Methods for detecting contaminating carboxylesterase activity

Abstract

The present invention relates to a method for detecting carboxylesterase activity of contaminating host cell proteins in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture, comprising the steps of contacting the sample with the hydrophilic substrate HPTS ester and, optionally, additionally, separately contacting the sample with the lipophilic substrate 4-MU ester and detecting the carboxylesterase and optionally lipase activity of at least one contaminating host cell protein using a hydrophilic and optionally a lipophilic substrate by detecting the fluorescence intensity of the released chromophore.Furthermore, a method for producing a recombinant protein of interest is provided, comprising using, during production, a method for detecting carboxylesterase activity of contaminating host cell proteins in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture using a hydrophilic and optionally further a lipophilic substrate to determine contaminating carboxylesterase activity and / or lipase activity in a sample comprising the recombinant protein of interest.

Description

[Technical field]

[0001] The present invention relates to a method for detecting carboxylesterase activity of contaminating host cell proteins in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture, comprising the steps of contacting the sample with the hydrophilic substrate HPTS ester and, optionally, additionally, separately contacting the sample with the lipophilic substrate 4-MU ester and detecting the carboxylesterase and optionally lipase activity of at least one contaminating host cell protein using a hydrophilic and optionally lipophilic substrate by detecting the fluorescence intensity of the released chromophore.Furthermore, a method is provided for producing a recombinant protein of interest, comprising using, during production, a method for detecting carboxylesterase activity of contaminating host cell proteins in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture using a hydrophilic and optionally further lipophilic substrate to determine contaminating carboxylesterase and / or lipase activity in the sample comprising the recombinant protein of interest. [Background technology]

[0002] Proteins as therapeutic agents have become more and more popular over the past few decades. Formulations containing therapeutic proteins, such as monoclonal antibodies, often contain high protein concentrations of 100 mg / ml or more, and often require the presence of 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 prone to degradation, which can have a detrimental effect on product quality. Degradation can affect product quality not only due to the resulting reduction in polysorbate concentration in the formulation, but also due to the formation of visible and non-visible particles from insoluble substances due to polysorbate degradation, such as fatty acid and polyoxyethylene side chains. Polysorbates can be degraded chemically or enzymatically. Chemical polysorbate degradation is mainly caused by oxidation reactions that lead to the formation of aldehydes, ketones, and fatty acids, among others. Enzymatic polysorbate degradation is characterized by hydrolysis of the ester bonds connecting polyethoxylated sorbitan with fatty acids (Dwivedi et al., 2018, International Journal of Pharmaceutics 552:442-436). Although oxidative degradation of polysorbates has been known for many years, enzymatic hydrolysis of polysorbates in antibody formulations has only recently been considered as one of the major degradation pathways. Recently, polysorbate degradation has emerged as a major challenge in the biopharmaceutical industry.

[0004] It has been reported that residual host cell proteins (HCPs) with hydrolytic activity, such as lipases and other esterases, including carboxylesterases, in the finished drug product (DP) can lead to polysorbate degradation. The identified enzymes were mainly assigned to the class of lipases, the subclass of esterases, which catalyze the hydrolysis of lipids. The role of lipases in the degradation of polysorbates in antibody formulations was further highlighted by Chiu et al., who showed that recovered cell culture fluid (HCCF) derived from lipoprotein lipase (LPL) knockout CHO cells reduced the degradation of PS20 and PS80 compared to wild-type CHO cells (Chui et al., 2017, Biotechnol. Bioeng. 114, 1006-1015).Activity-based protein profiling assays also detected group XV lysosomal phospholipase A2 isomer X1 (LPLA2), putative phospholipase B-like 2 (PLBL2), hepatic carboxylesterase, group VII phospholipase A2 (PLA2G7), lysophospholipase 2 (LYPLA2), lysosomal acid lipase (LIPA), sialic acid acetylesterase (SIAE), palmitoyl-protein thioesterase 1 (PPT1), and lipoprotein lipase (LPL), pointing to these enzymes as potentially crucial enzymes for PS degradation in the final pharmaceutical product (Hall et al., 2016, Journal of Pharmaceutical Sciences, 105(5): 1633-1642; Dixit et al., 2016, Journal of Pharmaceutical Sciences, 105(5): 1657-1666; Zhang et al., 2020, Journal of Pharmaceutical Sciences, 109(11): 3300-3307; Chiu et al., 2017, Biotechnology and bioengineering 114(5): 1006-1015; Li et al., 2020, Anal. Chem., 93(23): 8161-8169; Graf et al., 2021, Journal of Pharmaceutical Sciences, 110:3558-3567). Furthermore, some of these enzymes belong to the class of esterases, such as carboxylesterases, that prefer more hydrophilic substrates. The determination of the effect of a single purification step and purification conditions on polysorbate degradation takes weeks, making the necessary changes and adaptations in upstream and especially downstream production processes for therapeutic protein production difficult.

[0005] The content and degradation of polysorbates can be studied using different analytical methods. For the quantification of polysorbates, the most commonly used method is reversed-phase liquid chromatography (such as RP-HPLC), which can be further coupled to an evaporative light scattering detector (ELSD) and a charged particle detector (CAD). Other techniques that can determine the polysorbate content consist of the fluorescent micellar assay (FMA) or the chemical complexation of the sorbitan ring with cobalt or iron thiocyanate. However, to determine whether the modification of the purification process has been successful in reducing the hydrolytic activity that contributes to polysorbate degradation, the sample of interest needs to be spiked with polysorbate and its degradation needs to be analyzed as described above. Thus, polysorbate degradation is typically evaluated by monitoring the decrease in polysorbate content over time. However, polysorbate degradation is a slow process that can take up to several weeks or months. Furthermore, the analysis is complicated and time consuming.

[0006] To develop purification conditions that minimize enzymatic polysorbate degradation in pharmaceutical products, there is a need for a rapid, reliable, sensitive, automated, high-throughput assay that can be easily adapted to different samples and that provides predictive information about hydrolytic activities that contribute to polysorbate degradation in drug substances of pharmaceutical samples. Such an assay would be useful as a tool to guide process development for the production of drug substances that co-purify with the target protein, improving product quality by minimizing polysorbate degrading activities.

[0007] Although the detection of lipase and carboxylesterase hydrolysis 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 that are only co-purified with the recombinant protein (protein of interest) from eukaryotic cells within a short period of time. For example, Tsuzuki et al., (Biosci. Biotechnol. Biochem, 2001, 65(9): 2078-2082) use fluorescent substrates to analyze the activity of several lipases from microorganisms at high concentrations. Similarly, Yoo et al., (Cell Chemical Biology, 2020, 27: 143-157) disclose a fluorogenic substrate assay for detecting lipase activity, using Triton X-100 to solubilize the highly concentrated lipase rPfMAGLLP prior to analysis. WO2010 / 024924 discloses an assay for screening lipases expressed in E. coli using fluorogenic substrates. Further fluorometric assays for detecting the activity of hydrolases, including carboxylesterases, are reported by Wolfbeis and Koller (Analytical Biochemistry, 1983, 129: 365-370), but again only isolated and concentrated hydrolases are examined. However, none of these assays are described as being used to detect lipase activity of contaminating host cell proteins in samples containing recombinant proteins purified from eukaryotic cells.

[0008] Menden et al., 2019 (Journal of Enzyme Inhibition of Medicinal Chemistry, 34(1): 1474-1480) reported a lipase activity assay in which lipase activity of a defined enzyme extract of Candida rugosa lipase (CRL) isoform was detected using 4-methylumbelliferyl butyrate (4-MUB) and 4-methylumbelliferyl palmitate (4-MUP) as substrates to verify the mode of action of the inhibitor tropolone. The assay has reported limitations including intrinsic reduction in solubility of the hydrophobic fatty acid tail, long-term autocatalysis of the substrate 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 for use in determining polysorbate degradation in samples of harvested cell culture fluid using 4-methylumbelliferyl oleate (4-MuO) as a substrate. However, the moderate sensitivity still requires incubation times of 24 hours or more. 4-Methylumbelliferyl-based substrates for detecting lipase activity have been further described by Bhargava et al., 2021 (Pharm Res. 38(3): 397-413), WO2022 / 047415, and WO2022 / 049294. These recent efforts illustrate the need for assays to detect polysorbate degradation activity during biopharmaceutical development. However, all of these assays focus on lipophilic substrates and lipase activity using conditions that favor the activity of certain polysorbate-degrading enzymes, so other polysorbate-degrading activities may be overlooked.Bhargava et al., 2021 (Pharm Res. 38(3): 397-413) and WO2022 / 047415 also mention limitations to the disclosed esterase activity assays, such as residual HCPs may differ in their hydrolysis activity towards carboxylic ester bonds in MU-C8 versus their hydrolysis activity towards carboxylic ester bonds in PS20 or PS80, and that the pH 8.0 used does not represent the low pH used in parenteral pharmaceuticals.

[0009] Therefore, rapid and high-throughput methods are needed for sensitive detection of polysorbate-degrading enzymes, such as carboxylesterases, to broaden the spectrum and complement existing assays. Summary of the Invention

[0010] The present invention relates to a method for detecting carboxylesterase activity of contaminating host cell proteins in a sample comprising a recombinant protein of interest produced in a eukaryotic cell, the method comprising the steps of: (a) providing at least one sample comprising a recombinant protein of interest produced in a eukaryotic cell in a cell culture and at least one contaminating host cell protein; (b) contacting the at least one sample with a reaction solution (comprising a hydrophilic substrate) to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS); (iii) optionally, a non-buffering salt; (c) incubating the sample and the substrate in a reaction mixture; and (d) detecting carboxylesterase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate HPTS ester and detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or its salt (HPTS); measuring hydrolysis by detecting the fluorescence intensity of the released chromophore HPTS over time while incubating the sample and substrate in the reaction mixture according to step (c). In certain embodiments, the sample and substrate in the reaction mixture are incubated for any time between 2 minutes and 5 hours, 2 minutes and 3 hours, 2 minutes and 2 hours, or 2 minutes and 0.5 hours; and / or the reaction mixture has a volume of 300 μl or less. According to this method, multiple reaction mixtures can be analyzed in parallel, preferably in a volume of 300 μl or less.

[0011] The fluorescence of the released chromophore HPTS is preferably determined using an excitation wavelength in the range of 401-405 nm and an emission wavelength in the range of 510-516 nm. In a preferred embodiment, the substrate HPTS ester is selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof. In a more preferred embodiment, the substrate HPTS ester is selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt. Preferably, the substrate HPTS ester is used at a final concentration in the reaction mixture of 50 μM or less, or preferably 30 μM or less.

[0012] In a preferred embodiment, the method further comprises (bi) contacting at least one sample comprising a recombinant protein of interest produced in a eukaryotic cell in cell culture and at least one contaminating host cell protein of step (a) with a reaction solution comprising a lipophilic substrate in a separate reaction setting to form a reaction mixture, the reaction solution comprising (i) a buffer having a pH of about pH 4 to about pH 8, (ii) a lipophilic 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-C16) 4-MU ester, (iii) optionally a non-buffering salt, and (iv) (ci) incubating the sample and substrate in the reaction mixture of step (bi); and (di) detecting lipase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; the method may further comprise the steps of: (i) detecting the hydrolysis of the substrate 4-MU ester 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 (ci).

[0013] The lipophilic substrate is preferably selected from the group consisting of 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), 4-methylumbelliferyl undecanoate, and 4-methylumbelliferyl dodecanoate. Micelle formation improves lipase activity and solubilization of the lipophilic substrate. Thus, according to the present invention, the surfactant used in the reaction solution containing the lipophilic substrate has a final concentration in the reaction mixture that exceeds its critical micelle concentration in the reaction mixture. In certain embodiments, the surfactant is selected from the group consisting of CHAPS, CHAPSO, and Zwittergent, preferably CHAPS. When the surfactant is CHAPS, the surfactant is preferably 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. In certain embodiments, the surfactant is not polyethylene glycol tert-octylphenyl ether (Triton X-100) or polyethylene glycol nonylphenyl ether (NP-40).

[0014] Buffers used in the methods of the invention (using hydrophilic and / or lipophilic substrates) include formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl)methyl(me-thyl)]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 The buffer may include one or more buffer substances selected from the group consisting of: 1-(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 5 to about 7.5, preferably the buffer has a pH of about 5.5 to about 7.0. The buffer may also be 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.

[0015] Optionally, the non-buffering salt may be selected from the group consisting of NaCl, KCl, and CaCl2, and preferably, the non-buffering salt is NaCl or KCl. Further, the non-buffering salt may have a concentration in the reaction mixture of about 100 mM to about 200 mM, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM. In certain embodiments, 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; and / or the cumulative ionic strength of the buffer and non-buffering salt in the reaction mixture is about 450 mM or less in the reaction mixture, preferably about 400 mM or less, more preferably about 350 mM or less.

[0016] The at least one sample according to certain embodiments is a harvested cell culture fluid (HCCF), an in-process control (IPC) sample, a UF / DF filtrate, a drug substance sample, or a drug product sample. In a preferred embodiment, the recombinant protein of interest is produced in CHO cells and the at least one contaminating host cell protein is a CHO host cell protein (CHOP). According to the present invention, the recombinant protein of interest is not a carboxylesterase or lipase and / or an enzyme having carboxylesterase or lipase activity. Preferably, the recombinant protein of interest is selected from the group consisting of an antibody, an antibody fragment, an antibody-derived molecule, and a fusion protein.

[0017] The present invention relates to a method for producing a recombinant protein of interest, comprising the steps of: (i) culturing in a cell culture eukaryotic cells expressing the recombinant protein of interest; (ii) recovering the recombinant protein of interest; (iii) purifying the recombinant protein of interest; and (iv) optionally formulating the recombinant protein of interest into a pharma- ceutically acceptable formulation suitable for administration; and (v) obtaining at least one sample comprising the recombinant protein of interest in steps (ii), (iii) and / or (iv); detecting carboxylesterase activity in the sample comprising the recombinant protein of interest and at least one contaminating host cell protein, the method comprising the steps of: (a) detecting the recombinant protein of interest produced in the eukaryotic cells in the cell culture and the at least one contaminating host cell protein; (b) contacting the at least one sample obtained in step (v) with a reaction solution comprising a hydrophilic substrate to form a reaction mixture, the reaction solution comprising (i) a buffer having a pH of about pH 4 to about pH 8, (ii) a hydrophilic substrate which is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester), and (iv) optionally a non-buffering salt; (c) incubating the sample and the substrate in the reaction mixture; and (d) detecting carboxylesterase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate HPTS ester and detecting the fluorescence intensity of the released chromophore HPTS;Optionally, detecting lipase activity in a sample, comprising: (bi) contacting at least one sample comprising the recombinant protein of interest produced in a eukaryotic cell of step (a) and at least one contaminating host cell protein, in a separate reaction setting, with a reaction solution comprising a lipophilic substrate to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a lipophilic 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-C16) 4-MU ester; (iii) optionally a non-buffering salt; and (iv) an ester. (c) comprising a non-denaturing surfactant that does not have a carboxyl bond and is a non-ionic or zwitterionic surfactant; (ci) incubating the sample and substrate in a reaction mixture; (di) detecting lipase activity of at least one contaminating host cell protein 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 HPTS and / or 4-MU over time while incubating the sample and substrate in a reaction mixture according to step (c) or (ci), respectively;

[0018] The method for producing according to the present invention may further comprise obtaining at least one sample comprising the recombinant protein of interest in step (ii), where the sample is a harvested cell culture fluid (HCCF) or a cell lysate; in step (iii), where the sample is an in-process control (IPC) sample; and / or in step (iv), where the sample is a UF / DF sample, drug substance sample, or drug product sample. In a preferred embodiment, the method comprises obtaining at least one sample in step (iii), comprising the recombinant protein of interest produced in eukaryotic cells in cell culture and at least one contaminating host cell protein, comprising obtaining the at least one sample before and after affinity chromatography, before and after acid treatment, before and after depth filtration, and / or before and after ion exchange chromatography, preferably anion exchange chromatography or cation exchange chromatography.

[0019] In yet another aspect, the present invention relates to a kit for determining contaminating carboxylesterase and / or lipase activity in a sample comprising a recombinant protein of interest, comprising: (i) a buffer having a pH of about pH 4 to about pH 8; and (ii) hydrophilic and lipophilic substrates, wherein (a) the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester); (b) the lipophilic substrate comprises 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-C16) 4-MU ester (substrate 4-MU ester); and optionally (iii) a non-buffering salt; and / or (iv) a non-denaturing surfactant that does not have an ester bond and is a nonionic or zwitterionic surfactant.

[0020] In certain embodiments, the hydrophilic substrate HPTS ester in the kit is 1-octanoyloxypyrene-3,6,8-trisulfonic acid or its salt (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid or its salt, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid or its salt (1-octanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt (OPTS), 1-nonao ... and / or the lipophilic substrate 4-MU ester 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. The kit according to the invention may further comprise one or more microtiter plates having 96 wells or a multiple of 96 wells.

[0021] In yet another aspect, the present invention relates to the use of the hydrophilic substrate HPTS ester, or the hydrophilic substrate HPTS ester and the lipophilic substrate 4-MU ester, as substrates for detecting carboxylesterase activity, or carboxylesterase activity and lipase activity (respectively) of contaminating host cell proteins in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture in an assay, preferably wherein the recombinant protein is produced in CHO cells, the at least one contaminating host cell protein is a CHO host cell protein (CHOP), the hydrophilic substrate is a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester) and the lipophilic substrate is a saturated unbranched chain fatty acid (C6-C16) 4-MU ester.

