WO2025054112A2 - Methods to control polysorbate degradation in biotherapeutic formulations - Google Patents

Abstract

The disclosure provides methods for heat-treating pharmaceutical compositions containing at least one active biological ingredient (e.g., a protein), a serine hydrolase that cleaves polysorbate while in its native conformation, and polysorbate, thereby reducing the activity of the serine hydrolase in the pharmaceutical composition.

Description

METHODS TO CONTROL POLYSORBATE DEGRADATION IN BIOTHERAPEUTIC

FORMULATIONS

CROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63/581,328 filed September 8, 2023, the entire contents of which are incorporated by reference herein.

FIELD

[0002] This disclosure relates generally to methods for removing enzymatic activity from a pharmaceutical composition.

BACKGROUND

[0003] Polysorbates (PS) are the most widely used excipient in biotherapeutic formulations to protect therapeutic proteins from shear, aggregation, and surface adsorption (Ref. 1). Despite widespread use in the pharmaceutical industry', controlling PS degradation remains challenging. PS is a heterogenous mixture of fatty' acylated sorbitan or isosorbide (Ref. 2). In addition, different manufacturers can supply PS products with different levels of characteristic components, residues of process intermediates, degradants, and impurities. Furthermore, PS is susceptible to degradation, which could lead to the formation of visible and/or subvisible particles. These particles may consist of fatty' acids released from PS and/or protein aggregates and can potentially impact product quality. Causes for PS degradation include chemical stress and residual host cell-derived enzymes (Ref. 3). Recent studies have identified several PS- degradative enzymes (PSDE), all of which belong to the serine hydrolase superfamily and are often generically referred to as "lipases" (Ref. 4-13). In one example, a chemical proteomics approach was used to identify phospholipase A2, group VII (PLA2G7) in an ion-exchange column pool that exhibited considerable PS degradation, and the enzymatic activity of PLA2G7 on PS was confirmed using a recombinant protein (Ref. 8).

[0004] Host cell protein (HCP)-derived polysorbate degradation is an ongoing and growing challenge in the pharmaceutical industry as more commercial formulations move towards subcutaneous dosing. Subcutaneous dosing requires monoclonal antibody (mAb) formulations to be dosed at higher concentrations due to loss of absorption of drug product (DP) through subcutaneous layers. The required increase in mAb concentration subsequently is accompanied with increased HCP levels needed to be removed in the DP. The competitive market advantage of offering subcutaneous dosing versus l.V. dosing is enormous but as mentioned comes with additional challenges in titer, purification, and stability of DP.

[0005] Currently, the control strategy⁷ for polysorbate degrading enzymes remains heavily reliant on downstream process development aimed at the removal of such proteins through filtration and column purification (Ref. 2, 14). However, such techniques are not always successful. There is a need for other methods of controlling serine hydrolase enzymes to minimize their effect on degrading PS and increasing the frequency of high molecular weight particles in drug product.

SUMMARY

[0006] In one aspect, the present disclosure provides a method for reducing serine hydrolase activity in a pharmaceutical composition comprising: a) incubating the pharmaceutical composition at a temperature of from about 40°C to about 70°C; wherein the pharmaceutical composition comprises i) an active biological ingredient, ii) a serine hydrolase that cleaves polysorbate while in its native conformation, and iii) polysorbate; wherein the serine hydrolase is irreversibly misfolded after the incubating step; and wherein the active biological ingredient in the pharmaceutical composition is not irreversibly misfolded after the incubating step.

[0007] In some embodiments, the serine hydrolase is Phospholipase A2 Group VII (PLA2G7) or Sialic Acid Acetylesterase (SIAE).

[0008] In some embodiments, the active biological ingredient has a melting temperature that is about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, or about 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning fluorimetry (DSF). In some embodiments, the active biological ingredient has a melting temperature that is 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning fluorimetry’ (DSF).

[0009] In some embodiments, the active biological ingredient has a melting temperature that is about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, or about 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning calorimetry⁷ (DSC). In some embodiments, the active biological ingredient has a melting temperature that is 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C above the melting temperature of PLA2G7, S1AE. or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning calorimetry (DSC).

[0010] In some embodiments, the step of incubating the pharmaceutical composition is performed at a temperature from about 45°C to about 55°C. from about 47°C to about 53°C, or at about 50°C. In some embodiments, the step of incubating the pharmaceutical composition is performed at a temperature from 45°C to 55°C, from 47°C to 53°C, or at 50°C. In some embodiments, the step of incubating the pharmaceutical composition is performed at a temperature from about 45°C to about 65°C, from about 50°C to about 60°C, from about 53°C to about 57°C, or at about 55°C. In some embodiments, the step of incubating the pharmaceutical composition is performed at a temperature from 45°C to 65°C, from 50°C to 60°C, from 53°C to about 57°C, or at 55°C. In some embodiments, incubating the pharmaceutical composition is performed at a temperature from about 50°C to about 67°C, from about 55°C to about 65°C, from to 57°C to about 63°C, or at about 60°C. In some embodiments, incubating the pharmaceutical composition is performed at a temperature from 50°C to 67°C. from 55°C to 65°C, from 57°C to 63°C, or at 60°C.

