Compositions, methods and kits for detecting lipolytic activity

Abstract

The present disclosure provides compositions, methods, and kits for detecting lipolytic activity. In some embodiments, the compositions include an aqueous assay sample and an organic solvent, wherein the organic solvent includes 4-methylumbelliferyl oleate (4MuO). Also provided herein are methods for determining the stability of protein preparations.

Description

[Technical field]

[0001] The present disclosure provides compositions, methods and kits for detecting and / or quantifying lipolytic activity.Also provided herein is a method for determining the stability of protein preparations.In some embodiments, the composition for detecting lipolytic activity comprises an aqueous assay sample and an organic solvent, and the organic solvent comprises 4-methylumbelliferyl oleate (4MuO). [Background technology]

[0002] Cell cultures can be used for the production of commercially important proteins, such as therapeutic proteins. Besides the target protein, the cells produce host cell proteins (HCPs) (e.g., lipases), which can be found in the production pool of the target protein (e.g., cell culture supernatant or cell lysate). Downstream purification processes, which may include various chromatography steps (e.g., affinity chromatography in the case of monoclonal antibody production), can generally deplete most of the HCPs. However, due to the natural principles of equilibrium-based separation and purification methods, 100% depletion of HCPs may not be possible. HCP limits accepted by industry and health authorities are about 1-100 ppm, relative to the amount of active protein. HCPs are typically measured using a standard concentration range of 100 ppm, e.g., as specified in the USP <1132> As outlined in, they are measured and reported as sum parameters using enzyme-linked immunosorbent assays (ELISAs). Thus, they typically contain a range of different host-derived proteins at various concentrations. Drugs with the same total HCP content as quantified by ELISA may have different HCP profiles. This may lead to situations where slight process variations cause an increase in a specific HCP (e.g., lipase) at low concentrations, but may not be detected by application of a total HCP assay. Thus, a small percentage of HCPs may be present in the final protein product and may have catalytic activity. Residual catalytic activity may have adverse effects on the stability of excipients (e.g., surfactants such as polysorbates) and ultimately on product quality, such as the stability, quality, and safety of the protein product. When the protein is a therapeutic protein, particularly one used in humans, it may be paramount to minimize or eliminate these adverse effects.

[0003] Residual lipase activity in protein preparations (e.g., due to non-optimal depletion during downstream purification processes of the target protein) can result in hydrolysis of some excipients (e.g., surfactants). Consequences can include, for example, liberation of non-polar (and therefore insoluble) long-chain fatty acids and other degradants from the surfactants, as well as the formation of subvisible and visible particles due to the loss of stabilization provided by the surfactants. Degradation of surfactants can also have adverse effects on protein quality, for example, the generation of peroxides leading to protein degradation, or the generation of lauric acid-induced protein aggregation. Degradation of surfactants also reduces their concentration in the formulation, potentially causing, for example, insufficient protection of proteins against interfacial stresses (e.g., shaking, freeze / thaw, etc.), and also leading to potential differences in safety considerations for products with different in vivo propensities due to, for example, degraded surfactants. Thus, residual lipase activity can compromise the quality of the final protein preparation due to hydrolysis of excipients. For example, degradation products of excipients such as polysorbates can also pose safety issues in patients, such as injection site reactions (see, for example, Non-Patent Document 1). [Prior art documents] [Non-patent literature]

[0004] [Non-Patent Document 1] Singh et al., J Pharm Sci 107(11):2735-2741(2018) Summary of the Invention

[0005] In some embodiments, the disclosure provides a composition, comprising: (a) an aqueous assay sample comprising a protein preparation; and (b) an organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate (4MuO), wherein the aqueous assay sample has a pH of 5.0 to 7.0, wherein the aqueous assay sample is about 80% to about 99.9% of the composition, and wherein the organic solvent is about 0.1% to about 20% of the composition.

[0006] In some embodiments, the protein preparation is a cell culture supernatant. In some embodiments, the protein preparation is a partially purified protein preparation. In some embodiments, the protein preparation is a purified protein preparation.

[0007] In some embodiments, the protein preparation comprises a therapeutic protein.

[0008] In some embodiments, the protein preparation 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.

[0009] In some embodiments, the protein preparation further comprises additional host cell proteins.

[0010] In some embodiments, the aqueous assay sample further comprises a buffer, a salt, or both.

[0011] 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 is about 50 mM to about 400 mM in the aqueous assay sample. In some embodiments, the sodium chloride is about 100 mM to about 200 mM in the aqueous assay sample. In some embodiments, the calcium chloride is about 0.2 mM to about 10 mM in the aqueous assay sample. In some embodiments, the calcium chloride is about 1.0 mM to about 2.0 mM in the aqueous assay sample.

[0012] In some embodiments, the buffer has a 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 about 2 mM to about 200 mM in the aqueous assay sample. In some embodiments, the buffer is about 10 mM to about 100 mM in the aqueous assay sample. In some embodiments, the buffer is about 40 mM to about 60 mM in the aqueous assay sample. In some embodiments, the buffer is about 45 mM to about 55 mM in the aqueous assay sample.

[0013] In some embodiments, the organic solvent is an alcohol, a sulfoxide, a nitrile, or a combination thereof. In some embodiments, the organic solvent is dimethyl sulfoxide (DMSO). In some embodiments, the organic solvent comprises acetonitrile.

[0014] 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.

[0015] In some embodiments, the composition further comprises a lipase inhibitor, hi some embodiments, the lipase inhibitor is (S)-2-formylamino-4-methyl-pentanoic acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]-dodecyl ester (orlistat).

[0016] In some embodiments, the lipase inhibitor is in an organic solvent. In some embodiments, the lipase inhibitor is in the composition at about 1 μM to about 50 μM. In some embodiments, the lipase inhibitor is in the composition at about 5 μM to about 25 μM.

[0017] In some embodiments, the disclosure provides a composition, comprising: (a) an aqueous assay sample comprising (i) a purified protein preparation, (ii) a buffering agent, and (iii) a salt; and (b) an organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate (4MuO), the aqueous assay sample has a pH of 5.0 to 7.0, the aqueous assay sample is about 80% to about 99.9% of the composition, and the organic solvent is about 0.1% to about 20% of the composition.

[0018] In some embodiments, the disclosure provides a composition comprising: (a) about 90% to about 99.9% (volume / volume) aqueous assay sample comprising (i) a purified protein preparation comprising a protein and a lipid; (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% (volume / volume) organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethylsulfoxide (DMSO), acetonitrile, or a combination thereof, further comprising 4-methylumbelliferyl oleate (4MuO), wherein the aqueous assay sample has a pH of 5.0 to 7.0.

[0019] In some embodiments, the disclosure provides a method for detecting lipolytic activity in an aqueous assay sample, the method comprising: (a) combining an aqueous assay sample comprising a protein preparation with an organic solvent comprising 4-methylumbelliferyl oleate (4MuO); and (b) measuring the formation of oleate and 4-methylumbelliferone (4Mu) by fluorescence.

[0020] In some embodiments, the disclosure provides a method for detecting lipolytic activity in an aqueous assay sample, the method comprising: (a) combining an aqueous assay sample comprising a protein preparation with an organic solvent comprising 4-methylumbelliferyl oleate (4MuO) to form an assay composition; (b) combining a control sample comprising the protein preparation and a lipase inhibitor with an organic solvent comprising 4-methylumbelliferyl oleate (4MuO) to form a control composition; and (c) measuring the formation of oleate and 4-methylumbelliferone (4Mu) by fluorescence in the assay composition and the control composition.

[0021] In some embodiments, the disclosure provides a method for determining the stability of a protein preparation, comprising: (a) combining an aqueous assay sample containing the protein preparation with an organic solvent containing 4-methylumbelliferyl oleate (4MuO); (b) measuring the formation of oleate and 4-methylumbelliferone (4Mu) by fluorescence; and (c) determining the stability of the protein preparation based on the measured fluorescence.

[0022] In some embodiments, the aqueous assay sample has a pH of 5.0 to 7.0. In some embodiments, the aqueous assay sample and the organic solvent are combined in a ratio of about 80:20 to about 98:2. In some embodiments, the aqueous assay sample and the organic solvent are combined in a ratio of about 90:10 to about 99.9:0.1.

[0023] In some embodiments, the fluorescence is measured with fluorescence excitation at 330 nm and fluorescence emission at 495 nm. In some embodiments, the fluorescence is measured for up to 24 hours. In some embodiments, the fluorescence is measured for about 24 hours to about 400 hours. In some embodiments, the fluorescence is measured for more than about 100 hours.

[0024] In some embodiments, the aqueous assay sample is incubated with the lipase inhibitor for about 10 minutes to about 1 hour prior to step (a). In some embodiments, the aqueous assay sample is incubated with the lipase inhibitor for about 30 minutes prior to step (a). In some embodiments, the lipase inhibitor is (S)-2-formylamino-4-methyl-pentanoic acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]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.

[0025] In some embodiments, the disclosure provides a kit comprising, in two or more containers, (a) an organic solvent; (b) 4-methylumbelliferyl oleate (4MuO); and (c) a lipase inhibitor.

[0026] In some embodiments, the kit further comprises a buffering agent, a salt, or both, hi some embodiments, the kit further comprises a buffer exchange column.

[0027] In some embodiments, the disclosure provides a kit, the kit comprising: (a) an organic solvent comprising 4-methylumbelliferyl oleate (4MuO); (b) a column suitable for buffer exchange of a protein preparation; and (c) a lipase inhibitor. [Brief description of the drawings]

