Method for reducing lipase activity

Abstract

The present invention provides pharmaceutical formulations comprising an antibody that specifically binds to human interleukin-4 receptor alpha (hIL-4R alpha). In addition to the anti-IL-4R alpha antibody, the formulations may also contain one or more buffers, at least one amino acid, at least one sugar, and a surfactant comprising a polysorbate, polyethylene glycol, or poloxamer. The invention also provides a method for producing a pharmaceutical formulation having reduced lipase activity, which can comprise subjecting a pharmaceutical substance to anion exchange chromatography, agitation stress, thermal stress, and additional ion exchange or size exclusion chromatography under acidic conditions. In one aspect, the pharmaceutical formulations do not have significant sub-visible particle formation in the presence of lipase and exhibit a considerable degree of antibody stability during storage and after being subjected to thermal and other physical stresses.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS

[0002] This application incorporates by reference the priority and benefits of provisional patent application No. US 63 / 337,532 filed on May 2, 2022, provisional patent application No. 63 / 436,850 filed on January 3, 2023, and provisional patent application No. 63 / 499,441 filed on May 1, 2023.

[0003] Sequence Listing

[0004] This application contains a sequence listing that has been submitted electronically in .xml format and is hereby incorporated by reference in its entirety. The .xml copy created on January 31, 2023 is named 070816-03480.xml and is 180,926 bytes in size. Technical Field

[0005] 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 free fatty acid particle formation. Background Art

[0006] Among drug products, protein-based biotherapeutics are an important class of drugs that offer high levels of selectivity, potency, and efficacy, as evidenced by the significant increase in clinical trials with monoclonal antibodies (mAbs) over the past several years. A key aspect of clinically and commercially viable biotherapeutics is the stability of the drug product during the manufacturing process as well as during shelf life. Surfactants, such as polysorbates, are often used to enhance the physical stability of protein-based biotherapeutic products. More than seventy percent of commercially available monoclonal antibody therapeutics contain 0.001% to 0.2% polysorbate, a type of surfactant, to impart physical stability to protein-based biotherapeutics.

[0007] Enzymatic hydrolysis of polysorbates has been recognized as the major pathway for polysorbate degradation in biotherapeutic formulations. Polysorbate degradation may be caused by co-purification of esterases or lipases during the production of the drug product. Polysorbate hydrolysis results in the release of free fatty acids, which may drive undesirable particle formation in the drug product. Particles may be visible or subvisible; subvisible particles are typically less than 150 microns or 100 microns in diameter.

[0008] Therefore, there is a need for pharmaceutical products having reduced esterase or lipase activity and reduced free fatty acid particle formation and processes for preparing the same. Summary of the invention

[0009] Products and methods for reducing esterase activity, reducing lipase activity, reducing polysorbate degradation and reducing fatty acid particle formation in compositions or preparations have been developed. On the one hand, a sample comprising a concerned protein and an esterase or lipase is subjected to hydrophobic interaction chromatography to produce a formulated 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 not subjected to hydrophobic interaction chromatography. On the one hand, a sample comprising a concerned protein and an esterase or lipase is subjected to an anion exchange chromatography using a high loading pH 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 subjected to an anion exchange chromatography with a high loading pH.

[0010] In one aspect, a sample comprising a protein of interest and an esterase or lipase is formulated with a polysorbate comprising 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 aspect, a sample comprising 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 aspects, the protein of interest is an anti-IL4Rα antibody. In some aspects, the anti-IL-4Rα antibody is Dupilumab.

[0011] In one aspect, a formulated drug substance comprising a protein of interest, a fatty acid ester, and an esterase or lipase is subjected to stress, such as agitation stress or heat stress, to inactivate, degrade, and / or reduce the activity of the esterase or lipase, which may result in the formation of high molecular weight (HMW) species of the esterase or lipase and / or drug protein. The substance may then be subjected to filtration and / or separation, such as using cation exchange chromatography or size exclusion chromatography, to remove HMW species and produce a formulated drug substance and / or drug product having 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 aspects, the protein of interest is an anti-IL4Rα antibody. In some aspects, the anti-IL-4Rα antibody is dupilumab.

[0012] The present disclosure provides additional methods for producing pharmaceutical compositions with reduced esterase or lipase. In some aspects, these methods can include subjecting a sample comprising a protein of interest and an esterase or lipase to anion exchange chromatography, wherein the pH of the sample loaded onto the chromatography column is between about 7.8 and about 8.3.

[0013] In some additional aspects, methods for producing a pharmaceutical composition having reduced esterase or lipase activity may include (a) subjecting a sample comprising a protein of interest and an esterase or lipase to stress conditions to form a sample having inactivated esterase or lipase; and (b) formulating the sample with the inactivated esterase or lipase to produce a pharmaceutical composition having reduced esterase or lipase activity.

[0014] In one aspect, the 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.

[0015] In one aspect, the step of preparing comprises adding a fatty acid ester to the sample. In a specific aspect, the fatty acid ester is polysorbate 20, polysorbate 40, polysorbate 60 or polysorbate 80. In a specific aspect, the fatty acid ester is polysorbate 80, and the concentration of oleate in the polysorbate 80 is at least 80%. In a more specific aspect, the concentration of oleate in the polysorbate 80 is at least 98% or at least 99%.

[0016] On the one hand, the stress condition comprises stirring stress and / or thermal stress. In a specific aspect, the stirring stress comprises vibrating the sample at 50 to 500rpm, 200 to 300rpm, about 50rpm, about 75rpm, about 100rpm, about 125rpm, about 150rpm, about 200rpm, about 225rpm, about 250rpm, about 275rpm, about 300rpm, about 325rpm, about 350rpm, about 375rpm, about 400rpm, about 425rpm, about 450rpm, about 475rpm or about 500rpm. In another specific aspect, 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.

[0017] In another specific aspect, the thermal 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 a more specific aspect, the storage is 1 day to 6 months, 3 days to 3 months, 1 week to 2 months, 0.5 month to 1 month, about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, about 0.5 month, 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.

[0018] In one aspect, the method further comprises, prior to step (b), subjecting the sample with the inactivated esterase or lipase to filtration, enrichment or chromatographic separation to remove the inactivated lipase and / or protein HMW species. In a specific aspect, the chromatographic separation comprises cation exchange chromatography. In another specific aspect, the chromatographic separation comprises size exclusion chromatography.

[0019] In one aspect, the formulating step comprises adding an excipient to the sample.

[0020] The present disclosure provides additional methods for producing a pharmaceutical composition with reduced esterase or lipase activity. In some aspects, the method may include (a) subjecting the collected antibodies to affinity chromatography; (b) subjecting the antibodies collected 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 antibodies collected from step (b) to anion exchange chromatography in flow-through mode; (d) subjecting the antibodies collected from the eluate of step (c) to hydrophobic interaction chromatography in flow-through mode; (e) subjecting the antibodies collected from the flow-through fraction of step (d) to virus retention filtration; (f) subjecting the sample containing the antibody of interest and the esterase or lipase from step (e) to agitation stress or heat stress to form a pharmaceutical composition with reduced esterase or lipase activity.

[0021] In other aspects, the method may include (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 antibodies pooled from step (b) to anion exchange chromatography in flow-through mode; (d) subjecting the antibodies pooled from the flow-through fraction of step (c) to viral retention filtration; (e) subjecting the sample from step (d) containing the antibody of interest and an esterase or lipase to agitation stress or heat stress to form a pharmaceutical composition with reduced esterase or lipase activity.

[0022] In one aspect, the method further comprises subjecting the pharmaceutical composition to filtration, enrichment or chromatography to remove HMW species. In a specific aspect, the chromatographic separation comprises ion exchange chromatography, cation exchange chromatography or size exclusion chromatography.

[0023] In one aspect, the pH of the sample loaded onto the AEX column is between about 7.8 and about 8.3.

[0024] The present disclosure provides methods for producing pharmaceutical compositions with increased stability. In some aspects, the methods may include reducing the esterase or lipase activity in the composition by subjecting a sample comprising a protein of interest, an esterase or lipase, and a fatty acid ester to anion exchange (AEX) chromatography, wherein the pH of the sample loaded onto the AEX column is between about 7.8 and about 8.3.

[0025] In one aspect, the method further comprises the step of subjecting the sample formed after AEX chromatography to thermal stress or agitation stress to reduce the activity of the esterase or lipase. In a specific aspect, the method further comprises subjecting the stressed sample to chromatographic separation to remove HMW species of the esterase or lipase.

[0026] In one aspect, the method further comprises adding poloxamer 188 or PEG3350 to produce a formulation comprising a pharmaceutical composition having reduced esterase or lipase activity and increased stability. In a specific aspect, the formulation is substantially free of polysorbate. In another specific aspect, 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%. In yet another specific aspect, 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%.

[0027] The present disclosure provides additional methods for producing pharmaceutical compositions with increased stability. In some aspects, the method may include (a) subjecting the collected 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) formulating the recombinant protein isolated from step (b) with polysorbate 80, wherein the concentration of oleate in the polysorbate 80 is at least 80%.

[0028] In one aspect, the concentration of oleate is at least 98% or at least 99%.

[0029] In one aspect, 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.

[0030] In some aspects, the protein of interest or recombinant protein is an anti-IL4Rα antibody. In some aspects, the anti-IL-4Rα antibody is dupilumab.

[0031] When considered in conjunction with the following description and the accompanying drawings, these and other aspects of the present invention will be better recognized and understood. The following description, while indicating various aspects and many of its specific details, is given in an illustrative and non-limiting manner. Many substitutions, modifications, additions or rearrangements may be made within the scope of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 A chromatogram is shown showing the relative amounts of different molecular species ((A) lower mass polysorbate 20 (PS20-A), (B) higher mass polysorbate 20 (PS20-B), and (C) polysorbate 80 (PS80)) according to one aspect.

[0033] Figure 2A Shown is the formation of particles in a mAbl formulation comprising SR-PS80 as measured by membrane microscopy according to one aspect.

[0034] Figure 2B Shown is the formation of particles in a mAbl formulation comprising HP-PS20 as measured by membrane microscopy according to one aspect.

[0035] Figure 2C Shown is the formation of particles in a mAbl formulation comprising SR-PS80 as measured by MFI according to one aspect.

[0036] Figure 2D Shown is the formation of particles in a mAbl formulation comprising HP-PS20 as measured by MFI according to one aspect.

[0037] Figure 3A Shown is the recovery of polysorbate in mAbl formulations comprising SR-PS80 as measured by CAD-UHPLC according to one aspect.

[0038] Figure 3B Shown is the recovery of polysorbate in mAbl formulations comprising HP-PS20 as measured by CAD-UHPLC according to one aspect.

[0039] Figure 4 The correlation between particle formation and polysorbate recovery in mAbl formulations according to one aspect is shown.

[0040] Figure 5 The correlation between particle formation and polysorbate recovery in mAb5 formulations according to one aspect is shown.

[0041] Fig. 6AShown is the formation of particles in a mAbl formulation comprising SR-PS80 and subjected to HIC purification as measured by membrane microscopy according to one aspect.

[0042] Figure 6B Shown is the formation of particles in a mAbl formulation comprising HP-PS20 and subjected to HIC purification as measured by membrane microscopy according to one aspect.

[0043] Figure 6C Shown is the formation of particles in a mAbl formulation comprising SR-PS80 and subjected to HIC purification as measured by MFI according to one aspect.

[0044] Fig.6D Shown is the formation of particles in a mAbl formulation comprising HP-PS20 and subjected to HIC purification as measured by MFI according to one aspect.

[0045] Fig. 7A Shown is the recovery of polysorbate in mAbl formulations comprising SR-PS80 and subjected to HIC purification as measured by CAD-UHPLC according to one aspect.

[0046] Figure 7B Shown is the recovery of polysorbate in mAbl formulations comprising HP-PS20 and subjected to HIC purification as measured by CAD-UHPLC according to one aspect.

[0047] Figure 8 The correlation between phospholipase activity (in parts per million) and the percentage degradation of polysorbate 20 according to one aspect is shown.

[0048] Fig. 9 Shown is a visualization of a model generated from a multivariate study of AEX risk factors and responses according to one aspect.

[0049] Fig.10 Shown is the performance of AEX pool lipase activity (%) during validation batches compared to small-scale predictions from Monte Carlo simulations according to one aspect.

[0050] Fig.11 Shown are polysorbate recoveries from formulations comprising mAb5, mAb6, and mAb7 according to one aspect.

[0051] Fig.12 Shown are polysorbate recoveries from mAb5 formulations at different concentrations and incubation times according to one aspect.

[0052] Fig.13Homology modeling depicting the surface hydrophobicity of mAb5, mAb6, and mAb7 is shown according to one aspect.

[0053] Fig.14 Shown is the number of subvisible particles (≥10 μm) measured by membrane microscopy in protein drug products containing various types of polysorbate 80 according to one aspect.

[0054] Fig.15 Shown is the number of subvisible particles (≥10 μm) measured by microfluidics imaging (MFI) in protein drug products containing various types of polysorbate 80 according to one aspect.

[0055] Fig.16 The chemical structure of polyoxyethylene (20) sorbitan monooleate (the main fatty acid ester in polysorbate 80) according to one aspect is shown.

[0056] Fig.17 Shown are measured concentrations of free fatty acids in protein drug products containing various types of polysorbate 80 according to one aspect.

[0057] Fig.18A The polysorbate structure and major degradation pathways according to one aspect are shown.

[0058] Fig.18B The structure of Poloxamer 188 according to one aspect is shown.

[0059] Fig.19 Shown are surfactant recoveries at different temperatures for mAb5 formulations comprising PS20, PS80, or poloxamer 188 (P188) according to one aspect.

[0060] Fig. 20 Shown are the number of particles ≥10 μm (upper graph) and ≥25 μm (lower graph) identified by membrane microscopy in a 150 mg / mL anti-IL-4R antibody formulation containing lipase and PEG3350 or poloxamer 188 (at concentrations of 0.02%, 0.04%, or 0.1% w / v) and stored at 5°C for up to 36 months according to one aspect.

[0061] Fig.21A Shown is the effect of PEG3350 concentration on the stability (expressed as percentage of HMW species measured by SE-UPLC) of a 150 mg / mL anti-IL-4R antibody formulation under stirring stress over a period of 30 to 120 minutes at room temperature.

[0062] Fig.21BShown is the effect of poloxamer 188 concentration on the stability (expressed as percentage of HMW species measured by SE-UPLC) of a 150 mg / mL anti-IL-4R antibody formulation under stirring stress over a period of 30 to 120 minutes at room temperature.

[0063] Fig. 22 Shown is the effect of polysorbate 20, polysorbate 80, PEG3350, and poloxamer 188 (at varying concentrations) on the stability (expressed as percentage of HMW species measured by SE-UPLC) of a 150 mg / mL formulation of an anti-IL-4R antibody against thermal stress (45°C) over a period of up to 56 days. DETAILED DESCRIPTION

[0064] Therapeutic macromolecules must be formulated in a manner that not only makes the molecule suitable for administration to a patient, but also maintains its stability during storage. For example, unless the solution is properly formulated, therapeutic antibodies in liquid solutions are prone to degradation, aggregation, and / or undesirable chemical modifications. The stability of antibodies in liquid formulations depends not only on the type of excipients used in the formulation, but also on the amount and ratio of the excipients to each other. Therapeutic formulations may also be subject to the formation of particulate matter over time during storage. Particles may be visible or subvisible; subvisible particles are typically less than 150 microns or 100 microns in diameter. Formulations with high protein concentrations (e.g., concentrations of 30 mg / mL or higher) are more prone to aggregation and subvisible particle formation.

[0065] Polysorbate 20 (PS20) and polysorbate 80 (PS80) are the most commonly used nonionic 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, it has been reported that polysorbates (including polysorbate 20 and polysorbate 80) can be degraded in the presence of lipases, which leads to the formation of subvisible particles in the formulation over time.

[0066] PS is known to be susceptible to degradation through two major pathways: autooxidation 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 main pathway for PS degradation in high-concentration protein formulations, which leads to the accumulation of free fatty acids (FFA), which may drive undesirable particle formation in drug products. Oxidation is the second major pathway for PS degradation, which leads 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., Journal of Pharmaceutical Science 67(12):1676-1681 (1978); Yao et al., Pharmaceutical Research 26(10):2303-2313 (2009)). It has been recognized that PS hydrolysis is a greater threat to drug product quality because this process not only reduces PS concentration, but the low solubility of accumulated FFA (especially at storage temperatures of 2°C-8°C) is also associated with particle formation (Doshi et al., Journal of Pharmaceutical Science 110(2):687-692 (2021); Saggu et al., Journal of Pharmaceutical Science 110(3):1093-1102 (2021); Doshi et al., Mol Pharm. 12(11):3792-3804 (2015)).

[0067] Residual lipase or esterase present in the drug product is the main reason for PS hydrolysis (Chiu et al., Biotechnol Bioeng. 114(5):1006-1015 (2017); Hall et al., Journal of Pharmaceutical Science 105(5):1633-1642 (2016); Labrenz et al., Journal of Pharmaceutical Science 103(8):2268-2277 (2014); McShan et al., PDA J Pharm Sci Technol. 70(4):332-345 (2016); Zhang et al., Journal of Pharmaceutical Science 109(11):3300-3307 (2020); Zhang et al., Journal of Pharmaceutical Science 109(9):2710-2718 (2020)). After multiple downstream purification steps, the level of residual lipase in the final drug product is typically very low (<10 ppm), and the consequences of PS degradation may not be noticeable until after months or years of storage at typical storage temperatures (2°C-8°C).

[0068] Without intending to be bound by theory, it is believed that a putative phospholipase B-like 2 (PLBL2) that is highly conserved in hamsters, rats, mice, humans, and cattle co-purifies with some classes of proteins in certain processes. Other esterases or lipases may also co-purify with the protein of interest at concentrations that are too low to be reliably detected, but high enough to have measurable lipase activity, ultimately leading to loss of polysorbates, production of free fatty acids, and formation of visible or subvisible particles. Therefore, there is a need for pharmaceutical products with reduced lipase activity, reduced esterase activity, and reduced fatty acid particle formation, as well as methods for preparing the same.

[0069] Disclosed herein are novel pharmaceutical products, compositions and preparations with reduced lipase activity, reduced esterase activity and reduced fatty acid particle formation and methods for preparing the novel pharmaceutical products, compositions and preparations. The inventors have surprisingly found that a hydrophobic interaction chromatography (HIC) step can be added to the production process of the protein of interest to effectively remove esterase and lipase activity and prevent the formation of particles in the pharmaceutical product. In addition, optimized conditions for anion exchange (AEX) chromatography steps have been found to effectively reduce esterase and lipase activity by improving conventional pH loading conditions. Additionally, the inventors have found that polysorbate content (particularly polysorbate 80) can be optimized with high oleic acid concentrations so that even in a pharmaceutical product containing esterase or lipase activity, less fatty acid particles can be formed. Also studied the alternative surfactant of polysorbate, and surprisingly found that PEG3350 and poloxamer 188 can successfully stabilize protein, and have resistance to esterase or lipase activity simultaneously, thus prevent the formation of fatty acid particles.After producing drug substance, further surprisingly found that, by applying stress (for example, stirring stress or thermal stress), can successfully deactivate and / or remove esterase or lipase through co-purification, this may cause esterase or lipase degradation, thus reduce lipase activity.The application of stress can also cause the formation of high molecular weight (HMW) material.HMW material can use for example molecular weight filtration or chromatography (such as cation exchange chromatography or size exclusion chromatography) to remove from drug substance.These aspects of the present invention and other aspects are further elaborated in detail below.

[0070] Unless otherwise described, all technical and scientific terms used herein have the same meaning as those commonly understood by those of ordinary skill in the art to which the invention belongs. Although methods and materials similar or equivalent to any methods and materials described herein can be used in the practice or testing, specific methods and materials are now described.

[0071] 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, as understood by those of ordinary skill in the art, and where ranges are provided, the endpoints are included. As used herein, the terms "include," "includes," and "including" are intended to be non-limiting and are understood to mean "comprise," "comprises," and "comprising," respectively.

