Methods for detecting contaminating lipase activity

Abstract

The present invention provides a method for detecting contaminating lipase activity in a sample of recombinant protein by measuring the hydrolysis of 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 (C6-C 16 ) 4-MU esters. Additionally, a kit for measuring contaminating lipase activity in a sample of recombinant protein is provided.

Description

[Technical field]

[0001] The present invention relates to a method for detecting contaminating lipase activity in a sample of recombinant protein. More specifically, the method comprises the steps of contacting at least one sample (such as an IPC sample) with a reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 9; (ii) a non-denaturing detergent having no ester bonds, said detergent being a non-ionic or zwitterionic detergent; (iii) a substrate comprising 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-buffering salt; and detecting the contaminating lipase activity by measuring hydrolysis of the 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, comprising: (i) a buffer having a pH of about 4 to about 9; (ii) a non-denaturing detergent having no ester bonds, said detergent being a nonionic or zwitterionic detergent; and (iii) a substrate comprising 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 technology]

[0002] Proteins as therapeutic agents have become increasingly common in recent 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 surfactants. 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®) or polysorbate 80 (polyoxyethylene (20) sorbitan monooleate, Tween 80®).

[0003] Polysorbates are heterogeneous mixtures of sorbitol and its anhydrides with about 20 polymerized ethylene oxide moieties partially esterified with fatty acids. However, polysorbates are susceptible to degradation, which can adversely affect product quality. Degradation can affect product quality not only through the resulting reduced polysorbate concentration in the formulation, but also through the formation of visible and sub-visible particles from insoluble polysorbate decomposition products (such as fatty acids and polyoxyethylene side chains). Polysorbates can be degraded chemically or enzymatically. Chemical polysorbate degradation is mainly caused by oxidation reactions, which result in the generation of aldehydes, ketones, and fatty acids, among others. Enzymatic polysorbate degradation is characterized by hydrolysis of the ester bonds linking polyethoxylated sorbitan to fatty acids (Dwivedi et al., 2018, International Journal of Pharmaceutics 552:442-436). Although oxidative degradation of polysorbates has been known for a long time, enzymatic hydrolysis of polysorbates in antibody formulations has only recently been considered as one of the major degradation pathways.In recent years, polysorbate degradation has emerged as a major challenge in the biopharmaceutical industry.

[0004] It has been reported that residual hydrolytic activity of lipases or other enzymes of host cell proteins (HCPs) contained in the final drug product (DP) can lead to the degradation of polysorbates. The role of lipases in the degradation of polysorbates in antibody formulations has been further highlighted by Chiu et al., where cell culture fluid (HCCF) harvested from lipoprotein lipase (LPL) knockout CHO cells reduced the degradation of PS20 and PS80 compared to wild type (Chui et al., 2017, Biotechnol. Bioeng. 114, 1006-1015). As it takes weeks to measure the impact of individual purification steps and conditions on polysorbate degradation, it is difficult to make the necessary changes and adaptations to 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 reversed-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 that allow the measurement of the content of polysorbates consist of fluorescent micellar assay (FMA) or chemical complexation of the sorbitan ring with cobalt thiocyanate or iron(III) thiocyanate. However, to determine whether the changes in the purification process are successful in terms of 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. Thus, 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 weeks or months. Moreover, the analysis is complex and time-consuming.

[0006] In order to develop purification conditions that minimize the enzymatic degradation of polysorbates in said pharmaceutical products, there is a need for a sensitive, rapid, reliable, automated, high-throughput assay that can be easily adapted to various samples and provides predictive information about the hydrolytic activity responsible for the degradation of polysorbates in drug substances of pharmaceutical samples. Such an assay is useful as a tool to guide process development for producing drug substances with improved product quality due to the minimized degradative activity of polysorbates co-purified with the target protein.

[0007] Although detection of lipase hydrolytic activity in vitro using fluorescent substrates is known in the art, these prior art assays are not sensitive enough to reliably detect contaminating lipase activity in recombinant protein preparations (which are merely co-purified from eukaryotic cells with the recombinant protein (protein of interest)) in a short time. For example, Tsuzuki et al. (Biosci. Biotechnol. Biochem, 2001, 65(9): 2078-2082) analyzed the activity of several lipases from microorganisms at high concentrations using fluorescent substrates and found that DMSO increases the hydrolysis of highly hydrophobic substrates of certain lipases. DMSO is an organic solvent commonly used to solubilize substrates that is not a micelle-forming detergent. Sulciene et al. (Acta Paediatrica, supplement, 2018, 116:1049-1055) disclose the use of immobilized lipolytic enzymes from yeast to produce epoxidized oils and describe the detection of lipase activity of these concentrated lipase-nanoparticle conjugates using a fluorescent substrate, but do not disclose the exact conditions. Similarly, Yoo et al. (Cell Chemical Biology, 2020, 27: 143-157) disclose a fluorescent substrate assay for detecting lipase activity, using TritonX-100 to solubilize high concentrations of lipase rPfMAGLLP prior to analysis, but not as part of the reaction solution. WO 2010 / 024924 discloses an assay for screening lipases expressed in E. coli using a fluorescent substrate, but again, the assay is not used to detect contaminating lipase activity in recombinant protein samples purified from eukaryotic cells. None of these prior art assays yet measure 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) report a lipase activity assay to detect lipase activity of defined enzyme extracts of Candida rugosa lipase (CRL) isoforms using 4-methylumbelliferyl butyrate (4-MUB) and palmitate (4-MUP) as substrates to verify the mechanism of action of the inhibitor tropolone. Limitations to the assay are reported, including the inherent decrease in solubility with length of the hydrophobic fatty acid tails, and the autocatalysis of the substrates in the basic pH range. Additionally, no detergents are used in the assay. More recently, Jahn et al., 2020 (Pharm. Res. 37(118): 2-13) reported a chromophore-based lipase activity assay using 4-methylumbelliferyl oleate (4-MuO) as a substrate for use in measuring polysorbate degradation in harvested cell culture fluid samples. However, it has only moderate sensitivity and still requires incubation times of 24 hours or more.

[0009] Therefore, there remains a need for improved, sensitive methods for the rapid measurement of lipase activity in relevant samples. Summary of the Invention

[0010] The present invention relates to a method for detecting (contaminating) lipase activity in a sample comprising a recombinant protein, the method comprising the steps of: (a) providing at least one sample comprising 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 about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bonds, said 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, said 4-MU ester being a saturated unbranched fatty acid (C6-C 16) a 4-MU ester), and (iv) optionally comprising a non-buffering salt; (c) incubating the sample and the substrate in the reaction mixture; and (d) measuring hydrolysis of the 4-MU ester and detecting (contaminating) lipase activity by 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 substrate in the reaction mixture according to step (c). The method should be understood to refer to an in vitro method. In a particular embodiment, the sample and substrate in the reaction mixture are incubated for a time between 2 minutes and less than 5 hours, and 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 a contaminating lipase activity and / or originates from at least one contaminating protein with lipase activity, such as a host cell protein (HCP) from a eukaryotic cell.

[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 non-ionic or zwitterionic surfactant may be CHAPS, CHAPSO, Zwittergent (such as Zwittergent 3-12) or saponin. Preferably, the non-denaturing non-ionic or zwitterionic surfactant is CHAPS. CHAPS may be provided at a final concentration in the reaction mixture of about 8 mM to about 20 mM, preferably about 8 mM to about 15 mM, more preferably about 10 mM. Suitable buffers include 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-hydroxymethyl)methyl)amino, and the like. Preferably, the buffer comprises one or more buffer substances selected from the group consisting of N-tris(hydroxymethyl)methylglycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Tricine (N-tris(hydroxymethyl)methylglycine), NaHPO and NaHPO. Preferably, the buffer has a pH of about 5 to about 7.5, more preferably, the buffer has a pH of about 5.5 to about 7.5.In a particular embodiment, the buffer is a multicomponent 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] The optional non-buffering salt 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, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM in the reaction mixture. Alternatively, or in addition, 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 relates to 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 said recombinant protein; (iii) purifying said recombinant protein; and (iv) optionally formulating said recombinant protein into a pharma- ceutically acceptable formulation suitable for administration; and (v) in steps (ii), (iii) and / or (iv), obtaining at least one sample comprising said recombinant protein, said method comprising detecting a (contaminating) lipase in a sample comprising said recombinant protein. (a) providing the at least one sample comprising the recombinant protein produced in a eukaryotic cell of 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 about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bonds, 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 16) a 4-MU ester), and (iv) optionally a non-buffering salt; (c) incubating the sample and substrate in a reaction mixture; (d) detecting (contaminating) lipase activity by measuring hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; optionally detecting the fluorescence intensity of the released chromophore 4-MU over time while the sample and substrate are incubated in the reaction mixture according to step (c). In a particular embodiment, the method comprises obtaining at least one sample comprising said recombinant protein in step (ii), wherein said sample is a harvested cell culture fluid (HCCF) or a cell lysate; step (iii), wherein said sample is an in-process control (IPC) sample; and / or step (iv), wherein said sample is a drug substance sample or a drug product sample, preferably comprising obtaining at least one sample comprising said recombinant protein in step (iii), wherein step (iii) comprises obtaining at least one sample before or after affinity chromatography and / or before or after acid treatment, before or after depth filtration following acid treatment, and / or before or after ion exchange chromatography, preferably anion exchange chromatography or cation exchange chromatography.

[0014] In another aspect, the present invention provides 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 about pH 4 to about pH 9; (ii) a non-denaturing detergent having no ester bonds, said detergent being a non-ionic or zwitterionic detergent; (iii) a substrate comprising the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, said substrate being a saturated unbranched fatty acid (C6 to C8) 16) a 4-MU ester), and (iv) optionally a non-buffering salt, and / or (v) optionally water for dilution. In certain embodiments, the buffer, the surfactant, and the optional non-buffering salt are premixed as an assay buffer. Preferably, the assay buffer is at least about 3-fold concentrated, or about 3-fold to about 5-fold concentrated, relative to the final reaction mixture. Alternatively, the assay buffer is provided as a dry mix. Such a dry mix may be reconstituted with water to provide the assay buffer at least about 3-fold concentrated, or about 5-fold concentrated, relative to the final reaction mixture. Alternatively or additionally, the buffer, the surfactant, the substrate, and the optional non-buffering salt are premixed and added to the sample as a master mix, and the master mix is ​​provided at about 80% (v / v) to about 70% (v / v) of the reaction mixture, preferably at 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 the substrate dissolved in the organic solvent, and / or one or more microtiter plates having 96 wells or a multiple of 96 wells. The non-denaturing non-ionic or zwitterionic detergent may be CHAPS, CHAPSO, Zwittergent (such as Zwittergent 3-12) and saponin. Preferably, the non-denaturing non-ionic or zwitterionic detergent is CHAPS. In certain embodiments, the buffer 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}ethanesulfonic acid), The buffer solution preferably comprises one or more buffering substances selected from the group consisting of N-tris(hydroxymethyl)methylamino-propanesulfonic acid), Tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4 and NaH2PO4. Preferably, the buffer has a pH of about 5 to about 7.5, more preferably, the buffer has a pH of about 5.5 to about 7.5. In certain embodiments, the buffer is a multi-component buffer having a buffer 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 salts may be, for example, NaCl, KCl and CaCl2, preferably NaCl or KCl. [Brief description of the drawings]