[0028] [Figure 1A] Referring to Example 1A, Figures 1A and 1B show the effect of buffer concentration and pH of Tris and Bis-Tris buffers on porcine pancreatic lipase (PPL) activity and 4-methylumbelliferone (4Mu) fluorescence quenching, respectively. [Figure 1B]Referring to Example 1A, Figures 1A and 1B show the effect of buffer concentration and pH of Tris and Bis-Tris buffers on porcine pancreatic lipase (PPL) activity and 4-methylumbelliferone (4Mu) fluorescence quenching, respectively. [Figure 1C] Refers to Example 1A. Figure 1C shows the effect of buffer concentration and pH on lipase activity and autohydrolysis of 4-methylumbelliferyl oleate (4MuO). [Figure 1D] Referring to Example 1A, Figure ID shows the pH range over which the fluorescence of 4Mu was measured. Figure IE shows the different forms of 4Mu at various pHs. [Figure 1E] Referring to Example 1A, Figure 1E shows the different forms of 4Mu at various pH levels. [Figure 2A] Referring to Example 2A, Figures 2A and 2B show the effect of CaCl2 concentration on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 2B] Referring to Example 2A, Figures 2A and 2B show the effect of CaCl2 concentration on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 2C] Referring to Example 2A, Figure 2C shows the effect of CaCl concentration on the lipase activity and autohydrolysis of 4MuO. [Figure 3A] Referring to Example 3A, Figures 3A and 3B show the effect of NaCl concentration on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 3B] Referring to Example 3A, Figures 3A and 3B show the effect of NaCl concentration on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 3C] Refers to Example 3A. Figure 3C shows the effect of NaCl concentration on lipase activity and autohydrolysis of 4MuO. [Figure 4A] Referring to Example 4A, Figures 4A and 4B show the effect of organic solvents on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 4B]Referring to Example 4A, Figures 4A and 4B show the effect of organic solvents on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 4C] Refers to Example 4A. Figure 4C shows the effect of organic solvents on the lipase activity and autohydrolysis of 4MuO. [Figure 5A] Referring to Example 5, Figures 5A and 5B show the effect of surfactants on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 5B] Referring to Example 5, Figures 5A and 5B show the effect of surfactants on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 5C] Referring to Example 5, Figure 5C shows the effect of surfactants on the lipase activity and autohydrolysis of 4MuO. [Figure 6A] Referring to Example 6, Figures 6A and 6B show the effect of three different concentrations of the lipase inhibitor orlistat on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 6B] Referring to Example 6, Figures 6A and 6B show the effect of three different concentrations of the lipase inhibitor orlistat on lipase activity and fluorescence quenching of 4Mu, respectively. [Figure 7A] Refers to Example 7. Figures 7A and 7C show the effect of product inhibition of theoretically fully degraded PS20 on the lipase activity and autohydrolysis of 4Mu. [Figure 7B] Refers to Example 7. Figures 7B and 7D show the effect of product inhibition of theoretically fully degraded PS80 on the lipase activity and autohydrolysis of 4Mu. [Figure 7C] Refers to Example 7. Figures 7A and 7C show the effect of product inhibition of theoretically fully degraded PS20 on the lipase activity and autohydrolysis of 4Mu. [Figure 7D] Refers to Example 7. Figures 7B and 7D show the effect of product inhibition of theoretically fully degraded PS80 on the lipase activity and autohydrolysis of 4Mu. [Figure 8]Referring to Example 8. Figure 8 shows the effect of PS80 on the fluorescence quenching of 4Mu. [Figure 9A] Referring to Example 9, Figures 9A and 9B show the results of an HPLC-FMA assay to test the degradation of PS20 (Figure 9A) and PS80 (Figure 9B) in various concentrations of cell culture harvest fluid (CCHF). [Figure 9B] Referring to Example 9, Figures 9A and 9B show the results of an HPLC-FMA assay to test the degradation of PS20 (Figure 9A) and PS80 (Figure 9B) in various concentrations of cell culture harvest fluid (CCHF). [Figure 9C] Refers to Example 9. Figure 9C shows the theoretical readout of the 4MuO lipase assay. [Figure 9D] Referring to Example 9, Figure 9D shows the actual readout of the 4MuO lipase assay test of various concentrations of CCHF in degrading polysorbate. [Figure 10A] Referring to Example 9, Figures 10A-10H show the results of 4MuO lipase assay with PS80 at different CCHF concentrations. [Figure 10B] Referring to Example 9, Figures 10A-10H show the results of 4MuO lipase assay with PS80 at different CCHF concentrations. [Figure 10C] Referring to Example 9, Figures 10A-10H show the results of 4MuO lipase assay with PS80 at different CCHF concentrations. [Figure 10D] Referring to Example 9, Figures 10A-10H show the results of 4MuO lipase assay with PS80 at different CCHF concentrations. [Figure 10E] Referring to Example 9, Figures 10A-10H show the results of 4MuO lipase assay with PS80 at different CCHF concentrations. [Figure 10F] Referring to Example 9, Figures 10A-10H show the results of 4MuO lipase assay with PS80 at different CCHF concentrations. [Figure 10G]Referring to Example 9, Figures 10A-10H show the results of 4MuO lipase assay with PS80 at different CCHF concentrations. [Figure 10H] Referring to Example 9, Figures 10A-10H show the results of 4MuO lipase assay with PS80 at different CCHF concentrations. [Figure 11] Related to Example 1B. Figure 11 shows the effect of Tris and Bis-Tris pH on the lipolytic activity of PPL and CCHF, and the autohydrolysis (AH) of 4Mu. [Figure 12] Related to Example 2B. Figure 12 shows the effect of CaCl2 concentration on the lipolytic activity of PPL and CCHF. [Figure 13] Refers to Example 3B. Figure 13 shows the effect of NaCl concentration on the lipolytic activity of PPL and CCHF. [Figure 14] Refers to Example 4B. Figure 14 shows the effect of organic solvents on the lipolytic activity of PPL and CCHF. [Figure 15A] Refers to Example 5B. Figure 15A shows the effect of different surfactants on the lipolytic activity of PPL and CCHF. [Figure 15B] Referring to Example 5B, Figure 15B shows the quenching of 4Mu fluorescence by different surfactants. [Figure 16] Related to Example 6B. Figure 16 shows the effect on PPL activity of pre-incubating or co-incubating samples with the lipase inhibitor orlistat at three different concentrations. [Figure 17A] Referring to Example 10. Figure 17A shows the kinetics of lipase assays performed using protein-containing formulations with various positive and negative controls (samples, assays, formulations, and autohydrolysis). Table 10 herein summarizes the samples tested. [Figure 17B] Refers to Example 10. Figure 17B is a close-up of the assay kinetics. [Figure 18] Refers to Example 9. Figure 18 shows a summary of lipase assays performed with various concentrations of CCHF. [Figure 19A] Relating to Example 1B, Figures 19A and 19B show the calibration curve of 4Mu in the concentration range of 0.01 to 5 mM and the numerical results of the calibration curve of 4Mu, respectively. [Figure 19B] Relating to Example 1B, Figures 19A and 19B show the calibration curve of 4Mu in the concentration range of 0.01 to 5 mM and the numerical results of the calibration curve of 4Mu, respectively. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0029] The present disclosure relates to compositions, methods, and kits for detecting lipolytic activity.

[0030] As used herein, "a" or "an" can mean one or more. As used herein, the words "a" or "an" when used in conjunction with the word "comprising" can mean one or more. As used herein, "another" or "in addition" can mean at least a second or more.

[0031] Throughout this application, the term "about" is used to indicate that a value includes the inherent variation of error of the method / device used to determine the value or the variation that exists between study subjects. Typically, the term "about" is meant to encompass a variation of approximately 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%, or 20% or more, depending on the context. In some embodiments, a person skilled in the art will understand the level of variability indicated by the term "about" from the context in which it is used herein. It should also be understood that the use of the term "about" includes the specifically recited values.

[0032] Use of the term "or" in the claims is used to mean "and / or" unless expressly indicated to refer only to alternatives or the alternatives are mutually exclusive, but the present disclosure supports a definition that refers only to alternatives and "and / or."

[0033] As used herein, the terms "comprising" (and any variation or form of "comprising", such as "comprise" and "comprises"), "having" (and any variation or form of "having", such as "have" and "has"), "including" (and any variation or form of "including", such as "includes" and "include"), or the term "containing" (and any variation or form of "containing", such as "contains" and "contain") are inclusive or open-ended and do not exclude additional, unrecited elements or method steps. It is contemplated that any embodiment discussed herein can be implemented with respect to any method, composition, and / or kit of the present disclosure. Additionally, the compositions of the present disclosure can be used to achieve the methods and kits of the present disclosure.

[0034] The use of the term "for example" and its corresponding abbreviation "eg" (whether italicized or not) means that the particular term recited is an exemplary example and embodiment of the present disclosure and is not intended to be limited to the specific example referenced or cited, unless expressly stated otherwise.

[0035] As used herein, "between" is a range that includes the ends of the range. For example, numbers between x and y explicitly include the numbers x and y, as well as any numbers between x and y.

[0036] As used herein, "protein," "peptide," or "polypeptide" refers to a polymeric form of amino acids and can be of any length. Proteins can include, for example, antibodies, structural proteins, enzymes, membrane proteins, membrane-associated proteins, and / or transmembrane proteins, transporters, receptors, signaling proteins, and the like. Proteins and / or peptides of the present disclosure also encompass modified proteins conjugated to one or more non-peptide agents, such as, for example, drugs, targeting moieties, tags (e.g., visualization tags), and the like. Proteins of the present disclosure can be therapeutic proteins used, for example, in the diagnosis, treatment, and / or prevention of a disease or disorder. In some embodiments, the polysorbates described herein can improve the stability of a 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 preparations described herein include a protein (e.g., a therapeutic protein).

[0037] As used herein in the context of a protein preparation, "purification" refers to a process in which one or more substances (e.g., proteins) are isolated from a complex mixture, typically a cell, tissue, or organism. A "purified" protein sample or protein preparation may refer to a sample in which one or more water-insoluble components of a cell, tissue, or organism (e.g., cell membranes, lipids, aggregated proteins or nucleic acids, and other hydrophobic substances, etc.) have been reduced or removed, leaving only soluble components (e.g., soluble proteins, etc.). As used herein, "soluble" may refer to the ability of a substance to dissolve in a particular solvent (e.g., cell culture medium, buffer, water, or organic solvent). In the context of a protein, "soluble" may also refer to a protein that does not precipitate and / or aggregate in a particular solvent (e.g., cell culture medium, buffer, water, or organic medium).

[0038] An exemplary purification process may include growing a cell culture containing a protein of interest (e.g., a therapeutic protein), separating the cells from the culture medium, lysing the cells and separating the lysed cells to generate a cell culture supernatant containing soluble components and a pellet containing insoluble components as described herein, and subjecting the cell culture supernatant to buffer exchange, pH adjustment, centrifugation, filtration (including, e.g., ultrafiltration and / or diafiltration), chromatography, or any combination thereof to generate a purified protein preparation. In some embodiments, the purified protein preparations of the present disclosure are purified by the processes described herein. In some embodiments, the partially purified protein preparations of the present disclosure have been subjected to a portion of the purification process described herein. For example, the partially purified protein preparation may not have been subjected to all of the buffer exchange, pH adjustment, centrifugation, filtration, and / or chromatography steps used to generate the purified protein preparation. In some embodiments, the cell culture supernatant described herein comprises a therapeutic protein of the present disclosure. In some embodiments, the partially purified protein preparations described herein comprise a therapeutic protein of the present disclosure. In some embodiments, the purified protein preparations described herein comprise a therapeutic protein of the disclosure.

[0039] In some embodiments, the present disclosure relates to compositions and methods for the detection of lipolytic activity. Lipolytic activity, i.e., lipolysis, generally refers to the hydrolysis of lipids. Lipolytic reactions can be catalyzed by lipase enzymes, which are a subclass of esterase enzymes. Thus, "lipase" refers to an enzyme that hydrolyzes the ester bonds of lipids (e.g., triglycerides, phospholipids, cholesteryl esters, etc.). Lipases include, for example, triglyceride lipase, lipoprotein lipase, pancreatic lipase, hepatic lipase, gastric lipase, lingual lipase, endothelial lipase, and phosphatidylserine phospholipase. Lipases are naturally produced, for example, by mammalian pancreas, liver, lingual gland, stomach, thyroid, and / or mucosa, secreted by certain bacteria and fungi, and / or found in lysosomes. In some embodiments, lipases are endogenous to the cells from which the protein in protein purification was derived. In some embodiments, the lipase is endogenous to another biological component in the protein preparation (eg, a biological component that includes a stabilizing protein that is added to the protein preparation).

[0040] In some embodiments, the lipase is produced by cells in cell culture. In some embodiments, the lipase is produced by cells in cell culture for the production of a protein of interest. Non-limiting examples of cells suitable for the production of a protein of interest include bacteria, insects, yeast, mammalian, and / or transgenic cells. Non-limiting examples of cell lines include CHO, HEK293, HT-1080, PER.C6, CAP, VERO, BHK, HeLa, CV1, Cos, MDCK, 3T3, NS0, NS1, PC12, W138, Sp2 / 0, HKB-11, TM4, MMT060562, TR1, MRC5, FS4, myeloma cell lines, hybridoma cell lines, and hepatoma cell lines. In some embodiments, the cell line for producing a protein of interest is a stable cell line, e.g., the gene for the protein of interest is stably integrated into the genome of the cell. In some embodiments, the cell line for producing a protein of interest is a transient cell line, for example, the cells express a gene, but the gene is not integrated into the genome.

[0041] In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the protein of interest is purified from the cell culture to produce a purified protein preparation. In some embodiments, the lipase in the cell culture is not completely removed from the protein preparation during the purification process. Thus, in some embodiments, the lipase is present in the purified protein preparation.

