulation comprises at least or about 170 g / L of an anti-FcRn antibody, 0.02% polysorbate 20, 100 mM L-histidine / histidine HCl, and 100 mM L-arginine HCl in water at pH 6.0.

[0008] In some embodiments, the concentration of polysorbate remains within 20% of the starting concentration of polysorbate after storage. In some embodiments, the concentration of polysorbate remains within 10% of the starting concentration of polysorbate after storage. In some embodiments, the concentration of polysorbate remains within 5% of the starting concentration of polysorbate after storage. In some embodiments, the polysorbate is polysorbate 20 and the degradation product is a non-esterified species of polysorbate 20. In some embodiments, the non-esterified species of polysorbate 20 is sorbitan or isosorbide. In some embodiments, the polysorbate is polysorbate 20 and the polysorbate 20 degradation product is lauric acid. In some embodiments, the polysorbate is polysorbate 80 and the polysorbate 80 degradation product is oleic acid.

[0009] In some embodiments, the presence of polysorbate degradation products does not increase by more than 50% of the starting concentration of degradation products after storage. In some embodiments, the presence of polysorbate degradation products does not increase by more than 25% of the starting concentration of degradation products after storage. In some embodiments, the presence of polysorbate degradation products does not increase by more than 10% of the starting concentration of degradation products after storage. In some embodiments, the concentration of polyester species of polysorbate remains within 10% or 20% of the starting concentration of polyester species after storage for at least one day. In some embodiments, the concentration of monoester species of polysorbate remains within 10% or 20% of the starting concentration of monoester species after storage for at least one day. In some embodiments, storage for at least one day is 1, 3, or 7 days of storage. In some embodiments, storage for at least one day is about 14 days or 28 days or more of storage. In some embodiments, storage for at least one day is about 8 weeks or 12 weeks or more of storage. In some embodiments, storage for a period of at least one day is storage for about one year or more. In some embodiments, storage is at 2-8°C. In some embodiments, storage is at 25°C. In some embodiments, storage is at 40°C.

[0010] In some embodiments, the preparation is free or substantially free of cell culture host cell proteins with lipase and / or hydrolase activity. In some embodiments, the preparation is a pharmaceutical preparation, and the one or more excipients are pharmaceutically acceptable. In some embodiments, the protein having an Fc region is purified from the cell culture by Fc-binding affinity resin chromatography. In some embodiments, the Fc-binding affinity resin chromatography is a Protein A chromatography column or a Protein G chromatography column.

[0011] In another aspect, provided herein is a method for purifying a protein having an Fc region from a harvested cell culture, the method comprising: (i) loading the harvested cell culture containing the protein having an Fc region onto an Fc-binding affinity resin chromatography column; (ii) washing the column one, two, three, or more times with one or more wash buffers, at least one of which comprises a pH greater than 9; and (iii) eluting the protein having an Fc region from the column; optionally, the method is for making a high-concentration formulation of the protein with one or more excipients comprising polysorbate. In some embodiments, the method further comprises, after step (iii), formulating the protein of step (iii) into a formulation comprising adding one or more excipients, wherein the concentration of the protein in the formulation is greater than 50 g / L, and the one or more excipients comprise polysorbate.

[0012] In some embodiments, the polysorbate is polysorbate 20. In some embodiments, the polysorbate is polysorbate 80. In some embodiments, the polysorbate in the formulation is at a concentration of about 0.002% to 0.2%. In some embodiments, the polysorbate in the formulation is at a concentration of 0.01 to 0.05%.

[0013] In some embodiments, the Fc-binding affinity resin chromatography column is a Protein A chromatography column. In some embodiments, the Fc-binding affinity resin chromatography column is a Protein G chromatography column. In some embodiments, the Fc-binding affinity resin chromatography column comprises an engineered Fc-binding protein. In some embodiments, the Fc-binding affinity resin chromatography column load ratio in step (i) is 35 g / L or less. In some embodiments, the Fc-binding affinity resin chromatography column load ratio in step (i) is 25 g / L or less.

[0014] In some embodiments, at least one wash buffer comprises a pH of at least 10 or greater. In some embodiments, at least one wash buffer comprises a pH of about 10. In some embodiments, at least one wash buffer comprises a pH of at least 10.5 or greater. In some embodiments, at least one wash buffer comprises a pH of about 10.5. In some embodiments, at least one wash buffer comprises a pH of at least 11 or about 11.

[0015] In some embodiments, at least one wash buffer contains 1 M sodium chloride or a higher concentration of sodium chloride. In some embodiments, at least one wash buffer contains sodium chloride at a concentration less than 1 M or less than 0.5 M. In some embodiments, at least one wash buffer contains arginine. In some embodiments, at least one wash buffer does not contain arginine. In some embodiments, at least one wash buffer contains sodium phosphate or sodium carbonate. In some embodiments, at least one wash buffer contains 50 mM sodium carbonate.

[0016] In some embodiments, the concentration of the protein having an Fc region in the formulation is about 100 g / L or more, hi some embodiments, the concentration of the protein having an Fc region in the formulation is about 150 g / L, about 170 g / L, or about 200 g / L or more.

[0017] In some embodiments, the formulation is a pharmaceutical formulation.

[0018] In some embodiments, the protein having an Fc region is an antibody. In some embodiments, the antibody is a humanized or human monoclonal antibody. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an anti-FcRn antibody that specifically binds to FcRn. In some embodiments, the anti-FcRn antibody is a fully human monoclonal IgG1 antibody. In some embodiments, the anti-FcRn antibody comprises (a) (i) three heavy chain CDR amino acid sequences of SEQ ID NO: 27 (HCDR1), SEQ ID NO: 28 (HCDR2), and SEQ ID NO: 29 (HCDR3); and (ii) three light chain CDR amino acid sequences of SEQ ID NO: 30 (LCDR1), SEQ ID NO: 31 (LCDR2), and SEQ ID NO: 32 (LCDR3); or (b) (i) three heavy chain CDR amino acid sequences of SEQ ID NO: 49 (HCDR1), SEQ ID NO: 22 (HCDR2), and SEQ ID NO: 23 (HCDR3); and (ii) three light chain CDR amino acid sequences of SEQ ID NO: 50 (LCDR1), SEQ ID NO: 25 (LCDR2), and SEQ ID NO: 26 (LCDR3). In some embodiments, the anti-FcRn antibody comprises (a) a heavy chain variable region amino acid sequence of SEQ ID NO: 6 and a light chain variable region amino acid sequence of SEQ ID NO: 16; or (b) a heavy chain amino acid sequence of SEQ ID NO: 51; and a light chain amino acid sequence of SEQ ID NO: 52.

[0019] In some embodiments, the harvested cell culture is cell culture fluid harvested from host cells used to produce a protein having an Fc region, optionally by introducing a vector comprising the protein having an Fc region into the host cells and culturing the host cells under conditions suitable for expression of the protein, optionally wherein the host cells are mammalian cells suitable for production. In some embodiments, the host cells are CHO cells. In some embodiments, the host cells are harvested by centrifugation, optionally followed by filtration. In some embodiments, after step (iii), the eluate comprising the protein having an Fc region is subjected to filtration. In some embodiments, after step (iii), the eluate comprising the protein having an Fc region is subjected to low-pH viral inactivation.

[0020] In some embodiments, the eluate containing the protein having an Fc region is subjected to anion exchange (AEX) chromatography and / or cation exchange (CEX) chromatography. In some embodiments, after step (iii), and optionally after one, two, three, or all of the steps specified in any of the above embodiments, the eluate containing the protein having an Fc region is concentrated. In some embodiments, the protein having an Fc region is an anti-FcRn antibody that specifically binds to FcRn, and the formulation is a pharmaceutical formulation comprising at least or about 170 g / L of the anti-FcRn antibody, 0.02% polysorbate 20, 100 mM L-histidine / histidine HCl, and 100 mM L-arginine HCl in water at pH 6.0.

[0021] In another aspect, provided herein are formulations produced using the methods described herein. In some embodiments, the polysorbate comprises polysorbate 20 (PS20) or polysorbate 80 (PS80), and the concentration of PS20 or PS80 remains within 30% of the starting concentration of PS20 or PS80 after storage for at least one day, and / or the presence of PS20 or PS80 degradation products does not increase by more than two-fold the starting concentration of the degradation products after storage for at least one day. In some embodiments, the concentration of PS20 or PS80 remains within 20% of the starting concentration of PS20 or PS80 after storage. In some embodiments, the concentration of PS20 or PS80 remains within 10% of the starting concentration of PS20 or PS80 after storage. In some embodiments, the concentration of PS20 or PS80 remains within 5% of the starting concentration of PS20 or PS80 after storage. In some embodiments, the PS20 or PS80 degradation products are fatty acid esters. In some embodiments, the PS20 degradation products are non-esterified species of PS20. In some embodiments, the non-esterified species of PS20 are sorbitan or isosorbide. In some embodiments, the PS20 degradation product is lauric acid. In some embodiments, the PS80 degradation product is oleic acid. In some embodiments, the presence of PS20 or PS80 degradation products does not increase by more than 50% of the starting concentration of degradation products after storage. In some embodiments, the presence of PS20 or PS80 degradation products does not increase by more than 25% of the starting concentration of degradation products after storage. In some embodiments, the presence of PS20 or PS80 degradation products does not increase by more than 10% of the starting concentration of degradation products after storage. In some embodiments, the concentration of polyester species of PS20 or PS80 remains within 10% or 20% of the starting concentration of polyester species after storage for at least one day. In some embodiments, the concentration of monoester species of PS20 or PS80 remains within 10% or 20% of the starting concentration of monoester species after storage for at least one day. In some embodiments, storage for a period of at least 1 day is storage for 1, 3, or 7 days.In some embodiments, storage for at least one day is storage for about 14 days or 28 days or more. In some embodiments, storage for at least one day is storage for about 8 weeks or 12 weeks or more. In some embodiments, storage for at least one day is storage for about 1 year or more. In some embodiments, storage is at 2-8°C. In some embodiments, storage is at 25°C. In some embodiments, storage is at 40°C. In some embodiments, the formulation is free or substantially free of cell culture host cell proteins with lipase, esterase and / or hydrolase activity. [Brief explanation of the drawings]

[0022] [Figure 1A] ~ [Figure 1D] 1A-1D show the structures of polysorbate 20, polysorbate 80, lauric acid, and oleic acid, respectively.

[0023] [Figure 2A] FIG. 1 is a flow diagram showing the platform cell culture (upstream) process for proteins with Fc regions (e.g., anti-FcRn antibodies). [Figure 2B] FIG. 1 is a flow diagram showing the platform purification (downstream) process of proteins with Fc regions (e.g., anti-FcRn antibodies).

[0024] [Figure 3]

[0023] Figure 1 shows the stability of PS20 in drug substance using an established manufacturing process. Data was collected by spiking 200 μg / ml of PS-20 into UFDF material.

[0025] [Figure 4] 1 is a graph showing the stability of PS20 in placebo (formulation buffer), with a placebo target concentration of 220 μg / ml.

[0026] [Figure 5]13 is a graph showing the results of an in-process intermediate spiking study of a drug substance: PS-20 degradation kinetics for Protein A pool, VIN pool, AEX pool, CEX pool, and UFDF pool at 40° C.

[0027] [Figure 6] FIG. 1 is a flow diagram of the operating process of a Protein A affinity chromatography unit.

[0028] [Figure 7] 1 is a graph showing PS20 degradation over time when incubated with a Protein A pool (intermediate) under several different wash conditions.

[0029] [Figure 8] 1 is a graph showing residual host cell protein (HCP) levels in Protein A pools by wash condition.

[0030] [Figure 9] 1 is a graph showing experimental design using JMP software, predictive profiler.

[0031] [Figure 10] 1 is a graph showing a chart of the variation in residual HCP concentration for each buffer solution condition.

[0032] [Figure 11] 1 is a graph showing a chart of fluctuations in PS20 concentration on day 14 for each buffer condition.

[0033] [Figure 12] 1 is a graph showing a chart of changes in PS20 concentration on day 30 for each buffer condition.

[0034] [Figure 13] 1 is a graph showing a chart of the variation in SEC-HMW (%) concentration for each buffer condition.

[0035] [Figure 14] Graph showing predicted stability profiles of PS20 on days 7 and 14 with parameters centered.

[0036] [Figure 15] 1 is a graph showing a chart of the variation of high molecular weight (HMW) (by size exclusion chromatography) depending on the pH of the wash 2 buffer.

[0037] [Figure 16] 10 is a graph showing a variation chart of PS20 T14 depending on the pH of the wash 2 buffer and the loading ratio.

[0038] [Figure 17] 1 is a graph showing the stability of bulk drug substance (BDS) at storage (-70°C), accelerated (2-8°C), and stress (25°C) temperatures.

[0039] [Figure 18] 1 is a graph showing a comparison of Protein A eluate stability between old and new wash conditions.

[0040] [Figure 19] 1 is a graph showing a comparison of the stability of the ultrafiltration / diafiltration (UFDF) pool between old and new wash conditions.

[0041] [Figure 20] 1 is a graph showing a comparison of BDS stability between old and new wash conditions.

[0042] [Figure 21] 1 is a graph showing the polyester profile of PS20 in Protein A eluate at 40° C.

[0043] [Figure 22] 1 is a graph showing the monoester profile of PS20 in Protein A eluates at 40° C.

[0044] [Figure 23] 1 is a graph showing the deesterification profile of PS20 in Protein A eluates at 40° C.

[0045] [Figure 24] 1 is a graph showing the polyester profile of PS20 in BDS at 5° C. and 40° C.

[0046] [Figure 25] 1 is a graph showing the monoester profile of PS20 in BDS at 5° C. and 40° C.

[0047] [Figure 26] 1 is a graph showing the deesterification profile of PS20 in BDS at 5° C. and 40° C.

[0048] [Figure 27] 1 is a graph showing the lauric acid profile of PS20 in BDS at 5° C. and 40° C.

[0049] [Figure 28] 1 is a graph showing the non-ester profile by LC / MS for the stability of the intermediate pool at 40° C.

[0050] [Figure 29] FIG. 10 is a graph showing the monoester profile by LC / MS for the stability of the intermediate pool at 40° C.

[0051] [Figure 30] 1 is a graph showing polyester profiles by LC / MS for the stability of the intermediate pool at 40° C.

[0052] [Figure 31] FIG. 1 is a graph showing ester profiles by LC / MS for drug substance stability at 5° C. DETAILED DESCRIPTION OF THE INVENTION

[0053] Detailed Description Provided herein are formulations comprising a protein having an Fc region and a surfactant such as polysorbate (e.g., PS20 or PS80), which have improved surfactant stability, and methods for producing such formulations. Without wishing to be bound by theory, it is believed that lipase-like activity that co-purifies with proteins having an Fc region is responsible for the degradation of polysorbate at refrigerated or elevated temperatures. Thus, provided herein are methods for purifying proteins having an Fc region that allow for the removal of lipase-like activity, enabling the formulation of proteins having an Fc region at high concentrations with polysorbate (e.g., PS20 or PS80) or other fatty acid ester surfactants.

[0054] Surfactants / Polysorbates In some embodiments, the surfactant in the formulations described herein whose stability can be improved using the methods described herein is a polyester surfactant.

[0055] In some embodiments, the surfactant in the formulations described herein, whose stability can be improved using the methods described herein, is a polysorbate.

[0056] A description of exemplary polysorbates that may be used in the pharmaceutical compositions is provided in Wucher et al., Journal of Pharmaceutical Sciences, Vol. 111, No. 5, May 2022, pp. 1280-1291, which is specifically incorporated herein by reference for examples of polysorbates that may be present in the formulations described herein, as well as their properties and degradation products.

[0057] In some embodiments, the polysorbate is PS20. In some embodiments, the polysorbate is PS80. In some embodiments, the polysorbate has a shorter fatty acid chain (e.g., C5, C6, C7, C8, C9, C10, or C11 or shorter) (see Kerwin, J. of Pharm. Sciences 97:2924, 2008). In some embodiments, the polysorbate has a longer fatty acid chain (e.g., C12, C13, C14, C15, C16, C17, or C18 or longer). In some embodiments, the polysorbate is a polyester-type ligand. In some embodiments, the polysorbate is a polyamide. In some embodiments, the polysorbate is L-arginine-trehalose. In some embodiments, the polysorbate is L-histidine-trehalose. The structures of polysorbate 20 and polysorbate 80 are shown in Figures 1A and 1B, respectively. Polysorbate 20 and polysorbate 80 differ primarily in their esterified hydrophobic tails (see Figures 1A and 1B, Kerwin, J. of Pharm. Sciences 97:2924, 2008). Both polysorbate 20 and polysorbate 80 are heterogeneous mixtures of esterified fatty acids with reduced solubility. Generally, polysorbate 20 contains approximately 20-40% polyesters, approximately 30-60% monoesters, and several other impurities.

[0058] Polysorbates are degraded by oxidation, chemical reactions, hydrolysis, and enzymatic reactions. During degradation, polyesters degrade to form monoesters and free fatty acids, and monoesters degrade to form free fatty acids and other residues. Oxidation of polysorbates results in the formation of a mixture of various products, including fatty acid esters, fatty acids (approximately 15% of degradation products), aldehydes, ketones, acids, peroxides, and alkanes. Hydrolysis of polysorbates results in the formation of fatty acids and lower fatty acid esters (see Dwivedi et al., Int. J. of Pharmaceutics 552:422, 2018).

[0059] The important degradation products of polysorbate 20 are lauric acid in addition to fatty acid esters. The important degradation products of polysorbate 80 are oleic acid and fatty acid esters (see Dwivedi et al., Int. J. of Pharmaceutics 552:422, 2018). The structures of lauric acid and oleic acid are shown in Figure 1C and Figure 1D, respectively.