[0072] As used herein, the term "protein" or "protein of interest" may include any amino acid polymer with covalently linked amide bonds. Proteins include one or more amino acid polymer chains, commonly referred to as "polypeptides" in the art. "Polypeptide" refers to a polymer composed of amino acid residues, related naturally occurring structural variants, and synthetic non-naturally occurring analogs thereof, connected by peptide bonds. "Synthetic peptides or polypeptides" refer to non-naturally occurring peptides or polypeptides. Synthetic peptides or polypeptides can be synthesized, for example, using an automated peptide synthesizer. Various solid phase peptide synthesis methods are known to those skilled in the art. Proteins may include one or more polypeptides to form a single functional biomolecule. In another exemplary aspect, proteins may include antibody fragments, nanobodies, recombinant antibody chimeras, cytokines, chemokines, peptide hormones, etc. Proteins of interest may include any biological therapeutic protein, recombinant protein for research or therapy, trap protein and other chimeric receptor Fc fusion proteins, chimeric proteins, antibodies, monoclonal antibodies, polyclonal antibodies, human antibodies, bispecific antibodies, and antigen binding proteins.

[0073] Proteins can be produced using recombinant cell-based production systems such as insect baculovirus systems, yeast systems (e.g., Pichia sp.), and mammalian systems (e.g., CHO cells and CHO derivatives such as CHO-K1 cells). For a recent review of biotherapeutic proteins and their production, see 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 teaching of which is incorporated herein by reference). In some aspects, proteins include modifications, adducts, and other covalently linked moieties. These modifications, adducts and moieties include, for example, avidin, streptavidin, biotin, polysaccharides (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 epitope, fluorescent markers and other dyes, etc. Proteins can be classified according to composition and solubility, and thus can include simple proteins, such as globular proteins and fibrous proteins; conjugated proteins, such as nucleoproteins, glycoproteins, mucins, chromoproteins, phosphoproteins, metalloproteins and lipoproteins; and derivatized proteins, such as primary derivatized proteins and secondary derivatized proteins.

[0074] As used herein, the term "recombinant protein" refers to a protein produced by transcription and translation of a gene carried on a recombinant expression vector that has been introduced into a suitable host cell. In some aspects, the recombinant protein can be an antibody, such as a chimeric antibody, a humanized antibody or a fully human antibody. In some aspects, the recombinant protein can be an antibody of the isotype selected from the group consisting of: IgG, IgM, IgA1, IgA2, IgD or IgE. In some aspects, the antibody molecule is a full-length antibody (e.g., IgG1), or alternatively, the antibody can be a fragment (e.g., Fc fragment or Fab fragment).

[0075] Antibody

[0076] As used herein, the term "antibody" is generally intended to refer to immunoglobulin molecules comprising four polypeptide chains, two heavy (H) chains and two light (L) chains interconnected by disulfide bonds, and multimers thereof (e.g., IgM); however, immunoglobulin molecules consisting only of heavy chains (i.e., lacking light chains) are also included in the definition of the term "antibody". Each heavy chain includes a heavy chain variable region (heavy chain variable region, abbreviated herein as HCVR or VH) and a heavy chain constant region. The heavy chain constant region includes three domains CH1, CH2, and CH3. Each light chain includes a light chain variable region (light chain variable region, abbreviated herein as LCVR or VL) and a light chain constant region. The light chain constant region includes a domain (CL1). The VH region and the VL region can be further subdivided into hypervariable regions referred to as complementary determining regions (complementary determining regions, CDRs), which are interspersed with more conservative regions referred to as framework regions (framework regions, FRs). Each VH and VL is composed of three CDRs and four FRs arranged from amino-terminus to carboxyl-terminus in the following order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4.

[0077] In some aspects, the protein of interest is a human antibody. As used herein, the term "human antibody" is intended to include antibodies with variable regions and constant regions derived from human germline immunoglobulin sequences. Human antibodies of the present disclosure may include amino acid residues encoded by non-human germline immunoglobulin sequences (e.g., mutations introduced by random or site-specific mutagenesis in vitro or by somatic mutations in vivo), such as in CDR, especially in CDR3. However, as used herein, the term "human antibody" is not intended to include antibodies that will be derived from CDR sequences of other mammalian species such as mice and transplanted to human framework sequences.

[0078] Interleukin-4 (IL-4) and interleukin-13 (IL-13) are key cytokines that drive allergic reactions and T helper cell 2 (Th2) polarized inflammatory processes. IL-4 and IL-13 signaling is mediated by a heterodimeric receptor complex, in which IL-4 receptor α (IL-4Rα) is a receptor subunit shared by IL-4 and IL-13 signaling. Therefore, IL-4Rα is an attractive therapeutic target because it provides a single target that can be used to simultaneously block IL-4 and IL-13 signaling. In some aspects, the protein of interest is an anti-IL-4R antibody or an antigen-binding fragment thereof. Antibodies to hIL-4Rα are described in, for example, U.S. Patents Nos. 5,717,072, 7,186,809, and 7,605,237.

[0079] In some aspects, the anti-IL-4R antibody is a human IgG antibody. In various aspects, the anti-IL-4R antibody is a human antibody of isotype IgG1, IgG2, IgG3, or IgG4, or mixed isotypes. In some aspects, the anti-IL-4R antibody is a human IgG1 antibody. In some aspects, the anti-IL-4R antibody is a human IgG4 antibody. In any of the aspects discussed above or herein, the anti-IL-4R antibody may comprise a human kappa light chain. In any of the aspects discussed above or herein, the anti-IL-4R antibody may comprise a human lambda light chain.

[0080] In some aspects, the antibodies of the present disclosure may be recombinant human antibodies. As used herein, the term "recombinant human antibody" is intended to include all human antibodies prepared, expressed, created or separated by a recombinant manner, such as antibodies expressed using a recombinant expression vector transfected into a host cell, antibodies separated from a recombinant combination human antibody library, antibodies separated from an animal (e.g., mouse) that has been transformed with human immunoglobulin genes (see, e.g., Taylor et al. (1992) Nucleic Acids Res. 20: 6287-6295), or antibodies prepared, expressed, created or separated by any other means involving splicing human immunoglobulin gene sequences with other DNA sequences. Such recombinant human antibodies have variable and constant regions derived from human germline immunoglobulin sequences. However, in some aspects, such recombinant human antibodies are subjected to in vitro mutagenesis (or when using an animal that has been transformed with human Ig sequences, somatic mutagenesis in the receptor), and therefore, the V H Area and V L Although the amino acid sequence of the region is derived from human germline V H Sequence and V L Sequences that are not naturally present in the human antibody germline repertoire in vivo.

[0081] As used herein, the term "antibody" also includes the antigen binding fragment of a complete antibody molecule. As used herein, the term "antigen binding portion thereof", "antigen binding fragment" of an antibody, etc., include any naturally occurring, enzymatically obtainable, synthetic or genetically engineered polypeptide or glycoprotein, which specifically binds to an antigen to form a complex. The antigen binding fragment of an antibody can be derived from a complete antibody molecule, for example, using any suitable standard technique, such as proteolytic digestion or involving manipulation and expression of DNA encoding antibody variable domains and optionally constant domains. Such DNA is known and / or easy to obtain from, for example, commercial sources, DNA libraries (including, for example, phage-antibody libraries), or can be synthesized. DNA can be sequenced and manipulated chemically or by using molecular biology techniques, for example, to arrange one or more variable domains and / or constant domains into a suitable configuration, or to introduce codons, produce cysteine ​​residues, modify, add or delete amino acids, etc.

[0082] As used herein, "antibody fragments" include a portion of a complete antibody, such as an antigen binding region 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 separated complementary determining regions (CDR) regions, as well as trifunctional antibodies, tetrafunctional antibodies, linear antibodies, single-chain antibody molecules, and multispecific antibodies formed by antibody fragments. Fv fragments are combinations of the variable regions of immunoglobulin heavy and light chains, and ScFv proteins are recombinant single-chain polypeptide molecules in which immunoglobulin light and heavy chain variable regions are connected by peptide linkers. In some aspects, an antibody fragment includes enough amino acid sequence of a parent antibody, which is a fragment of the parent antibody so that it binds to the same antigen as the parent antibody; in some aspects, a fragment binds to an antigen having an affinity comparable to that of the parent antibody and / or competes with the parent antibody to bind to the antigen. Antibody fragments can be produced in any manner. For example, antibody fragments can be produced enzymatically or chemically by fragmentation of intact antibodies, and / or antibody fragments can be produced recombinantly by genes encoding partial antibody sequences. Alternatively or additionally, antibody fragments can be produced synthetically in whole or in part. Antibody fragments can optionally include single-chain antibody fragments. Alternatively or additionally, antibody fragments can include multiple chains linked together, for example, by disulfide bonds. Antibody fragments can optionally include multimolecular complexes. Functional antibody fragments typically include at least about 50 amino acids, and more typically include at least about 200 amino acids.

[0083] The term "bispecific antibody" includes antibodies that can selectively bind to two or more epitopes. Bispecific antibodies generally include two different heavy chains, wherein each heavy chain specifically binds to different epitopes on two different molecules (e.g., antigens) or on the same molecule (e.g., on the same antigen). If a bispecific antibody can selectively bind to two different epitopes (a first epitope and a second epitope), the affinity of the first heavy chain to the first epitope will generally be lower than the affinity of the first heavy chain to the second epitope by at least one to two or three or four orders of magnitude, and vice versa. The epitopes recognized by the bispecific antibody can be on the same or different targets (e.g., on the same or different proteins). Bispecific antibodies can be prepared, for example, by combining heavy chains that recognize different epitopes of the same antigen. For example, a nucleic acid sequence encoding a heavy chain variable sequence that recognizes different epitopes of the same antigen can be fused with a nucleic acid sequence encoding different heavy chain constant regions, and such sequences can be expressed in cells expressing immunoglobulin light chains.

[0084] A typical bispecific antibody has two heavy chains, each of which has 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 associate with each heavy chain, or can associate with each heavy chain and can bind to one or more of the epitopes bound by the heavy chain antigen binding region, or can associate with each heavy chain and enable one or both of the heavy chains to bind to one or two epitopes. bsAbs can be divided into two main categories: one with an Fc region (IgG-like); and the other lacking an Fc region, the latter being generally smaller than IgG and IgG-like bispecific molecules that include an Fc. IgG-like bsAbs can have different formats, such as, but not limited to, trifunctional antibodies, knob-in-hole IgG (kih IgG), crossMab, orth-Fab IgG, dual variable domain Ig (DVD-Ig), two-in-one or bifunctional Fab (DAF), IgG-single chain Fv (IgG-scFv) or κλ bodies. Different formats other than IgG-like include tandem scFv, diabody formats, single-chain diabodies, tandem diabodies (TandAb), dual affinity retargeting molecules (DART), DART-Fc, nanobodies or antibodies produced by dock-and-lock (DNL) methods (Gaowei Fan, Zujian Wang and Mingju Hao, Bispecific antibodies and their applications, 8 JOURNALOF HEMATOLOGY & ONCOLOGY 130; Dafne Müller and Roland E. Kontermann, Bispecific Antibodies, HANDBOOK OF THERAPEUTIC ANTIBODIES 265-310 (2014), the entire teachings of which are incorporated herein by reference). Methods for producing bsAbs are not limited to quadroma techniques based on somatic cell fusion of two different hybridoma cell lines, chemical coupling involving chemical crosslinkers, and genetic methods using recombinant DNA technology.

[0085] As used herein, "multispecific antibodies" refer to antibodies that have binding specificities for at least two different antigens. Although such molecules will typically bind only two antigens (i.e., bispecific antibodies, bsAbs), antibodies with additional specificities such as trispecific antibodies and KIH trispecific antibodies can also be addressed by the systems and methods disclosed herein.

[0086] As used herein, the term "monoclonal antibody" is not limited to antibodies produced by hybridoma technology. Monoclonal antibodies can be derived from a single clone by any available or known means in the art, including any eukaryotic clone, prokaryotic clone, or phage clone. Monoclonal antibodies useful in the present disclosure can be prepared using various techniques known in the art, including hybridoma technology, recombinant technology, and phage display technology, or a combination thereof.

[0087] As used herein, "isolated antibody" is intended to refer to an antibody that is substantially free of other antibodies having different antigenic specificities (eg, an isolated antibody that specifically binds to hIL-4Rα is substantially free of antibodies that specifically bind to antigens other than hIL-4Rα).

[0088] The term "specific binding" and the like means that the antibody or its antigen-binding fragment forms a relatively stable complex with the antigen under physiological conditions. Specific binding can be determined by a dissociation constant of at least about 1 x 10 -6 M or higher. Methods for determining whether two molecules specifically bind are well known in the art, including, for example, equilibrium dialysis, surface plasmon resonance, and the like. However, an isolated antibody that specifically binds to hIL-4Rα may have cross-reactivity with other antigens, such as 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 and one or more additional antigens is considered to "specifically bind" to hIL-4Rα. In addition, the isolated antibody may be substantially free of other cellular material and / or chemicals. However, in some cases, the isolated antibody may be co-purified with a phospholipase expressed by a mammalian cell line (e.g., CHO cells) that produces the anti-IL-4R antibody.

[0089] According to certain aspects of the present disclosure, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises a heavy chain complementary determining region HCDR1-HCDR2-HCDR3, and the heavy chain complementary determining region comprises the amino acid sequence shown in SEQ ID NO:3-4-5. According to certain aspects of the present disclosure, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises a light chain complementary determining region LCDR1-LCDR2-LCDR3, and the light chain complementary determining region comprises the amino acid sequence shown in SEQ ID NO:6-7-8. In certain aspects, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises a light chain complementary determining region (CDR) HCDR1-HCDR2-HCDR3-LCDR1-LCDR2-LCDR3, and it comprises the amino acid sequence shown in SEQ ID NO:3-4-5-6-7-8.

[0090] In certain aspects, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises heavy chain complementary determining regions HCDR1-HCDR2-HCDR3, respectively, comprising the amino acid sequence shown in SEQ ID NO:3-4-5, and having a heavy chain variable region (HCVR) 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 shown in SEQ ID NO:1. In certain aspects, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises light chain complementary determining regions LCDR1-LCDR2-LCDR3, respectively, comprising the amino acid sequence shown in SEQ ID NO:6-7-8, and having a light chain variable region (LCVR) 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 shown in SEQ ID NO:2. In certain aspects, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises: a heavy chain complementary determining region HCDR1-HCDR2-HCDR3 comprising the amino acid sequence of SEQ ID NO:3-4-5 and having a heavy chain variable region (HCVR) 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 complementary determining region LCDR1-LCDR2-LCDR3 comprising the amino acid sequence of SEQ ID NO:6-7-8 and having a light chain variable region (LCVR) 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.

[0091] In certain aspects, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises a heavy chain variable region (HCVR) having an amino acid sequence of SEQ ID NO: 1. In certain aspects, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises a light chain variable region (LCVR) having an amino acid sequence of SEQ ID NO: 2. In certain aspects, the anti-hIL-4R antibody or antigen-binding fragment thereof comprises a HCVR / LCVR amino acid sequence pair having an amino acid sequence of SEQ ID NO: 1 / 2, respectively. In some aspects, the anti-IL-4R antibody comprises a HCVR / LCVR having an amino acid sequence of SEQ ID NO: 1 / 2, respectively, and a human IgG1 heavy chain constant region. In some aspects, the anti-IL-4R antibody comprises a HCVR / LCVR having an amino acid sequence of SEQ ID NO: 1 / 2, respectively, and a human IgG4 heavy chain constant region. In some aspects, the anti-IL-4R antibody comprises a HCVR / LCVR having an amino acid sequence of SEQ ID NO: 1 / 2, respectively, and a human IgG heavy chain constant region. In some aspects, the anti-IL-4R antibody comprises a HCVR / LCVR having an amino acid sequence of SEQ ID NO: 1 / 2, respectively, and a human IgG1 or IgG4 heavy chain constant region. In some aspects, the anti-IL-4R antibody comprises a heavy chain having an amino acid sequence of SEQ ID NO: 9 and a light chain having an amino acid sequence of SEQ ID NO: 10. In some aspects, the anti-IL-4R antibody is dupilumab.

[0092] Other anti-IL-4R antibodies that can be used in the context of the methods of the present disclosure include, for example, antibodies known as and referred to in the art as AMG317 (Corren et al., 2010, Am J Respir Crit Care Med., 181(8):788-796) or MEDI 9314, or any anti-IL-4Rα antibody described in U.S. Pat. No. 7,186,809, U.S. Pat. No. 7,605,237, U.S. Pat. No. 7,638,606, U.S. Pat. No. 8,092,804, U.S. Pat. No. 8,679,487, U.S. Pat. No. 8,877,189, U.S. Pat. No. 10,774,141; U.S. Patent Application Publication No. US2021 / 0238294; or International Patent Publication Nos. WO2019 / 228405, WO2020 / 096381, WO 2020 / 135471, WO2020 / 135710 or WO 2020 / 239134, the contents of the above documents are incorporated herein by reference.

[0093] In some aspects, the anti-IL-4R antibody comprises one or more of the CDR, HCVR, and / or LCVR sequences shown in Table 1 below.

[0094] The amount of antibody or antigen-binding fragment thereof contained in the pharmaceutical formulations of the present disclosure may vary depending on the specific properties desired for the formulation and the specific circumstances and purposes for which the formulation is intended to be used. In certain aspects, the pharmaceutical formulation may contain from about 1 mg / mL to about 500 mg / mL of antibody; from about 5 mg / mL to about 250 mg / mL of antibody; from about 5 mg / mL to about 200 mg / mL of antibody; from about 15 mg / mL to about 200 mg / mL of antibody; from about 25 mg / mL to about 200 mg / mL of antibody; from about 50 mg / mL to about 200 mg / mL of antibody; from about 100 mg / mL to about 200 mg / mL of antibody; from about 125 mg / mL to about 175 mg / mL of antibody; or from about 150 mg / mL to about 200 mg / mL of antibody. For example, the formulation of the present disclosure can be a liquid formulation comprising 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. g / mL; about 110 mg / mL; 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 thereof that specifically binds to hIL-4Rα. In certain aspects, 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 aspects, 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 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.

[0095] The present disclosure encompasses antibodies having amino acid sequences that differ from those of the exemplary molecules disclosed herein, but that retain the ability to bind to a cognate antigen (e.g., hIL-4R). Such variant molecules may include one or more additions, deletions, or substitutions of amino acids compared to the parent sequence, but exhibit biological activities that are substantially equivalent to those of the antibodies discussed herein.

[0096] The present disclosure includes antigen binding molecules that are bioequivalent to any exemplary antibody described herein. In some aspects, the antigen binding molecule is a bioequivalent of dupilumab. For example, if two antibodies are administered in a single or multiple administrations at the same molar dose under similar experimental conditions, their absorption rate and absorption extent do not show significant differences, then they are considered bioequivalents and belong to drug equivalents or drug substitutes. If some antibodies are equivalent in their absorption extent but different in their absorption rate, however, since the difference in this absorption rate is intentional and embodied in the label instructions, it is not necessary for achieving effective in vivo drug concentrations (e.g., during long-term use) and is medically insignificant for the specific drug product under study, then these antibodies may also be considered equivalents or drug substitutes, and may be considered bioequivalents.

[0097] On the one hand, if there are no clinically meaningful differences in the safety, purity and potency of the two antibodies, then the two antibodies are bioequivalent. On the other hand, if patients can make one or more switches between the reference product and the biological product without increasing the risk of expected side effects (including clinically significant changes in immunogenicity or reduced effectiveness) compared to continued therapy without switching between the reference product and the biological product, then the two antibodies are bioequivalent.

