Methods for determining hydrolytic activity

Abstract

Reported herein is a method for determining the presence of hydrolase activity in a sample by incubating the sample in a buffer solution containing an artificial hydrolase substrate comprising an ester bond covalently linking an alcohol residue attached to a removal label and a carboxylic acid residue attached to a capture and detection label, and bovine serum albumin, followed by determining or quantifying, respectively, the released carboxylic acid in the incubation mixture, which has been depleted of free alcohol.

Description

[Technical field]

[0001] The present invention is in the field of hydrolases. More precisely, it reports a method for producing a recombinant therapeutic polypeptide preparation having a low level of host cell-associated hydrolytic activity, as well as a method for detecting such host cell-associated hydrolytic activity in a polypeptide preparation, and a method for selecting host cells which produce recombinant therapeutic polypeptides having a concomitant low level of host cell-associated hydrolytic activity. [Background technology]

[0002] Polysorbates are used as stabilizers against protein aggregation and to prevent surface adsorption, among other uses in biopharmaceutical protein formulations. Several studies have shown that residual hydrolytic activity / hydrolytically active host cell proteins (HCPs) are present in the final biopharmaceutical formulations. Such hydrolytic activity / hydrolytically active HCPs are responsible for the hydrolysis of polysorbates during long-term storage. Hydrolysis of polysorbates within therapeutic protein formulations results in increased levels of free fatty acids. This is a significant quality issue and a potential risk factor for product quality and shelf life. Both the concentration and activity of hydrolyzable HCPs in the final formulation are almost always low, so commonly used analytical methods are inadequate in terms of sensitivity and / or do not provide reasonable turnaround times for their detection.

[0003] Bhargava, AC et al. reported a high-throughput fluorescence-based esterase activity assay for evaluating polysorbate degradation risk during biopharmaceutical development (Pharm. Res. 38 (2021) 397-413 (Non-Patent Document 1)).

[0004] Jahn, M. et al. reported on the measurement of lipolytic activity to support process improvements for controlling lipase-mediated polysorbate degradation (Pharm. Res. 37 (2020) 118 (Non-Patent Document 2)).

[0005] WO 2021 / 050585 (Patent Document 1) reported compositions, methods and kits for detecting lipolytic activity.

[0006] Gluecklich, N. et al. reported on the evaluation of polysorbate-degrading fingerprints and lipase kinetics, as well as how the activity of polysorbate-degrading hydrolases is affected by the assay and assay conditions (Eur. J. Pharm. Sci. 166 (2021) 105980 (Non-Patent Document 3)).

[0007] Li, X. et al. reported profiling of active enzymes for polysorbate degradation in biotherapeutics by activity-based protein profiling (Anal. Chem. 93 (2021) 8161-8169 (Non-Patent Document 4)).

[0008] WO 2001 / 73442 reported a biotin-PEG substrate for lipase assays.

[0009] Li, X. et al. reported on the measurement and control of high-risk host cell proteins for polysorbate degradation in biologics (Antibod. Ther. 5 (2022) 42-54 (Non-Patent Document 5)).

[0010] Zhang, S. et al. (Anal. Biochem. 637 (2022) 114472; available online November 18, 2021 (Non-Patent Document 6)) reported monitoring polysorbate hydrolysis in therapeutic proteins using an ultrasensitive extraction-free fatty acid quantification method.

[0011] Thus, there is a need for assays with improved properties. [Prior art documents] [Patent documents]

[0012] [Patent Document 1] International Publication No. 2021 / 050585 [Patent Document 2] International Publication No. 2001 / 73442 [Non-patent literature]

[0013] [Non-Patent Document 1] Bhargava, AC et al. “Pharm.Res.” 38(2021)397-413 [Non-Patent Document 2] Jahn, M. et al. “Pharm. Res.” 37(2020)118 [Non-Patent Document 3] Gluecklich, N. et al. “Eur.J.Pharm.Sci.” 166(2021)105980 [Non-Patent Document 4] Li, X. et al. “Anal.Chem.” 93(2021)8161-8169 [Non-Patent Document 5] Li, X. et al. “Antibod.Ther.” 5(2022)42-54 [Non-Patent Document 6] Zhang, S. et al. “Anal.Biochem.” 637(2022)114472 Summary of the Invention

[0014] Herein, we report a new assay setup that uses tripartite artificial substrates for bond cleavage enzymes that mimic naturally occurring substrates of the bond cleavage enzymes and allow detection of cleavage products with high sensitivity in a short time, thus determining the bond cleavage activity in a sample.