[0060] The degradation mechanism can be determined, for example, using free fatty acid (FFA) stoichiometry or by incubation with antioxidants (see Tomlinson et al., Mol. Pharm. 12:3805, 2015). For FFA stoichiometry, polysorbate and FFA levels are measured at two time points; if approximately 100% of the FFAs are recovered, degradation is most likely hydrolytic. If approximately 15% of the FFAs (at 2-8°C) are recovered, degradation is likely oxidative. For analysis by incubation with antioxidants, samples are incubated with and without antioxidants, and the polysorbate content is measured (see Tomlinson et al., Mol. Pharm. 12:3805, 2015). If antioxidants reduce the rate of degradation, degradation has an oxidative component (although oxidation is inevitable at 40°C). Enzymatic degradation can be inhibited by lipase inhibitors. Degradation products formed as a result of oxidative degradation can be detected using stir-bar extraction coupled to GC-MS (see, e.g., Kishore et al., 2011, Pharmaceutical Research 28(28):1194-1210). Free fatty acids formed as a result of hydrolytic degradation can be detected by UPLC (after fluorescent labeling, see, e.g., Tomlinson et al., Mol. Pharmaceuticals 2015, 12(11):3805-3815) or LC-MS (Dwivedi et al., Int. J. of Pharmaceutics 552:422, 2018). In some embodiments, polysorbate degradation products in the formulations described herein are detected by any method known in the art or described herein.

[0061] Polysorbate degradation can occur as a result of enzymatic hydrolysis by lipase activity, where lipase is a host cell protein (HCP) (see Li et al., Antib Ther. 5:42, 2022). In some embodiments, a lipase assay is used to identify lipolytic activity in the formulations described herein. In some embodiments, stability testing, such as the polysorbate stability testing described herein, can be used as a surrogate for testing lipase activity.

[0062] Polysorbates in the formulation can be protected from oxidation by co-formulation with antioxidants such as butylated hydroxyanisole (BHA) and / or butylated hydroxytoluene (BHT) (see Schmidt et al., J. of Pharm. Sci. 109:1924, 2020). In some embodiments, the formulations described herein do not contain antioxidants. In some embodiments, the formulations described herein do not contain BHA and / or BHT. For example, polysorbate 80 can be protected from oxidation by co-formulation with antioxidants such as BHA and / or BHT. In some embodiments, the formulations described herein that include polysorbate 80 do not contain antioxidants. In some embodiments, the formulations described herein that include polysorbate 80 do not contain BHA and / or BHT. In some embodiments, the formulations described herein that include polysorbate 20 do not contain antioxidants. In some embodiments, the formulations described herein that include polysorbate 20 do not contain BHA and / or BHT. In some embodiments, the formulations described herein further include an antioxidant.

[0063] In some embodiments, the formulations described herein do not include a lipase inhibitor or other esterase inhibitor (see Roy et al., J. Pharm. Sci. 119:3313, 2021). In some embodiments, the formulations described herein that include polysorbate 20 do not include a lipase inhibitor. In some embodiments, the formulations described herein that include polysorbate 80 do not include a lipase inhibitor. In some embodiments, the formulations described herein further include a lipase inhibitor.

[0064] The stability of polysorbates in formulations can be monitored directly, for example, by measuring the levels of polysorbate or by measuring the levels of polysorbate degradation products, such as non-esterified species of PS20 (e.g., sorbitan or isosorbide), lauric acid, or oleic acid (see Dwivedi et al., Int. J. of Pharmaceutics 552:422, 2018).

[0065] Methods for measuring the level of polysorbate in a formulation are known in the art and described herein, including, for example, methods using a fluorescent micelle assay (FMA), high-performance liquid chromatography (HPLC) with a charged particle detector (CAD) or an evaporative light scattering detector (ELSD), or liquid chromatography / mass spectrometry (LC / MS) (see Dwivedi et al., Int. J. of Pharmaceutics 552:422, 2018). Methods for measuring the level of non-esterified species of polysorbate (e.g., PS20) are known in the art and described herein, including, for example, LCMS. In some embodiments, polysorbate levels in a formulation described herein are detected by any method known in the art or described herein.

[0066] FMA can be used to quantify PS20 and / or PS80 using HPLC (reaction coil) or plate reader configurations (see Dwivedi et al., Int. J. of Pharmaceutics 552:422, 2018). The fluorescence quantum yield of N-phenyl-1-naphthylamine (NPN) increases in hydrophobic environments. Fluorescence (emission) intensity increases with micelle concentration, i.e., polysorbate concentration. In some embodiments, proteins are precipitated with an organic solvent (acetonitrile, acetone, etc.), which is then removed prior to FMA measurement. POE sorbitan monolaurate can only be detected using mixed-mode HPLC with ELSD / CAD detection. In HPLC, the eluent is nebulized with an inert gas, causing volatile components to evaporate from the droplets. Nonvolatile components are detected by light scattering (ELSD) or ionized by positively charged nitrogen gas from a high-voltage platinum corona and quantified by electrometer (CAD).

[0067] Fc domain-containing proteins In some embodiments, the formulations described herein comprise any protein having an Fc region. In some embodiments, the formulations described herein comprise any protein having an Fc region (e.g., an antibody) that can be purified using the methods described herein.

[0068] In some embodiments, the protein having an Fc region is an antibody (or an Fc-containing fragment of an antibody). Antibodies can be any of the classes IgG, IgM, IgA, IgE, and IgD, which differ from one another by the nature of the heavy chain present in the molecule (see Schroeder et al., J. Allergy Clin. Immunol., 125:S41, 2010). In some embodiments, the antibody is an IgG antibody. The antibody can also be of any subclass, e.g., IgG1, IgG2, IgG3, or IgG4. In some embodiments, the antibody is an IgG1 antibody. In some embodiments, the antibody is an IgG4 antibody. In some embodiments, the antibody is a humanized or fully human monoclonal antibody. In some embodiments, the antibody is a human antibody. In some embodiments, the antibody is a murine antibody. In some embodiments, the antibody is a full-length antibody.

[0069] In some embodiments, the antibody is an anti-FcRn antibody. In some embodiments, the anti-FcRn antibody is batoclimab (also referred to herein as IMVT-1401 and HL161BKN), e.g., as described in International Patent Application Publications WO2015 / 167293, WO2023 / 235679, and WO2022 / 005113, each of which is incorporated herein in its entirety for examples of antibodies that may be present in the compositions described herein.

[0070] In some embodiments, the antibody is a therapeutic antibody. In some embodiments, the antibody is a therapeutic antibody approved by the FDA for the treatment of a disease or an antibody undergoing clinical trials for the treatment of a disease. In some embodiments, the antibody is a therapeutic antibody approved by the FDA for the treatment of a disease, or an antibody undergoing clinical trials for the treatment of a disease. In some embodiments, the antibody is a therapeutic antibody approved by the FDA for the treatment of a disease, or an antibody undergoing clinical trials for the treatment of a disease. In some embodiments, the antibody is a therapeutic antibody approved by the FDA for the treatment of a disease, or an antibody undergoing clinical trials for the treatment of a disease, such as Humira® (adalimumab), Remicade® (infliximab), Keytruda® (pembrolizumab), Susvimo® (ranibizumab), Aduhelm® (aducanumab-avwa), Evkeeza® (evinacumab-dgnb), Enspryng® (satralizumab-mwge), PHESGO (pertuzumab, trastuzumab, and hyaluronidase-zzxf), Vyepti® (eptinezumab-jmr), Beovu® (brolucizumab-dbll), Herceptin® (Hyperzine®), or a combination of these. Hylecta® (trastuzumab and hyaluronidase-oysk), Aimovig® (erenumab-aooe), Ajovy® (fremanezumab-vfrm), Emgaly® (galcanezumab-gnlm), Ilumya® (tildrakizumab-asmn), Takhzyro® (lanadelumab-flyo), Trogarzo® (ibalizumab-uiyk), Dupixent® (dupilimumab), Hemlibra® (emicizumab-kxwh), Kevzara® (sarilumab), Anthim® (obiltoxaximab) ), Zinbryta® (daclizumab), Cosentyx® (secukinumab), Nucala® (mepolizumab), Praluent® (alirocumab), Repatha® (evolocumab), Benlysta® (belimumab), Actemra® (tocilizumab), Ilaris® (canakinumab), Simponi® (golimumab), Cimzia® (certolizumab pegol), Raptiva (efalizumab), Xolair® (omalizumab), and Synagis® (palivizumab).See Wang et al., 2021, Antibody Therapeutics, Vol. 4, No. 4, pp. 262-273, which is incorporated herein by reference in its entirety. Wang et al. is specifically incorporated herein by reference for its disclosure of highly formulated antibodies and their excipients for use in the methods and formulations described herein.

[0071] In some embodiments, the protein having an Fc region is an Fc fusion protein. In some embodiments, the Fc fusion protein is a GAS6 binding protein-Fc fusion protein, such as batiracept or Enbrel® (etanercept).

[0072] Anti-FcRn antibody In certain aspects, the formulation comprising a protein having an Fc region produced by the methods described herein comprises an anti-FcRn antibody (or an Fc region-containing fragment thereof). In some embodiments, the protein having an Fc region present in the formulation described herein is an anti-FcRn antibody (or an Fc region-containing fragment thereof). In some embodiments, the anti-FcRn antibody is an antibody described in International Patent Publication No. WO / 2015 / 167293 or WO2023 / 235679, each of which is incorporated herein by reference in its entirety.

[0073] Exemplary CDR sequences of anti-FcRn antibodies are shown in Table 1. [Table 1]

[0074] In some embodiments, the antibody comprises three heavy chain CDR amino acid sequences of SEQ ID NO:27 (HCDR1), SEQ ID NO:28 (HCDR2), SEQ ID NO:29 (HCDR3); and three light chain CDR amino acid sequences of SEQ ID NO:30 (LCDR1), SEQ ID NO:31 (LCDR2), SEQ ID NO:32 (LCDR3). In some embodiments, the antibody comprises three heavy chain CDR amino acid sequences of SEQ ID NO:49 (HCDR1), SEQ ID NO:22 (HCDR2), SEQ ID NO:23 (HCDR3); and three light chain CDR amino acid sequences of SEQ ID NO:50 (LCDR1), SEQ ID NO:25 (LCDR2), SEQ ID NO:26 (LCDR3).

[0075] Exemplary heavy and light chain variable regions of anti-FcRn antibodies are shown in Table 2. [Table 2]

[0076] In some embodiments, the antibody comprises a heavy chain variable region comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 6, and a light chain variable region comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 16. In some embodiments, the antibody comprises heavy chain amino acids comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 46, and a light chain amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 48. In some embodiments, the antibody comprises heavy chain amino acids comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 51, and a light chain amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 52.

[0077] In some embodiments, the antibody comprises a heavy chain variable region amino acid sequence of SEQ ID NO: 6; and a light chain variable region amino acid sequence of SEQ ID NO: 16. In some embodiments, the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 46; and a light chain amino acid sequence of SEQ ID NO: 48. In some embodiments, the antibody comprises a heavy chain amino acid sequence of SEQ ID NO: 51; and a light chain amino acid sequence of SEQ ID NO: 52.

[0078] Exemplary heavy and light chains of anti-FcRn antibodies are shown in Table 3. [Table 3] TIFF2026508415000004.tif170170

[0079] In some embodiments, the antibody comprises a heavy chain comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 46, 53, or 55, and a light chain comprising an amino acid sequence at least 90%, at least 95%, at least 98%, or at least 99% identical to the sequence set forth in SEQ ID NO: 48, 54, or 56. In some embodiments, the antibody comprises a heavy chain comprising the sequence set forth in SEQ ID NO: 46, 53, or 55; and a light chain comprising the sequence set forth in SEQ ID NO: 48, 54, or 56.

[0080] In some embodiments, the anti-FcRn antibody a CDR1 comprising an amino acid sequence that is at least 90% identical to one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 21, 24, 27, 30, 33, 36, 39, 42, 49, and 50; a CDR2 comprising an amino acid sequence that is at least 90% identical to one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 22, 25, 28, 31, 34, 37, 40, and 43; and a CDR3 comprising an amino acid sequence that is at least 90% identical to one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 23, 26, 29, 32, 35, 38, 41, and 44; Includes:

[0081] In some embodiments, the anti-FcRn antibody a CDR1 comprising an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 21, 24, 27, 30, 33, 36, 39, 42, 49, and 50; a CDR2 comprising an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 22, 25, 28, 31, 34, 37, 40, and 43; and a CDR3 comprising an amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to one or more amino acid sequences selected from the group consisting of SEQ ID NOs: 23, 26, 29, 32, 35, 38, 41, and 44. Includes:

[0082] In some embodiments, the anti-FcRn antibody is an antibody having (i) the CDRs of HL161BKN, (ii) the heavy chain and / or light chain variable domain of HL161BKN, or (iii) the heavy chain and / or light chain of HL161BKN. In some embodiments, the anti-FcRn antibody is an antibody having (i) the CDRs of HL161B, (ii) the heavy chain and / or light chain variable domain of HL161B, or (iii) the heavy chain and / or light chain of HL161B. In some embodiments, the anti-FcRn antibody is an antibody having (i) the CDRs of HL161A, (ii) the heavy chain and / or light chain variable domain of HL161A, or (iii) the heavy chain and / or light chain of HL161A. In some embodiments, the anti-FcRn antibody is an antibody having (i) the CDRs of HL161C, (ii) the heavy and / or light chain variable domains of HL161C, or (iii) the heavy and / or light chains of HL161C. In some embodiments, the anti-FcRn antibody is an antibody having (i) the CDRs of HL161D, (ii) the heavy and / or light chain variable domains of HL161D, or (iii) the heavy and / or light chains of HL161D. In some embodiments, the anti-FcRn antibody is an antibody having (i) the CDRs of HL161ANS, (ii) the heavy and / or light chain variable domains of HL161ANS, or (iii) the heavy and / or light chains of HL161ANS. The sequences of each of these antibodies are provided herein.

[0083] In some embodiments, the anti-FcRn antibodies may contain one or more amino acid deletions, additions, or substitutions in the amino acid sequences described herein.

[0084] In some embodiments, anti-FcRn antibodies may contain amino acid sequences identical or homologous to the amino acid sequences described herein. The terms "identity" or "homology" refer to the relationship between the sequences of two or more polypeptides, as determined by comparing the sequences. The term "identity" also refers to the degree of sequence relatedness between polypeptides, as determined by the number of matches between strings of two or more amino acid residues. The percent "identity" between two sequences is a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap that need to be introduced for optimal alignment of the two sequences (i.e., the percent identity is equal to the number of identical positions / total number of positions × 100). Sequence comparison and determination of percent identity between two sequences can be accomplished using a mathematical algorithm. For sequence comparison, typically, one sequence serves as a reference sequence to which test sequences are compared. When using a sequence comparison algorithm, test and reference sequences are entered into a computer, subsequence coordinates are designated, if necessary, and sequence algorithm program parameters are designated. Default program parameters can be used, or alternative parameters can be designated. The sequence comparison algorithm then calculates the percent sequence identity of the test sequence relative to the reference sequence based on the program parameters. Additionally or alternatively, the amino acid sequences disclosed herein can be further used as a "query sequence" to search public databases, for example, to identify related sequences. For example, such searches can be performed using the BLAST program of Altschul et al. (J. Mol. Biol. 215:403-10, 1990).

[0085] Two sequences are "substantially identical" if they have a specified percentage of amino acid residues that are the same (i.e., 60% identity, optionally 65%, 70%, 75%, 80%, 85%, 90%, 95%, or 99% identity over a specified region, or if not specified, over the entire sequence), when compared and aligned for maximum correspondence over a comparison window or designated region, as measured using one of the following sequence comparison algorithms or by manual alignment and visual inspection. Optionally, the identity exists over a region at least about 10 amino acids in length, or over a region about 20, 50, 200, or more amino acids in length. In some embodiments, the anti-FcRn antibodies described herein comprise at least one amino acid sequence at least 90% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20-48. In some embodiments, the anti-FcRn antibodies described herein comprise at least one amino acid sequence that is at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identical to a sequence selected from the group consisting of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, and 20-48.