[0098] Bioequivalence can be demonstrated by both in vivo and in vitro methods. Bioequivalence measures include, for example: (a) tests conducted in humans or other mammals that measure the concentration of the antibody or its metabolites in blood, plasma, serum or other biological fluids over time; (b) in vitro tests that are correlated with and reasonably predictive of in vivo human bioavailability data; (c) tests conducted in humans or other mammals that measure the corresponding acute pharmacological effects of the antibody (or its target) over time; and (d) carefully designed clinical trials to establish the safety, efficacy or bioavailability or bioequivalence of the antigen binding protein.

[0099] Pharmaceutical preparations

[0100] As used herein, a "sample" can be obtained from any step of a bioprocess, such as cell culture fluid (CCF), harvested cell culture fluid (HCCF), any step in downstream processing, including a drug substance (DS) or drug product (DP) of a final formulated product.

[0101] As used in the present disclosure, the terms "composition", "formulation" and "formulated drug substance" (FDS) refer to a combination of two or more drug ingredients contained in a drug product. A composition, formulation or FDS can be, for example, a liquid composition comprising an active drug ingredient (such as an antibody) and an excipient (such as a stabilizer or surfactant). A composition, formulation or FDS can contain a variety of excipients. A composition, formulation or FDS can also contain other ingredients, such as host cell proteins that are co-purified with the protein of interest.

[0102] The term "drug product" (DP) as used in the present disclosure refers to a dosage form containing a certain amount of FDS for packaging, transportation or administration. For example, the drug product can be a pre-filled syringe containing a certain volume of FDS for administration to a patient.

[0103] As used herein, a "protein drug product", "biopharmaceutical product", "pharmaceutical preparation", "pharmaceutical composition" or "biotherapeutic agent" comprises an active ingredient that may be completely or partially biological in nature. On the one hand, a protein drug product may comprise a peptide, protein, fusion protein, antibody, antigen, vaccine, peptide-drug conjugate, antibody-drug conjugate, protein-drug conjugate, cell, tissue or a combination thereof. On the other hand, a protein drug product may comprise a recombinant, engineered, modified, mutated or truncated form of a peptide, protein, fusion protein, antibody, antigen, vaccine, peptide-drug conjugate, antibody-drug conjugate, protein-drug conjugate, cell, tissue or a combination thereof.

[0104] In some aspects, the pharmaceutical formulations of the present invention comprise: (i) a human antibody that specifically binds to hIL-4Rα; (ii) one or more buffers; (iii) a thermal stabilizer; (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 if such components do not significantly interfere with the viscosity and stability of the formulation. Specific exemplary components and formulations included in the present disclosure are described in detail below.

[0105] In certain aspects, the pharmaceutical preparation of the present disclosure can be a fluid preparation. As used herein, the term "fluid preparation" means a mixture of at least two components that are in a fluid state at about 2°C to about 45°C. Among other things, fluid preparations include liquid preparations. Depending on the specific ingredients of the fluid preparation, the fluid preparation can have low, medium or high viscosity.

[0106] Host cell proteins

[0107] As used herein, the term "host cell protein" (HCP) includes proteins derived from host cells in the production process of recombinant proteins. Host cell proteins can be process-related impurities, which can be derived from manufacturing processes and can include three main categories: cell substrate sources, cell culture sources, and downstream sources. Impurities derived from cell substrates include, but are not limited to, proteins and nucleic acids (host cell genomes, vectors, or total DNA) derived from host organisms. Impurities derived from cell cultures include, but are not limited to, inducers, antibiotics, serum, and other media components. Impurities derived from downstream sources include, but are not limited to, enzymes, chemical and biochemical processing reagents (e.g., cyanogen bromide, guanidine, oxidants and reductants), inorganic salts (e.g., heavy metals, arsenic, non-metallic ions), solvents, carriers, ligands (e.g., monoclonal antibodies), and other leachables.

[0108] Based on several measurable factors, the presence of host cell proteins in biotherapeutic products can be considered to have higher or lower risk. One such factor is the concentration or abundance (number) of HCP impurities in the biotherapeutic product. HCPs may have no significant effect at sufficiently low abundances, such as when measured by ELISA or mass spectrometry. The level at which HCPs may present significant risk can depend on the specific characteristics of the HCP, which can be considered to be an unacceptable level in the product and can be monitored as a critical quality attribute (CQA). Certain specific HCPs are known to pose risks at specific levels, such as the level of enzymatic activity of the HCP depending on an enzyme.

[0109] Relatedly, the criticality of the presence of an HCP may depend on the function of the HCP, particularly in relation to the components of the biotherapeutic product. For example, an HCP esterase or lipase that may or is known to degrade polysorbates in a biotherapeutic product of interest may be closely monitored, and the threshold for the amount of HCP impurities that may be allowed in the biotherapeutic product may be set lower. Other HCPs of particular concern may be, for example, proteases that may or are known to degrade proteins of interest in a biotherapeutic product, or immunogenic HCPs that may or are known to cause an immune response when administered to a subject.

[0110] Liquid chromatography

[0111] As used herein, the term "liquid chromatography" refers to a process in which a biological / chemical mixture carried by a liquid can be separated into different components due to the differential distribution of components when flowing through (or flowing into) a fixed liquid phase 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. The analyte separated using chromatography can have a unique retention time, which reflects the speed at which the analyte passes through the chromatographic column. Analytes can be compared by a chromatogram, which plots the retention time on one axis and plots the measured signal on another axis, wherein the measured signal can be generated by, for example, UV detection or fluorescence detection. In some aspects, a sample (e.g., a drug substance) comprising at least one esterase or lipase can be subjected to stress, such as stirring stress or thermal stress, and then subjected to a chromatography step to remove any HMW substances.

[0112] In certain aspects, it may be advantageous to subject a biological sample to affinity chromatography to produce a protein of interest. Chromatographic materials are capable of selectively or specifically binding to or interacting with a protein of interest. Non-limiting examples of such chromatographic materials include Protein A and Protein G. Also included are chromatographic materials that include, for example, a protein or portion thereof that is capable of binding to or interacting with a protein of interest.

[0113] Affinity chromatography can involve subjecting the biological sample to a chromatography column packed with a suitable protein A resin. As used herein, the term "protein A" encompasses protein A obtained from its natural source, protein A produced synthetically (e.g., by peptide synthesis or by recombinant techniques), and protein A that retains affinity to a protein with C H 2 / C H In certain aspects, protein A resins can be used for the production and separation of various antibody isotypes based on affinity by specifically interacting with the Fc portion of the molecule if it has such a region.

[0114] Protein A resins are available from several commercial sources. Suitable resins include, but are not limited to, MabSelect PrismA, MabSelect SuRe from Cytiva, TM , MabSelect SuRe LX, MabSelect, MabSelect SuRepcc, MabSelect Xtra, rProtein A Sepharose, ProSep HC, ProSep Ultra, ProSep Ultra Plus from EMD Millipore, MabCapture from ThermoFisher, and Amsphere from JSR Life Sciences TM A3.

[0115] Prior to loading the sample, the affinity column may be equilibrated with a suitable buffer. After loading the column, the column may be washed one or more times with a suitable wash buffer. The column may then be eluted using an appropriate elution buffer (e.g., glycine-HCl, acetic acid, or citric acid). The eluent may be monitored using techniques well known to those skilled in the art, such as a UV detector. The elution fraction of interest may be collected and then prepared for subsequent processing.

[0116] Cation exchange chromatography (CEX) uses cation exchange chromatography materials. Depending on the cation exchange chromatography materials used, cation exchange chromatography can be further subdivided into, for example, strong cation exchange (SCX) or weak cation exchange. Cation exchange chromatography materials with sulfonic acid groups (S) can be used as strong cation exchangers, while cation exchange chromatography materials with carboxymethyl (CM) can be used as weak cation exchangers. Strong cation exchangers include, for example, SOURCE S using methyl sulfate functional groups and SP Sepharose using sulfopropyl functional groups. Weak cation exchangers include, for example, CM-cellulose (Cellulose) using carboxymethyl functional groups. SCX may be preferred because, without causing the strong cation exchanger to lose its charge, a buffer with a wider pH range can be used, thereby allowing analytes with a wider pI range to be effectively separated.

[0117] Cation exchange chromatography materials are available under different names from a variety of 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 Hill, New York, USA); and 1,100-1,100-1. 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); CM1500, CM 3000 (available from BioChrom Labs, Terre Haute, Ind., USA); and CM-Sepharose Fast Flow (available from GE Healthcare, Life Sciences, Germany). In addition, commercially available cation exchange resins further 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 immobilized on agarose (e.g., S-Sepharose Fast Flow, available from GE Healthcare Life Sciences, Germany).

[0118] Cation exchange chromatography materials include mixed mode chromatography materials that combine ion exchange and hydrophobic interaction technology (e.g., Capto adhere, Capto MMC, MEP HyperCell, Eshmuno HCX, etc.), and mixed mode chromatography materials that combine anion exchange and cation exchange technology (e.g., hydroxyapatite, ceramic hydroxyapatite, etc.), etc. In some aspects, CEX can be used to remove HMW species from a sample, such as removing proteins that aggregate into HMW species under stirring or thermal stress.

[0119] In some aspects, a sample containing a protein of interest is subjected to at least one anion exchange (AEX) separation step. Anion exchange packed bed chromatography is based on ionic interactions between binding entities (target protein or impurities) and functional groups fixed on a chromatographic medium. Its performance can be a function of the mobile phase, functional groups, and resin backbone. Whether to use an anion exchange material or a cation exchange material depends in part on the local charge of the protein of interest. Anion exchange chromatography can be used in conjunction with other chromatographic procedures (such as affinity chromatography, size exclusion chromatography, hydrophobic interaction chromatography, and other chromatography modes known to those skilled in the art).

[0120] In the context of chromatographic separation, a chromatographic column is used to hold a chromatographic support material (resin or solid phase). A sample containing the protein of interest is loaded onto a specific chromatographic column. The chromatographic column can then be subjected to one or more washing steps using a suitable wash buffer. Components of the sample that have not adsorbed to the resin may flow directly through the chromatographic column. Components that have adsorbed to the resin can be differentially eluted using an appropriate elution buffer.

[0121] The anionic reagent can be selected from the group consisting of acetate, chloride, formate and a combination thereof. The cationic reagent can be selected from the group consisting of Tris, arginine, sodium ions and a combination thereof. The buffer can 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-propanol), piperazine, 1,3-diaminopropane and piperidine.

[0122] Packed anion exchange chromatography columns, anion exchange membrane devices, anion exchange monolithic devices, or depth filter media can be operated in bind and elute mode, flow-through mode, or mixed mode. In mixed mode, proteins bind to the chromatography material and can be eluted from such material using a buffer that is the same as or substantially similar to the loading buffer.

[0123] In the binding elution mode, the chromatographic column or membrane device is first treated with a buffer having an appropriate ionic strength and pH, so that certain proteins are adsorbed onto a resin-based matrix under such conditions. For example, during the feed loading process, the protein of interest can be adsorbed onto the resin due to electrostatic attraction. After washing the chromatographic column or membrane device with an equilibrium buffer or another buffer having different pH and / or conductivity, product recovery can be achieved by increasing the ionic strength (i.e., conductivity) of the elution buffer so that it competes with the solute for the charged sites on the anion exchange matrix. Changing the pH and thus the charge of the solute is another way to achieve solute elution. The change in conductivity or pH can be gradual (gradient elution) or step-by-step (step elution).

[0124] In flow-through mode, the column or membrane device is operated under selected pH and conductivity conditions such that the protein of interest does not bind to the resin or membrane, while the acidic species will be retained on the column or will exhibit a different elution profile than the protein of interest. In the context of this strategy, the acidic species will interact or bind to the chromatographic material under appropriate conditions, while the protein of interest and certain aggregates and / or fragments of the protein of interest will flow through the column.

[0125] In some aspects, the AEX step is performed in negative mode (flow-through mode), in which negatively charged process-related impurities are adsorbed to the immobilized positively charged ligand, while the protein of interest flows directly through.

[0126] In some aspects, the pH of the sample loaded onto the AEX column ("loading pH") can be selected to reduce esterase or lipase activity in the sample. In some aspects, the AEX loading pH can be about 7.8, about 7.9, about 8.0, about 8.1, about 8.2, about 8.3, between about 7.8 and about 8.3, between about 7.8 and about 8.0, between about 8.0 and about 8.3, or between about 8.0 and about 8.2.

[0127] Non-limiting examples of anion exchange resins include diethylaminoethyl (DEAE), quaternary aminoethyl (QAE) and quaternary amine (Q) groups. Other non-limiting examples include: Poros 50PI and Poros 50HQ, which are rigid polymer microbeads with a backbone composed of cross-linked poly[styrene-divinylbenzene]; Poros 50XQ; Capto Q Impres and Capto DEAE, which are high flow rate agarose microbeads; Capto Adhere; QSepharose Fast Flow; Toyopearl QAE-550, Toyopearl DEAE-650 and Toyopearl GigaCap Q-650, which are polymer-based microbeads; EMDTMAE Hicap, a synthetic polymer resin with a tentacle-like ion exchanger; Sartobind PA nano, which is a salt-tolerant chromatography membrane with primary amine ligands; Sartobind Q nano, which is a strong anion exchange chromatography membrane; CUNOBioCap, which is a zeta-plus depth filtration medium made of inorganic filter aids, refined cellulose, and ion exchange resins; XOHC, which is a depth filtration medium made of inorganic filter aids, cellulose, and mixed cellulose esters; and Unosphere Q. In some aspects, resins with larger pore sizes are selected to increase the surface area exposed to negatively charged species.

[0128] Additives such as polyethylene glycol (PEG), detergents, amino acids, sugars, chaotropes, etc. can be added to enhance separation performance to achieve better separation, recovery and / or product quality.

[0129] Size exclusion chromatography (SEC) or gel filtration separates components based on their molecular size. The separation is determined by the ratio of the time a substance spends in the porous stationary phase to the time it spends in the mobile phase. The probability of a molecule staying in a pore depends on the size of the molecule and the size of the pore. In addition, the ability of a substance to penetrate the pore is determined by the diffusion mobility of the macromolecule, which is higher for smaller macromolecules. Very large macromolecules may not be able to penetrate the pores of the stationary phase at all; and for very small macromolecules, the probability of penetration is close to 1. Although components with larger molecules pass through the stationary phase faster, components with smaller molecules have a longer path through the pores of the stationary phase and therefore remain in the stationary phase longer.

[0130] The analyte eluted from the SEC column can be separated into different fractions based on the elution time. For example, the analyte eluted earlier than the functional form of the protein of interest (e.g., monomeric form) can be roughly classified as a high molecular weight (HMW) substance. The HMW fraction can be further subdivided into, for example, very high molecular weight (vHMW) fractions and dimer fractions (representing the elution time of the dimer of the protein of interest). The analyte eluted later than the functional form of the protein of interest can be roughly classified as a low molecular weight (LMW) substance, and can be further subdivided into LMW fractions and rear tail fractions. Similarly, sample components (e.g., lipase) other than the protein of interest may also form higher and lower molecular weight substances that can be separated using SEC.

[0131] Chromatographic material can comprise size exclusion material, wherein said size exclusion material is resin or film.The matrix for size exclusion is preferably an inert gel medium, and said inert gel medium can be a complex of cross-linked polysaccharides, for example, cross-linked agarose and / or dextran in the form of spherical microbeads.The degree of cross-linking determines the size of the pores present in the swollen gel microbeads.Molecules greater than a certain size will not enter the gel microbeads, and thus will pass through the chromatographic bed as quickly as possible.Molecules with smaller sizes (such as detergents, proteins, DNA, etc.) will be blocked when passing through the chromatographic bed, because they will enter the gel microbeads to varying degrees according to size and shape.Therefore, molecules are usually eluted in the order of decreasing molecular size.In some aspects, SEC can be used to remove HMW substances from a sample, for example, to remove proteins that aggregate into HMW substances after stirring or thermal stress.

[0132] The term "hydrophobic interaction medium" means a combination of a support structure and a hydrophobic portion, wherein the hydrophobic portion is fixed to the support structure. The medium can be in the form of a chromatographic medium (e.g., microbeads or other particles in the form of a packed bed column), in the form of a membrane, or in any form that can accommodate a liquid including the protein of interest and contaminants. Thus, support structures include agarose microbeads (e.g., agarose gel), silica microbeads, cellulose membranes, cellulose microparticles, hydrophilic polymer microbeads, and the like. The hydrophobic portion is the functional end of the medium that binds to the hydrophobic surface of hydrophobic molecules and proteins. The degree of hydrophobicity of the medium can be controlled by selecting the hydrophobic portion. For example, the following portions can be attached to the medium matrix to produce a hydrophobic interaction medium with increasing hydrophobicity, i.e., from low hydrophobicity to high hydrophobicity: ether, butyl, octyl, and phenyl. The alkyl group can be a straight chain alkyl group or a branched chain alkyl group. For a review of hydrophobic interaction chromatography and media, see Kuczewski et al., "Using Hydrophobic Interaction Chromatography with Hydrophobic Interactions." Development of a polishing step using a hydrophobic interaction membrane adsorber with a -derived recombinant antibody”, Biotechnology and Bioengineering 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. Pat. No. 7,427,659 B2, Sept. 23, 2008; and Müller and Franzreb, “Suitability of commercial hydrophobic interaction sorbents for temperature-controlled protein liquid chromatography under low salt conditions”. conditions), Journal of Chromatography A (J. Chroma. A), 1260: 88-96 (2012).

[0133] Hydrophobic interaction media is used in a process called hydrophobic interaction chromatography (HIC), and is used to separate the protein of interest from pollutants associated with production and processes. When the protein of interest is manufactured in a host cell and / or purified from a host cell, the pollutants associated with production and processes are referred to as host cell proteins (HCPs). HCPs from Chinese hamster ovary (CHO) cells (a common host cell for manufacturing biotherapeutics) can be referred to as CHOP (Chinese hamster ovary protein). In some cases, a mixture containing a protein of interest (POI) and HCP is placed in a HIC medium in a buffer, the buffer being intended to promote the binding of the hydrophobic groups in the POI to the hydrophobic portion of the HIC medium. POI is attached to the HIC medium by binding to the hydrophobic portion, and some HCPs flow out with the wash buffer because they cannot bind. Then, the POI is eluted using a buffer that promotes the dissociation of the POI from the HIC hydrophobic portion, thereby separating the POI from unwanted HCPs.

[0134] In some cases, the HIC hydrophobic portion preferentially binds to some contaminants (such as HCPs), and the POIs are collected from the HIC flowthrough. This disclosure describes an example of using HIC in flowthrough mode, in which a group of HCP contaminants with esterase activity can remain bound to the hydrophobic interaction matrix.

[0135] In some cases, affinity chromatography designed to bind specific proteins with lipophilic properties can replace or work in conjunction with HIC. Since some esterases (such as lipases in general or phospholipases in particular) bind to triglycerides or phospholipids, molecules that mimic those lipids can be used to capture the esterase. For example, "myristoylated ADP-ribosylation factor 1" (also known as "myrARF1") can be used to capture lipases and allow the POI to remain unbound and flow directly through. To prepare a myrARF1 affinity column, myrARF1 can be bound to Q-Sepharose by 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)).

[0136] Mass spectrometry

[0137] As used herein, the term "mass spectrometer" includes a device that can identify specific molecular substances and measure their exact mass. The term is intended to include any molecular detector of a polypeptide or peptide. A mass spectrometer can include three main parts: an ion source, a mass analyzer, and a detector. The role of the ion source is to produce gas phase ions. Analyte atoms, molecules or clusters can be transferred to the gas phase and ionized simultaneously (as in electrospray ionization) or ionized by different processes. The choice of ion source depends on the application.