[0022] In yet another aspect, the present invention relates to the use of the hydrophilic substrate HPTS ester as a substrate for detecting in an assay the carboxylesterase activity of at least one contaminating host cell protein in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture, preferably wherein the recombinant protein is produced in CHO cells, the at least one contaminating host cell protein is a CHO host cell protein (CHOP), and the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or its salts (substrate HPTS ester). [Brief description of the drawings]

[0023] [Figure 1] (A) Schematic representation of the esterase family potentially involved in polysorbate degradation. (B) Schematic representation illustrating esterases detected by the novel OPTS and 4-MUD assays, showing carboxylesterases and minor overlapping and non-overlapping complementary esterase groups with lipases. (C) Schematic representation of the hydrolysis reaction of the substrate 4-methylumbelliferyl decanoate (4-MUD) to form the fluorophore 4-methylumbelliferone (4-MU). (D) Schematic representation of the hydrolysis reaction of the substrate 1-octanoyloxypyrene-3,6,8-trisulfonic acid (OPTS) trisodium salt to form the fluorophore 1-hydroxypyrene-3,6,8-trisulfonic acid (HPTS) trisodium salt. [Diagram 2] (A) Spectra for pH values ​​from 4 to 8 (top) for HPTS with an emission wavelength of 520 nm and excitation wavelengths of 300 to 500 nm, and for pH = 5.5 (bottom) for HPTS with an excitation wavelength of 415 nm and emission wavelengths of 420 to 620 nm, (B) Calibration curves for different pH values ​​(4 to 8) for HPTS concentrations ranging from 1.46 to 3000 nM (top), and for pH = 7 for HPTS concentrations ranging from 93.75 to 6000 nM (bottom). [Diagram 3] FIG. 1 shows kinetic measurements over 2 hours for 25 μM OPTS solution with AMT buffer at pH=5.5 (closed squares) and pH=8 (open triangles). [Figure 4] FIG. 1 shows kinetic measurements over 30 min for a reaction mixture with 1 mg×mL-1 PPL, 25 μM OPTS and AMT (pH=7). [Diagram 5] (A) Kinetic measurements of a reaction mixture with 1 mg×mL-1 PPL, 25 μM OPTS and AMT (pH=7) in the presence or absence of 10 mM CHAPS over 30 min, and (B) the same reaction mixture in the presence of the indicated concentrations of Tween 20. [Figure 6] (A) Kinetic measurements for reaction mixtures with 1 mg×mL-1 PPL, 100 μM OPTS in 150 mM Tris, 0.25% (w / v) Triton X-100, 0.125% (w / v) gum arabic, pH=8 (TTG buffer, as described for the esterase activity assay in WO2020 / 047416), and AMT buffer, pH=8, in the presence or absence of 10 mM CHAPS over 30 min. [Figure 7] Figure 1 shows kinetic measurements for nine different antibody preparations in 25 μM OPTS, AMT buffer (pH=5.5), 75 μL of each antibody solution (labeled 1-9) and HO to 300 μL. Kinetics were recorded over 30 min at 25° C. with n=2 measurements, results represent arithmetic mean values ​​and error bars represent maximum error. [Figure 8] Figure 1 shows kinetic measurements for nine different antibody preparations containing 30 μM 4-MUD substrate, AMT buffer (pH=5.5), 75 μL of each antibody solution (labeled 1-9) and HO to 300 μL. Kinetics were recorded in duplicate at 25° C. over 30 min, results represent arithmetic mean values ​​and error bars represent maximum error. [Figure 9]Kinetic measurements were performed using 25 μM OPTS (filled squares) or 30 μM 4-MUD substrate (open circles), AMT buffer (pH=5.5), 75 μL of each antibody solution (labeled 1-9), and HO to 300 μL. Kinetics were recorded in duplicate at 25° C. for 30 min, results represent arithmetic mean values, and error bars represent maximum error. [Figure 10] Kinetic measurements of 30 μM 4-MUD substrate, AMT buffer (pH=5.5), 75 μL of each antibody solution (labeled 1-9), 10 μM orlistat (squares) or 1 mM PMSF (circles) or 10 mM EDTA (triangles) and HO to 300 μL. Kinetics were recorded over 30 min at 25° C. with n=2, results represent arithmetic mean values ​​and error bars represent maximum error. [Figure 11] Kinetic measurements on lipase with 25 μM OPTS (black squares) or 30 μM 4-MUD substrate (white circles), AMT (pH=5.5), with or without different inhibitors (10 μM orlistat, 1 mM PMSF, 10 mM EDTA) and HO to 300 μL. Final lipase concentration is 0.007 mg×mL-1. Activity corresponds to the average activity units and error bars correspond to the maximum error for n=2 measurements. [Figure 12] Figure 1 shows pH profiles for lipase measured in 25 μM OPTS solution (closed squares) and 30 μM 4-MUD substrate solution (open circles) with AMT at the indicated pH and 0.007 mg×mL of lipase. Activity in units is plotted against buffer pH and error corresponds to the maximum error for n=2 measurements. [Figure 13] Figure 1 shows pH profiles for Ab9 preparations measured in 25 μM OPTS (closed squares) and 30 μM 4-MUD substrate solutions (open circles) with AMT at the indicated pH. Activity in units is plotted against buffer pH and errors correspond to the maximum error for n=2 measurements. [Figure 14](A) pH profiles for the two preparations measured in 25 μM OPTS solution (closed squares) and 30 μM 4-MUD substrate solution (open circles) with AMT at the indicated pH, and (B) scaling for the results measured with 4-MUD substrate. Activity in units is plotted against buffer pH and the error corresponds to the maximum error for n=2 measurements. [Figure 15] (A) pH profiles for the five preparations measured in 25 μM OPTS (solid squares) and 30 μM 4-MUD substrate (open circles) solutions with AMT at the indicated pH, and (B) scaling of the results with the coumarin substrate. Activity in units is plotted against the pH of the buffer and the error corresponds to the maximum error for n=2 measurements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0024] The term "including" or "comprising" means "including, but not limited to." The term "including" or "comprising" is intended to be open-ended so as to specify the presence of any stated feature, element, integer, step, or component, but not to exclude the presence or addition of one or more other features, elements, integers, steps, components, or groups thereof. Thus, the term "comprising" includes the more restrictive terms "consisting of" and "consisting essentially of." Furthermore, the singular and plural forms are not used in a restrictive manner. As used herein, the singular forms "a," "an," and "the" refer to both the singular and the plural, unless expressly stated to refer to the singular only.

[0025] As used herein, the term "sample" refers to any sample that contains a recombinant protein of interest produced in eukaryotic cells in cell culture: at least one sample can be, for example, a harvested cell culture fluid (HCCF) or cell lysate, an in-process control (IPC) sample, an ultrafiltration / dialysis sample (UF / DF sample), a drug substance (also referred to as bulk drug substance) sample, or a pharmaceutical product sample that contains a recombinant protein of interest, such as an antibody, an antibody fragment, an antibody-derived molecule, or a fusion protein (e.g., an Fc-fusion protein). As used herein, a recombinant protein of interest contained in a sample is not a carboxylase or lipase and / or does not contain carboxylase or lipase activity. Thus, any carboxylase or lipase activity detected in a sample is a contaminating carboxylase or lipase activity and / or is derived from at least one contaminating protein that has carboxylase or lipase activity, such as a host cell protein (HCP) derived from a eukaryotic cell.

[0026] As used herein, the term "contaminating" or "impurities" refers to the presence of undesired and / or unintended substances, such as endogenous proteins produced by host cells, also referred to as host cell proteins, in particular host cell proteins with esterase (carboxylesterase or lipase) activity, which are present only as minor components compared to the recombinant protein of interest, such as antibodies or antibody-like compounds. In the context of the present invention, hydrolytic activity, in particular lipase activity or carboxylesterase activity, is undesired due to its polysorbate degrading potential, which may be co-purified together with the recombinant protein of interest. This applies in particular to the final formulated protein preparation, which advantageously 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.

[0027] 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. As used herein, the term "carboxylesterase activity" refers to the activity of a substance, typically a protein (enzyme), that catalyzes the hydrolysis of ester bonds in carboxylic acid esters. Lipases or carboxylesterases are hydrolase enzymes that split esters into acids and alcohols in a chemical reaction involving water, also referred to as hydrolysis. Many lipases and carboxylesterases belong to the class of carboxylic ester hydrolases (EC 3.1.1). Carboxylesterases form a separate class, carboxylesterases (EC 3.1.1.1), whereas lipases include, but are not limited to, triacylglycerol lipase (EC 3.1.1.3), phospholipase A2 (EC 3.1.1.4), lysophospholipase (EC 3.1.1.5), acylglycerol lipase (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 phospholipase D (EC 3.1.4.50), or N-acetylphosphatidylethanolamine hydrolyzing phospholipase D (EC 3.1.4.54); or a glycosphingolipid deacylase (EC 3.5.1.69).

[0028] Carboxylesterases or lipases are contaminants in the context of the present invention that are undesirable, often difficult to remove, and may or may not mediate polysorbate degrading enzyme activity. Contaminating carboxylesterases may be immunogenic and / or have polysorbate degrading activity, such as sialic acid acetyl esterase (SIAE). Carboxylesterases prefer hydrophilic substrates and are detected using an assay with the substrate HPTS ester. Similarly, contaminating lipases may be immunogenic and / or have polysorbate degrading activity, such as PLBL2, LPL, LPLA2, PLA2G7, LYPLA2, and LIPA. Lipases prefer lipophilic substrates and are detected using an assay with the substrate 4-methylumbelliferyl (4-MU) ester. As used herein, the term "hydrolase activity" is a more general term that includes lipase activity and carboxylesterase activity, and also refers to the hydrolysis of compounds other than lipids or carboxylic acid esters, such as thioester hydrolases (EC 3.1.2), such as palmitoyl protein thioesterase 1 (PPT1), which may also be detected by the combination of assays disclosed herein.

[0029] 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 are post-translationally modified through reactions including, but not limited to, glycosylation, acetylation, phosphorylation, glycation, or protein processing. Modifications and changes, such as fusion with other proteins, amino acid sequence substitutions, deletions, or insertions, can be made in the structure of a polypeptide while the molecule retains its biologically functional activity. For example, certain amino acid sequence substitutions can be made in the polypeptide sequence or in the underlying nucleic acid coding sequence, yet still result in a protein with the same properties.

[0030] The term "recombinant protein" as used herein relates to a protein produced by recombinant methods such as molecular cloning methods, and may also be referred to as a recombinant protein of interest. A recombinant protein as used herein is a protein of interest, for example in a sample to be purified. Recombinant 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 different species than the recipient host cells, e.g. CHO cells or HEK293 cells, used for the production of the protein in cell culture, or based on artificial sequences such as fusion proteins. In the context of the present invention, a 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, bispecific or multispecific antibody), or a fusion protein (e.g. Fc-fusion protein). Thus, in one embodiment, the recombinant protein is selected from the group consisting of an antibody, an antibody fragment, an antibody-derived molecule, and a fusion protein.

[0031] As used herein, the term "eukaryotic cell" 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 cells" particularly encompass mammalian cells, such as Chinese Hamster Ovary (CHO) cells or HEK293 cell-derived cells, and yeast cells. As used herein, mammalian cells refer to cells of mammalian origin, particularly cell lines. In the present invention, "mammalian cells" particularly encompass human cells or rodent cells, and in most cases Chinese Hamster Ovary (CHO) cells or their derivatives. The cells referred to herein are cells maintained in culture and not primary cells, but relate to cell lines or cell line-derived cells, i.e. immortalized cells.

[0032] The term "drug substance (DS)" refers to an active pharmaceutical ingredient (API) formulated with excipients. The API mediates a therapeutic effect in the body, as opposed to excipients that aid in the delivery of the API. In the case of biotherapeutics, API formulated with excipients typically means the API in the final formulation buffer at least at the highest concentration used in the final dosage form, also referred to as the drug product. The term "drug product", as used herein and abbreviated as DP, refers to the final commercial dosage form of a drug substance, e.g., a tablet or capsule, or, in the case of biopharmaceuticals, typically a solution for injection in a suitable container, such as a vial or syringe. The drug product may also be in lyophilized form.

[0033] As used herein, the term "polysorbate 20" refers to a non-ionic polysorbate-type surfactant that is a laurate ester of sorbitol and its anhydrides copolymerized with about 20 moles of ethylene oxide for each mole of sorbitol and sorbitol anhydride (polyoxyethylene (20) sorbitan monolaurate; CAS number: 9005-64-5). It is also known as Tween 20. Its safety and relative non-toxicity allow it to be used as a surfactant / detergent and emulsifier in many national scientific analyses. Polysorbate 20 can be used as a detergent in immunoassays, Western blots, and ELISAs. Polysorbate 20 can further be used in pharmacological applications, such as pharmaceutical formulations, especially biopharmaceutical formulations, such as antibodies and Fc fusion proteins. In particular, polysorbate 20 helps prevent non-specific antibody binding. As used herein, the term "polysorbate 80" refers to a non-ionic polysorbate-type surfactant that is a mixture of partial esters of fatty acids, primarily oleic acid, with sorbitol and its anhydrides, ethoxylated with about 20 millimoles of ethylene oxide for each mole of sorbitol and sorbitol anhydride (polyoxyethylene (20) sorbitan monooleate; CAS number: 9005-65-6). It is also known as Tween 80 and is used interchangeably with polysorbate 20.

[0034] As used herein, the term "therapeutic protein" refers to proteins that can be used in human and / or animal medical treatment, including, but not limited to, antibodies, growth factors, blood clotting factors, vaccines, interferons, hormones, and fusion proteins. The term "produced" as used herein relates to the production of a recombinant protein of interest, 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 that allow the growth and / or production / expression of the protein. The recombinant protein may be produced by fed-batch cell culture or by continuous cell culture. Thus, the eukaryotic cell may be cultured in fed-batch cell culture, in continuous cell culture or in a combination thereof, although fed-batch cell culture is preferred.

[0035] As used herein, the term "expressing a recombinant protein" refers to a cell that contains a DNA sequence encoding a recombinant protein of interest 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. As used herein, the term "about" refers to a 10% variation from the specified value, for example, about 50% would retain a variance of 45-55%.

[0036] As used herein, the term "detecting the carboxylesterase activity of at least one contaminating host cell protein" refers to measuring the hydrolysis of a substrate HPTS ester by detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or its salt (HPTS). Thus, as used herein, "detecting the carboxylesterase activity of at least one contaminating host cell protein by measuring the hydrolysis of a substrate HPTS ester and detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or its salt (HPTS)" is used synonymously with "detecting the carboxylesterase activity of at least one contaminating host cell protein comprising (or through / via) measuring the hydrolysis of a substrate HPTS ester by detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or its salt (HPTS)". It is understood that the step of measuring the hydrolysis of the substrate HPTS ester is carried out by detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (HPTS).

[0037] Similarly, as used herein, the term "detecting lipase activity of at least one contaminating host cell protein" refers to and is performed by measuring the hydrolysis of a substrate 4-MU ester by detecting the fluorescence intensity of the released chromophore 4-MU. Thus, as used herein, "detecting lipase activity of at least one contaminating host cell protein by measuring the hydrolysis of a substrate 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU" is used synonymously with "detecting carboxylesterase activity of at least one contaminating host cell protein, comprising (or through / via) measuring the hydrolysis of a substrate 4-MU ester by detecting the fluorescence intensity of the released chromophore 4-MU." It is understood that measuring the hydrolysis of a substrate 4-MU ester is performed by detecting the fluorescence intensity of the released chromophore 4-MU.

[0038] Method for detecting carboxylesterase activity of contaminating host cell proteins The present invention relates to a method (in vitro method) for detecting carboxylesterase activity of contaminating host cell proteins in a sample comprising a recombinant protein of interest produced in a eukaryotic cell, the method comprising the steps of: (a) providing at least one sample comprising the recombinant protein of interest produced in a eukaryotic cell in cell culture and at least one contaminating host cell protein; (b) contacting the at least one sample with a reaction solution (comprising a hydrophilic substrate) to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a hydrophilic substrate which is a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester); and (iii) optionally a non-buffering salt. (c) incubating the sample and the substrate in a reaction mixture; (d) detecting the carboxylesterase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate HPTS ester and detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (HPTS); and optionally measuring the hydrolysis by detecting the fluorescence intensity of the released chromophore HPTS over time while incubating the sample and the substrate in a reaction mixture according to step (c). Thus, detecting the carboxylesterase activity of at least one contaminating host cell protein in step (d) comprises measuring the hydrolysis of the substrate HPTS ester by detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (HPTS), and optionally measuring the hydrolysis by detecting the fluorescence intensity of the released chromophore HPTS over time while incubating the sample and the substrate in a reaction mixture according to step (c). The reaction solutions used in the method of the present invention are aqueous reaction solutions. A person skilled in the art will understand that in step (b), at least one portion of the sample is used for each reaction mixture.Furthermore, more than one sample may be analyzed in parallel, and / or more than one portion of at least one sample may be analyzed in parallel. These more than one sample and / or more than one portion may be analyzed under the same conditions, or may be analyzed under distinct conditions, such as analyzing portions (or duplicates or triplicates) of at least one sample, each with a different pH value. This method is particularly suitable for small volume analysis and high throughput analysis. Thus, multiple reaction mixtures may be analyzed in parallel, such as in a microtiter plate with 96 wells or a multiple of 96 wells. Preferably, each reaction mixture volume is 300 μl or less. Preferably, each reaction mixture volume is 300 μl or less.

[0039] The assay readout may be 20 minutes or even faster. Thus, 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, or less than 0.5 hours. To obtain sufficient data points, it is recommended 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 5 hours, 2 minutes and 3 hours, 2 minutes and 2 hours, or 2 minutes and 0.5 hours, or between 2 minutes and less than 5 hours, less than 3 hours, less than 2 hours, or less than 0.5 hours. In certain embodiments, multiple reaction mixtures, such as 12 or more, 24 or more, 36 or more, 72 or more, or 96 or more, are analyzed in parallel and / or the reaction mixture has a volume of 300 μl or less. At least one sample may be an HCCF, an in-process control (IPC) sample, a UF / DF sample, a drug substance, or a drug product. According to the present invention, the recombinant protein of interest in the sample is not a carboxylesterase and / or does not contain carboxylesterase activity.

[0040] In certain embodiments, the fluorescence of the emitted chromophore HPTS is determined using an excitation wavelength in the range of 401-405 nm and an emission wavelength in the range of 510-516 nm, preferably using an excitation wavelength in the range of 402-404 nm and an emission wavelength in the range of 510-514 nm, and more preferably using an excitation wavelength of 403 nm and an emission wavelength of 512 nm.

[0041] The hydrolysis may be stopped at a certain time point before the detection of the fluorescence intensity of the released HPTS chromophore. Alternatively, and preferably, the fluorescence intensity of the released HPTS chromophore may be detected in real time without stopping the hydrolysis of the hydrophilic substrate HPTS ester. In certain embodiments, the fluorescence intensity of the released HPTS chromophore is detected without stopping the hydrolysis of the hydrophilic substrate HPTS ester. In certain embodiments, the hydrolysis is measured by detecting the fluorescence intensity of the released HPTS chromophore over time while incubating the sample and the substrate in the reaction mixture according to step (c).

[0042] The hydrophilic substrate containing the chromophore HPTS is in the form of a saturated unbranched fatty acid (C6-C12) HPTS ester, the acyl chain of the saturated unbranched fatty acid having carbon atoms C6-C12. As used herein, the abbreviation "HTPS" refers to 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof, preferably the trisodium salt. Similarly, as used herein, the abbreviation "substrate HPTS ester" refers to a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof, preferably the trisodium salt. As used herein, the abbreviation "OPTS" refers to 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, preferably the trisodium salt. Saturated unbranched fatty acid (C6-C12) HPTS esters, such as 1-octanoyloxypyrene-3,6,8-trisulfonic acid (OPTS) trisodium salt, are hydrolyzed in the presence of a carboxylesterase to a saturated unbranched fatty acid (C6-C12) such as octanoic acid and -hydroxypyrene-3,6,8-trisulfonic acid (HPTS) trisodium salt, as shown below:

[0043] [ka]

[0044] This substrate mimics the key features of polysorbates, namely, the fatty acid ester bond and the long-chain acyl chain. Polysorbate 20 is an ester of lauric acid, which is a saturated unbranched fatty acid. In contrast, polysorbate 80 is an ester of oleic acid, which is an unsaturated fatty acid. The hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or its salt (substrate HPTS ester). The shorter the saturated unbranched fatty acid chain (<C6), the more the substrate HPTS ester increases self-hydrolysis, while the longer the saturated unbranched fatty acid chain (>C12), the more the substrate HPTS ester increases lipophilicity and thus is not a suitable substrate for carboxylesterase. Therefore, the substrate HPTS ester is selected from the group consisting of 1-hexanoyloxypyrene-3,6,8-trisulfonic acid, 1-heptanoyloxypyrene-3,6,8-trisulfonic acid, 1-octanoyloxypyrene-3,6,8-trisulfonic acid (OPTS), 1-nonanoyloxypyrene-3,6,8-trisulfonic acid, 1-decanoyloxypyrene-3,6,8-trisulfonic acid, 1-undecanoyloxypyrene-3,6,8-trisulfonic acid, 1-dodecanoyloxypyrene-3,6,8-trisulfonic acid, and salts thereof. Preferably, the substrate HPTS ester is a C8-C10 ester such as an ester selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid or its salt (OPTS), 1-nonanoyloxypyrene-3,6,8-trisulfonic acid or its salt, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid or its salt. The salt can be any salt such as a sodium salt or a potassium salt.In a preferred embodiment, the chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or salt thereof is 1-hydroxypyrene-3,6,8-trisulfonic acid trisodium salt, and the substrate HPTS ester is 1-hexanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, 1-heptanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, 1-octanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, 1-decanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, 1-hex ..., 1-hexanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, 1-hexano It is selected from the group consisting of oxypyrene-3,6,8-trisulfonic acid trisodium salt, 1-undecanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, and 1-dodecanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, and more preferably selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid (OPTS) trisodium salt, 1-nonaoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt.

