Method for detecting foreign lipase activity

Abstract

To provide a method for detecting foreign lipase activity in a sample of recombinant protein, and a kit therefor.SOLUTION: A method includes the steps of: bringing at least one sample into contact with a reaction solution containing (i) a buffer solution having pH of from about 4 to about 9, (ii) a non-denaturing surfactant (nonionic or zwitterionic surfactant) having no ester bond, (iii) a substrate (saturated non-branched-chain fatty acid (C6 to C16) 4-MU ester) containing chromophore 4-methylumbelliferyl (4-MU) in the form of 4-MU ester, and (iv) optionally selectively, non-buffer salt; incubating the sample and the substrate in a reaction mixture; and measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU to detect foreign lipase activity.SELECTED DRAWING: None

Description

Technical Field

[0001] The present invention relates to a method for detecting contaminating lipase activity in a sample of a recombinant protein. More specifically, the method comprises contacting at least one sample (such as an IPC sample) with a reaction solution comprising: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond, said surfactant being a non-ionic or zwitterionic surfactant; (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, said 4-MU ester being a saturated unbranched fatty acid (C6-C16) 4-MU ester; and (iv) optionally, a non-buffered salt; and detecting the contaminating lipase activity by measuring the hydrolysis of said 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU. Further provided is a kit for measuring contaminating lipase activity in a sample containing a recombinant protein, such as an IPC sample, the kit comprising: (i) a buffer having a pH of from about 4 to about 9; (ii) a non-denaturing surfactant having no ester bond, said surfactant being a non-ionic or zwitterionic surfactant; and (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, said substrate being a saturated unbranched fatty acid (C6 to C16) 4-MU ester.

Background Art

[0002] Proteins as therapeutic agents have become increasingly common in the last few decades. Formulations containing therapeutic proteins, such as monoclonal antibodies, often contain high protein concentrations of 100 mg / mL or more and often require the presence of a surfactant. Due to their biocompatibility and low toxicity, the most widely used surfactants in the biopharmaceutical industry are polysorbates (PS), such as polysorbate 20 (polyoxyethylene (20) sorbitan monolaurate, Tween 20 (trademark)) or polysorbate 80 (polyoxyethylene (20) sorbitan monooleate, Tween 80 (trademark)).

[0003] Polysorbate is a heterogeneous mixture of sorbitol and its anhydrides with approximately 20 polymerized ethylene oxide moieties that are partially esterified with fatty acids. However, polysorbate is prone to decomposition, which can have an adverse effect on the quality of the product. Decomposition can affect the product quality not only through the resulting reduced polysorbate concentration in the formulation, but also through the formation of visible and sub-visible particles derived from insoluble substances of polysorbate degradation products (such as fatty acids and polyoxyethylene side chains). Polysorbate can be decomposed chemically or enzymatically. Chemical decomposition of polysorbate is mainly caused by oxidation reactions, which result in the formation of aldehydes, ketones and fatty acids, among other things. Enzymatic decomposition of polysorbate is characterized by the hydrolysis of the ester bond that binds polyethoxylated sorbitan and fatty acids (Dwivedi et al., 2018, International Journal of Pharmaceutics 552:442-436). Oxidative decomposition of polysorbate has been known for a long time, but enzymatic hydrolysis of polysorbate in antibody formulations has only recently come to be considered as one of the major degradation pathways. In recent years, polysorbate decomposition has emerged as a major issue in the biopharmaceutical industry.

[0004] The residual hydrolytic activity of lipases or other enzymes of host cell proteins (HCPs) contained in the final drug product (DP) has been reported to cause the degradation of polysorbates. The role of lipases in the degradation of polysorbates in antibody formulations has been further emphasized by Chiu et al., in which cell culture harvest fluid (HCCF) collected from lipoprotein lipase (LPL) knockout CHO cells decreased the degradation of PS20 and PS80 compared to the wild type (Chui et al., 2017, Biotechnol. Bioeng. 114, 1006 - 1015). Since it takes several weeks to measure the effect of individual purification steps and conditions on the degradation of polysorbates, it is difficult to make the necessary changes and adaptations to the upstream and especially downstream production processes in the production of therapeutic proteins.

[0005] The content and degradation of polysorbates can be studied using various analytical techniques. The most commonly used method for the quantification of polysorbates is reverse-phase liquid chromatography (such as RP-HPLC), which may be further combined with an evaporative light scattering detector (ELSD) and a charged aerosol detector (CAD). Other techniques capable of measuring the content of polysorbates consist of a fluorescence micelle assay (FMA) or the chemical complex formation of the sorbitan ring with cobalt thiocyanate or iron(III) thiocyanate. However, to determine whether a change in the purification process has been successful in reducing the hydrolytic activity responsible for polysorbate degradation, it is necessary to spike the sample of interest with polysorbate and analyze its degradation as described above. Therefore, typically, the degradation of polysorbates is evaluated by monitoring the decrease in polysorbate content over time. However, the degradation of polysorbates is a slow process and may take several weeks or months. Furthermore, the analysis is complex and time-consuming.

[0006] In order to develop purification conditions that minimize the enzymatic degradation of polysorbate in the pharmaceutical, there is a need for a sensitive, fast, and reliable automated high-throughput assay that can be easily adapted to various samples and provides predictive information about the hydrolytic activity that causes the degradation of polysorbate in the active ingredient of the pharmaceutical sample. Such an assay is useful as a tool to guide process development for producing an active ingredient with improved product quality, since the degradation activity of polysorbate co-purified with the target protein is minimized.

[0007] The in vitro detection of lipase hydrolysis activity using a fluorescent substrate is known in the art, but these prior art assays lack sufficient sensitivity to reliably detect contaminating lipase activity in recombinant protein preparations (those co-purified from eukaryotic cells only with the recombinant protein (the protein of interest)) in a short time. For example, Tsuzuki et al. (Biosci. Biotechnol. Biochem, 2001, 65(9): 2078-2082) analyzed the activities of several lipases derived from microorganisms at high concentrations using a fluorescent substrate and found that the hydrolysis of a highly hydrophobic substrate of a specific lipase was increased by DMSO. DMSO is an organic solvent commonly used to solubilize substrates, not a surfactant that forms micelles. Sulciene et al. (Acta Paediatrica, supplement, 2018, 116:1049-1055) disclosed the use of immobilized lipolytic enzymes derived from yeast for the production of epoxidized oils and described the detection of lipase activity of these concentrated lipase-nanoparticle conjugates using a fluorescent substrate, but did not disclose the exact conditions. Similarly, Yoo et al. (Cell Chemical Biology, 2020, 27: 143-157) disclosed a fluorescent substrate assay for detecting lipase activity and used Triton X-100 to solubilize a high concentration of lipase rPfMAGLLP before analysis, but did not use it as part of the reaction solution. International Publication No. WO 2010 / 024924 disclosed an assay for screening lipases expressed in E. coli using a fluorescent substrate, but again, the assay has not been used to detect contaminating lipase activity in recombinant protein samples purified from eukaryotic cells. None of these prior art assays have yet measured the contaminating lipase activity in recombinant protein samples produced in eukaryotic cells.

[0008] Menden et al., 2019 (Journal of Enzyme Inhibition of Medicinal Chemistry, 34(1): 1474-1480) reported a lipase activity assay that used 4-methylumbelliferyl butyrate (4-MUB) and palmitate (4-MUP) as substrates to detect the lipase activity of defined enzyme extracts of Candida rugosa lipase (CRL) isoforms and to verify the mechanism of action of the inhibitor tropolone. Limitations to the assay have been reported, including an essential decrease in solubility with increasing length of the hydrophobic fatty acid tail and autocatalysis of the substrate in the basic pH range. Further, no surfactant is used in the assay. More recently, Jahn et al., 2020 (Pharm. Res. 37(118): 2-13) reported a chromophore-based lipase activity assay for use in measuring polysorbate degradation in samples of harvested cell culture media using 4-methylumbelliferyl oleate (4-MuO) as a substrate. However, it has moderate sensitivity and still requires an incubation time of over 24 hours.

[0009] Accordingly, there is still a need for a highly sensitive and improved method for measuring lipase activity in related samples in a short time. SUMMARY OF THE INVENTION

[0010] The present invention is a method for detecting (contaminating) lipase activity in a sample containing a recombinant protein, comprising: (a) providing at least one sample containing a recombinant protein produced in a eukaryotic cell; (b) contacting the at least one sample with a reaction solution to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond, the surfactant being a non-ionic or zwitterionic surfactant; (iii) a substrate comprising the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, the 4-MU ester being a saturated unbranched fatty acid (C6-C 16a 4-MU ester), and (iv) optionally, a non-buffered salt; (c) incubating the sample and the substrate in the reaction mixture; and (d) detecting the lipase activity by measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU (which is the hydrolysis product of the 4-MU ester). Optionally, the hydrolysis is measured by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (c). The method is understood to refer to an in vitro method. In certain embodiments, the sample and the substrate in the reaction mixture are incubated for between 2 minutes and less than 5 hours, and for any time between 2 minutes and less than 3 hours, 2 minutes and less than 2 hours, or 2 minutes and less than 0.5 hours. The at least one sample may be a harvested cell culture fluid (HCCF), an in-process control (IPC) sample, a drug substance sample or a pharmaceutical product sample. The recombinant protein in the sample for detecting lipase activity is preferably a therapeutic protein such as an antibody, an antibody fragment, an antibody-derived molecule or a fusion protein (e.g., an Fc fusion protein). According to the present invention, the recombinant protein in the sample for detecting lipase activity is not a lipase and / or does not contain lipase activity. Thus, any lipase activity detected in the at least one sample is contaminant lipase activity and / or is derived from at least one contaminant protein having lipase activity, such as a host cell protein (HCP) derived from eukaryotic cells.

[0011] In a preferred embodiment, the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), 4-methylumbelliferyl undecanoate, and 4-methylumbelliferyl dodecanoate; more preferably, the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl decanoate (4-MUD), and 4-methylumbelliferyl dodecanoate. According to a preferred embodiment of the present invention, the surfactant has a final concentration in the reaction mixture that exceeds its critical micelle concentration in the reaction mixture. The non-denaturing nonionic or zwitterionic surfactant may be CHAPS, CHAPSO, Zwittergent (such as Zwittergent3-12), or saponin. Preferably, the non-denaturing nonionic or zwitterionic surfactant is CHAPS. CHAPS may be provided at a final concentration in the reaction mixture of from about 8 mM to about 20 mM, preferably from about 8 mM to about 15 mM, more preferably about 10 mM. Suitable buffers include one or more buffering substances selected from the group consisting of formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MES (2-(N-morpholino)ethanesulfonic acid), tris base, bis-tris, bis-tris-propane, bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4, and NaH2PO4. Preferably, the buffer has a pH of from about 5 to about 7.5, and preferably, the buffer has a pH of from about 5.5 to about 7.5.In certain embodiments, the buffer is a multi-component buffer having a buffering range of at least about pH 5 to at least about pH 7.5, preferably at least about pH 4 to at least about pH 8.

[0012] Optional non-buffering salts may be, for example, NaCl, KCl, and CaCl2, preferably NaCl or KCl. The non-buffering salt may be provided in the reaction mixture at a concentration of about 100 mM to about 200 mM, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM. The ionic strength of the non-buffering salt in the reaction mixture is preferably about 200 mM or less, more preferably about 170 mM or less, more preferably about 150 mM or less, for example about 100 mM to about 200 mM in the reaction mixture, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM. Alternatively, or additionally, the cumulative ionic strength of the buffer and non-buffering salt in the reaction mixture may be about 450, preferably about 400 mM or less, more preferably about 350 mM or less.

[0013] In another aspect, the present invention is a method for producing a recombinant protein of interest, comprising: (i) culturing eukaryotic cells that express the recombinant protein of interest in cell culture; (ii) collecting the recombinant protein; (iii) purifying the recombinant protein; and (iv) optionally formulating the recombinant protein into a pharmaceutically acceptable formulation suitable for administration; and (v) obtaining at least one sample containing the recombinant protein in step (ii), (iii) and / or (iv), wherein the method further comprises detecting (contaminating) lipase in the sample containing the recombinant protein, and this step comprises: (a) providing the at least one sample containing the recombinant protein produced in eukaryotic cells in step (v); (b) contacting the at least one sample with a reaction solution to form a reaction mixture, wherein the reaction solution comprises: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant is a non-ionic or zwitterionic surfactant); (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (the 4-MU ester is a saturated unbranched fatty acid (C6-C 16) (which is a 4-MU ester), and (iv) optionally, a step of including a non-buffered salt; (c) a step of incubating the sample and the substrate in a reaction mixture; (d) a step of detecting the (contaminating) lipase activity by measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; optionally, a step of detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (c). In a specific embodiment, the method includes obtaining at least one sample containing the recombinant protein in step (ii) where the sample is a harvested cell culture fluid (HCCF) or a cell lysate; in step (iii) where the sample is an in-process control (IPC) sample; and / or in step (iv) where the sample is a drug substance sample or a pharmaceutical product sample, preferably including obtaining at least one sample containing the recombinant protein in step (iii), and step (iii) includes obtaining at least one sample before and after affinity chromatography, and / or before and after acid treatment, before and after depth filtration following acid treatment, and / or before and after ion exchange chromatography, preferably anion exchange chromatography or cation exchange chromatography.

[0014] In another aspect, the present invention is a kit for measuring contaminating lipase activity in a sample containing a recombinant protein, such as an IPC sample, comprising (i) a buffer having a pH of from about pH 4 to about pH 9, (ii) a non-denaturing surfactant having no ester bond (the surfactant is a non-ionic or zwitterionic surfactant), (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (the substrate is a saturated unbranched fatty acid (C6 to C 16)which is a 4-MU ester), and (iv) optionally, a non-buffered salt, and / or (v) optionally, water for dilution. In certain embodiments, the buffer, the surfactant, and the optional non-buffered salt are pre-mixed as an assay buffer. Preferably, the assay buffer is concentrated at least about 3-fold, or about 3-fold to about 5-fold, relative to the final reaction mixture. Alternatively, the assay buffer is provided as a dry mixture. Such a dry mixture may be reconstituted with water to provide the assay buffer concentrated at least about 3-fold or 5-fold relative to the final reaction mixture. Alternatively or additionally, the buffer, the surfactant, the substrate, and the optional non-buffered salt are pre-mixed and added to the sample as a master mixture, and the master mixture is provided at about 80% (v / v) to about 70% (v / v), preferably about 75% of the reaction mixture.

[0015] In a preferred embodiment, the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), methylumbelliferyl undecanoate, and methylumbelliferyl dodecanoate. The kit may further comprise an organic solvent for dissolving the substrate, or a substrate dissolved in an organic solvent, and / or one or more microtiter plates having 96 wells or a multiple of 96 wells. The non-denaturing nonionic or zwitterionic surfactant may be CHAPS, CHAPSO, Zwittergent (such as Zwittergent3-12), and saponin. Preferably, the non-denaturing nonionic or zwitterionic surfactant is CHAPS. In certain embodiments, the buffer comprises one or more buffering substances selected from the group consisting of formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MES (2-(N-morpholino)ethanesulfonic acid), tris base, tris, bis-tris, bis-tris-propane, bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino-propane sulfonic acid), tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4, and NaH2PO4. Preferably, the buffer has a pH of from about 5 to about 7.5, and more preferably, the buffer has a pH of from about 5.5 to about 7.5. In certain embodiments, the buffer is a multi-component buffer having a buffering range of at least about pH 5 to at least about pH 7.5, preferably at least about pH 4 to at least about pH 8. The optional non-buffering salt may be, for example, NaCl, KCl, and CaCl2, preferably NaCl or KCl.

Brief Description of the Drawings

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[0017] Detailed Description "Comprising" or "comprised of" in the general embodiments encompasses "consisting of" in the more specific embodiments. Further, the singular and plural forms are not used restrictively. As used herein, the singular forms "a", "an", and "the" specify both the singular and plural forms unless explicitly stated to specify only the singular form.