[0138] In some aspects, the mass spectrometer can be a tandem mass spectrometer. As used herein, the term "tandem mass spectrometry" encompasses techniques for obtaining structural information about sample molecules by using multiple stages of mass selection and mass separation. A prerequisite is that the sample molecules are converted into the gas phase and ionized to form fragments in a predictable and controllable manner after the first mass selection step. MS / MS or MS 2This can be done by first selecting and isolating a precursor ion (MS 1 ) and then fragment it to obtain meaningful information. Tandem MS has been successfully performed by various analyzer combinations. For specific applications, which analyzer to combine can be determined by many different factors, such as sensitivity, selectivity and speed, as well as size, cost and availability. The two main categories of tandem MS methods are spatial tandem and time tandem, but there are also mixed situations, where the time tandem analyzer is spatially coupled or coupled to the spatial tandem analyzer. The spatial tandem mass spectrometer includes an ion source, a precursor ion activation device and at least two non-capture mass analyzers. A specific m / z separation function can be designed so that ions are selected in one section of the instrument, dissociated in the middle zone, and then the product ions are transmitted to another analyzer for m / z separation and data acquisition. In the time tandem, the mass spectrometer ions produced in the ion source can be captured, separated, fragmented and m / z separated in the same physical device.

[0139] The peptides identified by mass spectrometry can be used as surrogate representatives of intact proteins and their post-translational modifications or other modifications. The peptides can be used for protein characterization by correlating experimental and theoretical MS / MS data, the latter being generated from peptides that may be present in a protein sequence database. The characterization can include, but is not limited to, sequencing the amino acids of protein fragments, determining protein sequences, determining protein de novo sequencing, locating post-translational modifications or identifying post-translational modifications or performing comparability analysis or a combination thereof.

[0140] In some exemplary aspects, the mass spectrometer can work on nano-electrospray or nano-spray. As used herein, the term "nano-electrospray" or "nano-spray" refers to electrospray ionization at extremely low solvent flow rates, typically with sample solution flow rates of hundreds of nanoliters per minute or lower, and typically without external solvent delivery. The electrospray infusion device forming the nano-electrospray can use a static nano-electrospray emitter or a dynamic nano-electrospray emitter. The static nano-electrospray emitter can continuously analyze a small volume of sample (analyte) solution over a long period of time. The dynamic nano-electrospray emitter uses a capillary column and a solvent delivery system to chromatographically separate the mixture before entering the mass spectrometer for analysis.

[0141] In some aspects, mass spectrometry can be carried out under native conditions. As used herein, the term "native conditions" may include carrying out mass spectrometry under conditions of non-covalent interactions in the retained analyte. For a detailed review of native MS, reference is made to: Elisabetta Boeri Erba and Carlo Petosa, The emerging role of native mass spectrometry in characterizing the structure and dynamics of macromolecular complexes, 24 "Protein Science (PROTEIN SCIENCE)" 1176–1192 (2015).

[0142] As used herein, the term "database" refers to a compilation of protein sequences that may be present in a sample, such as in the form of a file in FASTA format. The related protein sequence can be derived from the cDNA sequence of the substance under study. Public databases that can be used to search for related protein sequences include databases hosted by, for example, Uniprot or Swiss-prot. Databases can be searched using "bioinformatics tools" as referred to in this article. Bioinformatics tools have the ability to search for unresolved MS / MS spectra for all possible sequences in the database, and provide resolved (annotated) MS / MS spectra as output results. Non-limiting examples of such tools include 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).

[0143] Formulation excipients

[0144] The pharmaceutical formulations of the present disclosure include one or more excipients. As used herein, the term "excipient" means any non-therapeutic agent added to a formulation to provide a desired consistency, viscosity or stabilizing effect.

[0145] The pharmaceutical preparation of the present disclosure can also include a buffer or buffer system for maintaining a stable pH and helping to stabilize the protein of interest. In some aspects, the buffer or buffer system includes at least one buffer, and the buffer range of the at least one buffer overlaps completely or partially with the range of pH 5.5 to 6.3. In various aspects, the pH of the preparation is 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2 or 6.3. In some aspects, the pH of the preparation is 5.9 ± 0.3. In some aspects, the pH of the preparation is 5.9 ± 0.2. In some aspects, the pH of the preparation is 5.9 ± 0.1. In some aspects, the buffer comprises a histidine buffer. In some aspects, the buffer comprises an acetate buffer. In some aspects, the buffer (e.g., histidine and / or acetate) is present in a concentration of about 1mM to about 40mM, about 5mM to about 30mM, about 10mM to about 15mM or about 15mM to about 25mM. In some aspects, the buffer comprises a histidine buffer having a concentration of 15mM to 25mM. In some aspects, the buffer comprises a histidine buffer having a concentration of 20mM ± 2mM. In some cases, the histidine buffer is present at a concentration of 15mM, 16mM, 17mM, 18mM, 19mM, 20mM, 21mM, 22mM, 23mM, 24mM or 25mM. In some aspects, the buffer comprises an acetate buffer having a concentration of 10mM to 15mM. In some aspects, the buffer comprises an acetate buffer having a concentration of 12.5mM ± 1.25mM. In some cases, the acetate buffer is present at a concentration of 10mM, 10.5mM, 11mM, 11.5mM, 12mM, 12.5mM, 13mM, 13.5mM, 14mM, 14.5mM or 15mM. In some aspects, the formulations of the present disclosure include both histidine buffer and acetate buffer at any concentration in the concentrations discussed above. In some cases, the formulation contains a histidine buffer at a concentration of 15mM to 25mM and an acetate buffer at a concentration of 10mM to 15mM. In some cases, the formulation contains a histidine buffer at a concentration of 20mM ± 2mM and an acetate buffer at a concentration of 12.5mM ± 1.25mM.

[0146] The pharmaceutical preparations of the present disclosure may also include one or more carbohydrates, such as one or more sugars. Sugar may be a reducing sugar or a non-reducing sugar. "Reducing sugars" include, for example, sugars with ketone or aldehyde groups and containing reactive hemiacetal groups, which allow sugars to act as reducing agents. 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 react substantially with amino acids or polypeptides to induce 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 salts thereof. In some aspects, sugar is sucrose. In some cases, sugar (e.g., sucrose) acts as a thermal stabilizer for the protein of interest.

[0147] The amount of sugar (e.g., sucrose) included in the pharmaceutical formulations of the present disclosure will vary depending on the specific circumstances in which the formulation is used and the intended use. In certain aspects, the formulation can 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 preparation of the present disclosure can include 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 aspects, the preparation contains about 5% sugar (e.g., sucrose). In some aspects, the preparation contains about 5% ± 0.5% sugar (e.g., sucrose). Each percentage mentioned above corresponds to a weight / volume (w / v) percentage.

[0148] In some aspects, the pharmaceutical preparation of the present disclosure comprises at least one amino acid. In some aspects, the amino acid is arginine. In some aspects, arginine is provided in the form of arginine hydrochloride. In some cases, amino acid (e.g., arginine) serves as a viscosity modifier for the preparation of the protein of interest.

[0149] The amount of amino acids included in the pharmaceutical formulations of the present disclosure may vary depending on the specific properties desired for the formulation and the specific circumstances and purposes for which the formulation is to be used. In certain aspects, the formulation may contain about 1 mM to about 200 mM of amino acids; about 5 mM to about 150 mM of amino acids; about 10 mM to about 100 mM of amino acids; about 20 mM to about 80 mM of amino acids; about 20 mM to about 30 mM of amino acids; about 45 mM to about 55 mM of amino acids; or about 70 mM to about 80 mM of amino acids. For example, the pharmaceutical preparation of the present disclosure can include about 5mM; about 10mM; about 15mM; about 20mM; about 25mM; about 30mM; about 35mM; about 40mM; about 45mM; about 50mM; about 55mM; about 60mM; about 65mM; about 70mM; about 75mM; about 80mM; about 85mM; about 90mM; about 95mM; or about 100mM of amino acid (e.g., arginine). In some aspects, the preparation contains about 25mM of amino acid (e.g., arginine). In some aspects, the preparation contains about 50mM of amino acid (e.g., arginine). In some aspects, the preparation contains about 75mM of amino acid (e.g., arginine).

[0150] Pharmaceutical preparations of the present disclosure may also include one or more organic cosolvents, the type and amount of the one or more organic cosolvents being able to stabilize the protein of interest under conditions of rough handling or stirring (such as, for example, orbital oscillation). In some respects, the organic cosolvent is a surfactant. As used herein, the term "surfactant" refers to a material that reduces the surface tension of a fluid and / or reduces the interfacial tension between oil and water. The surfactant may be ionic or nonionic. The specific nonionic surfactant that may be included in the preparation of the present disclosure includes, for example, polysorbate (such as PS20 and PS80), poloxamer (such as poloxamer 188) and polyethylene glycol (PEG) (such as PEG3350).

[0151] The amount of surfactant contained in the pharmaceutical formulations of the present disclosure may vary depending on the specific characteristics desired for the formulation and the specific circumstances and purposes for which the formulation is to be used. In some aspects, the formulation may contain at least about 0.01% of a surfactant. In some aspects, the formulation may contain less than 0.2% of a surfactant. In some aspects, the formulation may contain less than 0.5% of a surfactant. In some aspects, the formulation may contain about 0.01% to about 0.49% of a surfactant; about 0.01% to about 0.39% of a surfactant; about 0.01% to about 0.29% of a surfactant; about 0.01% to about 0.19% of a surfactant; about 0.01% to about 0.15% of a surfactant; about 0.01% to about 0.12%; about 0.01% to about 0.11% of a surfactant; about 0.01% to about 0.1% of a surfactant; or about 0.01% to about 0.09% of a 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 0.21%; 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 0.31%; 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. %; about 0.20%; 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 aspects, the formulation contains about 0.01% to 0.19% poloxamer 188. In some aspects, the formulation contains about 0.01% to about 0.49% poloxamer 188. In some aspects, the formulation contains about 0.01% to 0.19% PEG3350. In some aspects, the formulation contains about 0.01% to 0.49% PEG3350. Each percentage mentioned above corresponds to a weight / volume (w / v) percentage.

[0152] Polysorbate

[0153] In some aspects, the surfactant in the composition can be a polysorbate. As used herein, "polysorbate" refers to a common excipient used in formulation development to protect antibodies from various physical stresses such as stirring, freeze-thaw processes, and air / water interfaces (Emily Ha, Wei Wang, and Y. John Wang, Peroxide formation in polysorbate 80 and proteinstability, 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 of surfactants and monoclonal antibodies on sterilizing grade filters. Behavior of aSurfactant and a Monoclonal Antibody toSterilizing-Grade Filters), 99 Journal of Pharmaceutical Sciences 2620-2627 (2010), which may include a nonionic amphiphilic surfactant composed of a fatty acid ester of polyoxyethylene sorbitan. The ester 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 aspects, the polysorbate may be present in the formulation in an amount ranging from about 0.001% to 1% (weight / volume).Polysorbates can also contain mixtures of various fatty acid chains; for example, polysorbate 80 contains oleic, palmitic, myristic, and stearic fatty acids, with the monooleate portion accounting for approximately 58% of the polydisperse 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.

[0154] Polysorbates may be susceptible to self-oxidation in a pH-dependent and temperature-dependent manner, and exposure to UV light may also produce instability (Ravuri SkKishore et al., Degradation of Polysorbates 20 and 80: Studies on Thermal Autoxidation and Hydrolysis, 100 Journal of Pharmaceutical Sciences 721–731 (2011)), thereby generating free fatty acids and sorbitan head groups in solution. The free fatty acids generated by polysorbates may include any aliphatic fatty acid having 6 to 20 carbons. Non-limiting examples of free fatty acids include oleic acid, palmitic acid, stearic acid, myristic acid, lauric acid, or a combination thereof.

[0155] In some exemplary aspects, polysorbate can form free fatty acid particles. The size of free fatty acid particles can be at least about 1 μm or at least about 5 μm. In addition, these fatty acid particles can be divided into visible particles (about> 100 μm), sub-visible particles (about <100 μm, which can be further subdivided into microns (1-100 μm) and submicrons (100nm-1000nm)) and nanoparticles (about <100nm) (Linda Narhi, Jeremy Schmit and Deepak Sharma, Classification of protein aggregates, 101 "Journal of Pharmaceutical Sciences" 493-498) according to their size. In some exemplary aspects, fatty acid particles can be visible particles. Visible particles can be determined by visual inspection. In some aspects, fatty acid particles can be sub-visible particles. According to the United States Pharmacopeia (United States Pharmacopeia, USP), sub-visible particles can be monitored by light obstruction method. The increase of fatty acid particles may cause the product to no longer have acceptable quality, so the increase rate of fatty acid particles can be used as a measure of product shelf life. When free fatty acids are released into the preparation and exceed its soluble concentration, fatty acid particles may form, thereby precipitate out from solution. Therefore, measuring the degraded or released free fatty acids of polysorbate can indicate the formation of fatty acid particles, and as an extension, indicates the shelf life of the predicted product. In addition, preventing polysorbate from degrading, free fatty acids from releasing and / or fatty acid particles from forming may be very important for extending the product shelf life and improving product quality.

[0156] In some exemplary aspects, the concentration of the polysorbate in the formulation can be 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, In one aspect, the concentration of the polysorbate in the formulation is about 1% w / v.

[0157] In some exemplary aspects, 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.

[0158] In some exemplary aspects, polysorbate can be degraded by host cell proteins present in the composition. In some aspects, the host cell protein can be an esterase or a lipase. Residual esterase or lipase activity in the formulation can be assessed indirectly by measuring polysorbate degradation, free fatty acid release, or the concentration of visible or subvisible fatty acid particles.

[0159] The term "fatty acid ester" means any organic compound containing a fatty acid chain connected to a head group by an ester bond. When a hydroxyl group (e.g., a hydroxyl group in an alcohol or carboxylic acid) is replaced by an alkoxy group, an ester bond is formed. As used herein, a hydroxyl group can be part of a carboxylic acid (more specifically a fatty acid) and / or an alcohol (e.g., glycerol, sorbitol, sorbitan, isosorbide, etc.). The alcohol group is generally referred to as a head group in this article.

[0160] The example of fatty acid ester generally includes phospholipid, lipid (for example, head group is glycerol, including monoglyceride, diglyceride and triglyceride) and surfactant and emulsifier, including polysorbates such as polysorbate 20, polysorbate 60 and polysorbate 80, which belong to nonionic detergent. Surfactant and emulsifier can be used as cosolvent and stabilizer. The surfactant and emulsifier play a role by associating with both hydrophilic surface and lipophilic surface, to maintain the dispersion and structural stability of components such as protein. Surfactant is added to protein formulation, mainly to enhance the stability of protein against mechanical stress (such as air / liquid interface and solid / liquid interface shear). In the absence of surfactant, protein may be structurally unstable in solution in some cases, and forms polymer aggregates, eventually becoming sub-visible particles.

[0161] The term "fatty acid" or "fatty acid chain" means a carboxylic acid with an aliphatic tail chain. The aliphatic tail chain is a hydrocarbon chain consisting only of carbon and hydrogen, and in some cases, may also have substituents such as oxygen, sulphur, nitrogen and / or chlorine. The aliphatic tail chain can be saturated (as in saturated fatty acids), which means that all carbon-carbon bonds are single bonds (i.e., alkanes). The aliphatic tail chain can be undersaturated (as in unsaturated fatty acids), wherein one or more carbon-carbon bonds are double bonds (olefins) or triple bonds (alkynes).

[0162] Fatty acids are generally classified as short-chain fatty acids (those with fewer than 6 carbons in the aliphatic tail), medium-chain fatty acids (those with 6 to 12 carbons), long-chain fatty acids (those with 13 to 21 carbons), and very long-chain fatty acids (those with 22 or more carbons in the aliphatic tail). As mentioned above, fatty acids are also classified based on their degree of saturation, which is related to hardness and melting point. Common fatty acids include caprylic acid (8 carbons: 0 double bonds; expressed as 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 (18:1), linoleic acid (18:2), trans-linoleic acid (18:2), alpha-linolenic acid (18:3), arachidic acid (20:0), arachidonic acid (20:4), eicosapentaenoic acid (20:5), behenic acid (22:0), erucic acid (22:1), docosahexaenoic acid (22:6), tetracosanoic acid (24:0), and cerotic acid (26:0).

[0163] As mentioned above, polysorbates are fatty acid esters that can be used as nonionic 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 mainly includes the monolaurate of polyoxyethylene (20) sorbitan. Polysorbate 40 mainly includes the monopalmitate of polyoxyethylene (20) sorbitan. Polysorbate 60 mainly includes the monostearate of polyoxyethylene (20) sorbitan. Polysorbate 80 mainly includes the monooleate of polyoxyethylene (20) sorbitan.

[0164] The quality of commercial grade polysorbates varies from supplier to supplier. Thus, polysorbates are usually mixtures of various chemical entities, consisting primarily of polyoxyethylene (20) sorbitan monoesters (as described above), with isosorbide esters as contaminants in some cases. The head group (in this case polyoxyethylene (20) sorbitan) contains a sorbitan (a mixture of sorbitans including 1,4-sorbitan, 1,5-sorbitan and 1,4,3,6-dianhydrosorbitan) with three hydroxyl groups substituted to form ether bonds with three polyoxyethylene groups. The fourth hydroxyl group is substituted with a fatty acid to form a fatty acid ester.

[0165] In some commercially available polysorbate batches, the polysorbate contains isosorbide monoester. Isosorbide is a heterocyclic derivative of glucose, also prepared by dehydration of sorbitol. The isosorbide is a diol, i.e., has two alcoholic hydroxyl groups that can participate in the formation of one or two ester bonds. Thus, for example, some batches of polysorbate 20 may contain a large amount of isosorbide mono- and diesters of lauric acid, and some batches of polysorbate 80 may contain a large amount of isosorbide mono- and diesters of oleic acid.

[0166] In addition to the head group changes, polysorbate formulations also contain different amounts of other fatty acid esters. For example, analysis of polysorbate 20 from a particular source revealed that caprylic acid <10%, capric acid <10%, lauric acid 40-60%, myristic acid 14-25%, palmitic acid 7-15%, oleic acid <11%, stearic acid <7%, and linoleic acid <3%. Analysis of a batch of polysorbate 80 revealed that myristic acid <5%, palmitic acid <16%, oleic acid>58%, stearic acid <6%, and linoleic acid <18%. Analysis of polysorbate 80 from another source revealed an oleic acid content of about 70%, with the remainder being other fatty acid esters and impurities. Analysis of yet another source of polysorbate 80 revealed about 86-87% oleic acid. Analysis of another recently developed source of polysorbate 80 revealed ≥99% oleic acid.

[0167] In some aspects, the concentration of oleic acid in polysorbate 80 can be between about 50% and about 100%, between about 58% and about 100%, between about 60% and 100%, between about 80% and about 100%, between about 90% and about 100%, between about 95% and about 100%, between about 98% and about 100%, between about 99% and about 100%, between about 98.0% and about 99.9%, between about 98.5% and about 99.5%, between about 99.0% and 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 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%.

[0168] Biopharmaceutical drugs are usually formulated with nonionic detergents such as polysorbate 20 or polysorbate 80. These detergents help stabilize macromolecules such as antibodies and other proteins and help prevent the formation of supramolecular ternary complexes or other aggregates. Aggregates may become nanoparticles or subvisible particles in the range of 10 to 100 microns or 2 to 100 microns and interfere with drug product stability and shelf life. Therefore, in some cases, the stability of protein formulations depends on the stability of nonionic detergent additives. However, and as further discussed herein, polysorbate 20 and polysorbate 80 may promote the formation of aggregates, nanoparticles and subvisible particles in some cases.

[0169] The phrase "subvisible particles" means invisible particles (especially in liquids). In other words, solutions or other liquids containing subvisible particles but not containing visible particles will not appear turbid. Subvisible particles generally include those particles with a diameter of 100 microns or less, but in some cases include particles less than 150 microns (Narhi et al., "A critical review of analytical methods for subvisible and visible particles", Contemporary Pharmaceutical Biotechnology (Curr Pharm Biotechnol) 10 (4): 373-381 (2009)). Subvisible particles may be the result of foreign pollutants or protein aggregation. The shape of protein aggregates can be soft and amorphous, and therefore may be difficult to detect using conventional light shielding and microscopy methods (Singh and Toler, "Monitoring of subvisible particles in therapeutic proteins", Methods Mol Biol. 2012; 899: 379-401). Subvisible particles may include, among other things, silicone contaminants (oil droplets), free fatty acids (oil droplets), aggregated proteins (amorphous particles) and / or protein / fatty acid complexes (amorphous particles).

