Methods for reducing lipase activity

Abstract

A pharmaceutical formulation is provided that includes an antibody that specifically binds to human interleukin-4 receptor alpha (hIL-4Rα). In addition to an anti-IL-4Rα antibody, the formulation may contain one or more buffers, at least one amino acid, at least one sugar, and a surfactant including polysorbate, polyethylene glycol, or poloxamer. A method for producing a pharmaceutical formulation with reduced lipase activity is also provided, which may include subjecting the drug substance to anion exchange chromatography under acidic conditions, agitation stress, heat stress, and further ion exchange or size exclusion chromatography. In one embodiment, the pharmaceutical formulation does not show the formation of sub-visible particles that can be recognized in the presence of lipase during storage and after being subjected to heat and other physical stresses, showing a significant degree of antibody stability.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application incorporates by reference priority to and the benefit of Provisional Patent Application No. US 63 / 337,532, filed May 2, 2022, Provisional Patent Application No. 63 / 436,850, filed January 3, 2023, and Provisional Patent Application No. 63 / 499,441, filed May 1, 2023. Sequence Listing This application has been submitted electronically in .xml format and contains a Sequence Listing, which is incorporated herein by reference in its entirety. The .xml copy generated on January 31, 2023 is named 070816-03480.xml and is 180,926 bytes in size. Field The present invention relates to the field of therapeutic protein formulations. More specifically, the present invention relates to the field of pharmaceutical formulations with reduced esterase or lipase activity and / or reduced formation of free fatty acid particles. [Background technology]

[0002] Among pharmaceutical products, protein-based biotherapeutics are an important class of drugs, offering high levels of selectivity, potency and efficacy, as evidenced by the considerable increase in clinical trials with monoclonal antibodies (mAbs) over the past few years. One important aspect for clinically and commercially available biotherapeutics is the stability of the drug product with respect to the manufacturing process as well as shelf life. To enhance the physical stability of protein-based biotherapeutic products, surfactants such as polysorbates are often used. More than 70% of commercially available monoclonal antibody therapeutics contain 0.001%-0.2% polysorbate, a type of surfactant, to provide physical stability to protein-based biotherapeutics.

[0003] Enzymatic hydrolysis of polysorbates has been recognized as the major pathway of polysorbate degradation in biotherapeutic formulations. Polysorbate degradation can be caused by co-production of esterases or lipases during drug product manufacturing. Polysorbate hydrolysis results in the release of free fatty acids that can drive undesirable particulate formulations in drug products. Particles can be visible or subvisible, with subvisible particles typically being less than 150 or 100 microns in diameter.

[0004] Thus, a need exists for pharmaceutical products that have reduced esterase or lipase activity and reduced formation of free fatty acid particles, and methods for making such pharmaceutical products. Summary of the Invention

[0005] Products and methods have been developed for reducing esterase activity in a composition or formulation, reducing lipase activity in a composition or formulation, reducing polysorbate degradation, and reducing fatty acid particle formation.In one embodiment, a sample containing a protein of interest and an esterase or lipase is subjected to hydrophobic interaction chromatography to produce a compounded drug substance and / or drug product with reduced esterase and / or lipase activity, reduced polysorbate degradation, and / or reduced free fatty acid particle formation compared to a sample that has not been subjected to hydrophobic interaction chromatography.In one embodiment, a sample containing a protein of interest and an esterase or lipase is subjected to anion exchange chromatography using a high loading pH to produce a compounded drug substance and / or drug product with reduced esterase and / or lipase activity, reduced polysorbate degradation, and / or reduced fatty acid particle formation compared to a sample that has not been subjected to anion exchange chromatography with a high loading pH.

[0006] In one embodiment, a sample containing a protein of interest and an esterase or lipase is formulated with a polysorbate containing a high concentration of oleic acid, e.g., ≧98% oleic acid, to produce a formulated drug substance and / or drug product with reduced esterase and / or lipase activity, reduced polysorbate degradation, and / or reduced fatty acid particle formation, compared to a sample not formulated with polysorbate. Alternatively, in one embodiment, a sample containing a protein of interest and an esterase or lipase is formulated with poloxamer 188 or PEG3350 to produce a formulated drug substance and / or drug product with reduced esterase and / or lipase activity, reduced polysorbate degradation, and / or reduced fatty acid particle formation, compared to a sample not formulated with poloxamer 188 or PEG3350. In some embodiments, the protein of interest is an anti-IL4Rα antibody. In some embodiments, the anti-IL-4Rα antibody is dupilumab.

[0007] In one embodiment, a compounded drug substance comprising a protein of interest, a fatty acid ester and an esterase or lipase is subjected to stress, e.g., agitation stress or heat stress, to inactivate the esterase or lipase, degrade the esterase or lipase, and / or reduce the esterase or lipase activity, which may cause the formation of high molecular weight (HMV) species of the esterase or lipase and / or drug protein. The substance may then be subjected to filtration and / or separation, e.g., using cation exchange chromatography or size exclusion chromatography, to remove the HMW species and produce a compounded drug substance and / or drug product with reduced esterase or lipase activity, reduced polysorbate degradation, and / or reduced free fatty acid particle formation, compared to a sample that has not been subjected to stress and / or filtration / separation. In some embodiments, the protein of interest is an anti-IL4Rα antibody. In some embodiments, the anti-IL-4Rα antibody is dupilumab.

[0008] The present disclosure provides further methods for producing pharmaceutical compositions with reduced esterase or lipase. In some embodiments, these methods may include subjecting a sample containing a protein of interest and an esterase or lipase to anion exchange chromatography, where the pH of the sample loaded onto the chromatography column is about 7.8 to about 8.3.

[0009] In some further embodiments, a method for producing a pharmaceutical composition with reduced esterase or lipase activity may include (a) subjecting a sample containing a protein of interest and an esterase or lipase to stress conditions to form a sample in which the esterase or lipase is inactivated, and (b) combining said sample containing the inactivated esterase or lipase to produce a pharmaceutical composition with reduced esterase or lipase activity.

[0010] In one embodiment, said protein of interest is an antibody, an antibody-derived protein, an antibody fragment, a monoclonal antibody, a bispecific antibody, a fusion protein, an antibody-drug conjugate, or a therapeutic protein.

[0011] In one embodiment, the combining step comprises adding a fatty acid ester to the sample. In a particular embodiment, the fatty acid ester is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. In a particular embodiment, the fatty acid ester is polysorbate 80, and the concentration of oleic acid ester in the polysorbate 80 is at least 80%. In a more particular embodiment, the concentration of oleic acid ester in the polysorbate 80 is at least 98% or at least 99%.

[0012] In one embodiment, the stress conditions include agitation stress and / or heat stress. In a particular embodiment, the agitation stress includes shaking the sample at 50-500 rpm, 200-300 rpm, about 50 rpm, about 75 rpm, about 100 rpm, about 125 rpm, about 150 rpm, about 200 rpm, about 225 rpm, about 250 rpm, about 275 rpm, about 300 rpm, about 325 rpm, about 350 rpm, about 375 rpm, about 400 rpm, about 425 rpm, about 450 rpm, about 475 rpm, or about 500 rpm. In another specific embodiment, the agitation stress comprises shaking the sample for 1 to 96 hours, 24 to 48 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours.

[0013] In further specific embodiments, the heat stress comprises storing the sample at about 20° C. to about 60° C., about 25° C. to about 55° C., about 40° C. to about 50° C., about 44° C. to about 46° C., about 20° C., about 25° C., about 30° C., about 35° C., about 40° C., about 45° C., about 50° C., about 55° C., or about 60° C. In more specific embodiments, the storage is for 1 day to 6 months, 3 days to 3 months, 1 week to 2 months, 0.5 months to 1 month, about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, about 0.5 months, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 3 months, about 3.5 months, about 4 months, about 4.5 months, about 5 months, about 5.5 months, or about 6 months.

[0014] In one embodiment, the method further comprises subjecting the sample in which the esterase or lipase has been inactivated to filtration, concentration, or chromatographic separation to remove inactivated lipase and / or protein HMW species prior to step (b). In a particular embodiment, the chromatographic separation comprises cation exchange chromatography. In another particular embodiment, the chromatographic separation comprises size exclusion chromatography.

[0015] In one aspect, the combining step comprises adding an excipient to the sample.

[0016] The present disclosure provides further methods for producing a pharmaceutical composition with reduced esterase or lipase activity. In some embodiments, the method may include the steps of (a) subjecting the collected antibody to affinity chromatography, (b) subjecting the pooled antibody from the eluate of step (a) to viral inactivation at a pH of about 3 to about 4, and then adjusting the pH to about 5 to about 8, (c) subjecting the pooled antibody from step (b) to anion exchange chromatography in flow-through mode, (d) subjecting the pooled antibody from the eluate of step (c) to hydrophobic interaction chromatography in flow-through mode, (e) subjecting the pooled antibody from the flow-through fraction of step (d) to virus-retaining filtration, and (f) subjecting the sample from step (e) containing the antibody of interest and the esterase or lipase to agitation stress or heat stress to form a pharmaceutical composition with reduced esterase or lipase activity.

[0017] In other aspects, the method may comprise the steps of: (a) subjecting the harvested antibodies to affinity chromatography; (b) subjecting the pooled antibodies from the eluate of step (a) to viral inactivation at a pH of about 3 to about 4, and then adjusting the pH to about 5 to about 8; (c) subjecting the pooled antibodies from step (b) to anion exchange chromatography in flow-through mode; (d) subjecting the pooled antibodies from the flow-through fraction of step (c) to virus-retaining filtration; and (e) subjecting the sample from step (d) comprising the antibody of interest and an esterase or lipase to agitation or heat stress to form a pharmaceutical composition having reduced esterase or lipase activity.

[0018] In one embodiment, the method further comprises subjecting the pharmaceutical composition to filtration, concentration, or chromatographic separation to remove HMW species. In certain embodiments, the chromatographic separation comprises ion exchange chromatography, cation exchange chromatography, or size exclusion chromatography.

[0019] In one embodiment, the pH of the sample loaded onto the AEX column is from about 7.8 to about 8.3.

[0020] The present disclosure provides methods for producing pharmaceutical compositions with increased stability. In some embodiments, the methods may include reducing esterase or lipase activity in a composition by subjecting a sample containing a protein of interest, an esterase or lipase, and a fatty acid ester to anion exchange (AEX) chromatography, where the pH of the sample loaded onto the AEX column is about 7.8 to about 8.3.

[0021] In one embodiment, the method further comprises subjecting the sample formed after AEX chromatography to heat or agitation stress to reduce esterase or lipase activity, hi certain embodiments, the method further comprises subjecting the stressed sample to chromatographic separation to remove HMW species of the esterase or lipase.

[0022] In one embodiment, the method further comprises the addition of poloxamer 188 or PEG 3350 to produce a formulation comprising a pharmaceutical composition having reduced esterase or lipase activity and increased stability. In certain embodiments, the formulation is substantially free of polysorbates. In another specific embodiment, the concentration of the poloxamer 188 is 0.005% to 5%, 0.05% to 2.5%, 0.1% to 1%, about 0.05%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. In yet another specific embodiment, the concentration of PEG3350 is 0.005% to 5%, 0.05% to 2.5%, 0.1% to 1%, about 0.05%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.

[0023] The present disclosure provides further methods for producing pharmaceutical compositions with increased stability. In some embodiments, the methods may include: (a) subjecting the harvested recombinant protein to anion exchange chromatography in flow-through mode; (b) subjecting the flow-through fraction from step (a) to hydrophobic interaction chromatography in flow-through mode; and (c) combining the isolated recombinant protein from step (b) with polysorbate 80, wherein the concentration of oleic acid esters in the polysorbate 80 is at least 80%.

[0024] In one embodiment, the concentration of oleate is at least 98% or at least 99%.

[0025] In one embodiment, the pH of the sample loaded onto the anion exchange chromatography column is about 7.8 to about 8.3, about 7.9 to about 8.2, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, or about 8.3.

[0026] In some embodiments, the protein or recombinant protein of interest is an anti-IL4Rα antibody. In some embodiments, the anti-IL-4Rα antibody is dupilumab.

[0027] These and other aspects of the present invention will be better appreciated and understood when considered in conjunction with the following description and the accompanying drawings. The following description, while indicating various embodiments and many specific details thereof, is offered by way of illustration and not by way of limitation. Many substitutions, modifications, additions or rearrangements may be made within the scope of the present invention. [Brief description of the drawings]

[0028] [Figure 1] FIG. 1 shows chromatograms illustrating the relative amounts of different molecular species including (A) a lower quality polysorbate 20 (PS20-A), (B) a higher quality polysorbate 20 (PS20-B), and (C) polysorbate 80 (PS80), according to one embodiment.

[0029] [Figure 2A] FIG. 2A shows particle formation in a mAb1 formulation with SR-PS80 as measured by membrane microscopy, according to one embodiment.

[0030] [Figure 2B] FIG. 2B shows particle formation in a mAb1 formulation with HP-PS20 as measured by membrane microscopy, according to one embodiment.

[0031] [Figure 2C] FIG. 2C shows particle formation in a mAb1 formulation with SR-PS80 as measured by MFI, according to one embodiment.

[0032] [Figure 2D] FIG. 2D shows particle formation in a mAb1 formulation with HP-PS20 as measured by MFI, according to one embodiment.

[0033] [Figure 3A] FIG. 3A shows the recovery of polysorbate in mAb1 formulations with SR-PS80 as measured by CAD-UHPLC, according to one embodiment.

[0034] [Figure 3B] FIG. 3B shows the recovery of polysorbate in mAb1 formulations with HP-PS20 as measured by CAD-UHPLC, according to one embodiment.

[0035] [Figure 4] FIG. 4 shows the correlation between particle formation and polysorbate recovery in mAb1 formulations, according to one embodiment.

[0036] [Diagram 5] FIG. 5 shows the correlation between particle formation and polysorbate recovery in mAb5 formulations, according to one embodiment.

[0037] [Figure 6A] FIG. 6A shows particle formation in a mAb1 formulation containing SR-PS80 and subjected to HIC purification as measured by membrane microscopy, according to one embodiment.

[0038] [Figure 6B] FIG. 6B shows particle formation in a mAb1 formulation containing HP-PS20 and subjected to HIC purification as measured by membrane microscopy, according to one embodiment.

[0039] [Figure 6C] FIG. 6C shows particle formation in a mAb1 formulation containing SR-PS80 and subjected to HIC purification as measured by MFI, according to one embodiment.

[0040] [Figure 6D] FIG. 6D shows particle formation in a mAb1 formulation containing HP-PS20 and subjected to HIC purification as measured by MFI, according to one embodiment.

[0041] [Figure 7A] FIG. 7A shows the recovery of polysorbate in a mAb1 formulation containing SR-PS80 and subjected to HIC purification as measured by CAD-UHPLC, according to one embodiment.

[0042] [Figure 7B] FIG. 7B shows the recovery of polysorbate in a mAb1 formulation containing HP-PS20 and subjected to HIC purification as measured by CAD-UHPLC, according to one embodiment.

[0043] [Figure 8] FIG. 8 shows the correlation between phospholipase activity (parts per million) and percent degradation of polysorbate 20, according to one embodiment.

[0044] [Figure 9] FIG. 9 shows a visualization of a model generated for a multivariate study of AEX risk factors and response, according to one embodiment.

[0045] [Figure 10] FIG. 10 shows a comparison of AEX pool lipase activity (%) performance in validation batches compared to small scale predictions from Monte Carlo simulations, according to one embodiment.

[0046] [Figure 11] FIG. 11 shows polysorbate recovery from formulations containing mAb5, mAb6 and mAb7, according to one embodiment.

[0047] [Figure 12]FIG. 12 shows polysorbate recovery from mAb5 formulations at various concentrations and incubation times, according to one embodiment.

[0048] [Figure 13] FIG. 13 shows homology modeling illustrating the surface hydrophobicity of mAb5, mAb6 and mAb7, according to one embodiment.

[0049] [Figure 14] FIG. 14 shows the number of subvisible particles (≧10 μm) measured by membrane microscopy in a protein drug product containing various types of polysorbate 80 according to one embodiment.

[0050] [Figure 15] FIG. 15 shows the number of subvisible particles (≧10 μm) as measured by microflow imaging (MFI) in a protein drug product containing various types of polysorbate 80, according to one embodiment.

[0051] [Figure 16] FIG. 16 shows the chemical structure of polyoxyethylene (20) sorbitan monooleate, the most prominent fatty acid ester in polysorbate 80, according to one embodiment.

[0052] [Figure 17] FIG. 17 shows the measured concentrations of free fatty acids in protein drug products containing various types of polysorbate 80, according to one embodiment.

[0053] [Figure 18A] FIG. 18A illustrates polysorbate structures and major degradation pathways, according to one embodiment.

[0054] [Figure 18B] FIG. 18B illustrates the structure of poloxamer 188, according to one embodiment.

[0055] [Figure 19] FIG. 19 shows surfactant recovery for mAb5 formulations with PS20, PS80, or poloxamer 188 (P188) at various temperatures, according to one embodiment.

[0056] [Figure 20] FIG. 20 shows the number of particles ≧10 μm (top panel) and ≧25 μm (bottom panel) identified by membrane microscopy in 150 mg / mL formulations of anti-IL-4R antibody containing lipase and either PEG3350 or poloxamer 188 (at concentrations of 0.02%, 0.04%, or 0.1% w / v) stored at 5° C. for up to 36 months according to one embodiment.

[0057] [Figure 21A] FIG. 21A shows the effect of PEG3350 concentration on the stability (as percentage of HMW species as measured by SE-UPLC) of a 150 mg / mL anti-IL-4R antibody formulation against agitation stress at room temperature for 30 to 120 minutes.

[0058] [Figure 21B] FIG. 21B shows the effect of poloxamer 188 concentration on the stability (as percentage of HMW species as measured by SE-UPLC) of a 150 mg / mL formulation of anti-IL-4R antibody to agitation stress at room temperature for 30 to 120 minutes.

[0059] [Figure 22] FIG. 22 shows the effect of polysorbate 20, polysorbate 80, PEG3350, and poloxamer 188 (various concentrations) on the stability (as percentage of HMW species as measured by SE-UPLC) of a 150 mg / mL formulation of anti-IL-4R antibody to heat stress (45° C.) for up to 56 days. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0060] Therapeutic macromolecules must be formulated in a manner that not only renders the molecule suitable for administration to a patient, but also maintains its stability during storage. For example, therapeutic antibodies in liquid solutions are prone to degradation, aggregation and / or undesirable chemical modifications unless the solution is properly formulated. The stability of antibodies in liquid formulations depends not only on the types of excipients used in the formulation, but also on the amount of excipients and the ratio of excipients to each other. Therapeutic formulations may also be subject to the formation of particulate matter over time during storage. The particles may be visible or subvisible, with subvisible particles typically being less than 150 or 100 microns in diameter. Formulations with high protein concentrations, for example concentrations of 30 mg / mL or higher, tend to be more prone to aggregation and form subvisible particles.

[0061] Polysorbate 20 (PS20) and polysorbate 80 (PS80) are the most commonly used non-ionic surfactants in biopharmaceutical protein formulations to improve protein stability and protect protein products from aggregation and denaturation (Martos et al., J Pharm Sci. 106(7):1722-1735 (2017); Kiese et al., J Pharm Sci 97(19):4347-4366 (2008); Dwivedi et al., Int J Pharm 552(1-2):422-436 (2018)). However, polysorbates, including polysorbate 20 and polysorbate 80, can degrade in the presence of lipases, which over time results in the formation of subvisible particles in the formulation.

[0062] PS is known to be susceptible to degradation through two main pathways: autoxidation and hydrolysis (Dwivedi et al.; Kishore et al., Pharm Res. 28(5): 1194-1210 (2011); Larson et al., J Pharm Sci. 109(10):633-639 (2020);Kishore et al. J Pharm Sci. 100(2):721-731 (2011)). Enzymatic hydrolysis is considered to be the major pathway of PS degradation in high-concentration protein formulations, which results in the accumulation of free fatty acids (FFAs) that can drive undesirable particulate formation in drug products. Oxidation is the second major pathway of PS degradation, leading to the formation of peroxides, aldehydes, ketones, and short-chain esterified POE sorbitan / isosorbide species (Kishore et al. 2011a; Larson et al.; Kishore et al. 2011b; Donbrow et al., J Pharm Sci. 67(12):1676-1681 (1978); Yao et al. Pharm Res. 26(10):2303-2313 (2009)). PS hydrolysis has been considered a greater threat to drug product quality since this process not only reduces PS concentration but is also associated with particulate formation due to the low solubility of accumulated FFAs, especially at storage temperatures of 2°C to 8°C (Doshi et al., J Pharm Sci. 110(2):687-692 (2021);Saggu et al., J Pharm Sci. 110(3):1093-1102 (2021);Doshi et al. Mol Pharm. 12(11):3792-3804 (2015)).

[0063] Residual lipases or esterases present in pharmaceutical products are the major cause of PS hydrolysis (Chiu et al., Biotechnol Bioeng. 114(5):1006-1015 (2017);Hall et al. J Pharm Sci. 105(5):1633-1642 (2016);Labrenz et al. J Pharm Sci. 103(8):2268-2277 (2014);McShan et al. PDA J Pharm Sci Technol. 70(4):332-345 (2016);Zhang et al. J Pharm Sci. 109(11):3300-3307 (2020);Zhang et al. J Pharm Sci. 109(9):2710-2718 (2020)). Residual lipase levels in the final drug product are typically very low (<10 ppm) after multiple steps of downstream purification, and the consequences of PS degradation may not be noticed until after months or years of storage at typical storage temperatures (2°C to 8°C).

[0064] Without intending to be bound by theory, the putative phospholipase B-like 2 (PLBL2), which is highly conserved in hamsters, rats, mice, humans and cows, is co-purified with certain proteins under certain processes.Other esterases or lipases can also be co-purified with the protein of interest at concentrations that are too low to be reliably detected, but high enough to have measurable lipase activity, ultimately resulting in loss of polysorbate, production of free fatty acid, and formation of particles that may or may not be visible to the naked eye.Therefore, there is a need for pharmaceutical products with reduced lipase activity, reduced esterase activity, and reduced fatty acid particle formation, and methods for making such pharmaceutical products.