[0015] For example, if a bond-cleaving enzyme is present in the sample to be analyzed, the artificial substrate contains exactly one bond that is susceptible to cleavage by the bond-cleaving enzyme of the class of enzymes to which the bond-cleaving enzyme in question belongs. This susceptible bond is cleaved by the enzyme, and the intact artificial substrate and one of the cleavage products are removed, and then the other cleavage product is detected. Thus, the artificial substrate contains three tags. A part of the substrate is bound to a first tag that allows the removal of the intact artificial substrate and one of the cleavage products from the sample after incubation. A second part of the substrate is bound to two tags, the first tag for immobilization and the second tag for detection. As the first, second and third tags, any tag that is useful according to the knowledge of the person skilled in the art can be used.

[0016] Thus, one aspect of the invention provides a method for determining the presence of bond cleavage activity and / or a bond cleavage enzyme in a sample, comprising the steps of: (a) mixing and incubating a sample or an aliquot thereof and a buffer solution to form an incubated sample, the buffer solution comprises a buffer substance, an artificial bond-cleaving enzyme substrate, a salt, and optionally bovine serum albumin; the sample comprises a therapeutic polypeptide; the artificial bond-cleaving enzyme substrate comprises exactly one bond that is susceptible to cleavage by the bond-cleaving enzyme to be detected, the bond covalently linking a first portion of the substrate that is bound to a first label and a second portion of the substrate that is bound to a nucleic acid tag and to a second label that is different from the first label; cleaving the bond in the artificial bond cleavage enzyme substrate into a free first moiety and a free second moiety if the respective bond cleavage activity and / or the respective bond cleavage enzyme is present in the sample; (b) removing the free first moiety and the (uncleaved = intact) artificial bond cleavage enzyme substrate from the incubated sample to obtain a depleted sample, removing by contacting the incubated sample with a first solid phase that specifically binds the first label (for a time sufficient for the free first moiety and the (uncleaved) artificial bond cleavage enzyme substrate to bind to the solid phase and to the ligand that specifically binds the first label), and then separating the incubation mixture, depleted of the free first moiety and the (uncleaved) artificial bond cleavage enzyme substrate, from the first solid phase; (c) determining the presence of a bond cleavage activity and / or a bond cleavage enzyme in the sample by determining (the amount of) a second label in the depleted sample, determining the presence of bond cleavage activity and / or bond cleavage enzyme in the sample by capturing the second moiety on a second solid phase via the nucleic acid tag, followed by detection of the free second moiety captured on the solid phase with an antibody that specifically binds to the second label, which is linked to one or more detectable (ruthenium) labels (by electrochemiluminescence); or determining the presence of bond cleavage activity and / or bond cleavage enzyme in the sample by determining whether the amount of second label in the depleted sample exceeds the amount determined in a control sample in the absence / absence of bond cleavage activity and / or bond cleavage enzyme; The method includes:

[0017] In particular, a new assay setup is reported herein that uses tripartite artificial hydrolase substrates that mimic the hydrolysis-sensitive moieties of polysorbates and allow for the detection of hydrolytic cleavage products with high sensitivity in a short time, thus determining the hydrolase (hydrolysis) activity in a sample.

[0018] If a hydrolytic enzyme, such as an esterase, is present in the sample being analyzed, the artificial hydrolytic enzyme substrate is cleaved by the hydrolytic enzyme and the released alcohol moiety of the artificial substrate is subsequently detected.

[0019] In a preferred embodiment, the hydrolase is an esterase or a lipase.

[0020] In a preferred embodiment, detection is by electrochemiluminescence immunoassay.

