Compositions, methods, and kits for detecting lipolysis activity

Abstract

This disclosure provides compositions, methods, and kits for detecting lipolytic activity. In some embodiments, the composition comprises an aqueous assay sample and an organic solvent, wherein the organic solvent comprises 4-methylumbelliferyl oleate (4MuO). Methods for determining the stability of protein formulations are also provided herein.

Description

Technical Field

[0001] This disclosure provides compositions, methods, and kits for detecting and / or quantifying lipolytic activity. Methods for determining the stability of protein formulations are also provided herein. In some embodiments, the composition for detecting lipolytic activity comprises an aqueous assay sample and an organic solvent, wherein the organic solvent comprises 4-methylumbelliferyl oleate (4MuO). Background Technology

[0002] Cell cultures can be used to generate commercially important proteins, such as therapeutic proteins. In addition to target proteins, cells also produce host cell proteins (HCPs), such as lipases, which can be found in the production pool of target proteins, such as cell culture supernatants or cell lysates. Downstream purification processes, which can include various chromatographic steps (e.g., affinity chromatography in the case of monoclonal antibody production), are generally able to remove the vast majority of HCPs. However, due to the inherent principle of separation and purification methods—that is, based on equilibrium—it may not be possible to remove 100% of HCPs. Industrial and health authorities accept HCP limits of ~1-100 ppm relative to the amount of active protein. HCPs are typically measured and reported as a total parameter using enzyme-linked immunosorbent assays (ELISA), such as those used by USP. <1132> As described above, these typically contain a range of proteins from different host sources at varying concentrations. The same pharmaceutical substance may have different HCP profiles even with the same total HCP content quantified by ELISA. This can lead to situations where slight process variations result in low concentrations of specific HCPs, such as an increase in lipases, which may be undetectable by total HCP assays. Therefore, a small fraction of HCPs may be present in the final protein product and may possess catalytic activity. Residual catalytic activity can have detrimental effects on product quality, such as the stability of excipients, like surfactants such as polysorbates, and ultimately, on the stability, quality, and safety of the protein product. If the protein is therapeutic, especially for human use, minimizing or eliminating these detrimental effects may be of paramount importance.

[0003] Residual lipase activity in protein formulations, for example, due to suboptimal removal during downstream purification processes of the target protein, can lead to the hydrolysis of some excipients, such as surfactants. Consequences may include, for example, the formation of sub-visible and visible particles due to the release of nonpolar and therefore insoluble long-chain fatty acids and other degrading agents from the surfactant, and the loss of the stabilizing effect provided by the surfactant. Surfactant degradation can also adversely affect protein quality, for example, by generating peroxides that lead to protein degradation, or lauric acid that induces protein aggregation. Surfactant degradation also reduces their concentration in the formulation, potentially resulting in insufficient protection of the protein, such as protection against interfacial stresses (e.g., shaking, freezing / thawing, etc.), and also leading to potential differences in product safety considerations, such as different in vivo liabilities due to degraded surfactants. Therefore, residual lipase activity due to excipient hydrolysis can negatively impact the quality of the final protein formulation. Degradation products of excipients, such as polysorbate, can also pose safety concerns to patients, such as injection site reactions (see, for example, Singh et al., Journal of Pharmaceutical Science 107(11):2735-2741(2018)). Summary of the Invention

[0004] In some embodiments, this disclosure provides a composition comprising: (a) an aqueous assay sample containing a protein formulation; and (b) an organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate (4MuO); wherein the pH of the aqueous assay sample is from 5.0 to 7.0; and wherein the aqueous assay sample constitutes from about 80% to about 99.9% of the composition, and the organic solvent constitutes from about 0.1% to about 20% of the composition.

[0005] In some embodiments, the protein formulation is a cell culture supernatant. In some embodiments, the protein formulation is a partially purified protein formulation. In some embodiments, the protein formulation is a purified protein formulation.

[0006] In some embodiments, the protein formulation comprises a therapeutic protein.

[0007] In some embodiments, the protein formulation comprises a surfactant. In some embodiments, the surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate-20, polysorbate-80, or a combination thereof.

[0008] In some embodiments, the protein formulation further comprises additional host cell proteins.

[0009] In some embodiments, the aqueous test sample further comprises a buffer, a salt, or both.

[0010] In some embodiments, the salt is sodium chloride, calcium chloride, or a combination thereof. In some embodiments, the salt is sodium chloride and calcium chloride. In some embodiments, the sodium chloride concentration in the aqueous test sample is from about 50 mM to about 400 mM. In some embodiments, the sodium chloride concentration in the aqueous test sample is from about 100 mM to about 200 mM. In some embodiments, the calcium chloride concentration in the aqueous test sample is from about 0.2 mM to about 10 mM. In some embodiments, the calcium chloride concentration in the aqueous test sample is from about 1.0 mM to about 2.0 mM.

[0011] In some embodiments, the buffer has buffering capacity at about pH 6.0. In some embodiments, the buffer is Tris. In some embodiments, the buffer is Bis-Tris. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 2 mM to about 200 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 10 mM to about 100 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 40 mM to about 60 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 45 mM to about 55 mM.

[0012] In some embodiments, the organic solvent is an alcohol, sulfoxide, nitrile, or a combination thereof. In some embodiments, the organic solvent is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent comprises acetonitrile.

[0013] In some embodiments, the organic solvent comprises an alcohol. In some embodiments, the organic solvent comprises a mixture of acetonitrile and isopropanol. In some embodiments, the organic solvent is a C1-C6 alcohol. In some embodiments, the organic solvent is methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, or a combination thereof.

[0014] In some embodiments, the composition further comprises a lipase inhibitor. In some embodiments, the lipase inhibitor is (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetane]methyl]-dodecyl ester (orlistat).

[0015] In some embodiments, the lipase inhibitor is in an organic solvent. In some embodiments, the lipase inhibitor is in the composition at a concentration of about 1 μM to about 50 μM. In some embodiments, the lipase inhibitor is in the composition at a concentration of about 5 μM to about 25 μM.

[0016] In some embodiments, this disclosure provides a composition comprising: (a) an aqueous assay sample comprising (i) a purified protein formulation; (ii) a buffer; and (iii) a salt; and (b) an organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate (4MuO); wherein the pH of the aqueous assay sample is 5.0 to 7.0; and wherein the aqueous assay sample constitutes about 80% to about 99.9% of the composition, and wherein the organic solvent constitutes about 0.1% to about 20% of the composition.

[0017] In some embodiments, this disclosure provides a composition comprising: (a) about 90% to about 99.9% (vol / vol) of an aqueous assay sample, said aqueous assay sample comprising (i) a purified protein formulation containing proteins and lipids; (ii) a buffer; (iii) about 1.0 mM to about 2.0 mM calcium chloride; and (iv) about 100 mM to about 200 mM sodium chloride; and (b) about 10% to about 0.1% (vol / vol) of an organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethyl sulfoxide (DMSO), acetonitrile, or combinations thereof, further comprising 4-methylumbelliferyl oleate (4MuO); wherein the pH of the aqueous assay sample is 5.0 to 7.0.

[0018] In some embodiments, this disclosure provides a method for detecting lipolytic activity in an aqueous assay sample, the method comprising (a) combining an aqueous assay sample containing a protein formulation with an organic solvent containing 4-methylumbelliferone oleate (4MuO); and (b) measuring the formation of the oleate and 4-methylumbelliferone (4Mu) by fluorescence.

[0019] In some embodiments, this disclosure provides a method for detecting lipolytic activity in an aqueous assay sample, the method comprising (a) combining an aqueous assay sample containing a protein formulation with an organic solvent containing 4-methylumbelliferyl oleate (4MuO) to form an assay composition; (b) combining a control sample containing a protein formulation and a lipase inhibitor with an organic solvent containing 4-methylumbelliferyl oleate (4MuO) to form a control composition; and (c) determining the formation of oleate and 4-methylumbelliferyl oleate (4Mu) in the assay composition and the control composition by fluorescence measurement.

[0020] In some embodiments, this disclosure provides a method for determining the stability of a protein formulation, comprising (a) combining an aqueous assay sample containing the protein formulation with an organic solvent containing 4-methylumbelliferone oleate (4MuO); (b) measuring the formation of the oleate and 4-methylumbelliferone (4Mu) by fluorescence; and (c) determining the stability of the protein formulation based on the measured fluorescence.

[0021] In some embodiments, the pH of the aqueous test sample is from 5.0 to 7.0. In some embodiments, the aqueous test sample and the organic solvent are combined in a ratio of from about 80:20 to about 98:2. In some embodiments, the aqueous test sample and the organic solvent are combined in a ratio of from about 90:10 to about 99.9:0.1.

[0022] In some embodiments, fluorescence is measured by fluorescence excitation at 330 nm and fluorescence emission at 495 nm. In some embodiments, fluorescence measurement continues for up to 24 hours. In some embodiments, fluorescence measurement continues for about 24 hours to about 400 hours. In some embodiments, fluorescence measurement continues for more than about 100 hours.

[0023] In some embodiments, prior to step (a), the aqueous assay sample is incubated with a lipase inhibitor for about 10 minutes to about 1 hour. In some embodiments, prior to step (a), the aqueous assay sample is incubated with a lipase inhibitor for about 30 minutes. In some embodiments, the lipase inhibitor is (S)-2-formamido-4-methylvaleric acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetane]methyl]-dodecyl ester (orlistat). In some embodiments, the lipase inhibitor is added at about 1 μM to about 50 μM. In some embodiments, the lipase inhibitor is added at about 5 μM to about 25 μM.

[0024] In some embodiments, this disclosure provides a kit comprising: (a) an organic solvent; (b) 4-methylumbelliferyl oleate (4MuO); and (c) a lipase inhibitor in two or more containers.

[0025] In some embodiments, the kit further comprises a buffer, a salt, or both. In some embodiments, the kit further comprises a buffer exchange column.

[0026] In some embodiments, this disclosure provides a kit comprising: (a) an organic solvent containing 4-methylumbelliferyl oleate (4MuO); (b) a column suitable for exchanging protein formulation buffers; and (c) a lipase inhibitor. Attached Figure Description

[0027] Figure 1A-1E Related to Instance 1A. Figure 1A and 1B The effects of the buffer concentration and pH of Tris and Bis-Tris buffers on the activity of porcine pancreatic lipase (PPL) and the fluorescence quenching of 4-methylumbelliferone (4Mu) were shown. Figure 1CThe effects of buffer concentration and pH on lipase activity and the autohydrolysis of 4-methylumbelliferone oleate (4MuO) were shown. Figure 1D The pH range for measuring 4Mu fluorescence is shown. Figure 1E The different forms of 4Mu at various pH values ​​are shown.

[0028] Figure 2A-2C Related to Instance 2A. Figure 2A and 2B The effects of CaCl2 concentration on lipase activity and 4Mu fluorescence quenching were shown. Figure 2C The effect of CaCl2 concentration on lipase activity and autohydrolysis of 4MuO was shown.

[0029] Figures 3A-3C Related to Instance 3A. Figure 3A and 3B The effects of NaCl concentration on lipase activity and 4Mu fluorescence quenching were shown. Figure 3C The effect of NaCl concentration on lipase activity and autohydrolysis of 4MuO was shown.

[0030] Figures 4A-4C Related to Instance 4A. Figure 4A and 4B The effects of organic solvents on lipase activity and 4Mu fluorescence quenching were shown. Figure 4C The effects of organic solvents on lipase activity and the autohydrolysis of 4MuO were shown.

[0031] Figures 5A-5C Related to Example 5. Figure 5A and 5B The effects of surfactants on lipase activity and 4Mu fluorescence quenching were shown. Figure 5C The effects of surfactants on lipase activity and the autohydrolysis of 4MuO were shown.

[0032] Figures 6A-6B Related to Example 6. Figure 6A and 6B The effects of three different concentrations of the lipase inhibitor orlistat on lipase activity and 4Mu fluorescence quenching were shown.

[0033] Figures 7A-7D Related to Example 7. Figure 7A and 7C The study showed the inhibitory effect of theoretically completely degraded PS20 on lipase activity and the products of autohydrolysis of 4Mu. Figure 7B and 7D The study showed the inhibitory effect of theoretically completely degraded PS80 on lipase activity and the products of autohydrolysis of 4Mu.

[0034] Figure 8 Related to Example 8. Figure 8 The effect of PS80 on 4Mu fluorescence quenching is shown.

[0035] Figures 9A-9D Related to Example 9. Figure 9A and 9B The test PS20 was shown. Figure 9A ) and PS80 Figure 9B Results of HPLC-FMA determination of degradation in cell culture harvest broth (CCHF) at different concentrations. Figure 9C The theoretical readings for 4MuO lipase assays are shown. Figure 9D The actual readings of 4MuO lipase assays for the degradation of polysorbate by different concentrations of CCHF are shown.

[0036] Figures 10A-10H Related to Example 9. Figures 10A-10H The results of 4MuO lipase assays performed with PS80 at different CCHF concentrations are shown.

[0037] Figure 11 Related to Instance 1B. Figure 11 The effects of pH on the lipolytic activity of Tris and Bis-Tris and the autohydrolysis (AH) of 4Mu were shown.

[0038] Figure 12 Related to Instance 2B. Figure 12 The effect of CaCl2 concentration on the lipolysis activity of PPL and CCHF was shown.

[0039] Figure 13 Related to Instance 3B. Figure 13 The effect of NaCl concentration on the lipolysis activity of PPL and CCHF was shown.

[0040] Figure 14 Related to Instance 4B. Figure 14 The effect of organic solvents on the lipolysis activity of PPL and CCHF was shown.