[0170] Subvisible particles can be detected by any one or more methods in various methods. USP standards specify light shielding and optical microscopy procedures. Other methods include micro-flow imaging (MFI) analysis, Coulter counting and submicron particle tracking. For light obscuration (LO), particles will project shadows on a photodetector when passing through the light beam in the flow cell, and particles are counted based on these shadows. The size, shape and reverse intensity of the shadow depend on the size, shape and refractive index difference of the particle relative to the solution. The minimum size range detected using LO is about 2 microns. Commonly used LO devices are HIAC instruments (Beckman Coulter, Brea, Calif., Brea, California). Several methods for measuring and characterizing SVPs (e.g., light obscuration, flow microscopy, electrical sensing zone methods, and flow cytometry) are discussed in the following literature: for example, Narhi et al., "Subvisible (2-100 μm) Particle Analysis During Biotherapeutic Drug Product Development: Part 1, Considerations and Strategy", Journal of Pharmaceutical Sciences 104:1899-1908 (2015).

[0171] Light shielding is criticized for underestimating protein aggregates and other amorphous structures. Flow image analysis (such as microflow imaging (MFI)) (Brightwell Technologies, Ottawa, Ontario) is a more sensitive method for detecting irregularly shaped, fragile and transparent protein subvisible particles and distinguishing these types of particles from silicone microdroplets, bubbles and other foreign contaminants (Sharma et al., "Micro-flow imaging: Flow microscopy applied to sub-visible particulate analysis in protein formulations", Journal of the American Association of Pharmaceutical Scientists (AAPS J.) 12 (3): 455-464 (2010)). In general, since the SVP measurement and characterization performed by light shielding analysis is not as sensitive as MFI, the particle counts detected by MFI are often higher than the particle counts detected by light shielding analysis. In simple terms, MFI is a type of flow microscopy that continuously takes bright-field images and analyzes them in real time. By applying image analysis algorithms to these images, it is possible to distinguish between air bubbles, silicone oil droplets, and protein aggregates. Volumes as low as about 250 microliters and as high as tens of milliliters can be analyzed. Depending on the system used, particles in the range of 2 to 300 microns or 1 to 70 microns can be detected (ibid.).

[0172] In some aspects, visible or subvisible particles in a formulation can be detected and analyzed by Raman spectroscopy. As used herein, the term "Raman spectroscopy" refers to a spectroscopic method based on the Raman scattering method. Raman spectroscopy can provide a Raman spectrum that can identify the fingerprint region (2000 to 400 cm -1) can be compared with the Raman spectroscopy database to chemically identify the material being analyzed (CV Raman and KSK Krishnan, A new type of secondary radiation, 121 Nature, 501-502 (1928); Zai-Qing Wen, Raman spectroscopy of protein pharmaceuticals, 96 Journal of Pharmaceutical Science, 2861-287 (2007)).

[0173] The FDA and other government regulatory agencies have restricted the permissible content of subvisible particles in parenteral drug formulations. The main concern is the uncertainty associated with the potential immunogenicity of patients receiving the drug and subsequent adverse effects (Singh et al., "An industry perspective on the monitoring of subvisible particles as a quality attribute for protein therapeutics", Journal of Pharmaceutical Sciences 99 (8): 3302-21 (2010)). For small volumes of parenteral drugs (e.g., 100 mL or less), the pharmacopoeia stipulates that when determined by light obscuration analysis, subvisible particles (SVP) greater than or equal to 10 microns shall not exceed 6,000 SVPs per container, and SVPs greater than or equal to 25 microns shall not exceed 600 per container; and when determined by film microscopy, SVPs greater than or equal to 10 microns shall not exceed 3,000 SVPs per container, and SVPs greater than or equal to 25 microns shall not exceed 300 per container. (United States Pharmacopeia and National Formulary (USP 40-NF 28), <787> Subvisible Particulate Matter in Therapeutic Protein Injections. For ophthalmic drugs, the SVP limit is 50 per mL for 10 microns or larger, 5 per mL for 25 microns or larger, and 2 per mL for 50 microns or larger (ibid., <78922 Particulate Matter in Ophthalmic Solutions). Regulators are increasingly considering having manufacturers establish specifications for SVPs of 2 microns or larger (see Singh et al., "Industry Perspectives on Monitoring Subvisible Particles as a Quality Attribute of Protein Therapeutics," Journal of Pharmaceutical Sciences 99(8):3302-21 (2010)).

[0174] The term "esterase" means an enzyme that catalyzes the hydrolysis of ester bonds to produce acids and alcohols. Esterases are a diverse class of enzymes, including acetylesterases (e.g., acetylcholinesterase), phosphatases, nucleases, thioesterases, lipases, and other carboxylester hydrolases. As the name implies, carboxylester hydrolases (also known as carboxylesterases, carboxylester hydrolases, and EC 3.1.1.1) utilize water to hydrolyze carboxylates into alcohols and carboxylates. Lipase is a carboxylester hydrolase that catalyzes the hydrolysis of lipids (including triglycerides, fats, and oils) into fatty acids and alcohol head groups. For example, triglycerides are hydrolyzed by lipases (such as pancreatic lipases) to form monoacylglycerols and two fatty acid chains.

[0175] Phospholipase is a lipase that hydrolyzes phospholipids into fatty acids and other products. Phospholipase can be roughly divided into four major categories: phospholipase A (including phospholipase A1 and phospholipase A2), phospholipase B and phosphodiesterase (phosphodiesterase C and phosphodiesterase D). In addition to typical phospholipase, the phospholipase B sample enzyme that resides in the lysosomal lumen is considered to participate in lipid catalysis. For example, based on sequence homology and subcellular localization, it is assumed that phospholipase B sample 2 (PLBL2) has esterase activity (Jensen et al., " Biochemical characterization and liposomal localization localization of the mannose-6-phosphate protein p76 of mannose-6-phosphate protein p76 ", " Journal of Biological Chemistry " 402:449-458 (2007)).

[0176] As used herein, phrase " fatty ester hydrolysis percentage " means the molar ratio of the fatty acid ester that has been hydrolyzed.Because the hydrolysis of fatty acid ester causes the release of free fatty acids, so fatty ester hydrolysis percentage can be determined by the free fatty acids in the measurement sample.Therefore, fatty ester hydrolysis percentage can be determined by the sum of the mole number of the mole number of the mole number of the mole number of the free fatty acids divided by the fatty acid to add the fatty acid ester.With regard to the hydrolysis percentage of polysorbate 80 or polysorbate 20, described numerical value can be determined by the mole number of the free fatty acids and divided by the total mole number of the remaining polysorbate and the mole number sum of the free fatty acids.

[0177] The term "esterase inhibitor" means any chemical entity that reduces, inhibits or blocks the activity of an esterase. Applicants contemplate that the addition of an esterase inhibitor to a protein formulation containing a fatty acid ester surfactant may help maintain protein stability and the stability of the fatty acid esters, and help reduce the formation of SVPs. Common esterases known in the art include orlistat (tetrahydrolipistatin; an inhibitor of carboxylesterase 2 and lipoprotein lipase), diethylumbelliferyl phosphate (a cholesterol esterase [lipase A] inhibitor), URB602 ([1-1'-biphenyl]-3-ylcarbamic acid cyclohexyl ester; a monoacylglycerol lipase inhibitor), and 2-butoxyphenylboronic acid (an inhibitor of hormone-sensitive lipase). The addition of an esterase inhibitor during purification of the protein of interest or in the final formulation is expected to prevent or slow the hydrolysis of non-ionic detergents (such as polysorbate 20 and polysorbate 80), and thus to prevent or reduce the formation of subvisible particles.

[0178] Exemplary Formulations

[0179] According to one aspect 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 sequences disclosed in Table 1 below); (ii) acetate; (iii) histidine; (iv) sucrose; (v) arginine; and (v) a surfactant comprising polyethylene glycol or poloxamer.

[0180] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody that specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of the surfactant including polyethylene glycol or poloxamer can be 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%. On the other hand, 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, a stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 15 mg / mL to 200 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of the surfactant including polyethylene glycol or poloxamer can be 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%. On the other hand, 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 at a concentration of 15 mg / mL to 200 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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) PEG3350 or poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of the surfactant including PEG3350 or poloxamer 188 can be 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%. On the other hand, 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 at a concentration of 15 mg / mL to 200 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including PEG3350 can be 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 aspect, 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 at a concentration of 15 mg / mL to 200 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9 ± 0.2. In another aspect, the concentration of the surfactant including poloxamer 188 can be 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 aspect, 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, a stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 100 mg / mL to 200 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of the surfactant including polyethylene glycol or poloxamer can be 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%. On the other hand, 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 at a concentration of 100 mg / mL to 200 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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) PEG3350 or poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of PEG3350 or poloxamer 188 can be 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%. On the other hand, 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 at a concentration of 100 mg / mL to 200 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9 ± 0.2. In another aspect, the concentration of PEG3350 can be 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 aspect, 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 at a concentration of 100 mg / mL to 200 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9 ± 0.2. In another aspect, the concentration of poloxamer 188 can be 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 aspect, 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 some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 150 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of the surfactant including polyethylene glycol or poloxamer can be 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%. On the other hand, 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.

[0190] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 150 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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) PEG3350 or poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of the surfactant including PEG3350 or poloxamer 188 can be 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%. On the other hand, 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.

[0191] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 150 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including PEG3350 can be 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 aspect, 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.

[0192] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 150 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including poloxamer 188 can be 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 aspect, 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.

[0193] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 175 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of the surfactant including polyethylene glycol or poloxamer can be 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%. On the other hand, 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.

[0194] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 175 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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) PEG3350 or poloxamer 188 at a concentration of 0.01% w / v to 0.19% w / v, wherein the pH of the formulation is 5.7 to 6.1. On the other hand, the concentration of the surfactant including PEG3350 or poloxamer 188 can be 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%. On the other hand, 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.

[0195] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 175 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including PEG3350 can be 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 aspect, 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.

[0196] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 175 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including poloxamer 188 can be 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 aspect, 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.

[0197] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 175 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including PEG3350 can be 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 aspect, 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.

[0198] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 175 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including poloxamer 188 can be 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 aspect, 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.

[0199] In some cases, the stable liquid pharmaceutical formulation comprises: (i) a human antibody at a concentration of 175 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including PEG3350 can be 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 aspect, 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.

[0200] In some cases, a stable liquid drug formulation comprises: (i) a human antibody at a concentration of 175 mg / mL±10 mg / mL, wherein the antibody specifically binds to human IL-4Rα and comprises a heavy chain variable region (HCVR) 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 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 pH of the formulation is 5.9±0.2. In another aspect, the concentration of the surfactant including poloxamer 188 can be 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 aspect, 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.

[0201] In any of the various aspects of the pharmaceutical formulation discussed above or herein, the human IL-4R antibody may include a human IgG1 heavy chain constant region.

[0202] In any of the various aspects of the pharmaceutical formulation discussed above or herein, the human IL-4R antibody may include a human IgG4 heavy chain constant region.

[0203] In some aspects, the human IL-4R antibody can include 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.

[0204] Additional non-limiting examples of pharmaceutical formulations encompassed by the present disclosure are set forth elsewhere herein, including the working examples presented below.

[0205] Stability of drug formulations

[0206] The pharmaceutical preparations disclosed herein exhibit a high degree of stability. The term "stable" as used herein with respect to pharmaceutical preparations means that the protein of interest in the pharmaceutical preparation can still maintain an acceptable degree of structure and / or function and / or biological activity after being stored for a certain period of time. Even if the protein contained therein does not maintain 100% of its structure and / or function and / or biological activity after being stored for a certain period of time, the preparation can also be stable. In some cases, after being stored for a certain period of time, if the protein can maintain about 90%, about 95%, about 96%, about 97%, about 98% or about 99% of its structure and / or function and / or biological activity, maintenance can be regarded as "stable".

[0207] Stability can be measured in a variety of ways, in particular by determining the percentage of proteins in the formulation that form aggregates after storage at a specified temperature for a certain period of time or under stress conditions (e.g., stirring), wherein stability is inversely proportional to the percentage of aggregates formed. The percentage of aggregated protein can be determined by a variety of methods, such as size exclusion chromatography (e.g., size exclusion high performance liquid chromatography (SE-HPLC) or size exclusion ultra-performance liquid chromatography (SE-UPLC)). As used herein, the phrase "acceptable degree of stability" means that after storage at a given temperature for a certain period of time or under specific stress conditions, only about 15%, 10%, 5%, 4%, 3%, 2.5%, 2%, 1.5%, 1%, 0.5% or 0.1% of the protein in the formulation can be detected in the form of aggregates. The prescribed 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 longer. The storage temperature of the pharmaceutical formulation when evaluating stability can be any temperature from about -80°C to about 45°C, for example, stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-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 subjected may be agitation stress (e.g., vortexing) for a period of 10 minutes, 20 minutes, 30 minutes, 40 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 100 minutes, 110 minutes, 120 minutes, 150 minutes, 180 minutes, or longer. For example, a pharmaceutical formulation comprising 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 nine months of storage at 5°C. A pharmaceutical formulation may also be considered stable if less than about 12% of the protein is detected in aggregated form after 56 days of storage at 45°C. A 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 42 days of storage at 45°C.If a pharmaceutical formulation is stored at 45°C for 28 days, less than about 8%, or less than about 7.5%, or less than about 7% of the protein in aggregated form is detected, the formulation can also be considered stable. If a pharmaceutical formulation is stored at 45°C for 14 days, less than about 6% of the protein in aggregated form is detected, the formulation can also be considered stable. If a pharmaceutical formulation is stored at -20°C, -30°C or -80°C for three months, less than about 2%, 1.9%, 1.8%, 1.7%, 1.6%, 1.5%, 1%, 0.5% or 0.1% of the protein in aggregated form is detected, the formulation can also be considered stable. If a pharmaceutical formulation is stirred at room temperature for 120 minutes (e.g., by vortexing), less than 3% or less than 2.5% of the protein in aggregated form is detected, the formulation can also be considered stable.

[0208] Stability can also be determined in a variety of ways, in particular by determining the formation of particles in the formulation after the formulation is stored at a defined temperature for a certain period of time. For example, the formation of particles can be determined by microscopy techniques or microfluidic imaging techniques.

[0209] In some aspects, the preparations of the present disclosure include a detectable amount of lipase (e.g., PLBL2). Methods for detecting and quantifying the presence and activity of phospholipases are known in the art. In some aspects, phospholipases are detected by immunoassays (e.g., ELISA). In some aspects, phospholipases are detected by liquid chromatography-mass spectrometry (LC-MS).

[0210] Thus, a pharmaceutical formulation (containing an esterase or lipase) can be considered stable if no more than a certain number (e.g., 3000, 1000, 500, 250, 100 or 50) of fatty acid particles of size ≥10 μm or ≥25 μm are identified in a volume of 2.25 mL after storage at a defined temperature (e.g., 5°C) for a period of time (e.g., 6 months, 12 months, 18 months, 24 months or 36 months or longer). For example, a pharmaceutical formulation can be considered stable if no more than 3000 fatty acid particles are identified in a volume of 2.25 mL by microscopic examination after storage at 5°C for 24 months. On the other hand, a pharmaceutical formulation can be considered stable if no more than 1000 fatty acid particles are identified in a volume of 2.25 mL by microscopic examination after storage at 5°C for 24 months. A pharmaceutical preparation may also be considered stable if no more than 500 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 24 months. A pharmaceutical preparation may also be considered stable if no more than 250 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 24 months. A pharmaceutical preparation may also be considered stable if no more than 150 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 24 months. A pharmaceutical preparation may also be considered stable if no more than 1000 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 36 months. A pharmaceutical preparation may also be considered stable if no more than 500 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 36 months. A pharmaceutical preparation may also be considered stable if no more than 250 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 36 months. A pharmaceutical preparation may also be considered stable if no more than 150 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 36 months. A pharmaceutical preparation may also be considered stable if no more than 100 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 36 months. A pharmaceutical preparation may also be considered stable if no more than 50 fatty acid particles are identified by microscopic examination in a volume of 2.25 mL after storage at 5°C for 36 months.

[0211] Stability can also be measured in a variety of ways, in particular by determining the percentage of the remaining native protein of interest after the preparation is stored at a given temperature for a certain period of time. The percentage of the native protein of interest can be measured by a variety of methods, such as size exclusion chromatography (such as size exclusion high performance liquid chromatography (SE-HPLC)). As used herein, the phrase "acceptable degree of stability" means that after a certain period of time is stored at a given temperature, at least 90% of the native form of the protein in the preparation can be detected. In some aspects, after a certain period of time is stored at a given temperature, at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% of the native form of the protein can be detected in the preparation. The prescribed time 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 longer. The storage temperature of the pharmaceutical formulation when evaluating stability can be any temperature from about -80°C to about 45°C, for example, stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, about 35°C, about 37°C or about 45°C.

[0212] Stability can also be determined in a variety of ways, particularly by the percentage of the protein of interest that migrates to the more acidic component ("acidic form") during an ion exchange process (compared to its percentage in the major protein component ("major charged form"), where stability is inversely proportional to the proportion of protein in the acidic form. Although not wishing to be bound by theory, deamidation of a protein may cause the protein to be more negatively charged and therefore more acidic relative to 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-performance liquid chromatography (CEX-UPLC)). As used herein, the phrase "acceptable degree of stability" means that after storage at a given temperature for a certain period of time, at most 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 5%, 4%, 3%, 2%, 1%, 0.5% or 0.1% of the protein in the acidic form can be detected in the formulation. The specified 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 storage temperature of the pharmaceutical formulation when evaluating stability can be any temperature between about -80°C and about 45°C, for example, stored at about -80°C, about -30°C, about -20°C, about 0°C, about 4°C-8°C, about 5°C, about 25°C, or about 45°C.

[0213] Measuring the binding affinity of an antibody of interest to its target can also be used to assess stability. For example, if a formulation of the present disclosure is stored for a certain period of time (e.g., 14 days to 9 months) at a temperature such as -80°C, -30°C, -20°C, 5°C, 25°C, 37°C, 45°C, etc., the binding affinity of the anti-IL-4R antibody contained in the formulation to hIL-4Rα reaches at least 80%, 85%, 90%, 95% or more of the binding affinity of the antibody before storage, then the formulation can be considered stable. Binding affinity can be determined by any method such as ELISA or plasmon resonance. Biological activity can be determined by, for example, measuring the downstream activity of the IL-4R system in the presence of the antibody and comparing the activity to the activity of the IL-4R system in the absence of the antibody.

[0214] Reference to the stability of a pharmaceutical formulation "after" a specified period of time is intended to mean the measurement of a stability parameter (e.g., % native form, % HMW species, or % acidic form) at or about the end of the specified period of time, and is not intended to mean that the pharmaceutical formulation will necessarily maintain the same degree of stability for the measured parameter thereafter. For example, reference to a specific stability after 12 months means that the stability measurement was performed 12 months or about 12 months after the start of the study.

[0215] Container and method of application

[0216] The pharmaceutical preparations of the present invention can be contained in any container suitable for storing drugs and other therapeutic compositions. For example, the pharmaceutical preparations can be contained in sealed and sterilized plastic or glass containers with a defined volume, such as vials, ampoules, syringes, cartridges, bottles, or intravenous infusion (IV) bags. Different types of vials can be used to hold the preparations of the present invention, including, for example, transparent and opaque (e.g., amber) glass or plastic vials. Similarly, any type of syringe can be used to hold and / or administer the pharmaceutical preparations of the present invention. In some aspects, the pharmaceutical preparations are contained in prefilled syringes (PFS). In some aspects, the drug preparations are contained in prefilled post needle syringes.