[0045] The substrate HPTS ester is a hydrophilic substrate and is therefore easily soluble in water. Organic solvents and detergents tend to interfere with the assay with the hydrophilic substrate HPTS ester and / or quench the chromophore HPTS. Therefore, the hydrophilic substrate HPTS ester is preferably dissolved in an aqueous solution, such as in water or an aqueous buffer, more preferably in an aqueous solution that does not contain DMSO or DMF.

[0046] A suitable concentration of hydrophilic substrate (in the reaction mixture) in the method of the present invention may be about 1 μM to about 1 mM. Preferably, the hydrophilic substrate HPTS ester is used at a final concentration in the reaction mixture of 50 μM, more preferably 30 μM or less. 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 about 300 μM, preferably about 1 μM to about 50 μM, preferably about 1 μM to about 30 μM, more preferably about 3 μM to about 30 μM. In certain embodiments, the substrate is provided as a stock solution in an aqueous solution. As used herein, the term "aqueous solution" means a water-based solution, such as water or a buffer (such as a buffer used in the reaction solution), preferably free of organic solvents. The stock solution is added at about 1% to about 10% (v / v) of the reaction mix.

[0047] Furthermore, it is essential that the reaction solution containing the substrate HPTS ester does not contain any surfactants above its critical micelle concentration (CMC), and preferably the reaction solution containing the substrate HPTS ester does not contain any surfactants (e.g. non-ionic surfactants, non-ionic surfactants or zwitterionic surfactants). Surfactants (e.g. non-ionic surfactants or zwitterionic surfactants) include in particular non-denaturing surfactants (non-ionic or zwitterionic surfactants) without ester bonds as described herein, as well as surfactants containing ethoxylates and / or polyethylene glycol groups (such as 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)) and / or surfactants containing aromatic rings. Thus, in a specific embodiment, the reaction solution does not contain surfactants such as Triton X-100, NP-40 or CHAPS, in particular not above their respective CMCs. Preferably, no surfactant is present in the reaction solution or in the reaction solution containing the hydrophilic substrate and in the reaction mixture at or above its critical micelle concentration (CMC), more preferably no surfactant is present in the reaction solution or in the reaction solution and reaction mixture. In certain preferred embodiments, the reaction solution used in the method according to the invention, comprising (i) a buffer, (ii) a hydrophilic substrate, and optionally (iii) a non-buffering salt, does not contain a surfactant. The skilled artisan will understand that since the sample being analyzed may contain a surfactant, the reaction mixture may also contain a small amount of surfactant after contacting the reaction solution sample. The assay with the hydrophilic substrate HPTS ester is intended for in-process control samples for the production of recombinant proteins of interest as a rapid high-throughput assay for purification train development and optimization. Most in-process control samples do not contain surfactants such as polysorbate 20 or polysorbate 80. Samples that may contain surfactants are mainly the final drug substance and possibly the harvested cell culture fluid (HCCF) due to the antifoaming agent.However, considering the high hydrolase activity in HCCF (before purification), the sample needs to be diluted so that the concentration reached by the potential antifoaming agent will not interfere with the assay. Thus, the final reaction mixture (reaction solution containing sample and hydrophilic substrate) may contain low concentrations of polysorbate 20 or polysorbate 80 from the samples described herein. In terms of the final product, most drug substance samples have non-critical polysorbate concentrations (such as about 50 μg / ml or less of polysorbate 20) after dilution in the reaction mix. Alternatively, if required, UF / DF samples, i.e., before the addition of polysorbate, can be used to determine hydrolytic activity. The concentration of polysorbate 20 or polysorbate 80 in the reaction mixture is preferably below the CMC and / or does not exceed about 100 μg / ml, preferably about 75 μg / ml, more preferably about 50 μg / ml for polysorbate 20 (or about 60 μg / ml, preferably about 50 μg / ml, preferably about 30 μg / ml, more preferably about 20 μg / ml for polysorbate 80). However, the inventors also note that in this context the assay is particularly useful for high throughput analysis of small volumes of IPC samples for process optimization, which typically do not contain polysorbate.

[0048] It has further been observed that histidine may inhibit the method according to the invention using the hydrophilic substrate HPTS ester. Histidine is often used in formulation buffers. If histidine is present in the sample, it shall be dialyzed prior to the method according to the invention so as to remove it from the sample. Methods for preparative dialysis are well known in the art. For example, an antibody solution may be dialyzed against 500 volumes of 0.002% (w / v) NaCl solution using a pre-humidified dialysis cassette, replacing the 0.002% (w / v) NaCl solution at least once, with gentle stirring. Each dialysis step is maintained for about 1-2 hours. Histidine concentrations below 200 μM have been found to be acceptable. Thus, in one embodiment, a sample containing the protein of interest is dialyzed until a histidine concentration below 200 μM is reached. An alternative method would be the use of dextran-epichlorohydrin copolymer (Sephadex®) columns (size exclusion chromatography) or affinity chromatography. In certain embodiments, the sample containing the recombinant protein of interest produced in eukaryotic cells in cell culture and at least one contaminating host cell protein contains less than 200 μM histidine, preferably less than 100 μM histidine. Thus, as regards the final product, the drug substance sample may be dialyzed, if required, and the UF / DF sample, i.e., the UF / DF sample before the addition of histidine (and polysorbate), may be used to determine the hydrolytic activity. However, the inventors note that in this context the assay is particularly useful for high-throughput analysis of small volumes of IPC samples for process optimization, which typically do not contain histidine.

[0049] The method according to the invention may further comprise a step of analyzing at least one sample using the lipophilic substrate 4-MU ester. Thus, the method may further comprise a step of detecting lipase activity in a sample comprising a recombinant protein of interest produced in a eukaryotic cell and at least one contaminating host cell protein. In certain embodiments, the method according to the invention comprises the steps of (bi) contacting at least one sample comprising a recombinant protein of interest produced in a eukaryotic cell in cell culture and at least one contaminating host cell protein of step (a) with a reaction solution comprising a lipophilic substrate in a separate reaction setting to form a reaction mixture, the reaction solution comprising (i) a buffer having a pH of about pH 4 to about pH 8, (ii) a lipophilic 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-C16) 4-MU ester (substrate 4-MU ester), (iii) optionally , non-buffer salts, and iv) a non-denaturing surfactant that does not have an ester bond and is a non-ionic or zwitterionic surfactant; (ci) incubating the sample and substrate in the reaction mixture of step (bi); (di) further comprising the steps of detecting lipase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU; 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 (ci). Thus, detecting lipase activity of at least one contaminating host cell protein in step (di) comprises measuring hydrolysis of the substrate 4-MU ester by detecting the fluorescence intensity of the released chromophore 4-MU, and may comprise 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 (ci). Those skilled in the art will appreciate that in step (bi), at least one portion of the sample is used for each reaction mixture.More specifically, in step (b), a portion of at least one sample is used for each reaction mixture, and in step (bi), a portion of another at least one sample is used for each reaction mixture. Furthermore, more than one sample may be analyzed in parallel, and / or more than one portion (or fraction) of at least one sample may be analyzed in parallel using the substrate HPTS ester, and optionally using the substrate 4-MU ester. These more than one sample and / or more than one portion may be analyzed under the same conditions, or may be analyzed under separate conditions, such as analyzing portions (or duplicates or triplicates) of at least one sample, each with a different pH value. This method is particularly suitable for small volume analysis and high throughput analysis. Thus, multiple reaction mixtures may be analyzed in parallel, such as in a microtiter plate with 96 wells or a multiple of 96 wells. Preferably, each reaction mixture volume is 300 μl or less.

[0050] The analysis of at least one sample using the hydrophilic substrate HPTS-ester and the further lipophilic substrate 4-MU-ester broadens the spectrum of enzyme capture by the assay potentially involved in the degradation of polysorbate. The hydrophilic substrate and the hydrophilic substrate complement each other by detecting enzymes with hydrolytic activity and relatively lipophilic substrate specificity (such as lipases) and enzymes with hydrolytic activity and relatively hydrophilic substrate specificity (such as carboxylesterases). The substrates are intended to be used in parallel or sequentially using at least one identical sample. Those skilled in the art will understand that first, a portion of at least one sample is contacted with the reaction solution defined in step (b), and second, a portion of at least one sample is contacted with the reaction solution defined in step (bi).

[0051] As used herein, the term "different reaction set-up" refers to a separate reaction using a reaction solution containing the lipophilic substrate 4-MU ester, which is different from the reaction using the reaction solution containing the hydrophilic substrate HPTS ester in that it is carried out in a different reaction vessel or well (i.e., a physically separate reaction vessel or well). The different reaction sets are typically carried out using at least one identical sample for contacting and analysis, or alternatively, each reaction set-up is carried out using at least a portion of the same sample. The buffer (and optionally non-buffer salts) used in the reaction solution containing the hydrophilic substrate and in the reaction solution containing the lipophilic substrate are preferably the same. Thus, the reaction solution containing the hydrophilic substrate and the reaction solution containing the lipophilic substrate are the same, except for the substrate and surfactant present only in the reaction solution containing the lipophilic substrate.

[0052] The substrate containing the chromophore 4-MU is in the form of a 4-MU ester of a saturated unbranched fatty acid (C6-C16), the acyl chain of which has carbon atoms C6-C16. 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 lauric acid, a saturated unbranched fatty acid. In contrast, polysorbate 80 is an ester of oleic acid, an unsaturated fatty acid. The fluorescence of the released chromophore 4-MU is suitable for detection in a fluorescence spectrometer or a microplate spectrophotometer. In a particular embodiment, the fluorescence of the released chromophore 4-MU is determined using an excitation wavelength in the range of 330-340 nm and an emission wavelength of 450 nm.

[0053] Unsaturated fatty acids are larger than saturated fatty acids due to double bonds, and furthermore, branched chain fatty acids are larger compared to unbranched chain fatty acids. Lipase and / or carboxylesterase activity in a sample containing a recombinant protein of interest may be mediated by one or more lipases and / or carboxylases and may vary among various recombinant proteins of interest, such as individual antibodies (see FIG. 9). Thus, in most cases, the contaminating host cell proteins with lipase and / or carboxylase activity are unknown and may be a mixture of more than one protein. Many lipases, particularly triacylglycerol lipases, may be open or closed, but the active site is shielded from the solvent by a flap or lid, which is part of the polypeptide chain. Thus, the active site of many lipases resembles the inside of a cavity or cylinder, which most likely determines the substrate specificity. Thus, the lipophilic substrate 4-MU esters of medium length (C6-C16) saturated unbranched fatty acids (small fatty acids) used in the methods of the invention are likely to capture a broader enzyme spectrum than, for example, oleic acid with a long and unsaturated acyl chain. Similarly, the hydrophilic substrate HPTS esters of medium length (C6-C12) used in the methods of the invention are likely to capture a broader enzyme spectrum than, for example, fatty acids with long and / or unsaturated acyl chains. The preferred substrates HTPS esters and / or 4-MU esters, alone or in combination, capture an equally broad or broader enzyme spectrum than PS20 or PS80.

[0054] Furthermore, fatty acid esters with short acyl chains provide better solubility in water-based (aqueous) reaction mixtures compared to fatty acid esters with long chain lengths. As a result, more substrates can be used in the assay mix. More specifically, it was found that the solubility of the substrate 4-MU ester is strongly limited at chain lengths of C16 and above. While substrate HPTS esters are generally more water-soluble, increasing the chain length beyond C12 makes the substrate more lipophilic, which shifts the enzyme spectrum towards enzymes with more lipophilic substrate specificity, such as lipases, thereby narrowing the combined enzyme spectrum of substrate HPTS ester and substrate 4-MU ester.

[0055] In addition, for the substrate 4-MU ester, it was found that the decanoic acid ester (4-MUD) provides better resistance to autohydrolysis compared to, for example, the butyric acid ester (4-MUB). At the highest, a chain length of C5 was found to strongly increase autohydrolysis. Although the C10 fatty acid in 4-MUD was reported to be optimal for use in the assay (WO2022 / 049294), slightly longer or shorter saturated unbranched fatty acid esters, such as saturated unbranched fatty acid (C6-C16) 4-MU esters, or more preferably saturated unbranched fatty acid (C8-C12) 4-MU esters, may also be used in the method according to the invention. For the hydrophilic substrate HPTS ester, a similar observation was made with regard to autohydrolysis. Thus, the hydrophilic substrate HPTS ester used in the method according to the invention has an acyl chain of saturated unbranched fatty acid with carbon atoms C6-C12.

[0056] In addition, when a lipophilic 4-MU ester is used in the method according to the invention, it has an acyl chain of a saturated unbranched fatty acid with carbon atoms C6-C16. More preferably, the fatty acid is a medium chain fatty acid and the 4-MU ester is a 4-MU ester of a saturated unbranched fatty acid (C8-C12). In certain embodiments, the lipophilic 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 lipophilic 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 lipophilic substrate 4-MU ester is typically dissolved as a stock solution (such as a 100-fold concentrated stock solution compared to the concentration in the reaction mixture) in an organic solvent such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF), preferably DMSO. In certain embodiments, the lipophilic substrate is provided as a stock solution dissolved in an organic solvent selected from DMSO or DMF, preferably DMF.

[0057] In the present invention, a suitable lipophilic substrate concentration (in the reaction mixture) may be 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 about 300 μM, preferably about 1 μM to about 30 μM, more preferably about 3 μM to about 30 μM. In certain embodiments, the substrate is provided as a stock solution in an organic solvent, in which case the stock solution is added at about 1% to about 5% (v / v) of the reaction mix.

[0058] The method according to certain embodiments includes a step of contacting at least one sample with a reaction solution containing a non-denaturing surfactant that does not have an ester bond, where the surfactant is a non-ionic or zwitterionic surfactant (also referred to herein as "non-denaturing non-ionic or zwitterionic surfactant that does not have an ester bond"). As used herein, the term "surfactant" refers to a surface-active compound capable of forming micelles. Herein, surfactants can also be referred to as surface-active substances. Surfactants are amphiphilic surfactants, i.e., surfactants that 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 portions) have a certain length to form micelles. Thus, surfactants as used herein do not include organic solvents such as ethanol or dimethylsulfoxide (DMSO). The tails of most surfactants typically consist of one or more hydrocarbon chains, which may be branched, linear, or aromatic. Surfactants may contain one or more hydrophobic tails, and preferably, the surfactants contain one hydrophobic chain (single-tailed surfactants). Surfactants are generally classified according to the hydrophilic head group. Nonionic surfactants have no charged groups in their heads, ionic surfactants carry a net positive (cationic) or negative (anionic) charge, and zwitterionic surfactants contain two oppositely charged groups. Thus, nonionic or zwitterionic surfactants are naturally milder surfactants, since they do not carry a net charge in the hydrophilic head group. Furthermore, in many surfactants, the hydrophobic tail is linked to the hydrophilic head via an ester bond, as in PS20 or PS80. Additionally, nonionic or zwitterionic surfactants are non-denaturing surfactants.As used herein, the term "non-denaturing detergent" refers to the effect of a detergent on protein structure. Non-denaturing detergents do not disrupt protein-protein interactions, particularly water-soluble protein-protein interactions.

[0059] Surfactants containing ester bonds are potential substrates for lipases or carboxylesterases and may therefore interfere with the assay. Furthermore, since denaturation of proteins with lipase activity should be avoided, interference with lipase activity in the sample should also be avoided. Therefore, the surfactants used in the method according to the present invention are non-denaturing surfactants that do not have ester bonds, in which case the surfactants are non-ionic or zwitterionic surfactants. Examples of suitable non-denaturing zwitterionic surfactants 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), Zwittergents (Zwittergents of different lengths, such as n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-12)), and 3-[N,N-dimethyl(3-palmitoylaminopropyl)ammonio]-propanesulfonate, or other amidosulfobetaine surfactants. 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 (Brij 35) or other polyoxyethylene ethers; saponins (e.g., Digitonin), octylphenoxypolyethoxyethanol (IGEPAL CA-630), poloxamer 188, poloxamer 338, poloxamer 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 certain embodiments, the non-denaturing, non-ionic or zwitterionic surfactant that does not have an ester bond is not octoxynol-9, and 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 and is a non-ionic or zwitterionic surfactant, more preferably the surfactant is a non-denaturing surfactant (non-ionic or zwitterionic surfactant), preferably CHAPS, selected from the group consisting of CHAPS (CAS number: 75621-03-3), CHAPSO (CAS number: 82473-24-3), Zwittergent (Zwittergent 3-12; CAS number: 14933-08-5, etc.), and Saponin (CAS number: 8047-15-2). None of these exemplary suitable surfactants are substrates for lipases, since they do not exhibit ester bonds or acyl chains. These surfactants do not affect the sensitivity of the assay, since they do not compete with the substrate. In addition, the presence of the surfactant mediates the solubility of the substrate at the concentration in water used. One of skill in the art would know how to further identify suitable non-ester-bonded, non-denaturing, non-ionic or zwitterionic surfactants by determining their effect on the hydrolysis of 4-MU esters under assay conditions.

[0060] The presence of detergents (such as 10 mM CHAPS) has been shown to increase lipase activity and therefore improve the sensitivity of the assay. Without wishing to be bound by theory, it is hypothesized that detergents create an environment that promotes lipase activity by allowing the reconstitution and opening of lids or flaps, which have been described to target 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).

[0061] Thus, according to the present invention, the non-denaturing surfactant (non-ionic or zwitterionic) has a final concentration in the reaction mixture that is greater than its critical micelle concentration (CMC) in the reaction mixture. The CMC represents an important physicochemical characteristic of a given surfactant in aqueous solution. A micelle is a spherical aggregate whose hydrocarbon group is largely not in contact with water. As used herein, the term "critical micelle concentration" or "CMC" refers to the concentration of a surfactant above which micelles are formed (i.e., the maximum monomer concentration) and can be determined according to methods known in the art. For example, a suitable method for determining the CMC is the fluorescent micelle assay (FMA), which uses the partitioning of a fluorescent hydrophobic dye, N-phenyl-1-naphthylamine (NPN), into micelles of the surfactant. NPN exhibits a decrease in fluorescence quantum yield in an aqueous environment, which increases in a more hydrophobic environment, such as the core of a micelle. This assay was originally developed for CMC determination, but has also been used to determine the content of polysorbates in biopharmaceuticals, as shown in the Examples. An alternative method exploiting the enhanced fluorescence of 1,6-diphenyl-1,3,5-hexatriene (DPH) upon micellization is described by Chattopadhyay and Harikumar (FEBS Letters 391 (1996) 199-202).

[0062] The CMC for detergents can be derived from the literature, e.g., about 6 mM for CHAPS, about 8 mM for CHAPSO, and about 2 to 4 mM for Zwittergent 3-12. In certain embodiments, the non-denaturing zwitterionic detergent is CHAPS and is provided at a final reaction mixture concentration of about 8 mM to about 20 mM, preferably about 8 mM to about 15 mM, more preferably about 10 mM. In other embodiments, the non-denaturing zwitterionic detergent is CHAPSO and is provided at a final reaction mixture concentration 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 at a final reaction mixture concentration 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).

[0063] The hydrolysis may be stopped at a certain time point before the detection of the fluorescence intensity of the released chromophore 4-MU. Alternatively and preferably, the fluorescence intensity of the released chromophore 4-MU may be detected in real time without stopping the hydrolysis of the lipophilic substrate 4-MU ester. In certain embodiments, the fluorescence intensity of the released chromophore 4-MU is detected without stopping the hydrolysis of the lipophilic substrate 4-MU ester. In certain embodiments, the hydrolysis is measured by detecting the fluorescence intensity of the released chromophore 4-MU over time while incubating the sample and the substrate in the reaction mixture according to step (ci).