[0041] Figure 15A and 15B Related to Instance 5B. Figure 15A The effects of different surfactants on the lipolysis activity of PPL and CCHF were shown. Figure 15B The quenching of 4Mu fluorescence by different surfactants was shown.

[0042] Figure 16 Related to Instance 6B. Figure 16 The effect of pre-incubating or co-incubating samples with three different concentrations of the lipase inhibitor orlistat on PPL activity was shown.

[0043] Figure 17A and 17B Related to Example 10. Figure 17A The kinetics of lipase assays performed using protein-containing formulations with various positive and negative controls (samples, assays, formulations, and autohydrolysis) are shown. The samples tested are summarized in Table 10 of this paper. Figure 17B It is a scaled-down diagram of the measured dynamics.

[0044] Figure 18 Related to Example 9. Figure 18 This presents a summary of lipase assays performed using different concentrations of CCHF.

[0045] Figure 19A and 19B Related to Instance 1B. Figure 19A and 19B Numerical results of the 4Mu calibration curves and 4Mu calibration curves are shown for concentration ranges from 0.01 to 5 mM. Detailed Implementation

[0046] This disclosure relates to compositions, methods, and kits for detecting lipolytic activity.

[0047] As used herein, “a” or “an” may refer to one or more. As used herein, when used with the word “includes”, “a” or “an” may refer to one or more. As used herein, “additional” or “further” may refer to at least a second or more.

[0048] Throughout this application, the term "about" is used to indicate that a value includes inherent variation in the error of the method / apparatus used to determine the value, or variation present among study subjects. Generally, the term means, depending on the circumstances, a variability of approximately or less than 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20%. In some embodiments, those skilled in the art will understand the level of variability represented by the term "about" due to the context in which it is used herein. It should also be understood that the use of the term "about" also includes specifically stated values.

[0049] Although this disclosure supports the definitions of "substitute only" and "and / or", the term "or" used in the claims means "and / or" unless it is explicitly stated that "substitute only" or that the substitutes are mutually exclusive.

[0050] As used in this specification and claims, the terms “comprising” (and any form of inclusion, such as “comprise” and “comprises”), “having” (and any form of having, such as “have” and “has”), “including” (and any form of inclusion, such as “includes” and “include”), or “containing” (and any form of inclusion, such as “contains” and “contain”) are inclusive or open-ended and do not exclude additional, unstated elements or method steps. It is contemplated that any embodiments discussed in this specification can be implemented for any method, composition, and / or kit of this disclosure. Furthermore, the compositions of this disclosure can be used to implement the methods and kits of this disclosure.

[0051] The use of the term "for example" and its corresponding abbreviation "eg" (whether italic or not) means that the specific terms described are representative examples and embodiments of this disclosure, and that such representative examples and embodiments are not intended to be limited to the specific examples referenced or cited, unless otherwise expressly stated.

[0052] As used in this article, “between” is a range that includes both ends of a range. For example, numbers between x and y explicitly include the numbers x and y, as well as any numbers that fall within x and y.

[0053] As used herein, “protein,” “peptide,” or “polypeptide” refers to a polymer of amino acids of any length. Proteins may include, for example, antibodies, structural proteins, enzymes, membranes, membrane-associated and / or transmembrane proteins, transport proteins, receptors, signal transduction proteins, etc. The proteins and / or peptides of this disclosure also include modified proteins, for example, conjugated with one or more non-peptide substances, such as, for example, drugs, targeting moieties, tags such as visual tags, etc. The proteins of this disclosure may be therapeutic proteins, for example, therapeutic proteins for the diagnosis, treatment, and / or prevention of diseases or conditions. In some embodiments, the polysorbate described herein can improve the stability of the protein in a pharmaceutical formulation. In some embodiments, the therapeutic protein is an antibody. In some embodiments, the therapeutic protein is an antibody-drug conjugate. In some embodiments, the protein formulation described herein comprises a protein, for example, a therapeutic protein.

[0054] As used herein in the context of protein formulations, “purification” refers to the process of separating one or more substances, such as proteins, from a complex mixture, typically cells, tissues, or organisms. A “purified” protein sample or protein formulation can refer to a sample in which one or more water-insoluble components of the cells, tissues, or organisms (e.g., cell membranes, lipids, aggregated proteins or nucleic acids, and other hydrophobic substances) have been reduced or removed, leaving only soluble components (e.g., soluble proteins). As used herein, “soluble” can refer to the ability of a substance to dissolve in a solvent, such as cell culture medium, buffers, water, or an organic solvent. In the context of proteins, “soluble” can also refer to proteins that do not precipitate and / or aggregate in a solvent, such as cell culture medium, buffers, water, or an organic solvent.

[0055] An exemplary purification process may include: growing a cell culture containing a protein of interest, such as a therapeutic protein; isolating the cells from the culture medium; lysing the cells and separating the lysed cells to produce a cell culture supernatant containing soluble components and a precipitate containing the insoluble components described herein; and subjecting the cell culture supernatant to buffer exchange, pH adjustment, centrifugation, filtration (including, for example, ultrafiltration and / or percolation), chromatography, or any combination thereof, to produce a purified protein formulation. In some embodiments, the purified protein formulation of this disclosure is purified by the process described herein. In some embodiments, a partially purified protein formulation of this disclosure has undergone some of the purification processes described herein. For example, a partially purified protein formulation may not have undergone all the buffer exchange, pH adjustment, centrifugation, filtration, and / or chromatographic steps used to generate a purified protein formulation. In some embodiments, the cell culture supernatant described herein contains a therapeutic protein of this disclosure. In some embodiments, the partially purified protein formulation described herein contains a therapeutic protein of this disclosure. In some embodiments, the purified protein formulation described herein contains a therapeutic protein of this disclosure.

[0056] In some embodiments, this disclosure relates to compositions and methods for detecting lipolytic activity. Lipolytic activity, or lipolysis, generally refers to the hydrolysis of lipids. Lipolysis reactions can be catalyzed by lipases, a subclass of esterases. Thus, "lipase" refers to an enzyme that hydrolyzes the ester bonds of lipids, such as triglycerides, phospholipids, cholesterol esters, etc. Lipases include, for example, triglyceride lipases, lipoprotein lipases, pancreatic lipases, hepatic lipases, gastric lipases, tongue lipases, endothelial lipases, and phosphatidylserine phospholipases. Lipases are naturally occurring, for example, produced by the pancreas, liver, tongue glands, stomach, thyroid gland, and / or mucous membranes of mammals, secreted by certain bacteria and fungi, and / or found in lysosomes. In some embodiments, the lipase is cellularly endogenous to the protein being purified. In some embodiments, the lipase is endogenous to another biological component in the protein formulation, for example, a biological component containing a stable protein added to the protein formulation.

[0057] In some embodiments, the lipase is produced by cells in a cell culture. In some embodiments, the lipase is produced by cells in a cell culture for the production of the protein of interest. Non-limiting examples of cells suitable for producing the protein of interest include bacteria, insects, yeast, mammals, and / or transgenic cells. Non-limiting examples of cell lines include CHO, HEK 293, HT-1080, PER.C6, CAP, VERO, BHK, HeLa, CV1, Cos, MDCK, 3T3, NSO, NS1, PC12, W138, Sp2 / 0, HKB-11, TM4, MMT 060562, TR1, MRC 5, FS4, myeloma cell lines, hybridoma cell lines, and hepatocellular carcinoma cell lines. In some embodiments, the cell line for producing the protein of interest is a stable cell line, e.g., in which the gene for the protein of interest is stably integrated into the cell's genome. In some embodiments, the cell line for producing the protein of interest is a transient cell line, e.g., in which the cells express but do not integrate the gene into the genome.

[0058] In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the protein of interest is purified from a cell culture to produce a purified protein formulation. In some embodiments, lipases in the cell culture are not completely removed from the protein formulation during the purification process. Therefore, in some embodiments, lipases are present in the purified protein formulation.

[0059] As referred to herein, an "active" lipase is a lipase capable of lipolysis (also referred to herein as having "lipolytic activity"). Active lipases present in protein formulations can interfere with downstream processes involving proteins of interest, such as therapeutic proteins. In some embodiments, a protein formulation comprising a protein of interest, such as a therapeutic protein, and a lipase is included in a pharmaceutical formulation. In some embodiments, an excipient is added to the protein formulation. In some embodiments, the excipient stabilizes the protein formulation, for example by minimizing interfacial stress, reducing protein aggregation, and / or increasing protein solubility. In some embodiments, the excipient is a surfactant. In some embodiments, the excipient comprises a fatty acid, an ester, or both. In some embodiments, the excipient is readily hydrolyzed by an active lipase. In some embodiments, the presence of an active lipase in a protein formulation comprising a protein of interest and an excipient reduces the stability of the formulation. Therefore, it is advantageous to reliably detect lipolytic activity in protein formulations to minimize the negative effects of lipase hydrolysis of the excipient, such as increased particle size, safety concerns (e.g., due to increased injection site reactions), and decreased quality.

[0060] In some embodiments, this disclosure provides compositions in which lipolytic activity in protein formulations can be detected.

[0061] In some embodiments, this disclosure provides a composition comprising: (a) an aqueous assay sample containing a protein formulation; and (b) an organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate (4MuO); wherein the pH of the aqueous assay sample is 5.0 to 7.0; and wherein the aqueous assay sample constitutes about 80% to about 98% of the composition, and the organic solvent constitutes about 2% to about 20% of the composition.

[0062] In a further embodiment, this disclosure provides a composition comprising: (a) an aqueous assay sample comprising (i) a purified protein formulation; (ii) a buffer; and (iii) a salt; and (b) an organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate (4MuO); wherein the pH of the aqueous assay sample is from 5.0 to 7.0; and wherein the aqueous assay sample constitutes about 80% to about 98% (vol / vol) of the composition, and the organic solvent constitutes about 2% to about 20% (vol / vol) of the composition.

[0063] In a further embodiment, this disclosure provides a composition comprising: (a) about 90% to about 98% (vol / vol) of an aqueous assay sample, said aqueous assay sample comprising (i) a purified protein formulation; (ii) a buffer; (iii) about 1.0 mM to about 20 mM calcium chloride; and (iv) about 100 mM to about 200 mM sodium chloride; and (b) about 2% to about 10% (vol / vol) of an organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethyl sulfoxide (DMSO), acetonitrile, or combinations thereof, further comprising 4-methylumbelliferyl oleate (4MnO); wherein the pH of the aqueous assay sample is 5.0 to 7.0.

[0064] As used herein, "aqueous" (e.g., aqueous assay sample) refers to a solution or sample in which water is a solvent. Therefore, the aqueous assay samples of this disclosure may include, for example, cell culture media, buffer solutions, protein samples, etc. In some embodiments, the aqueous assay samples of this disclosure comprise protein formulations.

[0065] In some embodiments, the protein formulation is a cell culture supernatant. Cell culture supernatants are described herein and can be obtained, for example, from cell cultures used to produce the protein of interest. In some embodiments, the protein is a therapeutic protein. In some embodiments, the cell culture supernatant is produced after lysing cultured cells and separating soluble and insoluble components, for example by centrifugation. Examples of cells and cell lines suitable for culture and protein production are provided herein. In some embodiments, the cell culture supernatant contains the protein of interest, such as a therapeutic protein, along with additional host cell components. In some embodiments, the additional host cell components contain additional host cell proteins. In some embodiments, the additional host cell proteins contain lipases. In some embodiments, the lipases have lipolytic activity.

[0066] In some embodiments, the protein formulation is a partially purified protein formulation. Partially purified protein formulations are described herein and can be obtained, for example, after undergoing a partial purification procedure (e.g., the purification process described herein) from a cell culture of interest. In some embodiments, the protein is a therapeutic protein. In some embodiments, the partially purified protein formulation has undergone additional purification steps compared to the cell culture supernatant. In some embodiments, the partially purified protein formulation comprises an additional component of a therapeutic protein and a host cell. In some embodiments, the host cell component comprises a host cell protein. In some embodiments, the host cell protein comprises a lipase. In some embodiments, the lipase has lipolytic activity. In some embodiments, the therapeutic protein is 20% to 95% (w / w), 30% to 90% (w / w), or 40% to 80% (w / w) of all proteins in the partially purified protein formulation.

[0067] In some embodiments, the protein formulation is a purified protein formulation. Purified protein formulations are described herein and can be obtained, for example, after undergoing a purification process (e.g., the purification process described herein) of the protein of interest from a cell culture. In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the purified protein formulation comprises a therapeutic protein and an additional host cell component. In some embodiments, the host cell component comprises a host cell protein. In some embodiments, the host cell protein comprises a lipase. In some embodiments, the lipase has lipolytic activity. In some embodiments, the therapeutic protein is greater than 70% (w / w), greater than 80% (w / w), greater than 85% (w / w), greater than 90% (w / w), greater than 95% (w / w), or greater than 99% (w / w) of all proteins in the purified protein formulation.