[0011] In some embodiments, incubating the pharmaceutical composition is performed from about 1 hour to about 20 hours, from about 2 hours to about 20 hours, from about 3 hours to about 20 hours, from about 4 hours to about 20 hours, from about 5 hours to about 20 hours, from about 6 hours to about 20 hours, from about 7 hours to about 20 hours, from about 8 hours to about 20 hours, from about 9 hours to about 20 hours, from about 10 hours to about 20 hours, from about 12 hours to about 20 hours, from about 15 hours to about 20 hours, or from about 17 hours to about 20 hours. In some embodiments, incubating the pharmaceutical composition is performed from 1 to 20 hours, from 2 hours to 20 hours, from 3 hours to 20 hours, from 4 hours to 20 hours, from 5 hours to 20 hours, from 6 hours to 20 hours, from 7 hours to 20 hours, from 8 hours to 20 hours, from 9 hours to 20 hours, from 10 hours to 20 hours, from 12 hours to 20 hours, from 15 hours to 20 hours, or from 17 hours to 20 hours. In some embodiments. incubating the pharmaceutical composition is performed for at least about 5 minutes, at least about 7 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 7 hours, at least about 10 hours, at least about 15 hours, or at least about 20 hours. In some embodiments, incubating the pharmaceutical composition is performed for at least 5 minutes, at least 7 minutes, at least 10 minutes, at least 12 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 7 hours, at least 10 hours, at least 15 hours, or at least 20 hours. In some embodiments, incubating the pharmaceutical composition is performed for about 5 minutes, about 7 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 7 hours, about 10 hours, about 15 hours, or about 20 hours. In some embodiments, incubating the pharmaceutical composition is performed for 5 minutes, 7 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes, 45 minutes, 50 minutes, 55 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 5 hours, 7 hours, 10 hours, 15 hours, or 20 hours. [0012] In some embodiments, the pharmaceutical composition has a volume of from about 1 ml to about 5 ml, from about 2.5 ml to about 7.5 ml, from about 5 ml to about 10 ml, from about 7.5 ml to about 12.5 ml, from about 10 ml to about 15 ml, from about 12.5 ml to about 17.5 ml, or from about 15 ml to about 20 ml. In some embodiments, the pharmaceutical composition has a volume of from 1 ml to 5 ml, from 2.5 ml to 7.5 ml, from 5 ml to 10 ml, from 7.5 ml to 12.5 ml, from 10 ml to 15 ml. from 12.5 ml to 17.5 ml. or from 15 ml to 20 ml. In some embodiments, the pharmaceutical composition has a volume of about 20 ml or less, about 17 ml or less, about 15 ml or less, about 12 ml or less, about 10 ml or less, about 9 ml or less, about 8 ml or less, about 7 ml or less, about 6 ml or less, about 5 ml or less, about 4 ml or less, about 3 ml or less, about 2 ml or less, or about 1 ml or less. In some embodiments, the pharmaceutical composition has a volume of 20 ml or less. 17 ml or less. 15 ml or less, 12 ml or less, 10 ml or less. 9 ml or less, 8 ml or less, 7 ml or less, 6 ml or less, 5 ml or less, 4 ml or less, 3 ml or less, 2 ml or less, or 1 ml or less.

[0013] In some embodiments, the step of incubating is performed during upstream or downstream processing prior to any filling of the drug product into containers for storage or administration. In some embodiments, the step of incubating is performed post filling of the drug product into containers for storage or administration.

[0014] In some embodiments, the active biological ingredient is selected from the group consisting of: an enzyme; a hormone; a fusion protein, an Fc-containing protein; an immunoconjugate; a cytokine; and an antibody or antigen binding fragment thereof. In some embodiments, the antibody is selected from the group consisting of: a monoclonal antibody; a chimeric antibody; a humanized antibody; a human antibody; and a multispecific antibody . In some embodiments, the multispecific antibody is a bispecific antibody or a trispecific antibody. In some embodiments, the antigen binding fragment thereof is selected from the group consisting of: a Fab fragment; a Fab' fragment; a F(ab')2 fragment; an scFv; a di-scFv; a bi-scFv; a tandem (di, tri) scFv; an Fv; an sdAb; a tri-functional antibody; a BiTE; a diabody; and a triabody.

[0015] In some embodiments, the serine hydrolase is selected from the group consisting of: RB Binding Protein 9 (RBBP9), Palmitoyl-Protein Thioesterase 2 (PPT2), Phospholipase A2 Group VII (PLA2G7; known to cleave polysorbate), Cathepsin A (CTSA), and Acylaminoacyl-Peptide Hydrolase. In some embodiments, the serine hydrolase is selected from the group consisting of: PLA2G7, Sialic Acid Acetylesterase (SIAE), and Phospholipase B Domain Containing 2 (PLBL2).

[0016] In some embodiments, the polysorbate is selected from the group consisting of: polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

[0017] In some embodiments, the disclosure provides a pharmaceutical composition produced by any one of the methods embodied above. In some embodiments, the disclosure provides a pharmaceutical composition obtainable by any one of the methods embodied above.

[0018] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019] FIG. 1 A is a heat map of protein intensities in samples from a monoclonal antibody (mAbl) protein A pool (PAP). The log-transformed protein intensity for each protein in each sample is plotted. N = 2. FIG. IB is a line graph of heat inactivation of PLA2G7 in PAP. The intensity values corresponding to PLA2G7 are extracted from FIG. 1A and plotted against incubation temperature. FIG. 1C is a line graph of the heat inactivation of known polysorbate- degradative enzymes in harvested cell culture fluid (HCCF) for mAbl, including PLA2G7. N = 2.

[0020] FIG. 2A is a line graph that plots the fluorescence of unfolding for mAbl utilizing Spyro Orange extrinsic dye, a melting temperature comparison between extrinsic fluorescence and differential scanning calorimetry (DSC) reported Tms are given. FIG. 2B is a line graph plotting the 2nd derivative of unfolding for PL2GA7, SIAE and PLBL2, superimposed onto the mAbl unfolding profile. FIG. 2C is a line graph plotting the aggregation (diameter in nm) of PL2AG7, S1AE. PLBL2 and mAbl measured by Dynamic Light Scatering (DLS). The 95% confidence band is shaded around the line for each molecule. FIG. 2D is a line graph that fits the normalized DLS unfolding and aggregation profiles of PL2AG7, SIAE, PLBL2 and mAbl. The 95% confidence band is shaded around the line for each.