[0021] The invention is based, at least in part, on the discovery that using the method according to the invention, hydrolase (hydrolysis) activity in a sample can be determined within 48 hours without the need for sample pretreatment and without the need for chromatographic separation. Thus, the method of the invention allows the determination of hydrolase (hydrolysis) activity / presence of hydrolases at an improved speed compared to the FAMS assay, which requires 10-14 days (48 hours corresponds to the frequency with which a purification method for a biologic may be performed to generate a new sample), but with comparable sensitivity as well as improved because of reduced operational requirements compared to other assays, since no sample pre-preparation (e.g., buffer exchange, etc.) or chromatographic separation and equipment are required.

[0022] Furthermore, the assay according to the invention allows the determination of PS20 and PS80 degrading hydrolase (hydrolysis) activity / hydrolases using the same artificial enzyme substrate, which allows the determination of the total or sum of hydrolase (hydrolysis) activity / hydrolases in a sample.

[0023] Furthermore, the assay according to the present invention is suitable for high throughput screening (HTS).

[0024] The present invention is based, at least in part, on the discovery that the use of inactive bovine serum albumin results in an increase in the signal-to-noise ratio, thereby improving the sensitivity. Without being bound by this theory, it is believed that the use of inactive bovine serum albumin can increase the hydrolase (hydrolysis) enzyme activity in the sample, thereby increasing the sensitivity of the assay. In a preferred embodiment, the inactive bovine serum albumin is bovine serum albumin in which more than 80% of the cysteine ​​residues at position 34 are oxidized / in oxidized form. In a particular embodiment, more than 90% of the cysteine ​​residues at position 34 are oxidized / in oxidized form. In a particular embodiment, more than 95% of the cysteine ​​residues at position 34 are oxidized / in oxidized form. In one preferred embodiment, more than 80% of the bovine serum albumin is present in monomeric form (not bound to a second polypeptide at cysteine ​​residue 34). In a particular embodiment, more than 90% of the bovine serum albumin is present in monomeric form. In a particular embodiment, more than 95% of the bovine serum albumin is present in monomeric form.

[0025] The present invention is based, at least in part, on the discovery that the use of (3-(N-morpholino)propanesulfonic acid) (MOPS) as a buffer in the methods according to the invention results in better assay sensitivity. Without being bound by this theory, it is hypothesized that the pKa value of MOPS of 7.02 at 37° C. results in better buffering capacity at a pH value of 7.0.

[0026] The present invention is based, at least in part, on the discovery that the use of hybridized LNA molecules as immobilization tags for capturing the final analyte results in an increased signal-to-noise ratio, and thereby improved sensitivity, as compared to, for example, streptavidin / biotin-mediated capture.

[0027] The present invention is based, at least in part, on the discovery that a sample does not require pre-treatment in order to be suitable for the method according to the invention, i.e. the method according to the invention is independent of the sample matrix, such as, for example, the buffer or pH of the sample.

[0028] The present invention is based, at least in part, on the discovery that the addition of about 0.1% (w / v) of a surfactant, particularly a non-ionic surfactant, increases the sensitivity of the assay, which is particularly useful when purified samples, i.e., formulated drug substances, must be tested, so that even minute amounts of residual hydrolytic enzyme (hydrolytic) activity can be determined.