[0018] As used herein, the term "sample" refers to any sample containing a recombinant protein, wherein the recombinant protein is produced in eukaryotic cells in cell culture. The at least one sample may be, for example, a harvested cell culture fluid (HCCF) or cell lysate, an in-process control (IPC) sample, a bulk drug substance (also referred to herein as bulk drug) sample, or a pharmaceutical sample containing a recombinant protein (such as an antibody, antibody fragment, antibody-derived molecule or fusion protein (e.g., an Fc fusion protein)). As used herein, the recombinant protein contained in the sample is not a lipase and / or does not contain lipase activity. Thus, any lipase activity detected in the sample is adventitious lipase activity and / or is derived from an adventitious protein having lipase activity, such as a host cell protein (HCP) derived from eukaryotic cells, and is at least one such adventitious protein.

[0019] As used herein, the terms "adventitious" or "contaminating" refer to an unwanted and / or unintentional substance, compared to other predominantly produced substances having non-lipolytic activity (such as the protein of interest), or substances that can be regarded as only minor components of a protein preparation for medical treatment (such as an antibody or antibody-like compound) (e.g., the presence of at least one protein or substance having lipolytic activity and / or hydrolytic activity (such as lipase activity) associated with host cell proteins). In the context of the present invention, hydrolytic activity, particularly lipase activity, is undesirable due to its ability to degrade polysorbate that can co-purify with the recombinant protein. This applies in particular to the finally formulated protein preparation, which preferably contains such unwanted factors at less than 1% (w / w), preferably less than 0.1% (w / w), more preferably less than 0.01% (w / w) compared to the total protein content.

[0020] As used herein, the term "lipase activity" refers to the activity of a substance, typically a protein (enzyme), that catalyzes the hydrolysis of an ester bond in a lipid (such as a fatty acid ester). Lipase is a hydrolase that decomposes an ester into an acid and an alcohol in a chemical reaction with water, also called hydrolysis. Lipase may be, for example, a carboxylic acid ester hydrolase (EC 3.1.1) such as carboxylesterase (EC 3.1.1.1), triacylglycerol lipase (EC 3.1.1.3), phospholipase A2 (EC 3.1.1.4), lysophospholipase (EC 3.1.1.5), (EC 3.1.1.23), galactolipase (EC 3.1.1.26), phospholipase A1 (EC 3.1.1.32), lipoprotein lipase (EC 3.1.1.34) or hormone-sensitive lipase (EC 3.1.1.79); a phosphodiester hydrolase (EC 3.1.4) such as phospholipase D (EC 3.1.4.4), phosphoinositide phospholipase C (EC 3.1.4.11), glycosylphosphatidylinositol phospholipase D (EC 3.1.4.50) or N-acetylphosphatidylethanolamine-hydrolyzing phospholipase D (EC 3.1.4.54); or a sphingoglycolipid deacylase (EC 3.1.1.69).

[0021] The term "protein" is used interchangeably with "amino acid sequence" or "polypeptide" and refers to a polymer of amino acids of any length. These terms also include proteins that have been post-translationally modified through reactions (including, but not limited to, glycosylation, acetylation, phosphorylation, glycation or protein processing). Modifications and changes in the structure of a polypeptide (such as fusion to another protein, substitution, deletion or insertion of an amino acid sequence) can be made while the molecule maintains its biological functional activity. For example, substitutions of specific amino acid sequences can be made in a polypeptide or its underlying nucleic acid coding sequence to obtain a protein with the same properties.

[0022] As used herein, the term "recombinant protein" relates to a protein produced by recombinant techniques such as molecular cloning, and may be referred to as the recombinant protein of interest. As used herein, said recombinant protein is, for example, the protein of interest in a purified sample. Such methods gather genetic material from multiple sources or create sequences that do not naturally exist. Recombinant proteins typically are based on sequences from cells or organisms different from the recipient host cells (e.g., CHO cells or HEK293 cells) used for protein production in cell culture, or from different species, or are based on artificial sequences such as fusion proteins. In the context of the present invention, said recombinant protein is the protein of interest, preferably a therapeutic protein such as an antibody, antibody fragment, antibody-derived molecule (e.g., scFv, bispecific or multispecific antibody) or fusion protein (e.g., Fc fusion protein). Thus, in one embodiment, said recombinant protein is selected from the group consisting of antibodies, antibody fragments, antibody-derived molecules and fusion proteins.

[0023] As used herein, the term "eukaryotic cell" refers to a cell having a nucleus within a nuclear envelop, including animal cells, human cells, plant cells and yeast cells. In the present invention, "eukaryotic cells" specifically include mammalian cells such as cells derived from Chinese hamster ovary (CHO) cells or HEK293 cells, and yeast cells.

[0024] The terms "drug substance (DS)" or "bulk drug substance (BDS)" are used interchangeably herein and refer to the active pharmaceutical ingredient (API) formulated with excipients. Said API has a therapeutic effect in the body, as opposed to excipients that assist in the delivery of the API. In the case of biological therapeutics, the API formulated with excipients typically means at least the API in the final formulation buffer at the highest concentration used in the final dosage form, and is also referred to as a pharmaceutical product.

[0025] As used herein, the term "drug product", abbreviated as DP, refers to the final commercially available dosage form of the active pharmaceutical ingredient, e.g., tablets or capsules, or, in the case of a dosage form of a biological product, typically an injection solution contained in a suitable container (such as a vial or syringe). The drug product may also be in lyophilized form.

[0026] As used herein, the term "polysorbate 20" refers to a nonionic polysorbate-type surfactant derived from polyethoxylated sorbitan and lauric acid (polyoxyethylene (20) sorbitan monolaurate). It is also known as Tween20. Due to its stability and relatively low toxicity, it can be used as a surfactant and emulsifier in many domestic scientific analyses. Polysorbate 20 can be used as a detergent in immunoassays, Western blotting, and ELISA. Additionally, it can be used in pharmacological applications such as pharmaceutical formulations, particularly in biological products such as antibodies and Fc fusion proteins. It is particularly useful for preventing the binding of nonspecific antibodies.

[0027] As used herein, the term "polysorbate 80" refers to a nonionic polysorbate-type surfactant derived from polyethoxylated sorbitan and oleic acid (polyoxyethylene (20) sorbitan monooleate). It is also known as Tween80 and has uses similar to those of polysorbate 20.

[0028] As used herein, the term "therapeutic protein" refers to a protein that can be used in the medical treatment of humans and / or animals. These include, but are not limited to, antibodies, growth factors, blood coagulation factors, vaccines, interferons, hormones, and fusion proteins.

[0029] As used herein, the term "produced" relates to the production of a recombinant protein, preferably a therapeutic protein, in a eukaryotic cell, preferably a yeast or mammalian cell, in cell culture. Those skilled in the art know how to produce recombinant proteins intracellularly using fermentation. The production of a recombinant protein involves culturing a eukaryotic cell that expresses the recombinant protein of interest in cell culture. Culturing a eukaryotic cell that expresses a recombinant protein in cell culture involves maintaining the eukaryotic cell in a suitable medium under conditions that allow for growth and / or protein production / expression. The recombinant protein may be produced by fed-batch culture or continuous cell culture. Thus, the eukaryotic cell may be cultured in fed-batch culture or continuous cell culture, or a combination thereof, preferably fed-batch cell culture.

[0030] As used herein, the term "expressing a recombinant protein" refers to a cell containing a DNA sequence encoding a recombinant protein that is transcribed and translated into a protein sequence that includes post-translational modifications, i.e., that results in the production of the recombinant protein in cell culture.

[0031] As used herein, the term "about" refers to a variation of 10% of a specified value, e.g., about 50% has a variation from 45 to 55%.

[0032] Method for detecting lipase activity The present invention is a method (in vitro) for detecting lipase activity in a sample containing a recombinant protein, comprising: (a) providing at least one sample containing a recombinant protein produced in a eukaryotic cell; (b) contacting the at least one sample with a reaction solution to form a reaction mixture (the reaction solution comprising: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant being a non-ionic or zwitterionic surfactant); (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (the 4-MU ester being a saturated unbranched fatty acid (C6-C 16)a 4-MU ester), and (iv) optionally, a non-buffered salt); (c) incubating the sample and the substrate in the reaction mixture; (d) detecting lipase activity by measuring the hydrolysis of the 4-MU ester (the substrate) and detecting the fluorescence intensity of the released chromophore 4-MU (which is the hydrolysis product of the 4-MU ester); optionally, measuring hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (c). The method may further comprise analyzing the data obtained by measuring the hydrolysis of the at least one sample. The reaction solution used in the method of the present invention is an aqueous reaction solution. Those skilled in the art will also understand that the at least one sample contains a recombinant protein produced in eukaryotic cells in cell culture. Further, the method according to the present invention is for detecting contaminating lipase activity, and the lipase activity detected in step (d) is the contaminating lipase activity in the at least one sample, which contains the recombinant protein, more specifically the recombinant protein of interest.

[0033] The assay readout may be about 20 minutes or even earlier. Thus, in certain embodiments, the sample and substrate are incubated in the reaction mixture for less than 5 hours, less than 3 hours, less than 2 hours, or less than 0.5 hours. To obtain sufficient data points, it is desirable to incubate the sample and substrate in the reaction mixture for at least 1 minute, at least 2 minutes, or at least 5 minutes. Thus, the sample and substrate in the reaction mixture may be incubated for any time from 2 minutes to less than 5 hours, from 2 minutes to less than 3 hours, from 2 minutes to less than 2 hours, or from 2 minutes to less than 0.5 hours. The at least one sample may be HCCF, in-process control (IPC) sample, drug substance or pharmaceutical product. According to the present invention, the recombinant protein in the sample is not lipase and / or does not contain lipase activity. Further, the recombinant protein in the sample by the method of the present invention is not esterase or hydrolase and / or does not contain esterase activity or hydrolase activity. Thus, any lipase activity detected in the at least one sample is contaminant lipase activity and / or is derived from at least one contaminant protein having lipase activity, such as one or more host cell proteins (HCP) derived from eukaryotic cells. Thus, the at least one sample containing a recombinant protein produced in eukaryotic cells may potentially further contain at least one contaminant protein having lipase activity. In one embodiment, the method comprises, in step (a), providing at least one sample comprising a recombinant protein and a host cell protein (HCP) produced in cell culture in a eukaryote, preferably a mammalian cell; and in step (d), detecting the lipase activity of the HCP by measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU.

[0034] The substrate contains the chromophore 4-MU in the form of a saturated unbranched fatty acid (C6 to C 16 ) 4-MU ester (the acyl chain of the saturated unbranched fatty acid is C6 to C 16having a carbon atom). This substrate mimics a very important ester bond of polysorbate, namely the fatty acid ester bond.

[0035] Hydrolysis may be stopped at a specific time point prior to detection of the fluorescence intensity of the released chromophore 4-MU. Alternatively, and preferably, the fluorescence intensity of the released chromophore 4-MU may be detected in real time without stopping the hydrolysis of the 4-MU ester. In certain embodiments, the fluorescence intensity of the released chromophore 4-MU is detected without stopping the hydrolysis of the 4-MU ester. In certain embodiments, hydrolysis is measured by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and substrate in a reaction mixture according to step (c).

[0036] Real-time detection allows hydrolysis to be measured over time, and thus the specific reaction rate can be measured. In the method according to the invention, the hydrolysis of the 4-MU ester in the reaction mixture typically follows a pseudo-zero order reaction rate. Therefore, by detecting fluorescence in real time, it is possible to measure within the time frame of the pseudo-zero order reaction rate. Thus, in certain embodiments, the fluorescence intensity of the released chromophore 4-MU is detected over time and follows a pseudo-zero order reaction rate. Optionally, reaction mixtures that do not meet the pseudo-zero order reaction rate requirement are excluded from the analysis. The pseudo-zero order reaction rate can be evaluated by linear regression analysis. Preferably, the sample is experimented at least three times, and individual reaction mixtures are excluded from the analysis to exclude outliers if they do not meet the pseudo-zero order reaction rate, for example, due to bubbles in the wells. Excluding outliers as described strongly enhances the sensitivity of the analysis. A calibration curve using a specified concentration of 4-MU can be used to calculate the rate of hydrolysis (e.g., nmol / sec). The calibration curve at known 4-MU concentrations further allows the determination and comparison of reaction rates at different pH values.

[0037] As used herein, the term "reaction rate" refers to the rate of an enzyme that converts a substrate to at least one product within a specific time. In some reactions, the rate appears to be independent of the concentration of the reactants. This means that the rate of the equation is equal to the rate constant k of the reaction, which is called a zero-order reaction. The zero-order reaction kinetics is always an artefact of the conditions under which the reaction is carried out. For this reason, reactions that follow zero-order reaction kinetics are often called pseudo-zero-order reactions.

[0038] The method according to the invention may further comprise determining the rate of hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU as relative fluorescence units (RFU), and determining the amount (mol / sec) of the released chromophore 4-MU by comparing it with a calibration curve generated by using a defined concentration of 4-MU. Typically, the activity is determined by the release of 4-MU (nmol / min). Alternatively, or additionally, the relative value may be calculated in comparison to an internal standard, such as another sample, or preferably a commercially available lipase (such as porcine pancreatic lipase (PPL)) that serves as a positive control.

[0039] Incubation of the sample and the substrate in the reaction mixture allows any potentially present contaminating protein(s) with lipase activity to hydrolyze the 4-MU ester. Typically, the incubation is from several minutes to several hours. In one embodiment, hydrolysis is measured by detecting over time the fluorescence intensity of the released chromophore 4-MU while incubating the sample and the substrate in the reaction mixture according to step (c), i.e., in real time during incubation. For assay sensitivity, typically, detection is started immediately after step (b). The incubation time and thus the detection time may depend on the lipase activity present in the sample and typically do not exceed 5 hours, preferably do not exceed 3 hours. In certain embodiments, the sample and substrate in the reaction mixture are incubated for less than 5 hours, less than 3 hours, less than 2 hours, less than 1 hour, or less than 0.5 hours. To obtain sufficient data points, it is desirable to incubate the sample and substrate in the reaction mixture for at least about 1 minute, at least about 2 minutes, or at least about 5 minutes. Thus, the sample and substrate in the reaction mixture may be incubated for any time between about 2 minutes and less than 5 hours, between about 2 minutes and less than 3 hours, between about 2 minutes and less than 2 hours, or between about 2 minutes and less than 0.5 hours. Preferably, the sample and substrate in the reaction mixture are incubated at a temperature of about 25°C for between 20 minutes and 2 hours. Since the reaction temperature affects the reaction time, the reaction temperature should be kept constant during measurement, e.g., between 20 and 37°C, preferably between 22 and 28°C, more preferably between 24 and 26°C. In one embodiment, the sample and substrate in the reaction mixture are incubated at a constant temperature between 20 and 37°C, preferably between 22 and 28°C, more preferably between 24 and 26°C, for less than 5 hours, less than 3 hours, less than 2 hours, or less than 1 hour, or are incubated at a constant temperature between 20 and 37°C, preferably between 22 and 28°C, more preferably between 24 and 26°C, for any time between about 2 minutes and less than 5 hours, between about 2 minutes and less than 3 hours, between about 2 minutes and less than 2 hours, or between about 2 minutes and less than 1 hour.

[0040] The substrate containing the chromophore 4-MU is present in the form of a saturated unbranched fatty acid (C6-C 16 ) 4-MU ester, and the acyl chain of the saturated unbranched fatty acid has carbon atoms of C6-C 16 . This substrate mimics an important feature of polysorbate, namely the fatty acid ester bond and the long acyl chain. Polysorbate 20 is an ester of lauric acid, a saturated unbranched fatty acid. In contrast, polysorbate 80 is an ester of oleic acid, an unsaturated fatty acid.