[0021] FIGs. 3A-3C show PS degradation in PAP over four weeks of incubation at 25. 37, 40 and 50°C. FIG. 3 A is a line graph ploting the relative change of PS concentration in mAbl over four weeks. FIG. 3B is a line graph plotting the relative change of PS concentration of mAb2 over four weeks. FIG. 3C is a line graph ploting the relative change of PS concentration of formulation buffer with no protein over four weeks. N = 1.

[0022] FIG. 4A is a line graph of the decrease in PS-80 between heat inactivated mAbl drug product and control (non-heat inactivated) drug product. FIG. 4B is a bar graph showing sub- visible particle counts for heat inactivated mAbl drug product. FIG. 4C is a line graph of the percentage of high molecular weight species over time for heat inactivated mAbl drug product and control.

DETAILED DESCRIPTION

[0023] The present disclosure describes methods for heat inactivating serine hydrolases to reduce PS degradation and particle formation in drug product.

Definitions

[0024] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0025] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0026] As used herein, the articles “a” and “an” refer to one or to more than one (i.e., to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0027] As used herein, the term “about” refers to plus or minus up to 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides). The term “about”, when modifying the quantify (e.g., mg) of a substance or composition, a parameter of a substance or composition or a parameter used in characterizing a step in a method, or the like, refers to variation in the numerical quantity that can occur. For example, such variation can occur through typical measuring, handling and sampling procedures involved in the preparation, characterization and/or use of the substance or composition, through inadvertent error in these procedures, through differences in the manufacture, source, or purity of the ingredients employed to make or use the compositions or carry out the procedures.

[0028] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of ‘“from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and/or values.

[0029] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s).” “include(s).” ‘‘having,” “has.” “may.” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients/steps and permit the presence of other ingredients/steps. However, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0030] As used herein, the term “pharmaceutical composition” refers to a liquid composition containing an active biological ingredient (e.g., an antibody or antigen binding fragment thereof)), along with one or more additional excipients. Exemplary excipients may include additives, diluents, buffers, sugars, amino acids, chelating agents, surfactants, polyols, bulking agents, stabilizers, lyo-protectants, solubilizers, emulsifiers, salts, adjuvants, tonicity enhancing agents, delivery vehicles, and anti-microbial preservatives. Active biological ingredients may include, but are not limited to: antibodies; antigen binding fragments of antibodies; multi-specific antibodies (e.g., bispecific antibodies); chimeric proteins that are a fusion of a ligand-binding protein and an effector protein (e.g., etanercept); or any other protein or polypeptide used to treat a disease or disorder in a human or animal.

[0031] As used herein, the term “antibody” or “immunoglobulin” as used herein refers to a glycoprotein comprising at least two heavy chains (HCs) and two light chains (LCs) interconnected by disulfide bonds. Each HC is comprised of a heavy chain variable region or domain (VH) and a heavy chain constant region or domain. Each light chain is comprised of an LC variable region or domain (VL) and a LC constant domain. In certain naturally occurring IgG, IgD and IgA antibodies, the heavy chain constant region is comprised of three domains, CHI, CH2 and CH3. In general, the basic antibody structural unit for antibodies is a Y-shaped tetramer comprising two HC/LC pairs (2H). Each tetramer includes two identical pairs of polypeptide chains, each pair having one LC (about 25 kDa) and HC chain (about 50-70 kDa) (H+L). Each HC:LC pair comprises one VH: one VL pair. The one VH:one VL pair may be referred to by the term “Fab’'. Thus, each antibody tetramer comprises two Fabs, one per each arm of the Y-shaped antibody. In certain embodiments, the antibody may include post-translational modifications thereof (e.g., C-terminal Lysine clipping in the heavy chain, conversion of glutamine or glutamic acid to pyroglutamate or pyroglutamic acid) which may occur when recombinantly expressed in host cells (e.g., CHO cells), or during purification/storage.

[0032] As used herein, the term “antigen binding fragment” refers to a polypeptide or polypeptides comprising a fragment of a full-length antibody, which retains the ability to specifically bind to the antigen bound by the full length antibody, and/or to compete with the full length antibody for specifically binding to the antigen. Examples of antigen binding fragments include, but are not limited to: Fab fragments; Fab’ fragments; F(ab’)2 fragments; Fv regions; scFv proteins; and diabodies.

[0033] As used herein, the term “Fab fragment” refers to an antigen binder comprising one antibody light chain and the CHI and Vj-[ of one antibody heavy chain. The heavy chain of a Fab molecule cannot form a disulfide bond with another heavy chain molecule. A Fab fragment can be the product of papain cleavage of an antibody.

[0034] As used herein, the term “Fab¹ fragment” refers to an antigen binder comprising one antibody light chain and a portion or fragment of one antibody heavy chain that contains the Vp| and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond can be formed between the two heavy chains of two Fab' fragments to form a F(ab')2 molecule.

[0035] As used herein, the term “F(ab')2 fragment” refers to an antigen binder comprising two antibody light chains and two heavy chains containing the Vj-[ and the CHI domain up to a region between the CHI and CH2 domains, such that an interchain disulfide bond is formed between the two heavy chains. An F(ab')2 fragment thus is composed of two Fab' fragments that are held together by a disulfide bond between the two heavy chains. An F(ab')2 fragment can be the product of pepsin cleavage of an antibody. [0036] As used herein, the term ‘ v region” refers to an antigen binder comprising the variable regions from both the heavy and light chains of an antibody but lacks the constant regions.

[0037] As used herein, the term “ScFv” or “single-chain variable fragment” refers to a fusion protein comprising a V|q and Vp fused or linked together by a short linker peptide of ten to about 25 amino acids. The linker is usually rich in glycine for flexibility, as well as serine or threonine for solubility, and can either connect the N-terminus of the V [ with the C-terminus of the VL, or vice versa. This protein retains the specificity of the original immunoglobulin, despite removal of the constant regions and the introduction of the linker.