[0068] In some embodiments, the protein formulation comprises a therapeutic protein. Non-limiting examples of therapeutic proteins include antibodies (such as monoclonal or polyclonal antibodies) and antibody fragments; protein-based vaccines (such as, for example, hepatitis B surface antigen); blood factors (such as, for example, factor VIII and factor IX); thrombolytic agents (such as, for example, tissue plasminogen activator); hormones (such as, for example, insulin, glucagon, growth hormone, and gonadotropins); hematopoietic growth factors (such as, for example, erythropoietin and colony-stimulating factor); interferons (such as, for example, interferon-α, interferon-β, and interferon-γ); interleukin-based proteins (such as, for example, interleukin-12); and other proteins, such as tumor necrosis factor and therapeutic enzymes. Further examples of protein-based therapies include belimumab, ipilimumab, beracip, bentoximab, aflibercept, *Erwinia chrysanthemi* asparaginase, carboxypeptidase, olibutrazumab, pembrolizumab, bonatetumab, nivirumab, idarutumab, asofatase-alfa, dalatumab, elutuzumab, sebelipase alfase, atezolizumab, taliglucerase alfa, recibacubitumab, elosulfase alfa, metriptin, ramucirumab, stevoximab, pembrolizumab, idoxurumab, evamarumab, necetamol, obbituximab, abatacept, adalimumab, afaxetine, etanercept, infliximab, trastuzumab, uslinta, and denileukin. Diftitox and golimumab. Further examples of protein therapy are described, for example, in Dimitrov, Methods in Molecular Biology 899:1-26 (2012), Lagassé et al., F1000Res 6:113 (2017), and Protein Therapeutics, Eds: Vaughan et al., 2017: Wiley-VCH Verlag. Therapeutic proteins may comprise recombinant proteins, modified proteins, and fusion proteins, such as, for example, antibody-drug conjugates, antibody-cytokine fusions, Fc-fusions, bispecific antibodies, multispecific antibodies, affinity fusions, glycosylated proteins and peptides, and engineered receptor antagonists. In some embodiments, protein formulations comprising therapeutic proteins are used in pharmaceutical formulations.

[0069] In some embodiments, the protein formulation comprises a commercially important protein, such as an industrial enzyme. Commercially important proteins are used in a variety of industries, such as pharmaceuticals, chemical production, biofuels, food and beverages, and consumer products. For example, in some embodiments, the protein formulation is an enzyme used in a process to produce a desired product, or may be a product of interest. In some embodiments, the commercially important protein is used in the food, pharmaceutical synthesis, biofuel, chemical, or manufacturing industries. In some embodiments, the industrial enzyme comprises, but is not limited to, palatase lipozyme, lipopan, xylose isomerase, bromelain and noopazyme (for the food industry), cellulase and amylase (for the biofuel industry), resinase (for the paper processing industry), amidase (for the chemical industry), novozym-435 (for the cosmetic production of isopropyl myristate), or Bacillus subtilis protease (for detergents).

[0070] In some embodiments, the protein formulation includes a pharmaceutical excipient. The inclusion of a pharmaceutical excipient may be for example to assist in the processing of a drug delivery system before, during, or after manufacturing; to protect, support, or enhance stability, bioavailability, or patient acceptability; to assist in product identification and enhance overall safety; to assist in the effectiveness and / or delivery of the drug in use; and / or to assist in maintaining the integrity of the pharmaceutical product during storage. Non-limiting examples of pharmaceutical excipients include surfactants, fillers, diluents, viscosity agents, suspending agents, viscous agents, coatings, flavoring agents, disintegrants, coloring agents, lubricants, flow aids, preservatives, sweeteners, etc. In some embodiments, the pharmaceutical excipient is added to the protein formulation. In some embodiments, the pharmaceutical excipient is added to the protein formulation before, after, or during purification.

[0071] In some embodiments, the pharmaceutical excipient is a surfactant. As used herein, a "surfactant" refers to an agent that reduces the surface tension or interfacial tension between two liquids. In some embodiments, surfactants can stabilize compositions, such as the protein formulations described herein, by minimizing aggregation and / or precipitation and / or improving the solubility of one or more components of the composition (e.g., by reducing surface tension and inhibiting protein surface adsorption; see, for example, Agarkhed et al., AAPS Pharmaceutical Science and Technology (AAPSPharmSciTech) 14:1-9 (2013)). Surfactants in pharmaceutical compositions can also modulate the bioavailability of the active pharmaceutical ingredient (API); assist the API in maintaining its preferred polymorphic form; prevent aggregation or dissociation; and / or modulate the immunogenicity of the active ingredient. Surfactants can comprise cationic, anionic, nonionic, zwitterionic, acid-base facultative, and / or amphoteric surfactants. Non-limiting examples of surfactants include polysorbates (e.g., TWEEN surfactants such as TWEEN 20 and TWEEN 80, which are also referred to as polysorbate 20 and polysorbate 80, respectively), derived from ethoxylated dehydrated sorbitol esterified with fatty acids (e.g., lauric acid in polysorbate 20 and oleic acid in polysorbate 80); tyloxapine; poloxamer (e.g., PLURONIC F68LF, PLURONIC L-G2LF, PLURONIC L62D, LUTROL F68, and KOLLIPHOR P188); polyoxyethylene castor oil (e.g., KOLLIPHOR EL) and its derivatives; dehydrated sorbitol esters, also known as Spans; polyoxyethylene stearates; lecithin; phospholipids; polyoxyethylene surfactants such as, for example, TRITON (e.g., TRITON X-100) and BRIJ (e.g., BRIJ 35); and polyethoxylated fatty acids such as, for example, MYRJ. S40, MYRJ S100 and MYRJ 52.

[0072] In some embodiments, the surfactant comprises a fatty acid. In some embodiments, the surfactant comprises an ester. In some embodiments, the surfactant is a polysorbate. Polysorbates are a class of compounds derived from ethoxylated dehydrated sorbitol esterified with a fatty acid, and include, for example, polysorbate 20, polysorbate 40, polysorbate 60, polysorbate 80, polysorbate 21, polysorbate 61, polysorbate 65, polysorbate 81, and polysorbate 81. In some embodiments, the protein formulations provided herein comprise polysorbates. In some embodiments, the polysorbate in the protein formulation is polysorbate 20, polysorbate 80, or a combination thereof.

[0073] In some embodiments, the surfactant is about 0.001% w / v to about 2% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.005% w / v to about 2% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.01% w / v to about 2% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.02% to about 1.5% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.03% to about 1.0% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.04% to about 0.8% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.05% to about 0.6% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.06% to about 0.4% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.07% to about 0.2% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.08% to about 0.15% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.09% to about 0.10% w / v of the aqueous test sample. In some embodiments, the surfactant is about 0.01% to about 0.04% w / v of the aqueous test sample. In some embodiments, the surfactant is polysorbate. In some embodiments, the surfactant is polysorbate 20, polysorbate 80, or a combination thereof.

[0074] In some embodiments, the protein formulation further comprises one or more additional host cell proteins. As described herein, the protein formulation is prepared from a cell culture containing host cells of the protein of interest, such as a therapeutic protein. In some embodiments, the protein formulation comprises one or more additional host cell proteins. In some embodiments, the additional host cell proteins are soluble under substantially the same conditions as the protein of interest, such as the therapeutic protein. In some embodiments, the additional host cell proteins are not readily separable from the protein of interest, such as the therapeutic protein. In some embodiments, the additional host cell proteins comprise lipases. In some embodiments, one or more additional host cell proteins have lipolytic activity.

[0075] In some embodiments, the aqueous assay sample containing the protein formulation further comprises a buffer, a salt, or both. Generally, a salt in this disclosure refers to a substance whose anion is not OH-. - and O 2-Ionic compounds. In some embodiments, the salt reduces and / or prevents the degradation of one or more components in the composition. Those skilled in the art can choose suitable salts that can be included in the aqueous assay sample, including, for example, sodium salts, potassium salts, calcium salts, ammonium salts, etc. In some embodiments, the salt is potassium chloride (KCl), sodium chloride (NaCl), sodium carbonate (Na2CO3), sodium sulfate (Na2SO4), calcium chloride (CaCl2), ammonium chloride (NH4Cl), ammonium acetate (NH4CH3COO), ammonium sulfate ((NH4)2SO4), or combinations thereof. In some embodiments, the salt is NaCl, CaCl2, or combinations thereof. In some embodiments, the salt is both NaCl and CaCl2.

[0076] In some embodiments, the concentration of NaCl in the aqueous test sample is beneficial for accurate and / or effective detection of lipolytic activity in the sample. In some embodiments, the concentration of NaCl in the aqueous test sample is from about 10 mM to about 500 mM. In some embodiments, the concentration of NaCl in the aqueous test sample is from about 25 mM to about 400 mM. In some embodiments, the concentration of NaCl in the aqueous test sample is from about 50 mM to about 300 mM. In some embodiments, the concentration of NaCl in the aqueous test sample is from about 75 mM to about 250 mM. In some embodiments, the concentration of NaCl in the aqueous test sample is from about 100 mM to about 200 mM. In some embodiments, NaCl is in the aqueous assay buffer at a concentration of about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM.

[0077] In some embodiments, NaCl is present in the final composition (aqueous assay sample and organic solvent) at a concentration of about 10 mM to about 500 mM. In some embodiments, NaCl is present in the final composition at a concentration of about 25 mM to about 400 mM. In some embodiments, NaCl is present in the final composition at a concentration of about 50 mM to about 300 mM. In some embodiments, NaCl is present in the final composition at a concentration of about 75 mM to about 250 mM. In some embodiments, NaCl is present in the final composition at a concentration of about 100 mM to about 200 mM. In some embodiments, NaCl is present in the final composition at a concentration of about 100 mM to about 140 mM, for example, 120 mM. In some embodiments, NaCl is present in the final composition at a concentration of about 50 mM, about 60 mM, about 70 mM, about 80 mM, about 90 mM, about 100 mM, about 110 mM, about 120 mM, about 130 mM, 140 mM, 150 mM, 160 mM, 170 mM, 180 mM, 190 mM, or 200 mM.

[0078] In some embodiments, the CaCl2 concentration in the aqueous test sample is beneficial for accurate and / or effective detection of lipolytic activity in the sample. In some embodiments, the CaCl2 concentration in the aqueous test sample is from about 0.1 mM to about 20 mM. In some embodiments, the CaCl2 concentration in the aqueous test sample is from about 0.2 mM to about 10 mM. In some embodiments, the CaCl2 concentration in the aqueous test sample is from about 0.5 mM to about 5.0 mM. In some embodiments, the CaCl2 concentration in the aqueous test sample is from about 0.7 mM to about 3.0 mM. In some embodiments, the CaCl2 concentration in the aqueous test sample is from about 1.0 mM to about 2.0 mM. In some embodiments, the CaCl2 concentration in the aqueous assay sample is about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.5 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, or about 5.0 mM.

[0079] In some embodiments, CaCl2 is present in the final composition (aqueous assay sample and organic solvent) at a concentration of about 0.1 mM to about 20 mM. In some embodiments, CaCl2 is present in the final composition at a concentration of about 0.2 mM to about 10 mM. In some embodiments, CaCl2 is present in the final composition at a concentration of about 0.5 mM to about 5.0 mM. In some embodiments, CaCl2 is present in the final composition at a concentration of about 0.7 mM to about 3.0 mM. In some embodiments, CaCl2 is present in the final composition at a concentration of about 1.0 mM to about 2.0 mM. In some embodiments, CaCl2 is present in the final composition at amounts of about 0.5 mM, about 0.6 mM, about 0.7 mM, about 0.8 mM, about 0.9 mM, about 1.0 mM, about 1.1 mM, about 1.2 mM, about 1.3 mM, about 1.5 mM, about 1.5 mM, about 1.6 mM, about 1.7 mM, about 1.8 mM, about 1.9 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 3.5 mM, about 4.0 mM, about 4.5 mM, or about 5.0 mM.

[0080] In some embodiments, NaCl and CaCl2 reduce and / or prevent the degradation of one or more components in the aqueous assay sample. In some embodiments, NaCl and CaCl2 reduce and / or prevent the aggregation and / or precipitation of proteins, such as therapeutic proteins. In some embodiments, NaCl and CaCl2 reduce and / or prevent the degradation of one or more components in the composition that are not present in the aqueous assay sample, for example, in an organic solvent. In some embodiments, NaCl and CaCl2 reduce and / or prevent the autohydrolysis of 4-methylumbelliferyl oleate (4MuO). In some embodiments, in the aqueous assay sample, NaCl is present at a concentration of about 10 mM to about 500 mM, and CaCl2 is present at a concentration of about 0.1 mM to about 20 mM. In some embodiments, in the aqueous assay sample, NaCl is present at a concentration of about 25 mM to about 400 mM, and CaCl2 is present at a concentration of about 0.2 mM to about 10 mM. In some embodiments, in the aqueous assay sample, NaCl is present at a concentration of about 50 mM to about 300 mM, and CaCl2 is present at a concentration of about 0.5 mM to about 5.0 mM. In some embodiments, in the aqueous test sample, NaCl is about 75 mM to about 250 mM, and CaCl2 is about 0.7 to about 3.0 mM. In some embodiments, in the aqueous test sample, NaCl is about 100 mM to about 200 mM, and CaCl2 is about 1.0 to about 2.0 mM. In some embodiments, in the aqueous test sample, NaCl is about 150 mM, and CaCl2 is about 0.3 mM.

[0081] As used herein, a "buffer" is a substance used in solution to maintain the pH of the solution. A buffer can maintain a solution within a certain pH range (i.e., buffering capacity within a specific range) and prevent rapid pH changes when additional components are added to the solution. Generally, a buffer can be a weak acid or a weak base. In some embodiments, the buffer has buffering capacity at about pH 5.0, about pH 5.5, about pH 6.0, about pH 6.5, or about pH 7.0. Buffers with buffering capacity at about pH 5.0 to about pH 7.0 include, for example, citrates, acetates, phosphates, MES, Bis-Tris, ADA, ACES, PIPES, MOPSO, Bis-Tris propane, BES, MOPS, TES, HEPES, DIPSO, MOBS, TAPSO, and Tris. The buffering capacity of a buffer can be determined by a person skilled in the art. In some embodiments, a buffer in an aqueous assay sample is beneficial for the accurate and / or efficient detection of lipolytic activity in the sample. In some embodiments, the buffer reduces and / or prevents the degradation of one or more components in the aqueous assay sample. In some embodiments, the buffer reduces and / or prevents the aggregation of proteins, such as therapeutic proteins. In some embodiments, the buffer reduces and / or prevents the degradation of one or more components in the composition that are not present in the aqueous assay sample, for example, in the organic solvent of the composition. In some embodiments, the buffer reduces and / or prevents the autohydrolysis of 4-methylumbelliferyl oleate (4MuO). In some embodiments, the buffer is provided in the aqueous assay sample in the form of an aqueous buffer solution.