[0065] Disclosed herein are novel pharmaceutical products, compositions and formulations with reduced lipase activity, reduced esterase activity and reduced fatty acid particle formation, and methods for making them.The inventors have surprisingly discovered that adding a hydrophobic interaction chromatography (HIC) step to the manufacturing process of a protein of interest can effectively remove esterase and lipase activity and prevent particle formation in pharmaceutical products.Furthermore, by improving conventional pH loading conditions, the inventors have discovered conditions that are optimized for an anion exchange (AEX) chromatography step to effectively reduce esterase and lipase activity.The inventors have discovered that polysorbate content, especially polysorbate 80, can be optimized at high oleic acid concentrations to make fewer fatty acid particles form, even in pharmaceutical products that contain esterase and lipase activity. Alternative surfactants to polysorbates were also investigated, and it was surprisingly discovered that PEG3350 and poloxamer 188 are resistant to esterase or lipase activity, and can successfully stabilize proteins while preventing fatty acid particle formation. After preparation of the drug substance, it was further surprisingly discovered that the co-purified esterase or lipase can be successfully inactivated and / or removed by applying stress, such as stirring stress or heat stress, which can degrade the esterase or lipase and reduce lipase activity. The application of stress can also lead to the formation of high molecular weight (HMW) species. HMW species can be removed from the drug substance, for example, using molecular weight filtration or chromatography, such as cation exchange chromatography or size exclusion chromatography. These and other aspects of the invention are described in more detail below.

[0066] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing, specific methods and materials are described herein.

[0067] The term "a" should be understood to mean "at least one," and the terms "about" and "approximately" should be understood to allow for standard variations that would be understood by one of ordinary skill in the art, and when ranges are provided, the endpoints are included. As used herein, the terms "include," "includes," and "including" mean without limitation and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0068] As used herein, the term "protein" or "protein of interest" may include any amino acid polymer having covalently linked amide bonds. A protein includes one or more amino acid polymer chains, commonly known in the art as "polypeptides." A "polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof linked through peptide bonds. A "synthetic peptide or polypeptide" refers to a non-naturally occurring peptide or polypeptide. A synthetic peptide or polypeptide may be synthesized, for example, using an automated polypeptide synthesizer. A variety of solid-phase peptide synthesis methods are known to those of skill in the art. A protein includes one or many polypeptides that form a single functional biomolecule. In another exemplary embodiment, the protein may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, and the like. A protein of interest may also include any biotherapeutic protein, recombinant proteins used in research or therapy, trap proteins and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, bispecific antibodies, and antigen binding proteins.

[0069] Recombinant cell-based production systems, such as insect baculovirus systems, yeast systems (e.g., Pichia spp.), and mammalian systems (e.g., CHO cells and CHO derivatives, e.g., CHO-K1 cells) may be used to produce proteins. For a recent review discussing biotherapeutic proteins and their production, see, for example, Ghaderi et al., "Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation" (Darius Ghaderi et al., Production platforms for biotherapeutic glycoproteins. Occurrence, impact, and challenges of non-human sialylation, 28 BIOTECHNOLOGY AND GENETIC ENGINEERING REVIEWS 147-176 (2012)), the entire disclosure of which is incorporated herein by reference. In some embodiments, the proteins include modifications, additions, and other covalent linking moieties. These modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, glycans (e.g., N-acetylgalactosamine, galactose, neuraminic acid, N-acetylglucosamine, fucose, mannose, and other monosaccharides), PEG, polyhistidine, FLAG tags, maltose binding protein (MBP), chitin binding protein (CBP), glutathione-S-transferase (GST), myc epitopes, fluorescent labels and other dyes, etc. Proteins may be classified based on composition and solubility, and thus may include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucoproteins, chromoproteins, phosphoproteins, metalloproteins, and lipoproteins; and derived proteins, such as primary and secondary derived proteins.

[0070] As used herein, the term "recombinant protein" refers to a protein produced as a result of transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In certain embodiments, the recombinant protein may be an antibody, such as a chimeric, humanized, or fully human antibody. In certain embodiments, the recombinant protein may be an antibody of an isotype selected from the group consisting of: IgG, IgM, IgA1, IgA2, IgD, or IgE. In certain embodiments, the antibody molecule is a full-length antibody (e.g., IgG1), or the antibody may be a fragment (e.g., an Fc fragment or a Fab fragment). antibody

[0071] The term "antibody", as used herein, is generally intended to refer to immunoglobulin molecules that contain four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, as well as multimers thereof (e.g., IgM); however, immunoglobulin molecules consisting only of heavy chains (i.e., lacking light chains) are also included within the definition of the term "antibody". Each heavy chain contains a heavy chain variable region (abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region contains three domains, CH1, CH2, and CH3. Each light chain contains a light chain variable region (abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region contains one domain (CL1). The VH and VL regions can be further divided into regions of hypervariability called complementarity determining regions (CDRs), interspersed with more conserved regions called framework regions (FRs). Each VH and VL is composed of three CDRs and four FRs, arranged from amino-terminus to carboxy-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0072] In some embodiments, the protein of interest is a human antibody. The term "human antibody" as used herein is intended to include antibodies with variable and constant regions derived from human germline immunoglobulin sequences. The human antibodies of the present disclosure may contain amino acid residues not encoded by human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutation in vivo), for example in the CDRs, and particularly in CDR3. However, the term "human antibody" as used herein is not intended to include antibodies in which CDR sequences derived from the germline of another mammalian species, for example a mouse, are grafted onto human framework sequences.

[0073] Interleukin 4 (IL-4) and interleukin 13 (IL-13) are key cytokines in driving allergy and type 2 T helper cell (Th2) biased inflammatory processes. IL-4 and IL-13 signaling are mediated through heterodimeric receptor complexes, where IL-4 receptor alpha (IL-4Rα) is a receptor subunit shared by both IL-4 and IL-13 signaling. Therefore, IL-4Rα is an attractive therapeutic target because it provides a single target for blocking both IL-4 and IL-13 signaling. In some embodiments, the protein of interest is an anti-IL-4R antibody, or an antigen-binding fragment thereof. Antibodies against hIL-4Rα are described, for example, in U.S. Patent Nos. 5,717,072, 7,186,809, and 7,605,237.

[0074] In some embodiments, the anti-IL-4R antibody is a human IgG antibody. In various embodiments, the anti-IL-4R antibody is a human antibody of isotype IgG1, IgG2, IgG3 or IgG4, or mixed isotypes. In some embodiments, the anti-IL-4R antibody is a human IgG1 antibody. In some embodiments, the anti-IL-4R antibody is a human IgG4 antibody. In any of the embodiments discussed above or herein, the anti-IL-4R antibody may comprise a human kappa light chain. In any of the embodiments discussed above or herein, the anti-IL-4R antibody may comprise a human lambda light chain.

[0075] The antibodies of the present disclosure may, in some embodiments, be recombinant human antibodies. The term "recombinant human antibodies," as used herein, is intended to include all human antibodies that are prepared, expressed, generated, or isolated by recombinant means, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies isolated from a recombinant combinatorial human antibody library, antibodies isolated from animals (e.g., mice) that are transgenic for human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucl. Acids Res. 20:6287-6295), or antibodies prepared, expressed, generated, or isolated by any other means that involve splicing of human immunoglobulin gene sequences to other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in certain embodiments, such recombinant human antibodies are subjected to in vitro mutagenesis (or in vivo somatic mutagenesis, when animals transgenic for human Ig sequences are used), thus improving the V and V regions of the recombinant antibodies. H and V L The amino acid sequence of the region is H and V L These sequences are derived from and related to the human antibody germline repertoire, but may not naturally occur within the human antibody germline repertoire in vivo.

[0076] The term "antibody", as used herein, also includes antigen-binding fragments of full-length antibody molecules. The terms "antigen-binding portion" of an antibody, "antigen-binding fragment" of an antibody, etc., as used herein, include any naturally occurring, enzymatically obtainable, synthetic, or genetically engineered polypeptide or glycoprotein that specifically binds to an antigen to form a complex. Antigen-binding fragments of antibodies can be obtained from full-length antibody molecules using any suitable standard technique, such as, for example, proteolytic digestion, or recombinant genetic engineering techniques involving the manipulation and expression of DNA encoding antibody variable domains and, optionally, constant domains. Such DNA is known and / or readily available, for example, from commercial sources, DNA libraries (including, for example, phage-antibody libraries), or may be synthesized. The DNA may be sequenced and manipulated chemically or by using molecular biology techniques, for example, to place one or more variable and / or constant domains in the appropriate configuration, or to introduce codons, generate cysteine ​​residues, modify, add or delete amino acids, etc.

[0077] As used herein, an "antibody fragment" includes a portion of an intact antibody, such as an antigen-binding or variable region of an antibody. Examples of antibody fragments include, but are not limited to, Fab fragments, Fab' fragments, F(ab')2 fragments, scFv fragments, Fv fragments, dsFv diabodies, dAb fragments, Fd' fragments, Fd fragments, and isolated complementarity determining regions (CDRs), as well as triabodies, tetrabodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed from antibody fragments. An Fv fragment is a combination of the variable regions of an immunoglobulin heavy and light chains, and an scFv protein is a recombinant single-chain polypeptide molecule in which an immunoglobulin light and heavy chain variable region is linked by a peptide linker. In some embodiments, an antibody fragment contains sufficient amino acid sequence of the parent antibody of which it is a fragment to bind the same antigen as that bound by the parent antibody; in some embodiments, the fragment binds to an antigen with an affinity comparable to that of the parent antibody and / or competes with the parent antibody for binding to an antigen. Antibody fragments may be produced by any means. For example, antibody fragments may be produced enzymatically or chemically by fragmentation of an intact antibody and / or recombinantly from a gene encoding a partial antibody sequence. Alternatively, or in addition, antibody fragments may be produced synthetically, in whole or in part. Antibody fragments may optionally include single chain antibody fragments. Alternatively, or in addition, antibody fragments may include multiple chains linked together, for example by disulfide linkages. Antibody fragments may optionally include multimolecular complexes. Functional antibody fragments typically include at least about 50 amino acids, more typically at least about 200 amino acids.

[0078] The term "bispecific antibody" includes antibodies capable of selectively binding to two or more epitopes. Bispecific antibodies generally comprise two different heavy chains, where each heavy chain specifically binds to a different epitope, either epitopes on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). When a bispecific antibody is capable of selectively binding to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain for the first epitope will generally be at least one to two or three or four orders of magnitude lower than the affinity of the first heavy chain for the second epitope, or vice versa. The epitopes recognized by a bispecific antibody may be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies may be generated, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, nucleic acid sequences encoding heavy chain variable sequences that recognize different epitopes of the same antigen may be fused to nucleic acid sequences encoding different heavy chain constant regions, and such sequences may be expressed in a cell that expresses an immunoglobulin light chain.

[0079] A typical bispecific antibody has two heavy chains with three heavy chain CDRs followed by a CH1 domain, a hinge, a CH2 domain, and a CH3 domain, and an immunoglobulin light chain that does not confer antigen binding specificity but can either associate with each heavy chain, or can associate with each heavy chain and bind to one or more of the epitopes bound by the heavy chain antigen binding region, or can associate with each heavy chain and allow binding to one or both epitopes of one or both of the heavy chains. bsAbs are divided into two main classes, those that possess an Fc region (IgG-like) and those that lack an Fc region, the latter being usually smaller than Fc-containing IgG and IgG-like bispecific molecules. IgG-like bsAbs can have different formats such as, but not limited to, triomabs, knob-into-hole IgG (kih IgG), cross-Mab, ortho-Fab IgG, dual variable domain Ig (DVD-Ig), two-in-one or dual acting Fab (DAF), IgG single chain Fv (IgG-scFv), or kappa lambda bodies. Different non-IgG-like formats include tandem scFv, diabody formats, single chain antibodies, tandem diabodies (TandAb), dual affinity retargeting molecules (DART), DART-Fc, nanobodies, or antibodies produced by the dock-and-lock (DNL) method (Gaowei Fan, Zujian Wang & Mingju Hao, Bispecific antibodies and their applications, 8 JOURNAL OF HEMATOLOGY & ONCOLOGY 130; Dafne Mueller & Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014) the entire disclosure of which is incorporated herein). Methods for producing bsAbs are not limited to somatic cell fusion of two different hybridoma cell lines, chemical conjugation with chemical cross-linkers, and quadroma technology based on genetic approaches utilizing recombinant DNA technology.

[0080] As used herein, a "multispecific antibody" refers to an antibody that has binding specificities for at least two different antigens. Such molecules usually bind only two antigens (i.e., bispecific antibodies, bsAbs), but antibodies with additional specificities, such as trispecific antibodies and KIH trispecific antibodies, can also be addressed by the systems and methods disclosed herein.

[0081] The term "monoclonal antibody" as used herein is not limited to antibodies produced through hybridoma technology. Monoclonal antibodies may be obtained by any means available or known in the art from a single clone, including eukaryotic, prokaryotic, or phage clones. A wide variety of techniques known in the art may be used to prepare monoclonal antibodies useful in the present disclosure, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof.

[0082] An "isolated antibody," as used herein, is intended to refer to an antibody that is substantially free of other antibodies having different antigen specificities (e.g., an isolated antibody that specifically binds to hIL-4Rα is substantially free of antibodies that specifically bind to antigens other than hIL-4Rα).

[0083] The term "specifically binds" and the like means that an antibody or antigen-binding fragment thereof forms a complex with an antigen that is relatively stable under physiological conditions. Specific binding is at least about 1x10 -6The specific binding of an antibody to hIL-4Rα may be characterized by a dissociation constant of ≥ 1 M. Methods for determining whether two molecules specifically bind are well known in the art and include, for example, equilibrium dialysis, surface plasmon resonance, and the like. An isolated antibody that specifically binds to hIL-4Rα may, however, have cross-reactivity to other antigens, for example, IL-4Rα molecules (orthologs) from other species. In the context of the present disclosure, a multispecific (e.g., bispecific) antibody that binds to hIL-4Rα as well as one or more additional antigens is considered to "specifically bind" to hIL-4Rα. Furthermore, an isolated antibody may be substantially free of other cellular material and / or chemicals. However, in some instances, an isolated antibody may be co-purified with a phospholipase expressed by a mammalian cell line (e.g., CHO cells) in which the anti-IL-4R antibody is produced.

[0084] According to certain embodiments of the present disclosure, the anti-hIL-4R antibody, or antigen-binding fragment thereof, comprises heavy chain complementarity determining regions HCDR1-HCDR2-HCDR3, each comprising the amino acid sequence of SEQ ID NO:3-4-5. According to certain embodiments of the present disclosure, the anti-hIL-4R antibody, or antigen-binding fragment thereof, comprises light chain complementarity determining regions LCDR1-LCDR2-LCDR3, each comprising the amino acid sequence of SEQ ID NO:6-7-8. According to certain embodiments, the anti-hIL-4R antibody, or antigen-binding fragment thereof, comprises CDRs HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, each comprising the amino acid sequence of SEQ ID NO:3-4-5-6-7-8.

[0085] In certain embodiments, the anti-hIL-4R antibody, or antigen-binding fragment thereof, has a heavy chain variable region (HCVR) comprising heavy chain complementarity determining regions HCDR1-HCDR2-HCDR3, each comprising the amino acid sequence of SEQ ID NO:3-4-5, and having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:1. In certain embodiments, the anti-hIL-4R antibody, or antigen-binding fragment thereof, has a light chain variable region (LCVR) comprising light chain complementarity determining regions LCDR1-LCDR2-LCDR3, each comprising the amino acid sequence of SEQ ID NO:6-7-8, and having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:2. In certain embodiments, the anti-hIL-4R antibody, or antigen-binding fragment thereof, has a heavy chain variable region (HCVR) comprising heavy chain complementarity determining regions HCDR1-HCDR2-HCDR3 comprising the amino acid sequences of SEQ ID NOs:3-4-5, respectively, and having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:1; and a light chain variable region (LCVR) comprising light chain complementarity determining regions LCDR1-LCDR2-LCDR3 comprising the amino acid sequences of SEQ ID NOs:6-7-8, respectively, and having at least 90% sequence identity (e.g., at least 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% sequence identity) to the amino acid sequence of SEQ ID NO:2.

[0086] In certain embodiments, the anti-hIL-4R antibody, or antigen-binding fragment thereof, comprises a heavy chain variable region (HCVR) comprising the amino acid sequence of SEQ ID NO:1. In certain embodiments, the anti-hIL-4R antibody, or antigen-binding fragment thereof, comprises a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2. In certain embodiments, the anti-hIL-4R antibody, or antigen-binding fragment thereof, comprises a HCVR / LCVR amino acid sequence pair comprising the amino acid sequence of SEQ ID NO:1 / 2. In certain embodiments, the anti-IL-4R antibody comprises a HCVR / LCVR comprising the amino acid sequence of SEQ ID NO:1 / 2, respectively, and a human IgG1 heavy chain constant region. In some embodiments, the anti-IL-4R antibody comprises a HCVR / LCVR comprising the amino acid sequence of SEQ ID NO:1 / 2, respectively, and a human IgG4 heavy chain constant region. In some embodiments, the anti-IL-4R antibody comprises a HCVR / LCVR comprising the amino acid sequence of SEQ ID NO:1 / 2, respectively, and a human IgG heavy chain constant region. In some embodiments, the anti-IL-4R antibody comprises an HCVR / LCVR comprising the amino acid sequence of SEQ ID NO:1 / 2, respectively, and a human IgG1 or IgG4 heavy chain constant region. In some embodiments, the anti-IL-4R antibody comprises a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10. In some embodiments, the anti-IL-4R antibody is dupilumab.

[0087] Other anti-IL-4R antibodies that may be used in the context of the methods of the present disclosure include, for example, AMG317 (Corren et al., 2010, Am J Respir Crit Care Med., 181(8):788-796), or the antibody designated MEDI 9314 and known in the art, or those described in U.S. Pat. Nos. 7,186,809, 7,605,237, 7,638,606, 8,092,804, 8,679,487, 8,877,189, 10,774,141; U.S. Patent Application Publication No. US2021 / 0238294; or International Patent Publication Nos. WO2019 / 228405, WO2020 / 096381, WO2020 / 096382, WO2020 / 096383, WO2020 / 096384, WO2020 / 096385, WO2020 / 096386, WO2020 / 096387, WO2020 / 096389 ... In some embodiments, the antibody may be an anti-IL-4Rα antibody such as those described in WO 2020 / 135471, WO 2020 / 135710, or WO 2020 / 239134.

[0088] In some embodiments, the anti-IL-4R antibody comprises one or more CDR, HCVR, and / or LCVR sequences shown in Table 1 below.

[0089] The amount of antibody or antigen-binding fragment thereof included in the pharmaceutical formulation of the present disclosure may vary depending on the particular properties desired of the formulation, as well as the particular environment and purpose for which the formulation is intended to be used. In certain embodiments, the pharmaceutical formulation may contain about 1 mg / mL to about 500 mg / mL of antibody; about 5 mg / mL to about 250 mg / mL of antibody; about 5 mg / mL to about 200 mg / mL of antibody; about 15 mg / mL to about 200 mg / mL of antibody; about 25 mg / mL to about 200 mg / mL of antibody; about 50 mg / mL to about 200 mg / mL of antibody; about 100 mg / mL to about 200 mg / mL of antibody; about 125 mg / mL to about 175 mg / mL of antibody; or about 150 mg / mL to about 200 mg / mL of antibody. For example, the formulations of the present disclosure may have a concentration of about 1 mg / mL; about 2 mg / mL; about 5 mg / mL; about 10 mg / mL; about 15 mg / mL; about 20 mg / mL; about 25 mg / mL; about 30 mg / mL; about 35 mg / mL; about 40 mg / mL; about 45 mg / mL; about 50 mg / mL; about 55 mg / mL; about 60 mg / mL; about 65 mg / mL; about 70 mg / mL; about 75 mg / mL; about 80 mg / mL; about 85 mg / mL; about 90 mg / mL; about 95 mg / mL; about 100 mg / mL; about 105 mg / mL; about 110 mg / mL The liquid formulation may comprise about 115 mg / mL; about 120 mg / mL; about 125 mg / mL; about 130 mg / mL; about 135 mg / mL; about 140 mg / mL; about 145 mg / mL; about 150 mg / mL; about 155 mg / mL; about 160 mg / mL; about 165 mg / mL; about 170 mg / mL; about 175 mg / mL; about 180 mg / mL; about 185 mg / mL; about 190 mg / mL; about 195 mg / mL; or about 200 mg / mL of an antibody or antigen-binding fragment that specifically binds to hIL-4Rα. In certain embodiments, the pharmaceutical formulation is a liquid formulation that can contain 5±0.5 mg / mL to 200±20 mg / mL of antibody, 15±1.5 mg / mL to 200±20 mg / mL of antibody, 25±2.5 mg / mL to 200±20 mg / mL of antibody, 50±5 mg / mL to 200±20 mg / mL of antibody, 100±10 mg / mL to 200±20 mg / mL of antibody, 150±10 mg / mL of antibody, or 175±10 mg / mL of antibody. In some embodiments, the pharmaceutical formulation contains 140±5 mg / mL to 160±5 mg / mL of anti-IL-4R antibody.In some cases, the pharmaceutical formulation contains 165 mg / mL ± 5 mg / mL to 185 mg / mL ± 5 mg / mL of the anti-IL-4R antibody. In some cases, the pharmaceutical formulation contains 150 mg / mL ± 5 mg / mL of the anti-IL-4R antibody. In some cases, the pharmaceutical formulation contains 175 mg / mL ± 5 mg / mL of the anti-IL-4R antibody.

[0090] The present disclosure includes antibodies having amino acid sequences that differ from the exemplary molecules disclosed herein but that retain the ability to bind to a cognate antigen, e.g., hIL-4R. Such variant molecules may contain one or more additions, deletions, or substitutions of amino acids when compared to the parent sequence, but exhibit essentially the same biological activity as that of the antibodies discussed herein.

[0091] The present disclosure includes antigen-binding molecules that are biologically equivalent to any of the exemplary antibodies shown herein. In some embodiments, the antigen-binding molecule is biologically equivalent to dupilumab. Two antibodies are considered to be biologically equivalent if they are pharmaceutical equivalents or pharmaceutical substitutes whose absorption rates and extents do not show significant differences when administered at the same molar dose under similar experimental conditions, either in single or multiple doses. Some antibodies may be considered to be equivalent or pharmaceutical substitutes and still be considered biologically equivalent if their absorption rates are comparable but their absorption rates are not comparable, because such differences in absorption rates are intentional, reflected in the label, are not essential to achieving effective body drug concentrations, for example, during chronic use, and are considered medically insignificant with respect to the particular drug product being studied.