[0029] Thus, one aspect of the invention is a method for determining hydrolytic enzyme (hydrolytic) activity and / or the presence of hydrolytic enzymes in a sample, comprising the steps of: (a) mixing and incubating a sample or an aliquot thereof and a buffer solution to form an incubated sample, the buffer solution comprises a buffer substance, an artificial hydrolase substrate, a salt and bovine serum albumin; the sample comprises a therapeutic polypeptide; The artificial hydrolase substrate is a carboxylic acid residue attached to a first label, and An alcohol residue attached to the nucleic acid tag and to a second label that is different from the first label. Contains a single / exact ester bond covalently linking if hydrolase (hydrolysis) activity and / or hydrolases are present in the sample, the ester bonds in the artificial hydrolase substrate are cleaved into free alcohol and free carboxylic acid residues; (b) removing free carboxylic acid residues and (uncleaved) artificial hydrolase substrates from the incubated sample to obtain a depleted sample, removing by contacting the incubated sample with a first solid phase that specifically binds the first label (for a time sufficient for the free carboxylic acid and the (uncleaved) artificial arsenic hydrolase substrate to bind to the solid phase and to the ligand that specifically binds to the first label), and then separating the incubation mixture depleted of free carboxylic acid and the (uncleaved) artificial arsenic hydrolase substrate from the first solid phase; (c) determining the hydrolytic enzyme (hydrolytic) activity and / or the presence of hydrolytic enzymes in the sample by determining the (amount of) second label in the depleted sample, Determining the hydrolase (hydrolysis) activity and / or the presence of hydrolases in the sample is by capturing the free alcohol residues on a second solid phase via the nucleic acid tag, followed by detecting the free alcohol residues captured on the solid phase with an antibody that specifically binds to the second label, which is linked to one or more detectable (ruthenium) labels (by electrochemiluminescence); or Determining the presence of hydrolase (hydrolysis) activity and / or hydrolases in the sample by determining whether the amount of second label in the depleted sample exceeds the amount determined in a control sample in the absence / absence of hydrolase activity and / or the presence of hydrolases. The method includes:

[0030] One aspect of the invention is a method for determining hydrolytic enzyme activity in a sample, comprising the steps of: (a) mixing and incubating an aliquot of the sample and a buffer solution for a predetermined period of time to form an incubated sample; the buffer solution comprises a buffer substance, an artificial hydrolase substrate, a salt and bovine serum albumin; the sample comprises a therapeutic polypeptide; The artificial hydrolase substrate is a carboxylic acid residue attached to a first label, and An alcohol residue attached to the nucleic acid tag and to a second label that is different from the first label. Contains a single / exact ester bond covalently linking if hydrolase (hydrolysis) activity and / or hydrolases are present in the sample, the ester bonds in the artificial hydrolase substrate are cleaved into free alcohol and free carboxylic acid residues; (b) removing free carboxylic acid residues and (uncleaved) artificial hydrolase substrates from the incubated sample to obtain a depleted sample, removing by contacting the incubated sample with a first solid phase that specifically binds the first label (for a time sufficient for the free carboxylic acid and the (uncleaved) artificial arsenic hydrolase substrate to bind to the solid phase and to the ligand that specifically binds the first label), and then separating the incubation mixture depleted of the free carboxylic acid and the (uncleaved) artificial arsenic hydrolase substrate from the first solid phase; (c) optionally repeating steps (a)-(b) at least once with a further (new) aliquot of the sample for a period of time different from any previous period used in step (a); (d) determining (individually) the (amount of) second label in (each of) the depleted samples, the determination being by capturing the free alcohol residue on a second solid phase via the nucleic acid tag, followed by detection of the captured free alcohol residue with an antibody that specifically binds to the second label (by electrochemiluminescence) conjugated to one or more detectable (ruthenium) labels, (e) determining the hydrolase activity or hydrolase dynamics in the sample over time by quantifying the amount of second label present in the depleted sample, optionally individually, over a period of time, using the calibration curve; The method includes:

[0031] One aspect of the invention is a method for selecting (recombinant) mammalian cell clones, comprising the steps of: (1) measuring hydrolase activity separately for culture supernatant samples obtained by separately culturing at least two different (recombinant) mammalian cell clones by the following steps: (a) mixing and incubating a culture supernatant sample, or an aliquot thereof, with a buffer to form an incubated mixture, the buffer solution comprises a buffer substance, an artificial hydrolase substrate, a salt and bovine serum albumin; the sample comprises a therapeutic polypeptide; The artificial hydrolase substrate is a carboxylic acid residue attached to a first label, and An alcohol residue attached to the nucleic acid tag and to a second label that is different from the first label. Contains a single / exact ester bond covalently linking if hydrolase (hydrolysis) activity and / or hydrolases are present in the sample, the ester bonds in the artificial hydrolase substrate are cleaved into free alcohol and free carboxylic acid residues; (b) removing free carboxylic acid residues and (uncleaved) artificial hydrolase substrate from the incubated mixture to obtain a depleted mixture, removing by contacting the incubated mixture with a first solid phase that specifically binds the first label (for a time sufficient for the free carboxylic acid residues and the (uncleaved) artificial hydrolase substrate to bind to the solid phase and to a ligand that specifically binds to the first label), and then separating the incubation mixture depleted of free carboxylic acids and the (uncleaved) artificial hydrolase substrate from the first solid phase; (c) determining the hydrolytic enzyme (hydrolytic) activity in the sample by determining the (amount of) second label in the depleted sample, the determination being by capturing the free alcohol residue on a second solid phase via the nucleic acid tag, followed by detection of the captured free alcohol residue with an antibody that specifically binds to the second label (by electrochemiluminescence) conjugated to one or more detectable (ruthenium) labels, (2) selecting the mammalian cell clones having the lowest hydrolase (hydrolysis) activity as determined in step (c); The method includes:

[0032] In one preferred embodiment of all aspects and other embodiments of the invention, the hydrolase is an esterase and the hydrolase activity is an esterase activity.

[0033] In one preferred embodiment of all aspects and other embodiments of the invention, detection is by electrochemiluminescence immunoassay.

[0034] In certain embodiments of all of the aspects and other embodiments of the invention, the first and second labels are independently selected from the group consisting of an antigen and a partner of a specific binding pair.

[0035] In certain embodiments of all aspects and other embodiments of the present invention, the nucleic acid tag can stably hybridize with a second complementary nucleic acid molecule. In certain embodiments, the nucleic acid tag is a locked nucleic acid / LNA oligonucleotide. In one preferred embodiment, all nucleotides of the nucleic acid tag are locked nucleic acid monomers. In certain embodiments of all aspects and other embodiments of the present invention, the nucleic acid sequence tag is SEQ ID NO:01: tgctcctg; SEQ ID NO:02: caggagca; SEQ ID NO: 09: tgctcctgt; SEQ ID NO: 10: acaggagca; SEQ ID NO:11: gtgcgtct; SEQ ID NO:12: agacgcac; SEQ ID NO:13: gttggtgt; SEQ ID NO:14: acaccaac; SEQ ID NO:33: cttcc; SEQ ID NO:34: ggaag; SEQ ID NO:42: gctcc; SEQ ID NO:43: ggagc; SEQ ID NO:16: gttggt; SEQ ID NO:46: ccaac; SEQ ID NO:16: gttggt; SEQ ID NO:19: caccaac; SEQ ID NO:16: gttggt; SEQ ID NO:17: caacacaccaac; SEQ ID NO:16: gttggt; SEQ ID NO:18: acacaccaac; SEQ ID NO:16: gttggt; SEQ ID NO:14: acaccaac; SEQ ID NO:16: gttggt; SEQ ID NO:20: accaac; SEQ ID NO:40: ctgtca; SEQ ID NO:41: tgacag; SEQ ID NO:44: tgctcc; SEQ ID NO:45: ggagca; SEQ ID NO:35: tcttcc; SEQ ID NO:36: ggaaga; SEQ ID NO:21: gttggtg; SEQ ID NO:17: caacacaccaac; SEQ ID NO:21: gttggtg; SEQ ID NO:18: acacaccaac; SEQ ID NO:21: gttggtg; SEQ ID NO:14: acaccaac; SEQ ID NO:21: gttggtg; SEQ ID NO:19: caccaac; SEQ ID NO:21: gttggtg; SEQ ID NO:20: accaac; SEQ ID NO:01: tgctcctg; SEQ ID NO:02: caggagca; SEQ ID NO:11: gtgcgtct; SEQ ID NO:12: agacgcac; SEQ ID NO:13: gttggtgt; SEQ ID NO:17: caacacaccaac; SEQ ID NO:13: gttggtgt; SEQ ID NO:18: acacaccaac; SEQ ID NO:13: gttggtgt; SEQ ID NO:14: acaccaac; SEQ ID NO:13: gttggtgt; SEQ ID NO:19: caccaac; SEQ ID NO:13: gttggtgt; SEQ ID NO:20: accaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:17: caacacaccaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:18: acacaccaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:14: acaccaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:19: caccaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:20: accaac; SEQ ID NO:09: tgctcctgt; SEQ ID NO:15: caggagc; SEQ ID NO: 09: tgctcctgt; SEQ ID NO: 10: acaggagca; SEQ ID NO:09: tgctcctgt; SEQ ID NO:02: caggagca; SEQ ID NO:22: gttggtgtg; SEQ ID NO:30: cacaccaac; SEQ ID NO:37: ttctcttcc; SEQ ID NO:38: ggaagagaa; SEQ ID NO:23: gttggtgtgttg; SEQ ID NO:17: caacacaccaac; SEQ ID NO:23: gttggtgtgttg; SEQ ID NO:18: acacaccaac; SEQ ID NO:23: gttggtgtgttg; SEQ ID NO:14: acaccaac; SEQ ID NO:23: gttggtgtgttg; SEQ ID NO:19: caccaac; SEQ ID NO:23: gttggtgtgttg: SEQ ID NO:20: accaac; SEQ ID NO:31: gttggtgtgttggtg; SEQ ID NO:32: caccaacacaccaac; SEQ ID NO:28: aaaaaaaaa; SEQ ID NO:24: ttttttttt; SEQ ID NO:28: aaaaaaaaa; SEQ ID NO:25: tttttttt; SEQ ID NO:28: aaaaaaaaa; SEQ ID NO:26: tttttt; SEQ ID NO:28: aaaaaaaaa; SEQ ID NO:27: tttttt; SEQ ID NO:39: aaaaaa; SEQ ID NO:27: tttttt In one preferred embodiment, the nucleic acid tag has a sequence of TGGTTG in the 3' to 5' direction. In a particular embodiment, the nucleic acid tag specifically and stably hybridizes with the nucleic acid sequence CAACCA (in the 3' to 5' direction). In one preferred embodiment, the two distinct compatible binding partner pairs are pairs of all-LNA single-stranded oligonucleotides of SEQ ID NO: 16 and SEQ ID NO: 20.