[0041] Unsaturated fatty acids are bulkier than saturated fatty acids due to their double bonds, and furthermore branched-chain fatty acids are bulkier than unbranched-chain fatty acids. The lipase activity in a sample containing a recombinant protein is mediated by one or more lipases or other hydrolases and may vary depending on the various products, like individual antibodies (see Figure 2). Thus, in most cases, the contaminating protein(s) having lipase activity are unknown and can be a mixture of one or more proteins. Many lipases, such as triacylglycerol lipase, can be in an open or closed state, whereas their active sites are shielded from the solvent by a flap or lid that is part of the polypeptide chain. Thus, the active sites of many lipases resemble the inside of a cavity or barrel, which is likely to determine substrate specificity. Thus, esters of saturated unbranched fatty acids of medium length (non-bulky fatty acids) are likely to obtain a broader enzyme spectrum compared to, for example, oleic acid having a longer and unsaturated acyl chain as used in the method of the present invention. Preferably, the substrate obtains an equivalent or broader enzyme spectrum compared to PS20 or PS80.

[0042] Furthermore, fatty acid esters having shorter acyl chains provide better solubility in the aqueous reaction mixture compared to fatty acid esters with longer chain lengths. As a result, additional substrates can be used in the assay mixture. More specifically, at a chain length of C 16 and above, solubility was found to be strongly limited.

[0043] Furthermore, it has been found that the decanoate ester (4-MUD) provides better resistance to self-hydrolysis compared to, for example, the butyrate ester (4-MUB). Chain lengths up to C5 have been found to strongly increase self-hydrolysis. The C 10 fatty acids have been found to be optimal for use in the examples, but saturated unbranched fatty acids (C6-C 16 ) 4-MU esters, or more preferably saturated unbranched fatty acids (C8-C 12 ) 4-MU esters, such as slightly longer or shorter saturated unbranched fatty acid esters, can also be used in a similar manner in the method according to the invention. Thus, the 4-MU esters used in the method according to the invention have an acyl chain of a saturated unbranched fatty acid with carbon atoms of C6-C 16 . More preferably, the fatty acid is a medium-chain fatty acid, and the 4-MU ester is a saturated unbranched fatty acid (C8-C 12 ) 4-MU ester. In a particular embodiment, the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), 4-methylumbelliferyl undecanoate, and 4-methylumbelliferyl dodecanoate. In a particular preferred embodiment, the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl decanoate (4-MUD), and 4-methylumbelliferyl dodecanoate. In a more preferred embodiment, the substrate is 4-MUD. The substrate is typically dissolved as a stock solution (such as a stock solution 100 times the concentration in the reaction mixture) in an organic solvent such as dimethyl sulfoxide (DMSO) or dimethylformamide (DMF), preferably DMSO. In a particular embodiment, the substrate is provided as a stock solution dissolved in an organic solvent selected from DMSO or DMF, preferably DMF.

[0044] Suitable substrate concentrations in the present invention can be from about 1 μM to about 1 mM. Thus, in certain embodiments, the substrate is provided at a final concentration in the reaction mixture of from about 1 μM to about 1 mM, preferably from about 1 μM to 300 μM, preferably from 1 μM to 30 μM, more preferably from about 3 μM to 30 μM. In certain embodiments, the substrate is provided as a stock solution in an organic solvent, and the stock solution is added at from about 1% to about 5% (v / v) of the reaction mixture.

[0045] The method according to the present invention includes a step of bringing at least one sample into contact with a reaction solution containing a non-denaturing surfactant having no ester bond, and the surfactant is a nonionic or zwitterionic surfactant (also referred to herein as a "non-denaturing nonionic or zwitterionic surfactant having no ester bond"). As used herein, the term "surfactant" refers to a surface-active compound that can form micelles and reduce the surface tension between two liquids, between a gas and a liquid, and between a liquid and a solid. Surfactants are sometimes referred to herein as detergents. Surfactants are amphiphilic, i.e., they contain both a hydrophobic group (tail) and a hydrophilic group (head). Surfactants are typically organic compounds. In an aqueous phase, surfactants form aggregates such as micelles, where the hydrophobic tails form the core of the aggregate and the hydrophilic heads contact the surrounding aqueous liquid. Thus, the hydrophobic tails (also referred to as hydrophobic hydrocarbon moieties) have a certain length for forming micelles. Thus, the surfactants used herein do not include organic solvents such as ethanol or dimethyl sulfoxide (DMSO). The tails of most surfactants typically consist of one or more hydrocarbon chains that can be branched, straight-chain, or aromatic. The surfactant may include one or more hydrophobic tails, and preferably, the surfactant includes one hydrophobic chain (single-tail surfactant). Surfactants are generally classified according to their hydrophilic head groups. Nonionic surfactants do not have a charged group at their head, one ionic surfactant has a net one positive (cationic) or negative (anionic) charge, and one zwitterionic surfactant contains two oppositely charged groups. Thus, nonionic or zwitterionic surfactants do not have a net charge on their hydrophilic head groups and are milder in nature. Further, in many surfactants, the hydrophobic tail is bonded to the hydrophilic head via an ester bond, such as PS20 or PS80. Further, nonionic or zwitterionic surfactants are non-denaturing surfactants. As used herein, the term "non-denaturing surfactant" refers to the effect of the surfactant on the protein structure. Non-denaturing surfactants do not disrupt protein-protein interactions, especially for water-soluble proteins.

[0046] Surfactants containing ester bonds can interfere with the assay because they are potential substrates for lipase. Furthermore, protein denaturation due to lipase activity and thus interference with lipase activity in the sample should be avoided. Therefore, the surfactant used in the method according to the present invention is a non-denaturing surfactant having no ester bond, and the surfactant is a non-ionic or zwitterionic surfactant. Examples of suitable non-denaturing zwitterionic surfactants are 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-([3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate) (CHAPSO), CHAPS analogs (such as BIG-CHAP (N,N-bis(3-D-gluconamidopropyl)deoxycholamide)), Zwittergent (various lengths, for example, n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent3-12)) and 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]propanesulfonate or other amidosulfobetaine detergents, but are not limited thereto. Examples of suitable non-denaturing non-ionic surfactants are pyranoside-based surfactants (such as octyl β-D-glucopyranoside (OGP), nonyl β-D-glucopyranoside, dodecyl β-D-maltopyranoside (DDM) or octyl β-D-thioglucopyranoside), polyoxyethylene(23) lauryl ether (Brij35) or other polyoxyethylene ethers; saponin (for example, digitonin), octylphenoxy polyethoxyethanol (IGEPAL CA-630), poloxamer 188, 338, 407 or tergitol, but are not limited thereto. In a particular embodiment, the non-denaturing surfactant having no ester bond (non-ionic or zwitterionic surfactant) is not ethoxylated and / or does not contain a polyethylene glycol group and / or does not contain an aromatic ring.In certain embodiments, the non-denaturing non-ionic or zwitterionic surfactant having no ester bond is not octoxinol-9, and specifically is not polyethylene glycol tert-octylphenyl ether (Triton X-100; CAS No. 9002-93-1) and / or polyethylene glycol nonylphenyl ether (NP-40, CAS No. 9016-45-9). In a preferred embodiment, the surfactant is a non-denaturing surfactant having no ester bond, the surfactant is a non-ionic or zwitterionic surfactant, and more preferably, the surfactant is selected from the group consisting of CHAPS (CAS No. 75621-03-3 or its hydrate CAS No. 331717-45-4), CHAPSO (CAS No. 82473-24-3), Zwittergent (e.g., Zwittergent 3-12; CAS No. 14933-08-5) and saponin (CAS No. 8047-15-2), and is preferably a non-denaturing surfactant (non-ionic or zwitterionic surfactant) that is CHAPS. None of these exemplary and suitable surfactants exhibit an ester bond or an acyl chain, and thus are not substrates for lipase. These surfactants do not compete with the substrate and therefore do not affect the sensitivity of the assay. Further, the presence of the surfactant mediates the solubility of the substrate at the concentration of water used. One skilled in the art will know how to identify further suitable non-denaturing non-ionic or zwitterionic surfactants having no ester bond, for example, by measuring the effect on 4-MU ester hydrolysis under assay conditions, as shown for PS20 in Example 7.

[0047] The presence of a surfactant (such as 10 mM CHAPS) has been shown to increase lipase activity and thus improve the sensitivity of the assay. Without being bound by theory, it is hypothesized that the surfactant creates an environment that promotes lipase activity by allowing for the repositioning and opening of a lid or flap described as covering the active site (Grochulski P, Li Y, Schrag JD, et al. Protein Sci 1994; 3:82-91 and Grochulski P, Bouthillier F, Kazlauskas RJ, et al. Biochemistry 1994; 33:3494-500). To achieve this effect, the surfactant should be above its critical micelle concentration (CMC).

[0048] Accordingly, according to the present invention, the non-denaturing surfactant (non-ionic or zwitterionic) has a final concentration above the critical micelle concentration (CMC) in the reaction mixture. The CMC represents an important physicochemical property of a given surfactant in an aqueous solution. Micelles are spherical aggregates in which their hydrocarbon groups are mostly not in contact with water. As used herein, the term "critical micelle concentration" or "CMC" refers to the concentration of surfactant (i.e., the maximum monomer concentration) above which micelles are formed and can be measured according to methods known in the art. For example, a method suitable for measuring the CMC is the fluorescence micelle assay (FMA) that utilizes the partitioning of the fluorescent hydrophobic dye N-phenyl-1-naphthylamine (NPN) into surfactant micelles. NPN exhibits a low fluorescence quantum yield in an aqueous environment and this increases in a more hydrophobic environment such as the core of a micelle. This assay was originally developed for the measurement of the CMC and, as in the examples, has also been used to measure the polysorbate content in biopharmaceuticals. Another method that utilizes the enhancement of fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) upon micellization has been reported by Chattopadhyay and Harikumar (FEBS Letters 391 (1996) 199-202).

[0049] The CMC related to the surfactant can be derived from the literature. For example, for CHAPS, it is about 6 mM; for CHAPSO, it is about 8 mM; and for Zwittergent 3-12, it is about 2-4 mM. In certain embodiments, the non-denaturing zwitterionic surfactant is CHAPS, provided at a final concentration in the reaction mixture of from about 8 mM to about 20 mM, preferably from about 8 mM to about 15 mM, more preferably about 10 mM. In other embodiments, the non-denaturing zwitterionic surfactant is CHAPSO, provided at a final concentration in the reaction mixture of from about 10 mM to about 20 mM, preferably from about 10 mM to about 15 mM. In yet another embodiment, the non-denaturing zwitterionic surfactant is Zwittergent 3-12, provided at a final concentration in the reaction mixture of from about 4 mM to about 10 mM, preferably from about 6 mM to about 8 mM. In yet another embodiment, the non-denaturing non-ionic surfactant is saponin, provided at a final concentration in the reaction mixture of from about 0.001% to 0.01% (w / v).

[0050] The reaction solution used in the method according to the present invention further comprises a buffer having a pH of from about pH 4 to about pH 9. Preferably, the method is carried out using a buffer having a pH of from about pH 4 to about pH 8, preferably from about pH 5 to about pH 7.5, more preferably from about pH 5.5 to about pH 7.5. Those skilled in the art will understand that the pH of the buffer is within its buffering range when used in the method of the present invention. In principle, any buffer known in the art can be used as long as it has a buffering range with a pH from about pH 4 to about pH 9. The buffer may contain a single buffering substance or may be a multi-component buffer. Multi-component buffers typically have a broader buffering range. For example, the buffer may contain one or more buffering substances selected from the group consisting of formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MES (2-(N-morpholino)ethanesulfonic acid), tris base, tris, bis-tris, bis-tris-propane, bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4 and NaH2PO4. Preferably, the buffer is a phosphate buffer (Na2HPO4 and NaH2PO4), a tris buffer or a HEPES buffer. In a particular embodiment, the buffer has a concentration of from about 50 to 400 mM, preferably from about 50 to 300 mM, more preferably from about 50 to 200 mM.

[0051] The buffer solution may further be a multi-component buffer solution containing a plurality of buffering substances having overlapping buffering ranges in order to have a wider buffering range. The buffer solution may contain, for example, two, three, four, five or more buffering substances, preferably two or more buffering substances, more preferably three or more buffering substances. For example, the multi-component buffer solution may contain two to four buffering substances, three to four buffering substances, more preferably three buffering substances. In a specific embodiment, the multi-component buffer solution contains at least three buffering substances having overlapping buffering ranges, preferably containing at least one of Tris, MES and / or acetic acid, preferably containing acetic acid, MES and Tris in a ratio of 1:1:2.

[0052] Since the assay was found to be sensitive to ionic strength, for the design of an appropriate multi-component buffer solution, it is important not only that it contains buffering substances with overlapping buffering ranges, but also that the buffer solution changes the ionic strength only moderately (less than 15%, more preferably less than 10%) at different pHs (pH range 4-8) (Ellis KJ, Morrisson JF, 1982. Methods in Enzymology, 87: 405-426). For example, the AMT buffer solution containing acetic acid, MES and Tris allows the identification of conditions that reduce hydrolytic activity, such as pH conditions, by using buffer solutions at different pHs with only a moderate effect on ionic strength. This buffer solution further enables the measurement of the sample at the pH of the sample, the measurement of lipase activity under specific conditions present in the sample, and in addition the comparison of lipase activity in various states during purification. This assay enables the further enhancement of sensitivity by measuring the sample at the optimal pH.

[0053] Accordingly, the multi-component buffers disclosed herein enable the use of buffers having a variable pH from at least about pH 4 to at least about pH 8, or from at least about pH 4 to at least about pH 9. Alternatively, or additionally, the use of buffers having various pH values between about pH 4 and about pH 9 affects the ionic strength of the buffer by less than 15%, preferably less than 10%, or even less than 7.5% or 5%, for example from 0% to less than 15%, from 0% to less than 10%, from 0% to less than 7.5%, or 5% or less, or, for example, from 2% to less than 15%, from 2% to less than 10%, from 2% to less than 7.5% or from 2% to 5%. In one embodiment, the use of buffers having various pH values between about pH 4 and about pH 8 affects the ionic strength of the buffer by less than 15%, preferably less than 10%, or even less than 7.5% or 5%, for example from 0% to less than 15%, from 0% to less than 10%, from 0% to less than 7.5% or from 0% to 5% or less, or, for example, from 2% to less than 15%, from 2% to less than 10%, from 2% to less than 7.5% or from 2% to 5%. The use of the multi-component buffers disclosed herein further enables the adjustment of the pH of the buffer to the pH of the sample (without changing the buffer composition of the buffer). The use of the multi-component buffers disclosed herein further enables the adjustment of the pH of the buffer near the optimum of at least one contaminating protein having lipase activity (thereby enhancing the sensitivity of the method), and / or the comparison and identification of conditions that reduce the hydrolytic activity.

[0054] The reaction solution may further contain a non-buffering salt. In the present invention, any salt that dissociates in water and has no buffering effect may be suitable for adjusting the ionic strength of the reaction solution. Examples of suitable salts are NaCl, KCl, or CaCl2. In one example of the present invention, the non-buffering salt is selected from the group consisting of NaCl, KCl, and CaCl2, and preferably, the non-buffering salt is NaCl or KCl.

[0055] The concentration of the optional non-buffered salt may range from about 100 mM to about 200 mM. In certain embodiments of the invention, the non-buffered salt has a concentration in the reaction mixture of from about 100 mM to about 200 mM, preferably from about 130 mM to about 170 mM, more preferably from about 140 mM to about 150 mM. However, the ionic strength in the reaction mixture should not exceed a certain value as it has an adverse effect on lipase activity. For example, the ionic strength of the optional non-buffered salt is preferably about 200 mM or less, about 190 mM or less, about 180 mM or less, about 170 mM or less, about 160 mM or less, about 150 mM or less in the reaction mixture, and for example, in the reaction mixture, it is from about 100 mM to about 200 mM, preferably from about 130 mM to about 170 mM, more preferably from about 140 mM to about 150 mM. In certain embodiments, the cumulative ionic strength of the buffer and the non-buffered salt in the reaction mixture does not exceed about 450 mM. Thus, the cumulative ionic strength of the buffer and the non-buffered salt in the reaction mixture may be about 450 mM or less, about 400 mM or less, about 380 mM or less, about 360 mM or less, or about 350 mM or less. For example, the cumulative ionic strength of the buffer and the non-buffered salt in the reaction mixture may be from about 150 mM to about 450 mM or less, from about 150 mM to about 400 mM or less, from about 150 mM to about 380 mM or less, from about 150 mM to about 360 mM or less, or from about 150 mM to about 350 mM or less.