[0092] In one embodiment, two antibodies are bioequivalent if there are no clinically meaningful differences in their safety, purity, and potency. In one embodiment, two antibodies are bioequivalent if a patient can switch between the reference product and the biological product one or more times without a clinically significant change in immunogenicity or an expected increase in the risk of side effects, including a decrease in efficacy, compared to continuous therapy without such a switch.

[0093] Bioequivalence can be demonstrated by in vivo and in vitro methods. Bioequivalence measurements include, for example, (a) in vivo studies in humans or other mammals that measure the concentration of the antibody or its metabolites in blood, plasma, serum, or other biological fluids as a function of time; (b) in vitro studies that correlate with and reasonably predict human in vivo bioavailability data; (c) in vivo studies in humans or other mammals in which the relevant acute pharmacological effects of the antibody (or its target) are measured as a function of time; and (d) well-controlled clinical trials that establish the safety, efficacy, or bioavailability or bioequivalence of the antigen binding protein. Pharmaceutical Formulations

[0094] As used herein, a "sample" may be obtained from any step of a bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step in downstream processing, drug substance (DS), or drug product (DP), including the final formulated product.

[0095] The terms "composition," "formulation," and "formulated drug substance" (FDS) as used in this disclosure refer to a combination of two or more pharmaceutical ingredients for inclusion in a drug product. The composition, formulation, or FDS may be, for example, a liquid composition that includes an active pharmaceutical ingredient, such as an antibody, and an excipient, such as a stabilizer or surfactant. The composition, formulation, or FDS may include multiple excipients. The composition, formulation, or FDS may also include other components, such as host cell proteins that are co-purified with the protein of interest.

[0096] The term "drug product" (DP) as used in this disclosure refers to a dosage form that contains a quantity of FDS for packaging, shipping, or administration. For example, a drug product may be a pre-filled syringe that holds a volume of FDS for administration to a patient.

[0097] As used herein, a "protein pharmaceutical product", "biopharmaceutical product", "pharmaceutical formulation", "pharmaceutical composition" or "biotherapeutic agent" includes an active ingredient that may be fully or partially biological in nature. In one embodiment, a protein pharmaceutical product may include a peptide, a protein, a fusion protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof. In another embodiment, a protein pharmaceutical product may include a recombinant, engineered, modified, mutated, or truncated form of a peptide, a protein, a fusion protein, an antibody, an antigen, a vaccine, a peptide-drug conjugate, an antibody-drug conjugate, a protein-drug conjugate, a cell, a tissue, or a combination thereof.

[0098] In some embodiments, the pharmaceutical formulation of the present invention comprises: (i) a human antibody that specifically binds to hIL-4Rα; (ii) one or more buffering agents; (iii) a thermostabilizing agent; (iv) a surfactant (e.g., an organic cosolvent); and (v) a viscosity modifier. Additional components may be included in the formulations of the present disclosure, provided that such components do not significantly interfere with the viscosity and stability of the formulation. Certain exemplary components and formulations included within the present disclosure are described in detail below.

[0099] The pharmaceutical formulation of the present disclosure may be a fluid formulation in certain embodiments. As used herein, the expression "fluid formulation" refers to a mixture of at least two components that exists in a predominantly fluid state at about 2°C to about 45°C. Fluid formulations include, among others, liquid formulations. Fluid formulations may be low, medium, or high viscosities depending on their particular components. Host Cell Proteins

[0100] As used herein, the term "host cell proteins" (HCPs) includes proteins derived from host cells during recombinant protein production. Host cell proteins can be process-related impurities that may result from the production process and may include three major categories: cell substrate derived, cell culture derived, and downstream derived. Cell substrate derived impurities include, but are not limited to, proteins and nucleic acids (host cell genome, vector, or total DNA) derived from the host organism. Cell culture derived impurities include, but are not limited to, inducers, antibiotics, serum, and other media components. Downstream derived impurities include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidizing and reducing agents), inorganic salts (e.g., heavy metals, arsenic, non-metal ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other eluates.

[0101] The presence of host cell proteins in a biotherapeutic product may be considered a higher or lower risk based on several measurable factors. One such factor is the concentration or abundance (amount) of the HCP impurity in the biotherapeutic product. The HCP may have no discernible impact at a low enough abundance, for example as measured by ELISA or mass spectrometry. The level at which the HCP may be present at a significant risk that may be considered an unacceptable level in the product and monitored as a critical quality attribute (CQA) may depend on the specific identity of the HCP. A particular HCP may be known to present a risk at a particular level, for example depending on the enzymatic activity level of the HCP, which is an enzyme.

[0102] Relatedly, the significance of the presence of an HCP may depend on the function of that HCP, particularly as it relates to the components of the biotherapeutic product. For example, HCP esterases or lipases that may or are known to degrade polysorbates present in the biotherapeutic product of interest may be closely monitored, which may have a low threshold for how many HCP impurities are acceptable in the biotherapeutic product. Other HCPs of particular interest may be, for example, proteases that may or are known to degrade proteins of interest in the biotherapeutic product, or immunogenic HCPs that may or are known to cause an immune response when administered to a subject. Liquid Chromatography

[0103] As used herein, the term "liquid chromatography" refers to a process in which biological / chemical mixtures carried by a liquid can be separated into components as a result of the differential distribution of the components as they flow through a stationary liquid or solid phase. Non-limiting examples of liquid chromatography include reversed-phase liquid chromatography, ion-exchange chromatography, size-exclusion chromatography, affinity chromatography, hydrophobic interaction chromatography, hydrophilic interaction chromatography, or mixed-mode chromatography. Analytes separated using chromatography will be characterized by distinct retention times that reflect the speed at which the analytes move through the chromatography column. Analytes may be compared using chromatograms that plot retention time on one axis and measured signal on the other, where the measured signal may result from, for example, UV or fluorescence detection. In some embodiments, a sample, e.g., a drug substance, containing at least one esterase or lipase may be subjected to a stress, e.g., an agitation stress or a heat stress, followed by a chromatography step to remove any HMW species.

[0104] In certain embodiments, it may be suitable to subject biological samples to affinity chromatography for preparation of protein of interest.Chromatographic materials can selectively or specifically bind or interact with protein of interest.Non-limiting examples of such chromatographic materials include protein A and protein G.Also included are, for example, chromatographic materials that comprise proteins or parts thereof that can bind or interact with protein of interest.

[0105] Affinity chromatography can involve subjecting a biological sample to a column containing a suitable Protein A resin. As used herein, the term "Protein A" refers to Protein A recovered from its natural source, Protein A produced synthetically (e.g., by peptide synthesis or by recombinant technology), and Protein A produced synthetically (e.g., by peptide synthesis or by recombinant technology). H 2 / C H In certain embodiments, Protein A resins, if they possess an Fc partial region, are useful for affinity-based preparation and isolation of diverse antibody isotypes by specifically interacting with that portion of the molecule.

[0106] There are several commercial sources of Protein A resins. Suitable resins include, but are not limited to, MabSelect PrismA, MabSelect SuRe™, MabSelect SuRe LX, MabSelect, MabSelect SuRe pcc, MabSelect Xtra, rProtein A Sepharose from Cytiva, ProSep HC, ProSep Ultra, ProSep Ultra Plus from EMD Millipore, MabCapture from ThermoFisher, and Amsphere™ A3 from JSR Life Sciences.

[0107] Prior to sample loading, the affinity column may be equilibrated with a suitable buffer. After loading the column, the column may be washed one or more times using a suitable washing buffer. The column may then be eluted using a suitable elution buffer, such as glycine-HCl, acetic acid, or citric acid. The eluate may be monitored using techniques well known to those skilled in the art, such as a UV detector. The eluted fraction of interest may be collected and then prepared for further processing.

[0108] Cation exchange chromatography (CEX) uses cation exchange chromatography materials. Cation exchange chromatography can be further divided into, for example, strong cation exchange (SCX) or weak cation exchange, depending on the cation exchange chromatography material used. Cation exchange chromatography materials containing sulfonic acid groups (S) may be used for strong cation exchangers, while cation exchange chromatography materials containing carboxymethyl groups (CM) may be used for weak cation exchangers. Strong cation exchangers include, for example, SOURCE S, which uses a methyl sulfate functional group, and SP Sepharose, which uses a sulfopropyl functional group. Weak cation exchangers include, for example, CM-Cellulose, which uses a carboxymethyl functional group. SCX may be preferred because a wider range of pH buffers can be used without losing the charge of the strong cation exchangers, allowing for effective separation of analytes with a wide pI range.

[0109] Cation exchange chromatography materials are available under different names from several companies, such as Bio-Rex, Macro-Prep CM (available from BioRad Laboratories, Hercules, Calif., USA), weak cation exchanger WCX 2 (available from Ciphergen, Fremont, Calif., USA), Dowex MAC-3 (available from Dow chemical company, Midland, Mich., USA), Mustang C (available from Pall Corporation, East Hills, NY, USA), Cellulose CM-23, CM-32, CM-52, hyper-D, and partisphere (available from Whatman plc, Brentford, UK), Amberlite IRC 76, IRC 747, IRC 748, GT 73 (available from Tosoh Bioscience GmbH, Stuttgart, Germany), CM 1500, CM 3000 (BioChrom Labs, Terre Haute, Ind., USA), and CM-Sepharose Fast Flow (available from GE Healthcare, Life Sciences, Germany). Additionally, commercially available cation exchange resins include carboxymethylcellulose, Bakerbond ABX, sulfopropyl (SP) immobilized on agarose (e.g., SP-Sepharose Fast Flow or SP-Sepharose High Performance, available from GE Healthcare-Amersham Biosciences Europe GmbH, Freiburg, Germany), and sulfonyl (e.g., S-Sepharose Fast Flow, available from GE Healthcare, Life Sciences, Germany) immobilized on agarose.

[0110] Cation exchange chromatography materials include mixed-mode chromatography materials that perform a combination of ion exchange and hydrophobic interaction techniques (e.g., Capto adhere, Capto MMC, MEP HyperCell, Eshmuno HCX, etc.), mixed-mode chromatography materials that perform a combination of anion exchange and cation exchange techniques (e.g., hydroxyapatite, ceramic hydroxyapatite, etc.), etc. In some embodiments, CEX may be used to remove HMW species from a sample to remove proteins that have aggregated into HMW species, for example after agitation or heat stress.

[0111] In some embodiments, the sample containing the protein of interest is subjected to at least one anion exchange (AEX) separation step. Anion exchange packed bed chromatography is based on the ionic interaction between the binding entity (target protein or impurity) and the functional group immobilized on the chromatographic medium. The performance can be a function of the mobile phase, the functional group, and the resin backbone. The use of anion exchange material versus cation exchange material is based in part on the local charge of the protein of interest. Anion exchange chromatography can be used in combination with other chromatographic methods, such as affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and other modes of chromatography known to those skilled in the art.

[0112] In the context of chromatographic separation, a chromatographic column is used to contain a chromatographic support material (resin or solid phase). A sample containing the protein of interest is loaded onto the particular chromatographic column. The column may then be subjected to one or more washing steps using an appropriate washing buffer. Components of the sample that have not been adsorbed onto the resin will pass through the column. Components adsorbed to the resin can be differentially eluted using an appropriate elution buffer.

[0113] The anionic agent may be selected from the group consisting of acetate, chloride, formate and combinations thereof. The cationic agent may be selected from the group consisting of Tris, arginine, sodium and combinations thereof. The buffer may be selected from the group consisting of pyridine, piperazine, L-histidine, Bis-Tris, Bis-Tris propane, imidazole, N-ethylmorpholine, TEA (triethanolamine), Tris, morpholine, N-methyldiethanolamine, AMPD (2-amino-2-methyl-1,3-propanediol), diethanolamine, ethanolamine, AMP (2-amino-2-methyl-1-propaol), piperazine, 1,3-diaminopropane and piperidine.

[0114] Packed anion exchange chromatography columns, anion exchange membrane devices, anion exchange monolith devices, or depth filter media can be operated in either a bind-elute mode, a flow-through mode, or a hybrid mode in which proteins exhibit binding to the chromatographic material yet can be washed from such material using a buffer that is the same as or substantially similar to the loading buffer.

[0115] In the bind-elute mode, the column or membrane device is first conditioned with a buffer of appropriate ionic strength and pH under conditions in which a particular protein will be adsorbed to a resin-based matrix. For example, during feed loading, the protein of interest may be adsorbed to the resin by electrostatic attraction. After washing the column or membrane device with an equilibration buffer or another buffer with a different pH and / or conductivity, product recovery is achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer to compete with the solute for the charged sites of the anion exchange matrix. Changing the pH, thereby modifying the charge of the solute, is another way to achieve elution of the solute. The change in conductivity or pH may be gradual (gradient elution) or stepwise (step elution).

[0116] In flow-through mode, the column or membrane device is operated at a selected pH and conductivity such that the protein of interest does not bind to the resin or membrane, while acidic species are retained on the column or have a distinct elution profile when compared to the protein of interest. In the context of this strategy, the acidic species will, under appropriate conditions, interact with or bind to the chromatographic material, while the protein of interest as well as certain aggregates and / or fragments of the protein of interest will flow out of the column.

[0117] In some embodiments, the AEX step is performed in negative mode (flow-through mode), where negatively charged process-related impurities are adsorbed to the immobilized positively charged ligands and the protein of interest flows through.

[0118] In some embodiments, the pH of the sample loaded onto the AEX column (the "loading pH") may be selected to reduce esterase or lipase activity in the sample. In some embodiments, the AEX loading pH may be about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, about 7.8 to about 8.3, about 7.8 to about 8.0, about 8.0 to about 8.3, or about 8.0 to about 8.2.

[0119] Non-limiting examples of anion exchange resins include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Further non-limiting examples include: Poros 50PI and Poros 50HQ, which are rigid polymer beads with a backbone made of cross-linked poly[styrene-divinylbenzene]; Poros 50XQ; Capto Q Impres and Capto DEAE, which are high flow agarose beads; Capto Adhere; Q Sepharose Fast Flow; Toyopearl QAE-550, Toyopearl DEAE-650, and Toyopearl GigaCap Q-650, which are polymer-based beads; Fractogel® EMD TMAE Hicap, which is a synthetic polymer resin with tentacle ion exchangers; Sartobind STIC® PA Nano, which is a salt-tolerant chromatography membrane with primary amine ligands; Sartobind Q Nano, which is a strong anion exchange chromatography membrane; CUNO BioCap, which is a Zeta-Plus depth filter medium constructed with inorganic filter aids, purified cellulose, and ion exchange resins; XOHC, which is a depth filter medium constructed with inorganic filter aids, cellulose, and mixed cellulose esters; and Unosphere Q. In some embodiments, a resin is selected that has a relatively larger pore size to increase the surface area exposed to negatively charged species.

[0120] Additives such as polyethylene glycol (PEG), surfactants, amino acids, sugars, chaotropic agents, etc. may be added to enhance the performance of the separation and achieve better separation, recovery and / or product quality.

[0121] Size exclusion chromatography or gel filtration relies on the separation of components as a function of molecular size. Separation depends on the amount of time a substance spends in the porous stationary phase compared to its time in the fluid. The probability of a molecule being present in the pores depends on the size of the molecule and the pores. Furthermore, the ability of a substance to penetrate inside the pores is determined by the diffusion mobility of the macromolecules, which is higher for small macromolecules. Very large macromolecules may not be able to penetrate the pores of the stationary phase at all; for very small macromolecules, the probability of penetration is fairly consistent. Larger molecular size components move more quickly through the stationary phase, while smaller molecular size components move longer paths through the pores of the stationary phase and are therefore retained longer in the stationary phase.

[0122] Analytes eluting from a SEC column may be separated into fractions based on elution time. For example, analytes that elute earlier than the functional form of the protein of interest, e.g., the monomeric form, may be broadly classified as high molecular weight (HMW) species. The HMW fraction may be further divided, e.g., into an ultra-high molecular weight (vHMW) fraction and a dimer fraction (corresponding to the elution time of the dimer of the protein of interest). Analytes that elute later than the functional form of the protein of interest may be broadly classified as low molecular weight (LMW) species, which may be further divided into an LMW fraction and a late tail fraction. Similarly, sample components other than the protein of interest, e.g., lipases, may form higher and lower molecular weight species that may be separated using SEC.

[0123] The chromatographic material may include a resin or membrane size exclusion material. The matrix used for size exclusion is preferably an inert gel medium, which may be a complex of cross-linked polysaccharides, such as cross-linked agarose and / or dextran in the form of spherical beads. The degree of cross-linking determines the size of the pores present in the swollen gel beads. Molecules larger than a certain size will not enter the gel beads and therefore will move fastest through the chromatographic bed. Smaller molecules, such as detergents, proteins, DNA, etc., will enter the gel beads to varying degrees depending on their size and shape and will be retarded in passing through the bed. Thus, molecules are generally eluted in order of decreasing molecular size. In some embodiments, SEC may be used to remove HMW species from the sample to remove proteins that have aggregated into HMW species, for example after agitation or heat stress.

[0124] The term "hydrophobic interaction medium" refers to a combination of a support structure and a hydrophobic moiety, where the hydrophobic moiety is immobilized on the support structure. The medium may be in the form of a chromatographic medium, such as beads or other particles held in a packed bed column format, a membrane format, or any format that can accommodate the liquid containing the protein of interest and contaminants. Thus, support structures include agarose beads (e.g., sepharose), silica beads, cellulosic membranes, cellulosic beads, hydrophilic polymer beads, and the like. The hydrophobic moiety is the tip of the medium that binds to the hydrophobic surface of hydrophobic molecules and proteins. The degree of hydrophobicity of the medium can be adjusted by selecting the hydrophobic moiety. For example, the following moieties may be immobilized on the medium support to produce a hydrophobic interaction medium with increasing hydrophobicity, i.e., from low to high hydrophobicity. The alkyl group may be linear or branched.For a review of hydrophobic interaction chromatography and media, see Kuczewski et al., "Development of a polishing step using a hydrophobic interaction membrane adsorber with a PER.C6™-derived recombinant antibody," Biotech. Bioeng. 105(2):296-305 (2010); Roettger and Ladisch, "Hydrophobic interaction chromatography," Biotechnol Adv. 7(1):15-29 (1989); Shukla and Sanchayita, "Process for purifying proteins in a hydrophobic interaction chromatography flow-through fraction," U.S. Patent No. 7,427,659 B2, September 23, 2008; and Mueller and Franzreb, "Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions," J. Chroma. A 1260:88-96 (2012).

[0125] Hydrophobic interaction media are used in a process known as hydrophobic interaction chromatography (HIC) to separate proteins of interest from product and process-related contaminants. When a protein of interest is produced in and / or purified from a host cell, the product and process-related contaminants are referred to as host cell proteins (HCPs). HCPs derived from Chinese Hamster Ovary (CHO) cells, a common biotherapeutic production host cell, may be referred to as CHOP (Chinese Hamster Ovary Proteins). In some cases, a mixture containing a protein of interest (POI) and HCPs is applied to the HIC medium in a buffer designed to promote binding of hydrophobic groups in the POI to the hydrophobic moieties of the HIC medium. The POI sticks to the HIC medium by binding to the hydrophobic moieties, and some HCPs fail to bind and come out in the wash buffer. The POI is then eluted using a buffer that promotes dissociation of the POI from the HIC hydrophobic moieties, thereby separating the POI from the undesired HCPs.

[0126] In some cases, the HIC hydrophobic moieties preferentially bind some contaminants, e.g., HCPs, and the POI is collected from the HIC flow-through. This disclosure provides an example of the use of HIC in a flow-through mode, where the contaminant HCP population, including esterase activity, remains bound to the hydrophobic interaction medium.

[0127] In some cases, affinity chromatography designed to bind specific proteins with lipophilic properties may be used instead of or in conjunction with HIC. Some esterases, such as lipases in general, or phospholipases in particular, bind triglycerides or phospholipids, so molecules that mimic these lipids may be used to capture the esterase. For example, "myristoylated ADP-ribosylation factor 1" (also known as "myrARF1") may be used to capture the lipase, allowing the POI to remain unbound and in the flow-through. To prepare a myrARF1 affinity column, myrARF1 may be coupled to Q-Sepharose through N-hydroxysuccinimide activation (see Morgan et al., "Identification of phospholipase B from Dictyostelium discoideum reveals a new lipase family present in mammals, flies and nematodes, but not yeast," Biochem. J. 382: 441-449 (2004)). mass spectrometry

[0128] As used herein, the term "mass analyzer" includes devices capable of identifying and measuring the exact mass of a particular molecular species. The term is meant to include any molecular detection device capable of characterizing a polypeptide or peptide. A mass analyzer may include three main parts: an ion source, a mass analyzer, and a detection device. The role of the ion source is to generate gas phase ions. Analyte atoms, molecules, or clusters may be transferred to the gas phase and ionized either simultaneously (as in electrospray ionization) or through a separate process. The choice of ion source depends on the application.

[0129] In some embodiments, the mass analyzer is a tandem mass analyzer. As used herein, the term "tandem mass analyzer" includes techniques in which structural information about sample molecules is obtained by using multiple stages of mass selection and mass separation. The requirement is that the sample molecules are converted into the gas phase, ionized, and fragmented in a predictable and controllable manner after an initial mass selection step. Precursor ions are first selected and isolated (MS 1 ), which can then be fragmented to obtain meaningful information, using MS / MS or MS 2 Tandem MS has been successfully implemented with a wide variety of analyzer combinations. Which analyzers are combined for a particular application can be determined by many different factors, such as sensitivity, selectivity, and speed, but also size, cost, and availability. The two main categories of tandem MS methods are spatial tandem and temporal tandem, but there are also hybrids that couple temporal tandem analyzers in space or with spatial tandem analyzers. A spatial tandem mass analyzer includes an ion source, a precursor ion activation device, and at least two non-trapping mass analyzers. A specific m / z separation function may be designed such that ions are selected in one section of the instrument, dissociated in an intermediate region, and product ions are then transmitted to another analyzer for m / z separation and data acquisition. In a temporal tandem mass analyzer, it is also possible to trap, isolate, fragment, and m / z separate ions produced in the ion source in the same physical device.

[0130] Peptides identified by mass spectrometry can be used as surrogate representatives of the intact protein and its post-translational or other modifications. They can also be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter generated from possible peptides in protein sequence databases. Characterization includes, but is not limited to, amino acid sequencing of protein fragments, protein sequence determination, protein de novo sequence determination, post-translational modification location, or post-translational modification identification, or comparability analysis, or a combination thereof.