[0042] As referred to herein, an "active" lipase is a lipase that is capable of lipolysis (also referred to herein as having "lipolytic activity"). Active lipase present in a protein preparation may interfere with downstream processes involving a protein of interest (e.g., a therapeutic protein). In some embodiments, a protein preparation comprising a protein of interest (e.g., a therapeutic protein) and a lipase is included in a pharmaceutical formulation. In some embodiments, an excipient is added to the protein preparation. In some embodiments, the excipient stabilizes the protein preparation, for example, by minimizing interfacial stress, reducing protein aggregation, and / or improving 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 susceptible to hydrolysis by active lipase. In some embodiments, the presence of active lipase in a protein preparation comprising a protein of interest and an excipient reduces the stability of the preparation. It is therefore advantageous to reliably detect lipolytic activity in protein preparations to minimize negative effects such as increased particulates, safety concerns (e.g., due to increased injection site reactions), and reduced quality caused by lipase hydrolysis of excipients.

[0043] In some embodiments, the present disclosure provides compositions capable of detecting lipolytic activity in protein preparations.

[0044] In some embodiments, the disclosure provides a composition, comprising: (a) an aqueous assay sample comprising a protein preparation; and (b) an organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate (4MuO), the aqueous assay sample has a pH of 5.0 to 7.0, the aqueous assay sample is about 80% to about 98% of the composition, and the organic solvent is about 2% to about 20% of the composition.

[0045] In a further embodiment, the disclosure provides a composition comprising: (a) an aqueous assay sample comprising (i) a purified protein preparation, (ii) a buffering agent, and (iii) a salt; and (b) an organic solvent, wherein the organic solvent further comprises 4-methylumbelliferyl oleate (4MuO), the aqueous assay sample has a pH of 5.0 to 7.0, the aqueous assay sample is about 80% to about 98% (volume / volume) of the composition, and the organic solvent is about 2% to about 20% (volume / volume) of the composition.

[0046] In still further embodiments, the disclosure provides a composition comprising: (a) about 90% to about 98% (volume / volume) aqueous assay sample comprising (i) a purified protein preparation comprising a protein and a lipid; (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% (volume / volume) organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethylsulfoxide (DMSO), acetonitrile, or a combination thereof, further comprising 4-methylumbelliferyl oleate (4MuO), wherein the aqueous assay sample has a pH of 5.0 to 7.0.

[0047] As used herein, "aqueous" (e.g., an aqueous assay sample) refers to a solution or sample in which water is the solvent. Thus, an aqueous assay sample of the present disclosure can include, for example, cell culture media, buffers, protein samples, etc. In some embodiments, an aqueous assay sample of the present disclosure includes a protein preparation.

[0048] In some embodiments, the protein preparation is a cell culture supernatant. The cell culture supernatant is described herein and can be obtained, for example, from a cell culture for producing a protein of interest. In some embodiments, the protein is a therapeutic protein. In some embodiments, the cell culture supernatant is produced after lysing the cultured cells and separating soluble and insoluble components (e.g., by centrifugation). Examples of cells and cell lines suitable for culturing and protein production are provided herein. In some embodiments, the cell culture supernatant comprises a protein of interest (e.g., a therapeutic protein) and additional host cell components. In some embodiments, the additional host cell components comprise additional host cell proteins. In some embodiments, the additional host cell proteins comprise lipases. In some embodiments, the lipases have lipolytic activity.

[0049] In some embodiments, the protein preparation is a partially purified protein preparation. A partially purified protein preparation is described herein and can be obtained, for example, after a partial purification procedure (e.g., a purification process described herein) of a protein of interest from a cell culture. In some embodiments, the protein is a therapeutic protein. In some embodiments, the partially purified protein preparation has undergone an additional purification step compared to the cell culture supernatant. In some embodiments, the partially purified protein preparation comprises a therapeutic protein and additional components of a host cell. In some embodiments, the host cell components comprise host cell proteins. In some embodiments, the host cell proteins comprise a lipase. In some embodiments, the lipase has lipolytic activity. In some embodiments, the therapeutic protein is 20%-95% (w / w), 30%-90% (w / w), or 40%-80% (w / w) of all proteins in the partially purified protein preparation.

[0050] In some embodiments, the protein preparation is a purified protein preparation. A purified protein preparation is described herein, for example, can be obtained after performing a purification procedure (e.g., a purification process described herein) of a protein of interest from a cell culture. In some embodiments, the protein of interest is a therapeutic protein. In some embodiments, the purified protein preparation is a therapeutic protein and additional components of a host cell. In some embodiments, the host cell components include host cell proteins. In some embodiments, the host cell proteins include a lipase. In some embodiments, the lipase has lipolytic activity. In some embodiments, the therapeutic protein is more than 70% (w / w), more than 80% (w / w), more than 85% (w / w), more than 90% (w / w), more than 95% (w / w), or more than 99% (w / w) of all proteins in the purified protein preparation.

[0051] In some embodiments, the protein preparation 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 hepatitis B surface antigen), blood factors (such as factor VIII and factor IX), thrombolytic agents (such as tissue plasminogen activator), hormones (such as insulin, glucagon, growth hormone, and gonadotropins), hematopoietic growth factors (such as erythropoietin and colony-stimulating factors), interferons (such as interferon alpha, interferon beta, and interferon gamma), interleukin-based proteins (such as interleukin-12), and other proteins such as tumor necrosis factors and therapeutic enzymes. Further examples of protein-based therapeutic agents include belimumab, ipilimumab, belatacept, brentuximab vedotin, aflibercept, erwinia chrysanthemi asparaginase, glucarpidase, obinutuzumab, pembrolizumab, blinatumomab, nivolumab, idarucizumab, asofatase-alpha, daratumumab, elotuzumab, sebelipase alfase, atezolizumab, taliglucerase alfa, raxibacumab, elosulfase alfa, metreleptin, ramucirumab, siltuximab, pembrolizumab, dinutuximab, evolocumab, necitumumab, obiltoxaximab, abatacept, adalimumab, alefacept, etanercept, infliximab, trastuzumab, ustekinumab, denileukin diftitox, and golimumab. Further examples of protein therapeutics are described, for example, in Dimitrov, Methods Mol Biol 899:1-26 (2012), Lagasse et al., F1000Res 6:113 (2017), and Protein Therapeutics, Eds: Vaughan et al., 2017: Wiley-VCH Verlag.Therapeutic proteins can include recombinant proteins, modified proteins, and fusion proteins, such as antibody-drug conjugates, antibody-cytokine fusions, Fc fusions, bispecific antibodies, multispecific antibodies, affibody fusions, glycosylated proteins and peptides, and engineered receptor antagonists. In some embodiments, protein preparations containing therapeutic proteins are used in pharmaceutical formulations.

[0052] In some embodiments, the protein preparation comprises a commercially important protein, such as an industrial enzyme. Commercially important proteins can be used in various industries, such as pharmaceuticals, chemical production, biofuels, food and beverages, and consumer products. For example, in some embodiments, the protein preparation can be an enzyme used in a process to generate a desired product, or 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, industrial enzymes include, but are not limited to, paratase lipozyme, lipopan, xylose isomerase, bromelain, and nopazyme (used in the food industry), cellulase and amylase (used in the biofuel industry), resinase (used in the paper processing industry), amidase (used in the chemical industry), Novozyme-435 (used in cosmetic production of isopropyl myristate), or subtilisin (used in detergents).

[0053] In some embodiments, the protein preparation comprises a pharmaceutical excipient. Pharmaceutical excipients are included, for example, to aid in the processing of the drug delivery system before, during, or after manufacturing; to protect, support, or enhance stability, bioavailability, or patient acceptability; to aid in product identification and enhance overall safety; to aid in the efficacy and / or delivery of the drug during use; and / or to aid in maintaining the integrity of the pharmaceutical product during storage. Non-limiting examples of pharmaceutical excipients include surfactants, fillers, diluents, binders, suspending agents, thickening agents, coating agents, flavoring agents, disintegrants, coloring agents, lubricants, glidants, preservatives, sweeteners, and the like. In some embodiments, pharmaceutical excipients are added to the protein preparation. In some embodiments, pharmaceutical excipients are added to the protein preparation before, after, or during purification.

[0054] In some embodiments, the pharmaceutical excipient is a surfactant. As used herein, "surfactant" refers to an agent that reduces surface or interfacial tension between two liquids. In some embodiments, a surfactant can stabilize a composition (e.g., a protein preparation described herein) by minimizing aggregation and / or precipitation and / or improving solubility (e.g., by reducing surface tension and inhibiting surface adsorption of proteins; see, e.g., Agarkhed et al., AAPS PharmSciTech 14:1-9 (2013)) of one or more components of the composition. Surfactants in pharmaceutical compositions can also regulate the bioavailability of active pharmaceutical ingredients (APIs), help maintain the API in a preferred polymorphic form, prevent aggregation or dissociation, and / or regulate the immunogenic response of the active ingredient. Surfactants can include cationic surfactants, anionic surfactants, nonionic surfactants, zwitterionic surfactants, amphoteric surfactants, and / or ampholytic surfactants. Non-limiting examples of surfactants include polysorbates derived from ethoxylated sorbitan esterified with fatty acids (e.g., lauric acid in polysorbate 20 and oleic acid in polysorbate 80) (e.g., TWEEN surfactants such as TWEEN 20 and TWEEN 80, also known as polysorbate 20 and polysorbate 80, respectively), tyloxapol, poloxamers (e.g., PLURONIC F68LF, PLURONIC L-G2LF, PLURONIC L62D, LUTROL F68, and KOLLIPHOR P188), polyoxyethylene castor oil (e.g., KOLLIPHOR EL) and its derivatives, sorbitan esters (also known as Span), polyoxyl stearates, lecithin, phospholipids, polyoxyethylene surfactants such as TRITON (e.g., TRITON X-100) and BRIJ (e.g., BRIJ 35)), and polyethoxylated fatty acids (e.g., MYRJ 100, MYRJ 200, MYRJ 350, MYRJ 400, MYRJ 500, MYRJ 600, MYRJ 700, MYRJ 800, MYRJ 900, MYRJ 1000, MYRJ 1100, MYRJ 1200, MYRJ 1300, MYRJ 1400, MYRJ 1500, MYRJ 1600, MYRJ 1700, MYRJ 1800, MYRJ 1900, MYRJ 2000, MYRJ 2100, MYRJ 2200, MYRJ 2300, MYRJ S40, MYRJ S100, and MYRJ 52).

[0055] 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 sorbitan esterified with fatty acids, including, 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 preparation provided herein comprises a polysorbate. In some embodiments, the polysorbate in the protein preparation is polysorbate 20, polysorbate 80, or a combination thereof.

[0056] In some embodiments, the surfactant is about 0.001% (w / v) to about 2% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.005% (w / v) to about 2% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.01% (w / v) to about 2% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.02% to about 1.5% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.03% to about 1.0% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.04% to about 0.8% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.05% to about 0.6% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.06% to about 0.4% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.07% to about 0.2% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.08% to about 0.15% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.09% to about 0.10% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is about 0.01% to about 0.04% (w / v) of the aqueous assay sample. In some embodiments, the surfactant is a polysorbate. In some embodiments, the surfactant is polysorbate 20, polysorbate 80, or a combination thereof.

[0057] In some embodiments, the protein preparation further comprises one or more additional host cell proteins. As described herein, the protein preparation is prepared from a cell culture, i.e., a cell culture comprising a host cell of a protein of interest (e.g., a therapeutic protein). In some embodiments, the protein preparation 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 (e.g., a therapeutic protein). In some embodiments, the additional host cell proteins cannot be easily separated from the protein of interest (e.g., a therapeutic protein). In some embodiments, the additional host cell proteins comprise lipases. In some embodiments, one or more of the additional host cell proteins have lipolytic activity.