[0056] The method according to the invention uses a fluorescence spectrometer or a microplate spectrophotometer (preferably at λ ex 330 - 340 nm, λ emIt is suitable for the detection of fluorescence at 450 nm). Thus, the reaction mixture is contained (and preferably mixed therein) in a cuvette or a microtiter plate, preferably a microtiter plate with at least 96 wells, for measurement. Thus, the method according to the invention is particularly suitable for high-throughput analysis and / or automated analysis of samples. In certain embodiments, in the method according to the invention, at least 2, 3, 4, 5, 10 or more samples are analyzed simultaneously. Further, each sample is preferably measured at least 3 times. Thus, the method according to the invention is preferably carried out using a microtiter plate having 96 wells or a multiple of 96 wells. The microtiter plate is used not only for the measurement of hydrolysis in step (d), but also for contacting at least one sample with the reaction solution in step (d) and incubating the sample and the substrate in the reaction mixture in step (c). Thus, in certain embodiments, the sample is contacted, incubated and measured in the format of a microtiter plate having 96 wells or a multiple of 96 wells.

[0057] In certain embodiments, the sample is provided at about 30% (v / v) or less, preferably about 25% (v / v) or less of the reaction mixture. Thus, the sample is provided at about 20% (v / v) to about 30% (v / v) of the reaction mixture, preferably about 20% (v / v) to about 25% (v / v) of the reaction mixture. Optionally, the sample may be pre-diluted. At least one of the samples comprising the recombinant protein may be a harvested cell culture fluid (HCCF) or cell lysate, an in-process control (IPC) sample, a drug substance sample or a pharmaceutical product sample, preferably an IPC sample, a drug substance sample or a pharmaceutical product sample. Preferably, contacting at least one sample with the reaction solution to form the reaction mixture comprises mixing at least one sample with the reaction solution to obtain a homogeneous reaction mixture. Preferably, this is done by first adding the smaller volume (typically the sample) and then the larger volume (typically the reaction solution). Preferably, the components of the reaction solution are added as a master mixture, which is prepared as a concentrate and diluted to the working concentration before addition to the sample.

[0058] Furthermore, the buffer, non-denaturing surfactant (nonionic or zwitterionic), and optional non-buffered salt are preferably pre-mixed as an assay buffer at a concentration of at least about 3-fold, or about 3-fold to about 5-fold, relative to the reaction mixture. The assay buffer may be stored before use. Alternatively, the assay buffer is provided as a dry mixture. Such a dry mixture may be reconstituted with water to provide an assay buffer concentrated at least about 3-fold, or about 3-fold to about 5-fold, relative to the final reaction mixture. The substrate is added to the assay buffer before use to provide a reaction solution; preferably, the substrate is added to the assay buffer immediately before use. Thus, the buffer, surfactant, substrate, and optional non-buffered salt are preferably pre-mixed as a master mixture. The components of the master mixture are the same as the components in the reaction solution. The master mixture may be prepared as a concentrate that is diluted to the working concentration before adding to the sample.

[0059] In certain embodiments, the buffer, non-denaturing surfactant (nonionic or zwitterionic), substrate, and optional non-buffered salt are added as a master mixture, and the master mixture is provided at about 70% (v / v) or more, about 75% (v / v) or more. Thus, the master mixture may be provided at about 70% (v / v) to about 80% (v / v), preferably about 75% (v / v) to about 80% (v / v).

[0060] The at least one sample may be a harvested cell culture fluid (HCCF) or cell lysate, an in-process control (IPC) sample, a drug substance sample or a pharmaceutical product sample. The recombinant protein in the sample for detecting lipase activity is preferably a therapeutic protein such as an antibody, an antibody fragment, an antibody-derived molecule, a fusion protein (e.g., an Fc fusion protein), a growth factor, a cytokine or a hormone, preferably an antibody, an antibody fragment, an antibody-derived molecule or an Fc fusion protein. Thus, the recombinant protein is preferably a secreted protein. As used herein, the term “harvested cell culture fluid” or “HCCF” refers to the cell culture supernatant after harvest, i.e., after separation from the cells. According to the present invention, the recombinant protein in the sample for detecting lipase activity is not a lipase and / or does not contain lipase activity. Thus, any lipase activity detected in the at least one sample is adventitious lipase activity and / or is derived from at least one adventitious protein having lipase activity, such as a host cell protein (HCP) derived from eukaryotic cells. Further, the recombinant protein in the sample by the method of the present invention is not an esterase or a hydrolase and / or does not contain esterase or hydrolase activity.

[0061] Accordingly, advantageously, the method of the present invention can be used to detect lipase activity by measuring hydrolysis in a sample containing an antibody, antibody fragment, antibody-derived molecule or fusion protein (e.g., Fc fusion protein). Typically, the antibody is monospecific, although the antibody may be multispecific. Thus, the method according to the present invention may be used on a sample containing a monospecific antibody, a multispecific antibody, or fragments thereof, preferably an antibody (monospecific), a bispecific antibody, a trispecific antibody or fragments thereof, preferably an antigen-binding fragment thereof. Unless otherwise specified, the term "antibody" refers to a monospecific antibody. Exemplary antibodies within the scope of the present invention include, but are not limited to, anti-CD2 antibody, anti-CD3 antibody, anti-CD20 antibody, anti-CD22 antibody, anti-CD30 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD40 antibody, anti-CD44 antibody, anti-CD44v6 antibody, anti-CD49d antibody, anti-CD52 antibody, anti-EGFR1 (HER1) antibody, anti-EGFR2 (HER2) antibody, anti-GD3 antibody, anti-IGF antibody, anti-VEGF antibody, anti-TNFα antibody, anti-IL2 antibody, anti-IL-5R antibody, anti-IL-36R antibody or anti-IgE antibody, and preferably is selected from the group consisting of anti-CD20 antibody, anti-CD33 antibody, anti-CD37 antibody, anti-CD40 antibody, anti-CD44 antibody, anti-CD52 antibody, anti-HER2 / neu (erbB2) antibody, anti-EGFR antibody, anti-IGF antibody, anti-VEGF antibody, anti-TNFα antibody, anti-IL-2 antibody, anti-IL-36R antibody and anti-IgE antibody. In one embodiment, the antibody is an anti-IL-36R antibody, specifically spesolimab. In another embodiment, the antibody is not an anti-IL-36R antibody, particularly not spesolimab.

[0062] As used herein, the terms "antibody", "antibodies" or "immunoglobulin" refer to proteins selected from among the globulins that are naturally formed as a reaction of a host organism against a foreign substance (= antigen) from differentiated B-lymphocytes (plasma cells). There are various types of immunoglobulins, such as IgA, IgD, IgE, IgG, IgM, IgY, IgW. Preferably, the antibody is an IgG antibody, and more preferably, an IgG1 or IgG4 antibody. In this specification, the terms immunoglobulin and antibody are used interchangeably. Antibodies include monoclonal antibodies, monospecific antibodies and multispecific antibodies (such as bispecific or trispecific antibodies), single-chain antibodies, antigen-binding fragments of antibodies (for example, Fab or F(ab')2 fragments), disulfide-bonded Fv, etc. The antibody can be of any species and includes chimeric antibodies and humanized antibodies. A "chimeric" antibody is a molecule in which the antibody domains or antibody regions are derived from different species. For example, the variable regions of the heavy and light chains can be derived from a rat or mouse antibody, and the constant regions can be derived from a human antibody. In a "humanized" antibody, only a minimal sequence is derived from a non-human species. In many cases, only the CDR amino acid residues of a human antibody are replaced with the CDR amino acid residues of a non-human species such as a mouse, rat, rabbit or llama. Sometimes, several important framework amino acid residues that affect antigen-binding specificity and affinity are also replaced with non-human amino acid residues. Antibodies may be produced through chemical synthesis, via recombinant or transgenic means, via cell (e.g., hybridoma) culture, or by other means.

[0063] Typically, an antibody is a tetrameric polypeptide composed of two pairs of heterodimers formed by a heavy chain and a light chain, respectively. Stabilization of both the heterodimer and the tetrameric polypeptide structure occurs via interchain disulfide bridges. Each chain is composed of structural domains called "immunoglobulin domains" or "immunoglobulin regions", where the terms "domain" or "region" are used interchangeably. Each domain contains approximately 70-110 amino acids and forms a compact three-dimensional structure. Both the heavy chain and the light chain contain, at the N-terminus, a "variable domain" or "variable region" with a less conserved sequence responsible for antigen recognition and binding. The variable region of the light chain is also called "VL", and the variable region of the heavy chain is also called "VH".

[0064] Antigen-binding fragments include, but are not limited to, for example, "Fab fragments" (Fragment antigen-binding = Fab). Fab fragments consist of the variable regions of both chains held together by adjacent constant regions. These may be formed from conventional antibodies by protease digestion (e.g., by papain), but Fab fragments may also be produced by genetic engineering in the same way. Further antibody fragments include F(ab’)2 fragments, which are prepared by proteolytic cleavage with pepsin.

[0065] Using genetic engineering methods, it is possible to produce shortened antibody fragments consisting only of the variable regions of the heavy chain (VH) and the light chain (VL). These are called Fv fragments (Fragment variable = fragment of the variable part). Since these Fv fragments lack the covalent bonding of the two chains by the constant chain cysteine, the Fv fragments are often stabilized. It is advantageous to bind the variable regions of the heavy and light chains with short peptide fragments, for example, 10 to 30 amino acids, preferably 15 amino acids. In this way, a single peptide chain consisting of VH and VL linked by a peptide linker is obtained. This type of antibody protein is known as single-chain Fv (scFv). Examples of scFv antibody proteins are known to those skilled in the art. Thus, antibody fragments and antigen-binding fragments further include Fv fragments and especially scFv.

[0066] In recent years, various strategies have been developed for preparing scFv as multimeric derivatives. This is particularly intended to lead to recombinant antibodies with improved pharmacokinetic and biodistribution properties and increased binding affinity. To achieve multimerization of scFv, scFv has been prepared as a fusion protein having a multimerization domain. The multimerization domain may be, for example, the CH3 region of IgG or a coiled-coil structure (helical structure) such as a leucine zipper domain. However, there are also strategies that use the interaction between the VH / VL regions of scFv for multimerization (e.g., diabody, tribody, pentabody). For those skilled in the art, diabody means a bivalent homodimer scFv derivative. Shortening the linker within the scFv molecule to 5 - 10 amino acids results in the formation of a homodimer in which inter-chain VH / VL superposition occurs. The diabody may be further stabilized by the incorporation of disulfide bridges. Examples of diabody antibody proteins are known from the prior art.

[0067] To those skilled in the art, a minibody means a bivalent homodimeric scFv derivative. A minibody consists of a fusion protein containing the CH3 region of an immunoglobulin, preferably IgG, most preferably IgG1, as a dimerization region connected to the scFv via a hinge region (e.g., also from IgG1) and a linker region. Examples of minibody antibody proteins are known from the prior art.

[0068] To those skilled in the art, a tribody means a trivalent homotrimeric scFv derivative. An scFv derivative in which VH-VL are directly fused without a linker sequence results in the formation of a trimer.

[0069] Those skilled in the art are also familiar with so-called minibodies having a bivalent, trivalent or tetravalent structure and derived from scFvs. Multimerization is effected by bivalent, trivalent or tetravalent coiled-coil structures. In a preferred embodiment of the present invention, the gene of interest encodes any of the above-described desired polypeptides, preferably any of a monoclonal antibody, its derivatives or fragments.

[0070] An immunoglobulin fragment composed of the CH2 and CH3 domains of an antibody heavy chain is called an "Fc fragment", "Fc region" or "Fc" due to its tendency to crystallize (Fc = fragment crystallizable). These can be formed, for example, from conventional antibodies by protease digestion with papain or pepsin, but can also be produced by genetic engineering. The N-terminal portion of the Fc fragment can vary depending on the number of amino acids in the remaining hinge region.

[0071] An antibody containing an antigen-binding fragment and an Fc region may sometimes also be called a full-length antibody. A full-length antibody may be a monospecific, or a multispecific antibody such as a bispecific antibody or a trispecific antibody.

[0072] Preferred therapeutic antibodies according to the present invention are multispecific antibodies, specifically bispecific or trispecific antibodies. Bispecific antibodies typically combine antigen-binding specificities for a target cell (e.g., malignant B cells) and an effector cell (e.g., T cells, NK cells, or macrophages) within one molecule. Exemplary bispecific antibodies include, but are not limited to, diabodies, BiTE (Bi-specific T-cell Engager) formats, and DART (Dual-Affinity Re-Targeting) formats. The diabody format separates the heavy and light chain cognate variable domains of two antigen-binding specificities on two separate polypeptide chains, which are non-covalently associated. The DART format is based on the diabody format but provides additional stabilization through C-terminal disulfide bridging. Trispecific antibodies are monoclonal antibodies that combine three antigen-binding specificities. Trispecific antibodies may be constructed based on bispecific antibody technology that reconstitutes the antigen recognition domains of two different antibodies into one bispecific molecule. For example, trispecific antibodies targeting CD38 on cancer cells and CD3 and CD28 on T cells have been created. Multispecific antibodies are particularly difficult to manufacture with high product quality.

[0073] Another preferred therapeutic protein is a fusion protein such as an Fc fusion protein. Thus, the present invention can be advantageously used for the production of fusion proteins such as Fc fusion proteins. Furthermore, the method for increasing the production amount of the protein according to the present invention can be advantageously used for the production of fusion proteins such as Fc fusion proteins.

[0074] The effector portion of the fusion protein can be the complete sequence of a native or modified heterologous protein, or any part of the sequence. The sequence of the immunoglobulin constant domain can be obtained from any subtype of immunoglobulin, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 subtypes of immunoglobulin, or from a class such as IgA, IgE, IgD or IgM. Preferably, they are derived from human immunoglobulins, more preferably from human IgG, and even more preferably from human IgG1 and IgG2. Non-limiting examples of Fc fusion proteins include MCP1-Fc, ICAM-Fc, EPO-Fc and scFv fragments, etc., which are bound to the CH2 domain of the heavy chain immunoglobulin constant region containing an N-linked glycosylation site. Fc fusion proteins can be constructed by a genetic engineering approach, for example, by introducing the CH2 domain of the heavy chain immunoglobulin constant region containing an N-linked glycosylation site into another expression construct, such as another immunoglobulin domain, an enzymatically active protein portion, or an effector domain. Thus, the Fc fusion proteins according to the present invention also include single-chain Fv fragments bound to the CH2 domain of the heavy chain immunoglobulin constant region (e.g., those containing an N-linked glycosylation site).

[0075] The recombinant protein of the present invention is produced in eukaryotic cells. Preferably, the eukaryotic cells used to produce the recombinant protein are yeast cells (e.g., Saccharomyces Klyveromyces) or mammalian cells (e.g., hamster or human cells). The mammalian cells are preferably CHO cells, HEK293 cells or derivatives thereof. HEK293 cells include, but are not limited to, HEK293 cells, HEK293T cells, HEK293F cells, Expi293F cells or derivatives thereof. CHO cells commonly used for large-scale industrial production are often manipulated to improve their characteristics in the production process or to facilitate the selection of recombinant cells. Such manipulations include, but are not limited to, increasing apoptosis resistance, decreasing autophagy, increasing cell proliferation, modified expression of cell cycle regulatory proteins, chaperone manipulation, manipulation of the unfolded protein response (UPR), manipulation of the secretory pathway and manipulation of metabolism.