[0016] [Figure 1] The hydrolysis reaction of the substrate 4-MUD is shown. [Diagram 2] Detection of lipase activity in various bulk drug substances (BDS) at variable pH. The hydrolytic activity of bulk drug substances A-B and D-E (monoclonal antibody and antibody-like formats) samples was measured at various pH between pH 4 and pH 8 as indicated. All measurements were performed in black 96-well plates with 30 μM 4-MUD in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5) using a microplate reader (lambda em = 450 nm, lambda ex = 330 nm, top read mode). [Diagram 3] Solubility of 4-MUD in assay buffer. The solubility of 0 to 60 μM 4-MUD was tested using light scattering experiments in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 4-8) in a black 96-well plate using a microplate reader (λem=450 nm, λex=330 nm, top read mode). [Figure 4] Michaelis-Menten kinetics of hydrolytic activity in bulk drug substance (BDSD). Assays were performed in black 96-well plates with varying concentrations of 4-MUD (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) using a microplate reader (λem=450 nm, λex=330 nm, top read mode). [Diagram 5] The solubility of 4-MUD was tested using light scattering experiments in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 20 mM CHAPS, pH 7) in a 1 cm macrocuvette using a fluorescence spectrometer (λem=400 nm, λex=400 nm). [Figure 6] Autohydrolysis of 4-MUB and 4-MUD. Fluorescence was monitored for 1800 seconds at 30 μM 4-MUB or 4-MUD, respectively, in AMT assay buffer (75 mM acetate, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5) using a microplate reader (λem=450 nm, λex=330 nm, top read mode) in black 96-well plates. [Figure 7] Effect of CHAPS on lipase activity in bulk drug substance (BDS). (A) Hydrolytic activity of BDS G samples and buffer controls was measured with and without CHAPS and 4-MU (μM) released over time is provided. (B) Hydrolytic activity of BDS B, BDS G and BDS F (monoclonal antibody and antibody-like formats) samples was measured with and without CHAPS. Activity normalized to hydrolytic activity with CHAPS for blank subtracted data for each product is provided. Additionally, a representative IPC sample after UFDF / Diafiltration (UFDF Product D) is shown. All measurements were performed in black 96-well plates with 30 μM 4-MUD in AMT assay buffer (75 mM acetate, 75 mM MES, 150 mM Tris, 150 mM NaCl, pH 5.5) with or without 10 mM CHAPS using a microplate reader (λem=450 nm, λex=330 nm, bottom read mode). [Figure 8] Dependence of hydrolytic activity on ionic strength. The hydrolytic activity of an exemplary drug product (Product E) sample was measured in the presence of various concentrations of NaCl (7.8125 mM to 1000 mM). All measurements were performed in black 96-well plates with 30 μM 4-MUD in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5) using a microplate reader (λem=450 nm, λex=330 nm, top read mode). [Figure 9] Competitive inhibition of hydrolytic activity by PS20. Hydrolytic activity of pharmaceutical samples was measured in the presence of various concentrations of PS20 (0.0125 mg / mL to 3.2 mg / mL). All measurements were performed in black 96-well plates with 30 μM 4-MUD in AMT assay buffer (50 mM acetic acid, 50 mM MES, 100 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5) using a microplate reader (λem=450 nm, λex=330 nm, top read mode). [Figure 10] Comparison of hydrolytic activity using CHAPS, TritonX-100, or TritonX-100 and gum arabic in assay buffer. The hydrolytic activity of PPL (A), bulk drug substance (BDS) B (B), and BDS E (C) at 0.024 mg / mL PPL or 2.4 mg / mL BDS in the reaction mixture was measured under standard conditions (AMT buffer, 5.5) in either 10 mM CHAPS (top black line), 0.25% TritonX-100 (middle light gray line), or 0.25% TritonX-100 and 0.125% gum arabic (bottom dark gray line). [Figure 11]Comparison of lipase activity in various IPC samples of a product (monoclonal antibody) measured using a fluorescence spectrometer (A) and a microplate reader (B). All measurements were performed using phosphate assay buffer (81 mM Na2HPO4, 19 mM NaH2PO4, 140 mM NaCl, 10 mM CHAPS, pH 7.4) containing 3 μM 4-MUD. The fluorescence spectrometer used a 1 cm macro cuvette and the microplate reader used a black 96-well plate (top read mode), both with λem=450 nm and λex=340 nm. The various IPC samples are as follows: Protein A elution buffer (ProtA buffer) and product pool (Prot A Prod P), neutralized acid treatment product pool (Neutral.AT Prod Pool), depth filtration product pool (Depth.filtr.Prod P), cation exchange chromatography buffer (CIEX buffer) and product pool (CIEX Prod P), virus filtration product pool (Virus filtr.Prod P), 30kD ultrafiltration / diafiltration buffer (UF / DF buffer) and product pool (UF / DF Prod P), formulation buffer, and drug substance (DS). [Figure 12] Inhibition of polysorbate degradation by orlistat. Drug samples containing 0.2 mg / mL PS20 (product D) were incubated at room temperature for up to 56 days with several pull points. Residual PS20 content was measured using the HPLC-CAD method. 1 μM orlistat resulted in reduced PS20 degradation compared to the control reaction (DMSO only). [Figure 13]Inhibition of hydrolytic activity by orlistat. The hydrolytic activity of a drug sample (product D) was measured in the presence of various concentrations of orlistat (7.3 nM to 20 μM). All measurements were performed in black 96-well plates with 30 μM 4-MUD in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5) using a microplate reader (lambda em = 450 nm, lambda ex = 330 nm, top read mode). The results suggest that orlistat inhibits the hydrolytic activity responsible for the degradation of polysorbates, as well as the hydrolytic activity monitored using the 4-MUD assay. [Figure 14] Comparison of PS degradation in IPC samples spiked with PS (product B) and hydrolytic activity measured with the 4-MUD assay. PS degradation was measured using the FMA assay; hydrolytic activity (4-MUD assay) was performed in 1 cm macrocuvets using a fluorescence spectrometer (lambda em = 450 nm, lambda ex = 340 nm) with phosphate assay buffer (100 mM NaHPO4, 140 mM NaCl, 10 mM CHAPS, pH 7.4) containing 3 μM 4-MUD. The various IPC samples are as follows: Protein A column (MabSelect), depth filtration product (Cuno), cation exchange chromatography (Poros), bulk drug substance (BDS).

[0017] Detailed Description The general embodiments "including" or "including" encompass the more specific embodiment "consisting of." Furthermore, the singular and plural forms are not used in a limiting sense. As used herein, the singular forms "a," "an," and "the" designate both the singular and the plural, unless expressly stated to designate only the singular.

[0018] The term "sample" as used herein refers to any sample containing recombinant protein, said recombinant protein being 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 drug substance (also referred to herein as bulk drug substance) sample, or a pharmaceutical sample containing recombinant protein, such as an antibody, an antibody fragment, an antibody-derived molecule, or a fusion protein (e.g., 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 a contaminating lipase activity and / or is derived from and is at least one contaminating protein with lipase activity, such as a host cell protein (HCP) derived from a eukaryotic cell.

[0019] The term "contamination" or "contamination" as used herein refers to the presence of undesired and / or unintended substances (e.g. at least one protein or substance with lipolytic and / or hydrolytic activity (e.g. lipase activity) associated with host cell proteins) compared to other predominantly produced substances with non-lipolytic activity (such as the protein of interest) or that may be considered as only a minor component of a medical treatment protein (e.g. an antibody or antibody-like compound). In the context of the present invention, hydrolytic activity, especially lipase activity, is undesired due to its polysorbate degrading ability that may be co-purified with the recombinant protein. This applies in particular to the final formulated protein preparation, which preferably contains less than 1% (w / w), preferably less than 0.1% (w / w), more preferably less than 0.01% (w / w) of such undesired factors 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 ester bonds in lipids (such as fatty acid esters). Lipases are hydrolases that break down esters into acids and alcohols in a chemical reaction with water, also called hydrolysis. Lipases include, for example, carboxylesterases (EC 3.1.1.1), triacylglycerol lipases (EC 3.1.1.3), phospholipase A2 (EC 3.1.1.4), lysophospholipases (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); phospholipase D (EC 3.1.4.4), phosphoinositide phospholipase C (EC 3.1.4.11), glycosylphosphatidylinositol It may be a phosphodiester hydrolase (EC 3.1.4), such as a phospholipase D (EC 3.1.4.50) or an N-acetylphosphatidylethanolamine-hydrolyzing phospholipase D (EC 3.1.4.54); or a glycosphingolipid 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 (e.g., fusion to other proteins, substitutions, deletions or insertions of amino acid sequences) 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] The term "recombinant protein" as used herein relates to a protein produced by recombinant techniques such as molecular cloning, and may be referred to as a recombinant protein of interest. As used herein, said recombinant protein is a protein of interest, for example in a sample to be purified. Such methods bring together genetic material from multiple sources or create sequences that do not occur in nature. Recombinant proteins are typically based on sequences from different cells or organisms, or from different species, than the recipient host cells used to produce the protein in cell culture (e.g. CHO cells or HEK293 cells), or on artificial sequences such as fusion proteins. In the context of the present invention, said recombinant protein is a protein of interest, preferably a therapeutic protein, such as an antibody, an antibody fragment, an antibody-derived molecule (e.g. scFv, bi- or multispecific antibodies) or a fusion protein (e.g. Fc-fusion protein). Thus, in one embodiment, said recombinant protein is selected from the group consisting of an antibody, an antibody fragment, an antibody-derived molecule and a fusion protein.

[0023] The term "eukaryotic cell" as used herein refers to a cell having a nucleus within a nuclear envelope, and includes animal cells, human cells, plant cells and yeast cells. In the present invention, "eukaryotic cell" specifically encompasses 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 an active pharmaceutical ingredient (API) formulated with excipients. The API has a therapeutic effect in the body, as opposed to the excipients which aid in the delivery of the API. For biological therapeutics, the API formulated with excipients typically refers to the API in the buffer of the final formulation, at least at the highest concentration used in the final dosage form, also referred to as the drug product.

[0025] As used herein, the term "drug product", abbreviated as DP, refers to the final marketable dosage form of a drug substance, e.g., a tablet or capsule, or, in the case of a biological product dosage form, typically an injectable solution in a suitable container, such as a vial or syringe. The drug product may also be in lyophilized form.

[0026] The term "polysorbate 20" as used herein refers to a non-ionic polysorbate-type surfactant derived from polyethoxylated sorbitan and lauric acid (polyoxyethylene (20) sorbitan monolaurate). It is also known as Tween 20. Its stability and relative non-toxicity allow it to 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. It can also be used in pharmacological applications such as pharmaceutical formulations, especially biological preparations such as antibodies and Fc fusion proteins. It is particularly useful in preventing non-specific antibody binding.

[0027] The term "polysorbate 80" as used herein refers to a non-ionic polysorbate-type surfactant derived from polyethoxylated sorbitan and oleic acid (polyoxyethylene (20) sorbitan monooleate). It is also known as Tween 80 and has similar uses as polysorbate 20.

[0028] The term "therapeutic protein" as used herein refers to proteins that can be used for the medical treatment of humans and / or animals, including, but not limited to, antibodies, growth factors, blood clotting factors, vaccines, interferons, hormones and fusion proteins.

[0029] The term "produced" as used herein relates to the production of a recombinant protein, preferably a therapeutic protein, in a eukaryotic cell, preferably a yeast cell or a mammalian cell, in a cell culture. The skilled person knows how to produce a recombinant protein in a cell using fermentation. The production of a recombinant protein comprises culturing a eukaryotic cell expressing a recombinant protein of interest in a cell culture. Culturing a eukaryotic cell expressing a recombinant protein in a cell culture comprises maintaining the eukaryotic cell in a suitable medium and under conditions allowing the growth and / or production / expression of the protein. Said recombinant protein may be produced by fed-batch or continuous cell culture. Thus, the eukaryotic cell may be cultured in fed-batch 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 that contains a DNA sequence encoding a recombinant protein that is transcribed and translated into a protein sequence including post-translational modifications, i.e., resulting in the production of the recombinant protein in cell culture.