[0082] In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 5 mM to about 200 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 10 mM to about 100 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 20 mM to about 80 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 30 mM to about 70 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 40 mM to about 60 mM. In some embodiments, the buffer in the aqueous test sample is about 10 mM, about 12 mM, about 15 mM, about 18 mM, about 20 mM, about 22 mM, about 25 mM, about 28 mM, about 30 mM, about 32 mM, about 35 mM, about 38 mM, about 40 mM, about 42 mM, about 45 mM, about 48 mM, about 50 mM, about 52 mM, about 55 mM, about 58 mM, about 60 mM, about 62 mM, about 65 mM, about 68 mM, about 70 mM, about 72 mM, about 75 mM, about 78 mM, about 80 mM, about 82 mM, about 85 mM, about 88 mM, about 90 mM, about 92 mM, about 95 mM, about 98 mM, or about 100 mM. In some embodiments, the buffer is Bis-Tris. In some embodiments, the buffer is Tris.

[0083] In some embodiments, the buffer is present in the final composition (aqueous assay sample and organic solvent) at a concentration of about 5 mM to about 200 mM. In some embodiments, the buffer is present in the final composition (aqueous assay sample and organic solvent) at a concentration of about 10 mM to about 100 mM. In some embodiments, the buffer is present in the final composition at a concentration of about 20 mM to about 80 mM. In some embodiments, the buffer is present in the final composition at a concentration of about 30 mM to about 70 mM. In some embodiments, the buffer is present in the final composition at a concentration of about 40 mM to about 60 mM. In some embodiments, the buffer is in the final composition at about 10 mM, about 12 mM, about 15 mM, about 18 mM, about 20 mM, about 22 mM, about 25 mM, about 28 mM, about 30 mM, about 32 mM, about 35 mM, about 38 mM, about 40 mM, about 42 mM, about 45 mM, about 48 mM, about 50 mM, about 52 mM, about 55 mM, about 58 mM, about 60 mM, about 62 mM, about 65 mM, about 68 mM, about 70 mM, about 72 mM, about 75 mM, about 78 mM, about 80 mM, about 82 mM, about 85 mM, about 88 mM, about 90 mM, about 92 mM, about 95 mM, about 98 mM, or about 100 mM.

[0084] In some embodiments, the aqueous test sample comprises NaCl, CaCl2, and a buffer. In some embodiments, in the aqueous test sample, NaCl is present in a concentration of about 10 mM to about 500 mM, CaCl2 in a concentration of about 0.1 mM to about 20 mM, and the buffer in a concentration of about 5 mM to about 200 mM. In some embodiments, in the aqueous test sample, NaCl is present in a concentration of about 25 mM to about 400 mM, CaCl2 in a concentration of about 0.2 mM to about 10 mM, and the buffer in a concentration of about 10 mM to about 100 mM. In some embodiments, in the aqueous test sample, NaCl is present in a concentration of about 50 mM to about 300 mM, CaCl2 in a concentration of about 0.5 mM to about 5.0 mM, and the buffer in a concentration of about 20 mM to about 8 mM. In some embodiments, in the aqueous test sample, NaCl is present in a concentration of about 75 mM to about 250 mM, CaCl2 in a concentration of about 0.7 mM to about 3.0 mM, and the buffer in a concentration of about 30 mM to about 70 mM. In some embodiments, in the aqueous assay sample, NaCl is about 100 mM to about 200 mM, CaCl2 is about 1.0 to about 2.0 mM, and the buffer is about 40 mM to about 60 mM. In some embodiments, in the aqueous assay sample, NaCl is about 150 mM, CaCl2 is about 0.3 mM, and the buffer is about 45 mM to about 55 mM. In some embodiments, the buffer is Bis-Tris. In some embodiments, the buffer is Tris. Those skilled in the art will recognize that salts and buffers are commonly present in protein formulations, and therefore the above percentages are provided merely as examples.

[0085] In some embodiments, the pH of the aqueous assay sample is adjusted to maximize the fluorescence intensity of 4Mu. In some embodiments, the pH of the aqueous assay sample is adjusted to stabilize one or more components of the aqueous assay sample and / or organic solvent. In some embodiments, a slightly acidic to neutral pH (e.g., about 5.0 to about 7.0) minimizes the degradation of components in the aqueous assay sample. In some embodiments, a slightly acidic to neutral pH (e.g., about 5.0 to about 7.0) minimizes the aggregation of therapeutic proteins. In some embodiments, a slightly acidic to neutral pH (e.g., about 5.0 to about 7.0) minimizes the autohydrolysis of 4-methylumbelliferyl oleate (4MuO).

[0086] In some embodiments, the aqueous test sample has an acidic pH. In some embodiments, the pH of the aqueous test sample is 5.0 to 7.0. In some embodiments, the pH of the aqueous test sample is about 5.0, about 5.1, about 5.2, about 5.3, about 5.4, about 5.5, about 5.6, about 5.7, about 5.8, about 5.9, about 6.0, about 6.1, about 6.2, about 6.3, about 6.4, about 6.5, about 6.6, about 6.7, about 6.8, about 6.9, or about 7.0.

[0087] In some embodiments, the compositions disclosed herein comprise the aqueous assay sample described herein, and an organic solvent. As used herein, "organic solvent" refers to a carbon-based substance that can be used to dissolve one or more solutes. Examples of organic solvents include, but are not limited to, hydrocarbons, including, for example, aliphatic hydrocarbons, cyclic hydrocarbons, aromatic hydrocarbons, and halogenated hydrocarbons; ketones; amines; esters; alcohols; aldehydes; ethers; nitriles; sulfoxides, etc. In some embodiments, the organic solvent is capable of dissolving 4-methylumbelliferyl ketone oleate (4MuO).

[0088] In some embodiments, the organic solvent comprises an alcohol, sulfoxide, nitrile, or a combination thereof. In some embodiments, the organic solvent is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent comprises acetonitrile (ACN). In some embodiments, the organic solvent comprises an alcohol. In some embodiments, the organic solvent is a C1-C6 alcohol. In some embodiments, the C1-C6 alcohol is methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, tert-butanol, pentanol, or hexanol. In some embodiments, the organic solvent is methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, tert-butanol, or a combination thereof. In some embodiments, the organic solvent comprises a mixture of acetonitrile and an alcohol. In some embodiments, the organic solvent comprises a mixture of acetonitrile and isopropanol. In some embodiments, acetonitrile and isopropanol are mixed in a ratio of about 5:1, about 4:1, about 3:1, about 2:1, or about 1:1.

[0089] In some embodiments, 4-methylumbelliferyl oleate (4MuO) is present in the organic solvent. 4MuO has the following structure:

[0090]

[0091] In some embodiments, 4MuO is hydrolyzed to form oleic acid and 4-methylumbelliferone (4Mu) (Scheme I):

[0092]

[0093] Option I

[0094] In some embodiments, 4MuO does not fluoresce. In some embodiments, 4Mu fluoresces. In some embodiments, the fluorescence of 4Mu is used to detect the hydrolysis of 4MuO by lipases and is therefore an indicator of lipolysis activity. Those skilled in the art will understand that the hydrolysis of 4MuO (measured by 4Mu fluorescence) may indicate that lipolysis has occurred in the protein formulation, i.e., the surfactant may have been hydrolyzed, which may destabilize the protein formulation. In some embodiments, the fluorescence of 4Mu can be measured at an excitation wavelength of about 330 nm and an emission wavelength of about 495 nm. In some embodiments, the fluorescence of 4Mu can be measured at an excitation wavelength of about 327 nm and an emission wavelength of about 449 nm. In some embodiments, the fluorescence of 4Mu can be measured at an excitation wavelength of about 300 nm to about 350 nm and an emission wavelength of about 420 nm to about 500 nm. In some embodiments, the fluorescence measurement parameters of 4Mu (e.g., excitation and emission wavelengths) change when the pH changes. In some embodiments, the fluorescence measurement parameters of 4Mu change when the salt and / or buffer concentration changes. In some embodiments, 4MuO is a substrate of lipases. In some embodiments, 4MuO is hydrolyzed by a lipase in the protein formulation described herein. In some embodiments, the formation of 4Mu is measured by fluorescence. In some embodiments, the lipolytic activity of an assay sample containing the protein formulation described herein is measured by the fluorescence of 4Mu.

[0095] Various concentrations of 4MuO can be used in the compositions and methods described herein. Generally, the amount of 4MuO should be minimized to minimize the effects of autohydrolysis. In some embodiments, the 4MuO in the organic solvent is about 1 μM to about 1 mM, or about 10 μM to about 500 μM, or about 20 μM to about 200 μM, or about 50 μM to about 150 μM, or about 75 μM to about 125 μM, or about 100 μM.

[0096] In some embodiments, the composition comprises an aqueous assay sample containing the protein formulation described herein, and an organic solvent containing 4 MuO as described herein. In some embodiments, the composition does not contain an equal volume of aqueous assay sample and organic solvent. In some embodiments, the amount of organic solvent in the composition is less than the amount of aqueous assay buffer in the composition to minimize the potential adverse effects of the organic solvent on the protein formulation, particularly therapeutic proteins. For example, if the amount of organic solvent is too high, the therapeutic protein may aggregate. In some embodiments, the aqueous assay sample is about 70% to about 99.9% by volume of the composition, and the organic solvent is about 0.1% to about 30% by volume of the composition. In some embodiments, the aqueous assay sample is about 70% to about 99.5% by volume of the composition, and the organic solvent is about 0.5% to about 30% by volume of the composition. In some embodiments, the aqueous assay sample is about 70% to about 99% by volume of the composition, and the organic solvent is about 1% to about 30% by volume of the composition. In some embodiments, the aqueous assay sample is about 75% to about 99% by volume of the composition, and the organic solvent is about 1% to about 25% by volume of the composition. In some embodiments, the aqueous sample comprises about 80% to about 98% by volume of the composition, and the organic solvent comprises about 2% to about 20% by volume of the composition. In some embodiments, the aqueous sample comprises about 90% to about 98% by volume of the composition, and the organic solvent comprises about 2% to about 10% by volume of the composition. In some embodiments, the aqueous sample comprises about 95% to about 98% by volume of the composition, and the organic solvent comprises about 2% to about 5% by volume of the composition.

[0097] In some embodiments, the aqueous test sample is 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 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%, or about 99.9% by volume of the composition. In some embodiments, the protein formulation comprises about 70% to about 85%, about 75% to about 85%, or about 80% to about 85% by volume of the composition, and the non-protein formulation components such as buffers and / or salts of the aqueous assay sample comprise about 15% to about 30%, about 15% to about 25%, or about 15% to about 20% by volume of the composition. In some embodiments, the organic solvent is about 0.1%, about 0.2%, about 0.3%, about 0.4%, about 0.5%, about 0.6%, about 0.7%, about 0.8%, about 0.9%, about 1%, about 2%, about 3%, about 4%, about 5%, about 6%, about 7%, about 8%, about 9%, about 10%, about 11%, about 12%, about 13%, about 14%, about 15%, about 16%, about 17%, about 18%, about 19%, about 20%, about 25%, about 30%, about 35%, about 40%, about 45%, or about 50% by volume of the composition. Those skilled in the art will recognize that salts and buffers are commonly present in protein formulations, and therefore the above percentages are used only as examples.

[0098] In some embodiments, the composition further comprises a lipase inhibitor. In some embodiments, the lipase inhibitor reduces or eliminates lipolytic activity in the composition by inactivating lipase. In some embodiments, the lipase inhibitor is included in the composition to provide a negative control for detecting lipolytic activity, i.e., the composition containing the lipase inhibitor is expected to have no lipolytic activity. In some embodiments, the lipase inhibitor is added to the composition after detecting lipolytic activity, for example, by measuring fluorescence at 4 Mu. In some embodiments, the lipase inhibitor is in an aqueous assay sample. In some embodiments, the lipase inhibitor is water-soluble. In some embodiments, the lipase inhibitor is in an organic solvent. In some embodiments, the lipase inhibitor is not water-soluble.

[0099] In some embodiments, the concentration of the lipase inhibitor is sufficient to reduce or eliminate lipolytic activity in the composition. In some embodiments, the concentration of the lipase inhibitor is sufficient to reduce the lipolytic activity in the composition by about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100%. In some embodiments, the lipase inhibitor is present in the composition at a concentration of about 1 μM to about 50 μM. In some embodiments, the lipase inhibitor is present in the composition at a concentration of about 2 μM to about 40 μM. In some embodiments, the lipase inhibitor is present in the composition at a concentration of about 3 μM to about 35 μM. In some embodiments, the lipase inhibitor is present in the composition at a concentration of about 4 μM to about 30 μM. In some embodiments, the lipase inhibitor is present in the composition at a concentration of about 5 μM to about 25 μM. In some embodiments, the lipase inhibitor is in the composition in the form of about 1 μM, about 2 μM, about 3 μM, about 4 μM, about 5 μM, about 6 μM, about 7 μM, about 8 μM, about 9 μM, about 10 μM, about 11 μM, about 12 μM, about 13 μM, about 14 μM, about 15 μM, about 16 μM, about 17 μM, about 17 μM, about 18 μM, about 19 μM, about 20 μM, about 21 μM, about 22 μM, about 23 μM, about 24 μM, about 25 μM, about 30 μM, about 35 μM, about 40 μM, about 45 μM, or about 50 μM.