[0038] As used herein, the term “diabody” refers to an antigen binder comprising a small antibody fragment with two antigen-binding regions, which fragments comprise a heavy chain variable domain (V [) connected to a light chain variable domain (VL) in the same polypeptide chain (Vpj-Vp or VL-VJL). By using a linker that is too short to allow pairing between the two domains on the same chain, the domains are forced to pair with the complementarity domains of another chain and create two antigen-binding regions. Diabodies are described more fully in, e.g., EP 404,097; WO 93/11161; and Holliger et al. (1993) Proc. Natl. Acad. Sci. USA 90: 6444-6448. For a review of engineered antibody variants generally see Holliger and Hudson (2005) Nat. Biotechnol. 23: 1126-1136.

[0039] These and other potential constructs are described at Chan & Carter (2010) Nat. Rev. Immunol. 10:301. These antibody fragments are obtained using conventional techniques known to those with skill in the art, and the fragments are screened for utility' in the same manner as are intact antibodies. Antigen-binding fragments can be produced by recombinant DNA techniques, or by enzymatic or chemical cleavage of intact immunoglobulins.

[0040] The term “serine hydrolase” refers to a class of enzymes that contains a serine at the active site which is important for hydrolyzing a particular protein. In some embodiments of the disclosed invention, the serine hydrolase is capable of cleaving polysorbate. In some embodiments, the serine hydrolase is capable of cleaving polysorbate 80 (PS-80). In some embodiments, the serine hydrolase is from a CHO cell or from E. Coli In some embodiments, the serine hydrolase is PLA2G7 or SIAE.

[0041] The term “polysorbate” refers to a heterogenous mixture of fatty acylated sorbitan or isosorbide. Exemplary polysorbates include, but are not limited to, polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate). [0042] As used herein, the term “irreversibly misfolded” or “irreversibly denatured” refers to a protein which has been forced out of its native conformation due to heating beyond its melting temperature and is unable to return to its native conformation due to the kinetic forces preventing the protein from properly refolding to its native conformation. In its misfolded or denatured state, the protein is unable to catalyze enzymatic reactions or bind to other proteins as it would in its native conformation.

EXAMPLES

[0043] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.

Example 1 : Profiling Heat Sensitivity of serine hydrolase activity

[0044] The inventors profiled the heat sensitivity of serine hydrolase activity in a monoclonal antibody (mAbl) Protein A pool (PAP) with the active site-directed chemical reporter FP-biotin, which enables the sensitive detection of active serine hydrolases. The PAP was incubated at various temperatures between 40-75°C for 5 minutes. The samples were subsequently cooled on ice. buffer-exchanged with Tris-HCl pH 8.0, and treated with FP-biotin to label serine hydrolases having residual activity. Labelled proteins were then enriched with streptavidin magnetic beads, digested with trypsin, and identified with liquid chromatography-tandem mass spectrometry (LC- MS/MS).

[0045] The inventors identified 48 proteins with at least two unique peptides, including five serine hydrolases: RB Binding Protein 9 (RBBP9), Palmitoyl-Protein Thioesterase 2 (PPT2), Phospholipase A2 Group VII (PLA2G7; known to cleave polysorbate), Cathepsin A (CTSA), and Acylaminoacyl-Peptide Hydrolase (APEH) (FIG. 1 A). Five proteins, including the mAbl light and heavy chains, were consistently identified across the entire temperature range. These proteins were highly abundant and likely bound the streptavidin magnetic beads non-specifically. In contrast, many other proteins showed a decreased intensity as the incubation temperature increased. These proteins likely underwent heat-induced denaturation and either dissociated from the mAb or lost reactivity with FP-biotin. FIG. IB is a line graph of heat inactivation of PLA2G7 in PAP. The intensity values corresponding to PLA2G7 are extracted from FIG. 1A and plotted against incubation temperature.

[0046] Additionally, the inventors profiled the heat sensitivity' of serine hydrolase activity in harvested cell culture fluid (HCCF) for mAbl (FIG. 1C). While serine hydrolase activity decreased upon heating, the activity curves for several enzymes including PLA2G7 shifted to higher temperatures in HCCF. This observation points to a potential matrix effect on enzyme activity such as pH and the presence of stabilizing proteins in the sample. These findings suggest that brief heat treatment could serve as a control method for PS degradation and beyond either through the dissociation of heat-sensitive enzymes from mAbl to facilitate their removal or through inactivation of such enzymes in formulation.

Example 2: Profiling Heat Sensitivity of serine hydrolase activity for a single mAb [0047] Next, the inventors further characterized the thermal stability of mAbl and several PS- degradative enzymes. Each of mAbl, PLA2G7, Sialic Acid Acetylesterase (SIAE), and Phospholipase B Domain Containing 2 (PLBL2) was formulated in 10 mM L-histidine, 7% sucrose, pH 6.0. Differential scanning fluorimetry (DSF) and differential scanning calorimetry (DSC) measurements roughly agreed on the peak melting temperature (Tm) of mAbl and indicated 63.6°C and 69.6°C, respectively (FIG. 2A). On the other hand, PLA2G7 and SIAE unfolded at lower temperatures of 53.2°C and 57.8°C, respectively, while PLBL2 turned out to be as thermally stable as mAbl with its Tm at 65.4°C (FIG. 2B). In addition, label-free dynamic light scattering (DLS) measurements revealed a dramatic increase in hydrodynamic diameters of PLA2G7, SIAE, and PLBL2 at temperatures between 50°C and 60°C, which is indicative of protein aggregation (FIG. 2C). In contrast, normalized unfolding profiles indicated mAbl remained folded and well dispersed in this temperature range (FIG. 2D).

[0048] The difference between the Tm of mAbl and PLA2G7/SIAE using DSF and DSC to measure are shown below in Table 1.