[0031] As used herein, the term "about" refers to a 10% variation of the specified value, for example, about 50% would have a variation of 45 to 55%.

[0032] Methods for detecting lipase activity The present invention relates to an (in vitro) method for detecting lipase activity in a sample comprising a recombinant protein, the method comprising the steps of: (a) providing at least one sample comprising 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 about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bonds, said 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, said 4-MU ester being a saturated unbranched fatty acid (C6-C 16(c) incubating the sample and the substrate in the reaction mixture; (d) detecting lipase activity by measuring hydrolysis of 4-MU ester (the substrate) and detecting the fluorescence intensity of the released chromophore 4-MU (which is the hydrolysis product of 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 hydrolysis for the at least one sample. The reaction solution used in the method of the present invention is an aqueous reaction solution. The skilled person will also understand that the at least one sample comprises a recombinant protein produced in eukaryotic cells in cell culture. Furthermore, the method according to the invention is for detecting a contaminating lipase activity, wherein the lipase activity detected in step (d) is a contaminating lipase activity in said at least one sample comprising said recombinant protein, more particularly a recombinant protein of interest.

[0033] The readout of the assay may be as fast as 20 minutes or even faster. 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 between 2 minutes and less than 5 hours, between 2 minutes and less than 3 hours, between 2 minutes and less than 2 hours, or between 2 minutes and less than 0.5 hours. The at least one sample may be an HCCF, an in-process control (IPC) sample, a drug substance, or a drug product. According to the present invention, the recombinant protein in the sample is not a lipase and / or does not contain lipase activity. Furthermore, the recombinant protein in the sample according to the method of the present invention is not an esterase or hydrolase and / or does not contain esterase or hydrolase activity. Thus, any lipase activity detected in the at least one sample is a contaminating lipase activity and / or originates from at least one contaminating protein having lipase activity, such as one or more host cell proteins (HCPs) from eukaryotic cells. Thus, the at least one sample containing a recombinant protein produced in a eukaryotic cell may potentially further contain at least one contaminating protein having lipase activity. In one embodiment, the method comprises, in step (a), providing at least one sample containing a recombinant protein and a host cell protein (HCP) produced in a eukaryotic, preferably mammalian, cell culture; and, in step (d), detecting the lipase activity of the HCP by measuring hydrolysis of 4-MU esters and detecting the fluorescence intensity of the released chromophore 4-MU.

[0034] The substrate binds the chromophore 4-MU to a saturated unbranched fatty acid (C6 to C 16 ) in the form of 4-MU ester (the acyl chain of the saturated unbranched fatty acid is C6 to C 16This substrate mimics the crucial ester bond of polysorbates, i.e. the fatty acid ester bond.

[0035] The hydrolysis may be stopped at a certain time point before detecting 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 a particular embodiment, the fluorescence intensity of the released chromophore 4-MU is detected without stopping the hydrolysis of the 4-MU ester. In a particular embodiment, the 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 the hydrolysis to be measured over time, and therefore the specific reaction rate can be measured. In the method according to the invention, the hydrolysis of 4-MU ester in the reaction mixture typically follows pseudo-zero order kinetics. Therefore, detecting fluorescence in real time allows the measurement in the time frame of pseudo-zero order kinetics. Thus, in a particular embodiment, the fluorescence intensity of the released chromophore 4-MU is detected over time and follows pseudo-zero order kinetics. Optionally, reaction mixtures that do not meet the requirements of pseudo-zero order kinetics are excluded from the analysis. Pseudo-zero order kinetics can be evaluated by linear regression analysis. Preferably, samples are run at least three times, and individual reaction mixtures that do not meet the pseudo-zero order kinetics, for example due to air bubbles in the wells, are excluded from the analysis to eliminate outliers. Eliminating outliers as described strongly increases the sensitivity of the analysis. A calibration curve using a defined concentration of 4-MU can be used to calculate the rate of hydrolysis (e.g., nmol / sec). A calibration curve with known 4-MU concentrations further allows for the determination and comparison of reaction rates at different pH values.

[0037] The term "reaction rate" as used herein refers to the rate at which an enzyme converts a substrate into at least one product within a specific time. In some reactions, the rate is apparently independent of the concentrations of the reactants. This means that the rate of the equation is equal to the rate constant k of the reaction, and is called a zero-order reaction. Zero-order kinetics is always an artefact of the conditions under which the reaction is carried out. For this reason, reactions that follow zero-order 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 of released chromophore 4-MU (mol / sec) by comparing it with a calibration curve made by using defined concentrations of 4-MU. Typically, the activity is determined by the release of 4-MU (nmol / min). Alternatively, or in addition, the relative value may be calculated relative to another sample or, preferably, an internal standard, such as 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 at least one potentially contaminating protein with lipase activity to hydrolyze the 4-MU ester. Typically, incubation lasts from a few minutes to a few hours. In one embodiment, 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), i.e. in real time during incubation. Due to the sensitivity of the assay, detection typically begins immediately after step (b). The incubation time and therefore the detection time may depend on the lipase activity present in the sample, and typically does not exceed 5 hours, preferably does 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, about 2 minutes and less than 3 hours, about 2 minutes and less than 2 hours, or about 2 minutes and less than 0.5 hours. Preferably, the sample and substrate in the reaction mixture are incubated for between 20 minutes and 2 hours at a temperature of about 25° C. Since the reaction temperature affects the reaction time, the reaction temperature should be kept constant during the measurement, for example, 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 to 37°C, preferably between 22 to 28°C, more preferably between 24 to 26°C, for less than 5 hours, less than 3 hours, less than 2 hours, or less than 1 hour, or incubated at a constant temperature between 20 to 37°C, preferably between 22 to 28°C, more preferably between 24 to 26°C, for any time between about 2 minutes and less than 5 hours, about 2 minutes and less than 3 hours, about 2 minutes and less than 2 hours, or about 2 minutes and less than 1 hour.

[0040] Substrates containing the chromophore 4-MU are saturated unbranched chain fatty acids (C6-C 16 ) 4-MU ester, and the acyl chain of the saturated unbranched fatty acid is C6-C 16 This substrate mimics the key features of polysorbates, namely the fatty acid ester bond and the long acyl chain. Polysorbate 20 is an ester of the fatty acid lauric acid, which is a saturated unbranched fatty acid. In contrast, Polysorbate 80 is an ester of the fatty acid oleic acid, which is an unsaturated fatty acid.

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

[0042] Furthermore, fatty acid esters with shorter acyl chains provide better solubility in aqueous reaction mixtures compared to fatty acid esters of longer chain length, allowing additional substrate to be used in the assay mixture. 16 Above this chain length, solubility was found to be strongly limited.

[0043] Furthermore, it was found that the decanoic acid ester (4-MUD) offers better resistance to autohydrolysis compared to, for example, the butyric acid ester (4-MUB). Chain lengths up to C5 were found to strongly increase autohydrolysis. 10 The fatty acids found to be optimal for use in the examples were saturated unbranched fatty acids (C6-C 16 ) 4-MU ester, or more preferably saturated unbranched fatty acid (C8-C 12 Slightly longer or shorter saturated unbranched fatty acid esters, such as 4-MU esters, can also be used in the process according to the invention. Thus, the 4-MU esters used in the process according to the invention are 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 to C 12 ) 4-MU ester. In certain embodiments, 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 certain preferred embodiments, 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 100-fold stock solution relative to the concentration in the reaction mixture) in an organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF), preferably DMSO. In certain embodiments, 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 may 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 about 1 μM to about 1 mM, preferably about 1 μM to 300 μM, preferably 1 μM to 30 μM, more preferably 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 about 1% to about 5% (v / v) of the reaction mixture.

[0045] The method according to the invention comprises contacting at least one sample with a reaction solution comprising a non-denaturing surfactant without ester bonds, said surfactant being a non-ionic or zwitterionic surfactant (also referred to herein as a "non-denaturing non-ionic or zwitterionic surfactant without ester bonds"). The term "surfactant" as used herein refers to a surface-active compound capable of forming micelles and reducing 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 hydrophobic groups (tails) and hydrophilic groups (heads). Surfactants are typically organic compounds. In the aqueous phase, surfactants form aggregates such as micelles, where the hydrophobic tails form the core of the aggregates and the hydrophilic heads are in contact with the surrounding aqueous liquid. Thus, the hydrophobic tails (also referred to as hydrophobic hydrocarbon moieties) have a certain length to form micelles. Thus, surfactants as used herein do not encompass organic solvents such as ethanol or dimethyl sulfoxide (DMSO). The tail of most surfactants typically consists of one or more hydrocarbon chains, which can be branched, linear or aromatic. The surfactant may contain one or more hydrophobic tails, preferably the surfactant contains one hydrophobic chain (single-tail surfactants). Surfactants are generally classified according to the hydrophilic head group. A nonionic surfactant has no charged group in its head, an ionic surfactant has one net positive (cationic) or negative (anionic) charge, and a zwitterionic surfactant contains two oppositely charged groups. Thus, a nonionic or zwitterionic surfactant has no net charge in the hydrophilic head group and is milder in nature. Furthermore, in many surfactants, the hydrophobic tail is attached to the hydrophilic head via an ester bond, such as PS20 or PS80. Furthermore, a nonionic or zwitterionic surfactant is a non-denaturing surfactant. As used herein, the term "non-denaturing surfactant" refers to the effect of the surfactant on protein structure. Non-denaturing detergents do not disrupt protein-protein interactions, especially of water-soluble proteins.

[0046] Surfactants containing ester bonds are potential substrates for lipases and may therefore interfere with the assay. Furthermore, denaturation of proteins by lipase activity and therefore interference with lipase activity in the sample should be avoided. Therefore, the surfactants used in the method according to the invention are non-denaturing surfactants that do not have ester bonds, said surfactants being non-ionic or zwitterionic surfactants. Examples of suitable non-denaturing zwitterionic detergents include, but are not limited to, 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, e.g., n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent3-12)), and 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]propanesulfonate or other amidosulfobetaine detergents. Examples of suitable non-denaturing non-ionic surfactants include, but are not limited to, 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 (Brij35) or other polyoxyethylene ethers; saponins (e.g., digitonin), octylphenoxy polyethoxyethanol (IGEPAL CA-630), poloxamer 188,338,407 or tergitol. In certain embodiments, non-denaturing surfactants that do not have an ester bond (nonionic or zwitterionic surfactants) are not ethoxylates and / or do not contain a polyethylene glycol group and / or do not contain an aromatic ring.In a particular embodiment, the non-denaturing nonionic or zwitterionic surfactant without an ester bond is not octoxinol-9, in particular is not polyethylene glycol tert-octylphenyl ether (Triton X-100; CAS number 9002-93-1) and / or polyethylene glycol nonylphenyl ether (NP-40, CAS number 9016-45-9). In a preferred embodiment, the surfactant is a non-denaturing surfactant that does not have an ester bond, the surfactant is a non-ionic or zwitterionic surfactant, more preferably the surfactant is a non-denaturing surfactant (non-ionic or zwitterionic surfactant) 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), preferably CHAPS. None of these exemplary suitable surfactants exhibit ester bonds or acyl chains and therefore are substrates for lipase. These surfactants do not compete with the substrate and therefore do not affect the sensitivity of the assay. Furthermore, the presence of the surfactant mediates the solubility of the substrate at the concentration in water used. One of skill in the art will know how to identify non-denaturing non-ionic or zwitterionic surfactants without additional suitable ester bonds by measuring their effect on 4-MU ester hydrolysis under assay conditions, for example, as shown for PS20 in Example 7.