[0058] In some embodiments, the aqueous assay sample comprising the protein preparation further comprises a buffer, a salt, or both. Generally, the salts of the present disclosure are those in which the anion is OH - and O 2- Salt refers to an ionic compound that is not. In some embodiments, the salt reduces and / or prevents the decomposition of one or more components in the composition. Suitable salts that can be included in the aqueous assay sample include, for example, sodium salts, potassium salts, calcium salts, ammonium salts, and the like, and can be selected by those skilled in the art. 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 a combination thereof. In some embodiments, the salt is NaCl, CaCl2, or a combination thereof. In some embodiments, the salt is both NaCl and CaCl2.

[0059] In some embodiments, the concentration of NaCl in the aqueous assay sample facilitates accurate and / or efficient detection of lipolytic activity in the sample. In some embodiments, NaCl is about 10 mM to about 500 mM in the aqueous assay sample. In some embodiments, NaCl is about 25 mM to about 400 mM in the aqueous assay sample. In some embodiments, NaCl is about 50 mM to about 300 mM in the aqueous assay sample. In some embodiments, NaCl is about 75 mM to about 250 mM in the aqueous assay sample. In some embodiments, NaCl is about 100 mM to about 200 mM in the aqueous assay sample. In some embodiments, NaCl is 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 in the aqueous assay buffer.

[0060] In some embodiments, NaCl is about 10 mM to about 500 mM in the final composition (aqueous assay sample and organic solvent). In some embodiments, NaCl is about 25 mM to about 400 mM in the final composition. In some embodiments, NaCl is about 50 mM to about 300 mM in the final composition. In some embodiments, NaCl is about 75 mM to about 250 mM in the final composition. In some embodiments, NaCl is about 100 mM to about 200 mM in the final composition. In some embodiments, NaCl is about 100 mM to about 140 mM, e.g., 120 mM, in the final composition. In some embodiments, NaCl is 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 in the final composition.

[0061] In some embodiments, the concentration of CaCl2 in the aqueous assay sample facilitates accurate and / or efficient detection of lipolytic activity in the sample. In some embodiments, CaCl2 is about 0.1 mM to about 20 mM in the aqueous assay sample. In some embodiments, CaCl2 is about 0.2 mM to about 10 mM in the aqueous assay sample. In some embodiments, CaCl2 is about 0.5 mM to about 5.0 mM in the aqueous assay sample. In some embodiments, CaCl2 is about 0.7 mM to about 3.0 mM in the aqueous assay sample. In some embodiments, CaCl2 is about 1.0 mM to about 2.0 mM in the aqueous assay sample. In some embodiments, CaCl2 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 1.6 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 in the aqueous assay sample.

[0062] In some embodiments, CaCl2 is about 0.1 mM to about 20 mM in the final composition (aqueous assay sample and organic solvent). In some embodiments, CaCl2 is about 0.2 mM to about 10 mM in the final composition. In some embodiments, CaCl2 is about 0.5 mM to about 5.0 mM in the final composition. In some embodiments, CaCl2 is about 0.7 mM to about 3.0 mM in the final composition. In some embodiments, CaCl2 is about 1.0 mM to about 2.0 mM in the final composition. In some embodiments, CaCl2 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 1.6 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 in the final composition.

[0063] In some embodiments, NaCl and CaCl2 reduce and / or prevent degradation of one or more components in the aqueous assay sample. In some embodiments, NaCl and CaCl2 reduce and / or prevent aggregation and / or precipitation of a protein (e.g., a therapeutic protein). In some embodiments, NaCl and CaCl2 reduce and / or prevent degradation of one or more components in a composition (e.g., in an organic solvent) that is not present in the aqueous assay sample. In some embodiments, NaCl and CaCl2 reduce and / or prevent autohydrolysis of 4-methylumbelliferyl oleate (4MuO). In some embodiments, in the aqueous assay sample, NaCl is about 10 mM to about 500 mM and CaCl2 is about 0.1 mM to about 20 mM. In some embodiments, in the aqueous assay sample, NaCl is about 25 mM to about 400 mM and CaCl2 is about 0.2 mM to about 10 mM. In some embodiments, in the aqueous assay sample, NaCl is about 50 mM to about 300 mM, and CaCl2 is about 0.5 mM to about 5.0 mM. In some embodiments, in the aqueous assay 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 assay 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 assay sample, NaCl is about 150 mM, and CaCl2 is about 0.3 mM.

[0064] As used herein, a "buffer" refers to a substance used in a solution to maintain the pH of the solution. A buffer can maintain a solution at a particular pH range (i.e., buffering capacity in a given range) and prevent abrupt changes in pH when additional components are added to the solution. In general, a buffer can be a weak acid or a weak base. In some embodiments, a buffer has a 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 having a buffering capacity of about pH 5.0 to about pH 7.0 include, for example, citrate, acetate, phosphate, 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 one of skill in the art. In some embodiments, a buffer in an aqueous assay sample facilitates accurate and / or efficient detection of lipolytic activity in the sample. In some embodiments, the buffering agent reduces and / or prevents degradation of one or more components in the aqueous assay sample. In some embodiments, the buffering agent reduces and / or prevents aggregation of a protein (e.g., a therapeutic protein). In some embodiments, the buffering agent reduces and / or prevents degradation of one or more components in a composition not present in the aqueous assay sample (e.g., in an organic solvent of the composition). In some embodiments, the buffering agent reduces and / or prevents autohydrolysis of 4-methylumbelliferyl oleate (4MuO). In some embodiments, the buffering agent is provided in the aqueous assay sample as an aqueous buffer.

[0065] In some embodiments, the buffering agent is about 5 mM to about 200 mM in the aqueous assay sample. In some embodiments, the buffering agent is about 10 mM to about 100 mM in the aqueous assay sample. In some embodiments, the buffering agent is about 20 mM to about 80 mM in the aqueous assay sample. In some embodiments, the buffering agent is about 30 mM to about 70 mM in the aqueous assay sample. In some embodiments, the buffering agent is about 40 mM to about 60 mM in the aqueous assay sample. In some embodiments, the buffer 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 the aqueous assay sample. In some embodiments, the buffer is Bis-Tris, In some embodiments, the buffer is Tris.

[0066] In some embodiments, the buffering agent is about 5 mM to about 200 mM in the final composition (aqueous assay sample and organic solvent). In some embodiments, the buffering agent is about 10 mM to about 100 mM in the final composition (aqueous assay sample and organic solvent). In some embodiments, the buffering agent is about 20 mM to about 80 mM in the final composition. In some embodiments, the buffering agent is about 30 mM to about 70 mM in the final composition. In some embodiments, the buffering agent is about 40 mM to about 60 mM in the final composition. In some embodiments, the buffer 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 the final composition.

[0067] In some embodiments, the aqueous assay sample comprises NaCl, CaCl2, and a buffering agent. In some embodiments, in the aqueous assay sample, NaCl is about 10 mM to about 500 mM, CaCl2 is about 0.1 mM to about 20 mM, and the buffering agent is about 5 mM to about 200 mM. In some embodiments, in the aqueous assay sample, NaCl is about 25 mM to about 400 mM, CaCl2 is about 0.2 mM to about 10 mM, and the buffering agent is about 10 mM to about 100 mM. In some embodiments, in the aqueous assay sample, NaCl is about 50 mM to about 300 mM, CaCl2 is about 0.5 mM to about 5.0 mM, and the buffering agent is about 20 mM to about 8 mM. In some embodiments, in the aqueous assay sample, NaCl is about 75 mM to about 250 mM, CaCl2 is about 0.7 to about 3.0 mM, and the buffer is 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 found in protein preparations, and thus the above percentages are provided by way of example only.

[0068] 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 the organic solvent. In some embodiments, a slightly acidic to neutral pH (e.g., about 5.0 to about 7.0) minimizes 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 aggregation of therapeutic proteins. In some embodiments, a slightly acidic to neutral pH (e.g., about 5.0 to about 7.0) minimizes autohydrolysis of 4-methylumbelliferyl oleate (4MuO).

[0069] In some embodiments, the aqueous assay sample has an acidic pH. In some embodiments, the aqueous assay sample has a pH of 5.0 to 7.0. In some embodiments, the aqueous assay sample has a pH of 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.

[0070] In some embodiments, the composition of the present disclosure comprises an aqueous assay sample as 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 aliphatic, cyclic, aromatic, and halogenated hydrocarbons, ketones, amines, esters, alcohols, aldehydes, ethers, nitriles, sulfoxides, and the like. In some embodiments, the organic solvent can solubilize 4-methylumbelliferyl oleate (4MuO).

[0071] In some embodiments, the organic solvent comprises an alcohol, a sulfoxide, a 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.

[0072] In some embodiments, 4-methylumbelliferyl oleate (4MuO) is present in an organic solvent. 4MuO comprises the following structure: [ka]

[0073] In some embodiments, 4MuO hydrolyzes to form oleic acid and 4-methylumbelliferone (4Mu) (Scheme I). [ka]

[0074] In some embodiments, 4MuO is not fluorescent. In some embodiments, 4Mu is fluorescent. In some embodiments, 4Mu fluorescence is used to detect hydrolysis of 4MuO by lipase and is therefore an indicator of lipolytic activity. One skilled in the art will appreciate that hydrolysis of 4MuO (as measured by 4Mu fluorescence) indicates that lipolytic activity has occurred in the protein preparation (i.e., detergent has been hydrolyzed) and that the protein preparation is likely destabilized. In some embodiments, 4Mu fluorescence may be measured at an excitation wavelength of about 330 nm and an emission wavelength of about 495 nm. In some embodiments, 4Mu fluorescence may be measured at an excitation wavelength of about 327 nm and an emission wavelength of about 449 nm. In some embodiments, 4Mu fluorescence may 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 wavelength and emission wavelength) change when the pH changes. In some embodiments, the fluorescence measurement parameters of 4Mu change when the concentration of salt and / or buffer is changed. In some embodiments, 4MuO is a substrate for lipase. In some embodiments, 4MuO is hydrolyzed by lipase in the protein preparation described herein. In some embodiments, the formation of 4Mu is measured by fluorescence. In some embodiments, the lipolytic activity of the assay sample comprising the protein preparation described herein is measured by the fluorescence of 4Mu.

[0075] Various concentrations of 4MuO can be used in the compositions and methods described herein. In general, 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.

[0076] In some embodiments, the composition comprises an aqueous assay sample comprising a protein preparation as described herein and an organic solvent comprising 4MuO as described herein. In some embodiments, the composition does not comprise equal amounts 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 potential adverse effects of the organic solvent on the protein preparation (particularly the therapeutic protein). 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 assay sample is about 80% to about 98% by volume of the composition, and the organic solvent is about 2% to about 20% by volume of the composition. In some embodiments, the aqueous assay sample is about 90% to about 98% by volume of the composition, and the organic solvent is about 2% to about 10% by volume of the composition. In some embodiments, the aqueous assay sample is about 95% to about 98% by volume of the composition, and the organic solvent is about 2% to about 5% by volume of the composition.

[0077] In some embodiments, the aqueous assay sample is about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, 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 preparation comprises about 70% to about 85%, about 75% to about 85%, or about 80% to about 85% by volume of the composition, and non-protein preparation components of the aqueous assay sample (e.g., buffers and / or salts) 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 found in protein preparations, and therefore the above percentages are used by way of example only.

[0078] 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 the detection of lipolytic activity, i.e., a composition containing the lipase inhibitor is not expected to have lipolytic activity. In some embodiments, the lipase inhibitor is added to the composition after lipolytic activity has been detected, for example, by measuring the fluorescence of 4Mu. 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.