[0100] In some embodiments, the lipase inhibitor is (S)-2-formamido-4-methylvaleric acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetane]methyl]-dodecyl ester (orlistat). In some embodiments, the lipase inhibitor is an alkaloid, such as caffeine, theophylline, and theobromine. In some embodiments, the lipase inhibitor is a carotenoid, such as, for example, fucoxanthin. In some embodiments, the lipase inhibitor is a glycoside, such as cimicifugoside, kaempferol-3-O-rutinoside, rutin, kaempferol, quercetin, and luteolin. In some embodiments, the lipase inhibitor is a polyphenol, such as galangin, hesperidin, licohalcone A, CT-II, 7-phloroeckol, and isoliquiritigenin. In some embodiments, the lipase inhibitor is a saponin, such as sessiloside and chiianoside. In some embodiments, the lipase inhibitor is a terpene, such as crocin and crocinic acid. In some embodiments, the lipase inhibitor is derived from bacteria, such as lipristatin, valyl lactone, percyquinnin, panclicin, erbinolactone, styracin, and astatin. In some embodiments, the lipase inhibitor is a synthetic lipase inhibitor, such as a synthetic analog of a natural fat. A review of lipase inhibitors is available in Lunagariya et al., EXCLI Journal (EXCLIJ) 13:897-921 (2014).

[0101] In some embodiments, a composition is disclosed comprising: (a) about 90% to about 99.9% (vol / vol) of an aqueous assay sample, said aqueous assay sample comprising (i) a purified protein formulation containing proteins and lipids; (ii) a buffer; (iii) about 1.0 mM to about 2.0 mM calcium chloride; and (iv) about 100 mM to about 200 mM sodium chloride; and (b) about 10% to about 0.1% (vol / vol) of an organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethyl sulfoxide (DMSO), acetonitrile, or combinations thereof, further comprising 4-methylumbelliferyl oleate (4MuO); wherein the pH of the aqueous assay sample is 5.0 to 7.0.

[0102] In some embodiments, a composition is disclosed comprising: (a) about 90% to about 99.9% (vol / vol) of an aqueous assay sample, said aqueous assay sample comprising (i) a purified protein formulation containing a protein and a polysorbate surfactant; (ii) a buffer; (iii) about 1.0 mM to about 2.0 mM calcium chloride; and (iv) about 100 mM to about 200 mM sodium chloride; and (b) about 10% to about 0.1% (vol / vol) of an organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethyl sulfoxide (DMSO), acetonitrile, or combinations thereof, further comprising 4-methylumbelliferyl oleate (4MnO); wherein the pH of the aqueous assay sample is 5.0 to 7.0.

[0103] In some embodiments, a composition is disclosed comprising: (a) about 90% to about 99.9% (vol / vol) of an aqueous assay sample, said aqueous assay sample comprising (i) a partially purified protein formulation comprising proteins and lipids; (ii) a buffer; (iii) about 1.0 mM to about 2.0 mM calcium chloride; and (iv) about 100 mM to about 200 mM sodium chloride; and (b) about 10% to about 0.1% (vol / vol) of an organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethyl sulfoxide (DMSO), acetonitrile, or combinations thereof, further comprising 4-methylumbelliferyl oleate (4MuO); wherein the pH of the aqueous assay sample is 5.0 to 7.0.

[0104] In some embodiments, a composition is disclosed comprising: (a) about 90% to about 99.9% (vol / vol) of an aqueous assay sample, said aqueous assay sample comprising (i) a cell culture supernatant containing proteins and lipids; (ii) a buffer; (iii) about 1.0 mM to about 2.0 mM calcium chloride; and (iv) about 100 mM to about 200 mM sodium chloride; and (b) about 10% to about 0.1% (vol / vol) of an organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethyl sulfoxide (DMSO), acetonitrile, or combinations thereof, further comprising 4-methylumbelliferyl oleate (4MnO); wherein the pH of the aqueous assay sample is 5.0 to 7.0.

[0105] In other embodiments, the compositions provided herein are suitable for use in methods for detecting lipolytic activity in protein formulations. In some embodiments, this disclosure further provides a method for detecting lipolytic activity in aqueous assay samples.

[0106] In some embodiments, this disclosure provides a method for detecting lipolytic activity in an aqueous assay sample, the method comprising (a) combining an aqueous assay sample containing a protein formulation with an organic solvent containing 4-methylumbelliferone oleate (4MuO); and (b) measuring the formation of the oleate and 4-methylumbelliferone (4Mu) by fluorescence.

[0107] In some embodiments, the aqueous test sample is the aqueous test sample described herein. In some embodiments, the pH of the aqueous test sample is from 5.0 to 7.0.

[0108] In some embodiments, the aqueous assay sample further comprises a buffer, a salt, or both, as described herein. Examples of buffers and salts suitable for this method and their concentrations are also provided herein. In some embodiments, the salt is sodium chloride (NaCl), calcium chloride (CaCl2), or a combination thereof. In some embodiments, the salt is sodium chloride and calcium chloride. In some embodiments, sodium chloride is present in the aqueous assay sample at a concentration of about 50 mM to about 400 mM. In some embodiments, sodium chloride is present in the aqueous assay sample at a concentration of about 100 mM to about 200 mM. In some embodiments, sodium chloride is present in the aqueous assay sample at a concentration of about 0.2 mM to about 10 mM. In some embodiments, sodium chloride is present in the aqueous assay sample at a concentration of about 1.0 mM to about 2.0 mM.

[0109] In some embodiments, the buffer has buffering capacity at about pH 6.0. In some embodiments, the buffer is Tris. In some embodiments, the buffer is Bis-Tris. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 2 mM to about 200 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 10 mM to about 100 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 40 mM to about 60 mM. In some embodiments, the buffer is present in the aqueous test sample at a concentration of about 45 mM to about 55 mM.

[0110] In some embodiments, the protein formulation is the protein formulation described herein. In some embodiments, the protein formulation is a cell culture supernatant. In some embodiments, the protein formulation is a partially purified protein formulation. In some embodiments, the protein formulation is a purified protein formulation. Protein formulations purified to various degrees are described herein, such as cell culture supernatants, partially purified protein formulations, and purified protein formulations. In some embodiments, the protein formulation comprises a therapeutic protein, such as the therapeutic protein described herein. The methods disclosed herein advantageously allow for the simple and efficient determination of lipolytic activity in the protein formulation throughout its purification process. For example, the lipolytic activity of a cell culture supernatant (or product from a subsequent purification process) can be measured using this method to determine whether it is necessary to remove lipases during subsequent purification steps. Advantageously, this method can be used on the product throughout the purification process to determine whether the lipolytic activity in the protein formulation has been adequately eliminated.

[0111] In some embodiments, the protein formulation comprises additional host cell proteins. In some embodiments, the additional host cell proteins comprise lipases. Lipases are described herein.

[0112] In some embodiments, the protein formulation comprises a surfactant, such as the surfactant described herein. In some embodiments, the surfactant is a polysorbate. In some embodiments, the polysorbate is polysorbate 20, polysorbate 80, or a combination thereof.

[0113] In some embodiments, the organic solvent is the organic solvent described herein. In some embodiments, the organic solvent is an alcohol, sulfoxide, nitrile, or a combination thereof. In some embodiments, the organic solvent is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent comprises acetonitrile. In some embodiments, the organic solvent comprises an alcohol. In some embodiments, the organic solvent is a C1-C6 alcohol. In some embodiments, the organic solvent is methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, tert-butanol, or a combination thereof. In some embodiments, the organic solvent comprises a mixture of acetonitrile and isopropanol. In some embodiments, acetonitrile and isopropanol are mixed in a ratio of approximately 3:1.

[0114] In some embodiments, the organic solvent comprises 4-methylumbelliferone oleate (4MuO). The structure of 4MuO is provided herein. In some embodiments, 4MuO is hydrolyzed to form oleic acid and 4-methylumbelliferone (4Mu), as described, for example, in Scheme I. The structure of 4Mu is provided herein. In some embodiments, 4Mu fluoresces. In some embodiments, the fluorescence of 4Mu is measured at excitation at approximately 330 nm and emission at 495 nm.

[0115] In some embodiments, the method includes measuring fluorescence for up to 24 hours. In some embodiments, fluorescence is measured for about 24 hours to about 400 hours. In some embodiments, fluorescence is measured for more than about 24 hours. In some embodiments, fluorescence is measured for more than about 100 hours. In some embodiments, fluorescence is measured for more than about 300 hours. It should be understood that fluorescence measurement is not necessarily continuous and can be measured at predetermined time points. In some embodiments, fluorescence is measured at selected time points from about 12 hours to about 400 hours. In some embodiments, fluorescence is measured at time points of about 24 hours, about 48 hours, about 72 hours, about 96 hours, about 120 hours, about 144 hours, about 168 hours, about 192 hours, about 216 hours, about 240 hours, about 264 hours, about 288 hours, about 312 hours, about 336 hours, about 360 hours, about 384 hours, or about 400 hours. The time period for measuring fluorescence can be selected based on the level of lipase activity in the protein formulation. For example, due to the slow hydrolysis of 4MuO, low levels of lipolysis activity may require a longer detection period.

[0116] In some embodiments, a protein formulation containing a therapeutic protein is purified and then stored for a period of time (e.g., less than 4 hours, less than 8 hours, less than 1 day, about 1 day, about 2 days, about 3 days, about 4 days, about 5 days, about 6 days, about 1 week, more than 1 week, about 2 weeks, more than 2 weeks, about 3 weeks, more than 3 weeks, about 1 month, more than 1 month, about 2 months, more than 2 months, about 3 months, or more than 3 months). The protein formulation is then subjected to this method to test for lipolytic activity.

[0117] In some embodiments, the aqueous test sample and the organic solvent are combined in a ratio of about 70:30 to about 99:1. In some embodiments, the aqueous test sample and the organic solvent are combined in a ratio of about 75:25 to about 99:1. In some embodiments, the aqueous test sample and the organic solvent are combined in a ratio of about 80:20 to about 98:2. In some embodiments, the aqueous test sample and the organic solvent are combined in a ratio of about 85:15 to about 98:2. In some embodiments, the aqueous test sample and the organic solvent are combined in a ratio of about 90:10 to about 98:2. In some embodiments, the aqueous test sample and the organic solvent are combined in a ratio of about 95:5 to about 98:2.

[0118] In some embodiments, the aqueous assay sample containing the protein formulation is incubated with a lipase inhibitor for about 10 minutes to about 1 hour before combining with the organic solvent in step (a). In some embodiments, the aqueous assay sample is incubated with a lipase inhibitor for about 15 minutes to about 45 minutes, about 20 minutes to about 40 minutes, or about 30 minutes before step (a). In some embodiments, incubating the aqueous assay sample with a lipase inhibitor reduces or eliminates lipolysis activity. In some embodiments, incubating the aqueous assay sample with a lipase inhibitor provides a negative control for detecting lipolysis activity. In embodiments where the aqueous assay sample is incubated with a lipase inhibitor before step (a), low fluorescence is expected, indicating low or absent lipolysis activity. Lipase inhibitors are described herein. In some embodiments, the lipase inhibitor is (S)-2-formamido-4-methylvaleric acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetane]methyl]-dodecyl ester (orlistat). In some embodiments, the lipase inhibitor is present in the composition at a concentration of about 1 μM to about 50 μM. In some embodiments, the lipase inhibitor is present in the composition at a concentration of about 5 μM to about 25 μM.

[0119] In some embodiments, lipolytic activity is measured in parallel with that of a control sample and an aqueous assay sample. In some embodiments, this disclosure provides a method for detecting lipolytic activity in an aqueous assay sample, the method comprising (a) combining an aqueous assay sample containing a protein formulation with an organic solvent containing 4-methylumbelliferyl oleate (4MuO) to form an assay composition; (b) combining a control sample containing a protein formulation and a lipase inhibitor with an organic solvent containing 4-methylumbelliferyl oleate (4MuO) to form a control composition; and (c) determining the formation of oleate and 4-methylumbelliferyl oleate (4Mu) in the composition and the control composition by fluorescence measurement. In some embodiments, the protein formulation in (a) and the protein formulation in (b) are provided by the same protein formulation. For example, the protein formulation may be a cell culture from which two aliquots are taken. One aliquot may be a protein formulation of the aqueous assay sample, and the other aliquot may be a protein formulation of the control sample. In some embodiments, the protein formulation in (a) and the protein formulation in (b) contain substantially the same components. In some embodiments, the protein formulation in (a) and the protein formulation in (b) are expected to have the same level of lipolytic activity. In some embodiments, the aqueous assay sample and the control sample have substantially the same components, except for the lipase inhibitor in the control sample. In some embodiments, the control sample containing the lipase inhibitor is a negative control sample, i.e., fluorescence is not expected to be detected. In some embodiments, the method further utilizes a positive control sample, i.e., fluorescence is expected to be present. In some embodiments, the positive control sample contains a known amount of 4MuO. In some embodiments, the positive control sample contains a known amount of 4MuO and a known amount of active lipase.