[0086] In some embodiments, the antibody CDR1 comprising the amino acid sequence of SEQ ID NO: 21, CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23; CDR1 comprising the amino acid sequence of SEQ ID NO: 27, CDR2 comprising the amino acid sequence of SEQ ID NO: 28, and CDR3 comprising the amino acid sequence of SEQ ID NO: 29; CDR1 comprising the amino acid sequence of SEQ ID NO: 33, CDR2 comprising the amino acid sequence of SEQ ID NO: 34, and CDR3 comprising the amino acid sequence of SEQ ID NO: 35; a CDR1 comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 comprising the amino acid sequence of SEQ ID NO: 40, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 41; or CDR1 comprising the amino acid sequence of SEQ ID NO: 49, CDR2 comprising the amino acid sequence of SEQ ID NO: 22, and CDR3 comprising the amino acid sequence of SEQ ID NO: 23 The heavy chain variable region comprises:

[0087] In some embodiments, the antibody CDR1 comprising the amino acid sequence of SEQ ID NO: 24, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26; CDR1 comprising the amino acid sequence of SEQ ID NO: 30, CDR2 comprising the amino acid sequence of SEQ ID NO: 31, and CDR3 comprising the amino acid sequence of SEQ ID NO: 32; CDR1 comprising the amino acid sequence of SEQ ID NO: 36, CDR2 comprising the amino acid sequence of SEQ ID NO: 37, and CDR3 comprising the amino acid sequence of SEQ ID NO: 38; a CDR1 comprising the amino acid sequence of SEQ ID NO: 42, a CDR2 comprising the amino acid sequence of SEQ ID NO: 43, and a CDR3 comprising the amino acid sequence of SEQ ID NO: 44, or CDR1 comprising the amino acid sequence of SEQ ID NO: 50, CDR2 comprising the amino acid sequence of SEQ ID NO: 25, and CDR3 comprising the amino acid sequence of SEQ ID NO: 26 The light chain variable region comprises:

[0088] In some embodiments, the antibody comprises one or more heavy chain variable regions and one or more light chain variable regions selected from the group consisting of: a heavy chain variable region comprising a CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 21, a CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising a CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 24, a CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 25, and a CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 26; a heavy chain variable region comprising a CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 27, a CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 28, and a CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 29; and a light chain variable region comprising a CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 30, a CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 31, and a CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 32; a heavy chain variable region comprising a CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 33, a CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 34, and a CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 35; and a light chain variable region comprising a CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 36, a CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 37, and a CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 38; a heavy chain variable region comprising a CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 39, a CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 40, and a CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 41; and a light chain variable region comprising a CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 42, a CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 43, and a CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 44; and a heavy chain variable region comprising a CDR1 (HCDR1) comprising the amino acid sequence of SEQ ID NO: 49, a CDR2 (HCDR2) comprising the amino acid sequence of SEQ ID NO: 22, and a CDR3 (HCDR3) comprising the amino acid sequence of SEQ ID NO: 23; and a light chain variable region comprising a CDR1 (LCDR1) comprising the amino acid sequence of SEQ ID NO: 50, a CDR2 (LCDR2) comprising the amino acid sequence of SEQ ID NO: 25, and a CDR3 (LCDR3) comprising the amino acid sequence of SEQ ID NO: 26.

[0089] In some embodiments, the antibody comprises one or more heavy chain variable regions and / or one or more light chain variable regions comprising one or more amino acid sequences selected from the group consisting of the amino acid sequences of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 51 and 52.

[0090] In some embodiments, the antibody comprises a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, or 51 and / or a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12, 14, 16, 18, 20, or 52.

[0091] In some embodiments, the antibody comprises one or more heavy chain variable regions and one or more light chain variable regions selected from the group consisting of: a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 2 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 12; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 4 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 14; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 6 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 16; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 8 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 18; a heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 10 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 20; and A heavy chain variable region comprising the amino acid sequence of SEQ ID NO: 51 and a light chain variable region comprising the amino acid sequence of SEQ ID NO: 52.

[0092] In some embodiments, the antibody is a binding domain immunoglobulin fusion protein, a camelid antibody, a VHH-containing antibody, an IgD antibody, an IgE antibody, an IgM antibody, an IgG1 antibody, an IgG2 antibody, an IgG3 antibody, or an IgG4 antibody. In some embodiments, the antibody comprises a derivative in the antibody constant region. In some embodiments, the antibody is a synthetic antibody based on a protein scaffold capable of binding to FcRn.

[0093] In some embodiments, anti-FcRn antibodies contain conservative substitutions of amino acid residues in the variable and / or constant regions. As used herein, the term "conservative substitution" refers to a substitution of an amino acid residue with another amino acid residue having similar properties to those of the original amino acid residue. For example, lysine, arginine, and histidine have similar properties in that they have basic side chains, while aspartic acid and glutamic acid have similar properties in that they have acidic side chains. Glycine, asparagine, glutamine, serine, threonine, tyrosine, cysteine, and tryptophan have similar properties in that they have uncharged polar side chains, while alanine, valine, leucine, threonine, isoleucine, proline, phenylalanine, and methionine have similar properties in that they have nonpolar side chains. Tyrosine, phenylalanine, tryptophan, and histidine have similar properties in that they have aromatic side chains. Therefore, it will be clear to those skilled in the art that substitution of amino acid residues from the above groups exhibiting similar properties is unlikely to result in a significant change in the properties of the antibody.

[0094] Protein formulations with improved polysorbate stability In one aspect, provided herein is a formulation comprising a protein having an Fc region and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises a surfactant, and wherein the formulation has improved stability of the surfactant.

[0095] In one aspect, provided herein is a formulation comprising a protein having an Fc region and a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises one or more excipients comprising a polysorbate, and wherein the formulation has improved stability of the polysorbate.

[0096] In some embodiments, the formulations provided herein are pharmaceutical formulations. In some embodiments, the formulations are for use in biomedical applications. In some embodiments, the formulations are for use in diagnostic applications. In some embodiments, the formulations are for use in therapeutic applications, such as administration to a patient (e.g., a mammal, e.g., a human) to treat and / or prevent any condition or disease. The patient can be any mammal, such as a companion animal (e.g., a dog or cat) or a livestock animal (e.g., a horse, cow, or pig). In some embodiments, the patient is a human.

[0097] In some embodiments, the formulations are concentrated, e.g., at least 50 g / L, 60 g / L, 70 g / L, 75 g / L, 80 g / L, 85 g / L, 90 g / L, 95 g / L, 100 g / L, 105 g / L, 110 g / L, 115 g / L, 120 g / L, 125 g / L, 130 g / L, 135 g / L, 140 g / L, 145 g / L, 150 g / L, 155 g / L, 160 g / L, 165 g / L, 170 g / L, 175 g / L, 180 g / L, 185 g / L, 185 g / L, 190 g / L, 195 g / L, 200 g / L, 205 g / L, 210 g / L, 215 g / L, 220 g / L, 225 g / L, 230 g / L, 235 g / L, 240 g / L, 245 g / L, 250 g / L, 260 g / L, 265 g / L, 270 g / L, 275 g / L, 280 g / L, 285 g / L, 290 g / L, 300 g / L, 305 g / L, 310 g / L, 315 g / L, 320 g / L, 325 g / L, 330 g / L, 335 g / L, 340 g / L, 345 g / L, 350 g / L, 355 g / L, 360 g / L, 365 g / L, 370 g / L, 375 g / In some embodiments, the formulation comprises a protein having an Fc region at a concentration of about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L. In some embodiments, the formulation comprises a high concentration of a protein having an Fc region, for example, at least about 100 g / L or more. In some embodiments, the formulation comprises a high concentration of a protein having an Fc region, for example, at least about 125 g / L or more. In some embodiments, the formulation comprises a high concentration of a protein having an Fc region, for example, at least about 150 g / L or more. In some embodiments, the formulation comprises a high concentration of a protein having an Fc region, for example, at least about 170 g / L or 175 g / L or more. In some embodiments, the formulation comprises a high concentration of a protein having an Fc region, for example, at least about 200 g / L or more. In some embodiments, the formulation contains a protein having an Fc region at a high concentration, for example, about 100-110 g / L, about 110-120 g / L, about 120-130 g / L, about 130-140 g / L, about 140-150 g / L, about 150-160 g / L, about 160-170 g / L, about 170-180 g / L, about 180-190 g / L, or about 190-200 g / L.In some embodiments, the formulation comprises a protein having an Fc region at a concentration of about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L.

[0098] In some embodiments, the formulation comprises a protein having an Fc region and further comprises polysorbate (e.g., at any concentration, e.g., between 0.001% and 0.5%, e.g., about 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or 0.1%, or any concentration between these values).

[0099] In some embodiments, the formulation comprises a protein having an Fc region and further comprises polysorbate 20 (e.g., at any concentration, e.g., 0.001% to 0.5%, e.g., about 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or 0.1%, or any concentration between these values).

[0100] In some embodiments, the formulation comprises a protein having an Fc region and further comprises polysorbate 80 (e.g., at any concentration, e.g., 0.001% to 0.5%, e.g., about 0.005%, 0.01%, 0.02%, 0.03%, 0.04%, 0.05%, or 0.1%, or any concentration between these values).

[0101] In some embodiments, the formulation comprises a protein having an Fc region and a polysorbate, and further comprises at least one additive selected from mannitol, sorbitol, arginine, histidine, glycine, and salts thereof, and / or a buffer system selected from citrate or histidine. In some embodiments, the formulation does not comprise arginine. In some embodiments, the additive is arginine or a salt thereof, and the buffer system is histidine. In some embodiments, the formulation comprises 50-250 mM L-arginine or its hydrochloride salt. In some embodiments, the formulation comprises 50-250 mM L-histidine buffer. In some embodiments, the formulation comprises 0.01-0.05% polysorbate. In some embodiments, the formulation comprises 50-250 mM L-arginine or its hydrochloride salt, 50-250 mM L-histidine buffer, and 0.01-0.05% polysorbate. In some embodiments, the formulation comprises a protein having an Fc region and a polysorbate, and further comprises L-histidine / histidine HCl and L-arginine HCl. In some embodiments, the protein having an Fc region is at a high concentration in the formulation, for example, at a concentration equal to or greater than 50 g / L, 75 g / L, 100 g / L, 150 g / L, or 170 g / L. In some embodiments, the formulation has a pH of 4.0 to 8.0. In some embodiments, the formulation has a pH of 4.0 to 7.0.

[0102] The formulation may be suitable for administration via any desired route, including, for example, intravenous injection, subcutaneous injection, intramuscular injection, intraperitoneal injection, topical administration, intranasal administration, pulmonary administration, or rectal administration. In some embodiments, the formulation is a pharmaceutical formulation suitable for injection (e.g., subcutaneous injection) into a subject (e.g., a mammal, e.g., a human). In some embodiments, the formulation has a viscosity of 20 cP or less. In some embodiments, the formulation has an osmolality of 250 mOs / kg to 500 mOs / kg.

[0103] In some embodiments, the formulation comprises an anti-FcRn antibody at about 170 mg / mL or more. In some embodiments, the formulation comprises an anti-FcRn antibody and further comprises one or more excipients including a polysorbate (e.g., polysorbate 20 at a concentration of 0.01% to 0.2%, 0.01% to 0.05%, e.g., 0.02%). In some embodiments, the formulation comprises an anti-FcRn antibody and a polysorbate, and further comprises at least one additive selected from mannitol, sorbitol, arginine, histidine, glycine, and salts thereof, and / or a buffer system selected from citrate or histidine. In some embodiments, the additive is arginine or a salt thereof, and the buffer system is histidine. In some embodiments, the formulation comprises 50 to 250 mM L-arginine or its hydrochloride. In some embodiments, the formulation comprises a 50 to 250 mM L-histidine buffer. In some embodiments, the formulation comprises 0.01 to 0.05% polysorbate. In some embodiments, the formulation comprises an anti-FcRn antibody, 50 to 250 mM L-arginine or its hydrochloride, 50 to 250 mM L-histidine buffer, and 0.01 to 0.05% polysorbate. In some embodiments, the formulation comprises an anti-FcRn antibody and further comprises polysorbate 20 (e.g., polysorbate 20 at a concentration of 0.01% to 0.2%, 0.01% to 0.05%, e.g., 0.02%), L-histidine / histidine HCl, and L-arginine HCl. In some embodiments, the anti-FcRn antibody is present at a high concentration in the formulation, for example, at a concentration equal to or greater than 50 g / L, 75 g / L, 100 g / L, 150 g / L, or 170 g / L. In some embodiments, the formulation comprising an anti-FcRn antibody has a pH of 4.0 to 8.0 In some embodiments, the formulation comprising an anti-FcRn antibody has a pH of 4.0 to 7.0.

[0104] In some embodiments, the formulation comprises about 170 mg / mL of an anti-FcRn antibody in 100 mM L-histidine / histidine HCl, 100 mM L-arginine HCl, and 0.02% polysorbate 20 in water at pH 6.

[0105] In some embodiments, the formulation, e.g., an anti-FcRn antibody formulation, is for treating an autoimmune disease selected from the group consisting of myasthenia gravis, thyroid eye disease, warm autoimmune hemolytic anemia, neuromyelitis optica, immune thrombocytopenic purpura, pemphigus vulgaris, chronic inflammatory demyelinating polyneuropathy, lupus nephritis, and membranous nephropathy.

[0106] The preparation may include a protein having an Fc region purified from a cell culture (e.g., a mammalian host cell culture). In some embodiments, the protein having an Fc region is purified from a cell culture using an Fc-binding affinity resin chromatography column. In some embodiments, the protein having an Fc region is purified from a cell culture using Protein A affinity chromatography. In some embodiments, the protein having an Fc region is purified from a cell culture using Protein G affinity chromatography. In some embodiments, the Fc-binding affinity resin chromatography column comprises an engineered Fc-binding protein. In some embodiments, the protein having an Fc region is purified from a cell culture using any method of Fc-binding affinity resin chromatography described herein or known in the art.

[0107] In some embodiments, the concentration of polysorbate in the formulation remains substantially unchanged for a period of at least 1 day, e.g., 1 week, 2 weeks, 4 weeks, 8 weeks, 4 months, 8 months, 12 months, 2 years, or 3 years. In some embodiments, storage for at least 1 day is storage for 1, 3, or 7 days. In some embodiments, storage for at least 1 day is storage for about 14 days or 28 days or more. In some embodiments, storage for at least 1 day is storage for about 8 weeks or 12 weeks or more. In some embodiments, storage for at least 1 day is storage for about 1 year or more.

[0108] In some embodiments, the concentration of polysorbate remains substantially unchanged after the formulation is stored for an extended period of time at 2-8° C. In some embodiments, the concentration of polysorbate remains substantially unchanged after the formulation is stored for an extended period of time at 25° C. In some embodiments, the concentration of polysorbate remains substantially unchanged after the formulation is stored for an extended period of time at 40° C. In some embodiments, the concentration of polysorbate remains substantially unchanged after the formulation is stored for an extended period of time at −70° C.

[0109] In some embodiments, the concentration of polysorbate remains within 40% of the starting concentration of polysorbate after storage (e.g., at about 2-8°C or about 25°C, e.g., for at least 8 weeks of storage). In some embodiments, the concentration of polysorbate remains within 30% of the starting concentration of polysorbate after storage (e.g., at about 2-8°C or about 25°C, e.g., for at least 8 weeks of storage). In some embodiments, the concentration of polysorbate remains within 20% of the starting concentration of polysorbate after storage (e.g., at about 2-8°C or about 25°C, e.g., for at least 8 weeks of storage). In some embodiments, the concentration of polysorbate remains within 10% of the starting concentration of polysorbate after storage (e.g., at about 2-8°C or about 25°C, e.g., for at least 8 weeks of storage).

[0110] In some embodiments, the concentration of polysorbate remains within 5% of the starting concentration of polysorbate after storage (e.g., at about 2-8°C or about 25°C, e.g., for at least 8 weeks of storage).

[0111] In some embodiments, the concentration of polysorbate in the formulation remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 28 days of storage at about 40° C. The "starting concentration" is the concentration of polysorbate immediately after filling the formulation after purification (e.g., about 0.02%).

[0112] In some embodiments, the concentration of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration of polysorbate after storage at -70°C for at least 8 weeks.

[0113] In some embodiments, the concentration of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration of polysorbate after storage at about 2-8°C for at least 8 weeks.

[0114] In some embodiments, the concentration of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 25° C. for at least 8 weeks.

[0115] In some embodiments, the concentration of a particular species of polysorbate remains substantially constant during storage. For example, the concentration of a polyester and / or monoester of polysorbate may remain substantially unchanged over an extended period of time. In some embodiments, the degradation products are composed of free fatty acids (FFA), primarily lauric acid of the PS20 and / or POE head groups.

[0116] In some embodiments, the concentration of polyester species of the polysorbate remains within 10%, 20%, 30%, or 40% of the starting concentration of polyester species after storage for a period of at least 1 day. In some embodiments, the concentration of monoester species of the polysorbate remains within 10%, 20%, 30%, or 40% of the starting concentration of monoester species after storage for a period of at least 1 day.

[0117] In some embodiments, the concentration of polyester and / or monoester of polysorbate in the formulation remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 28 days of storage at about 40°C.

[0118] In some embodiments, the concentration of polyester and / or monoester of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0119] In some embodiments, the concentration of the polyester and / or monoester of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at 2-8°C for at least 8 weeks.

[0120] In some embodiments, the concentration of polyester and / or monoester of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at 25° C. for at least 8 weeks.

[0121] In some aspects, the amount of polysorbate degradation products (e.g., non-esterified species of lauric acid and / or PS20) does not increase or does not substantially increase in the formulation. In some embodiments, the presence of polysorbate degradation products does not increase by more than 50% of the starting concentration of degradation products after storage. In some aspects, the amount of polysorbate degradation products (e.g., non-esterified species of oleic acid and / or PS80) does not increase or does not substantially increase in the formulation. In some embodiments, the presence of polysorbate degradation products does not increase by more than 50% of the starting concentration of degradation products after storage. In some embodiments, the presence of polysorbate degradation products does not increase by more than 25% of the starting concentration of degradation products after storage. In some embodiments, the presence of polysorbate degradation products does not increase by more than 10% of the starting concentration of degradation products after storage.

[0122] In some embodiments, the concentration of non-esterified species of polysorbate in the formulation remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 28 days of storage at about 40°C.

[0123] In some embodiments, the concentration of non-esterified species of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0124] In some embodiments, the concentration of non-esterified species of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at 2-8°C for at least 8 weeks.

[0125] In some embodiments, the concentration of non-esterified species of polysorbate in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at 25° C. for at least 8 weeks.

[0126] In some embodiments described herein, lauric acid concentrations are measured in formulations containing PS20 because it is a significant degradation product of PS20. In some embodiments described herein, oleic acid concentrations are measured in formulations containing PS80 because it is a significant degradation product of PS80.

[0127] In some embodiments, the concentration of lauric acid in the formulation remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 28 days of storage at about 40°C.

[0128] In some embodiments, the concentration of lauric acid in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0129] In some embodiments, the concentration of lauric acid in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at 2-8°C for at least 8 weeks.

[0130] In some embodiments, the concentration of lauric acid in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at 25° C. for at least 8 weeks.