[0079] In some embodiments, the lipase inhibitor is at a concentration sufficient to reduce or eliminate lipolytic activity in the composition. In some embodiments, the lipase inhibitor is at a concentration sufficient to reduce lipolytic activity in the composition by about 50%, about 60%, about 70%, about 80%, about 90%, about 95%, about 99%, or about 100% in the composition. In some embodiments, the lipase inhibitor is at about 1 μM to about 50 μM in the composition. In some embodiments, the lipase inhibitor is at about 2 μM to about 40 μM in the composition. In some embodiments, the lipase inhibitor is at about 3 μM to about 35 μM in the composition. In some embodiments, the lipase inhibitor is at about 4 μM to about 30 μM in the composition. In some embodiments, the lipase inhibitor is at about 5 μM to about 25 μM in the composition. In some embodiments, the lipase inhibitor is at 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 in the composition.

[0080] In some embodiments, the lipase inhibitor is (S)-2-formylamino-4-methyl-pentanoic acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]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 fucoxanthin. In some embodiments, the lipase inhibitor is a glycoside, such as acteoside, kaempferol-3-O-rutinoside, rutin, kaempferol, quercetin, and luteolin. In some embodiments, the lipase inhibitor is a polyphenol, such as galangin, hesperidin, licochalcone A, CT-II, 7-phloroequol, and isoliquiritigenin. In some embodiments, the lipase inhibitor is a saponin, such as sesiroside and thianoside. In some embodiments, the lipase inhibitor is a terpene, such as crocin and crocetin. In some embodiments, the lipase inhibitor is derived from bacteria, such as lipstatin, valilactone, persikinin, panclicin, ebelactone, vibralactone, and estellastin. In some embodiments, the lipase inhibitor is a synthetic lipase inhibitor, such as a synthetic analogue of natural fat. Lipase inhibitors are reviewed in Lunagariya yet al., EXCLI J 13:897-921 (2014).

[0081] In some embodiments, the disclosure provides a composition comprising: (a) about 90% to about 99.9% (volume / volume) aqueous assay sample comprising (i) a purified protein preparation comprising a protein and a lipid; (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% (volume / volume) organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethylsulfoxide (DMSO), acetonitrile, or a combination thereof, further comprising 4-methylumbelliferyl oleate (4MuO), wherein the aqueous assay sample has a pH of 5.0 to 7.0.

[0082] In some embodiments, the disclosure provides a composition comprising: (a) about 90% to about 99.9% (volume / volume) aqueous assay sample comprising (i) a purified protein preparation comprising 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% (volume / volume) organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethylsulfoxide (DMSO), acetonitrile, or a combination thereof, further comprising 4-methylumbelliferyl oleate (4MuO), wherein the aqueous assay sample has a pH of 5.0 to 7.0.

[0083] In some embodiments, the disclosure provides a composition, comprising: (a) about 90% to about 99.9% (volume / volume) aqueous assay sample comprising (i) a partially purified protein preparation comprising a protein and a lipid; (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% (volume / volume) organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethyl sulfoxide (DMSO), acetonitrile, or a combination thereof, further comprising 4-methylumbelliferyl oleate (4MuO), wherein the aqueous assay sample has a pH of 5.0 to 7.0.

[0084] In some embodiments, the disclosure provides a composition comprising: (a) about 90% to about 99.9% (volume / volume) aqueous assay sample comprising (i) a cell culture supernatant comprising proteins and lipids, (ii) a buffering agent, (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% (volume / volume) organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethylsulfoxide (DMSO), acetonitrile, or a combination thereof, further comprising 4-methylumbelliferyl oleate (4MuO), wherein the aqueous assay sample has a pH of 5.0 to 7.0.

[0085] In further embodiments, the compositions provided herein are suitable for use in methods for detecting lipolytic activity in a protein preparation. In some embodiments, the present disclosure further provides methods for detecting lipolytic activity in an aqueous assay sample.

[0086] In some embodiments, the disclosure provides a method for detecting lipolytic activity in an aqueous assay sample, the method comprising: (a) combining an aqueous assay sample comprising a protein preparation with an organic solvent comprising 4-methylumbelliferyl oleate (4MuO); and (b) measuring the formation of oleate and 4-methylumbelliferone (4Mu) by fluorescence.

[0087] In some embodiments, the aqueous assay sample is an aqueous assay sample described herein. In some embodiments, the aqueous assay sample has a pH of 5.0 to 7.0.

[0088] 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 the 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, the sodium chloride is about 50 mM to about 400 mM in the aqueous assay sample. In some embodiments, the sodium chloride is about 100 mM to about 200 mM in the aqueous assay sample. In some embodiments, the calcium chloride is about 0.2 mM to about 10 mM in the aqueous assay sample. In some embodiments, the calcium chloride is about 1.0 mM to about 2.0 mM in the aqueous assay sample.

[0089] In some embodiments, the buffer has a 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 about 2 mM to about 200 mM in the aqueous assay sample. In some embodiments, the buffer is about 10 mM to about 100 mM in the aqueous assay sample. In some embodiments, the buffer is about 40 mM to about 60 mM in the aqueous assay sample. In some embodiments, the buffer is about 45 mM to about 55 mM in the aqueous assay sample.

[0090] In some embodiments, the protein preparation is a protein preparation described herein. In some embodiments, the protein preparation is a cell culture supernatant. In some embodiments, the protein preparation is a partially purified protein preparation. In some embodiments, the protein preparation is a purified protein preparation. Protein preparations of various degrees of purification, such as cell culture supernatants, partially purified protein preparations, and purified protein preparations, are described herein. In some embodiments, the protein preparation comprises a therapeutic protein (e.g., a therapeutic protein described herein). The method of the present disclosure advantageously allows for simple and efficient determination of lipolytic activity in a protein preparation throughout the purification process of the protein preparation. For example, the lipolytic activity of a cell culture supernatant (or a product of a subsequent purification process) can be measured using the method to determine whether lipase needs to be removed during a subsequent purification step. Advantageously, the method can be used on the product throughout the purification process to determine whether lipolytic activity in the protein preparation has been adequately eliminated.

[0091] In some embodiments, the protein preparation comprises an additional host cell protein. In some embodiments, the additional host cell protein comprises a lipase. The lipase is described herein.

[0092] In some embodiments, the protein preparation comprises a surfactant (e.g., a 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.

[0093] In some embodiments, the organic solvent is an organic solvent described herein. In some embodiments, the organic solvent is an alcohol, a sulfoxide, a 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 about 3:1.

[0094] In some embodiments, the organic solvent comprises 4-methylumbelliferyl oleate (4MuO). A structure of 4MuO is provided herein. In some embodiments, 4MuO is hydrolyzed to form oleic acid and 4-methylumbelliferone (4Mu), for example, as described in Scheme I. A structure of 4Mu is provided herein. In some embodiments, 4Mu is fluorescent. In some embodiments, the fluorescence of 4Mu is measured at about 330 nm excitation and 495 nm emission.

[0095] In some embodiments, the method includes measuring the fluorescence for up to 24 hours. In some embodiments, the fluorescence is measured from about 24 hours to about 400 hours. In some embodiments, the fluorescence is measured for more than about 24 hours. In some embodiments, the fluorescence is measured for more than about 100 hours. In some embodiments, the fluorescence is measured for more than about 300 hours. It is understood that the fluorescence measurement does not necessarily have to be a continuous measurement, and the fluorescence can be measured at a predetermined time point. In some embodiments, the fluorescence is measured at a selected time point from about 12 hours to about 400 hours. In some embodiments, the fluorescence is measured at 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 period over which fluorescence is measured may be selected depending on the level of lipase activity in the protein preparation, for example, low levels of lipolytic activity may require a longer detection period due to the slower hydrolysis of 4MuO.

[0096] In some embodiments, a protein preparation 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 preparation is then subjected to the method to detect lipolytic activity.

[0097] In some embodiments, the aqueous assay sample and the organic solvent are combined in a ratio of about 70:30 to about 99:1. In some embodiments, the aqueous assay sample and the organic solvent are combined in a ratio of about 75:25 to about 99:1. In some embodiments, the aqueous assay sample and the organic solvent are combined in a ratio of about 80:20 to about 98:2. In some embodiments, the aqueous assay sample and the organic solvent are combined in a ratio of about 85:15 to about 98:2. In some embodiments, the aqueous assay sample and the organic solvent are combined in a ratio of about 90:10 to about 98:2. In some embodiments, the aqueous assay sample and the organic solvent are combined in a ratio of about 95:5 to about 98:2.

[0098] In some embodiments, the aqueous assay sample is incubated with a lipase inhibitor for about 10 minutes to about 1 hour prior to step (a), and the aqueous assay sample containing the protein preparation and the organic solvent are combined. 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 prior to step (a). In some embodiments, incubating the aqueous assay sample with a lipase inhibitor reduces or eliminates lipolytic activity. In some embodiments, incubating the aqueous assay sample with a lipase inhibitor provides a negative control for detection of lipolytic activity. In embodiments, the aqueous assay sample is incubated with a lipase inhibitor prior to step (a), and the measured fluorescence is expected to be low in lipolytic activity (i.e., indicative of low or no lipolytic activity). Lipase inhibitors are described herein. In some embodiments, the lipase inhibitor is (S)-2-formylamino-4-methyl-pentanoic acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]-dodecyl ester (orlistat). In some embodiments, the lipase inhibitor is about 1 μM to about 50 μM in the composition. In some embodiments, the lipase inhibitor is about 5 μM to about 25 μM in the composition.

[0099] In some embodiments, the control sample is measured in parallel with the aqueous assay sample for lipolytic activity. In some embodiments, the present disclosure provides a method for detecting lipolytic activity in an aqueous assay sample, the method comprising: (a) combining an aqueous assay sample comprising a protein preparation with an organic solvent comprising 4-methylumbelliferyl oleate (4MuO) to form an assay composition; (b) combining a control sample comprising the protein preparation and a lipase inhibitor with an organic solvent comprising 4-methylumbelliferyl oleate (4MuO) to form a control composition; and (c) measuring the formation of oleate and 4-methylumbelliferone (4Mu) by fluorescence in the assay composition and the control composition. In some embodiments, the protein preparation in (a) and the protein preparation in (b) are provided from the same protein preparation. For example, a protein preparation can be obtained from a cell culture and two aliquots can be taken from it. One aliquot can be the protein preparation of the aqueous assay sample and the other aliquot can be the protein preparation of the control sample. In some embodiments, the protein preparation of (a) and the protein preparation of (b) contain substantially the same components. In some embodiments, the protein preparation of (a) and the protein preparation of (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., no fluorescence is expected to be detected. In some embodiments, the method further utilizes a positive control sample (i.e., a sample in which fluorescence is expected). In some embodiments, the positive control sample contains a known amount of 4Mu. In some embodiments, the positive control sample contains a known amount of 4MuO and a known amount of active lipase.

[0100] As discussed herein, lipases with lipolytic activity may interfere with components of a protein preparation. In some embodiments, lipases with lipolytic activity hydrolyze fatty acids and / or esters present in a protein preparation. In some embodiments, lipases with lipolytic activity hydrolyze surfactants present in a protein preparation. In some embodiments, hydrolysis of surfactants reduces the stability of the protein preparation. By measuring the amount of lipolytic activity using the methods provided herein, the level of hydrolysis that has occurred in the protein preparation can then be determined based on the measured amount of lipolytic activity, thereby determining the stability of the protein preparation. In some embodiments, the present disclosure provides a method for determining the stability of a protein preparation, comprising: (a) combining an aqueous assay sample containing the protein preparation with an organic solvent containing 4-methylumbelliferyl oleate (4MuO); (b) measuring the formation of oleate and 4-methylumbelliferone (4Mu) by fluorescence; and (c) determining the stability of the protein preparation based on the measured fluorescence. For example, increased fluorescence compared to a control indicates the presence of lipase, which hydrolyzes an excipient (e.g., a surfactant such as polysorbate) thereby forming non-polar, and therefore insoluble, long chain fatty acids, which can destabilize proteins in the protein preparation. In some embodiments, the method is used to determine the stability of a protein preparation in a pharmaceutical formulation.