[0131] In some exemplary embodiments, the mass analyzer may operate with nanoelectrospray or nanospray. The term "nanoelectrospray" or "nanospray" refers to electrospray ionization at very low solvent flow rates, typically less than a few hundred nanoliters per minute of sample solution, often without the use of external solvent delivery. The electrospray injection setup that forms the nanoelectrospray may use a static nanoelectrospray emitter or a dynamic nanoelectrospray emitter. A static nanoelectrospray emitter performs continuous analysis of small sample (analyte) solution volumes over an extended period of time. A dynamic nanoelectrospray emitter uses a capillary column and a solvent delivery system to perform chromatographic separation on a mixture before analysis by a mass analyzer.

[0132] In some embodiments, mass spectrometry can be carried out under non-denaturing conditions. As used herein, the term "non-denaturing conditions" can include carrying out mass spectrometry under conditions that preserve non-covalent interactions in analytes. For a detailed overview of non-denaturing MS, see Review: Elisabetta Boeri Erba & Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 PROTEIN SCIENCE 1176-1192 (2015).

[0133] As used herein, the term "database" refers to a compiled collection of protein sequences possibly present in a sample, for example in the form of a FASTA format file. Suitable protein sequences may be derived from the cDNA sequence of the species under study. Public databases that can be used to search for suitable protein sequences included, for example, databases hosted by Uniprot or Swiss-prot. Databases may be searched using what are referred to herein as "bioinformatics tools." Bioinformatics tools provide the ability to search uninterpreted MS / MS spectra against all possible sequences in the database(s) and provide interpreted (annotated) MS / MS spectra as output. Non-limiting examples of such tools are Mascot (www.matrixscience.com), Spectrum Mill (www.chem.agilent.com), PLGS (www.waters.com), PEAKS (www.bioinformaticssolutions.com), Proteinpilot (download.appliedbiosystems.com / proteinpilot), Phenyx (www.phenyx-ms.com), Sorcerer (www.sagenresearch.com), OMSSA (www.pubchem.ncbi.nlm.nih.gov / omssa / ), X!Tandem (www.thegpm.org / TANDEM / ), Protein Prospector (prospector.ucsf.edu / prospector / mshome.htm), Byonic (www.proteinmetrics.com / products / byonic) or Sequest (fields.scripps.edu / sequest). Excipients

[0134] The pharmaceutical formulations of the present disclosure include one or more excipients. The term "excipient" as used herein means any non-therapeutic agent added to a formulation to provide a desired consistency, viscosity, or stabilizing effect.

[0135] The pharmaceutical formulations of the present disclosure may also include a buffer or buffer system that serves to maintain a stable pH and aid in stabilizing the protein of interest. In some embodiments, the buffer or buffer system includes at least one buffer having a buffer range that overlaps fully or partially with the pH 5.5-6.3 range. In various embodiments, the pH of the formulation is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 or 6.3. In some embodiments, the formulation has a pH of 5.9±0.3. In some embodiments, the formulation has a pH of 5.9±0.2. In some embodiments, the formulation has a pH of 5.9±0.1. In certain embodiments, the buffer includes a histidine buffer. In certain embodiments, the buffer includes an acetate buffer. In certain embodiments, the buffer (e.g., histidine and / or acetate) is present at a concentration of about 1 mM to about 40 mM, about 5 mM to about 30 mM, about 10 mM to about 15 mM; or about 15 mM to about 25 mM. In some embodiments, the buffer includes a histidine buffer at a concentration of 15 mM to 25 mM. In some embodiments, the buffer includes a histidine buffer at a concentration of 20 mM ± 2 mM. In some cases, the histidine buffer is present at a concentration of 15 mM, 16 mM, 17 mM, 18 mM, 19 mM, 20 mM, 21 mM, 22 mM, 23 mM, 24 mM, or 25 mM. In some embodiments, the buffer includes an acetate buffer at a concentration of 10 mM to 15 mM. In some embodiments, the buffer includes an acetate buffer at a concentration of 12.5 mM ± 1.25 mM. In some cases, the acetate buffer is present at a concentration of 10 mM, 10.5 mM, 11 mM, 11.5 mM, 12 mM, 12.5 mM, 13 mM, 13.5 mM, 14 mM, 14.5 mM, or 15 mM. In some embodiments, the formulation of the present disclosure includes both histidine and acetate buffer at any of the concentrations discussed above. In some cases, the formulation contains a histidine buffer at a concentration of 15 mM to 25 mM, and an acetate buffer at a concentration of 10 mM to 15 mM. In some cases, the formulation contains a histidine buffer at a concentration of 20 mM ± 2 mM, and an acetate buffer at a concentration of 12.5 mM ± 1.25 mM.

[0136] The pharmaceutical formulation of the present disclosure may also include one or more carbohydrates, such as one or more sugars. The sugar may be a reducing sugar or a non-reducing sugar. "Reducing sugar" includes sugars that contain a reactive hemiacetal group, such as a ketone or aldehyde group, allowing the sugar to act as a reducing agent. Specific examples of reducing sugars include fructose, glucose, glyceraldehyde, lactose, arabinose, mannose, xylose, ribose, rhamnose, galactose and maltose. Non-reducing sugars may include an anomeric carbon that is an acetal and does not substantially react with an amino acid or polypeptide to initiate the Maillard reaction. Specific examples of non-reducing sugars include sucrose, trehalose, sorbose, sucralose, melezitose and raffinose. Sugar acids include, for example, saccharic acid, gluconate and other polyhydroxy sugars and their salts. In some embodiments, the sugar is sucrose. In some cases, the sugar (e.g., sucrose) acts as a heat stabilizer for the protein of interest.

[0137] The amount of sugar (e.g., sucrose) contained within the pharmaceutical formulations of the present disclosure will vary depending on the particular environment in which the formulation is used and the intended purpose. In certain embodiments, the formulation may contain about 0.1% to about 20% sugar; about 0.5% to about 20% sugar; about 1% to about 20% sugar; about 2% to about 15% sugar; about 3% to about 8% sugar; or about 4% to about 6% sugar. For example, the pharmaceutical formulations of the present disclosure may contain about 0.5%; about 1.0%; about 1.5%; about 2.0%; about 2.5%; about 3.0%; about 3.5%; about 4.0%; about 4.5%; about 5.0%; about 5.5%; about 6.0%; about 6.5%; about 7.0%; about 7.5%; about 8.0%; about 8.5%; about 9.0%; about 9.5%; about 10.0%; about 15%; or about 20% sugar (e.g., sucrose). In some embodiments, the formulation may contain about 5% sugar (e.g., sucrose). In some embodiments, the formulation may contain about 5%±0.5% sugar (e.g., sucrose). Each of the above percentages corresponds to a weight / volume (w / v) percentage.

[0138] In certain embodiments, the pharmaceutical formulation of the present disclosure comprises at least one amino acid. In some embodiments, the amino acid is arginine. In some embodiments, arginine is provided in the form of arginine hydrochloride. In some cases, the amino acid (e.g., arginine) acts as a viscosity modifier for the formulation of the protein of interest.

[0139] The amount of amino acids contained within the pharmaceutical formulations of the present disclosure can vary depending on the particular properties desired of the formulation, as well as the particular environment and purpose for which the formulation is intended to be used. In certain embodiments, the formulation can contain about 1 mM to about 200 mM amino acids; about 5 mM to about 150 mM amino acids; about 10 mM to about 100 mM amino acids; about 20 mM to about 80 mM amino acids; about 20 mM to about 30 mM amino acids; about 45 mM to about 55 mM amino acids; or about 70 mM to about 80 mM amino acids. For example, the pharmaceutical formulation of the present disclosure may comprise about 5 mM; about 10 mM; about 15 mM; about 20 mM; about 25 mM; about 30 mM; about 35 mM; about 40 mM; about 45 mM; about 50 mM; about 55 mM; about 60 mM; about 65 mM; about 70 mM; about 75 mM; about 80 mM; about 85 mM; about 90 mM; about 95 mM; or about 100 mM of amino acid (e.g., arginine). In some embodiments, the formulation contains about 25 mM of amino acid (e.g., arginine). In some embodiments, the formulation contains about 50 mM of amino acid (e.g., arginine). In some embodiments, the formulation contains about 75 mM of amino acid (e.g., arginine).

[0140] The pharmaceutical formulations of the present disclosure may also include one or more organic co-solvents of a type and amount that stabilizes the protein of interest under conditions of rough handling or agitation, e.g., orbital agitation. In some embodiments, the organic co-solvent is a surfactant. As used herein, the term "surfactant" refers to a substance that reduces the surface tension of the fluid in which it is dissolved and / or reduces the interfacial tension between oil and water. Surfactants may be ionic or non-ionic. Particular non-ionic surfactants that may be included in the formulations of the present disclosure include, for example, polysorbates, e.g., PS20 and PS80, poloxamers, e.g., poloxamer 188, and polyethylene glycols (PEGs), e.g., PEG3350.

[0141] The amount of surfactant contained in the pharmaceutical formulation of the present disclosure may vary depending on the particular properties desired of the formulation, as well as the particular environment and purpose for which the formulation is intended to be used. In certain embodiments, the formulation may contain at least about 0.01% surfactant. In certain embodiments, the formulation may contain less than 0.2% surfactant. In certain embodiments, the formulation may contain less than 0.5% surfactant. In certain embodiments, the formulation may contain from about 0.01% to about 0.49% surfactant; from about 0.01% to about 0.39% surfactant; from about 0.01% to about 0.29% surfactant; from about 0.01% to about 0.19% surfactant; from about 0.01% to about 0.15% surfactant; from about 0.01% to about 0.12%; from about 0.01% to about 0.11% surfactant; from about 0.01% to about 0.1% surfactant; or from about 0.01% to about 0.09% surfactant. For example, the formulations of the present disclosure may contain about 0.01%; about 0.02%; about 0.03%; about 0.04%; about 0.05%; about 0.06%; about 0.07%; about 0.08%; about 0.09%; about 0.1%; about 0.11%; about 0.12%; about 0.13%; about 0.14%; about 0.15%; about 0.16%; about 0.17%; about 0.18%; about 0.19%; about 0.20%; about 0.25%; about 0.26%; about 0.27%; about 0.28%; about 0.29%; about 0.30%; about 0.31%; about 0.32%; about 0.33%; about 0.34%; about 0.35%; about 0.36%; about 0.37%; about 0.38%; about 0.39%; about 0.40%; about 0.41%; about 0.42%; about 0.43%; about 0.44%; about 0.45%; about 0.46%; about 0.47%; about 0.48%; about 0.49%; about 0.50%; about 0.51%; about 0.52%; about 0.53%; about 0.54%; about 0.55%; about 0.56%; about 0.57%; about 0.58%; about 0.59%; about 0.60%; about 0.61%; about 0.62%; about 0.63%; about 0.64%; about 0.65%; about 0.66%; about 0. The formulation may contain 0%; about 0.21%; about 0.22%; about 0.23%; about 0.24%; about 0.25%; about 0.26%; about 0.27%; about 0.28%; about 0.29%; about 0.30%; about 0.35%; about 0.40%; about 0.45%; or about 0.50% of a surfactant (e.g., PS20, PS80, poloxamer 188, or PEG3350). In some embodiments, the formulation contains about 0.01% to 0.19% poloxamer 188. In some embodiments, the formulation contains about 0.01% to about 0.49% poloxamer 188. In some embodiments, the formulation contains about 0.01% to 0.19% PEG3350. In some embodiments, the formulation contains about 0.01% to 0.49% PEG3350. Each of the above percentages corresponds to a weight / volume (w / v) percent. Polysorbate

[0142] In some embodiments, the surfactant in the composition may be a polysorbate. As used herein, "polysorbate" refers to a common excipient used during formulation development to protect antibodies against a variety of physical stresses, such as agitation, freeze-thaw processes, and the air / water interface (Emily Ha, Wei Wang & Y. John Wang, Peroxide formation in polysorbate 80 and protein stability, 91 JOURNAL OF PHARMACEUTICAL SCIENCES 2252-2264 (2002);Bruce A. Kerwin, Polysorbates 20 and 80 Used in the Formulation of Protein Biotherapeutics: Structure and Degradation Pathways, 97 JOURNAL OF PHARMACEUTICAL SCIENCES 2924-2935 (2008);Hanns-Christian Mahler et al., Adsorption Behavior of a Surfactant and a Monoclonal Antibody to Sterilizing-Grade Filters, 99 Journal of Pharmaceutical Sciences 2620-2627). (2010), which may include non-ionic amphiphilic surfactants composed of fatty acid esters of polyoxyethylene sorbitan. The esters may include a polyoxyethylene sorbitan head group and a saturated monolaurate side chain (polysorbate 20; PS20) or an unsaturated monooleate side chain (polysorbate 80; PS80). In some embodiments, the polysorbate may be present in the formulation in the range of about 0.001% to 1% (weight / volume).Polysorbates may also contain a mixture of various fatty acid chains; for example, polysorbate 80 contains oleic, palmitic, myristic and stearic fatty acids, with the monooleate fraction constituting approximately 58% of the polysorbate mixture (Nitin Dixit et al., Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles, 105 JOURNAL OF PHARMACEUTICAL SCIENCES 1657-1666 (2016)). Non-limiting examples of polysorbates include polysorbate-20, polysorbate-40, polysorbate-60, polysorbate-65, and polysorbate-80.

[0143] Polysorbates are susceptible to autoxidation in a pH- and temperature-dependent manner, and exposure to UV light can also result in instability (Ravuri Sk Kishore et al., Degradation of Polysorbates 20 and 80: Studies on Thermal Autoxidation and Hydrolysis, 100 JOURNAL OF PHARMACEUTICAL SCIENCES 721-731 (2011)), which, along with the sorbitan head group, produces free fatty acids in solution. The free fatty acids produced from polysorbates can include any aliphatic fatty acid containing 6-20 carbons. Non-limiting examples of free fatty acids include oleic acid, palmitic acid, stearic acid, myristic acid, lauric acid, or combinations thereof.

[0144] In some exemplary embodiments, polysorbates can form free fatty acid particles. The free fatty acid particles are at least about 1 μm in size or at least about 5 μm in size. Furthermore, these fatty acid particles can be classified according to size into macroscopic (about >100 μm), submicroscopic (about <100 μm, which can be further subdivided into micron (1-100 μm) and submicron (100 nm-1000 nm)) and nanometer particles (about <100 nm) (Linda Narhi, Jeremy Schmit & Deepak Sharma, Classification of protein aggregates, 101 JOURNAL OF PHARMACEUTICAL SCIENCES 493-498). In some exemplary embodiments, fatty acid particles can be macroscopic particles. Macroscopic particles can be determined by visual inspection. In some embodiments, fatty acid particles can be submicroscopic particles. Subvisible particles can be monitored by light obscuration method according to the United States Pharmacopeia (USP). The increase of fatty acid particles can cause the product to no longer be of acceptable quality, and therefore the rate of fatty acid particle increase can be used as a measurement of product shelf life. Fatty acid particles can be formed when free fatty acids are released into the formulation and exceed the concentration at which they are soluble, thereby precipitating out of solution. Thus, measuring the concentration of polysorbate decomposition or released free fatty acid can be an indicator of fatty acid particle formation and thus predictive of product shelf life. Furthermore, preventing polysorbate decomposition, free fatty acid release, and / or fatty acid particle formation can be important to extend product shelf life and improve product quality.

[0145] In some exemplary embodiments, the concentration of polysorbate in the formulation is about 0.001% w / v, about 0.002% w / v, about 0.003% w / v, about 0.004% w / v, about 0.005% w / v, about 0.006% w / v, about 0.007% w / v, about 0.008% w / v, about 0.009% w / v, about 0.01% w / v, about 0.015% w / v, about 0.02% w / v, 0.025% w / v, about 0.03% w / v, about 0.035% w / v, about 0.04% w / v, about 0.045% w / v, about 0.05% w / v, about 0.06% w / v, about 0.07% w / v, about 0.08% w / v, about 0.09% The concentration of polysorbate in the formulation may be about 1% w / v, about 0.1% w / v, about 0.2% w / v, about 0.3% w / v, about 0.4% w / v, about 0.5% w / v, about 0.6% w / v, about 0.7% w / v, about 0.8% w / v, about 0.9% w / v, or about 1% w / v. In one embodiment, the concentration of polysorbate in the formulation is about 1% w / v.

[0146] In some exemplary embodiments, the concentration of free fatty acids in the formulation can be about 10 ng / mL, about 20 ng / mL, about 30 ng / mL, about 40 ng / mL, about 50 ng / mL, about 60 ng / mL, about 70 ng / mL, about 80 ng / mL, about 90 ng / mL, about 100 ng / mL, about 200 ng / mL, about 300 ng / mL, about 400 ng / mL, about 500 ng / mL, about 600 ng / mL, about 700 ng / mL, about 800 ng / mL, about 900 ng / mL, about 1 μg / mL, about 2 μg / mL, about 3 μg / mL, about 4 μg / mL, about 5 μg / mL, about 6 μg / mL, about 7 μg / mL, about 8 μg / mL, about 9 μg / mL, about 10 μg / mL, about 20 μg / mL, about 30 μg / mL, or about 40 μg / mL.

[0147] In some exemplary embodiments, polysorbates can be degraded by host cell proteins present in the composition. In some embodiments, the host cell proteins can be esterases or lipases. Residual esterase or lipase activity in the formulation can be indirectly assessed by measuring polysorbate degradation, release of free fatty acids, or the concentration of fatty acid particles visible or subvisible to the naked eye.

[0148] The term "fatty acid ester" refers to any organic compound containing a fatty acid chain linked to a head group through an ester bond. An ester bond is formed when a hydroxyl group (e.g., an alcohol or a carboxylic acid) is replaced by an alkoxy group. As used herein, the hydroxyl group can be part of a carboxylic acid, more specifically a fatty acid, and / or an alcohol, such as glycerol, sorbitol, sorbitan, isosorbide, etc. The alcohol group is generally referred to herein as the head group.

[0149] Examples of fatty acid esters generally include phospholipids, lipids (e.g., head group is glycerol, including monoglycerides, diglycerides, and triglycerides), and surfactants and emulsifiers, including polysorbates such as nonionic surfactants polysorbate 20, polysorbate 60, and polysorbate 80. Surfactants and emulsifiers are useful as cosolvents and stabilizers. They function by associating with both hydrophilic and lipophilic surfaces to maintain dispersion and structural stability of components such as proteins. Surfactants are primarily added to protein formulations to promote protein stability against mechanical stresses, such as air / liquid and solid / liquid interfacial shear. Without surfactants, proteins may in some cases become structurally unstable in solution and form multimeric aggregates that eventually become particles that are not visible to the naked eye.

[0150] The term "fatty acid" or "fatty acid chain" refers to a carboxylic acid having an aliphatic tail. The aliphatic tail is simply a hydrocarbon chain containing carbon and hydrogen, and in some cases oxygen, sulfur, nitrogen, and / or chlorine substitutions. The aliphatic tail may be saturated (as in saturated fatty acids), meaning that all carbon-carbon bonds are single bonds (i.e., alkanes). The aliphatic tail may be unsaturated (as in unsaturated fatty acids), where one or more carbon-carbon bonds are double bonds (alkenes), or triple bonds (alkynes).

[0151] Fatty acids are commonly referred to as short chain fatty acids, having less than 6 carbons in the aliphatic tail, medium chain fatty acids, having 6-12 carbons, long chain fatty acids, having 13-21 carbons, and very long chain fatty acids, having an aliphatic tail of 22 carbons or more. As mentioned above, fatty acids are also classified according to the degree of saturation, which correlates to hardness and melting point. Common fatty acids include caprylic acid (8 carbons:0 double bond; 8:0), capric acid (10:0), lauric acid (12:0), myristic acid (14:0), myristoleic acid (14:1), palmitic acid (16:0), palmitoleic acid (16:1), sapienic acid (16:1), stearic acid (18:0), oleic acid (18:1), elaidic acid (18:1), vaccenic acid (18:0), and oleic acid (18:1). 1), linoleic acid (18:2), linelaedic acid (18:2), alpha-linolenic acid (18:3), arachidic acid (20:0), arachidonic acid (20:4), eicosapentanoic acid (20:5), behenic acid (22:0), erucic acid (22:1), docosahexaenoic acid (22:6), lignoceric acid (24:0), and cerotic acid (26:0).

[0152] As mentioned above, polysorbates are fatty acid esters useful as non-ionic surfactants and protein stabilizers. Polysorbate 20, Polysorbate 40, Polysorbate 60, and Polysorbate 80 are widely used as stabilizers and emulsifiers in the pharmaceutical, cosmetic, and food industries. Polysorbate 20 mostly comprises the monolaurate ester of polyoxyethylene (20) sorbitan. Polysorbate 40 mostly comprises the monopalmitate ester of polyoxyethylene (20) sorbitan. Polysorbate 60 mostly comprises the monostearate ester of polyoxyethylene (20) sorbitan. Polysorbate 80 mostly comprises the monooleate ester of polyoxyethylene (20) sorbitan.

[0153] The quality of commercial grades of polysorbates varies between manufacturers. Polysorbates are therefore often a mixture of diverse chemical entities consisting mostly of polyoxyethylene (20) sorbitan monoesters (as discussed above) with, in some cases, isosorbide ester contaminants. The head group (in this case, polyoxyethylene (20) sorbitan) contains sorbitan (a mixture of sorbitol anhydrides, including 1,4-anhydrosorbitol, 1,5-anhydrosorbitol, and 1,4,3,6-dianhydrosorbitol), which is substituted with three of the alcohol groups to form ether bonds with three polyoxyethylene groups. The fourth alcohol group is substituted with a fatty acid to form a fatty acid ester.

[0154] In some commercial polysorbate batches, the polysorbate contains isosorbide monoesters. Isosorbide is a heterocyclic derivative of glucose, also prepared by dehydration of sorbitol. It is a diol, i.e. it has two alcohol groups, which can participate in the formation of one or two ester bonds. Thus, for example, some lots of polysorbate 20 may contain significant amounts of isosorbide laurate mono- and diesters, and some lots of polysorbate 80 may contain significant amounts of isosorbide oleate mono- and diesters.

[0155] In addition to head group variation, preparations of polysorbates contain varying amounts of other fatty acid esters. For example, analysis of one particular source of polysorbate 20 revealed <10% caprylic acid, <10% capric acid, 40-60% lauric acid, 14-25% myristic acid, 7-15% palmitic acid, <11% oleic acid, <7% stearic acid, and <3% linoleic acid. Analysis of a batch of polysorbate 80 revealed <5% myristic acid, <16% palmitic acid, >58% oleic acid, <6% stearic acid, and <18% linoleic acid. Analysis of another source of polysorbate 80 revealed approximately 70% oleic acid, with the remainder being other fatty acid esters and impurities. Analysis of yet another source of polysorbate 80 revealed approximately 86-87% oleic acid. Analysis of even more recently developed sources of polysorbate 80 revealed > 99% oleic acid.