[0131] In some embodiments, the concentration of oleic acid in the formulation remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 28 days of storage at about 40°C.

[0132] In some embodiments, the concentration of oleic acid in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0133] In some embodiments, the concentration of oleic acid in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at 2-8°C for at least 8 weeks.

[0134] In some embodiments, the concentration of oleic acid in the formulation remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at 25° C. for at least 8 weeks.

[0135] In some embodiments, the formulation is at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9% or 100% free of impurities.

[0136] In some embodiments, the preparations are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell proteins (e.g., proteins from cells in which proteins having an Fc region are produced). In some embodiments, the preparations described herein contain host cell proteins at a concentration of less than 20 pg / mg, less than 15 pg / mg, less than 10 pg / mg, less than 7.5 pg / mg, less than 5 pg / mg, less than 4 pg / mg, less than 3 pg / mg, less than 2.5 pg / mg, less than 2 pg / mg, less than 1.5 pg / mg, or less than 1 pg / mg. In some embodiments, the preparations described herein contain host cell proteins at a concentration of less than 5 pg / mg or less than 2 pg / mg. In some embodiments, host cell proteins are undetectable in the preparations described herein. Any method for detecting host cell proteins described herein or known in the art can be used. In some embodiments, the formulations are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell lipases and / or hydrolases. In some embodiments, the formulations are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell proteins with lipase activity. In some embodiments, host cell proteins with lipase activity are undetectable in the formulations described herein. In some embodiments, the formulations are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell proteins with hydrolase activity. In some embodiments, host cell proteins with hydrolase activity are undetectable in the preparations described herein.In some embodiments, the formulations are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell proteins with esterase activity. In some embodiments, host cell proteins with esterase activity are undetectable in the formulations described herein. Lipase, esterase, and / or hydrolase activity can be determined by measuring the stability of polysorbates.

[0137] In some embodiments, the formulations are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell DNA (e.g., DNA from cells in which a protein having an Fc region is produced). In some embodiments, the formulations described herein contain host cell DNA at a concentration of less than 2 pg / mg, less than 1.5 pg / mg, less than 1 pg / mg, less than 0.75 pg / mg, less than 0.5 pg / mg, less than 0.4 pg / mg, less than 0.3 pg / mg, less than 0.25 pg / mg, less than 0.2 pg / mg, less than 0.15 pg / mg, less than 0.1 pg / mg, or less than 0.05 pg / mg. In some embodiments, the formulations described herein contain host cell protein at a concentration of less than 0.5 pg / mg or less than 0.2 pg / mg. In some embodiments, host cell DNA is undetectable in the preparations described herein. Any method for detecting host cell DNA described herein or known in the art can be used.

[0138] In some embodiments, the formulations described herein do not contain an antioxidant. In some embodiments, the formulations do not contain BHA. In some embodiments, the formulations do not contain BHT. In some embodiments, the formulations described herein do not contain a lipase inhibitor. In other embodiments, the formulations described herein further contain an antioxidant. In some embodiments, the formulations described herein further contain a lipase inhibitor.

[0139] Method for producing protein formulations with improved polysorbate stability In another aspect, provided herein are methods for purifying proteins having an Fc region, which result in improved stability of surfactants (such as polysorbates) in the final formulation of the protein having an Fc region, and protein preparations produced by such methods. In some embodiments, the polysorbate is PS20. In some embodiments, the polysorbate is PS80.

[0140] In some embodiments, the formulations produced by the methods described herein contain a protein having an Fc region at a high concentration, for example, about 50-100 g / L, about 100-110 g / L, about 110-120 g / L, about 120-130 g / L, about 130-140 g / L, about 140-150 g / L, about 150-160 g / L, about 160-170 g / L, about 170-180 g / L, about 180-190 g / L, or about 190-200 g / L. In some embodiments, a formulation produced by the methods described herein comprises a protein having an Fc region at a concentration of about 50 g / L, about 100 g / L, about 110 g / L, about 120 g / L, about 130 g / L, about 140 g / L, about 150 g / L, about 160 g / L, about 170 g / L, about 180 g / L, about 190 g / L, or about 200 g / L. In some embodiments, a formulation produced by the methods described herein comprises 170 mg / mL of an anti-FcRn antibody in 100 mM L-histidine / histidine HCl, 100 mM L-arginine HCl, and 0.02% polysorbate 20 in water at pH 6. In some embodiments, a formulation produced by the methods described herein can be for injection, e.g., subcutaneous injection.

[0141] Proteins, including proteins having an Fc domain described herein, can be produced in a bioreactor by a fed-batch process using commercially available animal component-free media. Proteins, including proteins having an Fc domain described herein, can be isolated from any suitable host cell, including CHO cells, NS0 cells, Sp2 / 0 cells, HEK293 cells, and PERC6 cells. Cells expressing proteins, such as proteins having an Fc domain described herein, can be produced using any suitable method known in the art. For example, cells can be transfected with a vector expressing a protein having an Fc region. Cells can be harvested at an appropriate time point, and proteins can be purified from the cells as described herein or using any suitable method known in the art.

[0142] An overview of the upstream process is shown in Figure 2A. Generally, the upstream process involves thawing a cell bank vial containing cells engineered to express a protein with an Fc region, growing an inoculum of the cells in a flask, and then further growing the cell culture in a production bioreactor. The cell culture harvested from the production bioreactor is clarified using centrifugation, followed by depth filtration and bioburden reduction. The resulting harvest contains the protein with the Fc region and is the starting solution for further purification.

[0143] The downstream purification process is shown in Figure 2B. Generally, the downstream process involves chromatography to capture proteins having an Fc region from the clarified harvest. Any Fc-binding resin can be used in this chromatography step, including, for example, Protein A, Protein G, or a resin with an engineered Fc-binding protein. Examples of Fc-binding resins that can be used in the methods described herein are provided in Lei et al., December 2022, TrAC Trends in Analytical Chemistry, Vol. 157, p. 116744, which is incorporated herein by reference in its entirety for examples of chromatography resins that can be used in the methods described herein.

[0144] The chromatography step may be followed by low pH viral inactivation, anion exchange (AEX) chromatography, and / or cation exchange (CEX) chromatography. The resulting pool can then be filtered, for example, through a nanofilter, and / or subjected to ultrafiltration and diafiltration to produce a concentrated, buffer-exchanged UFDF pool. The recovered filtered pool can then be formulated.

[0145] In some embodiments, a method for purifying a protein having an Fc region from a harvested cell culture comprises: (i) loading the harvested cell culture containing the protein having an Fc region onto an Fc-binding affinity resin chromatography column; (ii) washing the column one, two, three or more times with one or more wash buffers, at least one of which has a pH greater than 9; and (iii) eluting the protein having an Fc region from the column; optionally, the method is for making a highly concentrated formulation of the protein with one or more excipients comprising polysorbate.

[0146] In some embodiments, the method comprises: (i) loading the cell culture comprising the protein having an Fc region onto an Fc-binding affinity resin chromatography column (e.g., a Protein A chromatography column); (ii) washing the column one, two, three or more times with one or more wash buffers, wherein at least one wash buffer comprises a pH greater than 9 (or equal to or greater than 10); (iii) eluting the protein having an Fc region from the column; (iv) optionally filtering and / or concentrating the eluate from step (iii) comprising the protein having an Fc region; and (v) formulating the protein having an Fc region from step (iv) into a pharmaceutical formulation comprising adding one or more excipients, wherein the concentration of the protein having an Fc region in the pharmaceutical formulation is greater than 50 g / L or equal to or greater than 100 g / L, and the one or more excipients comprise polysorbate (e.g., PS20 or PS80).

[0147] In some embodiments, the method comprises: (i) loading a cell culture comprising the protein having an Fc region onto a Protein A chromatography column; (ii) washing the column one, two, three or more times with one or more wash buffers, wherein at least one wash buffer comprises a pH equal to or greater than 10; (iii) eluting the protein having an Fc region from the column; (iv) optionally filtering and / or concentrating the eluate from step (iii) comprising the protein having an Fc region; and (v) formulating the protein having an Fc region of step (iv) into a pharmaceutical formulation comprising adding one or more excipients, wherein the concentration of the protein having an Fc region in the pharmaceutical formulation is greater than 75 g / L or equal to or greater than 100 g / L, and the one or more excipients comprise polysorbate 20.

[0148] The chromatography involves several steps, including, for example, equilibrating a column packed with MabSelect SuRe LX resin with an equilibration buffer before loading a solution containing the protein with an Fc region onto the column, and washing the column with three different buffers (Wash 1, Wash 2, and Wash 3) before elution. The stringency of the washes is increased from Wash 1 to Wash 2, and the column is prepared for elution with Wash 3. The protein with an Fc region is then eluted using a low pH elution buffer. See Figure 6 for an illustration of the Protein A chromatography process. Further details of the Protein A chromatography process are provided in the Examples section below.

[0149] Without wishing to be bound by theory, it is believed that the chromatography step is essential in determining the stability of surfactants (such as polysorbates) in the final protein formulation. In particular, the pH of the buffer used in the second wash step ("wash 2 buffer") is believed to be important in determining the stability of polysorbates. In some embodiments, the wash 2 buffer is a high pH buffer. In some embodiments, the wash 2 buffer has a pH greater than 9. In some embodiments, the wash 2 buffer has a pH of about 9.5 or greater. In some embodiments, the wash 2 buffer has a pH of about 10 or greater. In some embodiments, the wash 2 buffer has a pH of at least 10. In some embodiments, the wash 2 buffer has a pH of about 10.5 or greater. In some embodiments, the wash 2 buffer has a pH of about 11 or greater. In some embodiments, the wash 2 buffer has a pH of about 11.5 or greater.

[0150] Without wishing to be bound by theory, it is believed that a chromatography step using a high pH wash buffer may disrupt non-covalent or covalent interactions between a protein having an Fc region (purified according to the methods described herein) and host cell proteins (e.g., lipases, esterases and / or hydrolases).

[0151] Another factor that may contribute to the stability of polysorbate in the final protein formulation is the column loading ratio. Therefore, it is believed that a lower column loading ratio in the chromatography step improves the stability of polysorbate. In some embodiments, the Protein A column loading ratio is 40 g / L. 樹脂 In some embodiments, the Protein A column load ratio is 35 g / L or less. 樹脂 In some embodiments, the Protein A column loading ratio is 30 g / L or less. 樹脂 In some embodiments, the column load ratio is 25.0 g / L or less. 樹脂 In some embodiments, the column load ratio is 20 g / L or less. 樹脂 In some embodiments, the column load ratio is 15 g / L or less. 樹脂 In some embodiments, the Protein A column loading ratio is about 5 g / L or less. 樹脂 ~About 10g / L 樹脂 In some embodiments, the Protein A column loading ratio is about 10 g / L. 樹脂 ~Approx. 15g / L 樹脂 In some embodiments, the Protein A column loading ratio is about 15 g / L. 樹脂 ~About 20g / L 樹脂 In some embodiments, the Protein A column loading ratio is about 20 g / L. 樹脂 ~about 25g / L 樹脂 In some embodiments, the Protein A column loading ratio is about 25 g / L. 樹脂 ~About 30g / L 樹脂 In some embodiments, the Protein A column loading ratio is about 30 g / L. 樹脂 ~Approx. 35g / L 樹脂 In some embodiments, the Protein A column loading ratio is about 35 g / L. 樹脂 ~about 40g / L 樹脂 In some embodiments, the Protein A column loading ratio is about 40 g / L. 樹脂 ~About 45g / L 樹脂 In some embodiments, the Protein A column loading ratio is about 45 g / L.樹脂 ~about 50g / L 樹脂 is.

[0152] In some embodiments, the Protein A column loading ratio is about 1 g / L. 樹脂 In some embodiments, the Protein A column loading ratio is about 2 g / L. 樹脂 In some embodiments, the Protein A column loading ratio is about 3 g / L. 樹脂 In some embodiments, the Protein A column loading ratio is about 4 g / L. 樹脂 In some embodiments, the Protein A column loading ratio is about 5 g / L. 樹脂 In some embodiments, the Protein A column loading ratio is about 10 g / L. 樹脂 In some embodiments, the Protein A column loading ratio is about 15 g / L. 樹脂 In some embodiments, the Protein A column loading ratio is about 20 g / L. 樹脂 is.

[0153] In some embodiments, the Wash 2 buffer contains a high concentration of salt, such as sodium chloride (NaCl). In some embodiments, the Wash 2 buffer contains at least 0.5 M NaCl. In some embodiments, the Wash 2 buffer contains about 1 M NaCl. In some embodiments, the Wash 2 buffer does not contain a high concentration of salt (e.g., 0.3 M NaCl or less).

[0154] In some embodiments, the Wash 2 buffer comprises arginine. In some embodiments, the Wash 2 buffer comprises about 0.5 M arginine. In some embodiments, the Wash 2 buffer does not contain arginine.

[0155] The purification method described herein may include additional steps after the chromatography step. For example, the purification method may further include one or more virus inactivation steps. Methods for virus inactivation are known in the art.

[0156] In some embodiments, the methods described herein include at least one additional chromatography step. In some embodiments, the at least one additional chromatography step is anion exchange (AEX) chromatography. AEX chromatography can remove impurities such as protein size variants, host cell proteins, residual Protein A, and / or host cell DNA. Methods of AEX chromatography are known in the art. In some embodiments, the at least one additional chromatography step is cation exchange (CEX) chromatography. CEX chromatography can further remove impurities such as protein size variants, host cell proteins, residual resin from the chromatography column, and / or host cell DNA. Methods of CEX chromatography are known in the art.

[0157] In some embodiments, the method described herein further comprises at least one filtration step.For example, this method can further comprise a virus filtration step.Virus filtration sterically removes both enveloped and non-enveloped viruses.Methods of vial filtration are known in the art.

[0158] In some embodiments, the method described herein further comprises an ultrafiltration and / or diafiltration step. In some embodiments, the method described herein further comprises an ultrafiltration / diafiltration (UFDF) step. UFDF can be used to exchange the buffer solution of the solution with a diafiltration buffer, and concentrate the protein having an Fc region to a target concentration. UFDF methods are known in the art.

[0159] In another aspect, provided herein is a formulation comprising a protein having an Fc region produced by any of the methods described herein.

[0160] In some embodiments, the formulations produced by the methods described herein comprise a polysorbate. In some embodiments, the polysorbate is PS20. In some embodiments, the polysorbate is PS80.

[0161] In some embodiments, the concentration of polysorbate remains within 30% of the starting concentration of polysorbate after storage for at least 1 day, and / or the presence of polysorbate degradation products does not increase by more than 2-fold the starting concentration of degradation products after storage for at least 1 day, hi some embodiments, storage for at least 1 day is storage for 1, 3, or 7 days.

[0162] In some embodiments, storage for at least one day is storage for about 14 days or 28 days or more, in some embodiments, storage for at least one day is storage for about 8 weeks or 12 weeks or more, in some embodiments, storage for at least one day is storage for about 1 year or more.

[0163] In some embodiments, the concentration of polysorbate remains within 20% of the starting concentration of polysorbate after storage. In some embodiments, the concentration of polysorbate remains within 10% of the starting concentration of polysorbate after storage. In some embodiments, the concentration of polysorbate remains within 5% of the starting concentration of polysorbate after storage.

[0164] In some embodiments, the concentration of polysorbate in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 40°C.

[0165] In some embodiments, the concentration of polysorbate in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 28 days.

[0166] In some embodiments, the concentration of polysorbate in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 30 days.

[0167] In some embodiments, the concentration of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0168] In some embodiments, the concentration of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 2-8°C.

[0169] In some embodiments, the concentration of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 30 days.

[0170] In some embodiments, the concentration of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 8 weeks.

[0171] In some embodiments, the concentration of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 25° C. for at least 8 weeks.

[0172] In some embodiments, the concentration of polyester and / or monoester of polysorbate in a formulation produced by the methods described herein remains within about 5%, about 10%, about 15%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% of the starting concentration after at least 28 days of storage at about 40° C. In some embodiments, the concentration of polyester species of polysorbate remains within 10% or 20% of the starting concentration of polyester species after storage for at least 1 day. In some embodiments, the concentration of monoester species of polysorbate remains within 10% or 20% of the starting concentration of monoester species after storage for at least 1 day.

[0173] In some embodiments, the concentration of polyester and / or monoester of polysorbate in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 40°C.

[0174] In some embodiments, the concentration of polyester and / or monoester of polysorbate in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 30 days.

[0175] In some embodiments, the concentration of polyester and / or monoester of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0176] In some embodiments, the concentration of polyester and / or monoester of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 2-8°C.

[0177] In some embodiments, the concentration of polyester and / or monoester of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 30 days.

[0178] In some embodiments, the concentration of polyester and / or monoester of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 25° C. for at least 8 weeks.

[0179] In some aspects, the amount of non-esterified species of polysorbate (degradation products) does not increase in formulations produced by the methods described herein. In some embodiments, the presence of polysorbate degradation products does not increase by more than 25% of the starting concentration of degradation products after storage. In some embodiments, the presence of polysorbate degradation products does not increase by more than 10% of the starting concentration of degradation products after storage. In some embodiments, the polysorbate degradation products are non-esterified species of polysorbate. In some embodiments, the degradation products are non-esterified species of polysorbate 20. In some embodiments, the non-esterified species of polysorbate 20 is sorbitan or isosorbide. In some embodiments, the polysorbate 20 degradation product is lauric acid. In some embodiments, the presence of polysorbate 20 degradation products does not increase by more than 50% of the starting concentration of degradation products after storage. In some embodiments, the degradation products are non-esterified species of polysorbate 80. In some embodiments, the polysorbate 80 degradation product is oleic acid. In some embodiments, the presence of polysorbate 80 degradation products does not increase by more than 50% of the starting concentration of the degradation products after storage.