Using DSF to measure Tm, the combination of PLA2G7/SIAE have a 10.4°C lower Tm than mAbl, and SIAE has an 5.8 lower Tm than mAbl. Example 3: Testing effect of elevated temperatures on PS degradation

[0049] Having demonstrated the heat sensitivity of PLA2G7 activity and other PS-degradative enzymes, the inventors evaluated the impact of elevated temperatures on PS degradation. The mABl PAP was spiked with 0.2 mg/mL PS-80 and incubated at 5, 25, 37, 40, or 50°C for four weeks. Samples were pulled every week and PS-80 concentration was measured at week four with ultra-high performance liquid chromatography -charged aerosol detection assay (UPLC- CAD). The PS-80 concentration for mAb 1 decreased by a little over 20% for the samples under 5°C (FIG. 3A). PS degradation accelerated as the incubation temperature increased up to 40°C, presumably because of increased enzymatic activity at these temperatures. PS degradation slowed down at 50°C to a level that is comparable to 5°C samples; this is consistent with the observation that PLA2G7 lost ~ 90% of activity at 50°C relative to 40°C (see FIG. IB). In addition, the inventors executed a similar experiment with mAb2 PAP and observed the same trend (FIG. 3B). PS-80 concentration remained unchanged in buffer controls at all temperatures (FIG. 3C). These results demonstrate that elevated temperatures can inactivate PS-degradative enzymes and slow down PS degradation.

Example 4: Impact of heat inactivation on PS degradation and particle formation [0050] The inventors evaluated the impact of brief heat inactivation on PS degradation and particle formation. The inventors incubated mAbl drug product at 5°C or 50°C for 20 h and measured changes in PS-80 concentration, sub-visible particle count, and high-molecular weight species fraction over 28 days. PS-80 concentration was measured with a high performance liquid chromatography (HPLC)charged aerosol detector (CAD) assay and its decrease was slower for the heat-inactivated sample, with relative decreases of 31% versus 58% after 28 days (FIG. 4A). Sub-visible particle counts as measured with a HIAC™ light obstruction assay was lower for the heat-inactivated sample (FIG. 4B). In addition, the heat inactivation had negligible impact on the aggregation propensity as assessed by the fraction of high molecular weight species (FIG. 4C). These results suggest that heat inactivation of PS-degradative enzymes can mitigate the PS degradation and particle formation issue in biotherapeutic formulation with little to no impact on mAb stability.

Discussion

[0051] Heat inactivation of the drug product (DP) formulation at smaller volumes such as 2 mL glass crimped vials or pre-filled syringes may be more advantageous than performing the heat inactivation at larger scale such as 5L drug substance (DS) during processing. It may be easier to implement the inactivation step at the final stages of manufacturing post filling in comparison to engineering an additional step during upstream or downstream processing because determining the correct column or vessel size, hold-time and filter would require resources and development in comparison to determining the hold-time alone for heat inactivation of post-filled DP.

[0052] Heat inactivation may also be more effective in small volumes, as assurance of heat distribution is greater at smaller volumes. Heat inactivation of serine hydrolase in larger volumes would require longer hold times than inactivating in smaller glass vials. The observed similarity in percentage of high molecular weight particles between heat inactivated and control samples throughout the disclosed analysis shows that heat inactivation is viable post-DP fill.

[0053] The heat inactivation step could be part of the original processing and DP filling at release (e.g., filling glass vials, plastic or glass syringes, or other containers) or could potentially be implemented as an emergency contingency alleviating DP already in circulation. In emergent cases, most clinical storage sites and hospitals already have the local equipment (e.g., ovens) that could be utilized to heat inactivate DP that is at risk of polysorbate degradation and subsequent increase of subvisible particles. The heat inactivation of HCP during both the process and post DP filling stages offers the assurance that proteolytic activity will be decreased or diminished completely in manufactured DP, this potentially translates to purer DP with anticipated extended shelf-life.

Materials and Methods

[0054] FP-biotin was synthesized by WuXi AppTec. The recombinant proteins PLBD2, PLA2G7, and SIAE were expressed in CHO-Express cells and purified by Genscript Biotech. mAbl and mAb2 materials were internally prepared.

Activity-based protein profiling (ABPP) of heat-pulsed mAbl HCCF and PAP

[0055] The mAbl HCCF (0. 1 mL) or PAP (24.56 g/L, 0.2 mL) was heated at 40, 45, 50, 55, 60, 65. 70, or 75 °C for 5 min on a thermomixer with 500 rpm agitation and cooled on ice. The PAP sample was diluted with 50 mM Tris-HCl pH 8.0 (0.8 mL). To this was added FP-biotin (final concentration: 1 pM) at 25 °C, and the reaction mixture was incubated for 1 h on a thermomixer with 500 rpm agitation. The reaction mixture was filtered through a Zeba spin column that had been equilibrated with 0.2 % SDS in PBS pH 7. 1. The HCCF sample was diluted with 0.2 % SDS. To this were added magnetic streptavidin beads (20 pL), and the suspension was incubated at room temperature for 1 h on an end-over-end rotator. The beads were collected and washed three times with 0.2% SDS (1 mL) and once with PBS (1 mL). To this was added sequencing- grade modified trypsin (1 pg) in Tns-HCl pH 8.0 (50 pL). and the reaction mixture was incubated at 37°C for 18 h on a thermomixer with 500 rpm agitation. The supernatant was collected, cleaned up with a Cl 8 spin column, concentrated to dryness. and reconstituted with 0.1% formic acid in water (5 pL). LC-MS/MS analysis was performed on Easy-nLC 1200 and Q Exactive HF-X with Easy-Spray HPLC column ES900 and a 100-min gradient. Alternatively, the supernatant was loaded onto EvoTips and analysed on EvoSep One and Orbitrap Exploris 480 with EV 1106 column and an 88-min gradient. Mobile phases were (A) 0.1 % formic acid in water and (B) 0.1% formic in both cases. Acquired MS spectra were processed with MaxQuant v2.1.4.0 software using default settings.