[0064] Real-time detection allows the hydrolysis to be measured over time, so that the specific reaction rate can be determined. In the method according to the invention, the hydrolysis of the substrate HPTS ester or 4-MU ester in the reaction mixture typically follows pseudo-zero order kinetics. Thus, the detection of fluorescence in real time allows the measurement in a time frame with pseudo-zero order kinetics. Thus, in certain embodiments, the fluorescence intensity of the released chromophore HPTS or 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 in triplicate, and individual reaction mixtures that do not meet the requirements of pseudo-zero order kinetics, for example due to foaming in the wells, are excluded from the analysis to eliminate outliers. Eliminating outliers as described strongly increases the sensitivity of the assay. A calibration curve using defined concentrations of HPTS or 4-MU can be used to calculate the rate of hydrolysis (e.g., in nanomoles / second). Calibration curves with known concentrations of HPTS or 4-MU further allow the determination and comparison of reaction rates at different pH values.

[0065] As used herein, the term "reaction rate" 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 reactant concentration. This means that the rate of the reaction equation is equal to k, the rate constant of the reaction, and is called a zero-order reaction. Zero-order kinetics is always an artifact 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.

[0066] The method according to the invention may further comprise a step of determining the rate of hydrolysis by detecting the fluorescence intensity of the released chromophore HPTS or 4-MU as relative fluorescence units (RFU) and determining the amount of released chromophore HPTS or 4-MU (moles / sec) by comparing this with a calibration curve generated by using a defined HPTS or 4-MU concentration. Typically, the activity is measured by the release of HPTS or 4-MU in nanomoles / min. Alternatively or in addition, the relative value may be calculated relative to an internal standard, such as another sample, preferably a commercially available lipase, such as porcine pancreatic lipase (PPL) (or a commercially available crude extract containing PPL) used as a positive control.

[0067] Incubation of the sample and substrate in the reaction mixture allows at least one potentially contaminating host cell protein with carboxylesterase activity to hydrolyze the substrate HPTS ester and, in a separate sample, at least one potentially contaminating host cell protein with lipase activity to hydrolyze the substrate 4-MU ester. Incubation typically lasts from a few minutes to a few hours. In one embodiment, hydrolysis is measured by detecting the fluorescence intensity of the released chromophore HPTS or 4-MU over time, in real time, while incubating the sample and substrate in the reaction mixture according to step (c) or (ci), respectively, during incubation. Due to the high sensitivity of the assay, detection typically begins immediately after step (b) or (bi), respectively. Incubation may depend on the lipase activity present in the sample and thus on the detection time, which 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. In order to obtain sufficient data points, it is recommended 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 5 hours, 2 minutes and 3 hours, 2 minutes and 2 hours, or 2 minutes and 0.5 hours, or between about 2 minutes and less than 5 hours, less than 3 hours, less than 2 hours, or less than 0.5 hours. Preferably, the sample and substrate in the reaction mixture are incubated for 20 minutes to 2 hours at a temperature of about 25°C. Since the reaction temperature affects the reaction time, the reaction temperature shall be kept constant, such as a constant temperature between 20°C and 37°C, preferably between 22°C and 28°C, more preferably between 24°C and 26°C, during the measurement.In one embodiment, the sample and substrate in the reaction mixture are incubated at a constant temperature between 20°C and 37°C, preferably between 22°C and 28°C, more preferably between 24°C and 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°C and 37°C, preferably between 22°C and 28°C, more preferably between 24°C and 26°C, for 2 minutes to 5 hours, 2 minutes to 3 hours, 2 minutes to 2 hours, or 2 minutes to 0.5 hours, or for about 2 minutes to less than 5 hours, less than 3 hours, less than 2 hours, or less than 1 hour. In certain embodiments, the reaction mixture has a volume of 300 μl or less. In certain embodiments, a reaction mixture of multiple species, such as 12 or more species, 24 or more species, 36 or more species, 72 or more species, or 96 or more species, is analyzed in parallel, preferably in a volume of 300 μl or less.

[0068] The reaction solution used in the method according to the present invention (both for the hydrophilic substrate HPTS ester and for the lipophilic substrate 4-MU ester) further comprises a buffer having a pH of about pH 4 to about pH 8. Preferably, the method is carried out using a buffer having a pH of about pH 5 to about pH 7.5, more preferably about pH 5.5 to about pH 7.5. The skilled artisan will understand that the pH of the buffer is within the buffer range when used in the method of the present invention. Any buffer known in the art may be used and provided, provided that it has a buffer range within about pH 4 to about pH 8. The buffer may contain a single buffer substance or may be a buffer with multiple components. A buffer with multiple components typically has a wide buffer range. For example, buffers may include formic acid, acetic acid, lactic acid, citric acid, malic acid, maleic acid, glycine, glycylglycine, succinic acid, TES (2-{[tris(hydroxymethyl(me-thyl))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-hydroxyethyl)glycine), The solution may contain one or more buffer substances selected from the group consisting of N-(N-tris(hydroxymethyl)methyl)-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4, and NaH2PO4. Preferably, the buffer is a phosphate buffer (Na2HPO4 and NaH2PO4), a Tris buffer, or a HEPES buffer. In a particular embodiment, the buffer has a concentration of about 50 to 400 mM, preferably about 50 to 300 mM, and more preferably about 50 to 200 mM.

[0069] The buffer may further be a multi-component buffer, comprising more than one buffer substance with overlapping buffer ranges, to extend the buffer range. The buffer may comprise, for example, 2, 3, 4, 5 or more buffer substances, preferably 2 or more buffer substances, more preferably 3 or more buffer substances. For example, the multi-component buffer may comprise 2-4 buffer substances, 3-4 buffer substances, more preferably 3 buffer substances. In certain embodiments, the multi-component buffer comprises at least 3 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.

[0070] Since the assay was found to be highly sensitive to ionic strength, it is important for the design of a suitable multi-component buffer not only to include buffer substances with overlapping buffer ranges, but also that the buffer only moderately changes the ionic strength (less than 15%, more preferably less than 10%) at different pHs (range: pH 4-8) (Ellis KJ, Morrisson JF, 1982. Methods in Enzymology, 87: 405-426). For example, AMT buffers containing acetate, MES, and Tris allow the use of buffers that only moderately affect ionic strength at different pHs to identify conditions, including pH conditions that reduce hydrolytic activity. This buffer further allows the measurement at the pH of the sample to determine the lipase activity at the specific conditions present in the sample, as well as to compare the lipase activity at different conditions during purification. The assay allows the sensitivity to be further increased by measuring the sample at the pH optimum.

[0071] Thus, the multi-component buffers disclosed herein allow for the use of buffers in which the pH is variable from at least about pH 5 to at least about pH 7.5, or from at least about pH 4 to at least about pH 8. Alternatively, or in addition, the use of buffers in which the pH values ​​vary between about pH 4 and about pH 8 affects the ionic strength of the buffer by less than 15%, such as less than 0% to 15%, less than 0% to 10%, less than 0% to 7.5%, or less than 0% to 5%, or less than 2% to 15%, less than 2% to 10%, less than 2% to 7.5%, or less than 2% to 5%, preferably less than 10%, or even less than 7.5%, or less than 5%. In one embodiment, the use of buffers with pH values ​​varying 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 less than 0% to 15%, less than 0% to 10%, less than 0% to 7.5%, or less than 0% to 5%, or less than 2% to 15%, less than 2% to 10%, less than 2% to 7.5%, or less than 2% to 5%. The use of multi-component buffers as disclosed herein further allows for the pH of the buffer to be adjusted in light of the pH of the sample (without changing the buffer composition of the buffer). The use of multi-component buffers as disclosed herein further allows for the pH of the buffer to be adjusted to near the optimum amount of at least one contaminating protein having lipase activity (thereby increasing the sensitivity of the method) and / or to compare and identify conditions that reduce hydrolytic activity.

[0072] The reaction solution may further comprise a non-buffering salt. In the present invention, any salt that dissociates in water and does not exert a buffering effect may be suitable for adjusting the ionic strength of the reaction solution. Examples of suitable salts are NaCl, KCl, or CaCl2. In a particular example of the present invention, the non-buffering salt is selected from the group consisting of NaCl, KCl, and CaCl2, and preferably, the non-buffering salt is NaCl or KCl.

[0073] The concentration of any non-buffering salt may range from about 100 mM to about 200 mM. In certain embodiments 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 mix should not exceed a certain value due to negative effects on lipase activity. For example, the ionic strength of any 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, or about 150 mM or less in the reaction mixture, such as 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-buffer salts in the reaction mixture does not exceed about 450 mM. Thus, the cumulative ionic strength of the buffer and non-buffer salts in the reaction mixture can 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-buffer salts in the reaction mixture can 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.

[0074] The buffer solution according to step (b) of the method of the invention and the buffer solution according to step (bi) of the method of the invention can be the same or different. In a preferred embodiment, the buffer solution according to step (b) of the method of the invention and the buffer solution according to step (bi) of the method of the invention are the same. Thus, the buffer components, concentrations and one or more pH values ​​within the range are the same. The same applies to any non-buffering salts, their presence and their concentrations. This excludes differences other than substrate specificity when detecting carboxylesterase activity and lipase activity.

[0075] The method according to the invention detects fluorescence (preferably λ for HPTS) in a fluorescence spectrometer or a microplate spectrophotometer. Exis 401 to 405 nm, and λ Em For 4-MU, λ is 510-516 nm. Ex is 330 to 340 nm, and λ Em at 450 nm). The reaction mixture is therefore contained (and preferably mixed) in a cuvette or in a microtiter plate, preferably in 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 certain embodiments, 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 in triplicate. 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 not only used in step (d) or step (di) to measure hydrolysis, but also in step (d) or step (di) to contact at least one sample with a reaction solution and in step (c) or step (ci) to incubate the sample with the substrate in the reaction mixture. Thus, in certain embodiments, samples are contacted, incubated, and measured in a microtiter plate format having 96 wells or a multiple of 96 wells.

[0076] In certain embodiments, 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 may be 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 the recombinant protein of interest may be a harvested cell culture fluid (HCCF) or cell lysate, an in-process control (IPC) sample, a drug substance sample, or a pharmaceutical sample, preferably an IPC sample, a drug substance sample, or a pharmaceutical sample. Preferably, the step of 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. This is preferably done by first adding a small volume (typically the sample) and second adding a larger volume (typically the reaction solution). Preferably, the components of the reaction solution are added as a master mix, in which case the master mix may be prepared as a concentrate that is diluted to a working concentration prior to addition to the sample.

[0077] The at least one sample may be a harvested cell culture fluid (HCCF) or cell lysate, an in-process control (IPC) sample, a UF / DF sample, a drug substance sample, or a pharmaceutical product sample. The recombinant protein of interest in the sample for detecting carboxylesterase activity and, optionally, 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 of interest is preferably a secreted protein. As used herein, the term "recovered 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 of interest in the sample for detecting lipase activity is not a carboxylesterase or a lipase, and / or does not contain carboxylesterase or lipase activity. Thus, any carboxylesterase or lipase activity detected in at least one sample is a contaminating carboxylesterase or lipase activity and / or is derived from at least one contaminating host cell protein having carboxylesterase or lipase activity, such as CHO host cell protein (CHOP) derived from CHO cells. Furthermore, the recombinant protein of interest in the sample according to the method of the invention is not an esterase or hydrolase and / or does not contain esterase or hydrolase activity. In certain embodiments, the recombinant protein of interest is produced in CHO cells and the at least one contaminating host cell protein is CHO host cell protein (CHOP).

[0078] Thus, the method according to the invention may be advantageously used to detect carboxylesterase activity or lipase activity by measuring hydrolysis in a sample containing antibodies, antibody fragments, antibody-derived molecules, or fusion proteins (e.g. Fc-fusion proteins). Typically, antibodies are monospecific, but antibodies can also be multispecific. Thus, the method according to the invention may be used for samples containing monospecific antibodies, multispecific antibodies, or fragments thereof, preferably antibodies (monospecific), bispecific antibodies, trispecific antibodies, or fragments thereof, preferably antigen-binding fragments thereof. 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-CD44v6 antibodies, anti-CD49d antibodies, anti-CD52 antibodies, anti-EGFR1 (HER1) antibodies, anti-EGFR2 (HER2) antibodies, anti-GD3 antibodies, anti-IGF antibodies, anti-VEGF antibodies, anti-TNF alpha antibodies, anti-IL2 antibodies, The antibody may include, but is not limited to, an anti-IL-5R antibody, an anti-IL-36R antibody, or an anti-IgE antibody, and is preferably selected from the group consisting of an anti-CD20 antibody, an anti-CD33 antibody, an anti-CD37 antibody, an anti-CD40 antibody, an anti-CD44 antibody, an anti-CD52 antibody, an anti-HER2 / neu (erbB2) antibody, an anti-EGFR antibody, an anti-IGF antibody, an anti-VEGF antibody, an anti-TNF alpha antibody, an anti-IL2 antibody, an anti-IL-36R antibody, and an anti-IgE antibody.

[0079] As used herein, the term "antibody", "antibodies" or "immunoglobulin" refers to a protein selected from globulins formed naturally by differentiated B lymphocytes (plasma cells) as a response of the host organism to a foreign substance (= antigen). There are various classes of immunoglobulins: IgA, IgD, IgE, IgG, IgM, IgY, IgW. Preferably, the antibody is an IgG antibody, more preferably an IgG1 antibody or an IgG4 antibody. In the present specification, the terms immunoglobulin and antibody are used interchangeably. Antibodies include monoclonal antibodies, monospecific antibodies, multispecific (such as bispecific or trispecific) antibodies, single chain antibodies, antigen-binding fragments of antibodies (e.g., Fab fragments or F(ab')2 fragments), disulfide-linked Fv, etc. The antibody can be of any species and includes chimeric and humanized antibodies. A "chimeric" antibody is a molecule in which domains or regions of the antibody 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 the human antibody are replaced with those of a non-human species, such as mouse, rat, rabbit, or llama. In some cases, a small number of key 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, recombinantly, transgenic means, cell (e.g., hybridoma) culture, or by other means.

[0080] Typically, antibodies are tetrameric polypeptides composed of two pairs of heterodimers, each formed by a heavy chain and a light chain. The stabilization of both heterodimers as well as the tetrameric polypeptide structure occurs via interchain disulfide bridges. Each chain is composed of structural domains called "immunoglobulin domains" or "immunoglobulin regions", where the terms "domain" or "region" are used interchangeably. Each domain contains about 70-110 amino acids and forms a compact three-dimensional structure. Both heavy and light chains contain at their N-terminus "variable domains" or "variable regions", with less conserved sequences that contribute to antigen recognition and binding. The variable region of the light chain is also referred to as "VL" and the variable region of the heavy chain is also referred to as "VH".

[0081] Antigen-binding fragments include, but are not limited to, for example, "fragment antigen binding" (Fab). Fab fragments consist of the variable regions of both chains held together by adjacent constant regions. Fab fragments can be formed from conventional antibodies by protease digestion, e.g., with papain, but can also be produced by genetic engineering as well. Further antibody fragments include F(ab')2 fragments, which can be prepared by proteolytic cleavage with pepsin.

[0082] Using genetic engineering techniques, it is possible to generate truncated antibody fragments consisting only of the variable region of the heavy chain (VH) and the variable region of the light chain (VL). These are called Fv fragments (fragment variable). These Fv fragments are often stabilized since they lack the covalent bond between the two chains by the cysteines of the constant chains. It is advantageous to link the variable region of the heavy chain and the variable region of the light chain by a short peptide fragment, for example of 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, in particular scFv.

[0083] In recent years, various strategies have been developed to prepare scFv as multimeric derivatives. This is intended to result in recombinant antibodies with improved pharmacokinetic and biodistribution properties, as well as increased binding avidity. To achieve multimerization of scFv, scFv have been prepared as fusion proteins with multimerization domains. The 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 in which interactions between the VH / VL regions of scFv are used for multimerization (e.g., diabodies, tribodies, and pentabodies). By diabody, the skilled artisan means a scFv derivative that is a bivalent homodimer. The shortening of the linker in the scFv molecule to 5-10 amino acids results in the formation of homodimers, where interchain VH / VL superposition occurs. In addition, diabodies may be stabilized by the incorporation of disulfide bridges. Examples of diabody antibody proteins are known from the prior art.

[0084] By minibody the skilled person means a bivalent, homodimeric, scFv derivative. Minibody consists of a fusion protein containing the CH3 region of an immunoglobulin, preferably IgG, most preferably IgG1, as a dimerization region connected to the scFv via a hinge region (for example also derived from IgG1) and a linker region. Examples of minibody-antibody proteins are known from the prior art. By triabody the skilled artisan means a scFv derivative that is trivalent, homotrimeric. An scFv derivative in which the VH-VL are directly fused without a linker sequence results in the formation of trimers. The skilled person will also be familiar with the so-called mini-antibodies, which have a bivalent, trivalent or tetravalent structure and are derived from scFv. Multimerization is carried out by dimeric, trimeric or tetrameric coiled-coil structures. In a preferred embodiment of the present invention, the gene of interest codes for any of the desired above mentioned polypeptides, preferably a monoclonal antibody, derivative or fragment thereof.

[0085] Immunoglobulin fragments consisting of the CH2 and CH3 domains of an antibody heavy chain are called "Fc fragments", "Fc regions", or "Fc" because of their tendency to crystallize (Fc = fragment crystallizable). They may be formed by protease digestion from conventional antibodies, for example with papain or pepsin, or may be produced by genetic engineering. The N-terminal portion of the Fc fragment may vary depending on how many amino acids of the hinge region remain. An antibody comprising an antigen-binding fragment and an Fc region may also be referred to as a full-length antibody. Full-length antibodies may be monospecific antibodies, and multispecific antibodies such as bispecific antibodies or trispecific antibodies.

[0086] A preferred therapeutic antibody according to the present invention is a multispecific antibody, in particular a bispecific or trispecific antibody. A bispecific antibody typically combines antigen-binding specificities for target cells (e.g., malignant B cells) and effector cells (e.g., T cells, NK cells, or macrophages) in one molecule. Exemplary bispecific antibodies include, but are not limited to, diabodies, the BiTE (bi-specific T cell engager) format, and the DART (dual-affinity re-targeting) format. The diabody format separates the cognate variable domains of the heavy and light chains with the two antigen-binding specificities on two separate polypeptide chains, which are non-covalently associated. The DART format is based on the diabody format, but provides additional stabilization through a C-terminal disulfide bridge. A trispecific antibody is a monoclonal antibody that combines three antigen-binding specificities. A trispecific antibody can be constructed by 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 make into high quality products.

[0087] Another preferred therapeutic protein is a fusion protein, such as an Fc fusion protein. Thus, the present invention may be advantageously used for the production of a fusion protein, such as an Fc fusion protein. Furthermore, the method of increasing the production of a protein according to the present invention may also be advantageously used for the production of a fusion protein, such as an Fc fusion protein.

[0088] The effector portion of the fusion protein may be a complete sequence or any part of the sequence of a native or modified heterologous protein. The immunoglobulin constant domain sequence may be derived from any immunoglobulin subtype, such as subtype IgG1, IgG2, IgG3, IgG4, IgA1, or IgA2, or class, such as IgA, IgE, IgD, or IgM. Preferentially, the immunoglobulin constant domain sequence is derived from a human immunoglobulin, more preferably from human IgG, and even more preferably from human IgG1 and human IgG2. Non-limiting examples of Fc fusion proteins include MCP1-Fc, ICAM-Fc, EPO-Fc, and scFv, which are fragments that contain an N-linked glycosylation site and are coupled to the CH2 domain of the heavy chain immunoglobulin constant region. Fc fusion proteins can be constructed by genetic engineering techniques through the introduction of a CH2 domain of a heavy chain immunoglobulin constant region, which contains 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, which contains an N-linked glycosylation site, for example.