[0180] In some embodiments, the concentration of non-esterified species of polysorbate in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 40°C.

[0181] In some embodiments, the concentration of non-esterified species of polysorbate in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 28 days of storage at about 40°C.

[0182] In some embodiments, the concentration of non-esterified species of polysorbate in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 30 days.

[0183] In some embodiments, the concentration of non-esterified species of polysorbate in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0184] In some embodiments, the concentration of non-esterified species of PS20 in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 2-8°C.

[0185] In some embodiments, the concentration of non-esterified species of PS20 in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 30 days.

[0186] In some embodiments, the concentration of non-esterified species of PS20 in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 25° C. for at least 8 weeks.

[0187] In some embodiments, the concentration of lauric acid in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 40°C.

[0188] In some embodiments, the concentration of lauric acid in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 28 days.

[0189] In some embodiments, the concentration of lauric acid in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 30 days.

[0190] In some embodiments, the concentration of lauric acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0191] In some embodiments, the concentration of lauric acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 2-8°C.

[0192] In some embodiments, the concentration of lauric acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 30 days.

[0193] In some embodiments, the concentration of lauric acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 8 weeks.

[0194] In some embodiments, the concentration of lauric acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 25° C. for at least 8 weeks.

[0195] In some embodiments, the concentration of non-esterified species of PS80 in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after at least 21 days of storage at about 2-8°C.

[0196] In some embodiments, the concentration of non-esterified species of PS80 in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 30 days.

[0197] In some embodiments, the concentration of non-esterified species of PS80 in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 8 weeks.

[0198] In some embodiments, the concentration of non-esterified species of PS80 in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 25° C. for at least 8 weeks.

[0199] In some embodiments, the concentration of oleic acid in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 21 days.

[0200] In some embodiments, the concentration of oleic acid in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 28 days.

[0201] In some embodiments, the concentration of oleic acid in a formulation produced by the methods described herein remains within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 40° C. for at least 30 days.

[0202] In some embodiments, the concentration of oleic acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at -70°C for at least 8 weeks.

[0203] In some embodiments, the concentration of oleic acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 21 days.

[0204] In some embodiments, the concentration of oleic acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 30 days.

[0205] In some embodiments, the concentration of oleic acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 2-8°C for at least 8 weeks.

[0206] In some embodiments, the concentration of oleic acid in a formulation produced by the methods described herein remains within about 1%, within about 5%, within about 10%, within about 15%, within about 20%, within about 25%, within about 30%, within about 35%, within about 40%, within about 45%, or within about 50% of the starting concentration after storage at about 25° C. for at least 8 weeks.

[0207] In another aspect, the methods described herein reduce impurities in formulations produced by the methods described herein. In some embodiments, formulations produced by the methods described herein are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of impurities.

[0208] In some embodiments, the methods described herein result in the removal of host cell proteins (e.g., proteins from the cells in which the antibody is produced). In some embodiments, the methods described herein result in the removal of host cell lipases and / or hydrolases. In some embodiments, the preparations produced by the methods described herein are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell lipases and / or hydrolases. In some embodiments, the preparations produced by the methods described herein are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell proteins. In some embodiments, a formulation produced by the methods described herein comprises host cell protein at a concentration of less than 20 pg / mg, less than 15 pg / mg, less than 10 pg / mg, less than 7.5 pg / mg, less than 5 pg / mg, less than 4 pg / mg, less than 3 pg / mg, less than 2.5 pg / mg, less than 2 pg / mg, less than 1.5 pg / mg, or less than 1 pg / mg. In some embodiments, a formulation produced by the methods described herein comprises host cell protein at a concentration of less than 5 pg / mg or less than 2 pg / mg. In some embodiments, host cell protein is undetectable in a formulation produced by the methods described herein.

[0209] In some embodiments, the formulation is free or substantially free of host cell proteins with lipase and / or hydrolase activity, ie, the host cell proteins with lipase and / or hydrolase activity are reduced by about 20%, about 30%, about 40%, about 50%, about 60%, about 70%, about 80%, about 95%, about 98%, about 99%, or about 100% compared to a formulation produced using a buffer at pH 9 or less in a chromatographic wash step (e.g., Wash 2).

[0210] In some embodiments, the methods described herein result in the removal of host cell DNA (e.g., DNA from cells in which the antibody is produced). In some embodiments, the formulations produced by the methods described herein are at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, at least 99.5%, at least 99.8%, at least 99.9%, or 100% free of host cell DNA. In some embodiments, the formulations produced by the methods described herein contain host cell DNA at a concentration of less than 2 pg / mg, less than 1.5 pg / mg, less than 1 pg / mg, less than 0.75 pg / mg, less than 0.5 pg / mg, less than 0.4 pg / mg, less than 0.3 pg / mg, less than 0.25 pg / mg, less than 0.2 pg / mg, less than 0.15 pg / mg, less than 0.1 pg / mg, or less than 0.05 pg / mg. In some embodiments, the preparations produced by the methods described herein contain host cell protein at a concentration of less than 0.5 pg / mg or less than 0.2 pg / mg, hi some embodiments, host cell DNA is undetectable in the preparations produced by the methods described herein. Exemplary Embodiment I 1. A pharmaceutical formulation comprising (i) an antibody (or another protein having an fc region) at a concentration greater than 50 g / L and (ii) a pharmaceutically acceptable carrier, wherein the pharmaceutically acceptable carrier comprises polysorbate 20, and wherein the concentration of polysorbate 20 remains within 30% of the starting concentration of polysorbate 20 after storage for a period of at least 1 day, and / or the presence of polysorbate 20 degradation products does not increase by more than 2-fold the starting concentration of the degradation products after storage for a period of at least 1 day. The pharmaceutical formulation of embodiment 1, wherein the concentration greater than 2.50 mg / ml is a concentration equal to or greater than 100 g / L, 150 g / L, 170 g / L, or 200 g / L. 3. The pharmaceutical formulation of embodiment 1 or 2, wherein the antibody is a humanized or fully human monoclonal antibody. 4. The pharmaceutical formulation according to any one of embodiments 1 to 3, wherein the antibody is an IgG1 antibody. 5. The pharmaceutical formulation according to any one of embodiments 1 to 4, wherein the antibody is an anti-FcRn antibody that specifically binds to FcRn. 6. The pharmaceutical formulation of embodiment 5, wherein the anti-FcRn antibody is a fully human monoclonal IgG1 antibody. 7. Anti-FcRn antibodies (a) (i) the three heavy chain CDR amino acid sequences of SEQ ID NO: 27 (HCDR1), SEQ ID NO: 28 (HCDR2), and SEQ ID NO: 29 (HCDR3); and (ii) the three light chain CDR amino acid sequences of SEQ ID NO: 30 (LCDR1), SEQ ID NO: 31 (LCDR2), and SEQ ID NO: 32 (LCDR3); or (b) (i) three heavy chain CDR amino acid sequences of SEQ ID NO: 49 (HCDR1), SEQ ID NO: 22 (HCDR2), SEQ ID NO: 23 (HCDR3); and (ii) three light chain CDR amino acid sequences of SEQ ID NO: 50 (LCDR1), SEQ ID NO: 25 (LCDR2), SEQ ID NO: 26 (LCDR3). 7. The pharmaceutical formulation of embodiment 5 or 6, comprising: 8. Anti-FcRn antibodies (a) a heavy chain variable region amino acid sequence of SEQ ID NO: 6 and a light chain variable region amino acid sequence of SEQ ID NO: 16; or (b) the heavy chain amino acid sequence of SEQ ID NO: 51; and the light chain amino acid sequence of SEQ ID NO: 52. 8. The pharmaceutical formulation according to any one of embodiments 5 to 7, comprising: 9. The pharmaceutical formulation of any one of embodiments 1 to 8, wherein the polysorbate 20 is at a concentration of about 0.002% to 0.2%. 10. The pharmaceutical formulation of any one of embodiments 1 to 8, wherein the polysorbate 20 is at a concentration of 0.01 to 0.05%. 11. The pharmaceutical formulation of any one of embodiments 1-10, wherein the antibody is an anti-FcRn antibody that specifically binds to FcRn, and the pharmaceutical formulation comprises at least or about 170 g / L of the anti-FcRn antibody, 0.02% polysorbate 20, 100 mM L-histidine / histidine HCl, and 100 mM L-arginine HCl in water at pH 6.0. 12. The pharmaceutical formulation of any one of embodiments 1-11, wherein the concentration of polysorbate 20 remains within 20% of the starting concentration of polysorbate 20 after storage. 13. The pharmaceutical formulation of any one of embodiments 1-11, wherein the concentration of polysorbate 20 remains within 10% of the starting concentration of polysorbate 20 after storage. 14. The pharmaceutical formulation of any one of embodiments 1-11, wherein the concentration of polysorbate 20 remains within 5% of the starting concentration of polysorbate 20 after storage. 15. The pharmaceutical formulation of any one of embodiments 1-14, wherein the polysorbate 20 degradation products are non-esterified species of polysorbate 20. 16. The pharmaceutical formulation of embodiment 15, wherein the non-esterified species of polysorbate 20 is sorbitan or isosorbide. 17. The pharmaceutical formulation of any one of embodiments 1-14, wherein the polysorbate 20 degradation product is lauric acid. 18. The pharmaceutical formulation of any one of embodiments 1-17, wherein the presence of polysorbate 20 degradation products does not increase after storage by more than 50% of the starting concentration of the degradation products. 19. The pharmaceutical formulation of any one of embodiments 1-17, wherein the presence of polysorbate 20 degradation products does not increase after storage by more than 25% of the starting concentration of the degradation products. 20. The pharmaceutical formulation of any one of embodiments 1-17, wherein the presence of polysorbate 20 degradation products does not increase after storage by more than 10% of the starting concentration of the degradation products. 21. The pharmaceutical formulation of any one of embodiments 1-20, wherein the concentration of the polyester species of Polysorbate 20 remains within 10% or 20% of the starting concentration of the polyester species after storage for a period of at least 1 day. 22. The pharmaceutical formulation of any one of embodiments 1-21, wherein the concentration of the monoester species of Polysorbate 20 remains within 10% or 20% of the starting concentration of the monoester species after storage for a period of at least 1 day. 23. The pharmaceutical formulation of any one of embodiments 1-22, wherein storage for a period of at least 1 day is storage for 1, 3, or 7 days. 24. The pharmaceutical formulation of any one of embodiments 1-22, wherein storage for a period of at least 1 day is storage for about 14 days or 28 days or more. 25. The pharmaceutical formulation of any one of embodiments 1-22, wherein storage for a period of at least 1 day is storage for about 8 weeks or 12 weeks or more. 26. The pharmaceutical formulation of any one of embodiments 1-22, wherein storage for a period of at least 1 day is storage for about 1 year or more. 27. The pharmaceutical formulation of any one of embodiments 1-26, wherein storage is at 2-8°C. 28. The pharmaceutical formulation of any one of embodiments 1-26, wherein storage is at 25°C. 29. The pharmaceutical formulation of any one of embodiments 1-26, wherein storage is at 40°C. 30. The pharmaceutical formulation of any one of embodiments 1 to 29, wherein the antibody is purified from the cell culture by Protein A affinity chromatography. 31. A method for purifying a protein having an Fc region from a cell culture, comprising: (i) loading the antibody-containing cell culture onto a Protein A chromatography column; (ii) washing the column one, two, three or more times with one or more wash buffers, at least one of which has a pH greater than 9; (iii) eluting the antibody from the column; (iv) filtering and / or concentrating the eluate from step (iii) containing the antibody; and (v) formulating the antibody of step (iv) into a pharmaceutical formulation comprising adding one or more excipients, wherein the concentration of the antibody in the pharmaceutical formulation is greater than 50 g / L, and the one or more excipients comprise polysorbate 20. 32. The method of embodiment 31, wherein the Protein A column loading ratio in step (i) is 25 g / L or less. 33. The method of embodiment 31 or 32, wherein at least one wash buffer comprises a pH of at least 10 or above. 34. The method of embodiment 31 or 32, wherein at least one wash buffer comprises a pH of about 10.5. 35. The method of any one of embodiments 31-34, wherein at least one wash buffer comprises 1 M sodium chloride or a higher concentration of sodium chloride. 36. The method of any one of embodiments 31-34, wherein at least one wash buffer comprises a sodium chloride concentration of less than 1 M or less than 0.5 M. 37. The method of any one of embodiments 31-36, wherein at least one wash buffer comprises arginine. 38. The method of any one of embodiments 31-36, wherein at least one wash buffer does not contain arginine. 39. The method of any one of embodiments 31-38, wherein at least one wash buffer comprises sodium phosphate or sodium carbonate. 40. The method of any one of embodiments 31-38, wherein at least one wash buffer comprises 50 mM sodium carbonate. 41. The method of any one of embodiments 31 to 40, wherein the concentration of the antibody in the pharmaceutical formulation is about 100 g / L or more, or about 150 g / L or more. 42. The method of any one of embodiments 31 to 40, wherein the final concentration of the antibody in the pharmaceutical formulation is about 170 g / L or higher. 43. The method of any one of embodiments 31 to 40, wherein the final concentration of the antibody in the pharmaceutical formulation is about 200 g / L or higher. 44. The method of any one of embodiments 31 to 43, wherein the antibody is a humanized antibody or a human monoclonal antibody. 45. The method of embodiment 44, wherein the antibody is an IgG1 antibody. 46. ​​The method of any one of embodiments 31-45, wherein the antibody is an anti-FcRn antibody that specifically binds to FcRn. 47. The method of embodiment 46, wherein the anti-FcRn antibody is a fully human monoclonal IgG1 antibody. 48. The anti-FcRn antibody comprises: (a) (i) the three heavy chain CDR amino acid sequences of SEQ ID NO: 27 (HCDR1), SEQ ID NO: 28 (HCDR2), and SEQ ID NO: 29 (HCDR3); and (ii) the three light chain CDR amino acid sequences of SEQ ID NO: 30 (LCDR1), SEQ ID NO: 31 (LCDR2), and SEQ ID NO: 32 (LCDR3); or (b) (i) three heavy chain CDR amino acid sequences of SEQ ID NO: 49 (HCDR1), SEQ ID NO: 22 (HCDR2), SEQ ID NO: 23 (HCDR3); and (ii) three light chain CDR amino acid sequences of SEQ ID NO: 50 (LCDR1), SEQ ID NO: 25 (LCDR2), SEQ ID NO: 26 (LCDR3). 48. The method of embodiment 46 or 47, comprising: 49. The anti-FcRn antibody comprises: (a) a heavy chain variable region amino acid sequence of SEQ ID NO: 6 and a light chain variable region amino acid sequence of SEQ ID NO: 16; or (b) the heavy chain amino acid sequence of SEQ ID NO: 51; and the light chain amino acid sequence of SEQ ID NO: 52. 49. The method of any one of embodiments 46 to 48, comprising: 50. The method of any one of embodiments 31-49, wherein the cell culture is a harvested host cell used to produce an antibody, and the host cell is a mammalian cell suitable for antibody production. 51. The method of embodiment 50, wherein the mammalian cells are CHO cells. 52. The method of any one of embodiments 31-51, wherein step (iv) of the method comprises depth filtration. 53. The method of any one of embodiments 31-52, wherein step (iv) of the method comprises anion exchange (AEX) chromatography. 54. The method of any one of embodiments 31-53, wherein step (iv) of the method comprises cation exchange (CEX) chromatography. 55. The method of any one of embodiments 31 to 54, wherein step (iv) of the method comprises viral filtration. 56. The method of any one of embodiments 31-55, wherein step (iv) of the method comprises ultrafiltration and / or diafiltration. 57. The method of any one of embodiments 31-56, wherein the polysorbate 20 in the pharmaceutical formulation is at a concentration of about 0.002% to 0.2%. 58. The method according to any one of embodiments 31 to 56, wherein the polysorbate 20 in the pharmaceutical formulation is at a concentration of 0.01 to 0.05%. 59. The method of any one of embodiments 31-56, wherein the antibody is an anti-FcRn antibody that specifically binds to FcRn, and the pharmaceutical formulation optionally comprises any one, two, three or more of at least or about 170 g / L of the anti-FcRn antibody, 0.02% polysorbate 20, 100 mM L-histidine / histidine HCl, and 100 mM L-arginine HCl in water at pH 6.0. 60. A pharmaceutical formulation manufactured using the method of any one of embodiments 31-59. 61. The pharmaceutical formulation of embodiment 60, wherein the concentration of polysorbate 20 remains within 30% of the starting concentration of polysorbate 20 after storage for a period of at least 1 day, and / or the presence of polysorbate 20 degradation products does not increase by more than 2-fold the starting concentration of the degradation products after storage for a period of at least 1 day. 62. The pharmaceutical formulation of embodiment 61, wherein the concentration of polysorbate 20 remains within 20% of the starting concentration of polysorbate 20 after storage. 63. The pharmaceutical formulation of embodiment 61, wherein the concentration of polysorbate 20 remains within 10% of the starting concentration of polysorbate 20 after storage. 64. The pharmaceutical formulation of embodiment 61, wherein the concentration of polysorbate 20 remains within 5% of the starting concentration of polysorbate 20 after storage. 65. The pharmaceutical formulation of any one of embodiments 61-64, wherein the polysorbate 20 degradation products are non-esterified species of polysorbate 20. 66. A pharmaceutical formulation according to embodiment 65, wherein the non-esterified species of polysorbate 20 is sorbitan or isosorbide. 67. The pharmaceutical formulation of any one of embodiments 61-64, wherein the polysorbate 20 degradation product is lauric acid. 68. A pharmaceutical formulation according to any one of embodiments 61-67, wherein the presence of polysorbate 20 degradation products does not increase after storage by more than 50% of the starting concentration of the degradation products. 69. A pharmaceutical formulation according to any one of embodiments 61-67, wherein the presence of polysorbate 20 degradation products does not increase after storage by more than 25% of the starting concentration of the degradation products. 70. A pharmaceutical formulation according to any one of embodiments 61-67, wherein the presence of polysorbate 20 degradation products does not increase after storage by more than 10% of the starting concentration of the degradation products. 71. A pharmaceutical formulation according to any one of embodiments 61-70, wherein the concentration of the polyester species of polysorbate 20 remains within 10% or 20% of the starting concentration of the polyester species after storage for a period of at least 1 day. 72. The pharmaceutical formulation of any one of embodiments 61-71, wherein the concentration of the monoester species of Polysorbate 20 remains within 10% or 20% of the starting concentration of the monoester species after storage for a period of at least 1 day. 73. The pharmaceutical formulation according to any one of embodiments 61-72, wherein storage for a period of at least 1 day is storage for 1, 3 or 7 days. 74. The pharmaceutical formulation of any one of embodiments 61-72, wherein storage for a period of at least 1 day is storage for about 14 days or 28 days or more. 75. The pharmaceutical formulation of any one of embodiments 61-72, wherein storage for a period of at least 1 day is storage for about 8 weeks or 12 weeks or more. 76. The pharmaceutical formulation of any one of embodiments 61-72, wherein storage for a period of at least 1 day is storage for about 1 year or more. 77. The pharmaceutical formulation of any one of embodiments 61-76, wherein storage is at 2-8°C. 78. The pharmaceutical formulation of any one of embodiments 61-76, wherein storage is at 25°C. 79. The pharmaceutical formulation of any one of embodiments 61-76, wherein storage is at 40°C. Exemplary Embodiment II 1. A method for purifying an anti-FcRn antibody, comprising: (a) subjecting a solution containing an anti-FcRn antibody to Protein A chromatography, wherein the chromatography column is washed with a buffer having a pH greater than 9 or equal to or greater than 10; (b) filtering and / or concentrating the solution; and (c) formulating an anti-FcRn antibody for clinical administration, wherein the anti-FcRn antibody formulation comprises 170 mg / mL of anti-FcRn antibody in 100 mM L-histidine / histidine HCl, 100 mM L-arginine HCl, and 0.02% polysorbate 20 in water at pH 6.0. 2. The method of embodiment 1, wherein the buffer comprises salt at a concentration of at least 1 M. 3. The method of embodiment 1 or 2, wherein the pH is at least 10. 4. The method of any one of embodiments 1 to 3, wherein the pH of the buffer is 10.5. 5. The method of any one of embodiments 1 to 3, wherein the pH of the buffer is 11. 6. Protein A column load ratio is 25.0 g / L 樹脂 6. The method of any one of embodiments 1 to 5, wherein: 7. The method of any one of embodiments 1-6, wherein the wash buffer comprises at least 1 M sodium chloride. 8. The method of any one of embodiments 1 to 7, wherein the wash buffer comprises arginine. 9. The method of any one of embodiments 1 to 8, further comprising subjecting the solution to viral inactivation. 10. The method of any one of embodiments 1-9, wherein at least one chromatographic method is anion exchange (AEX) chromatography. 11. The method of any one of embodiments 1-9, wherein at least one chromatographic method is cation exchange (CEX) chromatography. 12. The method of any one of embodiments 1-11, wherein the filtration is ultrafiltration and / or diafiltration. 13. An anti-FcRn antibody formulation produced by the method according to any one of embodiments 1 to 12. 14. The formulation of embodiment 13, wherein the concentration of polysorbate 20 in the formulation remains within 25% of the starting concentration after storage at 40° C. for at least 28 days. 15. The formulation of embodiment 13, wherein the concentration of polysorbate 20 in the formulation remains within 5% of the starting concentration after storage at -70°C for at least 8 weeks. 16. The formulation of embodiment 13, wherein the concentration of polysorbate 20 in the formulation remains within 1% of the starting concentration after storage at -70°C for at least 8 weeks. 17. The formulation of embodiment 13, wherein the concentration of polysorbate 20 in the formulation remains within 10% of the starting concentration after storage at 2-8°C for at least 8 weeks. 18. The formulation of embodiment 13, wherein the concentration of polysorbate 20 in the formulation remains within 11% of the starting concentration after storage at -25°C for at least 8 weeks. [Example]