[0076] Preferably, CHO cells enabling an efficient cell line generation process are metabolically modified, for example, by glutamine synthetase (GS) knockout and / or dihydrofolate reductase (DHFR) knockout (each facilitating selection by methionine sulfoximine (MSX) or methotrexate).

[0077] Preferably, the CHO cells used to produce the recombinant protein are CHO-DG44 cells, CHO-K1 cells, CHO-DXB11 cells, CHO-S cells, CHO glutamine synthetase (GS) -deficient cells or derivatives of any of these cells. [Table 1]

[0078] Cells are most preferably established, adapted and fully cultured under serum-free conditions (and optionally in a medium containing no animal-derived proteins / peptides). Commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma) are representative suitable nutrient solutions. Any of the above media may be supplemented, as necessary, with various compounds, non-limiting examples of which are recombinant hormones and / or other recombinant growth factors (such as insulin, transferrin, epidermal growth factor, insulin-like growth factor, etc.), salts (such as sodium chloride, calcium, magnesium, phosphate, etc.), buffers (such as HEPES), nucleosides (such as adenosine, thymidine, etc.), glutamine, glucose or other equivalent energy sources, antibiotics and trace elements. Any other necessary supplements may also be included at appropriate concentrations known to those skilled in the art. For the growth and selection of genetically modified cells expressing selectable genes, an appropriate selection agent is added to the culture medium.

[0079] The recombinant protein of the method of the present invention is produced by cell culture in eukaryotic cells. After expression, the recombinant protein is harvested and further purified. The recombinant protein is recovered from the culture medium as a secreted protein in the harvested cell culture fluid (HCCF), or from the cell lysate (i.e., a liquid containing the contents of cells lysed by any means including, but not limited to, enzymatic, chemical, osmotic, mechanical, and / or physical disruption of the cell membrane and optionally the cell wall), and may be purified using techniques well known in the art. Samples obtained and / or analyzed in the various steps of purification are also referred to as in-process control (IPC) samples or process intermediates. The harvesting typically includes, for example, centrifugation and / or filtration of the harvested cell culture fluid or cell lysate, preferably for making the harvested cell culture fluid. Thus, the harvested cell culture fluid or cell lysate may also be referred to as the clarified harvested cell culture fluid or clarified cell lysate. Most cell components have been removed and it contains no living cells or cell debris. Clarification typically means centrifugation or filtration, preferably filtration. Further process steps may include affinity chromatography, specifically protein A column chromatography for antibodies or Fc-containing proteins, to separate the product from contaminants. Further process steps may include acid treatment to inactivate viruses, clarification of the product pool by depth filtration (preferably following acid treatment), and removal of cell contaminants such as HCP and DNA. Further process steps may include ion exchange chromatography, specifically anion exchange chromatography to further remove contaminating cell components and / or cation exchange chromatography to remove product-related contaminants such as aggregates, in this order or any other order that may be appropriate in individual cases. Further, preferably, the next process steps may include nanofiltration to further remove viruses, as well as ultrafiltration and diafiltration to concentrate the recombinant protein and exchange the buffer, respectively.

[0080] Since lipase activity may be associated with host cell protein contaminants, the method according to the invention is particularly useful in the analysis of process intermediates after (preferably before and after) a purification step for removing HCP, such as before and after affinity chromatography, before and after depth filtration combined with acid treatment and / or before and after anion exchange chromatography (process steps which can be adapted for more efficient removal of lipase activity in the process intermediate). In some embodiments, the method comprises obtaining at least one sample after affinity chromatography and / or after depth filtration combined with acid treatment (or after acid treatment and / or after depth filtration) and / or after ion exchange chromatography (e.g. anion exchange chromatography and / or cation exchange chromatography, preferably anion exchange chromatography). In some embodiments, the method comprises obtaining at least one sample before and after affinity chromatography and / or before and after depth filtration combined with acid treatment (or before and after acid treatment and / or before and after depth filtration) and / or before and after ion exchange chromatography (e.g. anion exchange chromatography and / or cation exchange chromatography, preferably anion exchange chromatography). One skilled in the art will know that a sample obtained after a step of a particular method can be the same as a sample obtained before the next step of the method, for example, a sample obtained after affinity chromatography (e.g. protein A chromatography) can be the same as a sample before acid treatment (or before depth filtration combined with (i.e. following) acid treatment). As explained above, due to the broad buffer range of the buffer, even lipase activity in samples having different pH values can be compared using the method according to the invention. Other samples that can be analyzed using the method according to the invention are samples of the active pharmaceutical ingredient or drug product. Samples of the active pharmaceutical ingredient or drug product contain the formulation buffer and thus often contain polysorbate. At very high concentrations, polysorbate can inhibit the reaction by competing with the substrate.However, depending on the sensitivity of the assay, lipase activity can be measured in samples of the active ingredient or pharmaceutical product with typical concentrations of polysorbate from 0.4 to 0.8 mg / mL.

[0081] In one aspect, a method for producing a recombinant protein of interest, comprising the step of detecting lipase activity in a sample containing the recombinant protein, the step comprising: (a) providing at least one sample containing a recombinant protein produced in a eukaryotic cell; (b) contacting the at least one sample with a reaction solution to form a reaction mixture (the reaction solution comprising: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant being a non-ionic or zwitterionic surfactant); (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (the 4-MU ester being a saturated unbranched fatty acid (C6-C 16(which is a 4-MU ester), and (iv) optionally, a non-buffered salt); (c) incubating the sample and the substrate in the reaction mixture; (d) detecting lipase activity by measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; Optionally, a method is provided that includes measuring hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (c). One of ordinary skill in the art will understand that the method is a method for detecting contaminating lipase activity, and that the lipase activity detected in step (d) is contaminating lipase activity in at least one sample that includes a recombinant protein, more specifically, the recombinant protein of interest. One of ordinary skill in the art will understand that the method further includes (i) culturing eukaryotic cells that express the recombinant protein of interest in cell culture; (ii) harvesting the recombinant protein; (iii) purifying the recombinant protein; and (iv) optionally, formulating the recombinant protein into a pharmaceutically acceptable formulation suitable for administration.Accordingly, a method for producing a recombinant protein of interest, comprising: (i) culturing a eukaryotic cell that expresses the recombinant protein of interest; (ii) collecting the recombinant protein; (iii) purifying the recombinant protein; and (iv) optionally formulating the recombinant protein into a pharmaceutically acceptable formulation suitable for administration; and (v) in step (ii), (iii) and / or (iv), obtaining at least one sample containing the recombinant protein, the method further comprising detecting (contaminating) lipase activity in the sample containing the recombinant protein, this step comprising: (a) providing at least one sample containing the recombinant protein produced in eukaryotic cells in step (v); (b) contacting the at least one sample with a reaction solution to form a reaction mixture, wherein the reaction solution comprises: (i) a buffer having a pH of about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant is a non-ionic or zwitterionic surfactant); (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (the 4-MU ester is a saturated unbranched fatty acid (C6-C. 16)which is a 4-MU ester), (iv) optionally, a step of including a non-buffered salt; (c) incubating the sample and the substrate in the reaction mixture; (d) detecting the contaminating lipase activity by measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; Optionally, a method is provided that includes measuring the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (c). The reaction solution used in the method of the present invention is an aqueous reaction solution. Further, the lipase activity detected in step (d) is the contaminating lipase activity in at least one sample, including the recombinant protein, more specifically the recombinant protein of interest. In certain embodiments, the recombinant protein of interest is a therapeutic protein such as an antibody, antibody fragment, antibody-derived molecule (e.g., scFv, bispecific or multispecific antibody) or fusion protein (e.g., Fc fusion protein). In one embodiment, the antibody is an anti-IL-36R antibody, particularly spesolimab. In another embodiment, the antibody is not an anti-IL-36R antibody and particularly not spesolimab.

[0082] In certain embodiments, a method for producing a recombinant protein of interest according to the present invention includes obtaining at least one sample containing the recombinant protein within the step of collecting the recombinant protein (within step (ii)) (the sample being the harvested cell culture fluid (HCCF) or cell lysate); within the step of purifying the recombinant protein (within step (iii)) (the sample being an in-process control (IPC) sample); and / or optionally within the step of formulating the recombinant protein into a pharmaceutically acceptable formulation suitable for administration (within step (iv)) (the sample being a drug substance sample or a pharmaceutical product sample). Preferably, a method for producing a recombinant protein of interest according to the present invention includes, within step (iii), obtaining at least one sample containing the recombinant protein (the sample being an in-process control (IPC) sample), for example, after affinity chromatography, after depth filtration following acid treatment (or after and / or before acid treatment), and / or after ion exchange chromatography (preferably anion exchange chromatography or cation exchange chromatography), obtaining at least one sample. More preferably, the method includes obtaining at least one sample before and after affinity chromatography, before and after depth filtration after acid treatment (or before and after and / or before and after acid treatment), and / or before and after ion exchange chromatography (preferably anion exchange chromatography or cation exchange chromatography). The step of detecting lipase activity in a sample containing the recombinant protein is carried out according to and as specified in the method for detecting lipase activity described herein.

[0083] Kit for measuring contaminating lipase activity by measuring hydrolysis in a sample Also, a kit for measuring contaminating lipase activity in a sample containing a recombinant protein, comprising: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant being a non-ionic or zwitterionic surfactant); (iii) a substrate containing a chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (the substrate being a saturated unbranched fatty acid (C6~C 16 ) 4-MU ester); (iv) optionally, a non-buffered salt, and / or (v) optionally, water for dilution. In one embodiment, the kit further comprises an internal standard, such as a commercially available lipase, e.g., porcine pancreatic lipase (PPL), which functions as a positive control and / or enables calculation of relative values compared to the internal standard. The kit may also include one or more microtiter plates having 96 wells, or a multiple of 96 wells. The components of the kit may be provided separately or pre-mixed, as solutions and / or dry components. In the case of the buffer, it may be provided as a dry compound that provides a buffer having a pH of from about pH 4 to about pH 9 upon dilution or reconstitution.

[0084] In certain embodiments, the buffer, surfactant, and optional non-buffered salts are premixed as an assay buffer. Preferably, the assay buffer is concentrated at least about 3-fold, or from about 3-fold to about 5-fold, relative to the final reaction mixture. Alternatively, the assay buffer is provided as a dry mixture. Such a dry mixture may be reconstituted with water to provide an assay buffer that is concentrated at least about 3-fold, or 5-fold, relative to the final reaction mixture. In one embodiment, the dry mixture of the assay buffer is a lyophilized assay buffer. The substrate is provided separately so that it can be added to the assay buffer prior to use to provide a reaction solution. Alternatively, the kit may include the buffer, surfactant, substrate, and optional non-buffered salts premixed as a master mixture. The master mixture may be adapted to be provided at from about 80% (v / v) to about 70% (v / v), preferably from about 80% to about 75% (v / v), of the reaction mixture. The assay buffer and reaction solution are aqueous solutions.

[0085] The components of the reaction solution are the same as those specified above for the method of the present invention. The substrate containing the chromophore 4-MU is in the form of a saturated unbranched fatty acid (C6 to C 16 ) 4-MU ester, and the aliphatic chain of the saturated unbranched fatty acid has from C6 to C 16 carbon atoms, or preferably from C8 to C 12 carbon atoms. Thus, the substrate may be 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), methylumbelliferyl undecanoate, or methylumbelliferyl dodecanoate. In certain embodiments, the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl decanoate (4-MUD), and 4-methylumbelliferyl dodecanoate, and in a preferred embodiment, the substrate is 4-MUD.

[0086] The kit may further comprise an organic solvent for dissolving the substrate, or the substrate may be dissolved in an organic solvent. The substrate may be provided as a dry substance and an optional additional organic solvent, or as a stock solution (e.g., a 100-fold stock solution relative to the concentration in the reaction mixture), dissolved in an organic solvent (e.g., dimethyl sulfoxide (DMSO) or dimethylformamide (DMF), preferably DMSO). Thus, in certain embodiments, the substrate is provided as a stock solution from about 100 μM to about 100 mM, preferably from about 100 μM to 30 mM, preferably from 100 μM to 3 mM, more preferably from about 300 μM to 3 μM. In certain embodiments, the substrate is provided as a stock solution in an organic solvent, and the stock solution is added at from about 1% to about 5% (v / v) of the reaction mixture.

[0087] Examples of suitable non-denaturing zwitterionic surfactants that do not have an ester bond are 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS), 3-([3-cholamidopropyl)dimethylammonio]-2-hydroxy-1-propanesulfonate (CHAPSO), CHAPS analogs (such as Big CHAP (N,N-bis-(3-D-gluconamidopropyl)deoxycholamide)), Zwittergent (of various lengths, for example n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent3-12)) and 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate or other amidosulfobetaine detergents, but are not limited thereto. Examples of suitable non-denaturing non-ionic surfactants are pyranoside surfactants (such as octyl β-D-glucopyranoside (OGP), nonyl β-D-glucopyranoside, dodecyl β-D-maltopyranoside (DDM) or octyl β-D-thioglucopyranoside), polyoxyethylene (23) lauryl ether (Brij 35) or other polyoxyethylene ethers; saponins (such as digitonin), octylphenoxypolyethoxyethanol (IGEPAL CA-630), poloxamer 188, 338, 407 or Triton, but are not limited thereto. In certain embodiments, the surfactant is a non-denaturing non-ionic or zwitterionic surfactant that does not have an ester bond, preferably the surfactant is not polyethylene glycol tert-octylphenyl ether (TritonX-100) and is not polyethylene glycol nonylphenyl ether (NP-40). In a preferred embodiment, the surfactant is a non-denaturing non-ionic or zwitterionic surfactant selected from the group consisting of CHAPS, CHAPSO, Zwittergent (such as Zwittergent3-12) and saponin, preferably CHAPS.

[0088] In certain embodiments, the buffer comprises one or more buffering substances selected from the group consisting of formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MES (2-(N-morpholino)ethanesulfonic acid), tris base, tris, bis-tris, bis-tris-propane, bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4, and NaH2PO4. In certain embodiments, the buffer has a pH of from about pH 4 to about pH 8, preferably the buffer has a pH of from about pH 5 to about pH 7.5, more preferably the buffer has a pH of from about pH 5.5 to about pH 7.5.

[0089] The buffer solution may contain a single buffering substance or may be a multi-component buffer solution as specified above for the method according to the invention. The multi-component buffer solution may contain one or more buffering substances having overlapping buffering ranges so as to have a wider buffering range. The buffer solution may contain, for example, two, three, four, five or more buffering substances, preferably two or more buffering substances, more preferably three or more buffering substances. For example, the multi-component buffer solution may contain two to four buffering substances, three to four buffering substances, more preferably three buffering substances. In certain embodiments, the multi-component buffer solution contains at least three buffering substances having overlapping buffering ranges, preferably contains at least one of Tris, MES and / or acetic acid, and more preferably contains acetic acid, MES and Tris in a ratio of 1:1:2. In certain embodiments, the buffer solution is a multi-component buffer solution having a buffering range of at least about pH 5 to at least about pH 7.5, preferably at least about pH 4 to at least about pH 8. In one embodiment, the use of multi-component buffer solutions with different pH values between about pH 4 and about pH 9 has an effect on the ionic strength of the buffer solution of less than 15%, preferably less than 10%, or even less than 7.5% or 5%, for example, from 0% to less than 15%, from 0% to less than 10%, from 0% to less than 7.5% or from 0% to less than 5%, or, for example, from 2% to less than 15%, from 2% to less than 10%, from 2% to less than 7.5% or from 2% to less than 5%. Optional non-buffering salts may be, for example, NaCl, KCl and CaCl2, preferably NaCl or KCl.