[0156] In some embodiments, the concentration of oleic acid in polysorbate 80 is about 50% to about 100%, about 58% to about 100%, about 60% to about 100%, about 80% to about 100%, about 90% to about 100%, about 95% to about 100%, about 98% to about 100%, about 99% to about 100%, about 98.0% to about 99.9%, about 98.5% to about 99.5%, about 99.0% to about 99.9%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 9 ... %, at least 96%, at least 97%, at least 98%, at least 98.5%, at least 99%, at least 99.5%, about 58%, about 60%, about 65%, about 70%, about 75%, about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, about 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, about 98.5%, about 99%, about 99.1%, about 99.2%, about 99.3%, about 99.4%, about 99.5%, about 99.6%, about 99.7%, about 99.8%, about 99.9%, or about 100%.

[0157] Biopharmaceutical agents are often formulated with non-ionic surfactants such as polysorbate 20 or polysorbate 80. These surfactants help stabilize macromolecules such as antibodies and other proteins and help prevent the formation of supramolecular ternary complexes or other aggregates. The aggregates can become nanoparticles, or sub-visible particles in the 10-100 micron range or 2-100 micron range, which can interfere with drug product stability and shelf life. Thus, the stability of protein formulations depends in some cases on the stability of non-ionic surfactant additives. However, and as discussed further herein, polysorbate 20 and polysorbate 80 can contribute in some cases to the formation of aggregates, nanoparticles, and sub-visible particles.

[0158] The phrase "subvisible particles" refers to particles that are not visible to the naked eye, especially in liquids. In other words, it does not include particles that are visible to the naked eye, but a solution or other liquid containing subvisible particles would not appear cloudy. Subvisible particles generally include particles that are 100 microns or less in diameter, but in some cases include particles less than 150 microns (Narhi et al., "A critical review of analytical methods for subvisible and visible particles," Curr Pharm Biotechnol 10(4):373-381 (2009)). Subvisible particles can be the result of foreign contaminants or protein aggregation. Protein aggregates can be soft and amorphous in shape, and therefore difficult to detect using conventional light obscuration and microscopy (Singh and Toler, "Monitoring of subvisible particles in therapeutic proteins," Methods Mol Biol. 2012; 899:379-401). Sub-visible particles may include, inter alia, silicon contaminants (oily droplets), free fatty acids (oily droplets), aggregated proteins (irregular particles), and / or protein / fatty acid complexes (irregular particles).

[0159] Particles that are invisible to the naked eye can be detected by any one or more of a variety of methods. USP standards specify light obscuration and light microscopy protocols. Other methods include microflow image (MFI) analysis, Coulter counting, and submicron particle tracking. For light obscuration (LO), particles are counted based on the shadow they cast on a light detection device as they pass through a light beam in a flow cell. The size, shape, and inverse intensity of the shadow depend on the size, shape, and difference in refractive index of the particle compared to the solution. The lower size range for detection using LO is about 2 microns. A commonly used LO device is the HIAC instrument (Beckman Coulter, Brea, Calif.). Several methods for measuring and characterizing SVPs (e.g., optical obscuration, flow microscopy, electronic sensing zone methods, and flow cytometry) are discussed, for example, in Narhi et al., “Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy,” J. Pharma. Sci. 104:1899-1908 (2015).

[0160] Optical obscuration is criticized for underestimating protein aggregates and other amorphous structures. Flow imaging analysis, e.g., micro-flow imaging (MFI) (Brightwell Technologies, Ottawa, Ontario), is a more sensitive method to detect irregularly shaped, fragile, transparent proteinaceous particles that are invisible to the naked eye and to distinguish these types of particles from silicon droplets, air bubbles, and other foreign contaminants (Sharma et al., “Micro-flow imaging: Flow microscopy applied to sub-visible particulate analysis in protein formulations,” AAPS J. 12(3): 455-464 (2010)). In general, SVP measurement and characterization by optical obscuration analysis is less sensitive than MFI, and particle counts detected by MFI will tend to be higher than particle counts detected by optical obscuration analysis. Briefly, MFI is a flow microscopy method in which continuous bright-field images are taken and analyzed in real time. Image analysis algorithms are applied to the images to distinguish between air bubbles, silicone oil droplets, and proteinaceous aggregates. Volumes as low as about 250 microliters and as high as tens of milliliters can be analyzed. Depending on the system used, sizes as small as 2-300 microns, or 1-70 microns, can be detected (ibid.).

[0161] In some embodiments, particles visible or invisible to the naked eye in a formulation can be detected and analyzed by Raman spectroscopy. As used herein, the term "Raman spectroscopy" refers to spectroscopy based on Raman scattering. Raman spectroscopy is a method of detecting particles in the fingerprint region (2000-400 cm -1) in a Raman spectrum in which the presence and location of bands in the nucleus can be identified, allowing chemical identification of the analyzed material by comparison with databases of Raman spectra (CV Raman and KS Krishnan, A new type of secondary radiation, 121 NATURE 501-502 (1928); Zai-Qing Wen, Raman spectroscopy of protein pharmaceuticals, 96 JOURNAL OF PHARMACEUTICAL SCIENCES 2861-287 (2007)).

[0162] The FDA and other government regulatory agencies impose limits on the amount of subvisible particles permitted in parenteral drug formulations, with the primary and apparent concern being the uncertainty surrounding potential immunogenicity and downstream negative effects in patients receiving the drug (Singh et al., "An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics," J. Pharma. Sci. 99(8):3302-21 (2010)). For small-volume parenteral drug products (e.g., 100 mL or less), the Pharmacopeia limits subvisible particles (SVPs) 10 microns or greater to 6,000 SVPs per container, and 25 microns or greater to 600 SVPs per container, as determined by light obscuration analysis; and limits SVPs 10 microns or greater to 3,000 SVPs per container, and 25 microns or greater to 300 SVPs per container, as determined by membrane microscopy (United States Pharmacopeia and National Formulary (USP 40-NF 28), <787> (Subvisible Particulate Matter in Therapeutic Protein Injections.) For ophthalmic drugs, the SVP limits are 50 per mL for 10 microns or greater, 5 per mL for 25 microns or greater, and 2 per mL for 50 microns or greater. (Id., <78922 Particulate Matter in Ophthalmic Solutions). Regulatory agencies increasingly expect manufacturers to establish specifications for SVPs 2 microns or greater. (See Singh et al., "An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics," J. Pharm. Sci. 99(8):3302-21 (2010)).

[0163] The term "esterase" refers to an enzyme that catalyzes the hydrolysis of ester bonds to produce acids and alcohols. Esterases are a diverse category of enzymes that include acetyl esterases (e.g., acetylcholinesterase), phosphatases, nucleases, thiolesterases, lipases, and other carboxylester hydrolases. As the name suggests, carboxylester hydrolases (also known as carboxylesterases, carboxylester hydrolases, and EC 3.1.1.1) hydrolyze carboxylesters to alcohols and carboxylates using water. Lipases are carboxylester hydrolases that catalyze the hydrolysis of lipids, fats, and oils, including triglycerides, to fatty acid and alcohol head groups. For example, triglycerides are hydrolyzed by lipases, such as pancreatic lipase, to form monoacylglycerol and two fatty acid chains.

[0164] Phospholipases are lipases that hydrolyze phospholipids into fatty acids and other products. Phospholipases belong to four broad categories: phospholipase A (including phospholipase A1 and phospholipase A2), phospholipase B, and phosphodiesterase, phosphodiesterase C, and phosphodiesterase D. In addition to classical phospholipases, phospholipase B-like enzymes resident in the lysosomal lumen are thought to be involved in lipid catalysis. For example, phospholipase B-like 2 (PLBL2) is hypothesized to have esterase activity based on sequence homology and subcellular localization (Jensen et al., “Biochemical characterization and liposomal localization of the mannose-6-phosphate protein p76,” Biochem. J. 402: 449-458 (2007)).

[0165] As used herein, the phrase "percent fatty acid ester hydrolysis" refers to the molar ratio of fatty acid esters that are hydrolyzed.Since hydrolysis of fatty acid esters results in the release of free fatty acids, the percent fatty acid ester hydrolysis can be determined by measuring the free fatty acids in a sample.Thus, the percent fatty acid ester hydrolysis can be determined by calculating the number of moles of free fatty acid divided by the sum of the number of moles of fatty acid plus the number of moles of fatty acid ester.In the case of the percent hydrolysis of polysorbate 80 or polysorbate 20, this number can be determined by calculating the number of moles of free fatty acid and dividing by the total number of moles of remaining polysorbate plus the number of moles of free fatty acid.

[0166] The term "esterase inhibitor" refers to any chemical entity that reduces, inhibits, or blocks the activity of esterase. Applicants envision that the inclusion of an esterase inhibitor in a protein formulation containing a fatty acid ester surfactant may help maintain protein stability and fatty acid ester stability and help reduce SVP formation. Common esterase inhibitors known in the art include orlistat (tetrahydrolipistatin; an inhibitor of carboxylesterase 2 and lipoprotein lipase), diethylumbelliferyl phosphate (cholesterol esterase (lipase A) inhibitor), URB602 ([1-1'-biphenyl]-3-tl-carbamic acid cyclohexyl ester; a monoacylglycerol lipase inhibitor), and 2-butoxyphenylboronic acid (an inhibitor of hormone-sensitive lipase). The inclusion of esterase inhibitors during purification of a protein of interest or in the final formulation is expected to prevent or slow the hydrolysis of non-ionic surfactants such as polysorbate 20 and polysorbate 80, which in turn is expected to prevent or reduce sub-visible particle formation. Exemplary Formulations

[0167] According to one embodiment of the present disclosure, the pharmaceutical formulation comprises: (i) a human antibody that specifically binds to hIL-4R (e.g., an antibody comprising one or more of the sequences disclosed in Table 1 below); (ii) acetate; (iii) histidine; (iv) sucrose; (v) arginine; and (v) a surfactant comprising polyethylene glycol or a poloxamer.

[0168] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) a surfactant comprising polyethylene glycol or poloxamer at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the surfactant, including polyethylene glycol or poloxamer, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0169] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 15 mg / mL to 200 mg / mL; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) a surfactant comprising polyethylene glycol or poloxamer at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the surfactant, including polyethylene glycol or poloxamer, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0170] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 15 mg / mL to 200 mg / mL; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) PEG3350 or poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the surfactant, including PEG 3350 or poloxamer 188, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0171] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 15 mg / mL to 200 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 25 mM ± 2.5 mM; and (vi) a surfactant comprising PEG3350 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including PEG 3350, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0172] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 15 mg / mL to 200 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 25 mM ± 2.5 mM; and (vi) a surfactant comprising poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including poloxamer 188, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0173] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 100 mg / mL to 200 mg / mL; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) a surfactant comprising polyethylene glycol or poloxamer at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the surfactant, including polyethylene glycol or poloxamer, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0174] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 100 mg / mL to 200 mg / mL; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) PEG3350 or poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the PEG 3350 or poloxamer 188 can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0175] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 100 mg / mL to 200 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 25 mM ± 2.5 mM; and (vi) a surfactant comprising PEG3350 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the PEG 3350 can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0176] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 100 mg / mL to 200 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 25 mM ± 2.5 mM; and (vi) a surfactant comprising poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the poloxamer 188 can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0177] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 150 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) a surfactant comprising polyethylene glycol or poloxamer at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the surfactant, including polyethylene glycol or poloxamer, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0178] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 150 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) PEG3350 or poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the surfactant, including PEG 3350 or poloxamer 188, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0179] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 150 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 25 mM ± 2.5 mM; and (vi) a surfactant comprising PEG3350 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including PEG 3350, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0180] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 150 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 25 mM ± 2.5 mM; and (vi) a surfactant comprising poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including poloxamer 188, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0181] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 175 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) a surfactant comprising polyethylene glycol or poloxamer at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the surfactant, including polyethylene glycol or poloxamer, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0182] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 175 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 10 mM to 15 mM; (iii) histidine at a concentration of 15 mM to 25 mM; (iv) sucrose at a concentration of 2.5% w / v to 7.5% w / v; (v) arginine at a concentration of 20 mM to 80 mM; and (vi) a surfactant comprising PEG3350 or poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.7 to 6.1. In another embodiment, the surfactant, including PEG 3350 or poloxamer 188, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0183] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 175 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 25 mM ± 2.5 mM; and (vi) a surfactant comprising PEG3350 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including PEG 3350, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0184] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 175 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 25 mM ± 2.5 mM; and (vi) a surfactant comprising poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including poloxamer 188, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0185] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 175 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 50 mM ± 2.5 mM; and (vi) a surfactant comprising PEG3350 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including PEG 3350, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0186] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 175 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 50 mM ± 2.5 mM; and (vi) a surfactant comprising poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including poloxamer 188, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0187] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 175 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 75 mM ± 2.5 mM; and (vi) a surfactant comprising PEG3350 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including PEG 3350, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0188] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds human IL-4Rα, and comprises a heavy chain variable region (HVCR) comprising the amino acid sequence of SEQ ID NO:1 and a light chain variable region (LCVR) comprising the amino acid sequence of SEQ ID NO:2, at a concentration of 175 mg / mL ± 10 mg / mL; (ii) acetate at a concentration of 12.5 mM ± 1.25 mM; (iii) histidine at a concentration of 20 mM ± 2 mM; (iv) sucrose at a concentration of 5% w / v ± 0.5% w / v; (v) arginine at a concentration of 75 mM ± 2.5 mM; and (vi) a surfactant comprising poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the formulation has a pH of 5.9 ± 0.2. In another embodiment, the surfactant, including poloxamer 188, can be at a concentration of about 0.01% w / v to 0.5% w / v, or about 0.01% w / v to 0.4%, or about 0.01% w / v to 0.3%, or about 0.01% w / v to 0.2%. In another embodiment, the pH of the formulation can be about 5.4 to 6.5, about 5.5 to 6.2, or about 5.6 to 6.2.

[0189] In any of the various embodiments of the pharmaceutical formulations discussed above or herein, the human IL-4R antibody may comprise a human IgG1 heavy chain constant region.

[0190] In any of the various embodiments of the pharmaceutical formulations discussed above or herein, the human IL-4R antibody may comprise a human IgG4 heavy chain constant region.

[0191] In some embodiments, the human IL-4R antibody may comprise a heavy chain comprising the amino acid sequence of SEQ ID NO:9 and a light chain comprising the amino acid sequence of SEQ ID NO:10.

[0192] Further non-limiting examples of pharmaceutical formulations encompassed by this disclosure are set forth elsewhere herein, including in the working examples provided below. Stability of pharmaceutical formulations

[0193] The pharmaceutical formulations of the present disclosure exhibit a high level of stability. The term "stable" as used herein with respect to pharmaceutical formulations means that the protein of interest within the pharmaceutical formulation retains an acceptable degree of structure and / or function and / or biological activity after storage for a defined period of time. A formulation may be stable even if the protein contained therein does not maintain 100% of its structure and / or function and / or biological activity after storage for a defined period of time. Under certain circumstances, maintenance of about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of the protein's structure and / or function and / or biological activity after storage for a defined period of time may be considered "stable".

[0194] Stability can be measured by determining the percentage of protein that forms aggregates in the formulation after storage at a defined temperature or under stress conditions (e.g., agitation) for a defined time, where stability is inversely proportional to the percentage of aggregates formed. The percentage of aggregated protein can be determined by size-exclusion chromatography (e.g., size-exclusion high performance liquid chromatography (SE-HPLC) or size-exclusion ultra-performance liquid chromatography (SE-UPLC)), among others. As used herein, the phrase "acceptable degree of stability" means that at most about 15%, 10%, 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5%, or 0.1% of the protein can be detected in aggregates in the formulation after storage at a given temperature or under specified stress conditions for a defined time. The defined time to measure stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, or more. The temperature at which the pharmaceutical formulation may be stored when assessing stability can be any temperature between about -80°C and about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C. The "stress conditions" to which the formulated protein of interest may be exposed may be agitation stress (e.g., vortexing) for periods of 10, 20, 30, 40, 50, 60, 70, 80, 90, 100, 110, 120, 150, 180, or more minutes. For example, a pharmaceutical formulation containing an anti-IL-4R antibody may be considered stable if less than about 2%, 1.75%, 1.5%, 1.25%, 1%, 0.75%, 0.5%, 0.25%, or 0.1% of the antibody is detected in aggregated form after storage at 5° C. for 9 months. A pharmaceutical formulation may also be considered stable if less than about 12% of the protein is detected in aggregated form after storage at 45° C. for 56 days.A pharmaceutical formulation may also be considered stable if less than about 10% or less than about 9% of the protein is detected in aggregated form after storage at 45° C. for 42 days. A pharmaceutical formulation may also be considered stable if less than about 8% or less than about 7.5% or less than about 7% of the protein is detected in aggregated form after storage at 45° C. for 28 days. A pharmaceutical formulation may also be considered stable if less than about 6% of the protein is detected in aggregated form after storage at 45° C. for 14 days. A pharmaceutical formulation may also be considered stable if less than about 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1%, 0.5%, or 0.1% of the protein is detected in aggregated form after storage at −20° C., −30° C., or −80° C. for 3 months. A pharmaceutical formulation may also be considered stable if less than 3% or less than 2.5% of the protein is detected in aggregated form after agitation (e.g., via vortexing) at room temperature for 120 minutes.

[0195] Stability can also be measured, inter alia, by determining the particulate composition within the formulation after storage at a defined temperature for a defined time. The particulate composition can be determined, for example, by microscopy techniques or by microflow imaging techniques.

[0196] In some embodiments, the formulation of the present disclosure comprises a detectable amount of lipase (e.g., PLBL2). Methods for detecting and quantifying the presence and activity of phospholipase are known in the art. In some embodiments, phospholipase is detected by immunoassay (e.g., ELISA). In some embodiments, phospholipase is detected by liquid chromatography-mass spectrometry (LC-MS).

[0197] Thus, a pharmaceutical formulation (containing an esterase or lipase) may be considered stable if, after storage at a defined temperature (e.g., 5° C.) for a period of time (e.g., 6, 12, 18, 24, or 36 months or longer), no more than a specified number of fatty acid particles of size ≧10 μm or ≧25 μm (e.g., 3000 particles, 1000 particles, 500 particles, 250 particles, 100 particles, or 50 particles) are identified within a volume of 2.25 mL. For example, a pharmaceutical formulation may be considered stable if, after 24 months of storage at 5° C., no more than 3000 fatty acid particles are identified within a volume of 2.25 mL through a microscope. In another embodiment, a pharmaceutical formulation may be considered stable if, after 24 months of storage at 5° C., no more than 1000 fatty acid particles are identified within a volume of 2.25 mL through a microscope. A pharmaceutical formulation may also be considered stable if, after 24 months of storage at 5° C., no more than 500 fatty acid particles are identified within a volume of 2.25 mL through a microscope. A pharmaceutical formulation may also be considered stable if, after 24 months storage at 5° C., no more than 250 fatty acid particles are identified through a microscope in a volume of 2.25 mL. A pharmaceutical formulation may also be considered stable if, after 24 months storage at 5° C., no more than 150 fatty acid particles are identified through a microscope in a volume of 2.25 mL. A pharmaceutical formulation may also be considered stable if, after 36 months storage at 5° C., no more than 1000 fatty acid particles are identified through a microscope in a volume of 2.25 mL. A pharmaceutical formulation may also be considered stable if, after 36 months storage at 5° C., no more than 500 fatty acid particles are identified through a microscope in a volume of 2.25 mL. A pharmaceutical formulation may also be considered stable if, after 36 months storage at 5° C., no more than 250 fatty acid particles are identified through a microscope in a volume of 2.25 mL. A pharmaceutical formulation may also be considered stable if, after 36 months storage at 5° C., no more than 150 fatty acid particles are identified through a microscope in a volume of 2.25 mL. A pharmaceutical formulation may also be considered stable if, after 36 months of storage at 5° C., no more than 100 fatty acid particles are identifiable through a microscope in a volume of 2.25 mL. A pharmaceutical formulation may also be considered stable if, after 36 months of storage at 5° C., no more than 50 fatty acid particles are identifiable through a microscope in a volume of 2.25 mL.

[0198] Stability can also be measured, inter alia, by determining the percentage of the non-denatured protein of interest remaining in the formulation after storage at a given temperature for a defined period of time. The percentage of the non-denatured protein of interest can be determined, inter alia, by size-exclusion chromatography (e.g., size-exclusion high performance liquid chromatography (SE-HPLC)). As used herein, the phrase "acceptable degree of stability" means that at least 90% of the non-denatured form of the protein can be detected in the formulation after storage at a given temperature for a defined period of time. In certain embodiments, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the non-denatured form of the protein can be detected in the formulation after storage at a given temperature for a defined period of time. The defined time period for measuring stability can be at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, or more. The temperature at which the pharmaceutical formulation may be stored when assessing stability can be any temperature between about -80°C and about 45°C, such as storage at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C.

[0199] Stability can also be measured, inter alia, by determining the percentage of the protein of interest that migrates to a more acidic fraction during ion exchange ("acid form") than the main fraction of the protein ("main charge form"), where stability is inversely proportional to the fraction of the protein in the acid form. Without wishing to be bound by theory, deamidation of a protein can make the protein more negatively charged and therefore more acidic than the non-deamidated protein (see, e.g., Robinson, N., Protein Deamidation, PNAS, April 16, 2002, 99(8):5283-5288). The percentage of "acidified" protein can be determined by ion exchange chromatography (e.g., cation exchange high performance liquid chromatography (CEX-HPLC) or cation exchange ultra-high performance liquid chromatography (CEX-UPLC)). As used herein, the phrase "acceptable degree of stability" means that up to 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.1% of the protein can be detected in the acid form in the formulation after storage at a given temperature for a defined time. The defined time for measuring stability can be at least 2 weeks, at least 28 days, at least 1 month, at least 2 months, at least 3 months, at least 4 months, at least 5 months, at least 6 months, at least 7 months, at least 8 months, at least 9 months, at least 10 months, at least 11 months, at least 12 months, at least 18 months, at least 24 months, at least 30 months, at least 36 months, or more. The temperature at which the pharmaceutical formulation may be stored when assessing stability can be any temperature between about -80°C and about 45°C, such as about -80°C, about -30°C, about -20°C, about 0°C, about 4°C to 8°C, about 5°C, about 25°C, about 35°C, about 37°C, or about 45°C.