[0101] In some embodiments, the present disclosure provides a kit suitable for providing the composition of the present invention.In some embodiments, the present disclosure provides a kit that can be used to achieve the method of the present invention.For example, in some embodiments, the present disclosure further provides a kit, comprising in two or more containers: (a) an organic solvent; (b) 4-methylumbelliferyl oleate (4MuO); and (c) a lipase inhibitor.

[0102] Any suitable container may be used in the kits 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 the 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 the organic solvent are in a first container and 4MuO and the organic solvent are in a second container. In some embodiments, the 4MuO is provided as a solid (e.g., a powder). In some embodiments, the 4MuO is provided in a solution (e.g., an organic solvent). In some embodiments, the lipase inhibitor is provided as a solid (e.g., a powder, such as a lyophilized powder). In some embodiments, the lipase inhibitor is provided in a solution (e.g., an organic solvent). In any of the above embodiments, (a) the organic solvent, (b) 4-methylumbelliferyl oleate (4MuO), and / or (c) the lipase inhibitor may be included in respective containers to receive a predetermined specific amount of the protein preparation, the amount of each component being sufficient to carry out the method of determining lipolytic activity described herein. In some embodiments, the kit further includes instructions for utilizing the kit to determine lipolytic activity, as described herein.

[0103] 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 preparation for use with the kit. In some embodiments, the user's protein preparation is in a buffer that is not suitable for use with the kit (e.g., a buffer that promotes autohydrolysis of 4MuO and / or 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 preparation into 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 GE HEALTHCARE, VIVAFLOW and VIVASPIN concentrators from SARTORIUS, BIO-SPIN and ECONO columns from BIO-RAD, and SPINOUT columns from G-BIOSCIENCES.

[0104] The column of 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 column can be used to exchange the buffer in protein preparations to a buffer more suitable for carrying out the method for determining lipolytic activity described herein.

[0105] In some embodiments, the disclosure provides a kit, the kit comprising: (a) an organic solvent comprising 4-methylumbelliferyl oleate (4MuO); (b) a column suitable for buffer exchange of a protein preparation; and (c) a lipase inhibitor.

[0106] Suitable organic solvents for the kit of the present disclosure include organic solvents described herein. In some embodiments, the organic solvent is an alcohol, a sulfoxide, a 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 isopropyl alcohol.

[0107] Suitable lipase inhibitors for the kit of the present disclosure include lipase inhibitors described herein. In some embodiments, the lipase inhibitor is (S)-2-formylamino-4-methyl-pentanoic acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]-dodecyl ester (orlistat). In some embodiments, the lipase inhibitor is used as a control when performing the method for determining lipolytic activity described herein.

[0108] Suitable salts for the kit of the present disclosure include the salts 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.

[0109] Suitable buffers for the kit of the present disclosure include those described herein. In some embodiments, the buffer is Tris. In some embodiments, the buffer is Bis-Tris.

[0110] In some embodiments, the kit further comprises instructions for performing an assay to determine lipolytic activity. In some embodiments, the assay comprises a method described herein.

[0111] All references cited herein, including patents, patent applications, articles, textbooks, and the like, and the references cited therein (to the extent they are not already cited), are hereby incorporated by reference in their entirety. EXAMPLES

[0112] Chemicals and reagents for the experiments described herein are as follows:

[0113] 2-Propanol (IPA, 99.9%), acetonitrile (ACN, HPLC Plus, ≥99.9%), calcium chloride (≥97%), Triton X-100 (laboratory grade), sodium phosphate monobasic 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 (bio-reagent, ≥99%), TRIZMA® hydrochloride (reagent grade, 99.0%), TRIZMA® 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 KOLLIPHOR® P188 (suitable for cell culture) were obtained from Sigma. Acetonitrile (LC-MS grade) was obtained from Thermo Fisher. Ethanol (EtOH, liquid chromatography gradient grade) and hydrochloric acid 25% (v / v) were obtained from Merck. Sucrose (USP / NF, EP, JP, high purity) was obtained from Pfanstiehl. A 1 M solution of sodium hydroxide was obtained from Honeywell FlukaTM. Sodium chloride (USP, multicompendial), L-histidine (USP, multicompendial), L-histidine monohydrochloride (FCC, multicompendial), PS20 (NF), and PS80 (NF) were obtained from JT Baker. Highly purified water (hereafter referred to as "water") was prepared by using a water purification system (Barnstead™ GenPure™ Pro, Thermo Fisher). pH was measured using a pH meter (780 pH meter, Metrohm) and a platinum pH electrode (Unitrode Pt1000, Metrohm).Cell culture harvest fluid (CCHF) containing a mixture of lipases as part of the HCP produced from a proprietary CHO cell line expressing a non-therapeutic monoclonal antibody (mAb1), as well as mAb1, were obtained from Lonza Biologics, Slough, UK.

[0114] Example 1A. Lipase Assay Development I: pH A lipase assay was developed based on the method described by Kurihara et al., Biol Pharm Bull 26:383-385 (2003), which utilizes 4-methylumbelliferyl oleate (4MuO) as a substrate for the lipase enzyme.

[0115] Porcine pancreatic lipase (PPL) was used in the development of the assay, in which 500 nM PPL and 4MuO were incubated for 22 h in one of four different buffer systems and tested for fluorescence quenching and autohydrolysis of 4MuO: (a) 10.4 mM 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.

[0116] The results in Figures 1A-1B and Table 1 show that autohydrolysis of 4MuO was reduced at pH 6 compared to pH 8. Figure 1C shows that the rate of autohydrolysis at pH 8 was nearly twice that at pH 6. Lipase was slightly more active (20% less) at pH 8 compared to pH 6. The buffer concentration did not appear to have a strong 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. [Table 1]

[0117] The autohydrolysis of 4MuO was further investigated. ais approximately 7.7. Figure 1D shows the pH range over which 4Mu was measured (graph reprinted from Zhi et al., J Spectrosc 1 (2013) doi:10.1155 / 2013 / 147128). Figure 1E shows the four forms of 4Mu across various pH ranges (reprinted from Zhi et al., 2013). Form II predominates, with a λ of 320 nm. ex and λ of 445 nm em The excitation and emission are shown in FIG.

[0118] Example 1B. Lipase Assay Development I: pH Further tests were performed to evaluate different buffering agents and pH of the lipase assay buffer. The following volume ratios were applied (all v / v): sample (75%), high concentration matrix buffer (HCMB) (20%), organic solvent (5%). The mixtures were incubated for a time frame of about 24 hours to about 300 hours. The components of the assay were transferred to a 96-well plate and analyzed for fluorescence intensity. For the 4 μM calibration curve, the reference standard 4 μM was prepared in organic solvent at concentrations of 0 μM, 0.2 μM, 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, 50 μM, and 100 μM, and the other assay components were spiked with 5% (v / v) 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. The slope of the calibration curve was found to be slightly shifted from the low calibration range (0.01-0.5 μM) to the high calibration range up to 5 μM. Therefore, the quantitative results of 4Mu were calculated using a calibration curve limited to 0.01-0.5 μM, with a concentration range of up to 0.5 μM. The calibration curves of 4Mu are shown in Figures 19A and 19B.

[0119] In this study, the "sample" was a placebo composition of buffer containing 20 mM L-histidine, pH 6 (pH adjustment was made by adding 25% (w / v) HCl or 1 M NaOH), 250 mM sucrose, 0.5% cell culture harvest fluid (CCHF), or porcine pancreatic lipase (PPL) at approximately 0.0025 mg / mL. HCMB contained TRIS (208 mM) at pH 7 and pH 8, or BIS-TRIS (208 mM) at pH 6 (pH adjustment was made by adding 25% (w / v) HCl or 1 M NaOH), NaCl (200 mM, 600 mM, or 1800 mM), CaCl2 (0.52 mM, 5.2 mM, or 52 mM). The organic solvent was either methanol (MeOH), dimethylsulfoxide (DMSO), or isopropanol (IPA) containing 100 μM 4MuO, with 5% (v / v) organic solvent / substrate added. The assay components were transferred to a 96-well plate and analyzed for fluorescence intensity.

[0120] For fluorescence analysis, samples were transferred to a 96-well microplate (Thermo Scientific™ Nunc™ F96 Microwell™ black polystyrene plate). Fluorescence measurements were performed with a Molecular Devices SpectraMax iD3 microplate reader and SMP7.1 software. The excitation maximum (λ ex ) and emission wavelength (λ em ) were 330 nm and 495 nm, respectively, and after optimization, for an intermediate concentration of 4 Mu, using SoftMax Pro7.1 (SMP7.1) wavelength optimization mode. Fluorescence signals were read at incubation times from about 24 h to about 300 h, and for each reading, a new 96-well microplate was prepared by transferring 200 μL of liquid (containing sample, HCMB, and organic solvent) into each well. Fluorescence measurements were performed at ambient temperature.

[0121] Figure 11 shows the results of the evaluation of two different buffers (TRIS or BIS-TRIS) at pH 6, pH 7, or pH 8. The conversion of 4MuO to 4Mu was measured by monitoring the fluorescence intensity of 4Mu. Samples containing either PPL or CCHF and 4MuO autohydrolysis were evaluated. The most significant effect of the pH change was observed for autohydrolysis (AH), which decreased approximately 5-fold when the pH was lowered from 8 to 6. The 4Mu concentration in samples of different pH was measured against a standard curve of 4Mu in a reference sample at the same pH. That is, the observed trends are not due to different responses of 4Mu fluorescence at different pHs, but rather to different reaction kinetics. PPL activity increased almost 2-fold when the pH was lowered from 8 to 6, whereas CCHF lipolytic activity was unaffected. The pH dependence of PPL has been previously described by Li et al., “Adsorption and catalytic activity of Porcine pancreatic lipase on rod-like 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 types of lipases that may be present in the HCPs contained in the DS pool. pH 6 was considered most suitable in view of the fact that many therapeutic protein formulations in which polysorbate degradation has been observed and reported are typically maintained at a slightly acidic pH, and therefore adapting the assay pH to about 6 is assumed to encompass the culprits (e.g., lipases) responsible for causing lipase-mediated hydrolytic polysorbate degradation. Therefore, a concentration of 200 mM BIS-TRIS was selected and added to HCMB to obtain a final assay concentration of 40 mM at pH 6.

[0122] Example 2A. Lipase Assay Development II: CaCl The calcium chloride concentration in the lipase assay buffer was investigated. Lipase assays using 500 nM PPL and 4MuO were performed at three different CaCl2 concentrations to investigate the fluorescence quenching and autohydrolysis of 4MuO: (a) 0.104 mM CaCl2, (b) 1.04 mM CaCl2, and (c) 10.4 mM CaCl2.

[0123] The results in Figures 2A-2C and Table 2 show that no autohydrolysis of 4MuO occurred and no fluorescence quenching by chloride anions was observed. Lipase activity was highest at 1.3 mM CaCl2. [Table 2]

[0124] Example 2B. Lipase Assay Development II: CaCl The calcium chloride concentration in the lipase assay buffer was investigated in further experiments. The samples, HCBM compositions, and experimental procedures were as described in Example 1B, and CaCl2 was included in the HCMB at 0.52 mM, 5.2 mM, or 52 mM as described in Example 1B.

[0125] The results of the evaluation of different CaCl2 concentrations in HCMB with samples containing either PPL or CCHF are shown in Figure 12. At the highest concentration of CaCl2, a slight decrease in PPL activity was observed. Considering the HCMB requirement to establish similar assay conditions for various drug substance / drug product matrices, 5 mM CaCl2 was selected and added to HCMB to obtain a final assay concentration of 1 mM CaCl2.