[0036] In certain embodiments of all of the aspects and other embodiments of the invention, the buffer solution further comprises sodium chloride.

[0037] In certain embodiments of all aspects and other embodiments of the present invention, the alcohol of the artificial hydrolase substrate and the carboxylic acid of the artificial hydrolase substrate each, independently of the other, contain one or more ethylene oxide (CH2-CH2-O) units.

[0038] In certain embodiments of all aspects and other embodiments of the present invention, the artificial hydrolase substrate is TIFF2025500334000002.tif16128 (in the formula, n and m are each independently 0 or a positive integer value; R1 comprises a first label, R2, R3 comprise a second label and a nucleic acid tag) Includes.

[0039] An independent aspect of the present invention is an artificial hydrolase substrate, which is a means for carrying out the assay according to the present invention.

[0040] In certain embodiments of all aspects and other embodiments of the invention, the buffer solution has a pH value of about 7.

[0041] In certain embodiments of all aspects and other embodiments of the invention, the incubation is performed at a temperature ranging from 25° C. to 45° C. In certain embodiments, the incubation is performed at a temperature ranging from 33° C. to 41° C. In certain embodiments, the incubation is performed at a temperature ranging from 37° C. to 40° C. In certain embodiments, the incubation is performed at a temperature of about 37° C.

[0042] In certain embodiments of all aspects and other embodiments of the invention, the incubation is for at least 15 minutes and up to 36 hours, in certain embodiments up to 22 hours, and in certain embodiments up to 8 hours.

[0043] In certain embodiments of all aspects and other embodiments of the present invention, the incubation mixture comprises bovine serum albumin at a final concentration of 300 μM or less. In certain embodiments, the incubation mixture comprises bovine serum albumin at a final concentration of 30 μM or less. In one preferred embodiment, the incubation mixture comprises bovine serum albumin at a final concentration of 10 μM or less. In one preferred embodiment, the incubation mixture comprises bovine serum albumin at a concentration of about 6 μM.