[0211] Example 1: Optimization of wash buffer for PS20 stability Degradation of PS20 and identification of the root cause of the degradation mechanism Drug substance production using bioreactors is by a fed-batch process using commercially available animal component-free media. An overview of the upstream unit operations is shown in Figure 2A. An overview of the downstream process is shown in Figure 2B.

[0212] The upstream process begins with the thawing of cell bank vials, followed by a series of inoculum growth steps in shake flasks and bioreactors (cell bags and stirred tanks) until there are enough cells to inoculate the production bioreactor. The production bioreactor is operated in fed-batch mode and is replenished with base, antifoam, and feed solution during the production process. The production bioreactor is harvested based on culture duration or viability. Harvest clarification is performed using centrifugation, followed by depth filtration and bioburden reduction using sterilizing-grade filters. The harvest is collected in a holding tank and subsequently purified using a series of chromatography and filtration steps.

[0213] Downstream processing utilizes Protein A chromatography to capture IMVT-1401 protein from the clarified harvest. The Protein A affinity chromatography step is followed by low-pH viral inactivation, anion exchange (AEX) chromatography using a multimodal resin, and cation exchange (CEX) chromatography. The CEX pool is nanofiltered to remove potential viruses. The viral filtrate is subjected to ultrafiltration and diafiltration (UFDF) to generate a UFDF pool. The collected UFDF pool is formulated and filtered directly through a sterilizing-grade filter into a pre-sterilized container and stored at ≤-60°C.

[0214] A reduction in the levels of polysorbate 20 was observed in the drug substance formulation when stored at 2-8°C or higher. Investigation revealed that handling procedures were not the underlying cause of the low concentrations of PS20. The reduction in PS20 levels may be due to degradation of PS20.

[0215] IMVT-1401 (batoclimab) drug substance is produced from a cell bank in an upstream process that produces the antibody in a manufacturing bioreactor, followed by downstream processing where the final drug substance is purified and formulated at a target concentration of 170 mg / mL in a histidine and arginine buffer containing 0.02% w / v polysorbate 20 at pH 6.0, and then stored frozen at ≤-60°C. The IMVT-1401 drug product has the same formulation as the drug substance. No formulation adjustments are made during drug product fabrication, which consists of thawing the drug substance, pooling, sterile filtering, and filling into container closures of either PFS or vials, which are stored at 2-8°C.

[0216] PS20 degradation mechanism in IMVT-1401 There are two main mechanisms of PS20 degradation: hydrolysis and oxidation. Hydrolysis is characterized by 1) the formation of fatty acids as a major by-product of degradation, 2) linear kinetics, 3) absence in placebo, and 4) enzymatic reactions in active (antibody, protein, and other modality) samples (see Dwivedi et al., Int. J. of Pharmaceutics 552:422, 2018; Weber Int. J. Pharm. X. 2023;6:100202). Oxidation can be characterized by 1) the formation of fatty acid esters, peroxides, aldehydes, ketones, fatty acid esters, and lesser fatty acids, 2) nonlinear kinetics, 3) associated with placebo and active samples, and 4) chemical reactions (Weber Int. J. Pharm. X. 2023;6:100202).

[0217] To confirm the PS20 degradation observations in the samples, short-term thermal kinetic studies of the drug substance at accelerated temperatures of 2-8 °C and 40 °C were performed (Figure 3). Substantial PS20 degradation was observed at both temperatures. The degradation was so rapid that no significant difference was observed between the two temperatures. Similar degradation was not observed in a placebo formulated with the same excipients and PS20 concentration minus the antibody at 170 mg / mL (Figure 4).

[0218] PS20 kinetic studies as a surrogate for process impurity mapping A PS20 kinetic study was used as a surrogate method to identify the introduction point of the problematic impurity in the manufacturing process. A process mapping study was performed across the critical purification steps of the drug substance process. Process intermediates, including the capture chromatography eluate (Protein A pool), viral inactivation pool (VIN pool), polishing chromatography eluate (AEX and CEX pools), and ultrafiltration / diafiltration (UFDF pool), were spiked with a freshly prepared PS20 stock solution at the same target PS20 concentration as the drug substance (0.02% w / v) and incubated at 40°C for the duration of the kinetic study.

[0219] In-process pools from various steps of the downstream purification process (Protein A chromatography, viral inactivation and neutralization (VIN), anion exchange chromatography (AEX), and cation exchange chromatography (CEX)) were formulated at a target concentration of 0.02% PS-20 and staged for short-term stability studies over several days at 2-8 °C (5 °C) and an accelerated temperature of 40 °C. As shown in Figure 5, PS-20 is substantially degraded over 3 days in all downstream intermediate steps. Compared to the AEX and CEX pools, faster degradation was observed in the Protein A pool. Degradation kinetics observed in pools from the initial API downstream unit operations of Protein A chromatography; >70% degradation of PS20 within 24 h; indicate that factors contributing to PS-20 degradation are potentially introduced from the API upstream process and co-purified with the product during the downstream purification process.

[0220] LC-MS was performed to further characterize the PS20 degradation mechanism. One potential mechanism of degradation was identified as hydrolysis via cleavage of the ester bond, resulting in the presence of lauric acid as a by-product. Furthermore, no significant PS20 degradation was observed in the formulation buffer. Therefore, it was concluded that residual host cell proteins (HCPs) from upstream processes were the major contributors to PS20 degradation in in-process and drug substance samples.

[0221] Because HCP impurities are introduced from the upstream cell culture process and co-elute with the product at all critical purification steps, we evaluated the separation of impurities from the product at the very first purification step, the Protein A capture step.

[0222] Process design for Protein A affinity chromatography unit (old process) The Protein A affinity chromatography step serves as the capture and initial purification step. Harvested cell culture fluid (HCCF) is loaded onto a column packed with MabSelect SuRe LX resin (Cytivia Life) and equilibrated with equilibration buffer. The Protein A column is operated in bind-and-elute mode, and IMVT-1401 binds to the resin while cell culture medium and host cell impurities pass through the column. To reduce nonspecific binding interactions with impurities, the column-bound IMVT-1401 is washed with intermediate wash steps (Wash 1, Wash 2, and Wash 3) before elution. The stringency of the washes is increased from Wash 1 to Wash 2, and the column is prepared for elution with Wash 3. IMVT-1401 is eluted using a low-pH elution buffer. The Protein A eluate is then processed through further downstream steps.

[0223] A unit operation flow diagram is shown in Figure 6. As shown in Figure 5, the Protein A eluate pool using this process did not result in the removal of problematic impurities involved in PS20 degradation.

[0224] Process development efforts were undertaken in stages to achieve stability of PS20 in the intermediate step (Protein A eluate pool) and ultimately in the drug substance.

[0225] Wash 2 buffer component screening; screening studies to evaluate wash buffer matrix, wash buffer pH and higher wash stringency using higher concentrations of sodium chloride (NaCl) and addition of arginine (one factor at a time, OFAT) Representative cell culture harvest material from a large-scale run was utilized as the load material. Development work was performed on a small scale utilizing a qualified scaled-down model of the Protein A chromatography unit operation. The process was carried out as described above, except for the Wash 2 buffer conditions, which were varied based on the experimental design. [Table 4]

[0226] Protein A eluate pools from each condition were assessed for concentration step yield, PS20 stability at 40°C (performed by spiking PS20 at a target concentration of 0.02% (w / v) and incubating at 40°C for the duration of the kinetic study), host cell protein concentration (HCP shown in Tables 5-7), purity by SE-HPLC, CE-SDS (reduced / non-reduced), and product quality attributes such as charge variants by cIEF, deamidation, and oxidation by Peptide Map. [Table 5] [Table 6] [Table 7]

[0227] In these experiments, control was performed similar to the large-scale process performance, and therefore a scaled-down model was considered suitable for development work.

[0228] The Wash 2 buffer condition (condition 6) with pH 10, 1 M NaCl, 0.5 M arginine in a Tris matrix showed significant improvement. Thus, over 70% of the PS20 was retained during the 14-day study, with only a 27% loss observed compared to the control (condition 1), which was completely degraded within one day of the stability study.

[0229] A slightly higher impurity clearance or lower HCP levels were observed in condition 6 compared to the control.

[0230] Transient exposure to high pH Wash 2 buffer did not affect important product quality attributes, and no effect was observed on oxidation and deamidation levels by peptide mapping.

[0231] The alkalinity (high pH) of the wash 2 buffer had a positive effect on the stability of PS20, while a pH below pH 10 had no effect.

[0232] No effect of salt at low pH was observed on the stability of PS20. No effect of arginine at low pH was observed on the stability of PS20. No effect of buffer was observed at low or intermediate pH conditions (Conditions 1 and 7).

[0233] These data indicate that problematic impurities responsible for rapid PS20 degradation can be removed by improving the intermediate wash (Wash 2) conditions. Higher pH (pH 10 or pH > 9), high salt (1 M salt), and the presence of arginine in the wash buffer significantly improved PS20 stability.

[0234] Wash 2 Buffer Component Screening: Design of Experiments (DoE) Study to Evaluate Wash Buffer pH, Sodium Chloride (NaCl), and Arginine Representative cell culture harvest material from a large-scale IMVT-1401 manufacturing run was utilized as the load material. Development work was performed on a smaller scale utilizing a qualified scale-down model of the Protein A chromatography unit operation (same as above). The process was performed as described above, except for the Wash 2 buffer conditions, which were modified based on the experimental design described below.

[0235] A total of nine conditions were tested to understand the effects of pH, sodium chloride concentration, arginine concentration, and any interactions between these factors. Sodium phosphate buffer was not evaluated because it is not a suitable matrix for high-pH solutions. Condition 6 (listed in Tables 6 and 7 above) was evaluated as a control using a Tris buffer matrix. However, for all other conditions, carbonate buffer was evaluated as the buffer matrix due to its better buffering capacity at higher pH. Because no effect on PS20 stability was observed at pH < 10, pH ≥ 10 was used in subsequent experiments. Two levels of sodium chloride were evaluated: 0.3 M (used in the previous process) and 1 M. Two levels of arginine concentration were evaluated: no arginine and a concentration of 0.5 M. The experimental conditions are detailed in Table 8. [Table 8]

[0236] The Protein A eluate pool from each condition was evaluated for concentration step yield, PS20 stability at 40°C (performed by spiking PS20 at a target concentration of 0.02% (w / v) and incubating at 40°C for the duration of the kinetic study), HCP concentration (shown in Table 9 and Figure 8), and product quality attributes (shown in Table 10). [Table 9] [Table 10] TIFF2026508415000012.tif75170 [Table 11]

[0237] The predictive profiler for each factor was derived by statistical analysis using JMP software and is shown in Figure 9. The pH of the wash 2 buffer showed a strong positive correlation with PS20 stability on days 14 (Figure 11) and 30 (Figure 12). The sodium chloride concentration showed a weak positive correlation with PS20 stability, while the arginine concentration showed no correlation.

[0238] These results suggest that the wash 2 buffer pH is the primary contributing factor to PS20 stability. While all conditions showed significant improvements in PS20 stability, the pH 11 wash condition exhibited a more stable PS20 profile compared to the pH 10 wash buffer (Figures 11 and 12). Because the PS20 stability study was conducted at a stress temperature of 40 °C, further reduction in PS20 was expected beyond day 14 due to potential oxidation. However, the results obtained at days 14 and 30 show similar overall trends. Furthermore, the negative correlation between HCP and buffer pH indicates that the high pH condition of the wash 2 buffer effectively removes problematic host cell proteins and enzymes derived from the host cell line or upstream bioreactor manufacturing process that could contribute to PS20 degradation.

[0239] The process step yields were negatively correlated with pH, ​​suggesting that IMVT-1401 may have dissociated from the column due to the high pH of the wash buffer. No significant differences in product quality were observed under any of the conditions tested, with respect to intact IgG, charge variants, asparagine deamidation (N25 and N319), and / or methionine oxidation (M256 and M432), indicating that high pH does not affect product purity. However, some differences were observed by SE-HPLC regarding impurity levels. Thus, higher HMW levels were observed at pH 11 compared to pH 10 (see Table 11, condition 2).

[0240] Therefore, it can be concluded that high pH is the primary factor for achieving PS20 stability. Based on a common HCP test kit, lower HCP levels were observed at higher pH. Lower total HCPs may indicate lower HCP heterogeneity. Higher sodium chloride concentrations (1 M) had a positive effect on HCP removal at lower pH, but no effect of sodium chloride was observed at higher pH. Arginine addition did not correlate with greater HCP removal, regardless of pH and salt concentration.

[0241] Design of Experiments (DoE) Study to Integrate an Optimized Wash 2 Buffer with Additional Protein A Chromatography Operating Parameters Based on the above experiments, it was concluded that pH and pH-sodium chloride interactions are the main factors affecting the stability of PS20. Wash 2 buffer solutions with pH 10-11 and 1 M sodium chloride concentration were effective in achieving PS20 stability, and the higher the pH, the more stable the PS20 profile during kinetic studies.

[0242] The sodium chloride concentration was kept constant at 1 M, and the buffer matrix was fixed as carbonate. The pH range from pH 10 to pH 11 was further evaluated to find the optimal operating range. Furthermore, the chromatographic parameters IMVT-1401 load ratio (g / L) 樹脂 ), Wash 2 buffer volume, Wash 3 buffer pH, and elution buffer pH were evaluated in a multivariate DoE study. Representative cell culture harvest material from a large-scale IMVT-1401 manufacturing run was utilized as the load material. A qualified scale-down model of the Protein A chromatography unit operation was utilized to conduct the DoE on a small scale. The process was performed as described above, except for factors that were modified based on the experimental design as described below.