[0090] In view of the above, it is understood that the present invention also encompasses the following items: Item 1 is a method for detecting lipase activity in a sample containing a recombinant protein, comprising: (a) providing at least one sample containing a recombinant protein produced in a eukaryotic cell; (b) contacting said at least one sample with a reaction solution to form a reaction mixture (said reaction solution comprising: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (said surfactant being a non-ionic or zwitterionic surfactant); (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (said 4-MU ester being a saturated unbranched fatty acid (C6~C 16 ) 4-MU ester), and (iv) optionally, a non-buffered salt), (c) incubating said sample and said substrate in said reaction mixture; (d) detecting lipase activity by measuring the hydrolysis of said 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; optionally, measuring hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating said sample and said substrate in said reaction mixture according to step (c). A method is provided that includes this step.

[0091] Item 2 specifies the method of Item 1 or Item 2, wherein the fluorescence intensity of the released chromophore 4-MU is detected without stopping the hydrolysis of the 4-MU ester, and / or the sample and the substrate in the reaction mixture are incubated for any time between 2 minutes and less than 5 hours, 2 minutes and less than 3 hours, 2 minutes and less than 2 hours, or 2 minutes and less than 0.5 hours.

[0092] Item 3 specifies the method of any one of the preceding items, wherein the fluorescence intensity of the released chromophore 4-MU is detected over time and follows pseudo-zero order reaction kinetics (optionally, reaction mixtures that do not meet the requirements of pseudo-zero order reaction kinetics are excluded from the analysis).

[0093] Item 4 identifies the method of any one of the preceding items, and (a) determines the hydrolysis rate by detecting the fluorescence intensity of the released chromophore 4-MU as relative fluorescence units (RFU), and compares it with a calibration curve created by using a defined concentration of 4-MU to determine the amount (mol / sec) of the released chromophore 4-MU, and / or (b) further includes the step of calculating a relative value compared with an internal standard.

[0094] Item 5 identifies the method of any one of the preceding items, and the lipase activity detected in at least one sample is contaminating lipase activity.

[0095] Item 6 identifies the method of any one of the preceding items, and the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), 4-methylumbelliferyl undecanoate, and 4-methylumbelliferyl dodecanoate.

[0096] Item 7 identifies the method of any one of the preceding items, and the substrate is provided at a final concentration of about 1 μM to about 1 mM in the reaction mixture.

[0097] Item 8 identifies the method of any one of the preceding items, and the substrate is provided as a stock solution in an organic solvent, and the stock solution is added at a concentration of about 1% to about 5% (v / v) of the reaction mixture, and / or the organic solvent is DMSO or DMF.

[0098] Item 9 identifies the method of any one of the preceding items, and the surfactant has a final concentration in the reaction mixture that exceeds its critical micelle concentration in the reaction mixture.

[0099] Item 10 identifies the method of any one of the preceding items, and the surfactant is selected from the group consisting of CHAPS, CHAPSO, and Zwittergent, preferably CHAPS, and / or, and not polyethylene glycol tert-octylphenyl ether (Triton X-100), and not polyethylene glycol nonylphenyl ether (NP-40).

[0100] Item 11 identifies the method of any one of the preceding items, and the surfactant is CHAPS and is provided at a final concentration in the reaction mixture of from about 8 mM to about 20 mM, preferably from about 8 mM to about 15 mM, more preferably about 10 mM.

[0101] Item 12 identifies the method of any one of the preceding items, and the buffer contains one or more buffering substances selected from the group consisting of formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MES (2-(N-morpholino)ethanesulfonic acid), tris base, tris, bis-tris, bis-tris-propane, bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4, and NaH2PO4.

[0102] Item 13 identifies the method of any one of the preceding items, and the buffer has a pH of from about 5 to about 7.5, preferably, the buffer has a pH of from about 5.5 to about 7.5.

[0103] Item 14 identifies the method of any one of the preceding items, and the buffer is a multi-component buffer having a buffering range of at least about pH 5 to at least about pH 7.5, preferably at least about pH 4 to at least about pH 8.

[0104] Item 15 identifies the method of Item 14, and the multi-component buffer contains at least three buffering substances having overlapping buffering ranges, preferably including at least one of Tris, MES, and / or acetic acid.

[0105] Item 16 identifies the method of Item 14 or Item 15, and the method includes: (a) using a buffer having a variable pH from at least about pH 4 to at least about pH 8; (b) using buffers having various pH values between about pH 4 and about pH 8, thereby having an impact on the ionic strength of less than 15%, preferably less than 10%, preferably from 0% to less than 15%, from 0% to less than 10%, from 0% to less than 7.5%, or from 0% to less than 5%; (c) adjusting the pH of the buffer to the pH of the sample; (d) adjusting the pH of the buffer to near the optimum of the at least one (contaminating) protein having lipase activity; or (e) including the step of comparing and identifying conditions that reduce the hydrolytic activity.

[0106] Item 17 identifies the method of any one of the preceding items, and at least 2, 3, 4, 5, 10 or more samples are analyzed simultaneously.

[0107] Item 18 identifies the method of any one of the preceding items, and the sample is contacted, incubated, and measured in a format of a plate having 96 wells or a multiple of 96 wells.

[0108] Item 19 identifies the method of any one of the preceding items, and the sample is provided at about 20% to about 30% (v / v) of the reaction mixture, preferably about 25% of the reaction mixture, and optionally, the sample can be pre-diluted.

[0109] Item 20 identifies the method of any one of the preceding items, and the buffer, surfactant, and optional non-buffered salt are pre-mixed as an assay buffer concentrated about 3-fold to about 5-fold with respect to the reaction mixture.

[0110] Item 21 identifies the method of any one of the preceding items, and the buffer, surfactant, substrate, and optional non-buffered salt are added to the sample as a master mixture, and the master mixture is provided at about 80% (v / v) to about 70% (v / v), preferably about 75% of the reaction mixture.

[0111] Item 22 identifies the method of any one of the preceding items, and the non-buffered salt is selected from the group consisting of NaCl, KCl, and CaCl2, and preferably, the non-buffered salt is NaCl or KCl.

[0112] Item 23 identifies the method of any one of the preceding items, and the non-buffered salt has a concentration in the reaction mixture of about 100 mM to about 200 mM, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM.

[0113] Item 24 identifies the method of any one of the preceding items, and the ionic strength of the non-buffered salt is about 200 mM or less in the reaction mixture, preferably about 150 mM or less in the reaction mixture, preferably about 100 mM to about 200 mM in the reaction mixture, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM.

[0114] Item 25 identifies the method of any one of the preceding items, and the cumulative ionic strength of the buffer salt and the non-buffered salt in the reaction mixture is about 450 mM or less in the reaction mixture, preferably about 400 mM, more preferably 350 mM or less.

[0115] Item 26 identifies the method of any one of the preceding items, and the fluorescence is detected using a fluorescence spectrometer or a microplate spectrophotometer.

[0116] Item 27 identifies the method of any one of the preceding items, and the at least one sample is a collected cell culture fluid (HCCF) or cell lysate, an in-process control (IPC) sample, a drug substance sample or a pharmaceutical product sample.

[0117] Item 28 identifies the method of any one of the preceding items, and (a) the recombinant protein is not a lipase and / or is not an enzyme having lipase activity, and / or (b) the recombinant protein is selected from the group consisting of an antibody, an antibody fragment, an antibody-derived molecule and a fusion protein.

[0118] Item 29 identifies the method of any one of the preceding items, and the eukaryotic cell used to produce the recombinant protein is a yeast cell or a mammalian cell, and the mammalian cell is preferably a CHO cell, a HEK293 cell or a derivative thereof.

[0119] Item 30 provides a kit for measuring contaminating lipase activity in a sample containing a recombinant protein, the kit comprising: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant is a non-ionic or zwitterionic surfactant); and (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (the substrate is a saturated unbranched fatty acid (C6 to C 16 ) 4-MU ester); and (iv) optionally, a non-buffered salt; and / or (v) optionally, water for dilution.

[0120] Item 31 identifies the kit of Item 30, and the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), 4-methylumbelliferyl undecanoate, and 4-methylumbelliferyl dodecanoate.

[0121] Item 32 identifies the kit of Item 30 or Item 31, and the kit further comprises an organic solvent for dissolving the substrate, preferably DMSO or DMF.

[0122] Item 33 identifies the kit of any one of Items 30 to 32, and the surfactant is not polyethylene glycol tert-octylphenyl ether (TritonX-100) and is not polyethylene glycol nonylphenyl ether (NP-40), or the surfactant is a non-denaturing zwitterionic surfactant selected from the group consisting of CHAPS, CHAPSO, and Zwittergent, preferably CHAPS.

[0123] Item 34 identifies the kit of any one of Items 30 to 33, and the buffer solution comprises one or more buffering substances selected from the group consisting of formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MES (2-(N-morpholino)ethanesulfonic acid), tris base, tris, bis-tris, bis-tris-propane, bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4, and NaH2PO4.

[0124] Item 35 identifies any one kit from Items 30 to 34, and the buffer has a pH of from about 5 to about 7.5, and preferably, the buffer has a pH of from about 5.5 to about 7.5.

[0125] Item 36 identifies any one kit from Items 30 to 35, and the buffer is a multi-component buffer having a buffering range of at least about pH 5 to at least about pH 7.5, preferably at least about pH 4 to at least about pH 8.

[0126] Item 37 identifies the kit of Item 36, and the multi-component buffer contains at least three buffering substances having overlapping buffering ranges, and preferably contains at least one of Tris, MES and / or acetic acid.

[0127] Item 38 identifies any one kit from Items 30 to 37, and the kit further includes one or more microtiter plates having 96 wells or a multiple of 96 wells.

[0128] Item 39 identifies any one kit from Items 30 to 38, and the buffer, surfactant and optional non-buffered salt are pre-mixed as an assay buffer concentrated at least about 3-fold, or about 3-fold to about 5-fold, with respect to the final reaction mixture, and / or provided as a dry mixture.

[0129] Item 40 identifies any one kit from Items 30 to 39, and the non-buffered salt is selected from the group consisting of NaCl, KCl and CaCl2, and preferably, the non-buffered salt is NaCl or KCl.

[0130] Item 41 is a method for producing a recombinant protein of interest, comprising: (i) culturing eukaryotic cells that express the recombinant protein of interest in cell culture; (ii) collecting the recombinant protein; (iii) purifying the recombinant protein; and (iv) optionally formulating the recombinant protein into a pharmaceutically acceptable formulation suitable for administration; and (v) obtaining at least one sample containing the recombinant protein in step (ii), (iii) and / or (iv), the method further comprising detecting (contaminating) lipase activity in the sample containing the recombinant protein, the step comprising: (a) providing at least one sample containing the recombinant protein produced in eukaryotic cells in step (v); (b) contacting the at least one sample with a reaction solution to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of from about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant being a non-ionic or zwitterionic surfactant); (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester (the 4-MU ester being a saturated unbranched fatty acid (C6-C 16) being a 4-MU ester), and (iv) optionally, a step of including a non-buffered salt; (c) a step of incubating the sample and the substrate in the reaction mixture; (d) a step of detecting the (interfering) lipase activity by measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; Optionally, a step of measuring the hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (c). In a preferred embodiment, the surfactant in step (b)(ii) is not polyethylene glycol tert-octyl phenyl ether (Triton X-100) and is not polyethylene glycol nonyl phenyl ether (NP-40). Preferably, the surfactant is a non-denaturing zwitterionic surfactant such as those selected from the group consisting of CHAPS, CHAPSO, and Zwittergent, preferably CHAPS.

[0131] Item 42 identifies a method for producing a recombinant protein of interest according to item 41, the method comprising obtaining, in step (ii), at least one sample comprising the recombinant protein, wherein the sample is a harvested cell culture fluid (HCCF) or cell lysate; in step (iii), the sample is an in-process control (IPC) sample; and / or in step (iv), the sample is a drug substance or pharmaceutical sample.

[0132] Item 43 identifies a method for producing a recombinant protein of interest according to item 41 or item 42, the method comprising obtaining, in step (iii), at least one sample comprising the recombinant protein, wherein the sample is an in-process control (IPC) sample.

[0133] Item 44 identifies a method for producing the recombinant protein of interest described in item 43, said method comprising obtaining at least one sample after affinity chromatography, following acid treatment and subsequent depth filtration (or after and / or before acid treatment and / or depth filtration), and / or after anion exchange chromatography, preferably obtaining at least one sample before and after affinity chromatography, before and after acid treatment and subsequent depth filtration (or before and after and / or before and after acid treatment and / or depth filtration), and / or before and after anion exchange chromatography.

[0134] Item 45 identifies a method for producing the recombinant protein of interest described in any one of items 41 to 44, said method comprising detecting lipase activity in a sample containing said recombinant protein according to any one of the methods of items 1 to 29.

Examples

[0135] Selection of 4-MU as a chromophore Many lipases are able to hydrolyze fatty acid esters of 4-methylumbelliferone (4-MU). Upon hydrolysis, not only fatty acids but also highly fluorescent 4-MU are released (Figure 1). The increase in fluorescence is directly proportional to the rate of hydrolysis and can thus be used to measure the hydrolysis activity or lipase activity in a given sample.

[0136] 4-Methylumbelliferyl was selected as the detector because its spectral properties combine a high quantum yield with sufficient insensitivity to changing ionic strength and pH (data not shown). These properties support robust assay performance. This further unlocks the key to a highly sensitive fluorescence-based detection principle that is sufficiently insensitive to interferences caused, for example, by light scattering (data not shown).

[0137] Lipase assay Two different buffers are used in the lipase assay. They are the phosphate assay buffer or the AMT buffer, a multi-component buffer. The phosphate buffer has a pH of 7.4 and contains 108 mM Na2HPO4, 25 mM NaH2PO4, 186.2 mM NaCl, and 13.3 mM CHAPS. The final concentrations in the reaction mixture at pH 7.4 are 81 mM Na2HPO4, 19 mM NaH2PO4, 140 mM NaCl, and 10 mM CHAPS. The AMT assay buffer, which has a broad buffering range from pH 4 to 8, is provided as a 4-fold concentrated stock solution containing 0.3 M acetic acid, 0.3 M MES, 0.6 M Tris, 0.6 M NaCl, and 40 mM CHAPS. The pH is adjusted as described (recommended pH range: 4 - 8) using HCl or NaOH, and the final concentrations in the reaction mixture are 75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, and 10 mM CHAPS.

[0138] The substrate is stored as a concentrated stock solution containing 3 mM 4-methylumbelliferyl decanoate (4-MUD; FM25973, Carbosynth) in DMSO and is diluted 1:10 with DMSO before use to obtain a 100-fold stock solution for use containing 0.3 mM in DMSO.

[0139] To ensure comparability of measurements, the assay buffer and substrate are mixed before use. The master mixture is prepared immediately before use, and the reaction is initiated by mixing a given sample (e.g., the drug substance) with the master mixture containing the substrate and assay buffer, and optionally additional water. Mixing in the reaction vessel was done by providing the smaller amount of the two components of the sample and master mixture to the reaction vessel before adding the larger amount. Thus, typically, the sample was added first.

[0140] The reaction mixture was prepared as follows. In the assay based on the phosphate buffer (constant pH 7.4), A) In a cuvette: In the reaction vessel, to 720 μL of the sample, a master mixture (2250 μL of phosphate assay buffer, 30 μL of 0.3 mM 4-MUD in DMSO) was added. B) Per well of a 96-well plate: In the reaction vessel, to 72 μL of the sample, a master mixture (225 μL of phosphate assay buffer, 3 μL of 0.3 mM 4-MUD in DMSO) was added. In the assay based on the AMT buffer (also called a three-component buffer) (variable pH), C) In a cuvette: In the reaction vessel, to 720 μL of the sample, a master mixture (750 μL of 4-fold AMT assay buffer, 1500 μL of H2O, 30 μL of 0.3 mM 4-MUD in DMSO) was added. D) Per well of a 96-well plate: In the reaction vessel, to 72 μL of the sample, a master mixture (75 μL of 4-fold AMT assay buffer, 150 μL of H2O, 3 μL of 0.3 mM 4-MUD in DMSO) was added.