[0047] The presence of detergents (such as CHAPS 10 mM) has been shown to increase lipase activity and therefore improve the sensitivity of the assay. Without being bound by theory, it is hypothesized that detergents create an environment that promotes lipase activity by allowing the repositioning and opening of a lid or flap that is described to cover 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 detergent should be above its critical micelle concentration (CMC).

[0048] Thus, according to the present invention, the non-denaturing surfactant (non-ionic or zwitterionic) has a final concentration in the reaction mixture that exceeds the critical micelle concentration (CMC). The CMC represents an important physicochemical property of a given surfactant in aqueous solution. Micelles are spherical aggregates whose hydrocarbon groups are largely not in contact with water. The term "critical micelle concentration" or "CMC" as used herein refers to the concentration of a surfactant above which micelles are formed (i.e., the maximum monomer concentration) and can be measured according to methods known in the art. For example, a suitable method for measuring the CMC is the fluorescent micelle assay (FMA), which uses the partitioning of the fluorescent hydrophobic dye N-phenyl-1-naphthylamine (NPN) into surfactant micelles. NPN exhibits a low fluorescence quantum yield in aqueous environments, which increases in more hydrophobic environments such as the core of the micelles. This assay was originally developed for measuring the CMC and has also been used to measure the content of polysorbates in biopharmaceuticals, as shown in the examples. Another method utilizing 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] CMCs for detergents can be derived from the literature, e.g., about 6 mM for CHAPS, about 8 mM for CHAPSO, and about 2-4 mM for Zwittergent 3-12. In a particular embodiment, the non-denaturing zwitterionic detergent is CHAPS and is provided in a final concentration in the reaction mixture of about 8 mM to about 20 mM, preferably about 8 mM to about 15 mM, and more preferably about 10 mM. In another embodiment, the non-denaturing zwitterionic detergent is CHAPSO and is provided in a final concentration in the reaction mixture of about 10 mM to about 20 mM, preferably about 10 mM to about 15 mM. In yet another embodiment, the non-denaturing zwitterionic detergent is Zwittergent 3-12 and is provided in a final concentration in the reaction mixture of about 4 mM to about 10 mM, preferably about 6 mM to about 8 mM. In yet another embodiment, the non-denaturing non-ionic detergent is saponin and is provided at a final concentration in the reaction mixture of about 0.001% to 0.01% (w / v).

[0050] The reaction solution used in the method according to the invention further comprises a buffer having a pH of about pH 4 to about pH 9. Preferably, the method is carried out with a buffer having a pH of about pH 4 to about pH 8, preferably about pH 5 to about pH 7.5, more preferably about pH 5.5 to about pH 7.5. The skilled person will understand that the pH of the buffer is within its buffer range when used in the method of the invention. In principle, any buffer known in the art can be used, provided that the pH has a buffer range of about pH 4 to about pH 9. The buffer may comprise a single buffering substance or may be a multi-component buffer. A multi-component buffer typically has a wider buffer range. For example, the buffer may be 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- The solution may comprise one or more buffer substances selected from the group consisting of 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 about 50 to 400 mM, preferably about 50 to 300 mM, more preferably about 50 to 200 mM.

[0051] The buffer may further be a multi-component buffer comprising multiple buffer substances with overlapping buffer ranges to have a wider buffer range. The buffer may, for example, comprise two, three, four, five or more buffer substances, preferably two or more buffer substances, more preferably three or more buffer substances. For example, the multi-component buffer may comprise two to four buffer substances, three to four buffer substances, more preferably three buffer substances. In a particular embodiment, the multi-component buffer comprises at least three buffer substances with overlapping buffer ranges, preferably comprising at least one of Tris, MES and / or acetate, preferably acetate, MES and Tris in a ratio of 1:1:2.

[0052] The assay was found to be sensitive to ionic strength, so for the design of a suitable multi-component buffer, it is important that it not only contains buffer substances with overlapping buffer ranges, but that said buffer only moderately changes the ionic strength (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, AMT buffers containing acetate, MES and Tris only moderately affect the ionic strength, making it possible to identify conditions, including pH conditions, that reduce the hydrolytic activity using buffers at different pHs. This buffer further allows measurements to be performed at the pH of the sample, measuring the lipase activity at specific conditions present in the sample, as well as comparing the lipase activity at various conditions during purification. The assay allows the sensitivity to be further increased by measuring the sample at its optimum pH.

[0053] Thus, the multi-component buffers disclosed herein allow for the use of buffers with 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 in addition, the use of buffers with 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 less than 5%, such as 0% to less than 15%, 0% to less than 10%, 0% to less than 7.5%, or less than 5%, or such as 2% to less than 15%, 2% to less than 10%, 2% to less than 7.5%, or 2% to less than 5%. In one embodiment, the use of buffers with different 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 less than 5%, such as 0% to less than 15%, 0% to less than 10%, 0% to less than 7.5% or 0% to less than 5%, or such as 2% to less than 15%, 2% to less than 10%, 2% to less than 7.5% or 2% to less than 5%. The use of the multi-component buffers disclosed herein further allows for adjusting 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 allows for adjusting the pH of the buffer to near the optimum of at least one contaminating protein with lipase activity (thereby increasing the sensitivity of the method) and / or for comparing and identifying conditions that reduce hydrolytic activity.

[0054] The reaction solution may further comprise 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 embodiment 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-buffering salt may range from about 100 mM to about 200 mM. In one embodiment of the present invention, the non-buffering salt has 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 in the reaction mixture. However, the ionic strength in the reaction mixture should not exceed a certain value, since it will adversely affect the lipase activity. For example, the ionic strength of the optional non-buffering 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, for example, about 100 mM to about 200 mM, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM in the reaction mixture. In certain embodiments, the cumulative ionic strength of the buffer and non-buffering salts in the reaction mixture does not exceed about 450 mM. Thus, the cumulative ionic strength of the buffer and non-buffering salts 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 non-buffering salts in the reaction mixture may be about 150 mM to about 450 mM or less, about 150 mM to about 400 mM or less, about 150 mM to about 380 mM or less, about 150 mM to about 360 mM or less, or about 150 mM to about 350 mM or less.

[0056] The method according to the invention can be carried out using a fluorescence spectrometer or a microplate spectrophotometer (preferably λ ex 330~340nm, λ emThe reaction mixture is therefore contained in (and preferably mixed in) a cuvette or a microtiter plate, preferably a microtiter plate with at least 96 wells, for measurement. The method according to the invention is therefore particularly suitable for high-throughput and / or automated analysis of samples. In a particular embodiment, in the method according to the invention, at least 2, 3, 4, 5, 10 or more samples are analyzed simultaneously. Furthermore, each sample is preferably measured at least three times. The method according to the invention is therefore preferably carried out using a microtiter plate with 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 for incubating the sample and substrate in the reaction mixture in step (c). In a particular embodiment, the sample is therefore contacted, incubated and measured in a format of a microtiter plate with 96 wells, or a multiple of 96 wells.

[0057] In a particular embodiment, the sample is provided at about 30% (v / v) or less of the reaction mixture, preferably about 25% (v / v) or less. 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. The at least one sample comprising a 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 drug product sample, preferably an IPC sample, a drug substance sample or a drug product sample. Preferably, contacting the at least one sample with the reaction solution to form a reaction mixture comprises mixing the at least one sample with the reaction solution to obtain a homogenous reaction mixture. Preferably, this is done by first adding a smaller volume (typically the sample) and then adding a larger volume (typically the reaction solution). Preferably, the components of the reaction solution are added as a master mix, which may be prepared as a concentrate and diluted to working concentration before addition to the sample.

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

[0059] In certain embodiments, the buffer, non-denaturing detergent (non-ionic or zwitterionic), substrate and optional non-buffering salts are added as a master mix, the master mix being provided at about 70% (v / v) or more, about 75% (v / v) or more, and thus the master mix 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 harvesting, 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 a contaminating lipase activity and / or is derived from at least one contaminating protein with lipase activity, such as a host cell protein (HCP) from a eukaryotic cell. Furthermore, the recombinant protein in the sample according to the method of the present invention is not an esterase or hydrolase and / or does not contain esterase or hydrolase activity.

[0061] Thus, advantageously, the method of the invention can be used to detect lipase activity by measuring hydrolysis in a sample containing an antibody, an antibody fragment, an antibody-derived molecule or a fusion protein (e.g., an Fc-fusion protein). Typically, antibodies are monospecific, but antibodies may also be multispecific. Thus, the method according to the invention may be used on a sample containing a monospecific antibody, a multispecific antibody, or a fragment thereof, preferably an antibody (monospecific), a bispecific antibody, a trispecific antibody, or a fragment thereof, preferably an antigen-binding fragment thereof. Unless otherwise stated, the term "antibody" refers to a monospecific antibody. Exemplary antibodies within the scope of the invention include anti-CD2 antibodies, anti-CD3 antibodies, anti-CD20 antibodies, anti-CD22 antibodies, anti-CD30 antibodies, anti-CD33 antibodies, anti-CD37 antibodies, anti-CD40 antibodies, anti-CD44 antibodies, anti-CD44v 6 antibodies, 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 The antibody may be selected from the group consisting of, but is not limited to, anti-IL-36R, anti-IL-5R, anti-IL-36R or anti-IgE antibodies, and is preferably selected from the group consisting of anti-CD20, anti-CD33, anti-CD37, anti-CD40, anti-CD44, anti-CD52, anti-HER2 / neu (erbB2), anti-EGFR, anti-IGF, anti-VEGF, anti-TNFα, anti-IL-2, anti-IL-36R and anti-IgE antibodies. In one embodiment, the antibody is an anti-IL-36R antibody, in particular spesolimab. In another embodiment, the antibody is not an anti-IL-36R antibody, in particular not spesolimab.

[0062] The term "antibody", "antibody" or "immunoglobulin" as used herein relates to a protein selected from among globulins formed naturally as a response of the host organism to foreign substances (= antigens) from differentiated B-lymphocytes (plasma cells). There are different types of immunoglobulins: IgA, IgD, IgE, IgG, IgM, IgY, IgW. Preferably, said antibody is an IgG antibody, more preferably an IgG1 or IgG4 antibody. In the present specification, the terms immunoglobulin and antibody are used interchangeably. Antibodies include monoclonal antibodies, monospecific and multispecific antibodies (such as bispecific or trispecific antibodies), single chain antibodies, antigen-binding fragments of antibodies (e.g. Fab or F(ab')2 fragments), disulfide-linked Fv, etc. Antibodies may be of any species and include chimeric 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 may be derived from a rat or mouse antibody, and the constant regions from a human antibody. In a "humanized" antibody, only minimal sequences are derived from the non-human species. Often, only the CDR amino acid residues of a human antibody are replaced with CDR amino acid residues of a non-human species, such as mouse, rat, rabbit, or llama. Sometimes, a number of 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, through recombinant or transgenic means, through cell (e.g., hybridoma) culture, or by other means.