[0243] Operational parameter design space characterization A median composite surface-centered three-level design was utilized for the Protein A multivariate DoE experiments. A 32-run design was performed to evaluate the five parameters and characteristics shown in Table 12. The detailed design is shown in Table 13. [Table 12] [Table 13]

[0244] The Protein A eluate was tested for several product quality attributes and stability studies of PS20. Evaluation criteria were determined based on previous process experience to assess practical impact on product quality attributes.

[0245] The predictive profile of each factor was derived by statistical analysis using JMP software, as shown in Figure 9. The pH of the wash 2 buffer showed a strong positive correlation with PS20 stability (PS20-14d; Figure 11). As shown in Figure 16, an additional factor, loading ratio, was identified as a contributing factor and had a negative correlation with PS20 stability.

[0246] These results suggest that the main contributing factors to PS20 stability are the wash 2 buffer pH and column loading ratio. High pH also affected HMW levels, but a significant increase in HMW was not observed up to pH 10.50 (Figure 13). Furthermore, the negative correlation between HCP and buffer pH and loading ratio (Figure 14) indicates that high pH conditions in the wash 2 buffer and lower loading ratios effectively remove problematic host cell proteins and enzymes derived from the host cell line or upstream bioreactor manufacturing process that may cause PS20 degradation. Lower total HCP may imply lower HCP heterogeneity.

[0247] The process step yields were negatively correlated with pH, ​​suggesting that IMVT-1401 may be dissociating from the column due to the high pH of the wash buffer. No significant differences in product quality were observed under any of the conditions tested, with respect to intact IgG, charge variants, asparagine deamidation (N25 and N319), and methionine oxidation (M256 and M432), indicating that high pH does not affect product purity. However, some differences were observed by SE-HPLC regarding impurity levels. Thus, higher HMW levels were observed at high buffer pH, pH 11, compared to pH 10.

[0248] Therefore, it is concluded that the high pH of the wash 2 buffer and the lower loading of the Protein A column are the major factors for achieving PS20 stability.

[0249] In the next set of experiments, the new Protein A process was integrated with the remaining downstream unit operations. The antibody concentration in the Protein A eluate was approximately 15 g / L, resulting in a final bulk drug substance (DS) concentration of 170 g / L for IMVT-1401. Therefore, as the molecule moves further along the downstream purification process and is ultimately concentrated and formulated into the final formulation buffer using ultrafiltration / diafiltration (UFDF), the antibody is concentrated more than 10-fold, achieving a final bulk drug substance (BDS) concentration of 170 g / L. It is possible that some problematic impurities may also be co-concentrated with the antibody and / or enriched in the process. It is important to demonstrate sufficient reduction of these unintended problematic HCPs at the Protein A step itself and to evaluate the potential further reduction or enrichment of these HCPs as further downstream processing occurs.

[0250] The viral inactivation (VI) step achieves inactivation of potential adventitious viruses by low pH treatment.

[0251] The combined Protein A eluate pool from all cycles was subjected to viral inactivation, which was performed immediately after collection of the eluate from the last Protein A chromatography cycle.

[0252] The eluate was further subjected to anion exchange (AEX) chromatography to remove impurities such as size variants, HCPs, residual Protein A, and HCD. Subsequently, cation exchange (CEX) chromatography was applied to remove impurities such as size variants, HCPs, residual Protein A, and HCD.

[0253] The CEX eluate was filtered through a 0.2 μm filter.

[0254] Viral filtration (VF) was used to sterically remove both enveloped and non-enveloped viruses that may be present in the CEX pool.

[0255] Finally, ultrafiltration / diafiltration (UFDF) was performed to exchange the buffer into diafiltration buffer and concentrate the IMVT-1401 product to the target concentration. The UFDF pool was diluted to the target protein concentration using DF buffer (100 mM histidine, 100 mM arginine, pH 6.0) before forward processing into bulk drug substance formulation.

[0256] The objectives of the formulation, final filtration, and filling unit operations are to add excipients, make final concentration adjustments, remove potential bioburden from the final formulated DS, and transfer the product to bulk storage containers. The final composition of the DS is 170 mg / mL IMVT-1401, 100 mM histidine, 100 mM arginine, and 0.02% (w / v) polysorbate 20.

[0257] Host cell proteins (HCPs) were significantly reduced throughout the downstream process. Not all host cell proteins are considered problematic with respect to PS20 stability. As shown in Table 14, improving the Wash 2 conditions at the Protein A step did not adversely affect yield (100% yield was observed). Additionally, as previously observed, slightly higher HMW levels (1.59%) were observed at the Protein A eluate step, but HMW levels were reduced throughout the downstream process, and no impact on HMW levels was observed between the old and new processes at the BDS step. Product quality in terms of purity (CESDS (R / NR)) and charge variants (cIEF) also showed no impact of the process changes, as shown in Table 14. [Table 14] TIFF2026508415000017.tif254170

[0258] The HCP values ​​listed above are total HCP concentrations per microgram of protein. The HCP test kit is a generic kit for CHO cell expression systems and is not specifically designed for the IMVT-1401-expressing cell line. Therefore, the removal of problematic host cell proteins that resulted in improved PS20 stability may not be detected by the current HCP kit. Therefore, PS20 kinetic studies were used as a surrogate for measuring PS20 stability and the absence of problematic host cell proteins.

[0259] PS20 stability was evaluated for the Protein A eluate, VIN / depth filtration pool, AEX pool, CEX pool, UF1 pool, and UFDF pool at an accelerated temperature of 40°C. Additionally, the UFDF pool was evaluated for PS20 stability at 2-8°C. PS20 was added to these intermediates at a target concentration of 200 μg / mL, and samples were held at the study temperature for the duration of the study and evaluated at specific time points per study. The results of the PS20 kinetic study are shown in Table 15. As shown, by day 14, Protein A eluate PS20 levels had declined by 13%, and by day 28, levels had declined overall by 22% compared to day 0. For the UFDF pool, at 40°C, PS20 levels declined by 11% by day 14 and remained stable at subsequent time points. Overall, stable PS20 levels were observed at 2-8°C and the accelerated temperature of 40°C.

[0260] The overall variation of the PS20 method is expected to be approximately 20%. Therefore, the loss of PS20 was within the expected range of variation during the Protein A eluate stage and subsequent concentration in the intermediate and UFDF steps. Therefore, the effectiveness of the new Protein A process in removing problematic impurities is demonstrated, even at high concentrations of IMVT-1401 (>170 mg / mL). [Table 15]

[0261] The UFDF pool was formulated at a target PS20 concentration of 200 μg / mL and filtered through a 0.2 μm filter to produce the final BDS. PS20 stability at the BDS stage was evaluated at three temperatures: storage (-70°C), accelerated (2-8°C), and stress (25°C) for 8 weeks, as shown in Table 16 and Figure 17. No loss of PS20 was observed over the 8-week period at any of the temperatures tested, and the 8-week values ​​were within the analytical variability of the method used. [Table 16]

[0262] The stability of PS20 between the old and new processes at the Protein A eluate, UFDF pool, and BDS stages is shown in Figures 18-20. As shown in Figure 18, the Protein A eluate produced using the new process demonstrated significant improvement in PS20 stability when compared to the old process. Thus, PS20 levels were below the limit of quantitation until day 1 of the kinetic study using the old process, whereas using the new process at a highly accelerated temperature of 40 °C, PS20 was retained until day 28, with only a 23% loss observed. Similarly, at the UFDF pool stage, PS20 was below the limit of quantitation until day 1 with the old process, whereas in the UFDF pool produced using the new optimized Protein A conditions, no substantial loss of PS20 was observed at the highly accelerated temperature after 28 days (Figure 19). Furthermore, PS20 stability was observed in the final drug substance for 8 weeks at the accelerated temperature (Figure 20).

[0263] Potential chemical modifications of IMVT-1401 (deamidation, oxidation, pyroglutamation, N-glycosylation, and glycation) were also monitored. No adverse effects on the antibody's chemical modifications were observed, and BDS produced using the new Protein A optimization were comparable to those produced using the old process.

[0264] Therefore, Protein A chromatography changes were successfully integrated with the remaining downstream processes to yield comparable quality BDS with a stable PS20 profile. These process changes were validated at large scale and demonstrated similar results, as discussed in Section 2.

[0265] PS20 profile characterization by liquid chromatography / mass spectrometry LC / MS was performed to further characterize polysorbate degradation. Polysorbate species include polyethylene (POE) sorbitan and isosorbide esters with various degrees of esterification (mono, di, tri, etc.), various fatty acid chain lengths, and various ethoxylated units. PS20 primarily contains laurate as the fatty acid chain. As noted above, there are two main degradation pathways for PS20: one is oxidative and the other is hydrolytic. The rapid increase in the concentration of non-esterified species (sorbitan and isosorbide) indicated that degradation most likely proceeds via hydrolysis, resulting in loss of the fatty acid tail (see Dwivedi et al., Int. J. of Pharmaceutics 552:422, 2018). Loss of lauric acid fatty chains can be measured by scanning in negative ion mode, where the expected degradation product / hydrolyzed lauric acid ions can be observed. Quantitative analysis of these species was performed for the old and new processes.

[0266] PS20 profile (esters) and lauric acid (LA) levels in Protein A eluates PS20 kinetic studies were performed on the Protein A eluates from the old and new processes over 14 days at 40°C using the method described above.

[0267] As shown below, the old process shows a complete reduction of key components of PS20, namely polyesters (Figure 21) and monoesters (Figure 22), as well as an increase in non-esterified species (Figure 23). This suggests degradation of PS20 by a hydrolytic mechanism. The degradation of PS20 was so rapid that by the time sample analysis could even be completed, most of the PS20 had been degraded. However, the Protein A eluate from the new process shows minimal loss of polyester and monoester species, as well as minimal increase in non-esterified species (Figures 21-23).

[0268] Additionally, the formation of lauric acid confirms hydrolysis as the mechanism of action. Due to the low solubility of lauric acid, accurate quantification of lauric acid over time in the stability study was not possible. However, significant differences in lauric acid levels were observed between the old and new processes. Immediately after spiking, nearly 17 ppm LA was observed with the old process, while <1 ppm was observed with the new process, with no increase over 14 days.

[0269] The profile below confirms that the new improved wash conditions and lower loading ratio in the Protein A process resulted in high PS20 stability.

[0270] PS20 profile in BDS using the new Protein A process The BDS produced using the new Protein A capture chromatography process was further evaluated for PS20 stability using LC / MS. PS20 kinetic studies at 5°C and 40°C were performed over 28 and 14 days, respectively. As shown in Figures 24-27, stable polyester (Figure 24) and monoester (Figure 25) profiles are observed even at the highly accelerated temperature of 40°C compared to 5°C.

[0271] No increase in non-esterified PS20 was observed at 5°C for 28 days, and an increase in the non-esterified profile was observed at 40°C (Figure 26). Some degradation of PS20 over time at highly accelerated temperatures is expected. No increase in lauric acid levels was observed at 5°C for 28 days, however, some increase was observed at 40°C (Figure 27).

[0272] Therefore, the stability of PS20 in BDS was confirmed by orthogonal LC / MS. These process changes were confirmed on a larger scale and demonstrated similar results, as discussed in Section

[0258] .

[0273] Host cell lines and relevant host cell proteins affecting PS20 hydrolysis The host cell line used for the production of IMVT-1401 is a CHO line.

[0274] As mentioned above, the degradation rate was very rapid, so PS20 degraded immediately after formulation or at T = 0 in the kinetic studies. It has not been confirmed which host cell proteins are specifically involved in hydrolysis or whether additional factors (e.g., trace metals) may contribute to the rapid degradation. Furthermore, HCP quantification was performed using a general kit that provides total HCP quantification, rather than quantification of specific HCPs that may be responsible for PS20 degradation.

[0275] pH range of decomposition activity The pH of the Protein A eluate ranged from 3.8 to 4.1. When the samples were neutralized to pH 7 before testing, no difference in the degradation pattern was observed. Therefore, HCP or impurities could act over a wide range of pH.

[0276] Formulation-independent Given that the impurity was active in all intermediate steps, the Protein A eluate, the CEX eluate, the UFDF pool and the final BDS, it can be concluded that this issue is formulation independent.

[0277] Antibody Characteristics IMVT-1401 is a fully human monoclonal antibody in the form of immunoglobulin G1 (IgG1) with two gamma heavy chains and two lambda light chains linked by disulfide bonds. Given that problematic HCPs co-eluted with IMVT-1401 at a critical purification step in the previous process and that stringent wash 2 conditions were required to minimize the impact of HCPs in the new process, it is possible that HCPs interact with IMVT-1401 and / or that specific properties of IMVT-1401 preferentially bind to select subsets of HCPs or resins, allowing host cell proteins to co-elute (see Li Xuanwen et al., Antib Ther. 2022, Vol. 5:42-54).

[0278] Example 2: Large-scale validation batch using the new Protein A chromatography process After developing and characterizing the Protein A chromatography process using a qualified scale-down model, the new Protein A chromatography process was integrated with the remaining downstream process steps at bench scale in a confirmatory experiment as described above. The bench-scale process was further scaled up for large-scale production. The overall process schematic is shown in Figures 2A and 2B.

[0279] The stability of PS20 in the scaled-up process intermediate process pools was evaluated at an accelerated temperature of 40°C. The UFDF pool was evaluated at an additional temperature of 2-8°C. PS20 was added to the process intermediates (Protein A eluate, AEX pool, CEX pool, and UFDF pool) at a target concentration of 200 μg / mL. Samples were held at the study temperature for the duration of the study and evaluated at specific time points per study. The results of the PS20 kinetic study are shown in Table 17. As shown, by day 21, Protein A eluate PS20 levels had declined by 25% compared to the initial concentration (day 0). For the AEX pool, CEX pool, and UFDF pool, PS20 levels had declined by ≤9% compared to the initial concentration (day 0). Furthermore, no differences in PS20 stability were observed for the UFDF pool samples at 2-8°C and the accelerated temperature of 40°C.

[0280] The overall variability of the PS20 analytical method is expected to be approximately 20%, with additional variability due to sample preparation. Therefore, the stability of PS20 in the various intermediate pools, from the Protein A pool to the UFDF pool, is likely to be very similar. Therefore, we have confirmed the effectiveness of the new Protein A process at large scale for removing problematic impurities. [Table 17]

[0281] The UFDF pool was formulated at a target PS20 concentration of 200 μg / mL and filtered through a 0.2 μm filter to produce the final BDS. PS20 stability at the BDS stage was evaluated at three temperatures: storage (-70°C), accelerated (2-8°C), and stress (25°C) for one month, as shown in Table 18. No loss of PS20 was observed over one month at any of the temperatures evaluated. [Table 18]

[0282] Example 3: PS20 profile characterization by liquid chromatography / mass spectrometry LC / MS is effective in monitoring the hydrolysis of PS20 as the polyester hydrolytically degrades into monoester and non-esterified species, altering the percent distribution of the measured ester peaks.

[0283] To further confirm the stability of PS20 in the intermediate process pools, spiking studies as described above were performed. The intermediate process pool was spiked with PS20 at a target concentration, and PS20 was added to the process intermediates (Protein A eluate, AEX pool, CEX pool, and UFDF pool) at a target concentration of 200 μg / mL. Samples were held at an accelerated temperature of 40°C for the duration of the study and evaluated at specific time points per study. The relative abundance of non-ester and polyester species in percent area is shown in Figures 28, 29, and 30.

[0284] For the Protein A pool, CEX pool, and UFDF pool, no actual increase in non-esterified species or a trend toward a decrease in polyester and monoester species was observed up to 21 days at the accelerated temperature of 40°C; therefore, no evidence of PS20 degradation due to hydrolysis was observed for any of the intermediate pools, confirming successful scale-up of the new, improved Protein A process. Because no differences in the overall percent distribution of non-esterified or ester species were observed between the Protein A pool, CEX pool, and UFDF pool, the profiles confirm that problematic impurities responsible for PS20 degradation were removed using the improved Wash 2 buffer and Protein A chromatography conditions.

[0285] To further confirm the stability of PS20 in the final bulk drug substance similar to

[0256] , BDS samples were held at an accelerated temperature of 5°C for the duration of the study and evaluated at specific time points per study. The relative abundance of non-ester and polyester species in percent area is shown in Figure 31.

[0286] No actual increase in non-esterified species or a trend toward a decrease in polyester and monoester species was observed up to 56 days at an accelerated temperature of 5 °C. Thus, no evidence of PS20 degradation due to hydrolysis was observed in the BDS stage, further confirming the effective removal of problematic impurities during scale-up and the subsequent improvement in PS20 stability.

[0287] conclusion The data presented herein demonstrate a unique method for achieving polysorbate (e.g., PS20) formulation stability at the drug substance stage without significant process modifications and impacting critical product quality attributes. Four main approaches were evaluated: 1) increasing the pH of the wash 2 buffer for impurity removal; 2) increasing the stringency of the wash buffer using a higher concentration of sodium chloride; 3) using arginine for host cell protein solubilization; and 4) evaluating chromatographic operating parameters such as column load ratio.

[0288] In the experiments described above, neither high salt nor added arginine was required to aid in the removal of host cell proteins that cause PS20 hydrolysis. Improved PS20 stability was achieved by using Wash 2 buffer at a higher pH and reducing the column load ratio to improve removal of problematic host cell proteins.

[0289] Systematic screening, scoping, and confirmatory studies were conducted to assess one factor at a time and potential interactions between various factors.