[0089] The recombinant protein of interest of the present invention is produced in eukaryotic cells in cell culture. Preferably, the eukaryotic cells used to make the recombinant protein of interest are yeast cells (e.g., Saccharomyces, Kluyveromyces) or mammalian cells (e.g., hamster cells or human cells). The yeast cells can be, but are not limited to, Saccharomyces cerevisiae, Pichia pastoris, Kluyveromyces (Klyveromyces) lactis, or Kluyveromyces (Klyveromyces) marxianus. The mammalian cells are preferably CHO cells, HEK293 cells, or their derivatives. HEK293 cells include, but are not limited to, HEK293 cells, HEK293T cells, HEK293F cells, Expi293F cells, or derivatives thereof. For large-scale industrial production, commonly used CHO cells are often engineered to improve their characteristics in the production process or to facilitate the selection of recombinant cells. Such engineering includes, but is not limited to, increasing resistance to apoptosis, reducing autophagy, increasing cell proliferation, altering the expression of cell cycle regulatory proteins, engineering chaperones, engineering the unfolded protein response (UPR), engineering the secretory pathway, and engineering metabolism.

[0090] Preferably, CHO cells are metabolically engineered, such as by glutamine synthetase (GS) knockout, and / or dihydrofolate reductase (DHFR) knockout, to facilitate selection with methionine sulfoximine (MSX) or methotrexate, respectively, to allow for an efficient cell line development process.

[0091] Preferably, the CHO cells used to produce the recombinant protein of interest 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.

[0092] Non-limiting examples of mammalian cells that may be used within the context of the present invention are also summarized in Table A. However, other mammalian cells may also be used in the present invention, including derivatives / progeny of these cells, including but not limited to human, mouse, rat, monkey, and rodent cell lines, particularly for the production of biopharmaceutical proteins, such as recombinant proteins of interest.

[0093] [Table 1] JPEG2025509270000003.jpg93161

[0094] It is most preferred if the cells are cultured completely under established, adapted, serum-free conditions, optionally in a medium free of any proteins / peptides of animal origin. Commercially available media such as Ham's F12 (Sigma, Deisenhofen, Germany), RPMI-1640 (Sigma), Dulbecco's Modified Eagle Medium (DMEM; Sigma), Minimum Essential Medium (MEM; Sigma), Iscove's Modified Dulbecco's Medium (IMDM; Sigma), CD-CHO (Invitrogen, Carlsbad, CA), serum-free CHO medium (Sigma), and protein-free CHO medium (Sigma) are exemplary suitable nutrient solutions. Any of the 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 salts, magnesium salts, phosphates, 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 selection gene, an appropriate selection agent is added to the culture medium.

[0095] The recombinant protein of interest 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 of interest may be harvested as a secreted protein in harvested cell culture fluid (HCCF), from the culture medium, or from a cell lysate (i.e., a fluid 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 including such disruption of the cell wall), and purified using techniques well known in the art. Samples obtained and / or analyzed in the various purification steps are also referred to as in-process control (IPC) samples or process intermediates. Harvesting typically involves centrifugation and / or filtration, such as to produce a harvested cell culture fluid or cell lysate, preferably a harvested cell culture fluid. Thus, the harvested cell culture fluid or cell lysate may also be referred to as a clarified harvested cell culture fluid or a clarified cell lysate. The harvested cell culture fluid or cell lysate is free of viable cells and cell debris, and most cellular components have been removed. Clarification typically means centrifugation or filtration, preferably filtration. Further process steps may include affinity chromatography for antibodies or Fc-containing proteins, in particular Protein A column chromatography, to separate the product from contaminants. Further process steps may include clarification of the product pool by acid treatment to inactivate viruses, preferably by acid treatment to remove cellular contaminants such as HCPs and DNA, followed by depth filtration. 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 the individual case. Additionally, subsequent process steps may preferably include nanofiltration to further remove viruses, as well as ultrafiltration and dialysis to concentrate the recombinant protein of interest and exchange buffer, respectively.

[0096] Since carboxylesterase activity and lipase activity are associated with host cell protein contaminants, the method according to the invention may be particularly useful for analyzing process intermediates after (preferably before and after) a purification step removing HCPs in order to adapt the relevant steps to more efficiently remove carboxylesterase activity and / or lipase activity in the process intermediates, such as before and after affinity chromatography, before and after depth filtration in combination with acid treatment, and / or before and after anion exchange chromatography. In some embodiments, the method comprises obtaining at least one sample after affinity chromatography and / or after depth filtration in combination with acid treatment (or after acid treatment and / or after depth filtration) and / or after ion exchange chromatography, such as 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, such as anion exchange chromatography and / or cation exchange chromatography, preferably anion exchange chromatography. The skilled person will be aware that the sample obtained after a certain method step may be the same as the sample obtained before a subsequent method step, such as the sample obtained after affinity chromatography (e.g., Protein A chromatography) may be the same sample as the sample before acid treatment (or before depth filtration combined with acid treatment, i.e. before acid treatment followed by depth filtration). As explained above, due to the wide buffering range of the buffer, lipase activity in samples with different pH values ​​can still be compared using the method according to the invention. Other samples that can be analyzed using the method according to the invention are samples after ultrafiltration / dialysis (UF / DF samples), drug substance samples, or pharmaceutical samples. Drug substance or drug product samples often contain polysorbates as they include formulation buffers.At very high concentrations, polysorbate may inhibit the hydrolysis reaction of the lipophilic substrate 4-MU ester due to competition with the substrate. However, due to the high sensitivity of the assay with the lipophilic substrate 4-MU ester, where the typical concentration of polysorbate is 0.4-0.8 mg / ml, lipase activity may also be determined in drug substance samples or drug product samples diluted in the final reaction mixture as explained above. The assay with the hydrophilic substrate HPTS ester is slightly more sensitive to polysorbate in the reaction, which should not exceed 50 μg / ml. Polysorbate is typically added following ultrafiltration / dialysis (following the capture and refinement steps) to form the drug substance. Therefore, alternatively, UF / DF samples may be used to detect carboxylesterase and / or lipase activity in the (final) purified product.

[0097] The method of producing the recombinant protein of interest typically includes an ultrafiltration dialysis step using tangential flow filtration (TTF) to produce a purified antibody product pool. The purified recombinant protein of interest is buffer exchanged and further concentrated by ultrafiltration. Polysorbate is then added to the concentrated antibody, including all other excipients, of the formulation.

[0098] In one aspect, a method for producing a recombinant protein of interest comprising the steps of: (i) culturing in cell culture eukaryotic cells expressing the recombinant protein of interest; (ii) recovering the recombinant protein of interest; (iii) purifying the recombinant protein of interest; (iv) optionally formulating the recombinant protein of interest into a pharma- ceutically acceptable formulation suitable for administration; and (v) obtaining at least one sample comprising the recombinant protein of interest in steps (ii), (iii), and / or (iv); detecting carboxylesterase activity in the sample obtained in step (v) (i.e., a sample comprising the recombinant protein of interest and at least one contaminating host cell protein), comprising: (a) detecting a carboxylesterase activity in the sample obtained in step (v) (i.e., a sample comprising the recombinant protein of interest and at least one contaminating host cell protein), the carboxylesterase activity being determined by ...b) detecting a carboxylesterase activity in the sample obtained in step (v) (i.e., a sample comprising the recombinant protein of interest and at least one contaminating host cell protein), the carboxylesterase activity being determined by: (c) detecting a carboxylesterase activity in the sample obtained in step (v) (i.e., a sample comprising the recombinant protein of interest and at least one contaminating host cell protein), the carboxylesterase activity being determined by: providing at least one sample obtained in step (v), the sample comprising at least one contaminating host cell protein and a contaminating host cell protein; (b) contacting the at least one sample with a reaction solution comprising a hydrophilic substrate to form a reaction mixture, the reaction solution comprising (i) a buffer having a pH of about pH 4 to about pH 8, (ii) a hydrophilic substrate which is a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester), and (iv) optionally a non-buffering salt; (c) incubating the sample and the substrate in the reaction mixture; and (d) detecting the carboxylesterase activity of the at least one contaminating host cell protein by measuring hydrolysis of the substrate HPTS ester and detecting the fluorescence intensity of the released chromophore HPTS;Optionally, (bi) contacting at least one sample comprising the recombinant protein of interest produced in eukaryotic cells in cell culture of step (a) and at least one contaminating host cell protein with a reaction solution comprising a hydrophilic substrate in a separate reaction setting to form a reaction mixture, the reaction solution comprising (i) a buffer having a pH of about pH 4 to about pH 8, (ii) a lipophilic 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-C16) 4-MU ester (substrate 4-MU ester), (iii) optionally a non-buffering salt, and (iv) an ester. (ci) incubating the sample and substrate in a reaction mixture; (di) detecting lipase activity of at least one contaminating host cell protein 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 HPTS or 4-MU over time while incubating the sample and substrate in a reaction mixture according to step (c) or (ci), respectively. Thus, in certain embodiments, the method comprises detecting lipase activity in a sample (the sample obtained in step (v)), comprising the steps of: (bi) contacting at least one sample comprising the recombinant protein of interest produced in eukaryotic cells in cell culture of step (a) and at least one contaminating host cell protein with a reaction solution comprising a hydrophilic substrate in a separate reaction set-up to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a lipophilic 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-C16) 4-MU ester (substrate 4-MU ester); (iii) optionally a non-buffering salt; and (iv) a non-denaturing surfactant that does not have an ester bond and is a non-ionic or zwitterionic surfactant; (ci) incubating the sample and the substrate in the reaction mixture;(di) detecting lipase activity of at least one contaminating host cell protein by measuring hydrolysis of 4-MU esters and detecting the fluorescence intensity of the released chromophore 4-MU; and optionally measuring hydrolysis by detecting the fluorescence intensity of the released chromophore HPTS or 4-MU over time while incubating the sample and substrate in a reaction mixture according to step (c) or (ci), respectively. In certain embodiments, the recombinant protein of interest is a therapeutic protein, such as an antibody, an antibody fragment, an antibody-derived molecule (e.g., scFv, bispecific or multispecific antibody), or a fusion protein (e.g., Fc-fusion protein). Those skilled in the art will understand that the steps of (i) culturing eukaryotic cells; (ii) recovering the recombinant protein of interest; (iii) purifying the recombinant protein of interest; and (iv) optionally formulating the recombinant protein of interest may comprise several substeps. For example, step (iii) purifying the recombinant protein of interest may include the substeps of purifying the recombinant protein of interest using affinity chromatography, acid treatment, depth filtration, and / or ion exchange chromatography;

[0099] In certain embodiments, a method of producing a recombinant protein of interest according to the invention comprises obtaining at least one sample comprising the recombinant protein of interest in a step of recovering the recombinant protein of interest (step (ii)), where the sample is a harvested cell culture fluid (HCCF) or a cell lysate; in a step of purifying the recombinant protein of interest (step (iii)), where the sample is an in-process control (IPC) sample; and / or in an optional step of formulating the recombinant protein of interest into a pharma- ceutically acceptable formulation suitable for administration (step (iv)), where the sample is a drug substance or drug product sample. Preferably, the method for producing a recombinant protein of interest according to the invention comprises a step of obtaining at least one sample comprising the recombinant protein of interest in step (iii), the sample being an in-process control (IPC) sample, such as a step comprising obtaining at least one sample after affinity chromatography, after depth filtration followed by acid treatment (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 and after affinity chromatography, before and after depth filtration followed by acid treatment (or before and after acid treatment and / or before and after acid treatment), and / or before and after ion exchange chromatography, preferably anion exchange chromatography or cation exchange chromatography. Other samples that may be analyzed using the method according to the invention are samples after ultrafiltration / dialysis (UF / DF samples), drug substance samples, or pharmaceutical product samples.Thus, in certain embodiments, the method comprises obtaining at least one sample comprising the recombinant protein of interest in step (ii), where the sample is a harvested cell culture fluid (HCCF) or a cell lysate; and / or in step (iii), where the sample is an in-process control (IPC) sample; and / or in step (iv), where the sample is a UF / DF sample, drug substance sample, or drug product sample; preferably obtaining at least one sample in step (iii) comprising the recombinant protein of interest produced in eukaryotic cells in cell culture and at least one contaminating host cell protein, including obtaining the at least one sample before and after affinity chromatography, before and after acid treatment, before and after depth filtration, and / or before and after ion exchange chromatography, preferably anion exchange chromatography or cation exchange chromatography. The step of detecting carboxylesterase activity or lipase activity in the sample comprising the recombinant protein of interest is carried out according to the method for detecting lipase activity described herein as specified therein.

[0100] In yet another aspect, the present invention relates to the use of the hydrophilic substrate HPTS ester and / or the lipophilic substrate 4-MU ester as substrates for detecting the carboxylesterase activity and / or lipase activity of at least one contaminating host cell protein in a sample comprising a recombinant protein of interest produced in eukaryotic cells in a cell culture in an assay, wherein the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester) and the lipophilic substrate is a saturated unbranched fatty acid (C6-C16) 4-MU ester. Preferably, the recombinant protein is produced in CHO cells and the at least one contaminating host cell protein is a CHO host cell protein (CHOP). In one embodiment, the use is the use of the hydrophilic substrate HPTS ester as a substrate for detecting in an assay the carboxylesterase activity of at least one contaminating host cell protein in a sample comprising a recombinant protein of interest produced in a eukaryotic cell in a cell culture, wherein the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester). In a preferred embodiment, the use is the use of the hydrophilic substrate HPTS ester and the lipophilic substrate 4-MU ester as substrates for detecting the carboxylesterase activity and lipase activity of at least one contaminating host cell protein in a sample comprising a recombinant protein of interest produced in a eukaryotic cell in a cell culture in an assay, wherein the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester) and the lipophilic substrate is a saturated unbranched fatty acid (C6-C16) 4-MU ester. The same applies to the components of the reaction solution, the sample and the conditions as specified above for the method of the invention.

[0101] Kit for determining contaminating lipase activity by measuring hydrolysis in a sample Also provided is a kit for determining contaminating carboxylesterase and / or lipase activity in a sample containing a recombinant protein of interest, comprising: (i) a buffer having a pH of about pH 4 to about pH 8; and (ii) a hydrophilic substrate and a lipophilic substrate, where (a) the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester); (b) the lipophilic substrate comprises 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-C16) 4-MU ester (substrate 4-MU ester); and optionally (iii) a non-buffering salt; and / or (iv) a non-denaturing detergent that does not have an ester bond and is a non-ionic or zwitterionic detergent. Optionally, the kit further comprises water for dilution. Hydrophilic substrate and lipophilic substrate are used in separate reaction settings.Only the reaction solution containing lipophilic substrate is prepared with non-denaturing surfactant.Therefore, the reaction solution containing hydrophilic substrate is prepared without non-denaturing surfactant and / or any surfactant, particularly any surfactant above its CMC.In certain embodiments, the kit further comprises a manual that instructs that the reaction solution containing lipophilic substrate is prepared with non-denaturing surfactant, and the reaction solution containing hydrophilic substrate is prepared without non-denaturing surfactant.

[0102] In one embodiment, the kit further comprises an internal standard used as a positive control and / or allows for the calculation of relative values ​​compared to an internal standard such as a commercially available carboxylase and / or lipase, e.g., porcine pancreatic lipase (PPL) crude extract. The kit may also comprise one or more microtiter plates having 96 wells or a multiple of 96 wells. The components of the kit may be provided as solution components and / or dry components, either individually or in premixed form. The buffer may be provided as a dry formulation that upon dilution or reconstitution results in a buffer having a pH of about pH 4 to about pH 8.

[0103] In certain embodiments, for reaction solutions containing hydrophilic substrates, the buffer and any non-buffering salts are premixed as an assay buffer, or for reaction solutions containing lipophilic substrates, the buffer and any non-buffering salts and / or any surfactant 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, compared to the final reaction mixture. Alternatively, the assay buffer is provided as a dry mix. Such a dry mix can be reconstituted with water to provide the at least about 3-fold or 5-fold concentrated assay buffer compared 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 the reaction solution. An alternative kit may include the buffer, substrate, and any non-buffering salts and / or any surfactant as a premixed master mix. The master mix may be adapted to provide from about 80% (v / v) to about 70% (v / v) of the reaction mixture, preferably from about 80% to about 75% of the reaction mix. The assay buffer and reaction solution are aqueous solutions.

[0104] Regarding the components of the reaction solution, the same applies as specified above for the method of the present invention. The hydrophilic substrate HPTS ester is a saturated unbranched fatty acid (C6-C12, preferably C8-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or its salt (substrate HPTS ester). Thus, the hydrophilic substrate HPTS ester can be 1-hexanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, 1-heptanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, 1-decanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, 1-undecanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, and 1-dodecanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof. In certain embodiments, the hydrophilic substrate HPTS ester is selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof. In certain preferred embodiments, the hydrophilic substrate HPTS ester is 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof (OPTS), preferably 1-octanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt.

[0105] The lipophilic substrate comprising the chromophore 4-MU is in the form of a saturated unbranched fatty acid (C6-C16) 4-MU ester (substrate 4-MU ester), where the aliphatic chain of the saturated unbranched fatty acid has carbon atoms C6-C16 or preferably C8-C12. Thus, the lipophilic substrate 4-MU ester can be 4-methylumbelliferyl octanoate, 4-methylumbelliferyl nonanoate, 4-methylumbelliferyl decanoate (4-MUD), methylumbelliferyl undecanoate, or methylumbelliferyl dodecanoate. In certain embodiments, the lipophilic 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 lipophilic substrate is 4-MUD.

[0106] The kit may further comprise an organic solvent for dissolving the lipophilic substrate, or the lipophilic substrate is dissolved in an organic solvent. The lipophilic substrate may be provided as a dry substance, optionally with an additional organic solvent, or may be dissolved as a stock solution (such as a stock solution at 100 times the concentration in the reaction mixture) in an organic solvent, such as dimethylsulfoxide (DMSO) or dimethylformamide (DMF), preferably DMSO. The kit may further comprise water for dissolving the hydrophilic substrate. The lipophilic substrate may be provided as a dry substance, but shall be frozen after solubilization in an aqueous solution, such as water or an aqueous buffer solution.

[0107] Examples of suitable non-denaturing zwitterionic detergents without ester bonds (for reaction solutions containing lipophilic substrates) 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), Zwittergents (Zwittergents of different lengths, such as n-dodecyl-N,N-dimethyl-3-ammonio-1-propanesulfonate (Zwittergent 3-12)), 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 (Brij 35) or other polyoxyethylene ethers; saponins (e.g., Digitonin), octylphenoxypolyethoxyethanol (IGEPAL CA-630), poloxamer 188, poloxamer 338, poloxamer 407, or tergitol. In certain embodiments, the detergent is a non-denaturing, non-ionic or zwitterionic detergent that does not have an ester bond, preferably the detergent is neither polyethylene glycol tert-octylphenyl ether (Triton X-100) nor 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.Surfactants are required only in reaction solutions containing lipophilic substrates and should be excluded from reaction solutions containing hydrophilic substrates.

[0108] 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), HEP The buffer solution comprises one or more buffer substances selected from the group consisting of ES (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 solution has a pH of about pH 4 to about pH 8, preferably, the buffer solution has a pH of about pH 5 to about pH 7.5, and more preferably, the buffer solution has a pH of about pH 5.5 to about pH 7.5.