[0120] As discussed herein, lipases with lipolytic activity can interfere with components of protein formulations. In some embodiments, lipases with lipolytic activity hydrolyze fatty acids and / or esters present in the protein formulation. In some embodiments, lipases with lipolytic activity hydrolyze surfactants present in the protein formulation. In some embodiments, the hydrolysis of surfactants reduces the stability of the protein formulation. By measuring the amount of lipolytic activity using the methods provided herein, the level of hydrolysis that has occurred in the protein formulation can then be determined based on the measured amount of lipolytic activity, thereby determining the stability of the protein formulation. In some embodiments, this disclosure provides a method for determining the stability of a protein formulation comprising (a) combining an aqueous assay sample containing the protein formulation with an organic solvent containing 4-methylumbelliferone oleate (4MuO); (b) measuring the formation of the oleate and 4-methylumbelliferone (4Mu) by fluorescence; and (c) determining the stability of the protein formulation based on the measured fluorescence. For example, an increase in fluorescence relative to a control would indicate the presence of lipases, indicating that excipients, such as surfactants like polysorbates, have been hydrolyzed to form nonpolar and therefore insoluble long-chain fatty acids, which may destabilize the protein in the protein formulation. In some embodiments, the method is used to determine the stability of protein formulations used in pharmaceutical formulations.

[0121] In some embodiments, this disclosure provides a kit suitable for providing compositions of the present invention. In some embodiments, this disclosure provides a kit that can be used to carry out the methods of the present invention. For example, in some embodiments, this disclosure further provides a kit comprising: (a) an organic solvent; (b) 4-methylumbelliferyl oleate (4MuO); and (c) a lipase inhibitor in two or more containers.

[0122] Any suitable container may be used in the kit described herein. In some embodiments, the container is a vial. In some embodiments, the container is a bottle. In some embodiments, each container is a compartment of a multi-compartment container. In some embodiments, the organic solvent and 4MuO are in a first container, and the lipase inhibitor is in a second container. In some embodiments, the organic solvent and lipase inhibitor are in a first container, and 4MuO is in a second container. In some embodiments, the lipase inhibitor and 4MuO are in a first container, and the organic solvent is in a second container. In some embodiments, the lipase inhibitor and organic solvent are in a first container, and 4MuO and organic solvent are in a second container. In some embodiments, 4MuO is provided as a solid, for example, a powder. In some embodiments, 4MuO is provided in a solution, for example, in an organic solvent. In some embodiments, the lipase inhibitor is provided as a solid, for example, a powder, such as a lyophilized powder. In some embodiments, the lipase inhibitor is provided in a solution, for example, in an organic solvent. In any of the foregoing embodiments, (a) an organic solvent; (b) 4-methylumbelliferyl oleate (4MuO); and / or (c) a lipase inhibitor may be included in their respective containers to receive a predetermined specific quantity of the protein formulation, wherein the quantity of each component is sufficient to practice the method described herein for determining lipolytic activity. In some embodiments, the kit further includes instructions for determining lipolytic activity using the kit as described herein.

[0123] In some embodiments, the kit further comprises a buffer, a salt, or both. Suitable buffers and salts are described herein. In some embodiments, the user of the kit provides a protein formulation to be used with the kit. In some embodiments, the user's protein formulation is in a buffer that is not suitable for use with the kit, such as a buffer that promotes the autohydrolysis of 4MuO and / or the degradation of lipase inhibitors. In some embodiments, the kit provides a buffer exchange column. In some embodiments, the buffer exchange column exchanges the buffer of the user's protein formulation with a buffer suitable for use with the kit provided herein. Examples of buffer exchange columns include, but are not limited to, ZEBA columns from THERMO FISHER, PD-10, SEPHADEX, HIPREP, and HITRAP columns from GEHEALTHCARE, VIVAFLOW and VIVASPIN concentrators from SARTORIUS, BIO-SPIN and ECONO columns from BIO-RAD, and SPINOUT columns from G-BIOSCIENCES.

[0124] The columns in the kit described herein can be used to exchange buffer systems. Columns used for this purpose are known to those skilled in the art. For example, the columns can be used to exchange buffers in protein formulations for buffers more suitable for carrying out the methods described herein for determining lipolytic activity.

[0125] In some embodiments, this disclosure provides a kit comprising: (a) an organic solvent containing 4-methylumbelliferyl oleate (4MuO); (b) a column suitable for exchanging protein formulation buffers; and (c) a lipase inhibitor.

[0126] Organic solvents suitable for the kits disclosed herein include those described herein. In some embodiments, the organic solvent is an alcohol, sulfoxide, nitrile, or a combination thereof. In some embodiments, the organic solvent is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent comprises acetonitrile. In some embodiments, the organic solvent comprises an alcohol. In some embodiments, the organic solvent is a C1-C6 alcohol. In some embodiments, the organic solvent is methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, or a combination thereof. In some embodiments, the organic solvent comprises a mixture of acetonitrile and isopropanol.

[0127] The lipase inhibitors used in kits applicable to this disclosure comprise the lipase inhibitors described herein. In some embodiments, the lipase inhibitor is (S)-2-formamido-4-methylvaleric acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetane]methyl]-dodecyl ester (orlistat). In some embodiments, the lipase inhibitor is used as a control when practicing the methods described herein for determining lipolytic activity.

[0128] The salts used in the kits applicable to this disclosure include those described herein. In some embodiments, the salt is sodium chloride, calcium chloride, or a combination thereof. In some embodiments, the salt is sodium chloride and calcium chloride.

[0129] The buffers used in the kits made in this disclosure comprise the buffers described herein. In some embodiments, the buffer is Tris. In some embodiments, the buffer is Bis-Tris.

[0130] In some embodiments, the kit further includes instructions for performing an assay to determine lipolytic activity. In some embodiments, the assay includes the methods described herein.

[0131] All references cited in this article, including patents, patent applications, papers, textbooks, etc., and the references cited therein, are incorporated in their entirety by reference to the extent that they have not yet been incorporated.

[0132] Example

[0133] The chemicals and reagents used in the experiments described in this article are as follows.

[0134] 2-Propanol (IPA, 99.9%), acetonitrile (ACN, HPLC Plus, ≥99.9%), calcium chloride (≥97%), Triton X-100 (laboratory grade), sodium dihydrogen phosphate monohydrate (ACS reagent, ≥95%), and dimethyl sulfoxide (DMSO, Reagent Plus, ≥99.5%) were obtained from Sigma-Aldrich (now Merck KGaA). 4-Methylumbelliferone (4Mu, ≥98%) and N-phenyl-1-naphthylamine (NPN, reagent grade, 98%) were obtained from Aldrich. Sodium chloride (Bioreagent, ≥99%)... Hydrochloride (reagent grade, 99.0%) Base (primary standard and buffer, ≥99.9%), porcine pancreatic lipase (PPL, type II, 100-500 U / mg), BIS-TRIS hydrochloride (≥99.0% (titration)), 4-methylumbelliferyl oleate (4MuO, suitable for fluorescence, ≥95% (HPCE)) and P 188 (suitable for cell culture) was obtained from Sigma. Acetonitrile (LC-MS grade) was obtained from Thermo Fisher. Ethanol (EtOH, gradient grade for HPLC) and 25% v / v hydrochloric acid were obtained from Merck. Sucrose (USP / NF, EP, JP, high purity) was obtained from Pfanstiehl. Sodium hydroxide 1M solution was obtained from Honeywell Fluka™. Sodium chloride (USP, multi-compendial), L-histidine (USP, multi-compendial), L-histidine monohydrochloride (FCC, multi-compendial), PS20 (NF), and PS80 (NF) were obtained from JTBaker. Highly purified water, referred to as “water” below, was prepared using a water purification system (Barnstead™ GenPure™ Pro, Thermo Fisher). pH was measured using a pH meter (780 pH meter, Metrohm) and a Pt pH electrode (Unitrode Pt1000, Metrohm). Cell culture harvest medium (CCHF) containing a mixture of lipases as part of HCP, produced from a proprietary CHO cell line expressing a non-therapeutic monoclonal antibody (mAb 1), and mAb 1 were obtained from Lonza Biologics, Slough, UK.

[0135] Example 1A. Lipase Assay Development I: pH

[0136] According to the method described by Kurihara et al. in Biol Pharm Bulletin 26383-385 (2003), lipase assays were developed using 4-methylumbelliferone oleate (4MuO) as a substrate for lipases.

[0137] Porcine pancreatic lipase (PPL) was used for assay development. In this experiment, 500 nM PPL and 4 MuO were incubated for 22 h in one of four different buffer systems, and the fluorescence quenching and autohydrolysis of 4 MuO were tested: (a) 10.4 mM Bis-Tris pH 8.1, (b) 10.4 mM Bis-Tris pH 6, (c) 41.6 mM Bis-Tris pH 6, (d) 104 mM Bis-Tris pH 6.

[0138] Figure 1A-1B The results in Table 1 show that the autohydrolysis of 4MuO is reduced at pH 6 compared to pH 8. Figure 1C The autohydrolysis rate was almost twice that at pH 6. Lipase activity was slightly higher (<20%) at pH 8 compared to pH 6. Buffer concentration appeared to have little effect on autohydrolysis and lipase activity. Lipolytic activity appeared to be highest in 41.6 mM Bis-Tris buffer. Fluorescence quenching was observed in 104 mM Bis-Tris buffer.

[0139] Table 1

[0140]

[0141] The autohydrolysis of 4MuO was further investigated. The pK of 4Mu... a It is approximately 7.7. Figure 1D The pH range for measuring 4 Mu is shown (Figure reprinted from Zhi et al., Journal of Spectroscopy 1(2013) doi:10.1155 / 2013 / 147128). Figure 1E Four forms of 4Mu are shown across various pH ranges (reprinted from Zhi et al., 2013). Form II is universal and, in λ ex For 320nm and λ em Excitation and emission are shown at 445 nm.

[0142] Example 1B. Lipase Assay Development I: pH

[0143] Further investigation was conducted to evaluate the pH of various buffers and lipase assay buffers. The following volume ratios (all v / v) were applied: sample (75%), high-concentration matrix buffer (HCMB) (20%), and organic solvent (5%). The mixture was incubated over a timeframe of approximately 24 hours to approximately 300 hours. Assays were transferred to 96-well plates, and fluorescence intensity was analyzed. For the 4Mu calibration curves, reference standards 4Mu were prepared at concentrations of 0 μM, 0.2 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM in organic solvents and incorporated at 5% (v / v) into other assays to final concentrations of 0 μM, 0.1 μM, 0.05 μM, 0.1 μM, 0.25 μM, 0.5 μM, 1 μM, 2.5 μM, and 5 μM. As can be seen, the slope of the calibration curve shifts slightly from the low calibration range (0.01–0.5 μM) to the high calibration range (up to 5 μM). Therefore, the quantification results of 4Mu in the concentration range up to 0.5 μM were calculated using the simplified calibration curve of 0.01–0.5 μM. Figure 19A and 19B The 4Mu calibration curve is shown in the figure.

[0144] In this study, the “sample” was a placebo-modified buffer containing 20 mM L-histidine, pH 6 (pH adjusted by adding 25% (w / v) HCl or 1 M NaOH), 250 mM sucrose, 0.5% cell culture harvest medium (CCHF), or approximately 0.0025 mg / mL porcine pancreatic lipase (PPL). HCMB contained TRIS (208 mM) at pH 7 and 8 or BIS-TRIS (208 mM) at pH 6 (pH adjusted by adding 25% (w / v) HCl or 1 M NaOH), NaCl (200 mM, 600 mM, or 1800 mM), and CaCl2 (0.52 mM, 5.2 mM, or 52 mM). Organic solvents were methanol (MeOH), dimethyl sulfoxide (DMSO), or isopropanol (IPA) containing 100 μM 4 MuO, with 5% (v / v) organic solvent / substrate added. The assay components were transferred to a 96-well plate and the fluorescence intensity was analyzed.

[0145] For fluorescence analysis, the sample was transferred to a 96-well microplate (Thermo Scientific). TM Nunc TM F96MicroWell TM(Black polystyrene plate). Fluorescence measurements were performed using a Molecular Devices SpectraMax iD3 reader and SMP 7.1 software. After optimizing the intermediate concentration of 4Mu using the wavelength optimization mode of SoftMax Pro 7.1 (SMP 7.1), the maximum excitation wavelength (λ) was determined. ex ) and emission wavelength (λ) em The fluorescence wavelengths were 330 nm and 495 nm, respectively. Fluorescence signals were read over an incubation period of approximately 24 to 300 hours, and a new 96-well microplate was prepared for each read by transferring 200 μL of liquid (containing the sample, HCMB, and organic solvent) into each well. Fluorescence measurements were performed at ambient temperature.

[0146] Evaluation results of two different buffers (TRIS or BIS-TRIS) at pH 6, pH 7, or pH 8 showed... Figure 11 The conversion of 4MuO to 4Mu was measured by monitoring the fluorescence intensity of 4Mu. Samples containing PPL or CCHF and the autohydrolysis of 4MuO were evaluated. The effect of pH change on autohydrolysis (AH) was observed to be the most significant, decreasing by approximately 5-fold when the pH was lowered from 8 to 6. The concentration of 4Mu in samples at different pH values ​​was measured against a calibration curve of 4Mu in a reference sample at the same pH; i.e., the observed trend was not due to different responses of 4Mu fluorescence at different pH values, but rather to different reaction rates. When the pH was lowered from 8 to 6, PPL activity increased almost 2-fold, while CCHF lipolysis activity remained unaffected. The pH dependence of PPL was previously reported in Li et al., “Adsorption and catalytic activity of porcine pancreatic lipase on rod-shaped SBA-15 mesoporous material,” Colloids and Surfaces A: Physicochem Eng Aspects, 2009; 341:79–85. However, this pH dependence may not be the same for any other type of lipase that may be present in the HCP contained in the DS pool. pH 6 was also considered most suitable from the perspective that many therapeutic protein formulations for which polysorbate degradation has been observed and reported generally remain at a slightly acidic pH, and therefore it was hypothesized that the assay pH be adjusted to ~6 to cover the cause responsible for inducing lipase-mediated hydrolysis of polysorbate (e.g., lipase). Therefore, a concentration of 200 mM BIS-TRIS was selected and added to HCMB to produce a final assay concentration of 40 mM at pH 6.