[0126] Example 3A. Lipase Assay Development III: NaCl The sodium chloride concentration in the lipase assay buffer was investigated. Lipase assays using 500 nM PPL and 4MuO were performed at three different NaCl concentrations to investigate the fluorescence quenching and autohydrolysis of 4MuO: (a) 40 mM NaCl, (b) 120 mM NaCl, and (c) 360 mM NaCl.

[0127] The results in Figures 3A-3C and Table 3 show that autohydrolysis of 4MuO did not occur and no fluorescence quenching by chloride anions was observed. Lipase activity was highest at 120 mM NaCl. [Table 3]

[0128] Example 3B. Lipase Assay Development III: NaCl The sodium chloride concentration in the lipase assay buffer was investigated in further experiments. The samples, HCBM composition, and experimental procedures were as described in Example 1B. As described in Example 1B, NaCl was included in the HCMB at 200 mM, 600 mM, or 1800 mM.

[0129] The results of the evaluation of different NaCl concentrations in HCMB with samples containing either PPL or CCHF are shown in Figure 13. At the highest concentration of NaCl, a slight decrease in PPL activity was observed. Considering the HCMB requirement to establish similar assay conditions for various drug substance / drug product matrices, 600 mM NaCl was selected and added to HCMB to give a final assay concentration of 120 mM NaCl.

[0130] Example 4A. Lipase Assay Development IV: Organic Solvents Organic solvents in the lipase assay buffer were examined. Lipase assays using 500 nM PPL and 4MuO were performed using three different organic solvents to examine the fluorescence quenching and autohydrolysis of 4MuO: (a) DMSO, (b) isopropanol (IPA), and (c) methanol (MeOH).

[0131] The results in Figures 4A-4C and Table 4 show that of the three solvents, the least autohydrolysis was observed with isopropanol, although there was slight fluorescence quenching. However, methanol produced comparable results. Furthermore, Glogauer et al., Microb Cell Fact 10:54 (2011) show that methanol may have the ability to improve activity. [Table 4]

[0132] Example 4B. Lipase Assay Development IV: Organic Solvents Organic solvents in lipase assay buffers were investigated in further experiments. Samples, HCBM compositions, and experimental procedures were as described in Example 1B. As described in Example 1B, organic solvents methanol (MeOH), dimethyl sulfoxide (DMSO), or isopropanol (IPA) containing 100 μM 4MuO were included in the assay reactions.

[0133] Figure 14 shows the results of evaluation of different organic solvents with samples containing either PPL or CCHF. This is different from previous results reported in Glogauer et al., “Identification and characterization of a new true lipase isolated through metagenomic approach,” Microb Cell Fact 2011;10:54 (where strong effects of different solvents were reported for LipC12 lipase isolated from E. coli by metagenomic approach). According to Glogauer et al., MeOH- and (to a lesser extent) IPA- showed an activating effect, increasing LipC12 lipase activity by more than 10-fold. However, the bacterial enzymes studied are likely to have very different properties, as evidenced by different optimum pH for activity, for example, compared to the results of Example 1B. Based on the results of Figure 14, MeOH was selected for addition to HCMB.

[0134] Example 5A. Lipase Assay Development V: Surfactants The performance of lipase assay buffers in the presence of surfactants was examined. Lipase assays using 500 nM PPL and 4MuO were performed using two different concentrations of two surfactants to examine the fluorescence quenching and autohydrolysis of 4MuO: (a) 0.012% (w / v) TRITON X-100, (b) 0.06% (w / v) TRITON X-100, (c) 0.032% (w / v) KOLLIPHOR P188, and (d) 0.16% (w / v) KOLLIPHOR P188. A control without surfactant was also tested.

[0135] The results in Figures 5A-5C and Table 5 show that samples containing surfactant had the lowest autohydrolysis, although fluorescence quenching was observed, and lipolytic activity appeared to decrease with surfactant. [Table 5]

[0136] Example 5B. Lipase Assay Development V: Surfactants Further studies were performed to evaluate the effect of surfactants on the lipolytic activity of PPL and CCHF. The surfactants polysorbate 20 (PS20) and polysorbate 80 (PS80), which are found in biopharmaceutical formulations, were evaluated. Poloxamer was also tested. Previous studies (Gupta et al., “Simplified para-nitrophenyl palmitate assay for lipases and esterases,” Anal Biochem 2002;311:98-99) suggested that surfactants could solubilize poorly soluble substrates such as p-nitrophenyl palmitate.

[0137] Samples contained 20 mM L-histidine, 250 mM sucrose, surfactant (either 0.02% or 0.06% (all w / v) polysorbate 20 (PS20), polysorbate-80 (PS80), or poloxamer, or as a no surfactant control), 0.5% CCHF or 0.0025 mg / mL PPL at pH 6. HCMB contained 208 mM BIS-TRIS, 600 mM NaCl, 5.2 mM CaCl2 at pH 6. 5% (v / v) organic solvent MeOH containing 100 μM 4MeO was added to the assay. Assay components were transferred to a 96-well plate and analyzed for fluorescence intensity as described above for Example 1B.

[0138] The results of the effect of detergents on lipolytic activity are shown in Figure 15. The presence of detergents had a significant effect on lipolytic activity. Polysorbate significantly reduced the formation of 4Mu by >90% in PPL and approximately 80% in CCHF. PS80 reduced the formation of 4Mu to a lesser extent than PS20.

[0139] There are several possible reasons for these observations: (1) polysorbates consisting of fatty acid esters are competitive substrates / inhibitors of lipases, and / or (2) the non-polar substrate 4MuO is incorporated into the micellar structure of polysorbates, decreasing its concentration in the aqueous medium, which therefore correlates with the substrate concentration and negatively affects the enzyme rate constant (e.g., according to the concept of Michaelis-Menten kinetics), and / or (3) the lipase enzyme may be structurally affected, leading to unfolding and thereby reducing its activity.

[0140] Interestingly, the concentration of polysorbate seemed to affect the lipolytic activity of CCHF. PPL was similarly active with both polysorbates at the two different selected concentrations, but the higher concentrations of polysorbate were less active. Poloxamer, on the other hand, affected the lipolytic activity of PPL and CCHF in very different ways. The activity of PPL was reduced to about 50% compared to the control, and as already seen with polysorbate, the selected concentrations of surfactants did not show any obvious variation in enzyme activity. The lipolytic activity of CCHF was positively affected by poloxamer at least at a surfactant concentration of 0.02% and was clearly unaffected at a concentration of 0.06%. Without being bound to any particular theory, one reason for the enzymatic behavior in the presence of surfactants as well as the concentration dependence could be a combination of two or more causes. For example, kinetic and mechanistic investigations of lipases are difficult due to the fact that the enzyme catalyzes reactions on substrates (e.g., triglycerides and other fats) that are poorly soluble in water. At the water / fat interface, the enzyme is activated by the movement of the lid domain; in the closed conformation, the active site is protected, while in the open conformation, the active site becomes accessible to substrates. See, e.g., Lowe, “The triglyceride lipases of the pancreas,” J Lipid Res 2002;43(12):2007-2016.

[0141] In particular, polysorbates have a negative effect on the lipolytic activity of model lipases PPL and CCHF. As shown in Figure 15A, the fluorescence intensity of 4Mu hydrolysis products is also affected (about 20% decrease) in the presence of surfactants (see Figure 15B). This also contributes to the decrease in the signal intensity of 4Mu products.

[0142] Example 6A. Lipase Assay Development VI: Lipase Inhibitors Orlistat, a lipase inhibitor, was selected for control experiments in the lipase assay to evaluate the performance of the assay with different concentrations of the inhibitor. Orlistat was developed as a mechanism-based inhibitor that inhibits the enzyme by covalently binding to the serine in the active site of pancreatic lipase. This mode of action has made it an orally administered drug for treating obesity and related conditions based on fat hydrolysis in the gastrointestinal tract. No studies have been published that systematically investigate which classes of lipases can be inhibited by orlistat. However, it has previously been reported that apart from mammalian lipases, bacterial lipases (e.g., those from Streptomyces rimosus) can be inhibited with millimolar concentrations of orlistat and that the inhibition occurs by covalent modification of the serine in the active site. For example, Hadvary et al., “The lipase inhibitor tetrahydrolipstatin binds covalently to the putative active site serine of pancreatic lipase,” J Biol Chem 1991;266(4):2021-2027, Heck et al., “Orlistat, a new lipase inhibitor for the management of obesity.Pharmacotherapy 2000;20(3):270-279, Asler et al., “Mass spectrometric evidence of covalently-bound tetrahydrolipstatin at the catalytic serine of Streptomyces rimosus lipase,” Biochim Biophys See Acta. 2007;1770:163-170. In these previous studies, a large volume ratio of organic solvent, up to 50% (v / v), was required in the final assay to introduce the hydrophobic inhibitor at this high concentration. Such high concentrations of organic solvent were not anticipated in the current lipase assay due to potential interference with other assay components (e.g., precipitation of pharma- ceutical active proteins), so we chose to use lower solvent and inhibitor concentrations as well.This does not necessarily have a negative effect on the assay readout, as mammalian lipases have been found to be inhibited at much lower concentrations (i.e., nanomolar concentrations) of orlistat. See, e.g., Lewis et al., “Direct measurement of lipase inhibition by Orlistat using a dissolution linked in vitro assay,” Clin Pharmacol Biopharm 2012;1(3):1-3.

[0143] Lipase activity was tested to examine fluorescence quenching and autohydrolysis of 4MuO after co-treatment and pre-treatment of samples containing 500 nM PPL with three concentrations of orlistat (a lipase inhibitor): (a) co-treatment with 25 μM orlistat, (b) co-treatment with 15 μM orlistat, (c) co-treatment with 5 μM orlistat, (d) pre-treatment with 25 μM orlistat, (e) pre-treatment with 15 μM orlistat, and (f) pre-treatment with 5 μM orlistat. A control sample without orlistat was also tested.

[0144] The results in Figures 6A-6B and Table 6 show that pretreatment with 25 μM orlistat (30 min) completely abolished lipolytic activity. Residual activity was observed at lower concentrations of orlistat. No fluorescence quenching was observed. [Table 6]

[0145] Example 6B. Lipase Assay Development VI: Lipase Inhibitors The concentration of the lipase inhibitor orlistat was evaluated in further experiments. The samples, HCMB compositions, and experimental procedures were performed similarly to Example 1B. The inhibitor was prepared in MeOH at concentrations of 200, 600, and 1000 μM. For pre-incubation experiments, the inhibitor solution was supplemented to the assay at 2.5% (v / v) and pre-incubation was performed for 30 minutes. Then, 200 μM of 4MuO in MeOH was added to the assay at 2.5% (v / v). For co-incubation experiments, 100 μM of 4MuO and orlistat (100 μM, 300 μM, and 500 μM) in MeOH were added at 5% (v / v). A high concentration PPL of 0.0335 mg / mL was used.

[0146] Three concentrations of orlistat were tested, namely 5 μM, 15 μM, and 25 μM (relative to the final assay volume), using a very high concentration of lipase in the "worst case", i.e. 0.0335 mg / mL PPL. At these concentrations, the final assay solutions were clear and showed no turbidity due to potentially insoluble inhibitors. Furthermore, the effect on lipase activity of orlistat directly in the assay with substrate in 5% (v / v) MeOH was tested, compared to pre-addition of orlistat to a sample containing enzyme in 2.5% (v / v) MeOH, incubated for 30 min, followed by addition of substrate in another 2.5% (v / v) volume fraction of MeOH.