[0217] The pharmaceutical agents of the present disclosure can be contained in a "normal tungsten" syringe or a "low tungsten" syringe. As will be understood by those of ordinary skill in the art, the process of manufacturing a glass syringe generally involves the use of a hot tungsten rod, which is used to pierce the glass, thereby forming a hole so that liquid can be drawn in and out of the syringe. This process results in trace amounts of tungsten being deposited on the inner surface of the syringe. Subsequent washing and other processing steps can be used to reduce the tungsten content in the syringe. As used herein, the term "normal tungsten" means that the tungsten content in the syringe is greater than 500 parts per billion (ppb). The term "low tungsten" means that the tungsten content in the syringe is less than 500 ppb. For example, according to the present disclosure, the tungsten content in the low tungsten syringe can be less than about 490ppb, 480ppb, 470ppb, 460ppb, 450ppb, 440ppb, 430ppb, 420ppb, 410ppb, 390ppb, 350ppb, 300ppb, 250ppb, 200ppb, 150ppb, 100ppb, 90ppb, 80ppb, 70ppb, 60ppb, 50ppb, 40ppb, 30ppb, 20ppb, 10ppb or less.

[0218] The rubber piston used in the syringe and the rubber stopper used to close the vial opening can be coated to prevent contamination of the medicinal ingredients of the syringe or vial and / or maintain its stability. Therefore, according to certain aspects, the drug preparation of the present disclosure can be contained in a syringe including a coated piston or a vial sealed with a coated rubber stopper. For example, the plunger or stopper can be coated with a fluorocarbon film. Examples of coated stoppers and / or pistons suitable for use with vials and syringes containing the drug preparation of the present disclosure are mentioned in, for example, U.S. Patents 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. Specific exemplary coated rubber stoppers and pistons that can be used in the context of the present disclosure can be identified by the trade name Commercially available from West Pharmaceutical Services, Inc. (Lionville, PA). According to certain aspects of the present disclosure, the pharmaceutical formulation can be contained in a low tungsten syringe equipped with a fluorocarbon-coated piston. In some aspects, the container is a syringe, such as the Ompi EZ-Fill TM Syringe or BD Neopak TMSyringe. In some cases, the syringe is a 1 mL long glass syringe with a 1 mL iWest plunger, 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 aspects, 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 a 10 mL syringe (e.g., a glass syringe).

[0219] The pharmaceutical preparation can be administered to the patient by parenteral routes, such as injection (e.g., subcutaneous, intravenous, intramuscular, intraperitoneal, etc.) or transdermal, mucosal, nasal, pulmonary and / or oral administration. A variety of reusable pen-type and / or automatic syringe delivery devices can be used for subcutaneous delivery of the pharmaceutical preparations of the present disclosure. Examples include, but are not limited to, AUTOPEN TM (Owen Mumford, Inc., Woodstock, UK), DISETRONIC TM Pen (Disetronic Medical Systems, Bergdorf, Switzerland), HUMALOG MIX 75 / 25 TM Pen, HUMALOG TM Pen, HUMALIN 70 / 30 TM PEN (Eli Lilly and Co., Indianapolis, IN), NOVOPEN TM I, II and III (Novo Nordisk, Copenhagen, Denmark), NOVOPEN JUNIOR TM (Novo Nordisk, Copenhagen, Denmark), BD TMPen (Becton Dickinson, Franklin Lakes, NJ), OPTIPEN TM 、OPTIPEN PRO TM 、OPTIPEN STARLET TM and OPTICLIK TM (sanofi-aventis, Frankfurt, Germany), to name a few. Examples of disposable pen and / or auto-injector delivery devices for subcutaneous delivery of the pharmaceutical compositions of the present disclosure include, but are not limited to, SOLOSTAR TM Pen (Sanofi-Aventis), FLEXPEN TM (Novo Nordisk) and KWIKPEN TM (Eli Lilly), SURECLICK TM Autoinjector (Amgen, Thousand Oaks, CA), PENLET TM (Haselmeier, Stuttgart, Germany), EPIPEN (Dey, LP), and HUMIRA TM In some cases, the drug formulation is contained in a syringe specially adapted for use with an auto-injector. Subcutaneous injections can be administered using a 20-30 gauge needle or a 25-30 gauge needle. In some cases, subcutaneous injections can be administered using a 25 gauge needle. In some cases, subcutaneous injections can be administered using a 27 gauge needle. In some cases, subcutaneous injections can be administered using a 29 gauge needle.

[0220] Another type of delivery device can include a safety system. Such devices can be relatively cheap, and once the injection is completed, the safety sleeve can be manually or automatically extended over the needle. Examples of safety systems can include the ERIS device of West Pharmaceutical or the UltraSafe device of Becton Dickinson Medical. In addition, it is also contemplated herein to use a large volume device ("LVD") or a pellet syringe to deliver the pharmaceutical preparation of the present disclosure. In some cases, the LVD or pellet syringe can be configured to inject the drug into the patient. For example, the LVD or pellet syringe can be configured to deliver a "large" volume of drug (typically about 2mL to about 10mL).

[0221] The pharmaceutical formulations of the present disclosure may also be contained in unit dosage form. As used herein, the term "unit dosage form" refers to physically discrete units suitable as unit doses for patients to be treated, each unit containing a predetermined amount of active compound calculated to produce the desired therapeutic effect in conjunction with the desired pharmaceutical carrier, diluent or excipient. In various aspects, 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 be varied so as to obtain an amount of active ingredient that is effective for the desired therapeutic response for a particular patient, composition, and mode of administration without adversely affecting the patient. The dosage level selected will depend on various pharmacokinetic factors, including the activity of the particular composition of the present disclosure employed; the route of administration; the time of administration; the excretion rate of the particular compound employed; the duration of treatment; other drugs, compounds, and / or materials used in combination with the particular composition employed; the age, sex, weight, condition, general health, and prior medical history of the patient being treated; and similar factors well known in the medical arts. As used herein, the term "diluent" refers to a solution suitable for altering or achieving one or more exemplary or appropriate concentrations as described herein.

[0222] In various aspects, the unit dosage form contains an amount of active ingredient (e.g., anti-IL-4R antibody) for a single use. In various aspects, 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 any combination of the above ranges or intervals. For example, a combination of any of the above values ​​(or values ​​included in the above ranges) as an upper and / or lower numerical range is also included. In certain aspects, the formulation is generally provided in a liquid form of a unit dosage form. In some aspects, the unit dosage form contains about 100 mg of active ingredient. In some aspects, the unit dosage form contains about 150 mg of active ingredient. In some aspects, the unit dosage form contains about 200 mg of active ingredient. In some aspects, the unit dosage form contains about 300 mg of active ingredient. In some aspects, the unit dosage form contains about 350 mg of active ingredient. In some aspects, the unit dosage form contains about 600 mg of active ingredient. In some aspects, the unit dosage form according to the present disclosure is suitable for subcutaneous administration to a patient.

[0223] The present disclosure also includes methods of preparing unit dosage forms.In one aspect, a method for preparing a pharmaceutical unit dosage form comprises combining the formulation of any of the aforementioned aspects in a suitable container (eg, those discussed herein).

[0224] Therapeutic use of pharmaceutical preparations

[0225] The pharmaceutical formulations of the present disclosure comprising anti-IL-4R antibodies can be used to treat, prevent, and / or ameliorate any disease or condition associated with IL-4R activity, among other things.

[0226] The therapeutic methods disclosed herein 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 any human or non-human animal that could benefit from inhibiting or reducing IL-4R and / or IL-4R-mediated activity. The present disclosure further includes the use of any of the pharmaceutical formulations disclosed herein in a medicament for treating, preventing and / or ameliorating any disease or condition associated with IL-4R activity.

[0227] In some aspects, the disease or disorder associated with IL-4R activity is an inflammatory condition, an allergic disease, a lung / respiratory disease, a gastrointestinal disease, or a skin disease. In some aspects, the disease or disorder is a type 2 inflammatory disease. In some aspects, 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); anaphylaxis; 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 keratoconjunctivitis; autoimmune hemolytic anemia; autoimmune lymphoproliferative syndrome; autoimmune uveitis; Barrett's esophagus; benign prostatic hyperplasia; bronchiectasis; bullous pemphigus; Churg-Strauss syndrome; 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; dermatitis herpetiformis; hypertrophic scarring; inflammatory bowel disease; Kawasaki disease; nasal polyposis; renal disease; Netherton's syndrome; preeclampsia; prurigo nodularis; pruritus (e.g., chronic pruritus of unknown cause); rhinitis (e.g., allergic rhinitis); sinusitis (e.g., allergic fungal sinusitis, chronic sinusitis with or without nasal polyposis); scleroderma; sickle cell disease; Sjogren's syndrome syndrome; tuberculosis; ulcerative colitis; and Whipple's Disease.

[0228] In some aspects, the disclosure provides a kit comprising a pharmaceutical formulation as discussed herein (e.g., a container or unit dosage form containing the formulation), and packaging or labeling (e.g., a package insert) with instructions for using the pharmaceutical formulation to treat a disease or condition as discussed above. In some cases, the instructions provide the use of a unit dosage form as discussed herein for treating a disease or condition.

[0229] Table 1 below shows a summary of the sequences cited herein and the corresponding SEQ ID NOs.

[0230] Table 1: Informal Sequence Listing

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248] Example

[0249] The following examples are presented to fully disclose and describe to those of ordinary skill in the art how to prepare and use the methods and compositions of the present invention and are not intended to limit the scope of their invention as recognized by the inventors. Every effort has been made to ensure accuracy with respect to the numbers used (e.g., amounts, temperatures, etc.), but some experimental errors and deviations should be taken into account. Unless otherwise indicated, parts are parts by weight, molecular weights are average molecular weights, temperatures are in degrees Celsius, and pressures are at or near atmospheric pressure.

[0250] Determination of subvisible particles

[0251] For the determination of subvisible particles, suitable methods include "Method 1" (light obscuration particle count test) and "Method 2" (microscope particle count test). Using light obscuration, the FDA's requirement for subvisible particles in parenteral drug products is that for particles with a diameter ≥10 microns, the number of particles in each container should be ≤6,000, and for particles with a diameter ≥25 microns, the number of particles in each container should be ≤600 particles. Using the microscopy method, the FDA's requirement for subvisible particles in parenteral drug products is that for particles with a diameter ≥10 microns, the number of particles in each container should be ≤3,000, and for particles with a diameter ≥25 microns, the number of particles in each container should be ≤300 particles. Currently, there are no regulatory requirements for particles with a diameter less than 10 microns, but the FDA has required measurements of particles between 2 and 10 microns.

[0252] Particles with diameters greater than 1 micron are measured using HIAC light obscuration and Brightwell Microfluidic Imaging (MFI) technology. HIAC combines light obscuration with laser scattering to detect and count particles in the 500nm-350μm size range in a flowing fluid. Particles are sized based on the voltage response generated in the detector, and the particles are classified into a pre-set size range based on the voltage response.

[0253] For HIAC determination, the sample containing 150mg / mL monoclonal antibody from a production line (GMP batch) is merged into the cumulative volume of 25mL. For each merged sample, three readings are carried out to each five milliliters of sample. Also use HIAC to check the laboratory sample of identical 150mg / mL antibody preparation. Samples from at least three vials (2.5mL / vials), seven 1mL syringes (1.14mL / syringes) or five 2.25mL syringes (2mL / syringes) are merged, and three readings are carried out to one milliliter of sample each time. HIAC 9703 and HIAC 8000A instruments (Hach Company, Loveland, Colo., Colorado) are used respectively, and light shielding readings are carried out with HRLD 400 probes (its every mL can read 18,000 cumulative counts at most) and MC05 probes (its every mL can read 10,000 cumulative counts at most).

[0254] The MFI method uses less material than the HIAC light shielding (i.e., the amount in 1 mL of formulation or 1 stability vial or syringe) and yields a higher number of particles than HIAC. Since MFI is based on microscopy, the method is more sensitive to translucent protein particles and can distinguish silicone oil droplets / bubbles from protein particles for pre-filled syringe samples. MFI (as in the HIAC analysis) was performed on laboratory samples containing 150 mg / mL of monoclonal antibody. For MFI, one reading was performed on one mL of sample at a time.

[0255] Determination of polysorbate degradation

[0256] Degradation of polysorbates is examined using one or more of several methods. The first method employs an enzymatic colorimetric method to quantify non-esterified fatty acids (NEFA). The NEFA-HR(2) kit (Wako Diagnostics, Richmond, Va.) is used to detect fatty acids in formulated drug substances containing polysorbates. Briefly, the sample is combined with ATP and coenzyme A (CoA) in the presence of acyl-CoA synthetase (ACS). (Free) fatty acids react with CoA to form acyl-CoA. The acyl-CoA product reacts with oxygen and acyl-CoA oxidase to generate trans-2,3-dehydroacyl-CoA and hydrogen peroxide. Peroxidase catalyzes the reaction of hydrogen peroxide with 4-aminoantipyrine and 3-methyl-N-ethyl-N-(β-hydroxyethyl)-aniline to form a blue-violet pigment (maximum absorbance 550 nm). The amount of NEFA in a sample is proportional to the amount of pigment. For a detailed description of the colorimetric determination of NEFA, 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, M.C., "Modified Colorimetric Ultramicro Method for Estimating NEFA in Serum." Ultramicro Method for Estimating NEFA in Serum), Journal of Clinical Pathology (J. Clin. Pathol.), 21(5): 567-70 (1968).

[0257] The test sample containing the protein of interest (and putative host cell protein contaminants) was placed on a 10 kDa molecular weight cutoff filter. The retentate was recovered in 10 mM histidine (pH 6.0) with a protein concentration greater than 100 g / L and then spiked with polysorbate to obtain a 100 g / L protein, 0.8% (w / v) polysorbate, 10 mM histidine (pH 6.0) (t 初始 The test sample is subjected to 45°C for 44 hours (t 最终 ). Some samples were spiked with oleic acid to evaluate the recovery efficiency of NEFA in the samples. The percentage of polysorbate degradation was calculated as follows:

[0258]

[0259] The second method for determining polysorbate degradation is based on mass spectrometry. Using LC / MS analysis, this detection method is able to measure and compare the initial percentage of esters and the remaining percentage of esters after incubation of polysorbate at 45°C at different time points. mAb1 produced according to method 6 (without HIC and with PS degradation activity) and mAb1 produced according to method 3 (with HIC step and without PS degradation activity) (see Example 4 and Table 9) were included in the detection as negative and positive controls, respectively.

[0260] Briefly, 15 mg of antibody sample (at a concentration of approximately 5-10 mg / mL, or 7 mg / mL ± 1.5 mg / mL) was placed in an ultrafilter (Amicon Ultra 50K, Millipore, Billerica, Mass.) and centrifuged at 14,000 × g for 15 minutes or until the remaining volume was slightly below the 100 μL mark on the device. 1 μL of 10% polysorbate was added to the centrifugal filter containing the concentrated protein, followed by vortex mixing. The sample was recovered by reverse centrifugation at 1000 g for 5 minutes to obtain the entire sample in the collection tube.

[0261] The recovered volume was measured and the concentration of polysorbate was calculated. 1 μL of each recovered sample was diluted 100-fold in a separate test tube and the protein concentration was measured using a Nanodrop 1000 (Thermo Fisher Scientific, Inc., Wilmington, Del., Delaware). The sample was then diluted in histidine buffer (10 mM, pH 6.0) and polysorbate stock solution to a protein concentration of 150 mg / mL and a polysorbate concentration of 0.2% (w / w).

[0262] The initial time (T0) sample (2 μL) was retained from each sample and stored at -80°C until use. The samples to be tested were sealed under argon, then incubated at 45°C to induce degradation, and taken out at the specified time points for testing. 2 μL was taken out from each sample at each time point and diluted to 100 μL with water. Each diluted time point sample was stored at -80°C. After collecting the samples at each time point, the top space of the sample tube was filled with argon, the sample container was resealed, and the sample was returned to the incubator to resume incubation.

[0263] The samples at each time point were analyzed using an anion exchange column (Oasis MAX column, particle size 30 μm, inner diameter × length 2.1 mm × 20 mm; Waters Corporation, Milford, Mass.) and then connected to reverse phase chromatography (ACQUITY BEH 130C4 column, particle size 1.7 μm, id×length 2.1 mm×50 mm; Waters Corporation, Milford, MA). The reverse phase chromatographic output was connected to a mass spectrometer (Thermo Q-Exactive mass spectrometer; Thermo Fisher Scientific, Wilmington, DE). Table 2 describes the chromatographic conditions.

[0264] Before the first injection, the system was equilibrated with 99% mobile phase A (0.1% formic acid / water) at a flow rate of 0.1 mL / min for 40 minutes. A blank injection was performed with water. The mass spectrometer 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,” Journal of Chromatography A 1176(1-2):135-42 (2007); Plante et al., “The use of charged aerosol detection with HPLC for the measurement of lipids,” Methods in Mol. Biol. 579:469-82 (2009)).

[0265] Table 2: Chromatographic conditions used to determine polysorbate degradation

[0266]

[0267]

[0268] To estimate the total amount of polyoxyethylene (POE), mass spectra were extracted using a mass range of 300-800 m / z to avoid interference from degraded proteins, and the peak cluster at about 8-15 minutes was integrated. For CAD chromatograms, the first cluster of POE peaks at about 8-15 minutes was directly integrated (again, the retention time may be slightly shifted). When other substances co-eluted with POE, the baseline was corrected to minimize their impact on the peak area.

[0269] To estimate the total amount of POE esters, mass spectra were extracted using a mass range of 300-2000 m / z and the peak cluster from about 17-40 minutes was integrated. For the CAD chromatograms, the POE ester peak cluster at about 17-40 minutes was directly integrated.

[0270] The percentage of POE ester was calculated according to the following formula:

[0271]

[0272] The percentage of remaining POE ester was calculated according to the following formula:

[0273]

[0274] Where n = 2 days, 4 days or 10 days.

[0275] Example 1. Particle specifications are unqualified

[0276] Two GMP batches of 150 mg / mL antibody formulations were evaluated for subvisible particles by HIAC light shielding after storage at 5°C for at least six months as described in U.S. Pat. No. 10,342,876 (the contents of which are incorporated herein by reference). The formulations contained 0.02% polysorbate 20 and 150 mg / mL anti-IL-4R antibody from Supplier A. The antibody was purified from CHO cell culture using a combination of affinity capture and ion exchange chromatography. The results are presented in Table 3.

[0277] Table 3: Number of particles with a size ≥ 10 μm after storage

[0278]

[0279] Example 2. The quality and purity of fatty acid esters affect SVP formation

[0280] The effect of the nature and quality of non-ionic detergents (polysorbate 20 and polysorbate 80) on subvisible particle formation in protein formulations was tested by formulating the antibodies into the following three conditions as described in U.S. Pat. No. 10,342,876, the contents of which are incorporated herein by reference: (i) using polysorbate 20 from Supplier A (PS20-A), (ii) using polysorbate 20 from Supplier B (PS20-B), or (iii) using polysorbate 80 (PS80). Table 4 shows the HIAC SVP (size ≥ 10 μm) data for the formulated drug substance from the following formulation: 20 mM histidine (pH 5.9), 12.5 mM acetate, 0.02% non-ionic detergent (polysorbate), 5% sucrose (w / v), 25 mM arginine, and 150 mg / mL antibody, stored in 2.5 mL fills in 5 mL Type 1 borosilicate glass vials equipped with West Company S2-F451 4432 / 50GRY B2-40 stoppers.

[0281] Here, over time, formulated drug substances containing polysorbate 80 ("mAb1") showed significantly less SVP formation than those containing polysorbate 20. In addition, formulations containing polysorbate 20 from supplier B (PS20-B), which is a higher quality polysorbate 20, showed less SVP formation than those containing polysorbate 20 from supplier A (PS20-A; a lower quality polysorbate 20). Comparative analysis of PS20-A and PS20-B showed that the total ester amount of PS20-B was 5-10% greater than the total ester amount of PS20-A, and that PS20-A contained more isosorbide laurate than PS20-B, as shown in Table 1. Figure 1 shown.