[0063] Typically, antibodies are tetrameric polypeptides composed of two pairs of heterodimers formed by heavy and light chains, respectively. Stabilization of both the heterodimer and tetrameric polypeptide structures occurs via interchain disulfide bridges. Each chain is composed of structural domains called "immunoglobulin domains" or "immunoglobulin regions", the terms "domain" or "region" being used interchangeably herein. Each domain contains about 70-110 amino acids and forms a compact three-dimensional structure. Both heavy and light chains contain at the N-terminus a "variable domain" or "variable region" whose sequence is not highly conserved, which is 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. They may be formed from conventional antibodies by protease digestion (e.g., with papain), but Fab fragments may also be produced by genetic engineering as well. 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 generate shortened antibody fragments consisting only of the variable regions of the heavy (VH) and light (VL) chains. These are called Fv fragments (Fragment variable). These Fv fragments are often stabilized because they lack the covalent bond between the two chains by the cysteines of the constant chains. It is advantageous to link the variable regions of the heavy and light chains by a short peptide fragment, for example 10 to 30 amino acids, preferably 15 amino acids. In this way, a single peptide chain is obtained, consisting of VH and VL linked by a peptide linker. 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 in particular scFv.

[0066] In recent years, various strategies have been developed to prepare scFv as multimeric derivatives. This is intended in particular to lead to recombinant antibodies with improved pharmacokinetic and biodistribution properties and increased binding affinity. To achieve multimerization of scFv, scFv have been prepared as fusion proteins with multimerization domains. Said multimerization domains can be, for example, coiled-coil structures (helical structures) such as the CH3 region of IgG or leucine zipper domains. However, there are also strategies that use the interaction between the VH / VL regions of scFv for multimerization (e.g. diabodies, tribodies, pentabodies). For the skilled artisan, diabodies refer to bivalent homodimeric scFv derivatives. Shortening the linker in the scFv molecule to 5-10 amino acids leads to the formation of homodimers in which interchain VH / VL superposition occurs. Diabodies may be further stabilized by the incorporation of disulfide bridges. Examples of diabody antibody proteins are known from the prior art.

[0067] For the skilled artisan, a minibody means a bivalent homodimeric scFv derivative. A minibody consists of a fusion protein containing the CH3 region of an immunoglobulin, preferably an IgG, most preferably an IgG1, as a dimerization region connected to an 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] For those skilled in the art, a tribody refers to a trivalent homotrimeric scFv derivative. An scFv derivative in which VH-VL are directly fused without a linker sequence leads to the formation of trimers.

[0069] The skilled artisan is also familiar with the so-called mini-antibodies, which have a bi-, tri- or tetravalent structure and are derived from scFvs. The multimerization is achieved by bi-, tri- or tetravalent coiled-coil structures. In a preferred embodiment of the present invention, the gene of interest encodes any of the desired polypeptides mentioned above, preferably any of the monoclonal antibodies, derivatives or fragments thereof.

[0070] Immunoglobulin fragments consisting of the CH2 and CH3 domains of the antibody heavy chain are called "Fc fragments", "Fc regions" or "Fc" because of their tendency to crystallize (Fc = fragment crystallizable). They can be formed, for example, by protease digestion with papain or pepsin from conventional antibodies, but can also be produced by genetic engineering. The N-terminal part of the Fc fragment can differ by some amino acids of the hinge region still present.

[0071] Antibodies comprising an antigen-binding fragment and an Fc region may also be referred to as full-length antibodies. Full-length antibodies may be monospecific and multispecific antibodies, such as bispecific or trispecific antibodies.

[0072] A preferred therapeutic antibody according to the invention is a multispecific antibody, in particular a bispecific or trispecific antibody. A bispecific antibody typically combines antigen-binding specificity for a target cell (e.g., malignant B cell) and an effector cell (e.g., T cell, NK cell, or macrophage) in one molecule. Exemplary bispecific antibodies are, 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 the two antigen-binding specificities on two separate polypeptide chains, but the two polypeptide chains are non-covalently linked. The DART format is based on the diabody format, but provides additional stabilization through a C-terminal disulfide bridge. A trispecific antibody is a monoclonal antibody that combines three antigen-binding specificities. A trispecific antibody may be constructed based on bispecific antibody technology, which reconstitutes the antigen recognition domains of two different antibodies into one bispecific molecule. For example, trispecific antibodies have been made that target CD38 on cancer cells and CD3 and CD28 on T cells. 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 in the production of a fusion protein, such as an Fc fusion protein. Furthermore, the method for increasing the protein production amount according to the present invention can be advantageously used in the production of a fusion protein, such as an Fc fusion protein.

[0074] The effector portion of the fusion protein may be the complete sequence or any part of the sequence of a native or modified heterologous protein. The sequences of the immunoglobulin constant domains may be derived from any subtype of immunoglobulin, such as IgG1, IgG2, IgG3, IgG4, IgA1 or IgA2 subtypes, or classes of immunoglobulins, 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 linked to the CH2 domain of the heavy chain immunoglobulin constant region containing an N-linked glycosylation site. Fc fusion proteins can be constructed by genetic engineering approaches by introducing the CH2 domain of a heavy chain immunoglobulin constant region containing an N-linked glycosylation site into another expression construct, e.g., containing other immunoglobulin domains, enzymatically active protein moieties, or effector domains. Thus, Fc fusion proteins according to the invention also include single-chain Fv fragments linked to a CH2 domain of a heavy chain immunoglobulin constant region (e.g., one containing an N-linked glycosylation site).

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

[0076] Preferably, CHO cells are metabolically engineered to allow for an efficient cell line development process, for example by glutamine synthetase (GS) knockout and / or dihydrofolate reductase (DHFR) knockout, facilitating selection with methionine sulfoximine (MSX) or methotrexate, respectively.

[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] Most preferably, the cells are established, adapted and cultured entirely under serum-free conditions (and optionally in media that does not contain any animal-derived proteins / peptides). Examples of suitable media include Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle's Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, Commercially available media such as Sigma-Aldrich (CA), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma) are representative of suitable nutrient solutions. Any of the above media may be supplemented with various compounds, as needed, non-limiting examples of which are recombinant hormones and / or other recombinant growth factors (insulin, transferrin, epidermal growth factor, insulin-like growth factor, etc.), salts (sodium chloride, calcium, magnesium, phosphate, etc.), buffers (HEPES, etc.), nucleosides (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 a selectable gene, an appropriate selection agent is added to the culture medium.

[0079] The recombinant protein of the method of the invention is produced in eukaryotic cells, in cell culture. After expression, the recombinant protein is harvested and further purified. The recombinant protein may be recovered from the culture as a secreted protein in harvested cell culture fluid (HCCF) or from a 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 purified using techniques well known in the art. Samples obtained and / or analysed at the various steps of purification are also called in-process control (IPC) samples or process intermediates. The harvesting typically involves centrifugation and / or filtration, for example, to produce a harvested cell culture fluid or a cell lysate, preferably a harvested cell culture fluid. The harvested cell culture fluid or cell lysate may therefore also be called a clarified harvested cell culture fluid or a clarified cell lysate. Not only have most cellular components been removed, but it does not contain living cells or cell debris. Clarification typically means centrifugation or filtration, preferably filtration. Further process steps may include affinity chromatography, in particular Protein A column chromatography for antibodies or Fc-containing proteins, to separate the product from contaminating substances. Further process steps may include acid treatment to inactivate viruses, clarifying the product pool by depth filtration (preferably following acid treatment), removing cellular contaminants such as HCPs and DNA. Further process steps may include ion exchange chromatography, in particular anion exchange chromatography to further remove contaminating cellular components, and / or cation exchange chromatography to remove product-related contaminants such as aggregates, in this order or in any other order that may be appropriate in each individual case. Furthermore, 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 protein contaminants of the host cell, the method according to the invention may be particularly useful for the analysis of process intermediates after (preferably before and after) purification steps to remove HCPs, such as before and after affinity chromatography, before and after depth filtration combined with acid treatment and / or before and after anion exchange chromatography (to adapt the relevant steps to a more efficient removal of lipase activity in the process intermediates). 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). The skilled person will know that the sample obtained after a particular method step can be the same as the sample obtained before the next method step, for example the sample obtained after affinity chromatography (e.g. Protein A chromatography) can be the same as the sample before acid treatment (or before depth filtration combined with acid treatment (i.e. following acid treatment)). As explained above, due to the wide buffering range of the buffer, even lipase activity in samples with 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 drug substance or drug product samples. Drug substance or drug product samples contain the formulation buffer and therefore often contain polysorbate. At very high concentrations, polysorbate can inhibit the reaction by competing with the substrate.However, the sensitivity of the assay allows lipase activity to be measured in samples of drug substance or drug product at typical concentrations of 0.4 to 0.8 mg / mL polysorbate.

[0081] In one embodiment, a method for producing a recombinant protein of interest, comprising detecting lipase activity in a sample comprising the recombinant protein, the method 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) a non-denaturing surfactant having no ester bonds, 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 16(c) incubating the sample and substrate in the reaction mixture; (d) detecting lipase activity by measuring hydrolysis of the 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 the sample and substrate in the reaction mixture according to step (c). A person skilled 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 comprising a recombinant protein, more particularly the recombinant protein of interest. One of skill in the art will appreciate that the method further comprises 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) optionally formulating the recombinant protein into a pharma- ceutically acceptable formulation suitable for administration.Thus, there is provided a method for producing a recombinant protein of interest, comprising the steps of: (i) culturing a eukaryotic cell expressing the recombinant protein of interest; (ii) harvesting said recombinant protein; (iii) purifying said recombinant protein; and (iv) optionally formulating said recombinant protein into a pharma- ceutically acceptable formulation suitable for administration; and (v) in steps (ii), (iii) and / or (iv), obtaining at least one sample comprising said recombinant protein, said method further comprising detecting (contaminating) lipase activity in the sample comprising said recombinant protein. (a) providing at least one sample comprising the recombinant protein produced in a eukaryotic cell of 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 about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bonds (the surfactant being a nonionic 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-C6H). 16(iv) optionally including a non-buffering salt; (c) incubating the sample and the substrate in the reaction mixture; (d) detecting (contaminating) lipase activity by measuring hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; optionally 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 invention is an aqueous reaction solution. Moreover, the lipase activity detected in step (d) is a contaminating lipase activity in at least one sample comprising the recombinant protein, more particularly a recombinant protein of interest. In a particular embodiment, the recombinant protein of interest is a therapeutic protein, such as an antibody, an antibody fragment, an antibody-derived molecule (e.g., scFv, bi- or multispecific antibody) or a fusion protein (e.g., Fc-fusion protein). In one embodiment, the antibody is an anti-IL-36R antibody, in particular spesolimab. In another embodiment, the antibody is not an anti-IL-36R antibody, in particular not spesolimab.

[0082] In a particular embodiment, the method for producing a recombinant protein of interest according to the invention comprises obtaining at least one sample comprising said recombinant protein within the step of harvesting said recombinant protein (within step (ii)), said sample being a harvested cell culture fluid (HCCF) or a cell lysate; within the step of purifying said recombinant protein (within step (iii)), said sample being an in-process control (IPC) sample; and / or, optionally, within the step of formulating said recombinant protein into a pharma- ceutically acceptable formulation suitable for administration (within step (iv)), said sample being a drug substance or drug product sample. Preferably, the method for producing a recombinant protein of interest according to the invention comprises within step (iii) obtaining at least one sample comprising said recombinant protein (within step (iv)), said sample being an in-process control (IPC) sample), e.g. obtaining at least one sample after affinity chromatography, after acid treatment followed by depth filtration (or after acid treatment and / or after acid treatment) and / or after ion exchange chromatography (preferably anion exchange chromatography or cation exchange chromatography). More preferably, the method comprises obtaining at least one sample before or after affinity chromatography, before or after depth filtration after acid treatment (or before or after acid treatment and / or before or after acid treatment) and / or before or after ion exchange chromatography (preferably anion exchange chromatography or cation exchange chromatography). The step of detecting lipase activity in the sample comprising the recombinant protein is carried out according to and as specified in the method for detecting lipase activity described herein.