[0147] Example 2A. Development of Lipase Assay II: CaCl2

[0148] Lipase assays were performed using 500 nM PPL and 4 MuO at three different CaCl2 concentrations to investigate the fluorescence quenching and autohydrolysis of 4 MuO: (a) 0.104 mM CaCl2; (b) 1.04 mM CaCl2; and (c) 10.4 mM CaCl2.

[0149] Figure 2A-2C The results in Table 2 show that no autohydrolysis of 4MuO occurred, and no fluorescence quenching caused by chloride anions was observed. Lipase activity was highest in 1.3 mM CaCl2.

[0150] Table 2

[0151]

[0152] Example 2B. Lipase Assay Development II: CaCl2

[0153] Further experiments investigated the concentration of calcium chloride in the lipase assay buffer. The sample, HCMB composition, and experimental procedures were as described in Example 1B, wherein CaCl2 was contained in the HCMB at 0.52 mM, 5.2 mM, or 52 mM, as described in Example 1B.

[0154] Figure 12 The results show the evaluation of different CaCl2 concentrations in HCMB using samples containing PPL or CCHF. It was observed that PPL activity decreased slightly at the highest CaCl2 concentration. Considering the need to establish similar assay conditions for various drug substances / drug product matrices for HCMB, 5 mM CaCl2 was added to HCMB to produce a final assay concentration of 1 mM CaCl2.

[0155] Example 3A. Lipase Assay Development III: NaCl

[0156] The concentration of sodium chloride in the lipase assay buffer was investigated. Lipase assays were performed at three different NaCl concentrations using 500 mM PPL and 4 MuO to investigate the fluorescence quenching and autohydrolysis of 4 MuO: (a) 40 mM NaCl; (b) 120 mM NaCl; and (c) 360 mM NaCl.

[0157] Figures 3A-3C The results in Table 3 show that no autohydrolysis of 4MuO occurred and no fluorescence quenching caused by chloride anions was observed. Lipase activity was highest in 120 mM NaCl.

[0158] Table 3

[0159]

[0160] Example 3B. Lipase Assay Development III: NaCl

[0161] Further experiments investigated the sodium chloride concentration in the lipase assay buffer. Samples, HCBM composition, and experimental procedures were as described in Example 1B. As described in Example 1B, NaCl was contained in the HCBM at 200 mM, 600 mM, or 1800 mM.

[0162] Figure 13 The results of evaluating different NaCl concentrations in HCMB using samples containing PPL or CCHF are shown. It was observed that the activity of PPL decreased slightly at the highest NaCl concentration. Considering the need to establish similar assay conditions for various drug substances / drug product matrices for HCMB, 600 mM NaCl was added to HCMB to produce a final assay concentration of 120 mM NaCl.

[0163] Example 4A. Development of Lipase Assay IV: Organic Solvents

[0164] Organic solvents in lipase assay buffers were investigated. Lipase assays using 500 nM PPL and 4 MuO were performed using three different organic solvents to investigate the fluorescence quenching and autohydrolysis of 4 MuO: (a) DMSO; (b) isopropanol (IPA); and (c) methanol (MeOH).

[0165] Figures 4A-4C The results in Table 4 show that isopropanol exhibited the lowest autohydrolysis among the three solvents, but with slight fluorescence quenching. Methanol, however, showed similar results. Furthermore, Glogauer et al., *MicrobCell Fact* 10:54 (2011), suggest that methanol may have the ability to enhance activity.

[0166] Table 4

[0167]

[0168] Example 4B. Development of Lipase Assay IV: Organic Solvents

[0169] Further experiments investigated the organic solvents in the lipase assay buffer. The sample, HCBM composition, and experimental procedures were as described in Example 1B. Organic solvents containing 100 μM 4MnO, such as methanol (MeOH), dimethyl sulfoxide (DMSO), or isopropanol (IPA), were included in the assay reaction described in Example 1B.

[0170] The results of evaluating different organic solvents using samples containing PPL or CCHF showed that Figure 14Unlike the previous findings reported by Glogauer et al., "Identification and characterization of novel true lipases isolated by metagenomic methods" in *Microb Cell Fact*, 2011; 10:54, which reported the strong influence of different solvents on LipC12 lipase isolated from *E. coli* by metagenomic methods, Glogauer et al. found that MeOH and lower levels of IPA showed activating effects, increasing LipC12 lipase activity by more than 10-fold. However, the bacterial enzymes studied may have very different properties, as evidenced by differences in the optimal pH for activity compared to the results in Example 1B. Figure 14 Based on the results, choose to add MeOH to HCMB.

[0171] Example 5A. Development of Lipase Assay V: Surfactant

[0172] The performance of lipase assay buffers in the presence of surfactants was investigated. Lipase assays using 500 nM PPL and 4MuO were performed with two different concentrations of two surfactants to investigate the fluorescence quenching and autohydrolysis of 4MuO: (a) TRITON X-100 0.012% w / v, (b) TRITON X-100 0.06% w / v, (c) KOLLIPHOR P188 0.032% w / v, and (d) KOLLIPHOR P188 0.16% w / v. A surfactant-free control was also tested.

[0173] Figures 5A-5C The results in Table 5 show that the samples containing surfactants exhibited the lowest autohydrolysis, but fluorescence quenching was observed. Lipid-degrading activity also appeared to decrease with the use of surfactants.

[0174] Table 5

[0175]

[0176] Example 5B. Lipase Assay Development V: Surfactant

[0177] Further investigation was conducted to evaluate the effect of surfactants on the lipolytic activity of PPL and CCHF. The surfactants polysorbate-20 (PS20) and polysorbate-80 (PS80), which can be found in pharmaceutical formulations used in biopharmaceuticals, were evaluated. Poloxamer was also tested, as previous work (Gupta et al., “Simplified determination of p-nitrobenzene palmitate by lipases and esterases”, *Analytical Biochemistry*, 2002; 311: 98-99) showed that surfactants dissolve poorly soluble substrates such as p-nitrobenzene palmitate.

[0178] The sample contained 20 mM L-histidine, pH 6, 250 mM sucrose, surfactant (polysorbate-20 (PS20), polysorbate-80 (PS80), or poloxamer, 0.02% or 0.06% (w / v), or as a surfactant-free control), 0.5% CCHF, or 0.0025 mg / mL PPL. HCMB contained 208 mM BIS-TRIS, pH 6, 600 mM NaCl, and 5.2 mM CaCl2. 5% (v / v) of the organic solvent MeOH containing 100 μM 4MeO was added to the assay. The assay fraction was transferred to a 96-well plate, and the fluorescence intensity was analyzed as described above for Example 1B.

[0179] The effects of surfactants on lipolysis activity are shown in Figure 15. The presence of surfactants has a significant impact on lipolysis activity. Polysorbate (PPL) significantly reduced 4Mu formation, decreasing it by >90% for PPL and ~80% for CCHF. Compared to PS20, PS80 showed a slightly more significant reduction in 4Mu formation.

[0180] Several possible reasons for these observations are: (1) polysorbates, composed of fatty acid esters, are competitive substrates / inhibitors of lipases, and / or (2) the nonpolar substrate 4MuO is incorporated into the micelle structure of polysorbates, thereby reducing its concentration in aqueous media and thus negatively affecting the enzyme's rate constant, which is related to substrate concentration (according to concepts such as Michaelis-Menten kinetics) and / or (3) the lipase is structurally affected, which may lead to unfolding and thus a decrease in activity.

[0181] Interestingly, the concentration of polysorbate (PP) appears to affect the lipolytic activity of CCHF, with higher PPP concentrations producing lower activity, while PPL showed similar activity at two different selected concentrations of both PPPs. On the other hand, poloxamer affected the lipolytic activity of both PPL and CCHF in a very different manner. PPL activity decreased by approximately 50% compared to the control, and as already seen with polysorbate, the concentration of the selected surfactant did not show a significant change in enzyme activity. At least at a surfactant concentration of 0.02%, the lipolytic activity of CCHF was positively affected by poloxamer, and was apparently unaffected at a concentration of 0.06%. While not bound by any particular theory, the enzyme behavior in the presence of surfactant and its concentration dependence may be due to a combination of factors. For example, investigating the kinetics and mechanisms of lipases is difficult because enzymes catalyze reactions on substrates that are poorly soluble in water, such as triglycerides and other fats. At the water / lipid interface, enzymes are activated by the movement of cap structures, which protect the active site in a closed form and allow substrate access in an open form. See, for example, Lowe, “Triglyceride lipases of the pancreas,” Journal of Lipid Res, 2002; 43(12): 2007-2016.

[0182] Notably, polysorbate has a negative effect on the lipolytic activity of model lipases PPL and CCHF. The fluorescence intensity of the 4Mu hydrolysis product was also affected in the presence of the surfactant (reduced by approximately 20%) (see [link to relevant documentation]). Figure 15B This also leads to a decrease in the signal strength of 4Mu products, such as... Figure 15A As shown.

[0183] Example 6A. Development of Lipase Assay VI: Lipase Inhibitors

[0184] Orlistat, a lipase inhibitor, was selected for a control study of lipase assays, and the assay performance was evaluated using different concentrations of the inhibitor. Orlistat was developed as a mechanism-based inhibitor that covalently binds to the serine residue of the active site of pancreatic lipase, thereby inhibiting the enzyme. This mode of action makes it suitable as an oral medication for the treatment of obesity and related symptoms based on gastrointestinal lipolysis. No published studies have systematically investigated which classes of lipases can be inhibited by orlistat. However, it has been previously reported that bacterial lipases, such as those from *Streptomyces rimosus*, can be inhibited at millimolecular concentrations of orlistat, and this inhibition occurs through covalent modification of the serine residue at the active site. See, for example, Hadváry et al., “Covalent binding of the lipase inhibitor tetrahydrolipotastine to serine at the putative active site of pancreatic lipase”, *J Biol Chem* 1991; 266(4):2021-2027; Heck et al., “Orlistat, a novel lipase inhibitor for the management of obesity”, *Pharmacotherapy* 2000; 20(3):270-279; Asler et al., “Mass spectroscopic evidence of tetrahydrolipotastine covalently bound to serine at the catalytic site of *Streptomyces cereus* lipase”, *Biochim BiophysActa* 2007; 1770:163–170. In previous studies of this kind, a large volumetric ratio of 50% (v / v) of organic solvent was required in the final assay to introduce this high concentration of hydrophobic inhibitor. Such high organic solvent concentrations were not anticipated in current lipase assays because they could impair the precipitation of other assay components, such as drug-active proteins. Therefore, a lower solvent concentration and a lower inhibitor concentration were chosen. This does not necessarily negatively impact the assay readings, as mammalian lipases have been found to be inhibited at even lower concentrations, i.e., nanomolar concentrations of orlistat. See, for example, Lewis et al., “Direct measurement of orlistat lipase inhibition using a dissolution-linked in vitro assay,” *Clin Pharmacol Biopharm*, 2012; 1(3). 1–3.

[0185] Samples containing 500 nM PPL were co-treated and pre-treated with three concentrations of orlistat (a lipase inhibitor), and lipase activity was tested to investigate 4 MuO fluorescence quenching and autohydrolysis: (a) co-treated with 25 μM orlistat, (b) co-treated with 15 μM orlistat, (c) co-treated with 5 μM orlistat, (d) pre-treated with 25 μM orlistat, (e) pre-treated with 15 μM orlistat, and (f) pre-treated with 5 μM orlistat. A control sample without orlistat was also tested.

[0186] Figures 6A-6B The results in Table 6 show that pretreatment with 25 μM orlistat (30 min) completely eliminated lipolytic activity. Residual activity was observed at lower orlistat concentrations. No fluorescence quenching was observed.

[0187] Table 6

[0188]

[0189] Example 6B. Development of Lipase Assay VI: Lipase Inhibitors

[0190] The concentration of the lipase inhibitor orlistat was evaluated in further experiments. Samples, HCMB composition, and experimental procedures were performed as described in Example 1B. The inhibitor was prepared in MeOH at concentrations of 200, 600, and 1000 μM. For the pre-incubation experiment, the inhibitor solution was added to the assay at 2.5% (v / v) and pre-incubated for 30 minutes. MeOH containing 200 μM 4MuO was then added to the assay at 2.5% (v / v). For the co-incubation experiment, MeOH containing 100 μM 4MuO and orlistat (100 μM, 300 μM, and 500 μM) was added at 5% (v / v). A high PPL concentration of 0.0335 mg / mL was used.

[0191] Three concentrations of orlistat, namely 5 μM, 15 μM, and 25 μM (relative to the final assay), were tested, with the “worst-case” being a very high lipase concentration of 0.0335 mg / mL PPL. At these concentrations, the final assay solution was clear and did not exhibit turbidity due to the potential insolubility of the inhibitor. Furthermore, the effect on lipase activity was investigated when orlistat was presented directly to the assay along with the substrate in 5% (v / v) MeOH, compared to pre-adding orlistat to the enzyme-containing sample in 2.5% (v / v) MeOH and incubating for 30 min before adding another 2.5% (v / v) volume fraction of substrate in MeOH.