[0200] Measurement of the binding affinity of the antibody of interest to the target may also be used to assess stability. For example, a formulation of the present disclosure may be considered stable if, after storage for a defined period of time (e.g., 14 days to 9 months) at, e.g., -80°C, -30°C, -20°C, 5°C, 25°C, 37°C, 45°C, etc., the anti-IL-4R antibody contained within the formulation binds to hIL-4Rα with at least 80%, 85%, 90%, 95%, or higher than the binding affinity of the antibody prior to said storage. Binding affinity may be determined by any method, e.g., ELISA or plasmon resonance. Biological activity may be determined, e.g., by measuring downstream activity of the IL-4R system in the presence of the antibody and comparing that activity to the activity of the IL-4R system in the absence of the antibody.

[0201] Reference to stability of a pharmaceutical formulation "after" a specified period of time is intended to mean that the measurement of the stability parameter (e.g., % undenatured, % HMW species, or % acidic) is made at or near the end of the specified period of time, and is not intended to mean that the pharmaceutical formulation maintains the same degree of stability of the parameter measured thereafter. For example, reference to a particular stability after 12 months means that the stability measurement was made at or about 12 months after the start of the study. Containers and methods of administration

[0202] The pharmaceutical formulations of the present disclosure may be contained in any container suitable for storing drugs and other therapeutic compositions. For example, the pharmaceutical formulations may be contained in a sealed and sterilized plastic or glass container having a defined volume, such as a vial, an ampoule, a syringe, a cartridge, a bottle, or an IV bag. Different types of vials may be used to contain the formulations of the present disclosure, including, for example, transparent and opaque (e.g., amber) glass or plastic vials. Similarly, any type of syringe may be used to contain and / or administer the pharmaceutical formulations of the present disclosure. In some embodiments, the pharmaceutical formulations are contained in a pre-filled syringe (PFS). In some embodiments, the pharmaceutical formulations are contained in a pre-filled staked needle syringe.

[0203] The pharmaceutical formulations of the present disclosure may be contained in "normal tungsten" or "low tungsten" syringes. As will be appreciated by those of ordinary skill in the art, the process of making glass syringes generally involves the use of a hot tungsten rod that functions to penetrate the glass, thereby creating a hole through which liquid can be drawn and discharged from the syringe. This process results in the deposition of trace amounts of tungsten on the inner surface of the syringe. Subsequent washing and other processing steps may be used to reduce the amount of tungsten in the syringe. As used herein, the term "normal tungsten" means that the syringe contains greater than 500 parts per billion (ppb) of tungsten. The term "low tungsten" means that the syringe contains less than 500 ppb of tungsten. For example, a low tungsten syringe in accordance with the present disclosure may contain less than about 490, 480, 470, 460, 450, 440, 430, 420, 410, 390, 350, 300, 250, 200, 150, 100, 90, 80, 70, 60, 50, 40, 30, 20, 10, or less ppb of tungsten.

[0204] The rubber plunger used in the syringe and the rubber stopper used to close the opening of the vial may be coated to prevent contamination of the pharmaceutical contents of the syringe or vial and / or to maintain its stability.Accordingly, the pharmaceutical formulation of the present disclosure may be contained in a syringe with a coated plunger or in a vial sealed with a coated rubber stopper according to certain embodiments.For example, the plunger or stopper may be coated with a fluorocarbon film.Examples of coated stoppers and / or plungers suitable for use in the vials and syringes containing the pharmaceutical formulation of the present disclosure are referred to, for example, in U.S. Patent Nos. 4,997,423; 5,908,686; 6,286,699; 6,645,635; and 7,226,554, the contents of which are incorporated herein by reference in their entirety. Certain exemplary coated rubber stoppers and plungers that may be used in the context of the present disclosure are available from West Pharmaceutical Services, Inc. (Lionville, PA) and are commercially available under the trade name "Fluro Tec®". In accordance with certain embodiments of the present disclosure, the pharmaceutical formulation may be contained within a low tungsten syringe that includes a fluorocarbon coated plunger. In some embodiments, the container is a syringe, such as an Ompi EZ-Fill™ syringe or a BD Neopak™ syringe. In some cases, the syringe is a 1 mL long glass syringe with a 1 mL iWest piston, a 27G thin-walled needle, and an FM30 needle shield or a BD260 needle shield. In some cases, the syringe is a 2.25 mL glass syringe (e.g., Nuova Ompi).In various embodiments, the syringe is a 0.5 mL, 0.6 mL, 0.7 mL, 0.8 mL, 0.9 mL, 1.0 mL, 1.1 mL, 1.2 mL, 1.3 mL, 1.4 mL, 1.5 mL, 1.6 mL, 1.7 mL, 1.8 mL, 1.9 mL, 2.0 mL, 2.1 mL, 2.2 mL, 2.3 mL, 2.4 mL, 2.5 mL, 2.6 mL, 2.7 mL, 2.8 mL, 2.9 mL, 3.0 mL, 3.5 mL, 4.0 mL, 4.5 mL, 5.0 mL, 5.5 mL, 6.0 mL, 6.5 mL, 7.0 mL, 7.5 mL, 8.0 mL, 8.5 mL, 9.0 mL, 9.5 mL, or 10 mL syringe (e.g., a glass syringe).

[0205] The pharmaceutical formulations may be administered to the patient via parenteral routes, such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.), or via transdermal, mucosal, nasal, pulmonary and / or oral administration. Many reusable pen and / or autoinjector delivery devices may be used to deliver the pharmaceutical formulations of the present disclosure subcutaneously. Examples include, but are not limited to, AUTOPEN™ (Owen Mumford, Inc., Woodstock, UK), DISETRONIC™ pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25™ pen, HUMALOG™ pen, HUMALIN 70 / 30™ pen (Eli Lilly and Co., Indianapolis, IN), NOVOPEN™ I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR™ (Novo Nordisk, Copenhagen, Denmark), BD™ pen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN™, OPTIPEN PRO™, OPTIPEN STARLET™, and OPTICLIK™ (sanofi-aventis, Frankfurt, Germany), to name just a few. Examples of disposable pen and / or autoinjector delivery devices having application for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, the SOLOSTAR™ pen (sanofi-aventis), FLEXPEN™ (Novo Nordisk), and KWIKPEN™ (Eli Lilly), the SURECLICK™ autoinjector (Amgen, Thousand Oaks, CA), PENLET™ (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and the HUMIRA™ pen (Abbott Labs, Abbott Park, IL), to name only a few.In some cases, the pharmaceutical formulation is contained in a syringe specifically adapted for use with an autoinjector. The subcutaneous injection may be administered using a 20-30 gauge needle, or a 25-30 gauge needle. In some cases, the subcutaneous injection may be administered using a 25 gauge needle. In some cases, the subcutaneous injection may be administered using a 27 gauge needle. In some cases, the subcutaneous injection may be administered using a 29 gauge needle.

[0206] Another type of delivery device may include a safety system. Such devices are relatively inexpensive and may be operated to manually or automatically extend a safety sleeve over the needle once the injection is complete. Examples of safety systems may include the ERIS device by West Pharmaceutical, or the UltraSafe device by Becton Dickinson. Additionally, the use of a large dose device ("LVD") or bolus injector to deliver the pharmaceutical formulations of the present disclosure is also contemplated herein. In some cases, the LVD or bolus injector may be configured to inject the drug into the patient. For example, the LVD or bolus injector may be configured to deliver a "large" amount of drug (typically about 2 mL to about 10 mL).

[0207] The pharmaceutical formulations of the present disclosure may also be contained in unit dosage forms. The term "unit dosage form" as used herein refers to physically discrete units suitable as unitary dosages for a patient to be treated, each unit containing a predetermined amount of active compound calculated to produce a desired therapeutic effect in association with a required pharmaceutical carrier, diluent, or excipient. In various embodiments, the unit dosage form is contained in a container as discussed herein. The actual dosage level of the active ingredient (e.g., anti-IL-4R antibody) in the formulations of the present disclosure may vary to obtain an amount of active ingredient that is effective to achieve a desired therapeutic response without side effects for a particular patient, composition, and mode of administration. The dosage level selected will depend on a variety of pharmacokinetic factors, including the activity of the particular composition of the present disclosure used, the route of administration, the duration of administration, the excretion rate of the particular compound used, the duration of treatment, other drugs, compounds and / or materials used in combination with the particular composition used, the age, sex, weight, condition, general health and previous medical history of the patient being treated, and similar factors well known in the pharmaceutical industry. The term "diluent" as used herein refers to a solution suitable for modifying or achieving the exemplary or appropriate concentration(s) as described herein.

[0208] In various embodiments, the unit dosage form contains the amount of active ingredient (e.g., anti-IL-4R antibody) intended for a single use. In various embodiments, the amount of active ingredient in the unit dosage form is about 0.1 mg to about 5000 mg, about 100 mg to about 1000 mg, and about 100 mg to about 500 mg, about 100 mg to about 400 mg, about 100 mg to about 200 mg, about 250 mg to about 350 mg, about 125 mg to about 175 mg, about 275 mg to about 325 mg, or ranges or intervals thereof. For example, ranges of values ​​using any combination of the values ​​recited above (or values ​​contained within the ranges recited above) as upper and / or lower limits are intended to be included. In certain embodiments, the formulation is often supplied as a liquid in unit dosage form. In some embodiments, the unit dosage form contains about 100 mg of active ingredient. In some embodiments, the unit dosage form contains about 150 mg. In some embodiments, the unit dosage form contains about 200 mg. In some embodiments, the unit dosage form contains about 300 mg. In some embodiments, the unit dosage form contains about 350 mg. In some embodiments, the unit dosage form contains about 600 mg. In some embodiments, the unit dosage forms described herein are suitable for subcutaneous administration to a patient.

[0209] The present disclosure also includes a method for preparing a unit dosage form. In one embodiment, the method for preparing a pharmaceutical unit dosage form includes combining a combination of any of the above-mentioned embodiments in a suitable container (e.g., a container as discussed herein). Therapeutic Uses of Pharmaceutical Formulations

[0210] The pharmaceutical formulations of the present disclosure comprising anti-IL-4R antibodies are useful, inter alia, for the treatment, prevention and / or amelioration of any disease or disorder associated with IL-4R activity.

[0211] Therapies of the present disclosure include administering to a subject any formulation comprising an anti-hIL-4R antibody as disclosed herein. The subject to which the pharmaceutical formulation is administered can be, for example, any human or non-human animal in need of such treatment, prevention and / or amelioration or who would otherwise benefit from inhibition or attenuation of IL-4R and / or IL-4R-mediated activity. The present disclosure further includes the use of any of the pharmaceutical formulations disclosed herein in the manufacture of a medicament for the treatment, prevention and / or amelioration of any disease or disorder associated with IL-4R activity.

[0212] In some embodiments, the disease or disorder associated with IL-4R activity is an inflammatory condition, an allergic condition, a pulmonary / respiratory disorder, a gastrointestinal disorder, or a skin disorder. In some embodiments, the disease or disorder is a type 2 inflammatory disorder. In some embodiments, the disease or disorder is an atopic disease. Non-limiting examples of diseases and disorders associated with IL-4R activity include allergies (e.g., food allergies, environmental allergies, grass allergies, peanut allergies, dairy allergies), allergic reactions, allergic bronchopulmonary aspergillosis, allergic fungal sinusitis (AFRS), allergic rhinitis, alopecia areata, asthma (including mild, moderate, or severe asthma or persistent asthma), arthritis (including septic arthritis), atopic dermatitis (including moderate or severe atopic dermatitis), atopic dermatitis of the hands and feet, atopic keratitis, autoimmune hemolytic anemia, autoimmune lymphoproliferative syndrome, autoimmune uveitis, Barrett's esophagus, benign prostatic hyperplasia, bronchiectasis, bullae, These conditions include: pemphigoid, Churg-Strauss syndrome, chronic idiopathic urticaria, cold-induced urticaria, chronic induced urticaria, chronic spontaneous urticaria (CSU), contact dermatitis (e.g. allergic contact dermatitis), chronic obstructive pulmonary disease (COPD), eosinophilic esophagitis, eosinophilic gastroenteritis, Graves' disease, herpes, hypertrophic scarring, inflammatory bowel disease, Kawasaki disease, nasal polyposis, nephrosis, Netherton syndrome, preeclampsia, prurigo nodularis, pruritus (e.g. chronic pruritus of unknown origin), rhinitis (e.g. allergic rhinitis), sinusitis (e.g. allergic fungal sinusitis, chronic sinusitis with or without nasal polyposis), scleroderma, sickle cell disease, Sjögren's syndrome, tuberculosis, ulcerative colitis, and Whipple's disease.

[0213] In some embodiments, the disclosure provides a kit comprising a pharmaceutical formulation (e.g., a container containing the formulation or unit dosage form) as discussed herein, and packaging or labeling (e.g., a package insert) comprising instructions for using the pharmaceutical formulation for the treatment of a disease or disorder as discussed above. In some cases, the instructions provide for the use of the unit dosage form, as discussed herein, for the treatment of a disease or disorder.

[0214] A summary of the sequences referred to herein and the corresponding SEQ ID NOs are provided in Table 1 below. Table 1. Sequence listing information [Table 1] JPEG2025515041000002.jpg220166JPEG2025515041000003.jpg220166JPEG2025515041000004.j pg220166JPEG2025515041000005.jpg220166JPEG2025515041000006.jpg219166JPEG20255150410 00007.jpg221166JPEG2025515041000008.jpg221166JPEG2025515041000009.jpg221166JPEG202 5515041000010.jpg220166JPEG2025515041000011.jpg223166JPEG2025515041000012.jpg162166 EXAMPLES

[0215] The following examples are presented so as to provide one of ordinary skill in the art with a complete disclosure and description of how to make and use the methods and compositions of the present invention, and are not intended to limit the scope of what the inventors regard as the invention. Efforts have been made to ensure accuracy with respect to numbers used (e.g., amounts, temperatures, etc.), but some experimental error and deviation should be accounted for. Unless otherwise indicated, parts are parts by weight, molecular weight is average molecular weight, temperature is in degrees Celsius, and pressure is at or near atmospheric. Determination of particles invisible to the naked eye

[0216] For the determination of subvisible particles, suitable methods include "Method 1" (Light Obscuration Particle Count Test) and "Method 2" (Microscopic Particle Count Test). Using light obscuration, the FDA requirements for subvisible particulates in parenteral drug products are ≦6,000 particles per container for particles ≧10 micrometers in diameter and ≦600 particles per container for particles ≧25 micrometers in diameter. Using microscopy, the FDA requirements for subvisible particulates in parenteral drug products are ≦3,000 particles per container for particles ≧10 micrometers in diameter and ≦300 particles per container for particles ≧25 micrometers in diameter. Currently, there is no specification for particles less than 10 micrometers in diameter, but the FDA requires that particles between 2 and 10 micrometers be measured.

[0217] HIAC optical obscuration and Brightwell Microflow Imaging (MFI) were used to measure particles larger than 1 micrometer in diameter. HIAC combines optical obscuration and laser light scattering to allow for the detection and counting of particles ranging from 500 nm to 350 μm in a moving fluid stream. Particles are sized based on the voltage response produced in the detection device and sorted into predetermined size ranges based on the voltage response.

[0218] For the HIAC assay, samples from the manufacturing line (GMP lots) containing monoclonal antibody at 150 mg / mL were pooled to a total volume of 25 mL. For each pooled sample, three readings of 5 milliliters per sample were taken. Laboratory samples of the same 150 mg / mL antibody formulation were also examined by HIAC. Samples from at least three vials (2.5 mL / vial), seven 1 mL syringes (1.14 mL / syringe), or five 2.25 mL syringes (2 mL / syringe) were pooled and three readings of 1 milliliter per reading were taken. Light obscuration readings were taken using HIAC 9703 and HIAC 8000A instruments (Hach Company, Loveland, Colo.) using the HRLD 400 probe (reads up to 18,000 cumulative counts per mL) and the MC05 probe (reads up to 10,000 cumulative counts per mL), respectively.

[0219] The MFI method used less material than HIAC light obscuration (i.e., 1 mL of formulation, or one stability vial or syringe) and produced higher particle counts than HIAC. Because MFI is based on a microscope, the method was more sensitive to transparent protein particles and was able to distinguish silicon oil droplets / air bubbles from protein particles for pre-filled syringe samples. MFI was performed on laboratory samples containing 150 mg / mL of monoclonal antibody (as in the HIAC analysis). For MFI, one reading of 1 milliliter was taken per reading. Determination of polysorbate degradation

[0220] Degradation of polysorbates was examined using one or more of several methods. The first method used an enzymatic colorimetric assay to quantify nonesterified fatty acids (NEFAs). The NEFA-HR(2) kit (Wako Diagnostics, Richmond, Va.) was used to detect fatty acids in formulated drug substances containing polysorbates. Briefly, samples were combined with ATP and coenzyme A (CoA) in the presence of acyl-CoA synthetase (ACS). Available (free) fatty acids reacted with CoA to form acyl-CoA. The acyl-CoA product reacted with oxygen and acyl-CoA oxidase to produce trans-2,3-dehydroacyl-CoA and hydrogen peroxide. Peroxidase catalyzed the reaction of hydrogen peroxide with 4-aminoantipyrine and 3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline to form a blue-purple pigment (maximum absorbance at 550 nm). The amount of NEFA in a sample is proportional to the amount of pigment. For a detailed description of the NEFA colorimetric assay, see Duncombe, "The Colorimetric Micro-Determination of Non-Esterified Fatty Acids in Plasma," Clin Chim Acta. 9:122-5 (1964);Itaya and Ui, "Colorimetric Determination of Free Fatty Acids in Biological Fluids," J. Lipid Res. 6:16-20 (1965);Novak, M., "Colorimetric Ultramicro Method for the Determination of Free Fatty Acids," J. Lipid Res. 6:431-3 (1965);and Elphick, MC, "Modified Colorimetric Ultramicro Method for Estimating NEFA in Serum," J. Clin. Pathol. 21(5):567-70 (1968).

[0221] Test samples containing the protein of interest (and putative host cell protein contaminants) were applied to a 10 kDa molecular weight cutoff filter. The retentate was collected at >100 g / L protein in 10 mM histidine, pH 6.0, and was spiked with polysorbate to yield a test sample of 100 g / L protein, 0.8% (w / v) polysorbate, 10 mM histidine, pH 6.0 (t 初期 The test samples were subjected to 45° C. for 44 hours (t 最終 Some samples were spiked with oleic acid to assess the recovery efficiency of NEFAs in the samples. The percent polysorbate degradation was calculated as follows: [ka]

[0222] The second method for determining polysorbate degradation was based on mass spectrometry. Using LC / MS analysis, this assay allowed the measurement and comparison of the initial percentage of esters and the remaining percentage of esters in polysorbates at different time points after incubation at 45° C. mAb1 produced according to Process 6 (without HIC and with PS degrading activity) and mAb1 produced according to Process 3 (with HIC step and without PS degrading activity) were included as negative and positive controls, respectively (see Example 4 and Table 9).

[0223] Briefly, 15 mg of antibody sample (on the order of 5–10 mg / mL, or 7 mg / mL ± 1.5 mg / mL) was applied to an ultrafiltration membrane (Amicon Ultra 50K, Millipore, Billerica, Mass.) and centrifuged at 14,000 x g for 15 min or until the remaining volume was just below the 100 μL marked on the device. 1 μL of 10% polysorbate was added to the spin filter containing the concentrated protein and vortexed. Samples were collected by inverted centrifugation at 1000 g for 5 min and the entire volume was collected in a collection tube.

[0224] The recovered volume was measured and the polysorbate concentration was calculated. One microliter of each recovered sample was diluted 100-fold in a separate test tube, and the protein concentration was measured with a Nanodrop1000 (Thermo Fisher Scientific, Inc., Wilmington, Del.). Samples were then diluted with histidine buffer (10 mM, pH 6.0) and polysorbate stock to achieve a protein concentration of 150 mg / mL and a polysorbate concentration of 0.2% (w / w).

[0225] A time zero (T0) sample (2 μL) was retained from each sample and stored at −80° C. until use. Samples to be tested were sealed under argon, incubated at 45° C. to induce degradation, and removed for testing at the specified time points. 2 μL was taken from each sample at each time point and diluted to 100 μL with water. Samples at each dilution time point were stored at −80° C. After collection of each time point, the headspace of the sample tube was filled with argon gas, the sample container was resealed, and the sample was returned to the incubator and incubation resumed.

[0226] Time point samples were analyzed using an anion exchange column (Oasis MAX column, 30 μm, 2.1 mm×20 mm; Waters Corporation, Milford, Mass.) followed by reversed-phase chromatography (ACQUITY UPLC® BEH 130 C4 column, 1.7 μm, 2.1 mm×50 mm; Waters Corporation, Milford, Mass.) at t=5 min. The reversed-phase output was coupled to a mass analyzer (Thermo Q-Exactive mass analyzer; Thermo Fisher Scientific, Inc., Wilmington, Del.). Chromatographic conditions are listed in Table 2.

[0227] The system was equilibrated with 99% mobile phase A (0.1% formic acid in water) for 40 min at a flow rate of 0.1 mL / min before the first injection. Water was used as a blank injection. Mass analyzer parameters were as follows: mass range 150-2000 m / z; heater temperature 250 °C; voltage 3.8 kv; sheath gas 40; auxiliary gas 10; capillary temperature 350 °C; and S-lens 50. When mass spectrometry-based identification was not required, charged aerosol detection (CAD) was used at an analytical flow rate and a desolvation temperature of 100° C. (Lisa et al., “Quantitation of triacylglycerols from plant oils using charged aerosol detection with gradient compensation,” J Chromatogr A. 1176(1-2):135-42 (2007); Plante et al., “The use of charged aerosol detection with HPLC for the measurement of lipids,” Methods Mol Biol. 579:469-82 (2009)). Table 2. Chromatographic conditions for polysorbate degradation determination. [Table 2]

[0228] To estimate the total amount of polyoxyethylene (POE), mass chromatograms were extracted using the 300-800 m / z range to avoid interference from degraded proteins, and the cluster peak from approximately 8-15 min was integrated. For CAD chromatograms, the first cluster peak of POE was integrated directly from approximately 8-15 min (again, retention times may shift slightly). If there were other species coeluting with POE, the baseline was adjusted to minimize their effect on the peak area.

[0229] To estimate the total amount of POE esters, mass chromatograms were extracted using the 300–2000 m / z range and the cluster of peaks from approximately 17–40 min was integrated. For the CAD chromatograms, the POE ester peak cluster was directly integrated from approximately 17–40 min.