[0083] Kit for determining contaminating lipase activity by measuring hydrolysis in a sample Also, a kit for measuring contaminating lipase activity in a sample containing a recombinant protein is provided, comprising: (i) a buffer solution having a pH of about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant is a nonionic 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 ) 4-MU ester), (iv) optionally a non-buffering 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 serves as a positive control and / or allows for the calculation of relative values ​​compared to the internal standard. The kit may also comprise one or more microtiter plates with 96 wells or a multiple of 96 wells. The components of the kit may be provided as solutions and / or dry components, separately or in premixed form. In the case of a buffer, it may be provided as a dry compound that upon dilution or reconstitution provides a buffer with a pH of about pH 4 to about pH 9.

[0084] In certain embodiments, the buffer, surfactant, and optional non-buffering salt are premixed as an assay buffer. Preferably, the assay buffer is at least about 3-fold concentrated, or about 3-fold to about 5-fold concentrated, relative to the final reaction mixture. Alternatively, the assay buffer is provided as a dry mix. Such a dry mix may be reconstituted with water to provide an assay buffer that is at least about 3-fold concentrated, or about 5-fold concentrated, relative to the final reaction mixture. In one embodiment, the dry mix of assay buffers is a lyophilized assay buffer. The substrate is provided separately to 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-buffering salt premixed as a master mix. The master mix may be adapted to be provided at about 80% (v / v) to about 70% (v / v) of the reaction mixture, preferably at about 80% to about 75% 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 a saturated unbranched fatty acid (C6 to C 16 ) 4-MU ester form, and the aliphatic chain of the saturated unbranched fatty acid is C6 to C 16 or preferably C8 to C 12 Thus, the substrate may be 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), methylumbelliferyl undecanoate or methylumbelliferyl dodecanoate. In a particular embodiment, 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 is dissolved in an organic solvent. The substrate may be provided as a dry substance and optional additional organic solvent, or may be dissolved in an organic solvent (e.g., dimethylsulfoxide (DMSO) or dimethylformamide (DMF), preferably DMSO) as a stock solution (e.g., 100-fold stock solution relative to the concentration in the reaction mixture). Thus, in certain embodiments, the substrate is provided as a stock solution of about 100 μM to about 100 mM, preferably about 100 μM to 30 mM, preferably 100 μM to 3 mM, more preferably 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 about 1% to about 5% (v / v) of the reaction mixture.

[0087] Examples of suitable non-denaturing zwitterionic detergents without ester bonds include, but are not limited to, 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, e.g., n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent3-12)), and 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate or other amidosulfobetaine detergents. Examples of suitable non-denaturing non-ionic surfactants include, but are not limited to, pyranoside surfactants (e.g., 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 (e.g., digitonin), octylphenoxypolyethoxyethanol (IGEPAL CA-630), poloxamer 188, 338, 407 or tergitol. 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 detergent is a non-denaturing non-ionic or zwitterionic detergent selected from the group consisting of CHAPS, CHAPSO, Zwittergent (such as Zwittergent 3-12) and saponin, preferably CHAPS.

[0088] In certain embodiments, the buffer 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), HEPE, The buffer solution may comprise one or more buffer substances selected from the group consisting of S (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Tricine (N-tris(hydroxymethyl)methylglycine), NaHPO, and NaHPO. In certain embodiments, the buffer has a pH of about pH 4 to about pH 8, preferably, the buffer has a pH of about pH 5 to about pH 7.5, and more preferably, the buffer has a pH of about pH 5.5 to about pH 7.5.

[0089] The buffer may comprise a single buffer substance or may be a multi-component buffer as specified above for the method according to the invention. The multi-component buffer may comprise one or more buffer substances with overlapping buffer ranges to have a wider buffer range. The buffer may comprise, for example, two, three, four, five or more buffer substances, preferably two or more buffer substances, more preferably three or more buffer substances. For example, the multi-component buffer may comprise two to four buffer substances, three to four buffer substances, more preferably three buffer substances. In a particular embodiment, the multi-component buffer comprises at least three buffer substances with overlapping buffer ranges, preferably comprising at least one of Tris, MES and / or acetate, more preferably acetate, MES and Tris in a ratio of 1:1:2. In a particular embodiment, the buffer is a multi-component buffer with a buffer 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 buffers with different 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 less than 5%, such as 0% to less than 15%, 0% to less than 10%, 0% to less than 7.5% or 0% to less than 5%, or such as 2% to less than 15%, 2% to less than 10%, 2% to less than 7.5% or 2% to less than 5%. Optional non-buffering salts can be, for example, NaCl, KCl and CaCl2, preferably NaCl or KCl.

[0090] In view of the above, it will be appreciated that the present invention also encompasses the following: Item 1 is a method for detecting lipase activity in a sample containing a recombinant protein, comprising the steps of: (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 about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bond (the surfactant is a nonionic 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 is a saturated unbranched fatty acid (C6 to C7 16 ) a 4-MU ester), and (iv) optionally comprising a non-buffering salt; (c) incubating the sample and the substrate in the reaction mixture; (d) detecting lipase activity by measuring hydrolysis of the 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 the sample and the substrate in the reaction mixture according to step (c).

[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 terminating 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 kinetics (optionally, reaction mixtures that do not meet the requirements for pseudo-zero order kinetics are excluded from the analysis).

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

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

[0095] Item 6 specifies the method of any one of the preceding items, 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.

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

[0097] Item 8 specifies the method of any one of the preceding items, wherein the substrate is provided as a stock solution in an organic solvent, 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 specifies the method of any one of the preceding items, wherein the surfactant has a final concentration in the reaction mixture that exceeds the critical micelle concentration in the reaction mixture.

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

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

[0101] Item 12 specifies the method of any one of the preceding items, wherein the buffer 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), trisodium phosphate, tert-butyl ... The buffer comprises one or more buffer substances selected from the group consisting of tricine (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 specifies the method of any one of the preceding items, wherein the buffer has a pH of about 5 to about 7.5, and preferably the buffer has a pH of about 5.5 to about 7.5.

[0103] Item 14 specifies the method of any one of the preceding items, wherein 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 specifies the method of item 14, wherein the multi-component buffer comprises at least three buffering substances having overlapping buffer ranges, preferably comprising at least one of Tris, MES and / or acetate.

[0105] Item 16 specifies the method of item 14 or item 15, comprising: (a) the use of a buffer with a variable pH from at least about pH 4 to at least about pH 8; (b) the use of a buffer with various pH values ​​between about pH 4 and about pH 8, thereby affecting the ionic strength by less than 15%, preferably less than 10%, preferably 0% to less than 15%, 0% to less than 10%, 0% to less than 7.5%, or 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) comparing and identifying conditions that reduce hydrolytic activity.

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

[0107] Item 18 specifies the method of any one of the preceding items, wherein the samples are contacted, incubated, and measured in a plate format having 96 wells or a multiple of 96 wells.

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

[0109] Item 20 specifies the method of any one of the preceding items, wherein the buffer, detergent, and optional non-buffering salt are premixed as an assay buffer that is about 3-fold to about 5-fold concentrated relative to the reaction mixture.

[0110] Item 21 specifies the method of any one of the preceding items, wherein the buffer, detergent, substrate and optional non-buffering salts are added to the sample as a master mix, and the master mix is ​​provided at about 80% (v / v) to about 70% (v / v) of the reaction mixture, preferably about 75% of the reaction mixture.

[0111] Item 22 specifies the method of any one of the preceding items, wherein 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.

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

[0113] Item 24 specifies the method of any one of the preceding items, wherein the ionic strength of the non-buffering 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, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM in the reaction mixture.

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

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

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

[0117] Item 28 specifies the method of any one of the preceding items, wherein (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 specifies the method of any one of the preceding items, wherein 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 is 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 non-denaturing surfactant having no ester bonds, said surfactant being a non-ionic or zwitterionic surfactant; and (iii) a substrate comprising the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, said substrate being a saturated unbranched fatty acid (C6 to C8). 16 ) a 4-MU ester; and (iv) optionally, a non-buffering salt; and / or (v) optionally, water for dilution.

[0120] Item 31 specifies the kit of item 30, 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.

[0121] Item 32 specifies the kit of item 30 or 31, further comprising an organic solvent, preferably DMSO or DMF, for dissolving the substrate.

[0122] Item 33 specifies the kit of any one of Items 30 to 32, wherein 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, and is preferably CHAPS.

[0123] Item 34 specifies the kit according to any one of items 30 to 33, wherein 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)), and 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 specifies the kit of any one of items 30 to 34, wherein the buffer has a pH of about 5 to about 7.5, preferably, the buffer has a pH of about 5.5 to about 7.5.

[0125] Item 36 specifies the kit of any one of items 30 to 35, wherein the buffer is a multi-component buffer having a buffer 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 specifies the kit of item 36, wherein the multi-component buffer comprises at least three buffering substances having overlapping buffer ranges, preferably comprising at least one of Tris, MES and / or acetate.

[0127] Item 38 specifies the kit of any one of items 30 to 37, further comprising one or more microtiter plates having 96 wells or a multiple of 96 wells.

[0128] Item 39 specifies the kit of any one of items 30 to 38, wherein the buffer, surfactant and optional non-buffering salt are premixed as an assay buffer that is at least about 3-fold, or about 3-fold to about 5-fold concentrated relative to the final reaction mixture, and / or provided as a dry mix.

[0129] Item 40 specifies the kit of any one of items 30 to 39, wherein 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.

[0130] Item 41 is 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 said recombinant protein; (iii) purifying said recombinant protein; and (iv) optionally formulating said recombinant protein into a pharma- ceutically acceptable formulation suitable for administration; and (v) in steps (ii), (iii) and / or (iv) obtaining at least one sample comprising said recombinant protein, said method comprising detecting (contaminating) lipase activity in a sample comprising said recombinant protein. The method further comprises the steps of: (a) providing at least one sample comprising the recombinant protein produced in a eukaryotic cell of 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 about pH 4 to about pH 9; (ii) a non-denaturing surfactant having no ester bonds, 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 16(c) incubating the sample and the substrate in the reaction mixture; (d) detecting (contaminating) lipase activity by measuring hydrolysis of the 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; and 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). In a preferred embodiment, the detergent in step (b)(ii) is not polyethylene glycol tert-octylphenyl ether (TritonX-100) and is not polyethylene glycol nonylphenyl ether (NP-40). Preferably, the detergent is a non-denaturing zwitterionic detergent, such as one selected from the group consisting of CHAPS, CHAPSO and Zwittergent, preferably CHAPS.

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

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

[0133] Item 44 specifies a method for producing a recombinant protein of interest according to item 43, said method comprising the steps of obtaining at least one sample after affinity chromatography, after depth filtration followed by acid treatment (or after acid treatment and / or after depth filtration) and / or after anion exchange chromatography, preferably obtaining at least one sample before or after affinity chromatography, before or after depth filtration followed by acid treatment (or before or after acid treatment and / or before or after depth filtration) and / or before or after anion exchange chromatography.

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

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

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

[0137] Lipase assay Two different buffers have been used for the lipase assay: phosphate assay buffer, or AMT buffer, a multicomponent buffer. The phosphate buffer contains 108 mM Na2HPO4, 25 mM NaH2PO4, 186.2 mM NaCl, 13.3 mM CHAPS at pH 7.4, resulting in final concentrations in the reaction mixture of 81 mM Na2HPO4, 19 mM NaH2PO4, 140 mM NaCl, 10 mM CHAPS at pH 7.4. AMT assay buffer, with a wide buffer range of pH 4-8, is provided as a 4x stock solution and contains 0.3 M acetate, 0.3 M MES, 0.6 M Tris, 0.6 M NaCl, 40 mM CHAPS, pH adjusted as stated (recommended pH range: 4-8) using HCl or NaOH to give final concentrations in the reaction mixture of 75 mM acetate, 75 mM MES, 150 mM Tris, 150 mM NaCl, and 10 mM CHAPS.