[0147] Figure 16 shows the results of the evaluation of lipase inhibitors. Due to the high concentration of PPL as described above, in the absence of orlistat, 4MuO was almost completely hydrolyzed within 24 hours. It can also be seen that orlistat inhibits PPL and its inhibitory effect correlates with the orlistat concentration, i.e., the highest inhibitory effect was observed at 25 μM orlistat. Pre-incubation of the enzyme with the inhibitor had a beneficial effect; i.e., at the highest orlistat concentration tested of 25 μM, the enzyme activity was reduced by about 50% in the pretreated samples compared to the samples where the substrate and inhibitor were added simultaneously. Therefore, orlistat was chosen to be supplemented in the negative control at a high concentration (i.e., 25 μM), and the negative control was pre-incubated with the inhibitor for about 30 minutes before adding the substrate.

[0148] Example 7. Lipase Assay Development VII: Fatty Acid Product Inhibition In the lipase assay, fatty acid product inhibition was tested using two concentrations of theoretically degraded PS20 and PS80, oleic acid and lauric acid: (a) 0.1% and (b) 0.001%. In this experiment, the concentration of PPL was 800 nM.

[0149] The results in Figures 7A-7D and Table 7 show that lauric acid 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. [Table 7]

[0150] Example 8. Lipase Assay Development VIII: Quenching Effect The quenching effect of polysorbate 80 was examined. Samples containing 800 nM PPL and fresh PS80 were tested against controls containing 800 nM PPL and no PS80. The results in Figure 8 show that PS80 reduces the RFU values ​​of each standard, suggesting a mild quenching effect.

[0151] Example 9. Evaluation of Lipase Assay: CCHF To assess the applicability of the assay developed in Examples 1-8 for testing lipolytic activity in drug substances / drug products, the assay was evaluated at a concentration of lipase that could result in degradation of polysorbate within the time frame that such degradation is observed in drug products (e.g., within one month).

[0152] Samples for this study contained 20 mM L-histidine, 250 mM sucrose, 0.02% (w / v) 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) at pH 6. Samples were stored at room temperature and at 2-8 °C for 1 month. Samples were analyzed by HPLC-FMA as described below and the lipase assay herein. For the positive control of the assay, samples were supplemented with 0.055 mg / mL PPL. HCMB contained 208 mM BIS-TRIS, 600 mM NaCl, 5.2 mM CaCl2 at pH 6. 5% (v / v) organic solvent MeOH containing 100 μM 4MuO was added to the assay. For the negative control containing orlistat, the inhibitor was prepared at 1000 μM in MeOH and supplemented to the assay at 2.5% (v / v) and pre-incubated for 30 minutes. Then, 200 μM 4MuO in MeOH was added to the assay at 2.5% (v / v). The assay components were transferred to a 96-well plate and analyzed for fluorescence intensity as described for Example 1B.

[0153] Quantitative analysis of intact PS20 and PS80 was performed using a high performance liquid chromatography (HPLC) system (Waters Alliance e2695) equipped with an isocratic pump, autosampler, braided reactor coil (1 mL; Supelco #57410-U), and a fluorescence detector (Waters 2475FLR detector). Samples were injected in a mobile phase containing 5 μM N-phenyl-1-naptylamine (NPN), 15 ppm (w / v) Brij® 35 dissolved in a solution of 150 mM NaCl, 50 mM TRIS, 5% (v / v) ACN, pH 8. Fluorescence was measured at λ ex = 350 nm and λ em = 420 nm. Empower3 Chromatography Data System (CDS) was used for peak integration and analysis.

[0154] Table 9 summarizes the samples tested. [Table 8]

[0155] Figures 9A and 9B show the results from HPLC-FMA, indicating that polysorbate degradation was observed at CCHF concentrations of 0.10% and above. PS20 samples were more susceptible to degradation, with the effect seen at approximately 0.10% CCHF. Higher concentrations showed a decrease in apparent polysorbate concentration. At 25°C, the trend was more pronounced compared to 2-8°C. PS20-containing samples were apparently more susceptible to degradation than PS80-containing samples, with a decrease in signal intensity only detectable at 0.5% CCHF. In the aforementioned experiments on surfactant evaluation, PS80 was found to have a slightly enhanced negative effect on lipolytic activity in CCHF-containing samples compared to PS20. Therefore, as a worst case, PS80-containing samples from the HPLC-FMA assay were subjected to a lipase assay.

[0156] Figure 9C shows the theoretical readout of the lipase assay developed herein, and Figure 9D shows the actual results. The actual results are comparable to the expected readout. Samples containing different concentrations of CCHF showed the formation of 4Mu, which correlated with the CCHF concentration. This enzyme activity was higher than that of the three negative controls (i.e., the autohydrolysis sample and the two CCHF samples spiked with orlistat). The assay was sensitive enough to detect lipase activity at concentrations where the HPLC-FMA method still did not detect polysorbate degradation (i.e., at CCHF concentrations below 0.1%).

[0157] 10A-10H show additional lipase assay measurements containing PS80 in different concentrations of CCHF ranging from 0.001% to 2%. The concentrations of CCHF and polysorbate shown were prior to the addition of 4MuO and buffer.

[0158] Figure 18 summarizes the results of the lipase assay. The 1% CCHF sample spiked with PPL (positive control of the assay) showed higher activity than samples of the same CCHF concentration not spiked with PPL. The formation of 4Mu correlated with the concentration of CCHF, and this lipolytic activity was higher than that of the autohydrolysis and other negative controls spiked with orlistat. Most notably, the assay was sensitive enough to distinguish lipase activity from autohydrolysis and the negative controls at CCHF concentrations below 0.1%, and lipase assays can detect lipolytic activity at concentrations where the HPLC-FMA method still does not detect polysorbate degradation (i.e., at CCHF concentrations of 0.01% and 0.05%).

[0159] Example 10. Evaluation of Lipase Assay: Protein-Containing Formulations Biopharmaceutical formulations that may be adversely affected by lipolytic activity include biopharmaceutical proteins. The lipase assays developed in Examples 1-8 were tested on protein-containing formulations.

[0160] Samples contained 20 mM L-histidine, 250 mM sucrose, 0.02% PS80, 10 mg / mL mAb1 (proprietary IgG1), 0.5% CCHF or 0.025 mg / mL PPL at pH 6. HCMB consisted of 208 mM BIS-TRIS, 600 mM NaCl, 5.2 mM CaCl2 at pH 6. 100 μM 4MuO in 5% (v / v) organic solvent MeOH was added to the assay. For negative controls containing orlistat, the inhibitor was prepared at 1000 μM in MeOH and supplemented to the assay at 2.5% (v / v) and pre-incubated for 30 min. Then, 200 μM 4-MuO in MeOH was added to the assay at 2.5% (v / v). The assay components were transferred to a 96-well plate and analyzed for fluorescence intensity as described for Example 1 B. Table 10 summarizes the samples tested. [Table 9]

[0161] Figure 17A shows the assay results at different time points (1 h, 24 h, 72 h, 168 h, and 336 h). As expected, the assay positive control containing both CCHF and PPL showed the highest release of 4Mu. The assay negative control showed strongly reduced activity, indicating that both PPL and CCHF lipases were inhibited by orlistat. A similar trend is seen for the sample and formulation positive controls (high activity), while the sample and formulation negative controls were inhibited by orlistat (low activity). The autohydrolysis control and formulation negative control show similar readouts at all time points that are indistinguishable in Figure 17A. Figure 17B shows a close-up of the negative control, indicating that the activity in both negative controls is similar.

[0162] An offset can be observed in the apparent activity of the assay negative control / sample negative control compared to the autohydrolysis control / formulation negative control. The same offset was observed in the blank sample without substrate (data not shown), indicating that the protein-containing sample already had a fluorescence response independent of 4Mu. Within the time frame in which lipolytic activity was tested (i.e., within 336 hours), the offset increased, indicating that the initially inhibited CCHF and PPL lipases were slowly reactivated, but were clearly absent in the formulation negative.

[0163] Lookene et al. reported that orlistat is not only a mechanism-based inhibitor but also a true substrate of lipoprotein lipase, with fast inhibition (i.e., formation of a covalent enzyme-orlistat complex at the active site serine) and slow hydrolysis of the complex (see, e.g., Lookene et al., “Interactions of lipoprotein lipase with the active-site inhibitor tetrahydrolipstatin (Orlistat)R,” Eur J Biochem. 1994;222:395-403). With regard to the present assay and its application to the assessment of lipolytic activity in drug substances / drug products, this is relevant: if the culprits (e.g., lipolytic enzymes) responsible for the degradation of polysorbates, which are part of the residual fraction of HCPs in drug substances / drug products, can be inhibited by orlistat, they can be reactivated. If no distinction of lipolytic activity between the sample and the sample negative control is possible, this may indicate either low lipolytic activity in the sample or the presence of lipolytic enzymes that are not inhibited by orlistat. In the former case, if the formation of 4Mu is not significantly increased compared to the autohydrolysis control, this indicates low lipolytic activity and low risk of degradation of polysorbate in the drug substance / drug product. If there is high formation of 4Mu in the sample compared to autohydrolysis, this indicates that lipolytic enzymes are present but cannot be inhibited by orlistat.

[0164] All references cited herein, including patents, patent applications, articles, textbooks, and the like, and the references cited therein (to the extent they are not already cited), are hereby incorporated by reference in their entirety.

Claims

1. 1. A composition comprising: a. an aqueous assay sample comprising a protein preparation, the protein preparation comprising a therapeutic protein that does not have lipolytic activity; b. an organic solvent, further comprising 4-methylumbelliferyl oleate; Including, the aqueous assay sample has a pH of 5.0 to 7.0; the aqueous assay sample is 80% to 99.9% of the composition, and the organic solvent is 0.1% to 20% of the composition; composition.

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

3. The composition of claim 1 or 2, wherein the protein preparation comprises a surfactant.

4. The composition of claim 3 , wherein the surfactant is a polysorbate.

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

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

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

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

9. The composition of claim 8, wherein the sodium chloride is between 50 mM and 400 mM in the aqueous assay sample.

10. 10. The composition of claim 8 or 9, wherein the calcium chloride is between 0.2 mM and 10 mM in the aqueous assay sample.

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

12. The composition of any one of claims 7 to 11, wherein the buffering agent is at 2 mM to 200 mM in the aqueous assay sample.

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

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

15. 15. The composition of claim 14, wherein the lipase inhibitor is (S)-2-formylamino-4-methyl-pentanoic acid (S)-1-[[(2S,3S)-3-hexyl-4-oxo-2-oxetanyl]methyl]-dodecyl ester.

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

17. a. a 90% to 99.9% (volume / volume) aqueous assay sample, i. a purified protein preparation comprising a therapeutic protein and a lipid that does not have lipolytic activity; ii. a buffering agent; iii. 1.0 mM to 2.0 mM calcium chloride, and iv. 100 mM to 200 mM sodium chloride; an aqueous assay sample comprising: b. 10% to 0.1% (volume / volume) of an organic solvent selected from methanol, ethanol, propanol, isopropanol, butanol, sec-butanol, isobutanol, tert-butanol, dimethylsulfoxide, acetonitrile, or a combination thereof, further comprising 4-methylumbelliferyl oleate; The composition of claim 1, wherein the aqueous assay sample has a pH of 5.0 to 7.

0.

18. 1. A method for detecting lipolytic activity in an aqueous assay sample, the method comprising: a. combining the aqueous assay sample comprising a protein preparation, the protein preparation comprising a therapeutic protein that does not have lipolytic activity, with an organic solvent comprising 4-methylumbelliferyl oleate; b. Measuring the formation of oleate and 4-methylumbelliferone by fluorescence; Including, the aqueous assay sample has a pH of 5.0 to 7.0; the aqueous assay sample is 80% to 99.9% of a composition comprising the aqueous assay sample and an organic solvent, and the organic solvent is 0.1% to 20% of the composition; method.

19. 20. The method of claim 18, further comprising: c) determining the stability of the protein preparation based on the measured fluorescence.

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