[0109] 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. A multi-component buffer may comprise more than one buffer substance with overlapping buffer ranges to extend the buffer range. The buffer may comprise, for example, 2, 3, 4, 5 or more buffer substances, preferably 2 or more buffer substances, more preferably 3 or more buffer substances. For example, the multi-component buffer may comprise 2-4 buffer substances, 3-4 buffer substances, more preferably 3 buffer substances. In certain embodiments, the multi-component buffer comprises at least 3 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 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. In one embodiment, the use of a multi-component buffer with pH values ​​varying 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 less than 0% to 15%, less than 0% to 10%, less than 0% to 7.5%, or less than 0% to 5%, or less than 2% to 15%, less than 2% to 10%, less than 2% to 7.5%, or less than 2% to 5%. Optional non-buffering salts can be, for example, NaCl, KCl, and CaCl2, preferably NaCl or KCl.

[0110] With the above in mind, it will be appreciated that the present invention also encompasses the following provisions: Clause 1 is a method for detecting carboxylesterase activity of a contaminating host cell protein in a sample comprising a recombinant protein of interest produced in a eukaryotic cell, the method comprising: (a) providing at least one sample comprising the recombinant protein of interest produced in a eukaryotic cell in cell culture and at least one contaminating host cell protein; (b) contacting the at least one sample with a reaction solution (including a hydrophilic substrate) to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS); (iii) optionally, a non-buffering salt; (c) incubating the sample and the substrate in a reaction mixture; (d) detecting carboxylesterase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate HPTS ester and detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (HPTS); and optionally measuring hydrolysis by detecting the fluorescence intensity of the released chromophore HPTS over time while incubating the sample and the substrate in a reaction mixture according to step (c).

[0111] Clause 2 specifies a method as described in clause 1, wherein the sample and substrate in the reaction mixture are incubated for any time between 2 minutes and 5 hours, 2 minutes and 3 hours, 2 minutes and 2 hours, or 2 minutes and 0.5 hours. Clause 3 specifies a method according to clause 1 or 2, wherein the reaction mixture has a volume of 300 μl and / or multiple (such as 12 or more, 24 or more, 36 or more, 72 or more, or 96 or more) reaction mixtures are analysed in parallel, preferably in a volume of 300 μl or less. Clause 4 specifies a method according to any one of clauses 1 to 3, wherein at least one sample portion is used for each reaction mixture. Clause 5 specifies a method according to any one of clauses 1 to 4, wherein the fluorescence of the emitted chromophore HPTS is determined using an excitation wavelength in the range of 401 to 405 nm and an emission wavelength in the range of 510 to 516 nm.

[0112] Clause 6 specifies the method according to any one of clauses 1 to 5, wherein the substrate HPTS ester is selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, preferably selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid (OPTS) trisodium salt, 1-nonaoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt. Clause 7 specifies the method according to any one of clauses 1 to 6, wherein the reaction solution (including the hydrophilic substrate) does not contain a surfactant.

[0113] Clause 8 specifies a method according to any one of clauses 1 to 7, wherein the hydrophilic substrate HPTS ester is dissolved in an aqueous solution, preferably an aqueous solution free of DMSO or DMF. Clause 9 specifies a method according to any one of clauses 1 to 8, wherein the hydrophilic substrate HPTS ester is used at a final concentration in the reaction mixture of 50 μM or less, preferably 30 μM or less. Clause 10 includes (bi) contacting at least one sample comprising the recombinant protein of interest produced in eukaryotic cells in cell culture from step (a) and at least one contaminating host cell protein with a reaction solution comprising a lipophilic substrate in a separate reaction setting to form a reaction mixture, the reaction solution comprising (i) a buffer having a pH of about pH 4 to about pH 8, (ii) a lipophilic 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-C16) 4-MU ester, (iii) optionally a non-buffering salt, and (iv) a non-ionic surfactant having no ester linkages. or a zwitterionic surfactant; (ci) incubating the sample and the substrate in the reaction mixture of step (bi); and (di) detecting lipase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate 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 (ci).

[0114] Clause 11 specifies the method of clause 10, 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. Clause 12 specifies a method according to clause 10 or 11, wherein the substrate is provided at a final concentration in the reaction mixture of about 1 μM to about 1 mM. Clause 13 specifies a method according to any one of clauses 10 to 12, wherein the substrate is provided as a stock solution in an organic solvent, the stock solution being added at about 1% to about 5% (v / v) of the reaction mix, and / or the organic solvent is DMSO or DMF. Clause 14 specifies a method according to any one of clauses 10 to 13, wherein the surfactant has a final concentration in the reaction mixture that is above its critical micelle concentration in the reaction mixture. Clause 15 specifies the method according to any one of clauses 10 to 14, wherein the surfactant is selected from the group consisting of CHAPS, CHAPSO and Zwittergent, preferably CHAPS, and / or is neither polyethylene glycol tert-octylphenyl ether (Triton X-100) nor polyethylene glycol nonylphenyl ether (NP-40).

[0115] Clause 16 specifies the method of any one of clauses 10 to 15, wherein the 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. Clause 17 specifies a method according to any one of clauses 1 to 16, wherein the fluorescence intensity of the released chromophore HPTS is detected without terminating the hydrolysis of the hydrophilic substrate HPTS ester, and optionally the 4-MU is detected without terminating the hydrolysis of the lipophilic substrate 4-MU ester; and / or the sample and substrate in the reaction mixture are incubated for any time between 2 minutes and less than 5 hours, less than 3 hours, less than 2 hours, or less than 0.5 hours. Clause 18 specifies a method according to any one of clauses 1 to 17, in which the fluorescence intensity of the released chromophores HPTS and optionally 4-MU is detected over time and follows pseudo-zero order kinetics, and optionally reaction mixtures that do not meet the requirements of pseudo-zero order kinetics are excluded from the analysis. Clause 19 specifies a method according to any one of clauses 1 to 18, further comprising the steps of (a) determining the rate of hydrolysis by detecting the fluorescence intensity of the released chromophore HPTS and optionally 4-MU as relative fluorescence units (RFU) and comparing this with a calibration curve generated by using defined 4-MU concentrations to determine the amount (moles / sec) of the released chromophore HPTS and optionally 4-MU, and / or (b) calculating the relative value by comparison with an internal standard.

[0116] Clause 20 provides that 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 20. The method of any one of clauses 1-19, further comprising one or more buffer substances selected from the group consisting of glycine (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), NaHPO, and NaHPO. Clause 21 specifies the method according to any one of clauses 1 to 20, 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.

[0117] Clause 22 specifies the method of any one of clauses 1 to 21, 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. Clause 23 specifies the method of clause 22, wherein 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. Clause 24 specifies a method according to clause 22 or 23, comprising: (a) use of a buffer in which the pH is variable from at least about pH 4 to at least about pH 8; (b) use of a buffer in which the pH value varies between about pH 4 to about pH 8, thereby affecting the ionic strength by less than 15%, preferably less than 10%, preferably 0%-15%, 0%-10%, 0%-7.5%, or 0%-5%; (c) adjusting the pH of the buffer in relation to the pH of the sample; (d) adjusting the pH of the buffer to near the optimum amount of at least one (contaminating) host cell protein having carboxylase and / or lipase activity; or (e) comparing and identifying conditions that reduce hydrolytic activity.

[0118] Clause 25 specifies a method according to any one of clauses 1 to 24, wherein at least 2, 3, 4, 5, 10 or more samples are analysed simultaneously. Clause 26 specifies the method of any one of clauses 1 to 25, wherein the non-buffering salt is selected from the group consisting of NaCl, KCl, and CaCl2, preferably the non-buffering salt is NaCl or KCl. Clause 27 specifies the method of any one of clauses 1 to 26, wherein the non-buffering salt has a concentration in the reaction mixture of from about 100 mM to about 200 mM, preferably from about 130 mM to about 170 mM, and more preferably from about 140 mM to about 150 mM. Clause 28 specifies the method according to any one of clauses 1 to 27, wherein the ionic strength of the non-buffering salt is not more than about 200 mM in the reaction mixture, preferably not more than about 150 mM in the reaction mixture, preferably from about 100 mM to about 200 mM, preferably from about 130 mM to about 170 mM, more preferably from about 140 mM to about 150 mM in the reaction mixture. Clause 29 specifies the method of any one of clauses 1 to 28, wherein the cumulative ionic strength of the buffer and non-buffer salts in the reaction mixture is about 450 mM or less, preferably about 400 mM, more preferably about 350 mM or less, in the reaction mixture.

[0119] Clause 30 specifies the method of any one of clauses 1 to 29, wherein the fluorescence is detected using a fluorescence spectrometer or a microplate spectrophotometer. Clause 31 specifies a method as described in clause 30, wherein the fluorescence is detected using a microplate spectrophotometer in bottom read mode or top read mode, preferably in bottom read mode. Clause 32 specifies a method according to any one of clauses 1 to 31, wherein the samples are contacted, incubated and measured in one or more microtiter plates having 96 wells or a multiple of 96 wells. Clause 33 specifies a method according to any one of clauses 1 to 32, wherein at least one sample portion is used for each reaction mixture. Clause 34 specifies a method according to any one of clauses 1 to 33, wherein at least one sample (or a portion of at least one sample) is provided as about 20% to about 30% (v / v) of the reaction mixture, preferably as about 25% of the reaction mixture, and optionally the sample (or at least one portion of the sample) is pre-diluted.

[0120] Clause 35 specifies the method of any one of clauses 1 to 34, wherein at least one sample is a harvested cell culture fluid (HCCF) or cell lysate, an in-process control (IPC) sample, a UF / DF sample, a drug substance sample, or a drug product sample. Clause 36 specifies the method of any one of clauses 1 to 35, wherein (a) the recombinant protein of interest is not a carboxylesterase or lipase, and / or an enzyme having carboxylesterase or lipase activity; and / or (b) the recombinant protein of interest is selected from the group consisting of an antibody, an antibody fragment, an antibody-derived molecule, and a fusion protein.

[0121] Clause 37 specifies the method according to any one of clauses 1 to 36, wherein the eukaryotic cell used to produce the recombinant protein of interest is a yeast cell or a mammalian cell, the mammalian cell being preferably a CHO cell, a HEK293 cell, or a derived cell thereof. Clause 38 specifies the method of any one of clauses 1 to 37, wherein the recombinant protein of interest is produced in CHO cells and the at least one contaminating host cell protein is a CHO host cell protein (CHOP).

[0122] Clause 39 provides a method of producing a recombinant protein of interest comprising the steps of: (i) culturing in a cell culture eukaryotic cells expressing the recombinant protein of interest; (ii) recovering the recombinant protein of interest; (iii) purifying the recombinant protein of interest; and (iv) optionally formulating the recombinant protein of interest into a pharma- ceutically acceptable formulation suitable for administration; and (v) obtaining at least one sample comprising the recombinant protein of interest in steps (ii), (iii), and / or (iv); detecting carboxylesterase activity in the sample comprising the recombinant protein of interest and at least one contaminating host cell protein, the method comprising the steps of: (a) detecting the recombinant protein of interest produced in the eukaryotic cells in the cell culture and at least one contaminating host cell protein; (b) contacting the at least one sample with a reaction solution comprising a hydrophilic substrate to form a reaction mixture, the reaction solution comprising (i) a buffer having a pH of about pH 4 to about pH 8, (ii) a hydrophilic substrate which is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester), and (iv) optionally a non-buffering salt; (c) incubating the sample and the substrate in the reaction mixture; and (d) detecting carboxylesterase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate HPTS ester and detecting the fluorescence intensity of the released chromophore HPTS;Optionally, detecting lipase activity in a sample, comprising: (bi) contacting at least one sample comprising the recombinant protein of interest produced in eukaryotic cells in cell culture of step (a) and at least one contaminating host cell protein, in a separate reaction setting, with a reaction solution comprising a lipophilic substrate to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a lipophilic 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-C16) 4-MU ester; (iii) optionally a non-buffering salt; and (i) a pH of about 10 to about 15% by weight of the lipophilic substrate; v) comprising a non-denaturing surfactant that does not have an ester bond and is a non-ionic or zwitterionic surfactant; (ci) incubating the sample and the substrate in a reaction mixture; (di) detecting lipase activity of at least one contaminating host cell protein by measuring hydrolysis of the substrate 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 HPTS or 4-MU over time while incubating the sample and substrate in a reaction mixture according to step (c) or (ci), respectively;

[0123] Clause 40 specifies a method of producing a recombinant protein of interest as defined in clause 39, comprising obtaining at least one sample comprising the recombinant protein of interest in step (ii), where the sample is a Harvested Cell Culture Fluid (HCCF) or a cell lysate; in step (iii), where the sample is an In-Process Control (IPC) sample; and / or in step (iv), where the sample is a UF / DF sample, a drug substance sample, or a drug product sample. Clause 41 specifies the method of producing a recombinant protein of interest according to clause 39 or 40, to include a step of obtaining in step (iii) at least one sample comprising the recombinant protein of interest, wherein the sample is an in-process control (IPC) sample.

[0124] Clause 42 specifies a method of producing a recombinant protein of interest according to clause 41 comprising the steps of obtaining at least one sample after affinity chromatography, after acid treatment followed by depth filtration (or after acid treatment and / or after depth filtration) and / or after anion exchange chromatography, preferably obtaining at least one sample before and after affinity chromatography, before and after acid treatment followed by depth filtration (or before and after acid treatment and / or before and after depth filtration) and / or before and after anion exchange chromatography. Clause 43 specifies a method for producing a recombinant protein of interest according to any one of clauses 39 to 42, comprising detecting carboxylesterase and optionally lipase activity in a sample comprising the recombinant protein of interest produced in eukaryotic cells in cell culture and at least one contaminating host cell protein according to a method according to any one of clauses 1 to 38.

[0125] Clause 44 provides a kit for determining contaminating carboxylesterase and / or lipase activity in a sample containing a recombinant protein of interest, comprising: (i) a buffer having a pH of about pH 4 to about pH 8; and (ii) a hydrophilic substrate and a lipophilic substrate, wherein (a) the hydrophilic substrate is a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester); (b) the lipophilic substrate comprises the chromophore 4-methylumbelliferyl (4-MU) in the form of a 4-MU ester, the 4-MU ester being a saturated unbranched chain fatty acid (C6-C16) 4-MU ester (substrate 4-MU ester); and optionally (iii) a non-buffering salt; and / or (iv) a non-denaturing detergent, which does not have an ester bond and is a non-ionic or zwitterionic detergent.

[0126] Clause 45 further specifies the kit according to clause 44, wherein the hydrophilic substrate HPTS ester is selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, preferably selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid (OPTS) trisodium salt, 1-nonaoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt. Clause 46 further specifies the kit according to clause 44 or 45, wherein the lipophilic substrate 4-MU ester 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.

[0127] Clause 47 further specifies the kit of any one of clauses 44 to 46, further comprising one or more microtiter plates having 96 wells or a multiple of 96 wells. Clause 48 specifies the kit according to any one of clauses 44 to 47, further comprising an organic solvent for dissolving the lipophilic substrate 4-MU ester, preferably DMSO or DMF. Clause 49 specifies a kit according to any one of clauses 44 to 48, wherein the surfactant is not polyethylene glycol tert-octylphenyl ether (Triton X-100) and is not polyethylene glycol nonylphenyl ether (NP-40), or the surfactant is a non-denaturing zwitterionic surfactant selected from the group consisting of CHAPS, CHAPSO, and Zwittergent, preferably CHAPS.

[0128] Clause 50 provides that 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}propanesulfonic acid), tricine (N-tris(hydroxymethyl)methylglycine), Na2HPO4, and NaH2PO4. Clause 51 specifies the kit according to any one of clauses 44 to 50, 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.

[0129] Clause 52 specifies a kit according to any one of clauses 44 to 51, 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. Clause 53 specifies the kit of clause 52, wherein 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. Clause 54 specifies a kit according to any one of clauses 44 to 53, wherein the non-buffering salt is selected from the group consisting of NaCl, KCl, and CaCl2, preferably the non-buffering salt is NaCl or KCl. Clause 55 provides for the use of the hydrophilic substrate HPTS ester and the lipophilic substrate 4-MU ester as substrates for detecting carboxylesterase activity and lipase activity of at least one contaminating host cell protein in a sample comprising a recombinant protein of interest produced in eukaryotic cells in a cell culture in an assay, preferably wherein the recombinant protein of interest is produced in CHO cells and the at least one contaminating host cell protein is a CHO host cell protein (CHOP), the hydrophilic substrate is a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester) and the lipophilic substrate is a saturated unbranched chain fatty acid (C6-C16) 4-MU ester.

[0130] Clause 56 specifies the use of the hydrophilic substrate HPTS ester as a substrate for detecting in an assay the carboxylesterase activity of at least one contaminating host cell protein in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture, wherein the hydrophilic substrate is a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester), preferably wherein the recombinant protein is produced in CHO cells and the at least one contaminating host cell protein is CHO host cell protein (CHOP). Clause 57 specifies the use according to clause 55 or 56, comprising the step of detecting carboxylesterase and optionally lipase activity in a sample comprising a recombinant protein of interest produced in a eukaryotic cell in a cell culture and at least one contaminating host cell protein, according to a method according to any one of clauses 1 to 38. EXAMPLES

[0131] A novel assay was developed to characterize the activity of esterases that preferentially convert hydrophilic substrates (e.g., carboxylesterases). This assay was used to probe a variety of biotechnologically or biopharmaceutical relevant solutions that may potentially contain trace amounts of enzymatic impurities, such as lipases and other esterases, as well as various stabilizing substances (buffer substances, additives to adjust ionic strength, and surfactants). In the examples, the following method was used:

[0132] Measurement by fluorescence spectroscopy The substrate 1-octanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt (OPTS) was chosen to make it possible to determine the enzyme activity. Its hydrolysis product, 1-hydroxypyrene-3,6,8-trisulfonic acid trisodium salt (HPTS), can be detected by fluorescence spectroscopy. The change in the fluorescence signal over time is directly related to the change in the concentration of the free fluorophore. The establishment of a calibration curve by subsequent linear fitting allowed the conversion (R 2≧0.99). Experiments for kinetic measurements with the OPTS / 4-MUD substrate assay were performed in duplicate. The values ​​for the reaction mixtures without the protein of interest, used as respective controls, were subtracted from the values ​​for the protein-containing batches. The buffer used for measurements with OPTS (hydrophilic substrate) contained 0.3 M HOAc, 0.3 M MES, 0.6 M Tris, and 0.6 M NaCl (4x concentrated AMT (acetate, MES, Tris) buffer). In addition, 0.04 M CHAPS was added for measurements with the 4-MUD substrate (lipophilic substrate). The buffer (AMT) was prepared at 4x concentration (4x concentrated AMT) and added to the reaction mix at 25% (v / v). All measurements were performed in multi-well plates with a preparation volume of 300 μL (75 μL 4x AMT buffer, 30 μL substrate, 75 μL enzyme-containing solution, x μL additive, HO to 300 μL). Unless stated otherwise, all measurements by fluorescence spectroscopy were performed using 96-well, clear-bottom, non-binding plates (Greiner, Austria) on a SpectraMax M3 PlateReader (Molecular Device, USA) and recorded using SoftMax Pro 7.0.3 software (Molecular Device, USA). Measurements by OPTS assay were typically performed at 25° C., λ ex = 403 nm, and λ em = 512 nm, and measurements by 4-MUD assay were performed at 25 °C, λ ex = 330 nm, and λ em = 450 nm. For assessment, the fluorescence signal detected for the reaction mix without the protein of interest (control) was subtracted from the fluorescence signal detected for the reaction mix with the protein of interest (e.g., DS). Substrate hydrolysis was measured by detecting the fluorescence intensity of the released chromophore (4-MU or HPTS) immediately after mixing in real time for a few minutes up to 5 hours depending on the fluorescence intensity. The change in the fluorescence signal over time was successively converted into hydrolytic activity by linear fitting using a previously established calibration curve (R 2≧0.99).