[0141] The hydrolysis of the substrate 4-MUD was measured in real time from immediately after mixing for several minutes to 5 hours according to the fluorescence intensity by detecting the fluorescence intensity of the released chromophore 4-MU. Fluorescence indicating the release of 4-MU (λ em = 450 nm, λ ex(at 330 or 340 nm) was monitored at 25 °C either in a cuvette using a fluorescence spectrometer or in a 96-well plate using a suitable microplate reader. A high correlation was observed between the use of the fluorescence spectrometer (Fluoromax 4, Horiba Jobin Yvon) and the plate reader SpectraMax M3 (Molecular Devices) (see Figure 9). To exclude potential self-hydrolysis, negative controls without samples were included. The gradient (e.g., RFU / s) was calculated using linear regression. By detecting fluorescence in real time, it becomes possible to measure within a time frame at a pseudo-zero order reaction rate. Samples were tested at least three times (run), and individual reaction mixtures were excluded from the analysis if they did not meet the pseudo-zero order reaction rate, e.g., due to bubbles in the well. It was found that this method of removing outliers strongly enhanced the sensitivity of the assay. The hydrolysis rate (e.g., nmol / sec) can be calculated using a calibration curve with a defined concentration of 4-MU. The calibration curve using known 4-MU concentrations further enabled the determination and comparison of reaction rates at various pH values.

[0142] Development of AMT buffer The lipase assay was initially set up using a phosphate assay buffer. A three-component AMT buffer was developed for the kinetic measurement of the hydrolysis activity in various pharmaceutical samples at varying pH. The AMT buffer containing acetic acid, MES, and Tris as buffering substances enables measurement over a wider pH buffering range, and thus a wider pH range, or even at different pHs. The buffering substances used are known to be non-fluorescent and poor metal chelators, with little potential for interference with enzyme activity. To match the phosphate assay buffer, CHAPS above CMC was added and the ionic strength was adjusted using NaCl. Since the assay was found to be sensitive to ionic strength, it was important to create buffers that not only contain buffering substances with overlapping buffering ranges but also vary the ionic strength only moderately (less than 15%, preferably even less than 10%) at various pHs (in the range of pH 4 to 8) (Ellis KJ, Morrisson JF, 1982. Methods in Enzymology, 87: 405-426). The AMT buffer, for example, enables the identification of conditions (including pH conditions) that reduce the hydrolysis activity. In addition to measuring the lipase activity present in a sample under specific conditions by performing the measurement at the pH of the sample, this buffer also enables the comparison of lipase activities in various states during purification. The assay enables further enhancement of sensitivity by measuring the sample at the optimal pH.

[0143] HPLC-CAD method The content of polysorbate in aqueous solution was quantified using HPLC-CAD. Using an aqueous mobile phase containing isopropanol or its equivalent, intact polysorbate was bound to a mixed-mode column based on a mixture of a reversed phase and an ion exchange polymer. Next, the polysorbate was eluted using a mobile phase having acetonitrile or its equivalent. More specifically, HPLC chromatography was performed using mobile phase A (MPA) of 10 mM ammonium formate, pH 4.5, 20% (v / v) 2-propanol and mobile phase B (MPB) containing 50% (v / v) acetonitrile and 50% (v / v) 2-propanol. Separation of the protein and matrix components, as well as the polysorbate degradation products, was achieved on a mixed-mode column (Oasis® Max Online column, 2.1 mm × 20 mm, 30 μm, 80 Å) using a flow of MPA at 1.0 mL / min, and the intact polysorbate species were eluted by a step gradient using MPB. The analyte was detected using a charged aerosol detector (CAD). CAD detection employs an inert gas flow system that sprays the analyte, removes the mobile phase, and induces the formation of charged particles. The measured induced current is proportional to the amount of polysorbate contained in the sample. The polysorbate was quantified using an external calibration standard series.

[0144] Fluorescent micelle assay Fluorescent molecules such as N-phenyl-1-naphthylamine (NPN) can be solubilized in aqueous solution in the presence of surfactants. When the surfactant exceeds its critical micelle concentration, the fluorescent agent enters the interior of the micelles, which are hydrophobic, and a large increase in the fluorescence quantum yield is observed. The amount of solubilized fluorescent agent is directly proportional to the concentration of micelles in the solution. More specifically, the sample was spiked with 5×10 -6 M NPN, and the fluorescence was detected using a fluorescence detector (λ em = 420 nm, λ ex = 350 nm). The polysorbate was quantified using an external calibration standard series.

[0145] Example 1: Lipase assay enables measurement of activity in various drug substances The lipase assay was developed to measure the lipase activity in various drug substances after purification and to assist in adapting and improving the purification steps during downstream processing in order to remove the lipase activity in the final drug substance that causes polysorbate degradation in the final pharmaceutical product. The contaminating lipase activity that co-purifies as a host cell protein present in some drug substances may vary not only depending on the purification process but also depending on the protein. Various lipases exhibit specific optimal pHs, which are usually related to their cellular localization. For example, lysosomal lipases typically have an acidic optimal pH.

[0146] Therefore, the lipase assay was used for the kinetic measurement of hydrolysis activity in various bulk drug substances (BDS) at varying pH. For this purpose, the AMT buffer was established to measure pH dependence within the range of pH 4 - 8. The BDS was used undiluted at 72 μL per well for each sample. The reaction rate at each pH could be measured in nmol / min / mL using a calibration curve of known 4-MU concentrations. As a negative control, a blank test was performed using the formulation buffer only to monitor non-enzymatic hydrolysis. Optionally, a positive control containing a commercially available lipase such as porcine pancreatic lipase (PPL) at 0.24 mg / mL or less was included. Pharmaceuticals (referred to as products A, B, D, and E of BDS (BDS A, B, D, and E)) had different amounts of detected hydrolysis activity (see Figure 2) and different pH profiles of hydrolysis activity. For example, while BDS A showed a distinct optimal pH at alkaline pH, BDS D and BDS E showed an optimal pH rather at acidic pH (Figure 2). Above pH 7.5, auto-hydrolysis may cause the remaining hydrolysis activity (see BDS B and BDS E). This can be verified by detecting the remaining hydrolysis activity in the presence of a lipase inhibitor such as orlistat (3.3 μM) or by testing in parallel a blank sample without lipase activity (sample buffer or medium) (data not shown). In this experiment, it was also demonstrated that even extremely low lipase activity, such as that of BDS B, is detectable at the optimal pH (pH 6).

[0147] Overall, different pH dependencies have been found among the various drug substance samples tested, indicating that different drug substances contain various contaminating proteins with lipase activity, and they sometimes have specific optimal pHs. This demonstrates that the assay is useful for detecting such different enzymes with lipase activity in the samples tested.

[0148] Furthermore, various formulation buffers were tested at various pHs to demonstrate that various formulation buffers can assist or inhibit lipase activity in pharmaceuticals (data not shown).

[0149] Example 2: Influence of substrate concentration To achieve a sufficiently high sensitivity of the observed reaction, it is important to have sufficient substrate in the solution. This requirement competes with the limited solubility of 4-MUD and its derivatives in the water-based master mixture and reaction mixture. When 4-MUD was added to water at high concentration, immediately visible particles were formed. The addition of a surfactant such as CHAPS to the assay mixture greatly increased the solubility of 4-MUD and thus prevented precipitation.

[0150] Therefore, an ideal substrate concentration for the intended use was determined by conducting a set of light scattering and activity experiments. In a 1 cm macro cuvette, using a fluorescence spectrometer (λ em = 400 nm, λ ex = 400 nm), the solubility of 4-MUD was tested using right-angle light scattering (RALS) experiments in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 7). The maximum solubility in ATM buffer containing 10 mM CHAPS was found to be approximately 40 μM of 4-MUD (Figure 3).

[0151] The Michaelis-Menten kinetics of the hydrolysis activity in the bulk drug substance (BDS D) was analyzed (72 μL per well, undiluted BDS D). The assay was performed in a black 96-well plate using a microplate reader (λ em = 450 nm, λ exUsing a wavelength of 330 nm and top-read mode, the concentration of 4-MUD was varied (1.5625 μM to 150 μM) in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5). The pH was adjusted to the pH of the bulk drug substance. As shown in Figure 4, the reaction rate was found to be sufficient to support rapid readings even at 4-MUD of 3 μM or less. Typically, the release of 4-MU is measured immediately after mixing and detected from minutes to hours, typically from about 20 minutes to about 2 hours.

[0152] As a result, it can be used at concentrations from 1 μM to 1000 μM, but a 4-MUD concentration between 3 μM and 30 μM was demonstrated to be a reasonable compromise for use as a standard concentration (Figure 4).

[0153] For special unit operations and / or troubleshooting activities, higher concentrations may be preferred, for example, to measure Michaelis-Menten kinetics (Figure 4). Therefore, as shown in Figure 5, the CHAPS concentration may be increased, or other suitable surfactants (e.g., Zwittergent) may be used.

[0154] Example 3: Influence of fatty acid chain length The acyl ester derivative 4-methylumbelliferyl decanoate (4-MUD) was selected because the acyl ester of decanoic acid provides a broader enzyme spectrum compared to, for example, those using oleic acid esters containing longer unsaturated acyl chains. Furthermore, the shorter chain length of the decanoic acid ester provided better solubility in the aqueous reaction mixture compared to, for example, oleic acid esters. As a result, more substrate can be used in the assay mixture. More specifically, C 16Or with longer chain lengths, it was found that the solubility becomes strongly restricted (data not shown). Furthermore, it was found that the decanoate esters provide better resistance to self-hydrolysis compared to, for example, butyrate esters (Figure 6). The self-hydrolysis of 4-methylveratryl butyrate (4-MUB) and 4-MUD was analyzed. In a black 96-well plate, using a microplate reader (λ em = 450 nm, λ ex = 330 nm, top read mode), in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5), 30 μM 4-MUB and 30 μM 4-MUD were used to monitor fluorescence for 1800 seconds and compare. Furthermore, the inventors measured that chain lengths up to C5 strongly increase self-hydrolysis (data not shown). Using substrates with a C8 chain but different chromophores showed more, but still acceptable, self-hydrolysis (data not shown). In summary, the specific substrate used is important and has been demonstrated to strongly improve and enhance the sensitivity of the assay. The C 10 fatty acid in 4-MUD used in the assay was found to be the optimal choice.

[0155] Example 4: Influence of surfactant in the reaction mixture The reaction conditions should be designed to maintain and support the activity of the relevant enzymes, such as the hydrolytic activity of the lipase. In particular, this requirement was achieved by modifying the reaction mixture of the assay.

[0156] First, the surfactant was tested above its CMC (critical micelle concentration). Thus, 10 mM CHAPS was added to the assay. Specifically, the assay was performed in a black 96-well plate using a microplate reader (λ em = 450 nm, λ exPerformed using 30 μM 4-MUD in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, pH 5.5) with or without 10 mM CHAPS in a bottom-read mode at = 330 nm. The samples tested were drug substance samples including samples after ultrafiltration / diafiltration of Product G, Product B, Product F, and Product D, and 72 μL was added per well respectively. The results are shown in FIGS. 7A and B, demonstrating that the presence of a surfactant such as CHAPS above the CMC improved the sensitivity of the assay. Without being bound by theory, a hypothesis has been proposed that the surfactant creates an environment that promotes lipase activity by enabling the rearrangement and opening of a lid or flap that is described as covering the active site (Grochulski P, Li Y, Schrag JD, et al. Protein Sci 1994; 3:82-91 and Grochulski P, Bouthillier F, Kazlauskas RJ, et al. Biochemistry 1994; 33:3494-500). Therefore, a concentration of 10 mM CHAPS was selected in the final reaction mixture. As a result, lipase hydrolysis increased and, in addition, the sensitivity of the assay improved (FIG. 7).

[0157] The surfactant can be another surfactant, but in order to maintain the native structure and activity of the protein having lipase activity, it is necessary to be a mild, specifically non-denaturing surfactant. Furthermore, it is important that the surfactant does not compete with or inhibit lipase / hydrolysis. CHAPS does not have an ester bond or an acyl chain and is therefore not a substrate for lipase. Therefore, it does not compete with the substrate and thus does not affect the sensitivity of the assay. Furthermore, CHAPS mediates the solubility of 4-MUD in water at the concentration used (FIG. 3).

[0158] Example 5: Measurement of samples with various pH values The activity of an enzyme is often affected by pH. Since the expected pH range of in-process control (IPC) samples reaches from 3.5 to 7.5 (Table 1), an influence on the observed activity of contaminating lipase was expected. To maximize the comparability between samples at various pH values, a first buffer component was identified to maintain the pH at a constant 7.4 in the reaction mixture. Table 1 summarizes various IPC samples from one downstream process to demonstrate the pH of samples from various purification steps. The pH of each sample and the pH of the corresponding reaction mixture are shown. Similar pH fluctuations were found in various antibodies or Fc fusion proteins during downstream processing. Experiments were conducted with several products, yielding similar results.

Table 2

[0159] Analyzing IPC samples at the same pH maintains certain reaction conditions and thus enhances the comparability of the resulting data. However, in some cases, it may be beneficial to change the pH, for example, to find conditions that reduce hydrolytic activity (see, e.g., Figure 2) to assist in formulation development. Therefore, a three-component AMT buffer system was developed.

[0160] Example 6: Influence of ionic strength To prevent aggregation of the protein in the sample and maintain the native structure of the protein, it is beneficial to provide ions. However, ionic strength can also affect enzyme activity. Therefore, the effect of ionic strength on the measured hydrolytic activity was tested. The hydrolytic activity of an exemplary pharmaceutical sample was measured in the presence of various concentrations of NaCl (1000 mM to 7.8125 mM). All measurements were performed in a black 96-well plate using a microplate reader (λ em = 450 nm, λ exPerformed using 30 μM 4-MUD and 72 μL of sample in an AMT assay buffer without NaCl (75 mM acetic acid, 75 mM MES, 150 mM Tris, 10 mM CHAPS, pH 5.5) at 340 nm, 25 °C, top read mode. The dependence of hydrolytic activity on ionic strength has been investigated using several mAbs and is exemplified for Product F in Figure 8.

[0161] As can be seen from Figure 8, lipase activity decreased at NaCl concentrations of 250 mM and above. Therefore, an NaCl concentration of 150 mM was found to be optimal in the AMT buffer.

[0162] Example 7: Inhibition of lipase assay by polysorbate Polysorbate in the final pharmaceutical product is likely to function as a competing substrate for the trackable fluorescent substrate (4-MUD). The concentration of polysorbate 20 (PS20) or polysorbate 80 in the pharmaceutical product may range between about 0.2 g / L and 1.0 g / L and is typically 0.2 g / L to 0.4 g / L of PS20 or PS80, or a mixture thereof. Therefore, an experiment was set up in which the ultrafiltration-diafiltration material of the antibody was used as the pharmaceutically active ingredient (API in water, without PS20), and various concentrations of PS20 (0.0125 - 3.2 mg / mL) were added to the reaction mixture.

[0163] The hydrolytic activity of 4-MUD in the sample was measured in the presence of 0.0125 - 3.2 mg / mL of PS20 (final reaction mixture concentration). All measurements were performed in a black 96-well plate using a microplate reader (λ em = 450 nm, λ ex = 330 nm, top read mode) with 30 μM 4-MUD in an AMT assay buffer (50 mM acetic acid, 50 mM MES, 100 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5). In this first experiment, the AMT buffer was used at a lower concentration, but it was later found to be too low for buffering at pH 4 and pH 8. Therefore, the concentration was increased to the above concentration.

[0164] The results indicate that PS20 is a competitive inhibitor for 4-MUD hydrolysis at high concentrations. In the reaction mixture, no inhibition was observed up to 0.2 g / L of PS20 (or 0.8 g / L and above in DP), but concentration-dependent inhibition was observed at concentrations of 0.4 g / L and above in the assay sample, and 4-MUD hydrolysis was clearly detectable even at a concentration of 0.4 g / L of PS20 (Figure 9). In contrast, Jahn et al. (Pharm. Res., 2020, 37:118, pages 1-13) already observed almost complete inhibition at 0.02% (w / v) (0.2 g / L) of PS20 in the sample, indicating higher sensitivity of the lipase assay according to the present invention.