[0282] Table 4: Number of particles ≥ 10 μm after storage with different detergents

[0283]

[0284] As shown in Figure 2, formulated drug substances containing HP-PS20 or SR-PS80 were further evaluated using membrane microscopy or MFI after storage for up to 36 months. In addition, the number of particles >10 μm was compared between formulations stored in glass vials and formulations stored in prefilled syringes, as shown in Figure 2. Figure 2A , 2B , 2C and 2D. In all cases, the number of particles increased significantly over time.

[0285] It was hypothesized that degradation of the fatty acid esters of polysorbate in the formulation may contribute to protein instability and, therefore, SVP formation. To evaluate polysorbate degradation, the stability of polysorbate 20 and polysorbate 80 in formulations containing 150 mg / mL antibody (mAb1) and 0.02% non-ionic detergent (polysorbate) without HIC preparation (Method 3, see below and Table 9) was compared. The relative amounts of remaining esters (mono- and diesters) were determined by mass spectrometry. Significant degradation of the ester component of polysorbate 20 was observed after storing the samples for six months at 5°C or two months at 45°C. Under the same conditions, a lower degree of degradation of polysorbate 80 was observed (see Table 5). These results correlate with the observations of SVP particle formation.

[0286] Under the same conditions, the degradation rates of polysorbate 20 and polysorbate 80 (as described above for Table 5) formulated with 150 mg / mL antibody (mAb1) were determined using mass spectrometry to measure the relative amounts of free fatty acids and fatty acid esters. The percent degradation of esters was determined by the following formula:

[0287]

[0288] Where T0 = initial time, T1 = time under experimental conditions (i.e., 2 months at 45°C; 6 months at 5°C), and POE = polyoxyethylene. Table 6 shows the percentage degradation of polysorbate 20 and polysorbate 80 in 150 mg / mL antibody formulations. In otherwise identical antibody formulations, for mAb1 (but not for all antibodies tested), the degradation rate of polysorbate 80 was consistently lower than that of polysorbate 20.

[0289] Table 5: Percentage of esters remaining after storage

[0290]

[0291] Table 6: Percent degradation of esters after storage

[0292]

[0293]

[0294] The degradation of polysorbates in formulations containing SR-PS80 or HP-PS20 stored in glass vials or prefilled syringes was further compared using CAD-UHPLC. Figure 3A and 3B In all cases, significant degradation of the polysorbate occurred over time. Figure 4 As shown in Figure 5, the presence of SVP correlated with the remaining polysorbate concentration in the formulation. The same relationship was found using another formulation containing a monoclonal antibody (mAb5), as shown in Figure 5. Figure 5 Using Raman spectroscopy, it was confirmed that the particles in the polysorbate-containing formulations were consistent with the characteristics of fatty acids. Therefore, in order to address the issue of SVP formation, the phenomenon of polysorbate degradation and free fatty acid particle formation was further investigated.

[0295] Example 3. Polysorbate Degradation Activity

[0296] To determine the etiology of polysorbate degradation, the buffered mAbl antibody (150 mg / mL) was separated into two fractions by 10 kDa filtration: a protein fraction and a buffer fraction, according to the method described in U.S. Pat. No. 10,342,876 (which is incorporated herein by reference). These two fractions, as well as the buffered antibody that was not separated by filtration, were spiked into 0.2% (w / v) super-purified polysorbate 20 (PS20-B) and subjected to stress at 45°C for up to 14 days. The study showed that the protein fraction, but not the buffer fraction, had an effect on the degradation of sorbitan laurate (i.e., the main component of polysorbate 20), as shown in Table 7, and that the degradation of polysorbate 20 was correlated with the concentration of the antibody, as shown in Table 8.

[0297] Table 7: Degradation of sorbitan laurate in each mAbl fraction

[0298] Fraction Remaining sorbitan laurate % (14 days at 45°C) Drug substances 75％ Protein fraction 75％ Buffer fraction 100％

[0299] Table 8: Degradation of polysorbate in each mAb1 fraction as a function of antibody concentration

[0300] Fraction Antibody concentration (mg / mL) Remaining polysorbate 20% (12 days at 45°C) Drug substances 150 82％ Protein fraction 75 92％ Buffer fraction 25 98％

[0301] Example 4. Hydrophobic Interaction Chromatography

[0302] The antibodies were produced in a CHO cell host and purified using one of two methods (see Table 9), as described in U.S. Pat. No. 10,342,876, which is incorporated herein by reference. In one case, an ion exchanger was used as a polishing step (capture step, ion exchange 1, ion exchange 2; "Method 3") to purify the antibodies. In another case, one of the polishing steps for purifying the antibodies was hydrophobic interaction chromatography, which may be used as an additional polishing step (capture step, ion exchange, hydrophobic interaction; "Method 6"). The antibodies purified by Method 3 or Method 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. At day 14, approximately 98% of the sorbitan laurate (i.e., intact ester) remained in the formulation containing the antibody purified using Method 6, whereas only approximately 28% of the sorbitan laurate remained in the formulation containing the antibody purified using Method 3. Thus, the HIC step may have removed activities that lead to polysorbate degradation.

[0303] Table 9: Polysorbate recovery using various antibody purification methods

[0304] Method No. Purification steps Full PS20% 1 Protein A Affinity Capture (PA) 54％ 2 PA>Cation Exchange (CEX) 25％ 3 PA>CEX>Anion Exchange (AEX) 86％ 4 PA>CEX>Hydrophobic Interaction (HIC) 90％ 5 PA>AEX 83％ 6 PA>AEX>HIC 92％

[0305] The effect of batch process steps in removing putative polysorbate degradation factors (putative esterase activity) was evaluated. Antibodies produced by CHO cells were subjected to successive purification steps, and the stability of polysorbate 20 was evaluated at each step. The results of a set of experiments are presented in Table 9, which reports the percentage of intact polysorbate 20 in each step or sequence of steps. The percentage of intact polysorbate 20 is predicted to be inversely proportional to the amount of contaminant esterase activity.

[0306] A number of different antibodies were tested for their associated polysorbate degradation activity (esterase) and the effect of HIC on said activity. In each case, polysorbate 20 degradation activity was detected and was nearly eliminated by incorporating a HIC purification step (Table 10).

[0307] Table 10: Polysorbate degradation with and without HIC purification

[0308]

[0309]

[0310] The role of HIC in the formation of subvisible particles was explored. Without intending to be bound by theory, it is hypothesized that the stability of nonionic detergents in protein (e.g., antibody) formulations is directly related to the formation of subvisible particles. The loss of surfactant activity can allow proteins to aggregate and form subvisible particles. Additionally or alternatively, fatty acids released by degraded sorbitan fatty acid esters may also contribute to the formation of subvisible particles in the form of immiscible fatty acid droplets. Therefore, the levels of subvisible particles with a diameter of ≥10 microns 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) produced using HIC (e.g., Method 6) or without HIC (e.g., Method 3) were counted. The results (presented in Tables 11 and 12) show that even though lower quality PS20-A was used in these experiments, the application of the HIC step significantly reduced the formation of SVPs in the drug substance (approximately one-tenth).

[0311] Table 11: Number of particles ≥ 10 μm with and without HIC purification

[0312]

[0313] Table 12: Number of particles ≥ 25 μm with and without HIC purification

[0314]

[0315] To further characterize particle formation over time, mAbl preparations containing either PS20 or PS80 and produced by HIC were Fig. 6A , 6B , 6C and 6D. Formulations containing PS20 or PS80 did not show an increase in particle formation over time as measured by membrane microscopy or MFI.

[0316] We further evaluated the degradation of polysorbate over time in mAb1 formulations produced by HIC. Fig. 7A and 7B Consistent with the SVP results above, the formulations subjected to the HIC method showed no significant polysorbate degradation even over 36 months of storage.

[0317] Example 5. Putative phospholipase B-like 2 activity

[0318] During HIC purification of an exemplary antibody produced in CHO cell culture, polysorbate degradation activity was tracked as described in U.S. Pat. No. 10,342,876, which is incorporated herein by reference. A partially purified CHO cell extract was applied to HIC (phenyl-agarose gel). The flow-through containing almost all of the antibody was collected and analyzed for polysorbate degradation activity. No polysorbate degradation activity was observed in the 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 stripped fraction contained 9.9% polysorbate degradation activity, the filtered permeate contained 1.3% polysorbate degradation activity and 5% antibody yield, and the filtered retentate contained 7.4% polysorbate degradation activity and 95% antibody yield.

[0319] Table 13: Percent reduction in polysorbate 20 degradation achieved by lipase inhibitor concentration

[0320] Lipase inhibitors 0mM 0.001mM 0.01mM 0.1mM Orlistat 0％ 0％ 27.8％ 67％ Diethylumbelliferone phosphate 0％ 48％ 100％ >95％ URB602 0％ 0％ 20％ 0％ 2-Butoxyphenylboronic acid 0％ 0％ 28％ 0％

[0321] Whether the polysorbate degradation activity is derived from lipase was tested by combining a lipase inhibitor with the polysorbate degradation activity fraction spiked with polysorbate 20. The data presented in Table 13 show that the polysorbate degradation activity was reduced due to the action of the lipase inhibitor relative to the control group (antibody with relevant polysorbate degradation activity plus polysorbate 20 without a lipase inhibitor). The lipase inhibitor reduced or eliminated the polysorbate degradation activity associated with the antibody.

[0322] The HIC stripping fractions (not the flow-through) of the recombinant antibody produced by CHO containing polysorbate degradation activity were subjected to additional HIC in the binding / elution mode, during which the antibody was eluted by means of a shallow gradient elution. The PS20 degradation activity of the eluted fractions was tested, and those fractions with the activity were subjected to (i) complete mass spectrometry analysis, (ii) native size exclusion chromatography UV analysis (SEC-UV) and (iii) trypsin digestion, followed by LC-MS and proteomics research analysis. Complete mass spectrometry analysis of the reversed phase liquid chromatography fractions revealed the presence of unknown species in the hydrophobic fraction L8 (the most hydrophobic fraction). The formulated antibody sample containing polysorbate 20 and spiked with L8 (1:100) showed 20% polysorbate degradation on the eighth day. Antibody monomers and free light chains were detected in the less hydrophobic fractions L3-L7 and L8. Antibody dimers were detected in the fractions L5-L8.

[0323] HIC stripping fractions L3-L9 were subjected to SEC-UV under native conditions. Fraction L8 was divided into three major peaks that appeared first and two minor peaks that appeared later, representing smaller species. The first peak on the column contained antibody dimers and other oligomers. The second peak contained antibody monomers. The third peak contained substances with polysorbate degradation activity. Therefore, the degradation activity can be separated from the antibody, and the molecular rotation of the degradation activity is less than that of the antibody monomer.

[0324] HIC fraction L8 was also subjected to shotgun proteomics analysis. Briefly, the L8 fraction was sequentially subjected to the following operations: (i) retained on a 10 kDa filter, (ii) reconstituted in 6 M guanidine hydrochloride, 100 mM Tris-HCl pH 7.5, (iii) treated in 10 mM tris(2-carboxyethyl)phosphine hydrochloride) (TCEP) for 30 minutes at 50°C, followed by treatment in 20 mM indole-3-acetic acid (IAA) at room temperature in the dark for 30 minutes, (iv) diluted eightfold, trypsin was added at a ratio of 1 part trypsin to 20 parts sample, and incubated at 37°C for four hours, and then (v) subjected to LC-MS / MS analysis. Proteomic searches of the resulting peptide sequences revealed five proteins associated with L8: (i) putative phospholipase B-like 2 (accounting for 15% of this peak fraction), (ii) peroxiredoxin-1, (iii) heat shock 27 kDa protein 1, (iv) anaphase-promoting complex subunit 1, and (v) U3 small nuclear ribonucleoprotein MPP10.

[0325] The amount of polysorbate degradation activity is related to the abundance of the phospholipase B-like 2 protein (PLBL2) present. The amount of PLBL2 is determined by nanoLC-MS or LC-MS at various purification steps, and the polysorbate degradation rate (PS20 spiked fraction) is determined. The abundance of PLBL2 is calculated based on the ratio of peptide intensities from lipase and drug substance (i.e., antibody). Figure 8 The results are presented in Table 14.

[0326] Table 14: Correlation between PLBL2 and polysorbate degradation

[0327] Fraction PS20 degradation% <![CDATA[Relative amount PLBL2 (ppm) 2 > ProA Pool 69.02％ 991 CEX Pool 55.24％ 403 AEX Pool 12.85％ 84 HIC Pool 8.67％ 0 HIC Pool 2 10.96％ 0 HIC peeling 83.75％ 1384 mAb1 Method 3 (Example 4) 4.60％ 0 <![CDATA[mAb1 Method 6 (Example 4) 1 > 29.12％ 92

[0328] 1 The degradation rate is adjusted according to the concentration.

[0329] 2 Phospholipase abundance was calculated based on the ratio of peptide intensities from lipase and drug substance.

[0330] Example 6. Reduction of lipase activity using AEX

[0331] The ability of an anion exchange (AEX) chromatography unit operation to reduce lipase levels in a pharmaceutical formulation was investigated. AEX was performed in flow-through mode, where negatively charged impurities are adsorbed to an immobilized positively charged ligand (chromatographic column) and the product flows through. A stepwise regression approach was used to generate transfer functions for the process parameters considered most likely to affect lipase activity. Fig. 9 A visualization of the resulting model is shown in . The model enables the rational selection of set points and ranges to maximize process desirability and robustness by using an optimization algorithm. Surprisingly, lipase activity was negatively correlated with AEX loading pH, providing a new approach to minimize lipase activity by optimizing AEX chromatographic conditions.

[0332] The performance of the optimized AEX process was verified in four pilot scale (500 L) validation batches as shown in Table 15. Lipase activity was measured by stressing the concentrated samples at 45°C for 44 hours and measuring the changes in non-esterified fatty acids (NEFA) after 0.8% w / v polysorbate 20 spiking. Fig.10As shown, the anion exchange process performance in these batches was compared to small-scale model predictions derived from Monte Carlo simulations of the process operating at set points with estimated variations in input parameters. The validation batch responses are represented by solid orange lines. All responses were within the prediction range generated by the scaled-down multivariate model for anti-IL-4R antibodies produced using the methods of the invention, demonstrating the robustness of the process to scale-up and the suitability of the small-scale model for predicting pilot-scale performance.

[0333] Table 15: Overview of the 500 L scale performance of the anion exchange step during the process validation batches

[0334]

[0335] SD, standard deviation

[0336] Example 7. Optimizing fatty acid composition to reduce particle formation

[0337] The protein of interest may present additional challenges for lipase removal based on its structural and physicochemical properties. Although mAb5 was formulated following typical HCP removal techniques, it still showed degradation of polysorbates over time during storage, such as Fig.11 and Fig.12 Homology modeling demonstrated that mAb5 is characterized by a large hydrophobic patch compared to mAb6 and mAb7, as shown in Fig.13 As shown, this may cause the co-elution of mAb5 with the hydrophobic lipase. Therefore, additional approaches for reducing polysorbate degradation and SVP formation were investigated.

[0338] The percentage distribution of fatty acid composition and high melting point components is a useful consideration for predicting particle formation. Free fatty acids with high melting points may form insoluble particles that can be detected at room temperature and during analytical evaluation. Table 16 shows the composition of fatty acids in different polysorbates and their corresponding melting points. Commercially available polysorbates, whose fatty acid composition can have a wide range of melting points, wherein PS80 is characterized by a higher concentration of low melting point fatty acids, particularly oleic acid.

[0339] Table 16: Fatty acid composition of commercially available polysorbates

[0340]

[0341] The storage stability of mAb1 drug products (DP) prepared without the HIC step was evaluated in samples including different grades of PS80, as described in U.S. Patent Application Publication No. 20190083618A1, which is incorporated herein by reference. The volume of each DP sample was 2.136 mL, containing one of several batches of PS80 at the same concentration of mAb1 (150 mg / mL) and 0.2% (w / v). Each PS80 batch had one of three different oleate percentage contents (70%, 87%, and ≥99%). Table 17 summarizes the percentage content of oleate in PS80 in each FDS sample.

[0342] Table 17:

[0343] sample Oleate content in PS80 (batch) % DP A 87％ DP B ≥99%(Batch 1) DP C ≥99%(Batch 2) DP D 70% (Batch 1) DPE 70% (Batch 2) DP F 70% (Batch 3)

[0344] The DP samples were stored in glass pre-filled syringes at 2-8° C. for up to 24 months. The particles in each DP sample were measured every six months for a total of 24 months by both membrane microscopy methods and microflow imaging (MFI).

[0345] Fig.14 Shown are the numbers of SVPs with diameter ≥10 μm per vessel as measured by membrane microscopy. Fig.15 The number of SVPs with a diameter ≥ 10 μm per vessel as measured by MFI is shown in a graph. Fig.14 and Fig.15 As shown, DP B and DP C (the two DP samples containing PS80 with ≥99% oleate content) showed the lowest number of SVPs across the entire 24-month period, as determined by membrane microscopy ( Fig.14 ) and MFI( Fig.15 ) measured by both methods. DP A containing PS80 having an oleate content of 87% showed the next lowest number of subvisible particles over the 24 month period (specifically, showing 800 to 1200 particles by 24 months). DP D, DP E, and DP F all showed well over 3000 particles per container (as measured by both methods) by at least the 18 month mark.

[0346] It is assumed that the lower number of particles in DP A, DP B and DP C (relative to the higher number of particles in DP D, DP E and DP F) is the result of using PS80 with a higher percentage of oleic acid (or long chain fatty acid) ester content. Oleic acid is a long chain fatty acid with one unsaturated bond (see Fig.16). Therefore, the melting point of the oleic acid is below room temperature, at about 13°C. The precursor to the formation of subvisible and visible free fatty acid (FFA) particles is the aggregation of individual FFA chains into aggregates, which then precipitate out as particles. During storage of the formulation at 5°C, oleic acid can be produced by enzymatic hydrolysis of fatty acid esters in, for example, polysorbate 80. This oleic acid can form SVPs, but due to its low melting point, such particles are more likely to exist in the protein formulation as an oily liquid at room temperature (about 22°C) where the analysis is performed, and therefore will not persist as subvisible particles at room temperature. In contrast, higher levels of non-oleate components in the formulation will result in the formation of their corresponding FFAs upon hydrolysis, and due to their higher melting points, the subvisible and visible amorphous particles formed thereby will persist at ambient temperatures during the analysis.

[0347] Additionally, due to the higher hydrophobicity of oleic acid esters, oleic acid esters are better solubilizers / stabilizers than esters of shorter chain fatty acids, which enables oleic acid esters to solubilize free fatty acids and protein particles, thereby maintaining product stability. Therefore, polysorbate 80 with a higher oleic acid content (>98%) can provide better stability for protein formulations and drug products compared to polysorbate 80 with a lower oleic acid content.

[0348] After storing the samples at 5°C for 18 months, the concentration of each type of free fatty acid in each sample DP (DP AF) was evaluated (in micrograms / mL). Samples DP AF were prepared as described above. At 18 months, the free fatty acid concentration was measured by LC-MS, as shown in Table 1. Fig.17 As shown. DP B and DP C (two DP samples containing PS80 with ≥99% oleate content) showed the highest oleic acid concentration and the lowest other FFA concentrations. This indicates the uniformity of FFA (i.e., oleic acid) in DP B and DP C. This further indicates that the use of polysorbates including high oleic acid concentrations can reduce the formation of free fatty acid particles at ambient temperature even in formulations containing lipases that cause free fatty acid production.

[0349] Example 8. Prevention of particle formation in lipase-containing formulations with PEG 3350 or Poloxamer 188

[0350] Esterases or lipases can hydrolyze polysorbates by enzymatic hydrolysis of ester bonds, e.g. Fig.18A Alternative surfactants such as PEG3350 and poloxamer 188 do not contain ester bonds and are therefore not targets for esterases, such as Fig.18B mAb5 was formulated with PS20, PS80 or poloxamer 188, and the recovery of the surfactants was compared. Fig.19Unlike the PS20 and PS80 formulations, the poloxamer formulations did not suffer a loss in recovery at all temperatures tested.