Heating mAbl drug product

[0056] mAbl drug product 100 mg/mL mAbl, lOmM L-histidine, 7% sucrose, 0.02% (w/v) polysorbate 80, pH 6.0 (15 ml) was incubated at 5°C or 50°C for 20 h and filtered through a 0.22 pm filter. Aliquots were made for HPLC-CAD, UP-SEC, and sub- visible particle analyses (day 0). Remaining samples were incubated at 40°C and aliquoted for analyses on days 13 and 28.

Differential scanning fiuonmetrv (DSF)

[0057] Sypro Orange dye (Molecular Probes; Cat # S6650, Lot # 1001304) was added to MilliQ water (1:250) dilution. Protein samples of PLA2G7, SIAE, PLBL2, or mAbl formulated in lOmM L-histidine, 7% (w/v) sucrose, pH 6.0 were aliquoted (19pl each) into individual wells in a 96-well plate. Additionally, 1 pL of diluted Sypro Orange dye mix was added to each sample well and mixed. Plates were then covered, sealed and centrifuged at 1200 rpm for 3 min. Finally, plates were loaded into a thermal cycler for extrinsic fluorescence measurements. Experimental set-up <Protein melt_sunny.prcl> with the following experimental set-up (0.2°C step, hold 5 sec and read. Start at 25°C and go up to 95°C) was added to the CFX Manager software program to run temperature ramp, incubation and temperature range requirements. Data was fit and analyzed with Excel and Origin 8.0 software programs.

Differential scanning calorimetry (DSC)

[0058] 1 mg/mL mAbl formulated in lOmM L-histidine, 7% (w/v) sucrose, 0.02% (w/v) polysorbate 80, pH 6.0 w as aliquoted in 24 well PEAQ DSC plate and ran against formulation buffer at a temperature ramp increase of 1°C / min between the 25°C to 100°C temperature range. Following the procedure. Tm melting points were deconvoluted and determined by associated MicroCai PEAQ DSC software.

Dynamic light scattering (PLS)

[0059] DLS measurements were performed in a Malvern Nano Zetasizer utilizing Non-Invasive Back Scatter (NIBS). lOOpL of each sample formulated at Img/mL in lOmM L-histidine, 7% (w/v) sucrose, pH 6.0 was added to a lOOpL quartz cuvette. DLS measurements were made between the 30°C to 90°C temperature range at 5°C increments with additional 5 minute incubation time between measurements.

References:

(1) Wuchner, K. et al. Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 1: Survey Report on Current State and Common Practices for Handling and Control of Polysorbates. J. Pharm. Sci. 2022, 111 (5). 1280-1291. doi.org/10.1016/j.xphs.2022.02.009.

(2) Wuchner, K. et al. Industry Perspective on the Use and Characterization of Polysorbates for Biopharmaceutical Products Part 2: Survey Report on Control Strategy Preparing for the Future. J. Pharm. Sci. 2022, 111 (11), 2955-2967. doi.org/10.1016/j.xphs.2022.08.021.

(3) Li, X. et al. The Measurement and Control of High-Risk Host Cell Proteins for Polysorbate Degradation in Biologies Formulation. Antib. Ther. 2022, 5 (1), 42-54. doi.org/10.1093/abt/tbac002.

(4) Kovner, D. et al. Characterization of Recombinantly-Expressed Hydrolytic Enzymes from Chinese Hamster Ovary Cells: Identification of Host Cell Proteins That Degrade Polysorbate. J. Pharm. Sci. 2023, 000. doi.org/10.1016/j.xphs.2023.01.003.

(5) Liu, G.-Y.; Nie, S.; Zheng, X.; Li, N. Activity-Based Protein Profiling Probe for the Detection of Enzymes Catalyzing Polysorbate Degradation. Anal. Chem. 2022, 94 (24), 8625- 8632. doi.org/10.1021/acs.analchem.2c00059.

(6) Zhang, S.; Xiao, H.; Li, N. Degradation of Polysorbate 20 by Sialate O-Acetylesterase in Monoclonal Antibody Formulations. J. Pharm. Sci. 2021, 110 (12), 3866-3873. doi.org/10.1016/j.xphs.2021.09.001.

(7) Graf, T. et al. Identification and Characterization of Polysorbate-Degrading Enzymes in a Monoclonal Antibody Formulation. J. Pharm. Sci. 2021, 110 (1 1), 3558-3567. doi.org/10.1016/j.xphs.202L 06.033. (8) Li, X. et al. Profiling Active Enzymes for Polysorbate Degradation in Biotherapeutics by Activity-Based Protein Profiling. Anal. Chem. 2021, 93 (23), 8161-8169. doi . org/ 10.1021 / acs . anal chem.1 c00042.

(9) Zhang, S. et al. Putative Phospholipase B-Like 2 Is Not Responsible for Polysorbate Degradation in Monoclonal Antibody Drug Products. J. Pharm. Sci. 2020, 109 (9). 2710-2718. doi.org/10. 1016/j.xphs.2020.05.028.

(10) Zhang, S. et al. Rapid Polysorbate 80 Degradation by Liver Carboxylesterase in a Monoclonal Antibody Formulated Drug Substance at Early Stage Development. J. Pharm. Sci. 2020, 109 (11), 3300-3307. doi.org/10. 1016/j.xphs.2020.07.018.

(11) Hall, T. et al. Polysorbates 20 and 80 Degradation by Group XV Lysosomal Phospholipase A2 Isomer XI in Monoclonal Antibody Formulations. J. Pharm. Sci. 2016, 105 (5), 1633-1642. doi.org/10.1016/j.xphs.2016.02.022.