[0192] Evaluation results of lipase inhibitors show Figure 16Due to the aforementioned high PPL concentration, 4MuO was almost completely hydrolyzed within 24 hours in the absence of orlistat. It was also observed that orlistat inhibited PPL, and the inhibitory effect was concentration-dependent, with the highest inhibitory effect observed at 25 μM orlistat. Pre-incubation of the enzyme with the inhibitor had a beneficial effect; compared to samples with both substrate and inhibitor added simultaneously, the enzyme activity in the pretreated samples was reduced by approximately 50% at the highest test orlistat concentration of 25 μM. Therefore, orlistat was added to the negative control at a high concentration, i.e., 25 μM, and the negative control was pre-incubated with the inhibitor for approximately 30 minutes before adding the substrate.

[0193] Example 7. Development of Lipase Assay VII: Inhibition of Fatty Acid Products

[0194] Oleic acid and lauric acid were used in the lipase assay to test the inhibition of fatty acid products at two concentrations of theoretically degraded PS20 and PS80: (a) 0.1% and (b) 0.001%. In this experiment, the concentration of PPL was 800 nM.

[0195] Figures 7A-7D The results in Table 7 show that lauric acid at concentrations above 8.5 μM inhibits lipase activity, with no significant difference between 8.5 μM and 850 μM. For oleic acid, significant product inhibition was observed at 780 μM.

[0196] Table 7

[0197]

[0198] Example 8. Lipase Assay Development VIII: Quenching

[0199] The quenching effect of polysorbate 80 was investigated. A control test was conducted against samples containing 800 nM PPL and fresh PS80, with the control sample containing 800 nM PPL and no PS80. Figure 8 The results showed that PS80 reduced the RFU value of each standard, indicating a mild quenching effect.

[0200] Example 9. Evaluation of Lipase Assay: CCHF

[0201] To evaluate the applicability of the assays developed in Examples 1-8 in studying lipolytic activity in pharmaceutical substances / products, the assays were evaluated within a timeframe, such as one month, at lipase concentrations that could lead to polysorbate degradation and in which such degradation would be observed in the pharmaceutical product.

[0202] The samples in this study contained 20 mM L-histidine, pH 6, 250 mM sucrose, 0.02% (w / v) of surfactant PS20 or PS80, and various concentrations of CCHF (0%, 0.001%, 0.005%, 0.01%, 0.05%, 0.1%, 0.5%, 1%, 2%, all v / v). The samples were stored at room temperature and 2–8 °C for one month. The samples were analyzed by HPLC-FMA as described below and by the lipase assays in this paper. For the positive control assay, 0.055 mg / mL of PPL was added to the sample. HCMB contained 208 mM BIS-TRIS, pH 6, 600 mM NaCl, and 5.2 mM CaCl2. 5% (v / v) MeOH organic solvent containing 100 μM 4MuO was added to the assay. For the negative control containing orlistat, the inhibitor was prepared in MeOH at 1000 μM, and 2.5% (v / v) was added to the assay, followed by a 30-minute pre-incubation. Then, MeOH containing 200 μM 4MuO was added to the assay at 2.5% (v / v). The assay fraction was transferred to a 96-well plate, and fluorescence intensity was analyzed as described in Example 1B.

[0203] Complete quantitative analysis of PS20 and PS80 was performed using a high-performance liquid chromatography (HPLC) system (Waters Alliancee 2695) equipped with an isocratic pump, autosampler, tight-fitting reactor spiral tube (1 mL; Supelco #57410-U), and fluorescence detector (Waters 2475FLR detector). Samples were injected with 5 μM N-phenyl-1-naphthylamine (NPN) at 15 ppm (w / v). 35% dissolved in a mobile phase of 150 mM NaCl, 50 mM TRIS, 5% (v / v) ACN, pH 8. In λ ex =350nm and λ em Fluorescence was measured at 420 nm. The Empower 3 Chromatography Data System (CDS) was used for peak integration and analysis.

[0204] Table 9 summarizes the tested samples:

[0205] Table 9

[0206]

[0207]

[0208] HCMB = High Concentration Matrix Buffer: 208mM Bis-Tris pH 6, 600mM NaCl, 5.2mM CaCl2

[0209] The results of HPLC-FMA showed that Figure 9A and 9B The study showed that polysorbate degradation was observed at CCHF concentrations greater than or equal to 0.10%. PS20 samples degraded more readily, with an effect observed at approximately 0.10% CCHF. Higher concentrations showed a decrease in apparent polysorbate concentration. This trend was more pronounced at 25°C compared to 2–8°C. Samples containing PS20 were clearly more prone to degradation than those containing PS80, with a decrease in signal intensity only detectable at 0.5% CCHF. As seen in the previously described surfactant evaluation experiments, PS80 had a slightly enhanced negative effect on lipolysis activity in CCHF-containing samples compared to PS20, and therefore, as a worst-case scenario, caused PS80-containing samples from HPLC-FMA assays to undergo lipase assays.

[0210] The theoretical readings for the lipase assay developed in this paper show that... Figure 9C In the middle, the actual results are shown Figure 9D The actual results were in line with the expected readings. Samples containing different concentrations of CCHF showed that the formation of 4Mu was correlated with CCHF concentration. This enzyme activity was higher than that of the three negative controls: autohydrolyzed and two CCHF samples doped with orlistat. The assay was sensitive enough to detect lipase activity at concentrations where polysorbate degradation had not yet been detected by HPLC-FMA, specifically at CCHF concentrations <0.1%.

[0211] Figures 10A-10H Other lipase assays performed with PS80 in CCHF at different concentration ranges from 0.001% to 2% are shown. Concentrations of CCHF and polysorbate are indicated before the addition of 4M UO and buffer.

[0212] The results of the lipase assay are summarized in Figure 18 In the assay, the 1% CCHF sample doped with PPL (as a positive control) showed higher activity than the sample without PPL doping at the same CCHF concentration. The formation of 4Mu was concentration-dependent, and this lipolytic activity was higher than that of autohydrolysis and other negative controls doped with orlistat. Most notably, the assay was sensitive enough to distinguish lipase activity from autohydrolysis and the negative control at <0.1% CCHF concentrations where polysorbate degradation was not detected by HPLC-FMA, i.e., lipolytic activity could be detected by the lipase assay at CCHF concentrations of 0.01% and 0.05%.

[0213] Example 10. Evaluation of Lipase Assay: Protein-containing formulations

[0214] Biopharmaceutical formulations that may be negatively affected by lipolysis contain biotherapeutic proteins. Lipase assays developed in Examples 1-8 were tested using formulations containing proteins.

[0215] The samples contained 20 mM L-histidine, pH 6, 250 mM sucrose, 0.02% PS80, 10 mg / mL mAb1 (proprietary IgG 1), 0.5% CCHF, or 0.025 mg / mL PPL. HCMB consisted of 208 mM BIS-TRIS, pH 6, 600 mM NaCl, and 5.2 mM CaCl2. 5% (v / v) MeOH containing 100 μM 4-MuO was added to the assay. For the negative control containing orlistat, the inhibitor was prepared at 1000 μM in MeOH, and 2.5% (v / v) was added to the assay, followed by a 30-minute pre-incubation. Then, 2.5% (v / v) MeOH containing 200 μM 4-MuO was added to the assay. The assay fraction was transferred to 96-well plates, and fluorescence intensity was analyzed as described in Example 1B. Table 10 summarizes the tested samples:

[0216] Table 10

[0217]

[0218] Measurement results at different time points (1 hour, 24 hours, 72 hours, 168 hours, and 336 hours) show... Figure 17A As expected, the assay positive control containing both CCHF and PPL showed the highest 4Mu release. The assay negative control showed a strong decrease in activity, indicating that both PPL and CCHF lipases were inhibited by orlistat. The same trend was observed in the sample and formulation positive controls (high activity), while the sample negative control and formulation negative control were inhibited by orlistat (low activity). The autohydrolysis control and formulation negative control showed similar activity at all time points. Figure 17A Readings that cannot be distinguished from each other. Figure 17B A scaled-down view of the negative controls is shown, indicating that the activities in the two negative controls are similar.

[0219] A shift was observed in the apparent activity of the assay / sample negative control compared to the autohydrolysis / formulation negative control. The same shift was observed in the substrate-free blank sample (data not shown), indicating that the protein-containing sample already exhibited a fluorescence response independent of 4Mu. Within the timeframe for studying lipolysis activity, i.e., 336 hours, the shift increased, suggesting that the lipase initially inhibited in CCHF and PPL was slowly reactivated, although this was not apparent in the formulation negative.

[0220] Lookene et al. previously reported that orlistat is not only a mechanism-based inhibitor but also a true substrate of lipoprotein lipase, exhibiting rapid inhibition through the formation of a covalent enzyme-orlistat complex at the active site serine residue, followed by the slow hydrolysis of this complex (see, for example, Lookene et al., “Interaction between lipoprotein lipase and the active site inhibitor tetrahydrolipotassium (orlistat) R,” *Eur J Biochem.* 1994; 222:395-403). This is significant for this assay and its application in evaluating lipolytic activity in pharmaceutical substances / products: if lipolytic enzymes, which are part of the HCP residual fraction in pharmaceutical substances / products and responsible for the degradation of polysorbates (e.g., lipolytic enzymes), can be inhibited by orlistat, they may be reactivated. If lipolytic activity cannot be distinguished between the sample and the negative control, this may indicate either low lipolytic activity in the sample or the presence of lipolytic enzymes that cannot be inhibited by orlistat. In the first scenario, if the formation of 4Mu is not significantly increased compared to the autohydrolysis control, this indicates low lipolytic activity and a low risk of polysorbate degradation in the drug substance / product. If a high level of 4Mu formation is observed in the sample compared to autohydrolysis, this indicates the presence of lipolytic enzymes that are not inhibited by orlistat.

[0221] All references cited in this article, including patents, patent applications, papers, textbooks, etc., and the references cited therein, are incorporated in their entirety by reference to the extent that they have not yet been incorporated.

Claims

1. A composition comprising: a. An aqueous assay sample containing a protein formulation, wherein the protein formulation comprises a therapeutic protein selected from antibodies, antibody fragments, antibody-drug conjugates, antibody-cytokine fusions, Fc fusions, bispecific antibodies, multispecific antibodies, and affinity fusions; and b. An organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate. The pH of the aqueous test sample is between 5.0 and 7.0; and The aqueous test sample comprises 80% to 99.9% of the composition, and the organic solvent comprises 0.1% to 20% of the composition.

2. The composition according to claim 1, wherein the protein preparation is a cell culture supernatant, a partially purified protein preparation, or a purified protein preparation.

3. The composition according to any one of claims 1 to 2, wherein the protein formulation comprises a surfactant.

4. The composition according to claim 3, wherein the surfactant is polysorbate.

5. The composition according to claim 4, wherein the polysorbate is polysorbate-20, polysorbate-80, or a combination thereof.

6. The composition according to any one of claims 1 to 5, wherein the protein formulation further comprises additional host cell proteins.

7. The composition according to any one of claims 1 to 6, wherein the aqueous test sample further comprises a buffer, a salt, or both.

8. The composition according to claim 7, wherein the salt is sodium chloride, calcium chloride, or a combination thereof.

9. The composition according to claim 8, wherein the sodium chloride in the aqueous test sample is 50 mM to 400 mM.

10. The composition according to claim 8 or 9, wherein the calcium chloride in the aqueous test sample is 0.2 mM to 10 mM.

11. The composition according to any one of claims 7 to 10, wherein the buffer is Tris or Bis-Tris.

12. The composition according to any one of claims 7 to 11, wherein the buffer is 2 mM to 200 mM in the aqueous test sample.

13. The composition according to any one of claims 1 to 12, wherein the organic solvent is an alcohol, sulfoxide, nitrile, or a combination thereof.

14. The composition according to any one of claims 1 to 13, wherein the composition further comprises a lipase inhibitor.

15. The composition according to claim 14, wherein the lipase inhibitor is (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetane]methyl]-dodecyl ester of (S)-2-formamido-4-methyl-valeric acid.

16. The composition according to claim 14 or 15, wherein the lipase inhibitor is in the organic solvent.

17. The composition according to claim 1, comprising: a. A water-based test sample of 90% to 99.9% (vol / vol), wherein the water-based test sample comprises i. Purified protein formulations containing therapeutic proteins and lipids; ii. Buffer; iii. Calcium chloride, 1.0 mM to 2.0 mM; and iv. 100 mM to 200 mM sodium chloride; and b. 10% to 0.1% (vol / vol) of an organic solvent, said organic solvent being selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethyl sulfoxide, acetonitrile, or combinations thereof, further comprising 4-methyl umbelliferone oleate. The pH of the aqueous test sample is between 5.0 and 7.

0.

18. A method for detecting lipolytic activity in an aqueous sample, the method comprising: a. Combining an aqueous assay sample containing a protein formulation with an organic solvent containing 4-methylumbelliferyl oleate, wherein the protein formulation comprises a therapeutic protein selected from antibodies, antibody fragments, antibody-drug conjugates, antibody-cytokine fusions, Fc fusions, bispecific antibodies, multispecific antibodies, and affinity fusions, wherein the pH of the aqueous assay sample is from 5.0 to 7.0; and b. The formation of oleate and 4-methylumbelliferone was measured by fluorescence.

19. The method of claim 18, further comprising c. determining the stability of the protein formulation based on measured fluorescence.

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