[0230] The percentage of POE esters was calculated according to the following equation: [ka]

[0231] The percentage of remaining POE esters was calculated according to the following equation: [ka]

[0232] where n=2, 4, or 10 days. Example 1. Failure to specify particulates

[0233] Two GMP lots of 150 mg / mL antibody formulations were evaluated for particles not visible to the naked eye through HIAC light obscuration after at least 6 months at 5° C. as described in U.S. Patent No. 10,342,876, which is incorporated herein by reference. The formulation contained 0.02% polysorbate 20 from Supplier A, and 150 mg / mL of anti-IL-4R antibody. The antibody was purified from CHO cell cultures using a combination of affinity capture and ion exchange chromatography. The results are presented in Table 3. Table 3. Number of particles ≥ 10 μm in size after storage [Table 3] Example 2. Quality and purity of fatty acid esters affect SVP formation

[0234] The effect of the nature and quality of the non-ionic surfactants (Polysorbate 20 and Polysorbate 80) on the formation of subvisible particles in protein formulations was tested by formulating an antibody in either (i) Polysorbate 20 from Supplier A (PS20-A), (ii) Polysorbate 20 from Supplier B (PS20-B), or (iii) Polysorbate 80 (PS80), as described in U.S. Pat. No. 10,342,876, incorporated herein by reference. Table 4 shows HIAC SVP (≧10 μm SVP) data from compounded drug substance of the following formulation: 20 mM histidine (pH 5.9), 12.5 mM acetate, 0.02% non-ionic surfactant (polysorbate), 5% sucrose (w / v), 25 mM arginine, and 150 mg / mL antibody stored as 2.5 mL fills in 5 mL Type 1 borosilicate glass vials with West S2-F451 4432 / 50 GRY B2-40 stoppers.

[0235] Here, the formulated drug substance containing polysorbate 80 ("mAb1") exhibited significantly less SVP formation over time than the formulation containing polysorbate 20. Additionally, the formulation containing polysorbate 20 from supplier B (PS20-B), a higher grade of polysorbate 20, exhibited less SVP formation than the formulation containing polysorbate 20 from supplier A (PS20-A; a lower grade of polysorbate 20). Comparative analysis of PS20-A and PS20-B shows that PS20-B has 5-10% more total esters than PS20-A, and PS20-A has more isosorbide laurate esters than PS20-B, as shown in FIG. 1. Table 4. Number of microparticles ≥ 10 μm in size after storage with various surfactants. [Table 4]

[0236] The formulated drug substances containing HP-PS20 or SR-PS80 were further evaluated using membrane microscopy or MFI after up to 36 months of storage, as shown in Figure 2. The number of particles >10 μm was further compared comparing formulations stored in glass vials to formulations stored in pre-filled syringes, as shown in Figures 2A, 2B, 2C, and 2D. In all cases, the number of particles increased substantially over time.

[0237] It was hypothesized that degradation of the fatty acid esters of polysorbate in the formulation would promote protein instability, resulting in SVP formation. To assess polysorbate degradation, the stability of polysorbate 20 and polysorbate 80 in a 150 mg / mL antibody (mAb1) formulation containing 0.02% non-ionic surfactant (polysorbate) prepared without HIC (Process 3, see below and Table 9) was compared. The relative amounts of remaining esters (mono- and di-esters) were determined by mass spectrometry. Significant degradation of the ester components of polysorbate 20 was observed after samples were stored at 5° C. for 6 months or at 45° C. for 2 months. For polysorbate 80, less extensive degradation was observed under the same conditions (see Table 5). These results correlate with the SVP particle formation observations.

[0238] The degradation rates of polysorbate 20 and polysorbate 80 (as described above with respect to Table 5) formulated with 150 mg / mL antibody (mAb1) were determined under the same conditions using mass spectrometry to measure the relative amounts of free fatty acids and fatty acid esters. The following formula was used to determine the percent ester degradation: [ka]

[0239] where T0 = time zero, T1 = time at experimental conditions (i.e., 2 months at 45°C; 6 months at 5°C), and POE = polyoxyethylene. Table 6 shows the percent degradation of polysorbate 20 and polysorbate 80 in 150 mg / mL antibody formulations. The degradation rate of polysorbate 80 was consistently lower for mAb1 (but not for all antibodies tested) than the degradation rate of polysorbate 20 in otherwise identical antibody formulations. Table 5. Percentage of ester remaining after storage [Table 5] Table 6. Percentage of ester degradation after storage [Table 6]

[0240] Polysorbate degradation in formulations containing SR-PS80 or HP-PS20 stored in glass vials or prefilled syringes was further compared using CAD-UHPLC, as shown in Figures 3A and 3B. In all cases, there was discernible polysorbate degradation over time. The presence of SVPs correlated with the residual polysorbate concentration in the formulation, as shown in Figure 4. The same relationship was found using formulations containing another monoclonal antibody, mAb5, as shown in Figure 5. Using Raman spectroscopy, it was confirmed that the microparticles in the polysorbate-containing formulations matched the characteristics of fatty acids. Therefore, to resolve the issue of SVP formation, the phenomenon of polysorbate degradation and free fatty acid particle formation was further investigated. Example 3. Polysorbate decomposition activity

[0241] To determine the etiological agent responsible for polysorbate degradation, buffered mAb1 antibody (150 mg / mL) was separated into two fractions: protein fraction and buffer fraction by 10 kDa filtration, as described in U.S. Patent No. 10,342,876, which is incorporated herein by reference. These two fractions, as well as intact buffered antibody, were spiked with 0.2% (w / v) ultra-purified polysorbate 20 (PS20-B) and stressed at 45°C for up to 14 days. Studies showed that protein fractionation had an effect on the degradation of sorbitan laurate (i.e., the main component of polysorbate 20) as shown in Table 7, while buffer fractionation had no effect, and polysorbate 20 degradation correlated with the concentration of antibody as shown in Table 8. Table 7. Decomposition of sorbitan laurate in each mAb1 fraction [Table 7] Table 8. Degradation of polysorbate in each mAb1 fraction correlates with antibody concentration. [Table 8] Example 4. Hydrophobic interaction chromatography

[0242] The antibodies were produced in CHO cell hosts and purified using one of two processes (see Table 9) as described in U.S. Patent No. 10,342,876, which is incorporated herein by reference. In one case, the antibodies were purified using an ion exchanger as a polishing step (capture step, ion exchange 1, ion exchange 2; "Process 3"). In the other case, one of the polishing steps used to purify the antibodies was hydrophobic interaction chromatography as an additional polishing step (capture step, ion exchange, hydrophobic interaction; "Process 6"). The antibodies purified by either Process 3 or Process 6 were formulated at 150 mg / mL in 20 mM histidine (pH 5.9), 12.5 mM acetate, 5% sucrose, 25 mM arginine, and 0.02% polysorbate 20, and subjected to forced degradation at 45° C. for up to 14 days. On day 14, approximately 98% of the sorbitan laurate (i.e., intact esters) remained in the formulation containing antibody purified using process 6, while only approximately 28% of the sorbitan laurate remained in the formulation containing antibody purified using process 3. Thus, the hydrophobic interaction chromatography (HIC) step appeared to remove activity that contributes to polysorbate degradation. Table 9. Polysorbate recovery using various antibody purification processes [Table 9]

[0243] The role of bulk process steps in removing putative polysorbate degraders (putative esterase activity) was evaluated. Antibody produced from CHO cells was subjected to successive purification steps and the stability of polysorbate 20 was evaluated at each step. Results from one set of experiments are presented in Table 9, which are reported in terms of the percent of intact polysorbate 20 at each step or step sequence. The percent of intact polysorbate 20 is expected to be inversely proportional to the amount of contaminating esterase activity.

[0244] A number of different antibodies were tested for associated polysorbate degrading activity (esterase) and the effect of HIC on that activity. In each case, polysorbate 20 degrading activity was detected, and this activity was virtually eliminated by incorporation of a HIC purification step (Table 10). Table 10. Polysorbate degradation with and without HIC purification [Table 10]

[0245] The role of HIC in the formation of sub-visible particles was investigated. Without meaning to be limited by theory, it was hypothesized that the stability of non-ionic surfactants in protein (e.g., antibody) formulations is directly correlated to the formation of sub-visible particles. Loss of surfactant activity may allow proteins to aggregate and form sub-visible particles. Additionally or alternatively, fatty acids liberated by degradable sorbitan fatty acid esters may also contribute to the formation of sub-visible particles as immiscible fatty acid droplets. Thus, the level of sub-visible particles ≧10 micrometers in diameter was counted in drug substances (150 mg / mL antibody in 20 mM histidine (pH 5.9), 12.5 mM acetate, 5% sucrose, 25 mM arginine, and 0.02% polysorbate 20) manufactured with (e.g., process 6) or without (e.g., process 3) HIC. The results (presented in Tables 11 and 12) show that application of the HIC step significantly reduced (by an order of magnitude less than 10-fold) the formation of SVPs in the drug substance, although lower quality PS20-A was used in these experiments. Table 11. Number of particles ≥ 10 μm in size with and without HIC purification [Table 11] Table 12. Number of particles ≥ 25 μm in size with and without HIC purification [Table 12]

[0246] mAb1 formulations containing PS20 or PS80 and produced with HIC were further characterized for particle formation over time, as shown in Figures 6A, 6B, 6C, and 6D. Formulations containing either PS20 or PS80 did not show an increase in particle formation over time, as measured by either membrane microscopy or MFI.

[0247] mAb1 formulations produced with HIC were further evaluated for polysorbate degradation over time, as shown in Figures 7 A and 7 B. Consistent with the SVP results described above, formulations subjected to the HIC process showed no appreciable polysorbate degradation, even over 36 months of storage. Example 5. Putative phospholipase B-like 2 activity

[0248] Polysorbate decomposition activity was followed during HIC purification of an exemplary antibody produced in CHO cell culture as described in U.S. Patent No. 10,342,876, which is incorporated herein by reference. Partially purified CHO cell extract was applied to HIC (phenyl-Sepharose). The flow-through, which contains almost all the antibody, was collected and analyzed for polysorbate decomposition activity. No polysorbate decomposition activity was observed in this flow-through fraction. The HIC bound fraction was stripped from the HIC medium and subsequently subjected to 100 kDa cut-off ultrafiltration / diafiltration. The unfiltered strip fraction contained 9.9% polysorbate decomposition activity, the filter permeate contained 1.3% polysorbate decomposition activity and 5% antibody yield, and the filter retentate contained 7.4% polysorbate decomposition activity and 95% antibody yield. Table 13. Percentage reduction in polysorbate 20 degradation by concentration of lipase inhibitors. [Table 13]

[0249] Whether the polysorbate degrading activity is lipase was tested by combining a lipase inhibitor with a polysorbate degrading active fraction spiked with polysorbate 20. Table 13 presents data showing that the lipase inhibitor reduced the polysorbate degrading activity compared to the control (antibody with associated polysorbate degrading activity plus polysorbate 20, no lipase inhibitor). The lipase inhibitor reduced or eliminated the polysorbate degrading activity associated with the antibody.

[0250] CHO-produced recombinant antibody HIC strip fractions (not flow-through) containing polysorbate degrading activity were subjected to further HIC in bind / elute mode, where the antibody was eluted with a shallow gradient. Elution fractions were tested for PS20 degrading activity, and fractions with activity were subjected to (i) intact mass analysis, (ii) non-denaturing size-exclusion chromatography UV analysis (SEC-UV), and (iii) tryptic digestion followed by LC-MS and proteomics interrogation analysis. Intact mass analysis of the reversed-phase liquid chromatography fractions revealed an unknown species in the hydrophobic fraction L8 (the most hydrophobic fraction). Formulated antibody samples containing polysorbate 20 and spiked (1:100) with L8 showed 20% polysorbate degradation by day 8. Antibody monomer and free light chain were detected in the less hydrophobic fractions L3-L7, as well as L8. Antibody dimers were detected in fractions L5 to L8.

[0251] HIC strip fractions L3-L9 were subjected to SEC-UV under non-denaturing conditions. Fraction L8 resolved into three major peaks, the first coming first, and two minor peaks corresponding to smaller species coming later. The first peak leaving the column contained antibody dimers and other oligomers. The second peak contained antibody monomers. The third peak contained species with polysorbate degrading activity. Thus, the degrading activity is separable from the antibody and is of smaller molecular turnover than the antibody monomers.

[0252] HIC fraction L8 was also subjected to shotgun proteomics analysis. Briefly, the L8 fraction was successively (i) retained on a 10 kDa filter, (ii) reconstituted in 6 M guanidine-HCl, 100 mM Tris-HCl, pH 7.5, (iii) treated in 10 mM Tris(2-carboxythylphosphine hydrochloride) (TCEP) at 50° C. for 30 min, followed by treatment in 20 mM indole-3-acetic acid (IAA) at room temperature in the dark for 30 min, (iv) diluted 8-fold with 1 part trypsin added to 20 parts sample, incubated at 37° C. for 4 h, and then (v) subjected to LC-MS / MS analysis. A proteomic search of the resulting peptide sequences revealed that five proteins were associated with L8: (i) putative phospholipase B-like 2 (representing 15% of the peak fraction), (ii) peroxiredoxin-1, (iii) heat shock 27-kDa protein 1, (iv) anaphase-promoting complex subunit 1, and (v) U3 small ribonucleoprotein MPP10.

[0253] The amount of polysorbate degradation activity correlated with the abundance of phospholipase B-like 2 protein (PLBL2) present. At various purification steps, the amount of PLBL2 was determined by nanoLC-MS or LC-MS to determine the rate of polysorbate degradation (PS20 spiked fractions). The abundance of PLBL2 was calculated based on the ratio of peptide intensities from lipase and drug substance (i.e. antibody). The results are presented in Figure 8 and Table 14. Table 14. Correlation between PLBL2 and polysorbate degradation [Table 14] 1 Concentration-regulated decomposition rate 2 Phospholipase abundance calculated based on ratio of peptide intensities from lipase and drug substance Example 6. Reduction of lipase activity using AEX

[0254] The ability of an anion exchange (AEX) chromatography unit operation to reduce the levels of lipase in a pharmaceutical formulation was investigated. AEX was performed in flow-through mode, where negatively charged impurities are adsorbed onto a fixed positively charged ligand (column) and the product flows through. Stepwise regression was used to generate a transfer function for the process parameters deemed most likely to affect lipase activity. A visualization of the resulting model is shown in FIG. 9. This model can be used for rational set-point and range selection using an optimization algorithm to maximize the desirability and robustness of the process. Surprisingly, lipase activity was inversely correlated to AEX loading pH, providing a novel method to minimize lipase activity by optimizing AEX chromatographic conditions.

[0255] The performance of the optimized AEX process was verified in four pilot-scale (500 L) confirmation batches, as shown in Table 15. After spiking with 0.8% w / v polysorbate 20, concentrated samples were stressed at 45° C. for 44 hours and lipase activity was measured by measuring the change in non-esterified fatty acids (NEFA). The anion exchange process performance in these batches was compared to small-scale model predictions obtained from a Monte Carlo simulation of the process run at set points with estimated variations in input parameters, as shown in FIG. 10. The confirmation batch responses are shown in solid orange lines. All responses are within the expected ranges generated from a scale-down multivariate model of an anti-IL-4R antibody produced using the process of the present invention, illustrating the suitability of the small-scale model to predict pilot-scale performance and process robustness to scale up. Table 15. Summary of 500 L scale performance of anion exchange step during process validation batches. [Table 15] Example 7. Optimized fatty acid composition to reduce particle formation

[0256] Proteins of interest may present additional challenges to removing lipases based on their structure and physicochemical properties. After typical HCP removal techniques, mAb5 was formulated and still exhibited polysorbate degradation over time during storage, as shown in Figures 11 and 12. Homology modeling established that mAb5 features a large hydrophobic patch compared to mAb6 and mAb7, as shown in Figure 13, which may cause co-elution of hydrophobic lipases and mAb5. Therefore, further approaches to reduce polysorbate degradation and SVP formation were investigated.

[0257] Fatty acid composition and percent distribution of high melting point components are useful considerations for predicting particulate formation. Free fatty acids with high melting points may be capable of forming insoluble particles at room temperature that are detectable during the assay. The composition of fatty acids in different polysorbates, along with their respective melting points, is shown in Table 16. Commercially available polysorbates may have fatty acid compositions with a wide range of melting points, with PS80 being characterized by a higher concentration of low melting point fatty acids, especially oleic acid. Table 16. Fatty acid composition of commercially available polysorbates [Table 16]

[0258] The storage stability of mAb1 drug product (DP) prepared without the HIC step was evaluated across samples containing different grades of PS80, as described in US Patent Application Publication No. 20190083618 A1, which is incorporated by reference herein. Each DP sample had a volume of 2.136 mL, which contained the same concentration of mAb1 (150 mg / mL) and one of several lots of PS80 at 0.2% (w / v). Each PS80 lot had one of three different content percentages of oleate esters (70%, 87%, and ≧99%). Table 17 summarizes the percentage content of oleate esters in PS80 in each FDS sample. Table 17. [Table 17]

[0259] DP samples were stored in glass prefilled syringes at 2–8° C. for up to 24 months. Particulates were measured in each DP sample every 6 months for a total of 24 months by both membrane microscopy and microflow imaging (MFI).

[0260] FIG. 14 shows the number of SVPs per container having a diameter of ≧10 μm as measured by membrane microscopy. FIG. 15 shows, in chart form, the number of SVPs per container having a diameter of ≧10 μm as measured by MFI. As shown in FIG. 14 and FIG. 15, DP B and DP C (the two DP samples containing PS80 with ≧99% content of oleic acid esters) showed the lowest number of SVPs over the entire 24-month period as measured by both membrane microscopy (FIG. 14) and MFI (FIG. 15). DP A, containing PS80 with 87% content of oleic acid esters, showed the second lowest number of particulates subvisible to the naked eye over the 24-month period (notably, showing 800-1200 particles by 24 months). DP D, E, and F all showed well over 3000 particles per container (as measured by both methods) at least by the 18-month mark.

[0261] The lower number of particles in DP A, B and C (compared to the higher number of particles in DP D, E and F) was hypothesized to be the result of the use of PS80 with a higher percentage content of oleic acid (or long chain fatty acid) esters. Oleic acid is a longer long chain fatty acid with one unsaturated bond (see FIG. 16). It therefore has a melting point below ambient temperature of about 13° C. The precursor to the formation of sub- and visible free fatty acid (FFA) microparticles is the aggregation of individual FFA chains into aggregates, which then precipitate in the form of particles. Oleic acid can arise during storage of the formulation at 5° C., for example, by enzymatic hydrolysis of the fatty acid esters of polysorbate 80. This oleic acid can form SVPs, but due to its low melting point, such particles are more likely to exist as an oily liquid in the protein formulation at room temperature (about 22° C.) where the analysis is performed, and therefore do not persist as sub-visible microparticles at room temperature. In contrast, a higher non-oleic acid ester content in the formulation leads to the formation of the corresponding FFAs upon hydrolysis, which have higher melting points, and thus the subvisible and visible amorphous particulates thus formed persist at ambient temperature during analysis.

[0262] Furthermore, oleate esters are better solubilizers / stabilizers than esters of shorter chain fatty acids because oleate esters have a higher hydrophobicity, which allows them to solubilize free fatty acids and protein particulates, thereby maintaining product stability. Thus, a higher content of oleate esters (>98%) may provide improved stability to protein formulations and drug products when compared to polysorbate 80, which has a lower content of oleate esters.

[0263] The concentration (micrograms / mL) of each type of free fatty acid in each sample DP (DP A-F) was evaluated after the samples were stored at 5°C for 18 months. Samples DP A-F were prepared as described above. Free fatty acid concentrations were measured at 18 months by LC-MS, as shown in Figure 17. DP B and C (the two DP samples containing PS80 with ≥99% content of oleic acid esters) showed the highest concentration of oleic acid and the lowest concentration of other FFAs. This indicates the homogeneity of FFAs (i.e., oleic acid) in DP B and C. This further indicates that the use of polysorbates containing high oleic acid concentrations can reduce the formation of free fatty acid particles at ambient temperature, even in formulations containing lipases that trigger free fatty acid production. Example 8. Prevention of particle formation in lipase-containing formulations with PEG3350 or poloxamer 188

[0264] Esterases or lipases can hydrolyze polysorbates by enzymatic hydrolysis of ester bonds, as shown in Figure 18A. Alternative surfactants, such as PEG3350 and poloxamer 188, do not contain ester bonds and are therefore not targets for esterases, as shown in Figure 18B. mAb5 was formulated with PS20, PS80, or poloxamer 188, and the recovery of the surfactants was compared, as shown in Figure 19. Unlike the PS20 and PS80 formulations, the poloxamer formulations showed no recovery loss at all temperatures tested.

[0265] To determine whether the use of alternative surfactants could reduce the formation of SVPs in formulated DPs, formulations of IL-4R antibodies were prepared using different surfactants and microparticle formation was measured over time as described in U.S. Provisional Application No. 63 / 337532, incorporated herein by reference. Anti-IL-4R antibodies comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 1 / 2 at a concentration of 150 mg / mL were formulated in 20 mM histidine, 12.5 mM sodium acetate, 25 mM arginine-HCl, 5% w / v sucrose, and various concentrations of either PEG3550 or poloxamer 188, pH 5.9. The formulations were stored in syringes at 5° C. for up to 36 months, and periodic measurements were made of the number of particles (≧10 μm and ≧25 μm) present in the formulations as determined by microscopic examination. As shown in Figure 20, several particles of ≥ 10 μm or ≥ 25 μm were identified in the formulations over the 36 month observation period. Furthermore, no discernible differences or changes in the number of subvisible particles were observed over the course of the storage period among the different PEG3350 or poloxamer 188 containing formulations.

[0266] These results demonstrate that alternative surfactants such as poloxamer 188 are resistant to degradation in lipase-containing formulations and that low concentrations of PEG3350 or poloxamer 188 can prevent particle formation in DP formulations over extended storage periods.

[0267] To evaluate the ability of alternative surfactants to promote therapeutic protein stability, formulations containing PEG3350 or poloxamer 188 were further subjected to evaluation of agitation stress stability and heat stress stability. Agitation stress stability was tested for IL-4R antibody formulations containing different concentrations of surfactant PEG3350 or poloxamer 188. Anti-IL-4R antibody containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 1 / 2 at a concentration of 150 mg / mL was formulated with 20 mM histidine, 12.5 mM sodium acetate, 25 mM arginine-HCl, 5% w / v sucrose, and various concentrations of PEG3550 or poloxamer 188, pH 5.9. The formulations were stored in glass vials and agitated by vortexing (speed setting = 4) for 30 minutes, 60 minutes, or 120 minutes. The percentage of high molecular weight (HMV) species was then determined by size-exclusion ultra-performance liquid chromatography (SE-UPLC).