(12) McShan, A. C. et al. Hydrolysis of Polysorbate 20 and 80 by a Range of Carboxylester Hydrolases. PDA J. Pharm. Sci. Technol. 2016. 70 (4), 332-345. doi.org/10.5731/pdajpst.2015.005942.

(13) Dixit, N. et al. Residual Host Cell Protein Promotes Polysorbate 20 Degradation in a Sulfatase Drug Product Leading to Free Fatty Acid Particles. J. Pharm. Sci. 2016, 105 (5), 1657- 1666. doi.org/10. 1016/j.xphs.2016.02.029.

(14) Roy, I. et al. Polysorbate Degradation and Particle Formation in a High Concentration MAb: Formulation Strategies to Minimize Effect of Enzymatic Polysorbate Degradation. J. Pharm. Sci. 2021, 110 (9), 3313-3323. doi.org/10.1016/j.xphs.2021.05.012.

(15) Lim, A. et al. Characterization of a Cathepsin D Protease from CHO Cell-Free Medium and Mitigation of Its Impact on the Stability of a Recombinant Therapeutic Protein. Biotechnol. Prog. 2018, 34 (1), 120-129. doi.org/10.1002/btpr.2530.

(16) Bachovchin, D. A. et al. Superfamily -Wide Portrait of Serine Hydrolase Inhibition Achieved by Library-versus-Library Screening. Proc. Natl. Acad. Sci. U. S. A. 2010. 107 (49), 20941-20946. doi.org/10. 1073/pnas. 1011663107.

(17) Liu, Y. et al. Activity -Based Protein Profiling: The Serine Hydrolases. Proc. Natl. Acad. Sci. 1999, 96 (26), 14694-14699. doi.org/10.1073/pnas.96.26.14694.

[0060] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0061] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g., publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

WHAT IS CLAIMED IS:

1. A method for reducing serine hydrolase activity⁷ in a pharmaceutical composition comprising: a) incubating the pharmaceutical composition at a temperature of from about 40°C to about 70°C; wherein the pharmaceutical composition comprises i) an active biological ingredient, ii) a serine hydrolase that cleaves polysorbate while in its native conformation, and iii) polysorbate; wherein the serine hydrolase is irreversibly misfolded after the incubating step; and wherein the active biological ingredient in the pharmaceutical composition is not irreversibly misfolded after the incubating step.

2. The method of claim 1, wherein the serine hydrolase is Phospholipase A2 Group VII (PLA2G7) or Sialic Acid Acetylesterase (SIAE).

3. The method of claim 1 or 2, wherein the active biological ingredient has a melting temperature that is about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C, about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, or about 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning fluorimetry (DSF).

4. The method of claim 1 or 2, wherein the active biological ingredient has a melting temperature that is 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C. or 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE. wherein the melting temperature is measured by differential scanning fluorimetry (DSF).

5. The method of claim 1 or 2, wherein the active biological ingredient has a melting temperature that is about 3°C, about 4°C, about 5°C, about 6°C, about 7°C, about 8°C. about 9°C, about 10°C, about 11°C, about 12°C, about 13°C, about 14°C, or about 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning calorimetry (DSC).

6. The method of claim 1 or 2, wherein the active biological ingredient has a melting temperature that is 3°C, 4°C, 5°C, 6°C, 7°C, 8°C, 9°C, 10°C, 11°C, 12°C, 13°C, 14°C, or 15°C above the melting temperature of PLA2G7, SIAE, or both PLA2G7 and SIAE, wherein the melting temperature is measured by differential scanning calorimetry (DSC).

7. The method of any one of claims 1-6, wherein the step of incubating the pharmaceutical composition is performed at a temperature from about 45°C to about 55°C, from about 47°C to about 53°C, or at about 50°C.

8. The method of claim 7, wherein the step of incubating the pharmaceutical composition is performed at a temperature from 45°C to 55°C, from 47°C to 53°C, or at 50°C.

9. The method of any one of claims 1-6, wherein the step of incubating the pharmaceutical composition is performed at a temperature from about 45°C to about 65°C, from about 50°C to about 60°C, from about 53°C to about 57°C, or at about 55°C.

10. The method of claim 9, wherein the step of incubating the pharmaceutical composition is performed at a temperature from 45°C to 65°C, from 50°C to 60°C, from 53°C to about 57°C, or at 55°C.

11. The method of any one of claims 1-6, wherein incubating the pharmaceutical composition is performed at a temperature from about 50°C to about 67°C, from about 55°C to about 65°C. from to 57°C to about 63°C, or at about 60°C.

12. The method of claim 11, wherein incubating the pharmaceutical composition is performed at a temperature from 50°C to 67°C, from 55°C to 65°C, from 57°C to 63°C, or at 60°C.

13. The method of any one of claims 1-12, wherein incubating the pharmaceutical composition is performed from about 1 hour to about 20 hours, from about 2 hours to about 20 hours, from about 3 hours to about 20 hours, from about 4 hours to about 20 hours, from about 5 hours to about 20 hours, from about 6 hours to about 20 hours, from about 7 hours to about 20 hours, from about 8 hours to about 20 hours, from about 9 hours to about 20 hours, from about 10 hours to about 20 hours, from about 12 hours to about 20 hours, from about 15 hours to about 20 hours, or from about 17 hours to about 20 hours.

14. The method of claim 13, wherein incubating the pharmaceutical composition is performed from 1 to 20 hours, from 2 hours to 20 hours, from 3 hours to 20 hours, from 4 hours to 20 hours, from 5 hours to 20 hours, from 6 hours to 20 hours, from 7 hours to 20 hours, from 8 hours to 20 hours, from 9 hours to 20 hours, from 10 hours to 20 hours, from 12 hours to 20 hours, from 15 hours to 20 hours, or from 17 hours to 20 hours.