[0133] Setting the calibration curve by linear fitting For the establishment of the calibration curve by linear fitting, a 1:1 serial dilution of the free fluorophore HPTS in HO was prepared from 12 μM to 7.8 nM. Before adding the AMT buffer, each dilution of the HPTS solution was pipetted into the plate (composition: 75 μL of 4x concentrated AMT (varying pH), 75 μL of HPTS solution, 150 μL of HO). Calibration curves were made separately for different pH values ​​of the AMT buffer used (pH values ​​from 4 to 8, step increments of 0.5). The final concentration range of HPTS used for the calibration curve was from 3 μM to 1.46 nM. After pipetting, the measurement was started immediately.

[0134] Kinetic measurements with OPTS / 4-MUD substrate and antibody preparations The target concentration of OPTS was 25 μM and the target concentration of 4-MUD was 30 μM. To prepare the master mix, 4x AMT buffer was mixed with the appropriate volume of H2O in a Falcon tube. Dialyzed antibody (Ab) preparation samples (drug substance) or control solutions (75 μl) were transferred to a 96-well plate. Any inhibitors (10 μM orlistat, 1 mM PMSF, and 10 mM EDTA) were added to 4x AMT buffer and master mix containing water and substrate (3 μl / sample of 3 mM 4-MUD in DMSO or 30 μM / sample of 250 μM OPTS in H2O, aliquoted and frozen). The 4-MUD substrate was stored in a concentrated stock solution containing 3 mM 4-methylumbelliferyl decanoate (4-MUD; FM25973, Carbosynth) in DMSO, resulting in a 100x concentrated stock solution for use containing 0.3 mM 4-MUD in DMSO. A 250 μM OPTS stock solution was prepared in H2O, aliquoted, and frozen until use. Although OPTS is more stable in DMSO compared to H2O (preventing autohydrolysis), the substrate was diluted in water (or alternatively, AMT buffer) to avoid DMSO interference with the assay. The reaction was then started by adding the master mix (reaction solution) to each of the protein or control solutions. Measurements were started immediately. The measurement time was 30 min, with a measurement interval of 15 s. Stock solutions of inhibitors were used as follows: 300 μM orlistat in DMSO (Sigma-Aldrich, USA), 100 mM PMSF in isopropanol (Roth, Germany), and 500 mM EDTA in H2O (Sigma-Aldrich, USA). To generate a pH profile of the protein solution, kinetic measurements were performed at the indicated pH values ​​as described above. Added AMT buffer was adjusted to the indicated pH values ​​over the range pH 4-8 (in steps of 0.5) and kinetics were recorded for each pH value.

[0135] Effect of tropolone on the fluorescence of free fluorophores The effect of tropolone on the fluorescence of HPTS and 4-MU was investigated at pH=5.5 using a target concentration of 10 mM tropolone. 1:1 serial dilutions were performed for HPTS and 4-MU with target concentrations ranging from 3 μM to 96 nM. DMSO was used as a control substance for tropolone. In addition, 1:1 serial dilutions of tropolone were performed with target concentrations ranging from 10 mM to 3.2 μM.

[0136] Example 1 Determination of λex and λem of the free fluorophore HPTS Aqueous solutions of free fluorophore (HPTS, Sigma-Aldrich) were prepared to record spectra for excitation and emission wavelengths (see FIG. 2A). For the intended use, the fluorophore was required to be able to be used in the pH range of preferably pH=4-8. To determine λex and λem, aqueous solutions of HPTS with AMT buffers at pH values ​​of 4-8 (in steps of 0.5) were prepared by mixing the respective buffers in a plate with aqueous HPTS solutions (composition: 75 μL of 4x concentrated AMT (varying pH), 75 μL of 6 μM HPTS solution, 150 μL of fully desalted H2O) at a final concentration of 1.5 μM. To identify a possible effect of pH on the intensity of the resulting HPTS fluorescence signal, a spectrum was recorded with a variable excitation wavelength of λex = 300-500 nm and a constant emission wavelength of λem = 520 nm, and an additional spectrum was recorded with a constant excitation wavelength of λex = 415 nm and a variable emission wavelength of λem = 420-620 nm.

[0137] The highest fluorescence signal was observed with a global maximum at λex=403 nm, and further intensity changes were observed with pH values ​​from 4 to 6.5. With increasing pH, a slight decrease in intensity was observed at pH>6.5. However, the increase in intensity at λex=403 nm still resulted in high sensitivity compared to the isosbestic point (λex=413 nm) even at pH=8, where no pH dependence was observed. For this reason, an excitation wavelength of λex=403 nm was chosen for all further explorations. However, it was noted that with increasing pH (>6.5), the sensitivity slightly decreased. The emission maximum is at λ em = 512 nm and therefore all measurements were recorded at this emission wavelength unless otherwise stated.

[0138] Example 2 The calibration curve allows quantification of free fluorophore The calibration curve allows to correlate the recorded fluorescent signal with the fluorophore concentration in the solution. Thus, the change in HPTS concentration over time can be calculated, since it is based on the enzyme activity. To create the calibration curve, serial dilutions of HPTS were prepared and measurements of these dilutions were performed for each pH value of AMT (acetate, MES, Tris) buffer. This buffer was used throughout the experiments because of its wide buffering range without large changes in ionic strength and osmolality. These individual calibration curves further allow the correction of the pH-dependent changes in the fluorescent signal. In the upper panel of Figure 2B, the calibration line is plotted for the HPTS concentration range of 1.46-3000 nM, and in the lower panel, the limits of the linear range are shown using an example for a calibration line at pH=7 and a HPTS concentration range of 93.75-6000 nM. It can be seen that with increasing pH, the slope of the calibration curve decreases. Concentrations ≧3 μM showed less than perfect linear fits (R2 is less than 0.99999) as the linear behavior changes to an asymptotic behavior. The deviation of the RFU values ​​from the calibration curve for HPTS concentrations of 6 μM (including 3 μM) is 7.4%. Therefore, the calibration curve up to 3 μM was used.

[0139] Example 3 Measurement methods TR and BR (effect on calibration line) The Multiwell PlateReader SpektraMax M3 (Molecular Devices, US), in which the fluorescence is not read orthogonally to the excitation light, allows two possible measurement modes: top read (TR) and bottom read (BR). Therefore, using a dilution series in water of HPTS in the range of 1.5 μM to 1.46 nM, pH 4-8, recorded by TR and BR, we investigated whether reading from above or below would give different results.

[0140] For measurements in the TR format, multiwell plates with a black opaque bottom were used (96-well, black-bottom plates; Greiner Bio-One, Austria), whereas for measurements in the BR format, the plates have a special transparent bottom that allows measurements from below (96-well, clear-bottom plates, Greiner Bio-One, Austria). Furthermore, non-binding and medium-binding plates (Greiner Bio-One) were investigated, of which the non-binding plates were found to be slightly better. The BR measurement format resulted in steeper calibration lines compared to the TR measurements (data not shown), which means that smaller concentration changes can be calculated from the BR measurements, corresponding to a higher sensitivity. In the following experiments, measurements were performed in the BR measurement format using non-binding plates for OPTS and 4-MUD.

[0141] Example 4 Hydrolysis of the fluorogenic substrate OPTS The fluorogenic substrate OPTS (1-octanoyloxypyrene-3,6,8-trisulfonic acid, Sigma-Aldrich) consists of a trisulfonate-substituted pyrene esterified with caprylic acid. Because OPTS is highly soluble (>2 mM) in HO, it represents a substrate for enzymes that convert hydrophilic substrates, such as carboxylesterases. A stock solution of the fluorescent substrate in water was prepared. An aqueous solution with an OPTS concentration of ≥ 2.0 mM could be prepared. The solubility was verified by light scattering experiments. The fluorophore HPTS can be released from the substrate (here, esterified caprylic acid) via enzymatic or chemical hydrolysis (acidic and alkaline hydrolysis). To evaluate the effect of pH on the aqueous solution of the substrate, 225 μL of 33.3 μM OPTS solution was added to 75 μL each of AMT buffer (pH=5.5 and pH=8) and the kinetics of fluorophore release was recorded over a period of 2 hours. The results are shown in FIG. 3.

[0142] Clear differences can be seen in the slopes of the fluorescent signals for different pH values. Based on the recorded calibration curve, the autohydrolysis rate at pH 5.5 was estimated to be 1.58 × 10 -7 The rate of autohydrolysis at pH 8.0, calculated in units (µmol × min-1), was 3.48 × 10 -6 The hydrolysis rate was calculated as units. Thus, the approximately 22-fold increase in hydrolysis rate at pH 8 compared to pH 5.5 indicates that hydrolysis of the fluorogenic substrate can be observed over time at basic pH, but is limited to a very low level at pH 5.5. Since autohydrolysis increases with decreasing chain length, substrate HPTS esters derived from saturated unbranched fatty acids with less than 6 carbon atoms are not expected to be suitable. Therefore, suitable substrates would be substrate HPTS esters of at least C6, such as 1-hexanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, 1-heptanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt, or 1-octanoyloxypyrene-3,6,8-trisulfonic acid trisodium salt. Furthermore, the pH should be maintained below pH 8.

[0143] Example 5 Enzymatic hydrolysis of the fluorogenic substrate OPTS An aqueous solution of commercially available porcine pancreatic lipase, PPL (Sigma-Aldrich, US; 100-500 units per mg of protein), was prepared. According to the manufacturer, this powder contains primarily triacylglycerol lipase, but since it is a homogenized powder from lyophilized porcine pancreas (crude extract), it is likely to contain other carboxylesterases and lipases. This extract was chosen for functional testing since these enzymes hydrolyze fatty acid esters. To record the reaction kinetics of the substrate OPTS with PPL, a 10 mg / mL aqueous solution of PPL was prepared by adding 10 mg of PPL to 1 mL of HO. The suspension was vortexed for 2 min and centrifuged at 14,000 × g for 5 min before removing the supernatant.

[0144] The reaction in a mixture of 75 μL AMT buffer (pH=7), 165 μL H2O, and 30 μL of 10 mg×mL-1 aqueous PPL solution was started by the addition of 30 μL of 250 μM OPTS solution. The increase in fluorescence was recorded for 30 min with 15 s measurement intervals (n=2). A preparation without PPL (H2O only) was used as a control (blank). Figure 4 shows the results of two measurements (blank corrected). The data shown in Figure 4 indicate that the enzymes present in the PPL extract are capable of enzymatically hydrolyzing OPTS to HPTS. The calculated reaction rate is 2.89 ± 0.08 × 10-5 units (micromoles × min-1).

[0145] Maximum speed v max To study the concentration, OPTS solutions of different concentrations were prepared by performing a 1:1 serial dilution of 10 mM OPTS in water. 30 μL of each of these solutions was added to 75 μL of AMT buffer (pH=7) and made up to 300 μL with HO. At OPTS concentrations <125 μM, no linearity was observed between concentration and fluorescence intensity (R2≦0.999). Therefore, all further measurements were performed at an OPTS concentration of 25 μM.

[0146] Example 6 Effect of surfactants on the fluorescent substrate OPTS Detergents are added to a number of samples that may be aimed at investigating hydrolytic activity by OPTS. In a complementary assay (for lipophilic substrates) using 4-MUD as substrate, detergents such as CHAPS are added above the critical micelle formation concentration to solubilize the substrate and open the lipase lid that would otherwise cover the lipase active site. For this purpose, CHAPS was added to a 25 μM solution of OPTS with AMT buffer (pH = 7) and 1 mg × mL-1 of PPL to a final concentration of 10 mM. The reaction in the batch was then started without the addition of CHAPS. CHAPS was found to have a negative effect on the enzymatic hydrolysis of OPTS by PPL (Figure 5A). The 2.53 × 10 -5 The activity of the unit is 6.16 × 10 in the presence of 10 mM CHAPS. -7 The interference with enzymatic hydrolysis has different reasons. Thus, mixed micelles with CHAPS and OPTS may form a shield for the substrate, making it inaccessible to the enzyme's active site (Lasic, Martin et al. 1989), and the free fluorophore HPTS may be affected by the presence of CHAPS. Experimental evidence indicated that the accessibility of the substrate OPTS is strongly reduced by CHAPS due to mixed micelle formation. Furthermore, the determination of esterase activity in buffers containing detergents is not desired, since the assay is designed to measure enzymes that are able to turnover hydrophilic substrates more than lipases (which may be more active in the presence of detergents due to their open lid configuration). This is in clear contrast to the already developed 4-MUD assay (WO2022 049294) that detects lipase activity, which is highly sensitive in the presence of CHAPS. Triton X-100 was further shown to inhibit the assay (see Figure 10 of WO2022 049294).

[0147] Further inhibition by the non-ionic surfactant polysorbate 20 (Tween 20) was also examined. Polysorbate 20 is one of the most frequently used surfactants in biopharmaceuticals and is typically used at a CMC of 10 2 ~10 3 A concentration of polysorbate 20 above 100 μg / ml (referred to as 100 μg / ml) is used, with typical concentrations used being 0.2-0.4 mg / ml. Using PPL, target concentrations between 200 μg / ml and 0.098 μg / ml were investigated. No inhibition was observed up to 50 μg / ml at pH 7.0, but higher concentrations reduced the fluorescent signal (Figure 5B). Subsequent experiments showed that this was due to the negative effect of polysorbate 20 on the chromophore HPTS. Thus, polysorbate 20 should not exceed 100 μg / ml, and preferably not exceed 50 μg / ml, since at higher concentrations it slightly inhibits the fluorescent signal of HPTS. The polysorbate 20 concentration of the drug substance samples analyzed in this study was below 50 μg / ml after dilution in the reaction mixture.

[0148] The OPTS assay is intended for in-process control samples for the production of a recombinant protein of interest as a rapid, high-throughput assay for purification train development and optimization. Most in-process control samples do not contain surfactants such as polysorbate 20. Therefore, this observation does not limit the applicability of the assay. Samples that may contain surfactants are mainly the final drug substance and harvested cell culture fluid (HCCF) possibly due to antifoaming agents. However, due to high hydrolase activity in HCCF (before purification), the samples need to be diluted so that the concentration reached by potential antifoaming agents will not interfere with the assay. Regarding the final product containing polysorbate, as long as the polysorbate concentration does not exceed the CMC after sample dilution in the reaction mix, In this case, polysorbate should not negatively affect the assay similar to that in this example. Final polysorbate 20 concentrations between 50 μg / ml and 100 μg / ml still only slightly affect the sensitivity of the assay. If the polysorbate concentration in the reaction mixture (i.e. after dilution) is significantly above the CMC (such as above 100 μg / ml), the UF / DF sample, i.e., the UF / DF sample before the addition of polysorbate, may be used to determine the hydrolytic activity using the OPTS assay. However, we note that in this context the assay is particularly useful for high-throughput analysis of small volumes of IPC samples for process optimization, which typically do not contain polysorbate.

[0149] Finally, the activity of the OPTS assay in buffer, as described in WO2022 / 047416 (page 37), was investigated. For this purpose, 1 mg×mL-1 PPL and a final concentration of 100 μM OPTS in 150 mM Tris, 0.25% (w / v) Triton X-100, 0.125% (w / v) gum arabic (TTG buffer), pH=8, were investigated and compared with 100 μM OPTS, 1 mg / ml PPL (without surfactant) in AMT buffer, pH 8. The OPTS concentration was increased from 25 μM to 100 μM (matching the substrate concentration of the fluorogenic substrate containing 100 μM 4-MU used in WO2022 / 047416). Triton X-100 (molecular weight = 625 g per mole) has a critical micelle concentration (CMC) of 0.22-0.24 mM, so 0.25% (w / v) Triton X-100 (4 mM) is well above the CMC. TTG buffer was found to have a negative effect on the enzymatic hydrolysis of OPTS by PPL (Figure 6). In a buffer without detergent (AMT buffer), a 3-4 times more rapid enzyme-mediated hydrolysis of OPTS was observed compared to TTG buffer containing Triton X-100. Although a Triton X-100 concentration above the CMC was used, the incomplete inhibition can be explained by an excess amount of OPTS resulting in some substrate potentially accessible outside the formed micelles (autohydrolysis signal was subtracted from the measured signal). The aggregation number of Triton X-100 is approximately 80 (Stubicar N. et al., Micelles Determined by Light and Small-Angle X-Ray Scattering Techniques. In Mittal, KL (eds) Surfactants in Solution, Springer Boston, MA, pages 181-195), resulting in the formation of approximately 50 μM micelles, so that a substrate concentration of 100 μM would be present in molar excess. Furthermore, the buffers and conditions described in WO2022 / 047416 are not suitable for the OPTS assay of the present invention.In particular, the determination of esterase activity in a buffer containing a detergent (especially above its CMC) is undesirable. Since this assay was designed to measure enzymes capable of turning over hydrophilic substrates more than lipases, the exclusion of detergents is advantageous for detecting carboxylesterases, which are more active in the presence of detergents (due to the open lid configuration and the accessibility of the substrates in the micelles), potentially further reducing background detection of lipase activity. Therefore, the buffer used to measure the hydrolysis of hydrophilic substrates should be detergent-free or at least not exceed the detergent's CMC. Therefore, the conditions for the OPTS assay described herein, especially the detergent-exclusion conditions, make the assay specific for carboxylesterases acting on hydrophilic substrates.

[0150] The results show that the buffers described in WO2022 / 047416 cannot be used for the present OPTS buffer due to the presence of surfactants. Furthermore, at pH 8.0, autohydrolysis is increased, resulting in a reduction in assay sensitivity, and autohydrolysis was found to be more important for OPTS compared to 4-MUD, especially at pH 8.0 (see FIG. 3). The majority of samples analyzed using the OPTS assay are also in the acidic range, rather than the alkaline range.

[0151] Example 7 Determination of hydrolytic activity in antibody solutions using the OPTS assay The purpose of the OPTS assay is to characterize esterase (e.g., carboxylesterase) activity in samples containing recombinantly produced proteins, such as antibodies. Test samples were drug substance samples derived from nine different antibody preparations, including IgG1 and IgG4 monoclonal antibodies as well as bispecific IgG-like formats, produced in CHOK1 or DG44 cells. Drug substance samples contain stabilizing substances (buffer substances, additives to adjust ionic strength, and detergents) and small amounts of contaminating host cell proteins that potentially exhibit hydrolase activity. To verify that enzymatic activity is involved, antibody samples were pre-heat treated at 50°C in a ThermoMixer C (Eppendorf, Germany) and cooled (passively) to room temperature before performing kinetic measurements as described. Activity in all samples decreased with increasing length of thermal incubation, indicating enzymatic activity. Nine different antibody samples (Ab1-Ab9) were tested using the OPTS assay. As controls, measurements were performed with the same buffers used in the antibody samples.

[0152] For some of the antibody solutions, the controls showed slightly higher activity compared to the samples (data not shown), all of which contained histidine at approximately 25 mM. Histidine has previously been reported to mediate hydrolytic activity in aqueous solutions similar to that of esterases (Delort, Nguyen-Trung et al. 2006, Mason, McCracken et al. 2010, Amano, Kobayashi et al. 2014, Xu, Chen et al. 2017), so samples were dialyzed against 0.002% NaCl using a two-step dialysis with a molecular weight cutoff (MWCO) of 20K. For preparative dialysis, a pre-humidified dialysis cassette was filled with 2 ml of antibody solution and placed in a beaker filled with 1 L of NaCl (0.002% w / v) solution under gentle stirring. After 1 h, 1 L of the NaCl solution was replaced with fresh NaCl (0.002% w / v) solution and dialyzed for another 2 h with gentle stirring. -7 The influence of this buffer component can be largely ignored, since activities in the range of units were found (data not shown). In the following, the assay was carried out using dialysis of the drug substance sample. The same solution (final solution outside the dialysis cassette) was used as a control sample to subtract values ​​from the test samples.