[0044] In certain embodiments of all aspects and other embodiments of the invention, the incubation mixture comprises a divalent metal ion. In one preferred embodiment, the divalent metal ion is Mg 2+ In certain embodiments, the divalent metal ion is added in the form of a chloride salt. In certain embodiments, the divalent metal ion is (added as) MgCl.

[0045] In certain embodiments of all aspects and other embodiments of the invention, the incubation mixture further comprises a surfactant at a final concentration of 0.05% to 0.15% (w / v). In one preferred embodiment, the final concentration of the surfactant is about 0.1% (w / v). In certain embodiments, the surfactant is a non-ionic surfactant.

[0046] In certain embodiments of all aspects and other embodiments of the present invention, the surfactant is Triton X-100. In certain embodiments, the surfactant is an aromatic hydrocarbon lipophilic or hydrophobic group with a hydrophilic polyethylene oxide chain. In certain embodiments, the surfactant has, on average, 9.5 ethylene oxide units and the hydrocarbon is a 4-(1,1,3,3-tetramethylbutyl)-phenyl group.

[0047] In certain embodiments of all aspects and other embodiments of the invention, the surfactant is Triton CG-110. In other specific embodiments, the surfactant is an alkyl polyglucoside. In one preferred embodiment, the surfactant is a mixture of 58.0-62.0 (w / v)% D-glucopyranose, oligomeric decyl octyl glycoside, and 38.0-42.0 (w / v)% water.

[0048] In certain embodiments of all aspects and other embodiments of the invention, the incubation mixture comprises 20-30 mM sodium chloride with a monomer content of 95% or more, 3-10 μM BSA / oxidized Cys34, 100-200 mM MOPS, 3-10 mM magnesium chloride, 0.05-0.15% (w / v) surfactant, the therapeutic polypeptide has a concentration of 0.1-250 mg / mL, and has a pH value of 6.8-7.2, and the incubation is performed at 35-40° C. for 8-36 hours.

[0049] In one preferred embodiment of all aspects and other embodiments of the invention, the incubation mixture comprises final concentrations of about 300 mM TRIS, about 5 mM MgCl2, about 25 mM NaCl, about 0.1% (w / v) triton CG-110 and about 6 μM bovine serum albumin.

[0050] In one preferred embodiment of all aspects and other embodiments of the invention, the buffer solution comprises about 150 mM MOPS, about 5 mM MgCl2, about 25 mM NaCl, about 0.1% (w / v) triton CG-110 and about 6 μM bovine serum albumin.

[0051] In one preferred embodiment of all aspects and other embodiments of the invention, the first label is biotin or a variant thereof or the first label is (strept)avidin or a variant thereof.

[0052] In one preferred embodiment of all aspects and other embodiments of the invention, the second label is digoxigenin or a variant thereof.

[0053] In certain embodiments of all aspects and other embodiments of the invention, the mammalian cell clone is a recombinant mammalian cell clone comprising one or more nucleic acids encoding a therapeutic polypeptide. In certain embodiments, the therapeutic polypeptide is a heterologous therapeutic polypeptide. In certain embodiments, the therapeutic polypeptide is an antibody. In one preferred embodiment, the (recombinant) mammalian cell clone is a stable transfected CHO cell clone expressing an antibody, preferably a bispecific antibody. In certain embodiments, the cell clone produces a multispecific antibody. In certain embodiments, one of the binding specificities of the multispecific antibody is for a first antigen and the other is for a different second antigen. In certain embodiments, the multispecific antibody binds to two different epitopes of the same antigen. In certain embodiments, the second epitopes on the same antigen are non-overlapping epitopes. In certain embodiments, the antibody is a bispecific antibody. In certain embodiments, the bispecific antibody is a trivalent bispecific antibody or a bivalent bispecific antibody. In certain embodiments, multispecific antibodies comprise Fab fragments in which either the heavy and light chain variable or constant regions have been exchanged, i.e., in one chain, the heavy chain VH variable domain is either directly or via a peptide linker attached to the light chain CL constant domain, and in the respective other chain, the light chain VL variable domain is either directly or via a peptide linker attached to the heavy chain CH1 constant domain.