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

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

[0140] The reaction mixtures were prepared as follows: For the phosphate buffer (constant pH 7.4) based assay: A) cuvette: in a reaction vessel, 720 μL sample was added to the master mix (2250 μL phosphate assay buffer, 30 μL 0.3 mM 4-MUD in DMSO), B) per well of a 96-well plate: in a reaction vessel, 72 μL sample was added to the master mix (225 μL phosphate assay buffer, 3 μL 0.3 mM 4-MUD in DMSO). For assays based on AMT buffer (also called ternary buffer) (variable pH), C) cuvette: in a reaction vessel, 720 μL sample was added to the master mix (750 μL 4x AMT assay buffer, 1500 μL HO, 30 μL 0.3 mM 4-MUD in DMSO); D) per well of a 96-well plate: in a reaction vessel, 72 μL sample was added to the master mix (75 μL 4x AMT assay buffer, 150 μL HO, 3 μL 0.3 mM 4-MUD in DMSO).

[0141] Hydrolysis of the substrate 4-MUD was measured in real time by detecting the fluorescence intensity of the released chromophore 4-MU immediately after mixing and for a period ranging from a few minutes to 5 hours depending on the fluorescence intensity. Fluorescence (λ em = 450 nm, λ ex= 330 or 340 nm) was monitored at 25 °C either in cuvettes using a fluorescence spectrometer or in 96-well plates using a suitable microplate reader. A high correlation was found between the use of a fluorescence spectrometer (Fluoromax 4, Horiba Jobin Yvon) and the plate reader SpectraMax M3 (Molecular Devices) (see Figure 9). A negative control without sample was included to exclude potential autohydrolysis. A linear fit was used to calculate the slope (e.g. RFU / s). Detecting fluorescence in real time allows measurements within a time window with pseudo-zero order kinetics. Samples were run at least in triplicate and individual reaction mixtures were excluded from the analysis if they did not meet the pseudo-zero order kinetics, e.g. due to air bubbles in the wells. This method of removing outliers was found to strongly increase the sensitivity of the assay. Using a calibration curve with a defined concentration of 4-MU, the rate of hydrolysis (e.g. nmol / s) can be calculated. A calibration curve using known 4-MU concentrations further permitted the determination and comparison of reaction rates at various pH values.

[0142] Development of AMT buffer solution The lipase assay was initially set up using a phosphate assay buffer. A three-component AMT buffer was developed for the kinetic measurement of hydrolytic activity in various pharmaceutical samples at varying pH. The AMT buffer, which contains acetate, MES and Tris as buffer substances, allows for a wider pH buffering range and therefore wider pH ranges, or even measurements at different pHs. The buffer substances used are known to be non-fluorescent and poor metal chelators, and are unlikely to interfere with enzyme activity. Comparable to the phosphate assay buffer, CHAPS was added above the CMC and the ionic strength was adjusted with NaCl. The assay was found to be sensitive to ionic strength, so it was important to create buffers that only moderately changed ionic strength (less than 15%, preferably even less than 10%) at various pHs (ranging from pH 4 to 8), as well as buffers containing buffer substances with overlapping buffering ranges (Ellis KJ, Morrisson JF, 1982. Methods in Enzymology, 87: 405-426). The AMT buffer allows, for example, to identify conditions (including pH conditions) that reduce hydrolytic activity. This buffer also allows measurements to be taken at the pH of the sample to measure the lipase activity present in the sample at specific conditions, as well as to compare the lipase activity at various states during purification. The assay allows for an additional increase in sensitivity by measuring the sample at its optimum pH.

[0143] HPLC-CAD method The content of polysorbate in aqueous solution was quantified using HPLC-CAD. Intact polysorbate was bound to a mixed-mode column based on a mixture of reversed phase and ion-exchange polymers using an aqueous mobile phase containing isopropanol or its equivalent. Polysorbate was then eluted using a mobile phase with acetonitrile or its equivalent. More specifically, HPLC chromatography was performed using mobile phase A (MPA) of 10 mM ammonium formate, pH 4.5, 2-propanol 20% (v / v) and mobile phase B (MPB) containing acetonitrile 50% (v / v) and 2-propanol 50% (v / v). Separation of protein and matrix components, as well as polysorbate degradants, was achieved on a mixed-mode column (Oasis® Max Online column, 2.1 mm×20 mm, 30 μm, 80 Å) using a flow of 1.0 mL / min MPA and elution of intact polysorbate species with a step gradient using MPB. The analytes were detected using a charged aerosol detector (CAD). CAD detection employs an inert gas flow system that nebulizes the analytes, 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. 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 solutions in the presence of surfactants. When the surfactant exceeds its critical micelle concentration, a large increase in the fluorescence quantum yield is observed as the fluorescer enters the hydrophobic interior of the micelles. The amount of solubilized fluorescer is directly proportional to the concentration of micelles in the solution. More specifically, samples were prepared using 5×10 NPN -6 M, and the fluorescence was measured using a fluorescence detector (λ em = 420 nm, λ ex = 350 nm. Polysorbates were quantified using an external calibration standard series.

[0145] Example 1: Lipase assay allows to measure activity in various drug substances The lipase assay was developed to measure lipase activity in various drug substances after purification, in order to remove lipase activity in the final drug substance that causes polysorbate degradation in the final drug product, and to help adapt and improve the purification steps during downstream processing. The contaminating lipase activities that co-purify as host cell proteins present in some drug substances may vary not only from purification step to protein, but also from protein to protein. Various lipases show specific pH optima, which are usually related to their cellular localization, for example, lysosomal lipases typically have an acidic pH optimum.

[0146] The lipase assay was therefore used for the kinetic measurement of hydrolytic activity in different bulk drug substances (BDS) at varying pH. For this, the AMT buffer was established to measure pH dependence in the range of pH 4-8. The BDS was used undiluted at 72 μL per well for each sample. A calibration curve of known 4-MU concentrations allowed the reaction rate at each pH to be measured in nmol / min / mL. As a negative control, a blank was performed with the formulation buffer only to monitor non-enzymatic hydrolysis. Optionally, a positive control was included using a commercial lipase such as porcine pancreatic lipase (PPL) at 0.24 mg / mL or less. The pharmaceutical products (referred to as BDS products A, B, D and E (BDS A, B, D and E)) differed in the amount of hydrolytic activity detected (see Figure 2) as well as the pH profile of the hydrolytic activity. For example, BDS A showed a clear pH optimum at alkaline pH, whereas BDS D and BDS E showed a pH optimum at rather acidic pH (Figure 2). At pH 7.5 and above, autohydrolysis may be responsible for residual hydrolytic activity (see BDS B and BDS E). This can be verified by detecting residual hydrolytic activity in the presence of a lipase inhibitor such as orlistat (3.3 μM) or by testing blank samples (sample buffer or medium) without lipase activity in parallel (data not shown). This experiment also demonstrated that even extremely low lipase activity, such as that of BDS B, is detectable at the optimum pH (pH 6).

[0147] Overall, the pH dependence was found to be different among the various drug substance samples tested, indicating that different drug substances contain different contaminating proteins with lipase activity, sometimes with unique pH optima, demonstrating that the assay is useful for detecting such different enzymes with lipase activity in the tested samples.

[0148] Additionally, different formulation buffers were tested at different pHs to demonstrate that different formulation buffers can support or inhibit lipase activity in pharmaceutical products (data not shown).

[0149] Example 2: Effect of substrate concentration To achieve a sufficiently high sensitivity of the observed reaction, it is important to have enough substrate in solution. This need competes with the limited solubility of 4-MUD and its derivatives in water-based master and reaction mixtures. Addition of 4-MUD to water at high concentrations instantly resulted in the formation of visible particles. Addition of a surfactant such as CHAPS to the assay mixture greatly increased the solubility of 4-MUD and therefore prevented precipitation.

[0150] Therefore, a set of light scattering and activity experiments was performed to determine the ideal substrate concentration for the intended use. em = 400 nm, λ ex The solubility of 4-MUD was tested using right angle light scattering (RALS) experiments in ATM assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 7) using a NA = 400 nm. The maximum solubility of 4-MUD in ATM buffer with 10 mM CHAPS was found to be approximately 40 μM (Figure 3).

[0151] The Michaelis-Menten kinetics of hydrolytic activity in bulk drug substance (BDS D) was analyzed (72 μL per well, undiluted BDS D). The assay was performed in black 96-well plates using a microplate reader (λ em = 450 nm, λ exVarying concentrations of 4-MUD (1.5625 μM to 150 μM) were performed in AMT assay buffer (75 mM acetic acid, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5) using a 500 Hz NMR spectrometer (NMR spectroscopy; NMR spectroscopy; NMR spectroscopy; NMR spectroscopy; δ = 330 nm, top read mode). The pH was adjusted to that of the bulk drug substance. As shown in Figure 4, the rate of reaction was found to be sufficient to support a fast readout even at or below 3 μM 4-MUD. Typically, the release of 4-MU is measured immediately after mixing and detected for minutes to hours, typically from about 20 minutes to about 2 hours.

[0152] The results showed that a concentration of 4-MUD between 3 μM and 30 μM proved to be a reasonable compromise for use as a standard concentration, although concentrations between 1 μM and 1000 μM could also be used (Figure 4).

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

[0154] Example 3: Effect of fatty acid chain length The acyl ester derivative 4-methylumbelliferyl decanoate (4-MUD) was chosen because the acyl ester of decanoic acid obtains a broader enzyme spectrum compared to, for example, the oleic acid ester, which contains a longer unsaturated acyl chain. Furthermore, the shorter chain length of the decanoic acid ester provides better solubility in the aqueous reaction mixture compared to, for example, the oleic acid ester. As a result, more substrate can be used in the assay mixture. More specifically, C 16It was found that at chain lengths of 0.1 to 1.5 mm or longer, the solubility becomes strongly limited (data not shown). Furthermore, it was found that the decanoic acid ester offers better resistance to autohydrolysis compared to, for example, the butyric acid ester (FIG. 6). The autohydrolysis of 4-methylbelliferyl butyrate (4-MUB) and 4-MUD was analyzed. The elution was performed in a black 96-well plate using a microplate reader (λ em = 450 nm, λ ex Fluorescence was monitored and compared for 1800 s using 30 μM 4-MUB and 30 μM 4-MUD in AMT assay buffer (75 mM acetate, 75 mM MES, 150 mM Tris, 150 mM NaCl, 10 mM CHAPS, pH 5.5) using a 100 nm NMR spectroscopy (NMR spectroscopy, ... 10 Fatty acids were found to be the optimal choice.

[0155] Example 4: Effect of surfactants 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 lipase, among others, and this requirement was achieved by modifying the reaction mixture of the assay.

[0156] First, surfactants were tested above their CMC (critical micelle concentration). Therefore, 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 exThe assay was performed with 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 using a 500 nm NMR spectroscopy (NMR spectroscopy, ... Without being bound by theory, it is hypothesized that detergents create an environment that promotes lipase activity by allowing rearrangement and opening of a lid or flap that has been described to cover 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 chosen in the final reaction mixture, which resulted in increased lipase hydrolysis as well as improved sensitivity of the assay (Figure 7).