[0165] The inhibition of the lipase assay was expected because of the correlation it shows between the hydrolysis activity of 4-MUD detected in the assay and the degradation of polysorbate. However, for the measurement of lipase activity to be possible even in pharmaceuticals, it is advantageous that concentrations typically applied to antibody formulations, such as from 0.1 g / L to 0.4 g / L, do not interfere with the assay or only slightly interfere.

[0166] Overall, the data indicate that in 4-MUD experiments analyzing samples containing polysorbate concentrations commonly used for formulated active pharmaceutical ingredients, no significant effect on activity is expected.

[0167] Example 8: Influence of other detergents on lipase activity Jahn et al. (Pharm. Res., 2020, 37:118, pages 1-13) reported an inactivation effect on PPL activity caused by the presence of surfactants, especially Triton X-100. Therefore, the hydrolysis activities of PPL (0.024 mg / mL in the reaction mixture), as well as BDS B and BDS E (2.4 mg / mL in the reaction mixture), were measured in an ATM buffer at pH 5.5 containing either 10 mM CHAPS, 0.25% Triton X-100, or 0.25% Triton X-100 and 0.125% gum arabic. The results shown in Figures 10A-C demonstrate that CHAPS performs better than Triton X-100 and Triton X-100 and gum arabic. Gum arabic is an emulsifier, and in no experiment has the influence of gum arabic on activity been observed.

[0168] Example 9: Compatibility of the 4-MUD assay with IPC sample analysis The suitability of the 4-MUD assay for IPC sample analysis was tested for several exemplary processes. Therefore, for in-process control samples from various downstream processing steps, it was analyzed whether the solubility was sufficient, the pH was within the expected range, and the kinetic measurement values met the requirements (pseudo-zero-order reaction rate).

[0169] Table 2 shows the applicability of the processed samples regarding the possible influence of particles, ionic strength, pH, and several other influencing factors. The samples were analyzed three times, and reaction mixtures that did not meet the requirements of the pseudo-zero-order reaction rate were excluded from the analysis.

Table 3

[0170] The suitability of the assay could be demonstrated, and as a result, the assay could be applied to all process steps tested, including the steps following ultrafiltration / diafiltration (UF / DF), and the final formulated bulk drug substance (BDS).

[0171] In a spectrometer (Figure 11A) and a plate reader (Figure 11B), this was further measured for specific antibodies using a lipase assay.

[0172] The results of the 4-MUD plate reader assay generally correlated well with those of the spectrometer. Both readings were able to demonstrate that the lipase activity in the product-containing samples was low compared to the elution buffer alone, and that the lipase activity in the drug substance was low compared to the formulation buffer alone. The 4-MUD assay can be performed in a microtiter plate format for high-throughput purposes and can thus be automated.

[0173] Example 10: Effect of lipase inhibitors There are several substances that can inhibit the enzymatic hydrolysis of ester bonds. Therefore, inhibitors that can reduce the degradation of polysorbates in other processing steps of pharmaceutical, drug substance, and biopharmaceutical development should also inhibit the hydrolysis activity monitored by the 4-MUD assay.

[0174] Therefore, samples were incubated with or without 1 μM orlistat, an irreversible lipase inhibitor (Borgstrom 1988), and tested for stability at pH 5.5 for 2 months at room temperature (about 22°C). A pharmaceutical sample (Product D) containing 0.2 mg / mL of PS20 was incubated at room temperature for up to 56 days with several pull points. The content of PS20 was measured at the indicated time points using the HPLC-CAD method. As shown in Figure 12, 1 μM orlistat resulted in a decreased degradation of PS20 compared to the control reactant (DMSO only).

[0175] The hydrolysis activity of the same pharmaceutical sample (Product D) was measured using a lipase assay in the presence of various concentrations of orlistat (7.3 nM to 20 μM). All measurements were performed in a black 96-well plate using a microplate reader (λ em = 450 nm, λ exUsing a 4-MUD concentration of 30 μM in AMT assay buffer (75 mM acetate, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5) with a top-read mode at 330 nm, as shown in Figure 13, orlistat inhibited lipase activity in a concentration-dependent manner as measured by the 4-MUD assay. The results suggest that orlistat inhibits the hydrolysis activity monitored using the 4-MUD assay (Figure 13) in addition to the hydrolysis activity that causes the degradation of polysorbate (Figure 12). Furthermore, the inhibition was detectable at much lower inhibitor concentrations within minutes rather than over several days using the HPLC-CAD method.

[0176] Example 11: Degradation of polysorbate in spike experiments compared to 4-MUD activity To test the correlation between the hydrolysis activity measured by the 4-MUD assay and the degradation of polysorbate, a polysorbate spike experiment was conducted. To evaluate the degradation rate of polysorbate, in-process control (IPC) samples were spiked with polysorbate, and as a result, the polysorbate content was monitored over time by fluorescence micelle assay (FMA). The IPC samples tested included samples after protein A purification (MabSelect), after depth filtration (Cuno), after ion exchange chromatography (Poros), and the drug substance (BDS).

[0177] The degradation of polysorbate was measured using the FMA assay and compared with the hydrolysis activity measured by a lipase assay (in a 1-cm macro cuvette using a fluorescence spectrometer with λ em = 450 nm and λ ex = 340 nm) using phosphate assay buffer containing 3 μM 4-MUD (81 mM Na2HPO4, 19 mM NaH2PO4, 140 mM NaCl, 10 mM CHAPS, pH 7.4). The results (Figure 14) suggest a correlation between the hydrolysis activity measured by the lipase assay and the degradation of polysorbate in the relevant IPC steps of Product B.

Claims

1. 1. A method for detecting lipase activity in a sample containing a recombinant protein, comprising the steps of: (a) providing at least one sample comprising a recombinant protein produced in a eukaryotic cell; (b) contacting the at least one sample with a reaction solution to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 9; (ii) surfactants that are non-denaturing surfactants that do not have an ester bond and are nonionic or zwitterionic surfactants; and (iii) a substrate comprising the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, wherein the 4-MU ester is a saturated unbranched fatty acid (C ６ ~C １６ ) a substrate which is a 4-MU ester a process comprising: (c) incubating the sample and the substrate in the reaction mixture; and (d) detecting lipase activity by measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU. A method comprising: The method of claim 1, wherein the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), 4-methylumbelliferyl undecanoate, and 4-methylumbelliferyl dodecanoate.

2. The method described in claim 1, wherein the reaction solution further contains (iv) a non-buffering salt.

3. A method according to claim 1 or 2, comprising measuring hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (c).

4. 4. The method of any one of claims 1 to 3, wherein the sample and the substrate in the reaction mixture are incubated for any of the following times: from 2 minutes to less than 5 hours, from 2 minutes to less than 3 hours, from 2 minutes to less than 2 hours, or from 2 minutes to less than 0.5 hours. (a) the final concentration of the surfactant in the reaction mixture is greater than its critical micelle concentration in the reaction mixture; and / or (b) the surfactant is (i) selected from the group consisting of CHAPS, CHAPSO, and Zwittergent; or (ii) CHAPS and provided at a final concentration in the reaction mixture of about 8 mM to about 20 mM; or (iii) is not polyethylene glycol tert-octylphenyl ether (Triton X-100) and is not polyethylene glycol nonylphenyl ether (NP-40); The method according to any one of claims 1 to 4.

6. The method described in claim 5, wherein the surfactant is CHAPS.

7. The method of claim 5, wherein the surfactant is CHAPS and is provided at a final concentration in the reaction mixture of about 8 mM to about 15 mM.

8. The method of claim 5, wherein the surfactant is CHAPS and is provided at a final concentration in the reaction mixture of about 10 mM.

9. The buffer solution may contain formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MES (2-(N-morpholino)ethanesulfonic acid), Tris base, Tris, Bis-Tris, Bis-Tris-propane, Bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Tricine (N-tris(hydroxymethyl)methylglycine), Na ２ HPO ４ and NaH ２ P.O. ４ The method of any one of claims 1 to 8, further comprising one or more buffer substances selected from the group consisting of:

10. The buffer solution (a) having a pH of about 5 to about 7.5; and / or (b) a multi-component buffer having a buffering range of at least about pH 5 to at least about pH 7.5; The method according to any one of claims 1 to 9.

11. The method described in claim 10, wherein in (a), the buffer solution has a pH of about 5.5 to about 7.

5.

12. The method described in claim 10, wherein in (b), the buffer is a multi-component buffer having a buffering range of at least about pH 4 to at least about pH 8. (a) The non-buffering salts are NaCl, KCl, and CaCl. ２ and / or (b) the non-buffering salt has a concentration in the reaction mixture of about 100 mM to about 200 mM; and / or (c) the ionic strength of the non-buffering salt is about 200 mM or less in the reaction mixture; and / or (d) the cumulative ionic strength of the buffer and the non-buffering salt is less than or equal to about 450 mM in the reaction mixture; The method according to any one of claims 1 to 12.

14. The method of claim 13, wherein in (a), the non-buffering salt is NaCl or KCl.

15. The method of claim 13, wherein in (b), the non-buffering salt has a concentration of about 130 mM to about 170 mM in the reaction mixture.

16. The method described in claim 13, wherein in (b), the non-buffering salt has a concentration of about 140 mM to about 150 mM in the reaction mixture.

17. The method described in claim 13, wherein in (c), the ionic strength of the non-buffering salt is less than about 150 mM in the reaction mixture.

18. The method described in claim 13, wherein in (d), the cumulative ionic strength of the buffer and the non-buffering salt is less than about 400 mM in the reaction mixture.

19. The method described in claim 13, wherein in (d), the cumulative ionic strength of the buffer and the non-buffering salt is less than about 350 mM in the reaction mixture.

20. (a) the at least one sample is a harvested cell culture fluid (HCCF), an in-process control (IPC) sample, an active pharmaceutical ingredient sample, or a drug product sample; and / or (b) the recombinant protein is not a lipase and / or is not an enzyme with lipase activity; and / or (c) the recombinant protein is selected from the group consisting of an antibody, an antibody fragment, an antibody-derived molecule, and a fusion protein; The method according to any one of claims 1 to 19.

21. A method for producing a recombinant protein of interest, comprising the steps of: (i) culturing eukaryotic cells expressing the recombinant protein of interest in cell culture; (ii) harvesting the recombinant protein; (iii) purifying the recombinant protein; and (iv) obtaining at least one sample comprising said recombinant protein in step (ii) and / or (iii). A method comprising: The method further comprises detecting lipase activity in a sample containing the recombinant protein, the step comprising the steps of: (a) providing at least one sample comprising the recombinant protein produced in the eukaryotic cell of step (i); (b) contacting the at least one sample with a reaction solution to form a reaction mixture, the reaction solution comprising: (1) a buffer solution having a pH of about pH 4 to about pH 9; (2) A surfactant that is a non-modified surfactant that does not have an ester bond and is a nonionic or zwitterionic surfactant; and (3) A substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, wherein the 4-MU ester is a saturated unbranched fatty acid (C ６ ~C １６ ) a substrate which is a 4-MU ester a process comprising: (c) incubating the sample and the substrate in the reaction mixture; and (d) detecting lipase activity by measuring the hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU. A method comprising: The method of claim 1, wherein the substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), 4-methylumbelliferyl undecanoate, and 4-methylumbelliferyl dodecanoate.

22. The process of claim 1, wherein: (i) culturing eukaryotic cells expressing the recombinant protein of interest in cell culture; (ii) harvesting the recombinant protein; (iii) purifying the recombinant protein; (iv) formulating the recombinant protein into a pharmaceutically acceptable formulation suitable for administration; and (v) obtaining at least one sample comprising said recombinant protein in steps (ii) and / or (iii) and / or (iv).

22. The method of claim 21, comprising:

23. The method described in claim 21 or 22, wherein the surfactant is not polyethylene glycol tert-octylphenyl ether (Triton X-100) and is not polyethylene glycol nonylphenyl ether (NP-40).

24. A method described in any one of claims 21 to 23, wherein the reaction solution further contains (4) a non-buffering salt.

25. A method according to any one of claims 21 to 24, comprising measuring hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (c).

26. at least one sample containing said recombinant protein, step (ii), wherein the sample is a harvested cell culture fluid (HCCF) or a cell lysate; step (iii), wherein the sample is an in-process control (IPC) sample; and / or Step (iv), wherein the sample is a drug substance sample or a drug product sample. obtaining The method according to any one of claims 21 to 25.

27. The method of claim 26, comprising obtaining at least one sample comprising the recombinant protein in step (iii), wherein step (iii) comprises obtaining at least one sample before or after affinity chromatography, before or after acid treatment, before or after depth filtration, and / or before or after ion exchange chromatography.

28. The method described in claim 27, wherein the ion exchange chromatography is anion exchange chromatography or cation exchange chromatography.

29. 29. The method of any one of claims 1 to 28, wherein the lipase activity detected in the at least one sample is a contaminating lipase activity.

30. 1. A kit for measuring contaminating lipase activity in a sample containing a recombinant protein, comprising: (i) a buffer having a pH of about pH 4 to about pH 9; (ii) a surfactant that is a non-denaturing surfactant that does not have an ester bond and is a nonionic or zwitterionic surfactant; and (iii) a substrate comprising the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, wherein the 4-MU ester is a saturated unbranched fatty acid (C ６ ~C １６ ) a substrate which is a 4-MU ester A kit comprising: The substrate is selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), 4-methylumbelliferyl undecanoate, and 4-methylumbelliferyl dodecanoate.

31. The kit of claim 30, further comprising (iv) a non-buffering salt and / or (v) water for dilution.

32. A kit described in claim 30 or 31, further comprising an organic solvent for dissolving the substrate.

33. (a) the surfactant is selected from the group consisting of CHAPS, CHAPSO, Zwittergent, and saponin; (b) the surfactant is not polyethylene glycol tert-octylphenyl ether (Triton X-100) and is not polyethylene glycol nonylphenyl ether (NP-40); and / or (c) The buffer solution is selected from the group consisting of formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl]amino}ethanesulfonic acid), MOPS (3-(N-morpholino)propanesulfonic acid), PIPES (piperazine-N,N'-bis(2-ethanesulfonic acid)), MES (2-(N-morpholino)ethanesulfonic acid), Tris base, Tris, Bis-Tris, Bis-Tris-propane, Bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Tricine (N-tris(hydroxymethyl)methylglycine), Na ２ HPO ４ and NaH ２ P.O. ４ comprising one or more buffer substances selected from the group consisting of The kit according to any one of claims 30 to 32.

34. The kit described in claim 33, wherein in (a), the surfactant is CHAPS.

35. (a) the buffer has a pH of about 5 to about 7.5; (b) the buffer is a multi-component buffer having a buffering range of at least about pH 5 to at least about pH 7.5; and / or (c) the non-buffering salts are NaCl, KCl, and CaCl ２ selected from the group consisting of The kit according to any one of claims 30 to 34.

36. The kit described in claim 35, wherein in (a), the buffer solution has a pH of about 5.5 to about 7.

5.

37. The kit described in claim 35, wherein in (b), the buffer is a multi-component buffer having a buffering range of at least about pH 4 to at least about pH 8.

38. The kit described in claim 35, wherein in (c), the non-buffering salt is NaCl or KCl.

39. (a) the kit further comprises one or more microtiter plates having 96 wells or a multiple of 96 wells; and / or (b) the buffer and the surfactant are premixed as an assay buffer at about 3 to about 5 times the concentration of the final reaction mixture and / or are provided as a dry mix; The kit according to any one of claims 30 to 38.

40. (a) the kit further comprises one or more microtiter plates having 96 wells or a multiple of 96 wells; and / or (b) the buffer, the surfactant, and the non-buffering salt are premixed as an assay buffer at about 3-fold to about 5-fold concentration relative to the final reaction mixture and / or provided as a dry mix; 32. The kit of claim 31.

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