[0351] To investigate whether the use of alternative surfactants could reduce SVP formation in formulated DP, formulations of IL-4R antibodies were prepared using alternative surfactants as described in U.S. Provisional Application No. 63 / 337,532, which is incorporated herein by reference, and particle formation was measured over time. Anti-IL-4R antibodies comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 1 / 2 were formulated at a concentration of 150 mg / mL with 20 mM histidine, 12.5 mM sodium acetate, 25 mM arginine-HCl, 5% w / v sucrose, and varying concentrations of PEG3350 or poloxamer 188 at pH 5.9. Formulations were stored in syringes at 5°C for up to 36 months, and the number of particles (≥10 μm and ≥25 μm) present in the formulations was measured periodically by microscopy. Fig. 20 As shown, very few particles ≥10 μm or ≥25 μm were identified in the formulations over the 36-month observation period. In addition, no significant differences or changes in the number of subvisible particles were observed in the different formulations containing PEG 3350 or poloxamer 188 during storage.

[0352] These results suggest that alternative surfactants like 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 long storage periods.

[0353] To evaluate the ability of alternative surfactants to promote therapeutic protein stability, formulations containing PEG3350 or poloxamer 188 were further subjected to assessments of stirring stress stability and thermal stress stability. Stirring stress stability of IL-4R antibody formulations containing different concentrations of surfactant PEG3350 or poloxamer 188 were tested. Anti-IL-4R antibodies comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 1 / 2 were formulated at a concentration of 150 mg / mL with 20 mM histidine, 12.5 mM sodium acetate, 25 mM arginine-HCl, 5% w / v sucrose and different concentrations of PEG3350 or poloxamer 188 at pH 5.9. The formulations were stored in glass vials and stirred by vortexing (speed setting 4) for 30 minutes, 60 minutes, or 120 minutes. The percentage of high molecular weight (HMW) species was then determined by size exclusion ultra-performance liquid chromatography (SE-UPLC).

[0354] like Fig.21AAs shown, formulations of antibodies with at least 0.01% (w / v) PEG3350 can prevent the observed increase in HMW species due to stirring (quantified by SE-UHPLC). Lower amounts of PEG3350 (0.001% or 0.005%) were not sufficient to prevent the formation of HMW. Similarly, Fig.21B As shown, while formulations of the antibody with at least 0.01% (w / v) poloxamer 188 prevented the observable increase in HMW species, HMW species were observed at lower amounts of poloxamer 188 (0.001% or 0.005%).

[0355] Additionally, IL-4R antibody formulations containing PEG3350 or poloxamer 188 were tested for thermal stress stability and compared to various IL-4R antibody formulations containing polysorbates. Anti-IL-4R antibodies comprising the HCVR / LCVR amino acid sequence pair of SEQ ID NO: 1 / 2 were formulated at a concentration of 150 mg / mL with 20 mM histidine, 12.5 mM sodium acetate, 25 mM arginine-HCl, 5% w / v sucrose and varying concentrations of polysorbate 20, polysorbate 80, PEG3350 or poloxamer 188 at pH 5.9. These formulations were subjected to thermal stress (45°C) for a period of 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.

[0356] like Fig. 22 As shown, antibody formulations containing PEG3350 or poloxamer 188 at a concentration of 0.01% or 0.02% showed similar thermal stability to antibody formulations containing lower amounts of polysorbate (up to 0.1% w / v). By 28 days, formulations containing PEG3350 or poloxamer 188 showed a lower percentage of HMW species than the control formulation containing 0.2% polysorbate 20.

[0357] Additional studies were conducted comparing 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). At pH 5.9, 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 a surfactant. Formulations containing poloxamer 188 or PEG3350 showed a stability advantage over formulations containing polysorbate 80.

[0358] Table 18: Thermal stability of formulations with 0.2% PS80

[0359]

[0360]

[0361] Table 19: Thermal stability of formulations with 0.01% Poloxamer 188

[0362]

[0363] Table 20: Thermal stability of formulations with 0.1% Poloxamer 188

[0364]

[0365]

[0366] Table 21: Thermal stability of formulations with 0.5% Poloxamer 188

[0367]

[0368] Table 22: Thermal stability of formulations with 0.01% PEG3350

[0369]

[0370]

[0371] Table 23: Thermal stability of formulations with 0.1% PEG3350

[0372]

[0373] Table 24: Thermal stability of formulations with 0.5% PEG3350

[0374]

[0375] CEX, cation exchange; FCP, final concentration pool; HMW, high molecular weight; LMW, low molecular weight; MFI, microfluidic imaging; NR, not required; OD, optical density; RH, relative humidity; RP, reversed phase; SE, size exclusion; UPLC, ultra-performance liquid chromatography

[0376] Example 9. Reduction of lipase activity using agitation stress and heat stress

[0377] 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 was surprisingly found that subjecting the drug substance to stress conditions (such as agitation stress or thermal stress) can be used to reduce esterase and lipase activity and improve the long-term stability of the therapeutic molecule as well as the surfactant in the drug substance and subsequent drug product.

[0378] The mAb1 drug substance produced in the absence of the HIC step is subjected to stirring stress. The 200mg / mL mAb1 DS of 30mL is used. Two 125mL polycarbonate bottles are each filled with 10mL DS, and stirred at 250rpm for 0 hour, 24 hours or 48 hours on an orbital shaker. The remaining 10mL DS is transferred to a 15mL Falcon tube and used as a control without stress. Every kind of DS is taken to 2mL and analyzed as (control) DS (stirring 0 hour) before or after stress (stirring 24 or 48 hours).

[0379] At pH 5.9, 150 mg / mL final drug substance (FDS) was prepared using control or stressed DS, which contained 150 mg / mL mAb1, 20 mM histidine, 12.5 mM acetate, 5% sucrose, 0.2% high purity PS20 and 25 mM arginine HCl. FDS was sterilized by filtration and then stored at 45°C for 0, 4 or 8 weeks. In order to accelerate stability studies, storage at 45°C was selected. The physical stability of each sample collected above was analyzed using visual inspection for visible particles and aggregates, SE-UPLC for high and low molecular weight materials, microfluidic imaging for sub-visible particles (2-300 μm) analysis, and CAD-UPLC for determining PS20 levels.

[0380] As shown in Table 25, the mAbl drug substance subjected to the agitation stress and filtration steps showed less particle formation and more polysorbate retention over time.

[0381] Table 25: Thermal stability of formulations subjected to stirring stress

[0382]

[0383]

[0384] a MFI USP specifications: size ≥10 μm, ≤3000 particles / container; size ≥25 μm, ≤300 particles / container CAD, charged aerosol detector; CEX, cation exchange; FCP, final concentration pool; HMW, high molecular weight; LMW, low molecular weight; MFI, microflow imaging; NR, not required; OD, optical density; RH, relative humidity; RP, reversed phase; SE, size exclusion; UPLC, ultra-performance liquid chromatography.

[0385] Additional forms of stress were also studied. The mAb1 drug substance produced without the HIC step was subjected to thermal stress. The DS was stored at 45°C for 0, 0.5 or 1 month to degrade, aggregate and / or inactivate the lipase. At pH 5.9, a 150 mg / mL FDS was prepared using controls and stressed DS, which contained 150 mg / mL mAb1, 20 mM histidine, 12.5 mM acetate, 5% sucrose, 0.2% high purity polysorbate 20 and 25 mM arginine HCl. The FDS was sterilized by filtration and then stored under the conditions described in Table 26.

[0386] Table 26: Incubation / storage conditions of FDS from DS subjected to heat stress

[0387] Stress conditions Incubation time No storage t＝0 45℃ 0.5 months, 1 month, 1.5 months, 2 months 25℃ 3 months, 6 months 5℃ 3 months, 6 months, 12 months, 18 months, 24 months, 36 months and 3 backups

[0388] The physical stability of the above-mentioned samples subjected to thermal stress and control unstressed samples was analyzed using visual inspection for visible particles and aggregates, SE-UPLC for high and low molecular weight materials, microfluidic imaging for sub-visible particles (2-300 μm) analysis, and CAD-UPLC for determining PS20 levels. The results are described in the following tables 27-32. Subjecting DS to thermal stress before deployment significantly reduced lipase activity, thereby improving polysorbate recovery and reducing free fatty acid particle formation. The samples that were not subjected to stress showed a significant increase in sub-visible particles over time, which was not observed in the samples subjected to stress. Subjecting DS to thermal stress did significantly increase HMW levels, but the levels remained stable during further incubation.

[0389] Table 27: Stability of formulations from DS under 0 month heat stress

[0390]

[0391]

[0392] Table 28: Stability of formulations from DS under 0.5 month heat stress

[0393]

[0394] Table 29: Stability of formulations from DS under 1 month heat stress

[0395]

[0396]

[0397] Table 30: Stability of formulations from DS under 0 month heat stress

[0398]

[0399] Table 31: Stability of formulations from DS under 0.5 month heat stress

[0400]

[0401]

[0402] Table 32: Stability of formulations from DS under 1 month heat stress

[0403]

[0404] Compared to biotherapeutics (e.g., therapeutic antibodies), HCPs are expected to be disproportionately inactivated, degraded, and aggregated in response to stress due to reduced stability. HMW species in DS subjected to agitation stress or thermal stress, whether formed by HCPs, drug proteins, or a combination, can be removed using additional processing steps such as filtration or chromatography. DS subjected to thermal stress was subjected to cation exchange (CEX) chromatography to remove HMW species formed during stress conditions. As shown in Table 33, HMW species were effectively removed from DS subjected to stress.

[0405] Table 33: Clearance of HMW Species from Stressed Drug Substances

[0406]

[0407]

[0408] At pH 5.9, the DS having HMW removed was formulated into FDS, which contained 150mg / mL mAb1, 20mM histidine, 12.5mM acetate, 5% sucrose, 0.2% PS80 and 25mM arginine HCl. As shown in Table 34, the stability of the FDS from the DS subjected to heat stress having HMW removed was compared with the stability of the FDS from the DS not subjected to stress having HMW removed. After only one week (0.25 months), compared with the DS not subjected to stress having HMW removed, the FDS from the DS not subjected to stress having HMW removed showed a greatly improved PS80 recovery rate. Further improvement can be measured at a later time point; it is expected that free fatty acid particles can be formed measurably by the free fatty acids accumulated after the PS80 degradation in the DS not subjected to stress having HMW removed. It should be noted that the difference in lipase activity between stressed and unstressed DS after removal of HMW species may also be masked by the removal of HCP lipase in the unstressed DG due to the chromatography step. These results indicate that subjecting the drug substance to agitation stress or thermal stress (optionally followed by removal of HMW species as disclosed) results in improved pharmaceutical compositions having the advantages of reduced lipase activity, reduced polysorbate degradation, and reduced free fatty acid particle formation.

[0409] Table 34: Thermal stability of formulations from heat stressed DS with HMW removed

[0410]

[0411] The present invention is not limited to the scope of the specific aspects described herein. In fact, various modifications of the present invention, in addition to the modifications 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.

[0412] List the implementation methods

[0413] The following enumerated embodiments set forth below provide additional aspects of the present disclosure.

[0414] 1. A method for producing a pharmaceutical composition having reduced lipase, the method comprising:

[0415] A sample comprising the protein of interest and lipase is subjected to anion exchange chromatography, wherein the pH of the sample loaded onto the chromatography column is between about 7.8 and about 8.3.

[0416] 2. A method for producing a pharmaceutical composition having reduced lipase activity, the method comprising:

[0417] (a) subjecting a sample comprising a protein of interest and a lipase to a stress condition to form a sample having an inactivated lipase; and

[0418] (b) formulating the sample having inactivated lipase to produce a pharmaceutical composition having reduced lipase activity.

[0419] 3. The method according to embodiment 2, wherein the 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.

[0420] 4. The method according to embodiment 2 or 3, wherein the preparing 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.

[0421] 5. The method according to embodiment 4, wherein the fatty acid ester is polysorbate 80, and the concentration of oleate in the polysorbate 80 is at least 80%.

[0422] 6. The method of example 5, wherein the concentration of oleate in the polysorbate 80 is at least 98% or at least 99%.

[0423] 7. The method according to any one of embodiments 2 to 6, wherein the stress conditions include stirring stress and / or thermal stress.

[0424] 8. A method according to embodiment 7, wherein the stirring stress comprises shaking the sample at 50 to 500 rpm, 200 to 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.

[0425] 9. A method according to embodiment 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.

[0426] 10. A method according to embodiment 7, wherein the thermal 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.

[0427] 11. A method according to embodiment 10, wherein the storage is 1 day to 6 months, 3 days to 3 months, 1 week to 2 months, 0.5 month to 1 month, about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, about 0.5 month, 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.

[0428] 12. The method according to any one of embodiments 2 to 11, further comprising, before step (b), subjecting the sample with inactivated lipase to filtration, enrichment or chromatography to remove high molecular weight (HMW) species.

[0429] 13. A method according to embodiment 12, wherein the chromatographic separation comprises cation exchange chromatography.

[0430] 14. A method according to embodiment 12, wherein the chromatographic separation comprises size exclusion chromatography.

[0431] 15. The method of any one of embodiments 2 to 14, wherein the formulating step comprises adding an excipient to the sample.

[0432] 16. A method for producing a pharmaceutical composition having reduced lipase activity:

[0433] (a) subjecting the collected antibodies to affinity chromatography;

[0434] (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;

[0435] (c) subjecting the antibodies pooled from step (b) to anion exchange chromatography in flow-through mode;

[0436] (d) subjecting the pooled antibodies from the eluate of step (c) to hydrophobic interaction chromatography in flow-through mode;

[0437] (e) subjecting the antibodies pooled from the flow-through fraction of step (d) to virus-retaining filtration;

[0438] (f) subjecting the sample from step (e) comprising the antibody of interest and the lipase to agitation stress or heat stress to form a pharmaceutical composition having reduced lipase activity.

[0439] 17. A method for producing a pharmaceutical composition having reduced lipase activity:

[0440] (a) subjecting the collected antibodies to affinity chromatography;

[0441] (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;

[0442] (c) subjecting the antibodies pooled from step (b) to anion exchange chromatography in flow-through mode;

[0443] (d) subjecting the antibodies pooled from the flow-through fraction of step (c) to virus-retaining filtration;

[0444] (e) subjecting the sample from step (d) comprising the antibody of interest and the lipase to agitation stress or heat stress to form a pharmaceutical composition having reduced lipase activity.

[0445] 18. The method of embodiment 16 or 17, further comprising subjecting the pharmaceutical composition to filtration, enrichment or chromatography to remove HMW species.

[0446] 19. The method of embodiment 18, wherein the chromatographic separation comprises ion exchange chromatography, cation exchange chromatography, or size exclusion chromatography.

[0447] 20. The method of any one of embodiments 16 to 19, wherein the pH of the sample loaded onto the AEX column is between about 7.8 and about 8.3.

[0448] 21. A method for producing a pharmaceutical composition with increased stability, the method comprising:

[0449] The lipase activity in the composition is reduced by subjecting a sample comprising the protein of interest and the lipase to anion exchange (AEX) chromatography, wherein the pH of the sample loaded onto the AEX column is between about 7.8 and about 8.3.

[0450] 22. The method according to embodiment 21, further comprising the step of subjecting the sample formed after AEX chromatography to stirring stress or thermal stress to reduce lipase activity.

[0451] 23. The method of any one of embodiments 16 to 22, further comprising adding poloxamer 188 or PEG3350 to produce a formulation comprising a pharmaceutical composition having reduced lipase activity and increased stability.

[0452] 24. The method of embodiment 23, wherein the formulation is substantially free of polysorbate.

[0453] 25. The method of example 23 or 24, 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%.

[0454] 26. A method according to embodiment 23 or 24, 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%.

[0455] 27. A method for producing a pharmaceutical composition with increased stability, the method comprising:

[0456] (a) subjecting the collected recombinant protein to anion exchange chromatography in flow-through mode;

[0457] (b) subjecting the flow-through fraction from step (a) to hydrophobic interaction chromatography in flow-through mode; and

[0458] (c) formulating the recombinant protein isolated from step (b) with polysorbate 80, wherein the concentration of oleate in the polysorbate 80 is at least 80%.

[0459] 28. The method of embodiment 27, wherein the concentration of oleate is at least 98% or at least 99%.

[0460] 29. A method according to any one of embodiments 1 or 16 to 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 pharmaceutical composition having reduced lipase, the method comprising: A sample comprising the protein of interest and lipase is subjected to anion exchange chromatography, wherein the pH of the sample loaded onto the chromatography column is between about 7.8 and about 8.

3.

2. A method for producing a pharmaceutical composition having reduced lipase activity, the method comprising: (c) subjecting a sample comprising a protein of interest and a lipase to a stress condition to form a sample having an inactivated lipase; as well as (d) formulating the sample having inactivated lipase to produce a pharmaceutical composition having reduced lipase activity.

3. The method of claim 2, wherein the 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 claim 2 or 3, wherein the formulating 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 oleate in the polysorbate 80 is at least 80%.

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

7. The method according to any one of claims 2 to 6, wherein the stress conditions comprise stirring stress and / or thermal stress.

8. The method of claim 7, wherein the stirring stress comprises shaking the sample at 50 to 500 rpm, 200 to 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 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 claim 7, wherein the thermal 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 claim 10, wherein the storage is 1 day to 6 months, 3 days to 3 months, 1 week to 2 months, 0.5 month to 1 month, about 1 day, about 2 days, about 3 days, about 1 week, about 2 weeks, about 0.5 month, 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 according to any one of claims 2 to 11, further comprising, before step (b), subjecting the sample with inactivated lipase to filtration, enrichment or chromatography to remove high molecular weight (HMW) species.

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

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

15. The method according to any one of claims 2 to 14, wherein the formulating step comprises adding an excipient to the sample.

16. A method for producing a pharmaceutical composition having reduced lipase activity: (g) subjecting the collected antibodies to affinity chromatography; (h) 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; (i) subjecting the pooled antibodies from step (b) to anion exchange chromatography in flow-through mode; (j) subjecting the pooled antibodies from the eluate of step (c) to hydrophobic interaction chromatography in flow-through mode; (k) subjecting the antibodies pooled from the flow-through fraction of step (d) to virus-retaining filtration; (1) subjecting the sample from step (e) comprising the antibody of interest and the lipase to agitation stress or heat stress to form a pharmaceutical composition having reduced lipase activity.

17. A method for producing a pharmaceutical composition having reduced lipase activity: (f) subjecting the collected antibodies to affinity chromatography; (g) 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; (h) subjecting the pooled antibodies from step (b) to anion exchange chromatography in flow-through mode; (i) subjecting the antibodies pooled from the flow-through fraction of step (c) to virus-retaining filtration; (j) subjecting the sample from step (d) comprising the antibody of interest and the lipase to agitation stress or heat stress to form a pharmaceutical composition having reduced lipase activity.

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

19. The method of claim 18, wherein the 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 between about 7.8 and about 8.

3.

21. A method for producing a pharmaceutical composition with increased stability, the method comprising: The lipase activity in the composition is reduced by subjecting a sample comprising the protein of interest and the lipase to anion exchange (AEX) chromatography, wherein the pH of the sample loaded onto the AEX column is between about 7.8 and about 8.

3.

22. The method according to 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. The method of any one of claims 16 to 22, further comprising adding 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 formulation is substantially free of polysorbates.

25. The method of claim 23 or 24, 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 or 24, 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, the method comprising: (d) subjecting the collected recombinant protein to anion exchange chromatography in flow-through mode; (e) subjecting the flow-through fraction from step (a) to hydrophobic interaction chromatography in flow-through mode; and (f) formulating the recombinant protein isolated from step (b) with polysorbate 80, wherein the concentration of oleate in the polysorbate 80 is at least 80%.

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

29. The method of any one of claims 1 or 16 to 28, wherein the pH of the sample loaded onto an 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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