[0268] As shown in Figure 21A, antibody formulations containing at least 0.01% (w / v) PEG3350 prevented an observable increase in HMW species (quantified by SE-UHPLC) upon agitation. Lower amounts of PEG3350 (0.001% or 0.005%) were insufficient to prevent the formation of HMW. Similarly, as shown in Figure 21B, at least 0.01% (w / v) poloxamer 188 prevented an observable increase in HMW species, although HMW species were observed at lower amounts of poloxamer 188 (0.001% or 0.005%).

[0269] The heat stress stability of IL-4R antibody formulations containing PEG3350 or poloxamer 188 was further tested and compared to various IL-4R antibody formulations containing polysorbates. Anti-IL-4R antibody containing the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 1 / 2 at a concentration of 150 mg / mL was formulated in 20 mM histidine, 12.5 mM sodium acetate, 25 mM arginine-HCl, 5% w / v sucrose, and various concentrations of polysorbate 20, polysorbate 80, PEG3550 or poloxamer 188, pH 5.9. These formulations were subjected to heat stress (45° C.) for periods up to 56 days, and the percentage of high molecular weight (HMW) species was determined by size-exclusion ultra-performance liquid chromatography (SE-UPLC) at 7, 14, 28, 42, and 56 days.

[0270] As shown in Figure 22, antibody formulations with PEG3350 or poloxamer 188 at concentrations of 0.01% or 0.02% exhibited similar thermal stability to antibody formulations containing lower amounts of polysorbate (up to 0.1% w / v). By day 28, the PEG3350 or poloxamer 188-containing formulations exhibited a lower percentage of HMW species than the control formulation containing 0.2% polysorbate 20.

[0271] Further studies were performed to compare the thermal stability of antibody formulations containing 0.2% polysorbate 80 (Table 18), 0.01% poloxamer 188 (Table 19), 0.1% poloxamer 188 (Table 20), 0.5% poloxamer 188 (Table 21), 0.01% PEG3350 (Table 22), 0.1% PEG3350 (Table 23), or 0.5% PEG3350 (Table 24). Each formulation contained 150 mg / mL anti-IL4R antibody, 25 mM L-arginine-HCl, 20 mM L-histidine, 12.5 mM sodium acetate, 5% (w / v) sucrose, and surfactant at pH 5.9. Formulations containing poloxamer 188 or PEG3350 showed stability advantages compared to formulations containing polysorbate 80. Table 18. Thermal stability of formulations containing 0.2% PS80 [Table 18] Table 19. Thermal stability of formulations containing 0.01% Poloxamer 188 [Table 19] Table 20. Thermal stability of formulations containing 0.1% Poloxamer 188 [Table 20] Table 21. Thermal stability of formulations containing 0.5% Poloxamer 188 [Table 21] Table 22. Thermal stability of formulations containing 0.01% PEG3350 [Table 22] Table 23. Thermal stability of formulations containing 0.1% PEG3350 [Table 23] Table 24. Thermal stability of formulations containing 0.5% PEG3350 [Table 24] Example 9. Reduction of lipase activity using agitation and heat stress

[0272] In order to further reduce esterase and lipase activity, free fatty acid particle formation and / or polysorbate degradation in therapeutic products, additional methods for reducing esterase and lipase activity in formulated drug substances were investigated. It has surprisingly been discovered that subjecting the drug substance to stress conditions, such as agitation stress or heat stress, can reduce esterase and lipase activity and increase the long-term stability of therapeutic molecules and surfactants in the drug substance and subsequent drug product.

[0273] mAb1 drug substance produced without the HIC step was subjected to agitation stress. 30 mL of 200 mg / mL mAb1 DS was used. Two 125 mL polycarbonate bottles were filled with 10 mL of DS each and agitated at 250 rpm on an orbital shaker for 0, 24, or 48 hours. The remaining 10 mL of DS was transferred to a 15 mL Falcon tube and served as a non-stressed control. 2 mL of each DS was analyzed as either pre-stress (control) DS (0 hours of agitation) or post-stress DS (24 or 48 hours of agitation).

[0274] The control or stressed DS was used to prepare 150 mg / mL final drug substance (FDS) containing 150 mg / mL mAb1, 20 mM histidine, 12.5 mM acetate, 5% sucrose, 0.2% high purity PS20, and 25 mM arginine HCl at pH 5.9. The FDS was filter sterilized and then stored at 45°C for 0, 4, or 8 weeks. Storage at 45°C was chosen to accelerate the stability study. The physical stability of all samples collected above was analyzed using visual inspection for macroscopic particles and aggregates, SE-UPLC for high and low molecular weight species, microflow imaging for sub-macroscopic particulate (2-300 μm) analysis, and CAD-UPLC for determination of PS20 levels.

[0275] mAb1 drug substance subjected to agitation stress and filtering steps exhibited less particle formation and greater polysorbate retention over time, as shown in Table 25. Table 25. Thermal stability of formulations subjected to agitation stress. [Table 25]

[0276] Additional forms of stress were also investigated. mAb1 drug substance produced without a HIC step was subjected to heat stress. The DS was stored at 45° C. for 0, 0.5, or 1 month to degrade, aggregate, and / or inactivate lipase. The control and stress DS were used to prepare a 150 mg / mL FDS containing 150 mg / mL mAb1, 20 mM histidine, 12.5 mM acetate, 5% sucrose, 0.2% high-purity polysorbate 20, and 25 mM arginine HCl at pH 5.9. The FDS was filter sterilized and then stored under the conditions shown in Table 26. Table 26. Incubation / storage conditions for FDS from heat-stressed DS. [Table 26]

[0277] The physical stability of the heat stressed and control non-stressed samples described above was analyzed using visual inspection for visible particles and aggregates, SE-UPLC for high and low molecular weight species, microflow imaging for sub-visible particulate (2-300 μm) analysis, and CAD-UPLC for determination of PS20 levels. The results are shown in Tables 27-32 below. Subjecting the DS to heat stress prior to formulation resulted in a significant decrease in lipase activity, improved polysorbate recovery, and reduced free fatty acid particle formation. The non-stressed samples showed a significant increase in sub-visible particles over time that was not observed in the stressed samples. Subjecting the DS to heat stress resulted in a significant increase in HMW levels, however the levels remained stable during further incubation. Table 27. Stability of DS-derived formulations at 0 months of heat stress [Table 27] Table 28. Stability of DS-derived formulations at 0.5 months of heat stress [Table 28] Table 29. Stability of DS-derived formulations at 1 month of heat stress [Table 29] Table 30. Stability of DS-derived formulations at 0 months of heat stress [Table 30] Table 31. Stability of DS-derived formulations at 0.5 months of heat stress [Table 31] Table 32. Stability of DS-derived formulations at 1 month of heat stress [Table 32]

[0278] HCPs have reduced stability compared to biotherapeutics, such as therapeutic antibodies, and are therefore expected to be disproportionately inactivated, degraded, and aggregated in response to stress. HMW species in the DS subjected to agitation or heat stress, whether formed from HCPs, drug proteins, or combinations, can be removed using further processing steps, such as filtration or chromatography. The heat-stressed DS was subjected to cation exchange (CEX) chromatography to remove HMW species formed during stress conditions. HMW species were efficiently depleted from the stress DS, as shown in Table 33. Table 33. Clearance of HMW species from stress drug substances [Table 33]

[0279] HMW-depleted DS was formulated in FDS containing 150 mg / mL mAb1, 20 mM histidine, 12.5 mM acetate, 5% sucrose, 0.2% PS80, and 25 mM arginine HCl at pH 5.9. As shown in Table 34, the stability of FDS from HMW-depleted heat-stressed DS was compared to FDS from HMW-depleted non-stressed DS. After only one week (0.25 months), FDS from HMW-depleted non-stressed DS showed greatly improved PS80 recovery compared to HMW-depleted non-stressed DS. Further improvements can be measured at later time points; measurable formation of free fatty acid particles from accumulated free fatty acids is expected to follow PS80 degradation in HMW-depleted non-stressed DS. It should be noted that the difference in lipase activity between stressed and unstressed DS after HMW depletion may also be masked by HCP lipase depletion from the unstressed DS as a result of the chromatography step. These results demonstrate that the disclosed methods of subjecting drug substances to agitation or heat stress, followed by optional HMW depletion, produce improved pharmaceutical compositions with reduced lipase activity, reduced polysorbate degradation, and reduced free fatty acid particle formation. Table 34. Thermal stability of formulations derived from HMW-depleted heat-stressed DS. [Table 34]

[0280] The present invention is not intended to be limited in scope by the specific embodiments described herein. Indeed, various modifications of the invention in addition to those described herein will become apparent to those skilled in the art from the foregoing description. Such modifications are intended to fall within the scope of the appended claims. Numbered Examples

[0281] The following numbered examples presented below provide further aspects of the present disclosure. 1. A method for producing a pharmaceutical composition having reduced lipase, comprising subjecting a sample containing a protein of interest and lipase to anion exchange chromatography, wherein the pH of the sample loaded onto the chromatography column is from about 7.8 to about 8.3. 2. A method for preparing a pharmaceutical composition having reduced lipase activity comprising: (a) subjecting a sample containing a protein of interest and a lipase to stress conditions to form a sample in which the lipase is inactivated; (b) formulating said lipase-inactivated sample to produce a pharmaceutical composition having reduced lipase activity; The above method. 3. The method of Example 2, wherein said protein of interest is an antibody, an antibody-derived protein, an antibody fragment, a monoclonal antibody, a bispecific antibody, a fusion protein, an antibody-drug conjugate, or a therapeutic protein. 4. The method of Examples 2 or 3, wherein said combining step comprises adding a fatty acid ester to said sample, said fatty acid ester being polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80. 5. The method of Example 4, wherein the fatty acid ester is polysorbate 80 and the concentration of oleic acid ester in the polysorbate 80 is at least 80%. 6. The method of Example 5, wherein the concentration of oleic acid ester in said polysorbate 80 is at least 98% or at least 99%. 7. The method of any one of Examples 2-6, wherein the stress conditions include agitation stress and / or heat stress. 8. The method of Example 7, wherein the agitation stress comprises shaking the sample at 50-500 rpm, 200-300 rpm, about 50 rpm, about 75 rpm, about 100 rpm, about 125 rpm, about 150 rpm, about 200 rpm, about 225 rpm, about 250 rpm, about 275 rpm, about 300 rpm, about 325 rpm, about 350 rpm, about 375 rpm, about 400 rpm, about 425 rpm, about 450 rpm, about 475 rpm, or about 500 rpm. 9. The method of Example 7, wherein the agitation stress comprises shaking the sample for 1 to 96 hours, 24 to 48 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours. 10. The method of Example 7, wherein the heat stress comprises storing the sample at about 30°C to about 60°C, about 35°C to about 55°C, about 40°C to about 50°C, about 44°C to about 46°C, about 30°C, about 35°C, about 40°C, about 45°C, about 50°C, about 55°C, or about 60°C. 11. The method of Example 10, wherein the storage is for 1 day to 6 months, 3 days to 3 months, 1 week to 2 months, 0.5 months to 1 month, about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, about 0.5 months, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 3 months, about 3.5 months, about 4 months, about 4.5 months, about 5 months, about 5.5 months, or about 6 months. 12. The method of any one of Examples 2-11, further comprising subjecting the lipase-inactivated sample to filtration, concentration, or chromatographic separation to remove high molecular weight (HMW) species prior to step (b). 13. The method of example 12, wherein said chromatographic separation comprises cation exchange chromatography. 14. The method of example 12, wherein said chromatographic separation comprises size exclusion chromatography. 15. The method of any one of Examples 2-14, wherein the compounding step comprises adding an excipient to the sample. 16. A method for preparing a pharmaceutical composition having reduced lipase activity, comprising: (a) subjecting the collected antibodies to affinity chromatography; (b) subjecting the antibodies pooled from the eluate of step (a) to viral inactivation at a pH of about 3 to about 4, and then adjusting the pH to about 5 to about 8; (c) subjecting the pooled antibodies from step (b) to anion exchange chromatography in flow-through mode; (d) subjecting the pooled antibodies from the eluate of step (c) to hydrophobic interaction chromatography in flow-through mode; (e) subjecting the antibody pooled from the flow-through fraction of step (d) to virus-retaining filtration; (f) subjecting the sample from step (e) containing the antibody of interest and the lipase to an agitation stress or a heat stress to form a pharmaceutical composition having reduced lipase activity. The above method. 17. A method for preparing a pharmaceutical composition having reduced lipase activity, comprising: (a) subjecting the collected antibodies to affinity chromatography; (b) subjecting the antibodies pooled from the eluate of step (a) to viral inactivation at a pH of about 3 to about 4, and then adjusting the pH to about 5 to about 8; (c) subjecting the pooled antibodies from step (b) to anion exchange chromatography in flow-through mode; (d) subjecting the antibody pooled from the flow-through fraction of step (c) to virus-retaining filtration; (e) subjecting the sample from step (d) containing the antibody of interest and lipase to an agitation stress or heat stress to form a pharmaceutical composition having reduced lipase activity; The above method. 18. The method of Example 16 or 17, further comprising subjecting the pharmaceutical composition to filtration, concentration, or chromatographic separation to remove HMW species. 19. The method of Example 18, wherein said chromatographic separation comprises ion exchange chromatography, cation exchange chromatography, or size exclusion chromatography. 20. The method of any one of claims 16 to 19, wherein the pH of the sample loaded onto the AEX column is from about 7.8 to about 8.3. 21. A method for producing a pharmaceutical composition with increased stability, comprising subjecting a sample containing a protein of interest and a lipase to anion exchange (AEX) chromatography to reduce lipase activity in the composition, wherein the pH of the sample loaded onto the AEX column is from about 7.8 to about 8.3. 22. The method of Example 21, further comprising subjecting the sample formed after AEX chromatography to agitation or heat stress to reduce lipase activity. 23. The method of any one of claims 16-22, further comprising the addition of poloxamer 188 or PEG 3350 to produce a formulation comprising a pharmaceutical composition having reduced lipase activity and increased stability. 24. The method of Example 23, wherein the formulation is substantially free of polysorbate. 25. The method of Example 23 or 24, wherein the concentration of the poloxamer 188 is 0.005% to 5%, 0.05% to 2.5%, 0.1% to 1%, about 0.05%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. 26. The method of Example 23 or 24, wherein the concentration of the PEG3350 is 0.005% to 5%, 0.05% to 2.5%, 0.1% to 1%, about 0.05%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%. 27. A method for producing a pharmaceutical composition having increased stability, comprising: (a) subjecting the harvested recombinant protein to anion exchange chromatography in flow-through mode; (b) subjecting the flow-through fraction from step (a) to hydrophobic interaction chromatography in flow-through mode; (c) formulating the isolated recombinant protein from step (b) with polysorbate 80, wherein the concentration of oleic acid esters in said polysorbate 80 is at least 80%; The above method. 28. The method of Example 27, wherein the concentration of the oleic acid ester is at least 98% or at least 99%. 29. The method of any one of Examples 1 or 16-28, wherein the pH of the sample loaded onto the anion exchange chromatography column is about 7.8 to about 8.3, about 7.9 to about 8.2, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, or about 8.3.

Claims

1. A method for producing a lipase-reduced pharmaceutical composition, comprising subjecting a sample containing a target protein and lipase to anion exchange chromatography, wherein the pH of the sample loaded into the chromatography column is approximately 7.8 to approximately 8.

3.

2. A method for producing a pharmaceutical composition in which lipase activity is reduced, comprising: (a) Exposing a sample containing the target protein and lipase to stress conditions to form a sample in which the lipase has been inactivated, (b) The method comprising preparing a pharmaceutical composition having reduced lipase activity by incorporating the sample in which the lipase has been inactivated.

3. The method of claim 2, wherein the target protein is an antibody, an antibody-derived protein, an antibody fragment, a monoclonal antibody, a bispecific antibody, a fusion protein, an antibody-drug conjugate, or a therapeutic protein.

4. The method of claim 2 or 3, wherein the compounding step comprises adding a fatty acid ester to the sample, wherein the fatty acid ester is polysorbate 20, polysorbate 40, polysorbate 60, or polysorbate 80.

5. The method of claim 4, wherein the fatty acid ester is polysorbate 80, and the concentration of oleic acid ester in the polysorbate 80 is at least 80%.

6. The method of claim 5, wherein the concentration of oleic acid ester in the polysorbate 80 is at least 98% or at least 99%.

7. The method of claim 2, wherein the stress conditions include stirring stress and / or thermal stress.

8. The method of claim 7, wherein the stirring stress includes shaking the sample at 50–500 rpm, 200–300 rpm, about 50 rpm, about 75 rpm, about 100 rpm, about 125 rpm, about 150 rpm, about 200 rpm, about 225 rpm, about 250 rpm, about 275 rpm, about 300 rpm, about 325 rpm, about 350 rpm, about 375 rpm, about 400 rpm, about 425 rpm, about 450 rpm, about 475 rpm, or about 500 rpm.

9. The method of claim 7, wherein the stirring stress includes shaking the sample for 1 to 96 hours, 24 to 48 hours, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 6 hours, about 12 hours, about 18 hours, about 24 hours, about 30 hours, about 36 hours, about 42 hours, about 48 hours, about 60 hours, about 72 hours, about 84 hours, or about 96 hours.

10. The method of claim 7, wherein the thermal stress includes storing the sample at approximately 30°C to approximately 60°C, approximately 35°C to approximately 55°C, approximately 40°C to approximately 50°C, approximately 44°C to approximately 46°C, approximately 30°C, approximately 35°C, approximately 40°C, approximately 45°C, approximately 50°C, approximately 55°C, or approximately 60°C.

11. The method of claim 10, wherein the storage period is 1 day to 6 months, 3 days to 3 months, 1 week to 2 months, 0.5 months to 1 month, about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, about 0.5 months, about 3 weeks, about 4 weeks, about 1 month, about 5 weeks, about 6 weeks, about 7 weeks, about 8 weeks, about 2 months, about 3 months, about 3.5 months, about 4 months, about 4.5 months, about 5 months, about 5.5 months, or about 6 months.

12. The method of claim 2, further comprising subjecting the lipase-inactivated sample to filtration, concentration, or chromatographic separation to remove high molecular weight (HMW) species before step (b).

13. The method of claim 12, wherein the chromatographic separation includes cation exchange chromatography.

14. The method of claim 12, wherein the chromatographic separation includes size exclusion chromatography.

15. The method of claim 2, wherein the compounding step includes adding an excipient to the sample.

16. A method for producing a pharmaceutical composition in which lipase activity is reduced, comprising: (a) The step of subjecting the collected antibodies to affinity chromatography, (b) The antibody pooled from the eluate in step (a) is subjected to viral inactivation at a pH of approximately 3 to approximately 4, and then the pH is adjusted to approximately 5 to approximately 8. (c) The antibody pooled from step (b) is subjected to anion exchange chromatography in flow-through mode, (d) The antibody pooled from the eluate of step (c) is subjected to hydrophobic interaction chromatography in flow-through mode, (e) A step of subjecting the antibody pooled from the flow-through fraction of step (d) to virus retention filtration, (f) a step comprising the steps of (e) subjecting a sample derived from a step comprising the antibody of interest and lipase to stirring stress or thermal stress to form a pharmaceutical composition in which lipase activity is reduced.

17. A method for producing a pharmaceutical composition in which lipase activity is reduced, comprising: (a) The step of subjecting the collected antibodies to affinity chromatography, (b) The antibody pooled from the eluate in step (a) is subjected to viral inactivation at a pH of approximately 3 to approximately 4, and then the pH is adjusted to approximately 5 to approximately 8. (c) The antibody pooled from step (b) is subjected to anion exchange chromatography in flow-through mode, (d) The antibody pooled from the flow-through fraction of step (c) is subjected to virus retention filtration, The method comprising: (e) a step comprising

18. The method of claim 16 or 17, further comprising subjecting the pharmaceutical composition to filtration, concentration, or chromatographic separation to remove HMW species.

19. The method of claim 18, wherein the chromatographic separation includes ion exchange chromatography, cation exchange chromatography, or size exclusion chromatography.

20. The method of claim 16 or 17, wherein the pH of the sample loaded into the AEX column is approximately 7.8 to approximately 8.

3.

21. A method for producing a pharmaceutical composition with increased stability, comprising reducing the lipase activity in the composition by subjecting a sample containing a target protein and lipase to anion exchange (AEX) chromatography, wherein the pH of the sample loaded into the AEX column is about 7.8 to about 8.

3.

22. The method of claim 21, further comprising the step of subjecting the sample formed after AEX chromatography to stirring stress or thermal stress to reduce lipase activity.

23. A method according to any one of claims 16, 17, or 21, further comprising the addition of poloxamer 188 or PEG3350 to produce a formulation comprising a pharmaceutical composition having reduced lipase activity and increased stability.

24. The method of claim 23, wherein the aforementioned formulation is substantially polysorbate-free.

25. The method of claim 23, wherein the concentration of poloxamer 188 is 0.005% to 5%, 0.05% to 2.5%, 0.1% to 1%, about 0.05%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.

26. The method of claim 23, wherein the concentration of PEG3350 is 0.005% to 5%, 0.05% to 2.5%, 0.1% to 1%, about 0.05%, about 0.01%, about 0.02%, about 0.03%, about 0.04%, about 0.05%, about 0.06%, about 0.07%, about 0.08%, about 0.09%, about 0.1%, about 0.15%, about 0.2%, about 0.25%, about 0.3%, about 0.4%, about 0.5%, about 1%, about 1.5%, about 2%, about 2.5%, about 3%, about 3.5%, about 4%, about 4.5%, or about 5%.

27. A method for producing a pharmaceutical composition with increased stability: (a) The step of subjecting the collected recombinant protein to anion exchange chromatography in flow-through mode, (b) A step of subjecting the flow-through fraction derived from step (a) to hydrophobic interaction chromatography in flow-through mode, (c) The method comprising the step of compounding the recombinant protein isolated from step (b) with polysorbate 80, wherein the concentration of oleic acid ester in the polysorbate 80 is at least 80%.

28. The method of claim 27, wherein the concentration of the oleic acid ester is at least 98% or at least 99%.

29. The method according to any one of claims 1, 16, 17, or 21, wherein the pH of the sample loaded into the anion exchange chromatography column is about 7.8 to about 8.3, about 7.9 to about 8.2, about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, or about 8.3.

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