[0153] FIG. 7 shows the hydrolytic activity for different antibody solutions after dialysis using the OPTS assay. -7 3.28 x 10 for unit Ab5 -6 A wide range of hydrolytic activity (units) was observed, which may be explained by the presence of different types or amounts of esterases in the protein solutions. All results shown in Figure 7 were determined at a pH of 5.5, which corresponds to the pH of the solution under investigation. Carboxylesterases typically exhibit their maximum hydrolytic activity at about pH 6. Therefore, it is believed that no carboxylesterase activity was detected in protein solutions 1, 4, 8, and 9. The substrate OPTS, which has a chain length of C8, has a chain length that is right in the transition zone between short and long chain fatty acid esters, therefore, it is expected that although carboxylesterase activity is primarily measured, some lipase activity may also be detected.

[0154] To further characterize the detected activity, inhibition of hydrolytic activity was examined in all nine antibody samples using the lipase inhibitor orlistat (10 μM), the protease inhibitor PMSF (1 mM), and the metal chelator EDTA (10 mM). Following dialysis, the inhibitors were added to the samples and measured at 15 s intervals for 30 min before adding the freshly mixed master mix with substrate. Neither orlistat, PMSF, nor EDTA significantly reduced the hydrolytic activity measured in any of the samples (data not shown).

[0155] Example 8 Determination of hydrolytic activity in protein solutions using the 4-MUD assay The data generated using the OPTS assay were compared to a previously established micelle-containing assay using 4-MUD as a substrate, which was developed to detect the hydrolytic activity of lipases (Figure 8). The 4-MUD assay was used to study the same protein solution samples that were studied by the OPTS assay. To allow a reliable comparison of results, care was taken to keep the experimental conditions the same: same volume of antibody sample, measurements were performed at pH 5.5 and 25°C, and kinetics were recorded over 30 min with 15 s time intervals in the BR measurement mode. The reaction mixtures differed only in the substrate and substrate concentration (30 μM 4-MUD) and the use of CHAPS (Roth, Germany) as detergent, as well as different substrate-specific excitation wavelengths, λex, and emission wavelengths, λem.

[0156] The kinetic results determined using the 4-MUD assay are shown in Figure 8. Again, the difference is more than an order of magnitude (1.8 x 10 in protein solution 4). -7 Units ~ 2.3 x 10 in protein solution 8 -6 A wide range of hydrolytic activity was observed, with the target concentration of polysorbate 20 (Serva, Germany) being 100 μg×mL−1 for protein solutions 6, 7, 8, and 9. A qualitative comparison of the hydrolytic activity detected with OPTS or 4-MUD as substrate is shown in FIG. 9. Comparing the hydrolytic activity detected using the OPTS and 4-MUD assays, there is no clear trend derivable from the data shown in FIG. 9. Samples that show a comparable high level of activity in the OPTS assay (e.g., Ab5) show very low activity in the 4-MUD assay. In contrast, protein solution 4 shows high activity in the 4-MUD assay but only very low hydrolytic activity in the OPTS assay. Thus, considering that the OPTS assay primarily detects carboxylesterase activity and the 4-MUD assay primarily detects lipase activity, carboxylesterases are believed to be primarily present in Samples 2, 5, 6, and 7, and lipases are believed to be primarily present in Samples 1, 4, and 9.

[0157] In sample Ab3, the hydrolytic activity detected using the OPTS and 4-MUD assays was in the same range, while in protein solution 8, almost no hydrolytic activity was detected in either of the assays. Protein solution 5 showed the most significant difference between the two substrates, showing almost no hydrolytic activity using the 4-MUD assay, while the highest hydrolytic activity of all the samples tested was detected using the OPTS assay. This indicates that the hydrolytic activity of enzymes using hydrophilic substrates such as carboxylesterases appears to be significantly greater than that of lipases.

[0158] Example 9 Effect of inhibitors on hydrolytic activity in the 4-MUD assay The effects of the inhibitors orlistat (10 μM), PMSF (1 mM), and EDTA (10 mM), previously examined in samples analyzed using the OPTS assay, were also examined in all nine antibody samples using the 4-MUD assay. The results in Figure 10 show the hydrolytic activity in the presence of inhibitors compared to the same samples without added inhibitors (100%).

[0159] FIG. 10 shows that significant inhibition of hydrolytic activity by the lipase inhibitor orlistat (10 μM) occurred in all nine antibody samples examined. The resulting residual activity was in the range of 18-21% of the hydrolytic activity without the addition of an inhibitor. For PMSF, inhibition of hydrolytic activity was observed in all protein solutions except antibody solution Ab1. The residual activity ranged from 72% in protein solution 6 to 28% in antibody solution Ab2. This indicates that the hydrolytic activity observed in this sample was mainly due to the enzyme with a nucleophilic serine in the active site. In contrast, the addition of EDTA showed almost no inhibition in any of the samples. It is therefore concluded that the hydrolytic activity detected in antibody solutions Ab1 to Ab9 is not metal-dependent. The results of the different activities of the corresponding antibody solutions in the OPTS assay and the 4-MUD assay are summarized in Table B below, where (++) means a strong effect, (+) means a weak effect, and (-) means no significant effect.

[0160] [Table 2]

[0161] As can be seen from Table B, none of the inhibitors had a significant effect on the hydrolytic activity detected when OPTS was used as a substrate. In contrast, the specific lipase inhibitor orlistat showed strong inhibition of the hydrolytic activity detected when 4-MUD was used as a substrate. Also, the serine protease inhibitor PMSF showed significant inhibition of the hydrolytic activity detected when 4-MUD was used as a substrate in antibody sample Ab2 and antibody sample Ab5. The fact that the addition of EDTA showed no inhibition of the hydrolytic activity in either the OPTS or 4-MUD assays suggests that the hydrolytic enzyme present in the tested protein solutions was not a metalloenzyme. The inhibitory effect of orlistat is due to acylation of the serine in the catalytic triad of the enzyme's active site (Al-Suwailem, Al-Tamimi et al. 2006), and PMSF is also an inhibitor that binds to a serine in the active site (James 1978). Thus, these two inhibitors share the common feature of reducing the accessibility of the active site to the substrate to be converted, and thus belong to the class of competitive inhibitors.

[0162] In addition, α-tropolone, a recently described allosteric inhibitor of lipase, was also investigated. Due to its good solubility in water, this inhibitor would be suitable for further characterization of the enzyme activity detected by the OPTS assay. However, the strong effect of tropolone at 10 μM on both the free fluorophores HPTS (detected in the OPTS assay) and 4-methylumbelliferone (4-MU) (detected in the 4-MUD assay) suggests that the previously described effect of tropolone on the hydrolysis activity is most likely due to its effect on the fluorescence activity of the free fluorophore (fluorophore quenching) and not an inhibitory effect.

[0163] Example 10 Lipase hydrolytic activity detected using the OPTS assay In addition, a commercially available purified lipase with unknown substrate specificity was analyzed using the 4-MUD and OPTS assays. First, the hydrolysis of both soluble carboxyl esters (OPTS) and hydrophobic carboxyl esters (4-MUD) as well as the effects of orlistat, PMSF, and EDTA on the hydrolysis activity of the lipase were explored. The results are shown in Figure 11.

[0164] Lipases generally showed significantly higher activity in the micelle-based 4-MUD assay compared to the OPTS assay, where only low to no activity was found, indicating that polar hydrophilic esters are not substrates for this hydrolase. Furthermore, the OPTS assay showed no significant effect of inhibitors on hydrolytic activity. For hydrolytic activity detected using the 4-MUD substrate, orlistat almost completely inhibited the effectiveness of the detected hydrolysis, whereas PMSF and EDTA had only minor effects. Since the lipase tested is not a serine protease, inhibition was not expected when using PMSF, a serine protease inhibitor. Furthermore, the lipase was investigated for its pH dependence in the OPTS substrate assay and the 4-MUD substrate assay. For this purpose, the hydrolytic activity was examined at pH=4-8. The results are shown in FIG.

[0165] Figure 12 shows that using OPTS as a substrate, very low hydrolytic activity was detected, which increased slightly above pH 6.0. When 4-MUD was used as a substrate, the hydrolytic activity detected was high at low pH values ​​and was maximal at pH 5.5. Furthermore, the activity detected was greatly reduced at more basic pHs. Since the lipase is a lysosomal lipase, the finding that the pH optimum is pH 5.5 is consistent with the lysosomal pH being around pH 4.5-5.5. The distinct results obtained using OPTS and 4-MUD as substrates are further confirmation of previous findings that the assay detects the hydrolytic activity of different enzymes, such as a carboxylesterase using OPTS as a substrate and a lipase using 4-MUD as a substrate. Again, when the hydrolytic activity detected by antibody solution Ab9 using OPTS as a substrate was compared with that detected using 4-MUD as a substrate, an increase in activity was observed when OPTS was used as a substrate and an increase in activity was observed when 4-MUD was used as a substrate (FIG. 13), with an overall stronger activity observed with the 4-MUD substrate. For antibody solution Ab9, the maximum activity is observed in the acidic range, pH=4.5-5.

[0166] In contrast, antibody solution Ab2 showed the opposite behavior, exhibiting low activity with 4-MUD substrate and high activity with OPTS (Figures 14A and 14B). In both cases, activity increased with increasing pH. In Figure 15, OPTS shows a similar behavior for antibody solution Ab5, but the activity with the 4-MUD substrate is significantly lower. Furthermore, the pH profile when using 4-MUD is slightly different, showing a maximum in the acidic range (pH = 4-4.5), a lower activity between pH 5-6.5, and an increase at higher pH. However, the pH profile measured by OPTS shows a similar behavior to that of protein solution 2. The finding that there is almost no activity in the 4-MUD assay suggests that lipase is absent or present only at very low concentrations in antibody solution Ab5 (as is the case in antibody solutions Ab6 and Ab8; see Figure 9).

[0167] Comparison of pH profiles for the commercial lipases tested (Figure 12) and antibody solutions Ab2 (Figure 14) or Ab5 (Figure 15) show no similarity. However, antibody solution Ab9 shows a similar profile for the OPTS assay and 4-MUD substrate assay data at pH 4-8. This could be an indication for the presence of this lipase in antibody solution Ab9. Since the hydrolytic activity in the OPTS assay and the coumarin substrate assay are of the same order of magnitude, it is likely that other enzymes are present that also contribute to the hydrolytic activity.

Claims

1. 1. A method for detecting carboxylesterase activity of contaminating host cell proteins in a sample containing a recombinant protein of interest produced in a eukaryotic cell, comprising: (a) providing at least one sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture and at least one contaminating host cell protein; (b) contacting at least one sample with a reaction solution (including a hydrophilic substrate) to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a hydrophilic substrate which is a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester); and (iii) optionally, a non-buffering salt; the steps of: (c) incubating the sample and the substrate in a reaction mixture; (d) detecting the carboxylesterase activity of at least one contaminating host cell protein by measuring the hydrolysis of the substrate HPTS ester and detecting the fluorescence intensity of the released chromophore 1-hydroxypyrene-3,6,8-trisulfonic acid or salt thereof (HPTS). Including; The method may comprise measuring hydrolysis by detecting the fluorescence intensity of the released chromophore HPTS over time while incubating the sample and substrate in a reaction mixture according to step (c).

2. (a) the sample and substrate in the reaction mixture are incubated for any time between 2 minutes and 5 hours, 2 minutes and 3 hours, 2 minutes and 2 hours, or 2 minutes and 0.5 hours; and / or (b) multiple reaction mixtures are analyzed in parallel; and / or (c) the reaction mixture has a volume of 300 μl or less; The method of claim 1.

3. The method according to claim 1 or 2, wherein the fluorescence of the released chromophore HPTS is determined using an excitation wavelength in the range of 401-405 nm and an emission wavelength in the range of 510-516 nm.

4. The method of any one of claims 1 to 3, wherein the substrate HPTS ester is selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof.

5. (bi) contacting at least one sample comprising the recombinant protein of interest produced in eukaryotic cells in cell culture from step (a) and at least one contaminating host cell protein with a reaction solution comprising a lipophilic substrate in a separate reaction setting to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a lipophilic 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-C16) 4-MU ester. (iii) optionally, a non-buffering salt, and (iv) Non-denaturing surfactants that do not have an ester bond and are nonionic or zwitterionic surfactants. the steps of: (ci) incubating the sample and the substrate in the reaction mixture of step (bi); and (di) detecting the lipase activity of at least one contaminating host cell protein by measuring the hydrolysis of the substrate 4-MU ester and detecting the fluorescence intensity of the released chromophore 4-MU. Further comprising: measuring hydrolysis 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 (ci); The method according to any one of claims 1 to 4.

6. (a) the lipophilic 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; (b) the surfactant has a final concentration in the reaction mixture that is above its critical micelle concentration in the reaction mixture; and / or (c) a surfactant, (i) selected from the group consisting of CHAPS, CHAPSO, and Zwittergent, preferably CHAPS; or (ii) CHAPS, 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; or (iii) neither polyethylene glycol tert-octylphenyl ether (Triton X-100) nor polyethylene glycol nonylphenyl ether (NP-40); The method according to claim 5.

7. Buffer solutions may 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, Tris, Bis-Tris, Bis-Tris-propane, Bicine (N,N-bis(2-hydroxyethyl)glycine), HEPES (4-2-hydroxyethyl-1-piperazineethanesulfonic acid), TAPS (3-([tris(hydroxymethyl)methyl]amino}propanesulfonic acid), Tricine (N-tris(hydroxymethyl)methylglycine), Na 2 H.P.O. 4 , and NaH 2 P.O. 4 The method according to any one of claims 1 to 6, further comprising one or more buffer substances selected from the group consisting of:

8. The buffer solution is (a) having a pH of about 5 to about 7.5, preferably the buffer has a pH of about 5.5 to about 7.0; and / or (b) 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.

9. (a) The non-buffering salts are NaCl, KCl, and CaCl. 2 and / or (b) the non-buffering salt has a concentration in the reaction mixture of about 100 mM to about 200 mM, preferably about 130 mM to about 170 mM, more preferably about 140 mM to about 150 mM; and / or (c) the ionic strength of the non-buffering salts is about 200 mM or less in the reaction mixture, preferably about 150 mM or less in the reaction mixture; and / or (d) the cumulative ionic strength of the buffer and non-buffer salts in the reaction mixture is about 450 mM or less, preferably about 400 mM or less, and more preferably about 350 mM or less in the reaction mixture; The method according to any one of claims 1 to 8.

10. (a) the at least one sample is a harvested cell culture fluid (HCCF), an in-process control (IPC) sample, a UF / DF filtrate, a drug substance sample, or a drug product sample; (b) the recombinant protein of interest is produced in CHO cells and the at least one contaminating host cell protein is a CHO host cell protein (CHOP); (c) the recombinant protein of interest is not a carboxylesterase or lipase, and / or an enzyme having carboxylesterase or lipase activity; and / or (d) the recombinant protein of interest 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 9.

11. 1. A method for producing a recombinant protein of interest, comprising the steps of: (i) culturing in a cell culture a eukaryotic cell expressing the recombinant protein of interest; (ii) recovering the recombinant protein of interest; (iii) purifying the recombinant protein of interest; and (iv) optionally formulating the recombinant protein of interest into a pharma- ceutically acceptable formulation suitable for administration; and (v) obtaining at least one sample comprising the recombinant protein of interest in steps (ii), (iii) and / or (iv); Including; detecting carboxylesterase activity in a sample containing the recombinant protein of interest and at least one contaminating host cell protein, (a) providing at least one sample obtained in step (v), comprising a recombinant protein of interest produced in eukaryotic cells in cell culture and at least one contaminating host cell protein; (b) contacting at least one sample with a reaction solution comprising a hydrophilic substrate to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a hydrophilic substrate which is a saturated unbranched chain fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester); and (iv) optionally, a non-buffer salt; the steps of: (c) incubating the sample and the substrate in a reaction mixture; and (d) detecting the carboxylesterase activity of at least one contaminating host cell protein by measuring the hydrolysis of the substrate HPTS ester and detecting the fluorescence intensity of the released chromophore HPTS. The method further includes the steps of: Optionally, detecting lipase activity in the sample, (bi) contacting at least one sample comprising the recombinant protein of interest produced in a eukaryotic cell and at least one contaminating host cell protein of step (a) with a reaction solution comprising a lipophilic substrate in a separate reaction setting to form a reaction mixture, the reaction solution comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a lipophilic 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-C16) 4-MU ester; (iii) optionally, a non-buffering salt, and (iv) Non-denaturing surfactants that do not have an ester bond and are nonionic or zwitterionic surfactants. the steps of: (ci) incubating the sample and the substrate in a reaction mixture; (di) detecting lipase activity of at least one contaminating host cell protein by measuring hydrolysis of 4-MU esters and detecting the fluorescence intensity of the released chromophore 4-MU. Including; The method may further comprise measuring hydrolysis by detecting the fluorescence intensity of the released chromophores HPTS and / or 4-MU over time while incubating the sample and substrate in a reaction mixture according to step (c) or (ci), respectively.

12. At least one sample containing a recombinant protein of interest is - step (ii), wherein the sample is harvested cell culture fluid (HCCF) or a cell lysate; - the sample is an in-process control (IPC) sample, step (iii); and / or The sample is a UF / DF sample, a drug substance sample, or a pharmaceutical sample, step (iv). obtaining a 12. The method of claim 11, comprising obtaining in step (iii) at least one sample comprising the recombinant protein of interest, preferably produced in eukaryotic cells in cell culture, and at least one contaminating host cell protein, comprising obtaining the at least one sample before and after affinity chromatography, before and after acid treatment, before and after depth filtration and / or before and after ion exchange chromatography, preferably anion exchange chromatography or cation exchange chromatography.

13. A kit for determining contaminating carboxylesterase and / or lipase activity in a sample containing a recombinant protein of interest, comprising: (i) a buffer having a pH of about pH 4 to about pH 8; (ii) a hydrophilic substrate and a lipophilic substrate, (a) the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester); (b) the lipophilic substrate comprises 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-C16) 4-MU ester (substrate 4-MU ester); a hydrophilic substrate and a lipophilic substrate; and optionally (iii) non-buffering salts; and / or (iv) Non-denaturing surfactants that do not have an ester bond and are nonionic or zwitterionic surfactants. Kit including:

14. (a) the hydrophilic substrate HPTS ester is selected from the group consisting of 1-octanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof (OPTS), 1-nonaoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof, and 1-decanoyloxypyrene-3,6,8-trisulfonic acid or a salt thereof; and / or (b) the lipophilic substrate 4-MU ester 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; The kit of claim 13.

15. 15. The kit of claim 13 or 14, further comprising one or more microtiter plates having 96 wells or a multiple of 96 wells.

16. 1. Use of the hydrophilic substrate HPTS-ester and the lipophilic substrate 4-MU-ester as substrates for detecting the carboxylesterase activity and lipase activity of at least one contaminating host cell protein in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture in an assay, preferably wherein the recombinant protein is produced in CHO cells and the at least one contaminating host cell protein is CHO host cell protein (CHOP), the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester); The use wherein the lipophilic substrate is a saturated unbranched fatty acid (C6-C16) 4-MU ester.

17. 1. Use of the hydrophilic substrate HPTS-ester as a substrate for detecting in an assay the carboxylesterase activity of at least one contaminating host cell protein in a sample comprising a recombinant protein of interest produced in eukaryotic cells in cell culture, preferably wherein the recombinant protein is produced in CHO cells and the at least one contaminating host cell protein is CHO host cell protein (CHOP), The use, wherein the hydrophilic substrate is a saturated unbranched fatty acid (C6-C12) ester of 1-hydroxypyrene-3,6,8-trisulfonic acid or a salt thereof (substrate HPTS ester).

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