[0290] in conclusion: 1) At lower pH, arginine had some effect on HCP removal when added to the wash 2 buffer, but no effect on PS20 stability was observed in the Protein A eluate. 2) No additional effect of arginine was observed at high pH in removing problematic host cell proteins involved in PS20 degradation. 3) pH below 9 did not have any effect on the stability of PS20. 4) pH ≥ 10 had the most potent effect, improving the stability of PS20 several-fold. 5) High salt (up to 1 M NaCl) had no effect on the stability of PS20 below pH 9. 6) Problematic HCPs can be removed or significantly reduced in the Protein A step using high pH wash buffers, which can then reduce the impurity load in the remaining chromatography steps. 7) Decreasing the column load ratio (operating parameters) in Protein A chromatography along with the wash 2 buffer pH improves the stability of PS20.

[0291] PS20 stability is observed at low concentrations of monoclonal antibody, but it often exhibits instability at high product concentrations (e.g., >50 g / L or >150 g / L). Integrating high pH washes into the overall purification process allowed monoclonal antibody concentrations to be increased (e.g., >50 g / L or >150 g / L).

[0292] In summary, a higher pH without the addition of salt or arginine was sufficient to allow separation of HCPs from the Protein A capture eluate without affecting monoclonal antibody stability or significantly reducing process yield. Furthermore, use of the described process allows for the use of higher protein concentrations and potentially higher polysorbate (e.g., PS20 and PS80) concentrations without concern for degradation of these polyesters by residual HCPs with lipase or esterase-like activity.

[0293] These process changes were validated at large scale and demonstrated effective removal of residual HCPs with lipase or esterase-like activity, resulting in a stable surfactant (PS20) profile.

[0294] Incorporation by Reference All patents, patent applications and publications (eg, scientific publications) cited herein are hereby incorporated by reference in their entirety for all purposes.

Claims

1. A formulation comprising (i) a protein having an Fc region at a concentration greater than 50 g / L, and (ii) one or more excipients including a polysorbate, wherein the concentration of the polysorbate remains within 30% of the starting concentration of the polysorbate after storage for a period of at least one day, and / or the presence of polysorbate degradation products does not increase by more than two-fold the starting concentration of the degradation products after storage for a period of at least one day.

2. 2. The formulation of claim 1, wherein the concentration greater than 50 mg / ml is a concentration equal to or greater than 100 g / L.

3. 2. The formulation of claim 1, wherein the concentration greater than 100 mg / ml is a concentration equal to or greater than 150 g / L, 170 g / L, or 200 g / L.

4. The formulation according to any one of claims 1 to 3, wherein the protein having an Fc region is an antibody.

5. 5. The formulation of claim 4, wherein the antibody is a humanized or fully human monoclonal antibody.

6. 6. The formulation of claim 4 or claim 5, wherein the antibody is an IgG1 antibody.

7. The formulation of any one of claims 4 to 6, wherein the antibody is an anti-FcRn antibody that specifically binds to FcRn.

8. The formulation of claim 7, wherein the anti-FcRn antibody is a fully human monoclonal IgG1 antibody.

9. The anti-FcRn antibody (a) (i) the three heavy chain CDR amino acid sequences of SEQ ID NO:27 (HCDR1), SEQ ID NO:28 (HCDR2), and SEQ ID NO:29 (HCDR3); and (ii) the three light chain CDR amino acid sequences of SEQ ID NO:30 (LCDR1), SEQ ID NO:31 (LCDR2), and SEQ ID NO:32 (LCDR3); or (b) (i) three heavy chain CDR amino acid sequences of SEQ ID NO: 49 (HCDR1), SEQ ID NO: 22 (HCDR2), and SEQ ID NO: 23 (HCDR3); and (ii) three light chain CDR amino acid sequences of SEQ ID NO: 50 (LCDR1), SEQ ID NO: 25 (LCDR2), and SEQ ID NO: 26 (LCDR3).

9. The formulation of claim 7 or 8, comprising:

10. The anti-FcRn antibody (a) a heavy chain variable region amino acid sequence of SEQ ID NO: 6 and a light chain variable region amino acid sequence of SEQ ID NO: 16; or (b) a heavy chain amino acid sequence of SEQ ID NO: 51; and a light chain amino acid sequence of SEQ ID NO:

52. The formulation according to any one of claims 7 to 9, comprising:

11. The formulation of any one of claims 1 to 10, wherein the polysorbate is polysorbate 20.

12. The formulation of any one of claims 1 to 10, wherein the polysorbate is polysorbate 80.

13. 13. The formulation of any one of claims 1 to 12, wherein the polysorbate is at a concentration of about 0.002% to 0.2%.

14. 13. The formulation of any one of claims 1 to 12, wherein the polysorbate is at a concentration of 0.01 to 0.05%.

15. 11. The formulation of any one of claims 7 to 10, wherein the pharmaceutical formulation comprises at least or about 170 g / L of the anti-FcRn antibody, 0.02% polysorbate 20, 100 mM L-histidine / histidine HCl, and 100 mM L-arginine HCl in water at pH 6.

0.

16. 16. The formulation of any one of claims 1 to 15, wherein the concentration of the polysorbate remains within 20% of the starting concentration of the polysorbate after storage.

17. 16. The formulation of any one of claims 1 to 15, wherein the concentration of the polysorbate remains within 10% of the starting concentration of the polysorbate after storage.

18. 16. The formulation of any one of claims 1 to 15, wherein the concentration of the polysorbate remains within 5% of the starting concentration of the polysorbate after storage.

19. 19. The formulation of any one of claims 1 to 18, wherein the polysorbate is polysorbate 20 and the degradation products are non-esterified species of polysorbate 20.

20. 20. The formulation of claim 19, wherein the non-esterified species of polysorbate 20 is sorbitan or isosorbide.

21. 19. The formulation of any one of claims 1 to 18, wherein the polysorbate is polysorbate 20 and the polysorbate 20 degradation product is lauric acid.

22. 19. The formulation of any one of claims 1 to 18, wherein the polysorbate is polysorbate 80 and the polysorbate 80 degradation product is oleic acid.

23. 23. The formulation of any one of claims 1 to 22, wherein the presence of polysorbate degradation products does not increase after storage by more than 50% of the starting concentration of said degradation products.

24. 23. The formulation of any one of claims 1 to 22, wherein the presence of polysorbate degradation products does not increase after storage by more than 25% of the starting concentration of said degradation products.

25. 23. The formulation of any one of claims 1 to 22, wherein the presence of polysorbate degradation products does not increase after storage by more than 10% of the starting concentration of said degradation products.

26. 26. The formulation of any one of claims 1 to 25, wherein the concentration of polyester species of the polysorbate remains within 10% or 20% of the starting concentration of the polyester species after storage for a period of at least 1 day.

27. 26. The formulation of any one of claims 1 to 25, wherein the concentration of monoester species of the polysorbate remains within 10% or 20% of the starting concentration of the monoester species after storage for a period of at least 1 day.

28. 28. The formulation of any one of claims 1 to 27, wherein storage for a period of at least 1 day is storage for 1, 3 or 7 days.

29. 28. The formulation of any one of claims 1 to 27, wherein storage for a period of at least 1 day is storage for about 14 days or 28 days or more.

30. 28. The formulation of any one of claims 1 to 27, wherein storage for a period of at least 1 day is storage for about 8 weeks or 12 weeks or more.

31. 28. The formulation of any one of claims 1 to 27, wherein storage for a period of at least one day is storage for about one year or more.

32. The formulation according to any one of claims 1 to 31, wherein the storage is at 2 to 8°C.

33. The formulation according to any one of claims 1 to 31, wherein the storage is at 25°C.

34. The formulation of any one of claims 1 to 31, wherein the storage is at 40°C.

35. 35. A formulation according to any one of claims 1 to 34, which is free or substantially free of cell culture host cell proteins having lipase and / or hydrolase activity.

36. 36. The formulation of any one of claims 1 to 35, wherein the formulation is a pharmaceutical formulation and the one or more excipients are pharmaceutically acceptable.

37. 37. The formulation of any one of claims 1 to 36, wherein the protein having an Fc region is purified from cell culture by Fc-binding affinity resin chromatography.

38. 38. The formulation of claim 37, wherein the Fc-binding affinity resin chromatography is a Protein A chromatography column or a Protein G chromatography column.

39. 1. A method for purifying a protein having an Fc region from a harvested cell culture, comprising: (i) loading the harvested cell culture containing the protein having an Fc region onto an Fc-binding affinity resin chromatography column; (ii) washing the column one, two, three or more times with one or more wash buffers, at least one of which comprises a pH greater than 9; and (iii) eluting the protein having an Fc region from the column and optionally having one or more excipients comprising polysorbate, for making a highly concentrated formulation of said protein.

40. 40. The method of claim 39, further comprising, after step (iii), formulating the protein of step (iii) into a formulation comprising adding one or more excipients, wherein the concentration of the protein in the formulation is greater than 50 g / L, and wherein the one or more excipients comprise polysorbate.

41. 41. The method of claim 39 or 40, wherein the polysorbate is polysorbate 20.

42. 41. The method of claim 39 or 40, wherein the polysorbate is polysorbate 80.

43. 43. The method of any one of claims 39 to 42, wherein the polysorbate in the formulation is at a concentration of about 0.002% to 0.2%.

44. 43. The method of any one of claims 39 to 42, wherein the polysorbate in the formulation is at a concentration of 0.01 to 0.05%.

45. The method of any one of claims 39 to 44, wherein the Fc-binding affinity resin chromatography column is a Protein A chromatography column.

46. The method of any one of claims 39 to 44, wherein the Fc-binding affinity resin chromatography column is a Protein G chromatography column.

47. The method according to any one of claims 39 to 46, wherein the Fc-binding affinity resin chromatography column load ratio in step (i) is 35 g / L or less.

48. The method according to any one of claims 39 to 46, wherein the Fc-binding affinity resin chromatography column load ratio in step (i) is 25 g / L or less.

49. 49. The method of any one of claims 39 to 48, wherein the at least one wash buffer comprises a pH of at least 10 or above.

50. 49. The method of any one of claims 39 to 48, wherein the at least one wash buffer comprises a pH of at least or above, or about 10.

5.

51. 51. The method of any one of claims 39 to 50, wherein the at least one wash buffer comprises 1 M sodium chloride or a higher concentration of sodium chloride.

52. 51. The method of any one of claims 39 to 50, wherein the at least one wash buffer comprises a sodium chloride concentration of less than 1 M or less than 0.5 M.

53. 53. The method of any one of claims 39 to 52, wherein the at least one wash buffer comprises arginine.

54. 53. The method of any one of claims 39 to 52, wherein the at least one wash buffer does not contain arginine.

55. 55. The method of any one of claims 39 to 54, wherein the at least one wash buffer comprises sodium phosphate or sodium carbonate.

56. 56. The method of claim 55, wherein the at least one wash buffer comprises 50 mM sodium carbonate.

57. 57. The method of any one of claims 39 to 56, wherein the concentration of the protein having an Fc region in the formulation is about 100 g / L or more.

58. 58. The method of any one of claims 39 to 57, wherein the concentration of the protein having an Fc region in the formulation is about 150 g / L, about 170 g / L, or about 200 g / L, or higher.

59. 59. The method of any one of claims 39 to 58, wherein the formulation is a pharmaceutical formulation.

60. The method of any one of claims 39 to 59, wherein the protein having an Fc region is an antibody.

61. 61. The method of claim 60, wherein the antibody is a humanized antibody or a human monoclonal antibody.

62. 62. The method of claim 60 or 61, wherein the antibody is an IgG1 antibody.

63. The method of any one of claims 60 to 62, wherein the antibody is an anti-FcRn antibody that specifically binds to FcRn.

64. 64. The method of claim 63, wherein the anti-FcRn antibody is a fully human monoclonal IgG1 antibody.

65. The anti-FcRn antibody (a) (i) the three heavy chain CDR amino acid sequences of SEQ ID NO:27 (HCDR1), SEQ ID NO:28 (HCDR2), and SEQ ID NO:29 (HCDR3); and (ii) the three light chain CDR amino acid sequences of SEQ ID NO:30 (LCDR1), SEQ ID NO:31 (LCDR2), and SEQ ID NO:32 (LCDR3); or (b) (i) three heavy chain CDR amino acid sequences of SEQ ID NO: 49 (HCDR1), SEQ ID NO: 22 (HCDR2), and SEQ ID NO: 23 (HCDR3); and (ii) three light chain CDR amino acid sequences of SEQ ID NO: 50 (LCDR1), SEQ ID NO: 25 (LCDR2), and SEQ ID NO: 26 (LCDR3).

65. The method of claim 63 or 64, comprising:

66. The anti-FcRn antibody (a) a heavy chain variable region amino acid sequence of SEQ ID NO: 6 and a light chain variable region amino acid sequence of SEQ ID NO: 16; or (b) a heavy chain amino acid sequence of SEQ ID NO: 51; and a light chain amino acid sequence of SEQ ID NO:

52.

66. The method of any one of claims 63 to 65, comprising:

67. 67. The method of any one of claims 39 to 66, wherein the harvested cell culture is cell culture medium harvested from host cells used to produce the protein having an Fc region, optionally wherein the production is by introducing into the host cells a vector comprising the protein having an Fc region and culturing the host cells under conditions suitable for expression of the protein, and optionally wherein the host cells are mammalian cells suitable for the production.

68. 67. The method of claim 66, wherein the host cell is a CHO cell.

69. 69. The method of claim 67 or 68, wherein the host cells are harvested by centrifugation, optionally followed by filtration.

70. 70. The method of any one of claims 39 to 69, wherein after step (iii), the eluate containing the protein having an Fc region is subjected to filtration.

71. 71. The method of any one of claims 39 to 70, wherein after step (iii), the eluate containing the proteins having an Fc region is subjected to low pH viral inactivation.

72. 72. The method of any one of claims 39 to 71, wherein the eluate containing the protein having an Fc region is subjected to anion exchange (AEX) chromatography and / or cation exchange (CEX) chromatography.

73. 73. The method of any one of claims 39 to 72, wherein after step (iii), and optionally after one, two, three or all of the steps of claims 70 to 72, the eluate comprising the protein having an Fc region is subjected to concentration.

74. The method of any one of claims 39 to 73, wherein the protein having an Fc region is an anti-FcRn antibody that specifically binds to FcRn, and the formulation is a pharmaceutical formulation comprising at least or about 170 g / L of the anti-FcRn antibody, 0.02% polysorbate 20, 100 mM L-histidine / histidine HCl, and 100 mM L-arginine HCl in water at pH 6.

0.

75. A formulation produced using the method of any one of claims 39 to 74.

76. 76. The formulation of claim 75, wherein the polysorbate comprises polysorbate 20 (PS20) or polysorbate 80 (PS80), and the concentration of the PS20 or PS80 remains within 30% of the starting concentration of the PS20 or PS80 after storage for a period of at least 1 day, and / or the presence of degradation products of PS20 or PS80 does not increase by more than 2-fold the starting concentration of the degradation products after storage for a period of at least 1 day.

77. 77. The formulation of claim 76, wherein the concentration of PS20 or PS80 remains within 20% of the starting concentration of PS20 or PS80 after storage.

78. 77. The formulation of claim 76, wherein the concentration of PS20 or PS80 remains within 10% of the starting concentration of PS20 or PS80 after storage.

79. 77. The formulation of claim 76, wherein the concentration of PS20 or PS80 remains within 5% of the starting concentration of PS20 or PS80 after storage.

80. 80. The formulation of any one of claims 75 to 79, wherein the degradation product of PS20 or PS80 is a fatty acid or an ester thereof.

81. 80. The formulation of any one of claims 75 to 79, wherein the PS20 degradation products are non-esterified species of PS20.

82. 82. The formulation of claim 81, wherein the non-esterified species of PS20 is sorbitan or isosorbide.

83. 80. The formulation of any one of claims 75 to 79, wherein the PS20 degradation product is lauric acid.

84. 80. The formulation of any one of claims 75 to 79, wherein the PS80 degradation product is oleic acid.

85. 85. The formulation of any one of claims 75 to 84, wherein the presence of PS20 or PS80 degradation products does not increase by more than 50% of the starting concentration of said degradation products after storage.

86. 85. The formulation of any one of claims 75 to 84, wherein the presence of PS20 or PS80 degradation products does not increase by more than 25% of the starting concentration of said degradation products after storage.

87. 85. The formulation of any one of claims 75 to 84, wherein the presence of PS20 or PS80 degradation products does not increase by more than 10% of the starting concentration of said degradation products after storage.

88. 85. The formulation of any one of claims 75 to 84, wherein the concentration of polyester species in PS20 or PS80 remains within 10% or 20% of the starting concentration of said polyester species after storage for a period of at least 1 day.

89. 85. A formulation according to any one of claims 75 to 84, wherein the concentration of monoester species of PS20 or PS80 remains within 10% or 20% of the starting concentration of said monoester species after storage for a period of at least 1 day.

90. 90. The formulation of any one of claims 75 to 89, wherein storage for a period of at least 1 day is storage for 1, 3 or 7 days.

91. 90. The formulation of any one of claims 75 to 89, wherein storage for a period of at least one day is storage for about 14 days or 28 days or more.

92. 90. The formulation of any one of claims 75 to 89, wherein storage for a period of at least 1 day is storage for about 8 weeks or 12 weeks or more.

93. 90. The formulation of any one of claims 75 to 89, wherein storage for a period of at least one day is storage for about one year or more.

94. The formulation of any one of claims 75 to 93, wherein the storage is at 2 to 8°C.

95. 94. The formulation of any one of claims 75 to 93, wherein the storage is at 25°C.

96. 94. The formulation of any one of claims 75 to 93, wherein the storage is at 40°C.

97. 97. A formulation according to any one of claims 75 to 96, which is free or substantially free of cell culture host cell proteins having lipase, esterase and / or hydrolase activity.

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