15. The method of any one of claims 1-12, wherein incubating the pharmaceutical composition is performed for at least about 5 minutes, at least about 7 minutes, at least about 10 minutes, at least about 12 minutes, at least about 15 minutes, at least about 20 minutes, at least about 25 minutes, at least about 30 minutes, at least about 35 minutes, at least about 40 minutes, at least about 45 minutes, at least about 50 minutes, at least about 55 minutes, at least about 1 hour, at least about 2 hours, at least about 3 hours, at least about 4 hours, at least about 5 hours, at least about 7 hours, at least about 10 hours, at least about 15 hours, or at least about 20 hours.

16. The method of claim 15, wherein incubating the pharmaceutical composition is performed for at least 5 minutes, at least 7 minutes, at least 10 minutes, at least 12 minutes, at least 15 minutes, at least 20 minutes, at least 25 minutes, at least 30 minutes, at least 35 minutes, at least 40 minutes, at least 45 minutes, at least 50 minutes, at least 55 minutes, at least 1 hour, at least 2 hours, at least 3 hours, at least 4 hours, at least 5 hours, at least 7 hours, at least 10 hours, at least 15 hours, or at least 20 hours.

17. The method of any one of claims 1-12, wherein incubating the pharmaceutical composition is performed for about 5 minutes, about 7 minutes, about 10 minutes, about 12 minutes, about 15 minutes, about 20 minutes, about 25 minutes, about 30 minutes, about 35 minutes, about 40 minutes, about 45 minutes, about 50 minutes, about 55 minutes, about 1 hour, about 2 hours, about 3 hours, about 4 hours, about 5 hours, about 7 hours, about 10 hours, about 15 hours, or about 20 hours.

18. The method of claim 17, wherein incubating the pharmaceutical composition is performed for 5 minutes, 7 minutes, 10 minutes, 12 minutes, 15 minutes, 20 minutes, 25 minutes, 30 minutes, 35 minutes, 40 minutes. 45 minutes, 50 minutes, 55 minutes. 1 hour. 2 hours, 3 hours, 4 hours, 5 hours, 7 hours, 10 hours, 15 hours, or 20 hours.

19. The method of any one of claims 1-18, wherein the pharmaceutical composition has a volume of from about 1 ml to about 5 ml, from about 2.5 ml to about 7.5 ml, from about 5 ml to about 10 ml, from about 7.5 ml to about 12.5 ml, from about 10 ml to about 15 ml, from about 12.5 ml to about 17.5 ml, or from about 15 ml to about 20 ml.

20. The method of claim 19, wherein the pharmaceutical composition has a volume of from 1 ml to 5 ml, from 2.5 ml to 7.5 ml, from 5 ml to 10 ml, from 7.5 ml to 12.5 ml, from 10 ml to 15 ml, from 12.5 ml to 17.5 ml, or from 15 ml to 20 ml.

21. The method of any one of claims 1-18, wherein the pharmaceutical composition has a volume of about 20 ml or less, about 17 ml or less, about 15 ml or less, about 12 ml or less, about 10 ml or less, about 9 ml or less, about 8 ml or less, about 7 ml or less, about 6 ml or less, about 5 ml or less, about 4 ml or less, about 3 ml or less, about 2 ml or less, or about 1 ml or less.

22. The method of claim 19, wherein the pharmaceutical composition has a volume of 20 ml or less, 17 ml or less, 15 ml or less, 12 ml or less, 10 ml or less, 9 ml or less, 8 ml or less, 7 ml or less, 6 ml or less, 5 ml or less, 4 ml or less, 3 ml or less, 2 ml or less, or 1 ml or less.

23. The method of any one of claims 1-22, wherein the step of incubating is performed during upstream or downstream processing prior to any filling of the drug product into containers for storage or administration.

24. The method of any one of claims 1-22, wherein the step of incubating is performed post filling of the drug product into containers for storage or administration.

25. The method of any one of claims 1-24, wherein the active biological ingredient is selected from the group consisting of: an enzyme; a hormone; a fusion protein, an Fc-containing protein; an immunoconjugate; a cytokine; and an antibody or antigen binding fragment thereof.

26. The method of claim 25. wherein the antibody is selected from the group consisting of: a monoclonal antibody; a chimeric antibody; a humanized antibody; a human antibody; and a multispecific antibody.

27. The method of claim 26. wherein the multispecific antibody is a bispecific antibody or a trispecific antibody.

28. The method of claim 25, wherein the antigen binding fragment thereof is selected from the group consisting of a Fab fragment; a Fab' fragment; a F(ab')2 fragment; an scFv; a di-scFv; a bi-scFv; a tandem (di, tri) scFv; an Fv; an sdAb; a tri-functional antibody; a BiTE; a diabody; and a triabody.

29. The method of any one of claims 1-28, wherein the serine hydrolase is selected from the group consisting of: RB Binding Protein 9 (RBBP9), Palmitoyl -Protein Thioesterase 2 (PPT2), Phospholipase A2 Group VII (PLA2G7; known to cleave polysorbate), Cathepsin A (CTSA), and Acylaminoacyl-Peptide Hydrolase.

30. The method of claim 29, wherein the serine hydrolase is selected from the group consisting of: PLA2G7, Sialic Acid Acetylesterase (SIAE), and Phospholipase B Domain Containing 2 (PLBL2).

31. The method of any one of claims 1-30, wherein the polysorbate is selected from the group consisting of: polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate), polysorbate 40 (polyoxyethylene (20) sorbitan monopalmitate), polysorbate 60 (polyoxyethylene (20) sorbitan monostearate), and polysorbate 80 (polyoxyethylene (20) sorbitan monooleate).

32. A pharmaceutical composition produced by any one of the methods in claims 1-31.

33. A pharmaceutical composition obtainable by any one of the methods in claims 1-31.