[0157] The detergent can be another detergent, but it needs to be a mild, specifically non-denaturing detergent, to maintain the native structure and activity of the protein with lipase activity. Furthermore, it is important that the detergent does not compete 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 therefore does not affect the sensitivity of the assay. Furthermore, CHAPS mediates the solubility of 4-MUD in water at the concentrations used (Figure 3).

[0158] Example 5: Measurement of samples with different pH values The activity of enzymes is often influenced by pH. As the expected pH range of the in-process control (IPC) samples reaches from 3.5 to 7.5 (Table 1), the impact of contaminating lipases on the observed activity was expected. To maximize the comparability between samples of different pH values, a first buffer component was specified to maintain a constant pH of 7.4 in the reaction mixture. Table 1 summarizes various IPC samples from one downstream process to demonstrate the pH of samples originating from different purification steps. The pH of each sample and the pH of the corresponding reaction mixture are shown. Similar pH variations were found with various antibodies or Fc fusion proteins during downstream processing. Experiments were performed with several products, yielding similar results. [Table 2]

[0159] Analyzing IPC samples at the same pH maintains constant reaction conditions and therefore enhances the comparability of the data obtained. However, in some cases it is beneficial to modify the pH, for example to find conditions that reduce hydrolytic activity (see, for example, Figure 2) and to aid in formulation development. Therefore, a three-component AMT buffer system was developed.

[0160] Example 6: Effect of ionic strength Providing ions is beneficial to prevent protein aggregation in a sample and to maintain the native structure of the protein. 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 black 96-well plates using a microplate reader (λ em = 450 nm, λ exThe assay was performed using 30 μM 4-MUD and 72 μL of sample in NaCl-free AMT assay buffer (75 mM acetate, 75 mM MES, 150 mM Tris, 10 mM CHAPS, pH 5.5) using a chromatogram of 10 μM 4-MUD at 340 nm and 25 °C in top read mode. The dependence of hydrolytic activity on ionic strength was investigated using several mAbs and is exemplarily shown for product F in Figure 8.

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

[0162] Example 7: Inhibition of Lipase Assay by Polysorbates Polysorbates in the final drug product likely act as competitive substrates for the traceable fluorescent substrate (4-MUD). The concentration of polysorbate 20 (PS20) or polysorbate 80 in the drug product may range between about 0.2 g / L and 1.0 g / L, typically 0.2 g / L to 0.4 g / L of PS20 or PS80, or a mixture thereof. Therefore, we set up experiments using antibody UF-DF material as the active pharmaceutical ingredient (API in water, no PS20) and adding various concentrations of PS20 (0.0125-3.2 mg / mL) to the reaction mixture.

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

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

[0165] Inhibition of the lipase assay was expected since it shows a correlation between the hydrolytic activity of 4-MUD detected in the assay and the degradation of polysorbates. However, to be able to measure lipase activity also in pharmaceutical products, it is advantageous that the concentrations typically applied in antibody formulations, e.g. 0.1 g / L to 0.4 g / L, do not interfere or only slightly interfere with the assay.

[0166] Overall, the data indicate that 4-MUD experiments analyzing samples containing polysorbate concentrations commonly used in formulated drug substances are not expected to have a significant effect on activity.

[0167] Example 8: Effect 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 detergents, especially TritonX-100. Thus, the hydrolytic activity 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) was measured in ATM buffer at pH 5.5 containing either 10 mM CHAPS, 0.25% TritonX-100, or 0.25% TritonX-100 and 0.125% gum arabic. The results shown in Figure 10A-C demonstrate that CHAPS outperforms TritonX-100 as well as TritonX-100 and gum arabic. Gum arabic is an emulsifier, and no effect of gum arabic on activity was observed in any of the experiments.

[0168] Example 9: Suitability of the 4-MUD assay for analysis of IPC samples The suitability of the 4-MUD assay for IPC sample analysis was tested for several exemplary processes, where in-process control samples from various downstream processing steps were analyzed for sufficient solubility, pH within expected ranges, and kinetic measurements meeting requirements (pseudo-zero order kinetics).

[0169] Table 2 shows the applicability of the treated samples with regard to the possible influence of particles, ionic strength, pH and several other influencing factors. Samples were analyzed in triplicate and reaction mixtures that did not meet the requirements for pseudo-zero order kinetics were excluded from the analysis. [Table 3]

[0170] The suitability of the assay could be demonstrated such that it could be applied to all tested process steps, including those subsequent to ultrafiltration / diafiltration (UF / DF), and to the final formulated bulk drug substance (BDS).

[0171] This was further measured for certain antibodies using a lipase assay in a spectrophotometer (Figure 11A) and a plate reader (Figure 11B).

[0172] The results of the 4-MUD plate reader assay generally correlated well with the spectrophotometer results. Both readouts were able to demonstrate lower lipase activity in the product-containing samples compared to the elution buffer alone and lower lipase activity in the drug substance compared to the formulation buffer alone. The 4-MUD assay can be performed in a microtiter plate format for high throughput purposes and therefore can 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 during other processing steps of drug, drug substance and biopharmaceutical development should also inhibit the hydrolytic activity monitored by the 4-MUD assay.

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

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

[0176] Example 11: Degradation of Polysorbate in Spiking Experiments Compared to 4-MUD Activity Polysorbate spiking experiments were performed to test the correlation between hydrolytic activity measured by the 4-MUD assay and degradation of polysorbate. To assess the rate of degradation of polysorbate, relevant in-process (IPC) samples were spiked with polysorbate and the resulting polysorbate content was monitored over time by fluorescent micellar assay (FMA). 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] Degradation of polysorbates was measured using the FMA assay and lipase assay (in a 1 cm macrocuvette using a fluorescence spectrometer (λ em = 450 nm, λ ex = 340 nm) was used to compare the hydrolytic activity measured using phosphate assay buffer (81 mM Na2HPO4, 19 mM NaH2PO4, 140 mM NaCl, 10 mM CHAPS, pH 7.4) containing 3 μM 4-MUD. The results (Figure 14) suggest a correlation of the hydrolytic activity measured by lipase assay with the degradation of polysorbate in the relevant IPC step 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 selected from buffers comprising: - phosphate buffer, or - acetic acid / 2-(N-morpholino)ethanesulfonic acid (MES) / Tris buffer, (ii) CHAPS, and (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of 4-methylumbelliferyl decanoate (4-MUD) at a concentration of about 3 μM to about 30 μM a process comprising: (c) incubating the sample and the substrate in the reaction mixture; and (d) detecting lipase activity by measuring the hydrolysis of 4-MUD and detecting the fluorescence intensity of the released chromophore 4-MU. A method comprising:

2. The phosphate buffer has a concentration of Na2HPO4 and NaH2PO4 of about 50 mM to about 400 mM, respectively; or the acetate / MES / Tris buffer has a concentration of acetate, MES, and Tris of about 50 mM to about 400 mM, respectively; The method of claim 1.

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

4. The method of claim 3, wherein the non-buffering salt is NaCl at a concentration of about 100 mM to about 200 mM.

5. A method according to any one of claims 1 to 4, comprising measuring hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU over time whilst incubating the sample and the substrate in the reaction mixture according to step (c).

6. 6. The method of any one of claims 1 to 5, 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 CHAPS in the reaction mixture is greater than its critical micelle concentration in the reaction mixture; and / or (b) CHAPS is provided at a final concentration in the reaction mixture of about 8 mM to about 20 mM; The method according to any one of claims 1 to 6.

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

9. The method of claim 7, wherein CHAPS is provided at a final concentration in the reaction mixture of about 10 mM.

10. The method of claim 1, wherein the buffer has a buffering range of at least about pH 5 to at least about pH 7.

5.

11. The method of claim 1, wherein the buffer has a buffering range of at least about pH 4 to at least about pH 8. (a) the at least one sample is a harvested cell culture fluid (HCCF), an in-process control (IPC) sample, a drug substance sample or a drug product sample; and / or (b) the recombinant protein is not a lipase and / or is not an enzyme having 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 11.

13. A method for producing a recombinant protein of interest, comprising the steps of: (i) culturing a eukaryotic cell expressing a 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). Including, 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 a 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 having a pH of about pH 4 to about pH 9 selected from buffers including: - phosphate buffer, or - acetic acid / 2-(N-morpholino)ethanesulfonic acid (MES) / Tris buffer, (2) CHAPS, and (3) A substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of 4-methylumbelliferyl decanoate (4-MUD) at a concentration of about 3 μM to about 30 μM. a process comprising: (c) incubating the sample and the substrate in the reaction mixture; and (d) detecting lipase activity by measuring the hydrolysis of 4-MUD and detecting the fluorescence intensity of the released chromophore 4-MU. A method comprising:

14. The process of claim 13, (i) culturing a eukaryotic cell expressing a recombinant protein of interest in cell culture; (ii) harvesting the recombinant protein; (iii) purifying the recombinant protein; and (iv) formulating the recombinant protein into a pharma- ceutically 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). The method of claim 13, comprising:

15. The method of claim 15, wherein the phosphate buffer has a concentration of Na2HPO4 and NaH2PO4 of about 50 mM to about 400 mM, respectively; or the acetate / MES / Tris buffer has a concentration of acetate, MES, and Tris of about 50 mM to about 400 mM, respectively; 15. The method according to claim 13 or 14.

16. The method according to any one of claims 13 to 15, wherein the reaction solution further contains (4) a non-buffering salt.

17. The method of claim 16, wherein the non-buffering salt is NaCl at a concentration of about 100 mM to about 200 mM.

18. A method according to any one of claims 13 to 17, comprising measuring hydrolysis by detecting the fluorescence intensity of the released chromophore 4-MU over time whilst incubating the sample and the substrate in the reaction mixture according to step (c).

19. at least one sample containing the recombinant protein, - step (ii), wherein the sample is a harvested cell culture fluid (HCCF) or a cell lysate; and / or - step (iii), wherein said sample is an in-process control (IPC) sample; The method of any one of claims 13 to 18, comprising the step of obtaining

20. The method of any one of claims 1 to 19, wherein the lipase activity detected in the at least one sample is a contaminating lipase activity.

21. 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 selected from buffers comprising: - phosphate buffer, or - acetic acid / 2-(N-morpholino)ethanesulfonic acid (MES) / Tris buffer; (ii) CHAPS; and (iii) a substrate containing the chromophore 4-methylumbelliferyl (4-MU) in the form of 4-methylumbelliferyl decanoate (4-MUD) at a concentration of about 3 μM to about 30 μM Including the kit.

22. The phosphate buffer solution according to claim 21, wherein the phosphate buffer has a concentration of Na2HPO4 and NaH2PO4 of from about 50 mM to about 400 mM, respectively; or the acetate / MES / Tris buffer has a concentration of acetate, MES, and Tris of about 50 mM to about 400 mM, respectively; The kit of claim 21.

23. The kit of claim 21 or 22, further comprising (iv) a non-buffering salt.

24. The kit of claim 23, wherein the non-buffering salt is NaCl at a concentration of about 100 mM to about 200 mM.

25. The kit of claim 21, further comprising an organic solvent for dissolving the substrate.

26. The kit of any one of claims 21 to 25, wherein the buffer has a buffering range of at least about pH 5 to at least about pH 7.

5.

27. The kit of any one of claims 21 to 25, wherein the buffer has a buffering range of at least about pH 4 to at least about pH 8.

28. (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 CHAPS are premixed as an assay buffer that is about 3-fold to about 5-fold concentrated relative to the final reaction mixture, and / or are provided as a dry mix; The kit according to any one of claims 21 to 27.

29. (a) the kit further comprises one or more microtiter plates having 96 wells or a multiple of 96 wells; and / or (b) the buffer, CHAPS and 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.

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