[0054] In certain embodiments of all aspects and other embodiments of the present invention, the ester bond between the alcohol residue and the carboxylic acid residue in the artificial hydrolase substrate is susceptible to cleavage by the hydrolase.

[0055] In certain embodiments of all aspects and other embodiments of the present invention, the incubating is to allow the ester bond in the artificial hydrolase substrate to be cleaved by the hydrolase / hydrolase activity in the sample.

[0056] In certain embodiments of all aspects and other embodiments of the invention, the hydrolase / hydrolase activity is based on the presence of one or more hydrolases selected from the group consisting of lipoprotein lipase, palmitoyl protein thioesterase, acid ceramidase, the C-terminal domain of fatty acid synthase, putative phospholipase b-like 2, lysosomal acid lipase, lysosomal phospholipase, and sialate O-acetylesterase.

[0057] In certain embodiments of all aspects and other embodiments of the invention, the hydrolase / hydrolase activity is based on the presence of one or more hydrolases selected from the group consisting of lipoprotein lipase, palmitoyl protein thioesterase, acid ceramidase, the C-terminal domain of fatty acid synthase, putative phospholipase b-like 2, lysosomal acid lipase, lysosomal phospholipase, and sialate O-acetylesterase.

[0058] In certain embodiments of all aspects and other embodiments of the present invention, the incubated mixture comprises an uncleaved artificial hydrolase substrate and cleavage products of the artificial hydrolase substrate, hi certain embodiments, the cleavage products of the artificial hydrolase substrate are free alcohol residues and free carboxylic acid residues.

[0059] In certain embodiments of all aspects and other embodiments of the present invention, the depleted incubation mixture is depleted of free carboxylic acid residues and uncleaved artificial hydrolase substrates.

[0060] In certain embodiments of all aspects and other embodiments of the invention, the hydrolytic enzyme is a lipase.

[0061] In certain embodiments of all aspects and other embodiments of the invention, the hydrolase is selected from the group consisting of lipoprotein lipase (Accession No. G3H6V7), palmitoyl protein thioesterase 1 (Accession No. G3HN89), acid ceramidase (Accession No. G3GZB2), C-terminal domain of fatty acid synthase (Accession No. G3GXD7), putative phospholipase b-like 2 (Accession No. G316T1), lysosomal acid lipase (Accession No. G3HQY6), lysosomal phospholipase, phospholipase A2, group VII (PLA2G7) and group XV (PLA2G15), sialate O-acetyl esterase. In certain embodiments, the hydrolase is lipoprotein lipase, putative phospholipase b-like 2, and / or lysosomal phospholipase. In a preferred embodiment, the hydrolase is a lysosomal phospholipase, specifically lysosomal phospholipase A2 (LPLA2) (Accession Number: G3HKV9).

[0062] In certain embodiments of all aspects and other embodiments of the present invention, the sample is not pretreated. In certain embodiments, the sample is used without buffer exchange. In certain embodiments, the sample is used without chromatographic pre-separation. In one preferred embodiment, the sample is unpurified culture supernatant or unpurified crude preparative chromatographic column eluate.

[0063] In certain embodiments of all aspects and other embodiments of the invention, the method is for determining the total hydrolytic / hydrolytic enzyme activity in a sample.

[0064] In addition to the various aspects and embodiments described and claimed, the subject matter of the present disclosure is also directed to other aspects and embodiments having other combinations of features disclosed and claimed herein. Thus, the specific features presented herein, particularly as aspects or embodiments, can be combined with each other in other ways within the scope of the subject matter of the present disclosure, so that the subject matter of the present disclosure includes any suitable combination of features disclosed herein. The foregoing description of specific embodiments of the subject matter of the present disclosure is presented for illustrative and explanatory purposes. It is not intended to be exhaustive or to limit the subject matter of the present disclosure to the disclosed embodiments. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0065] Detailed Description of the Invention Herein, a new assay setup is reported that uses a tripartite artificial hydrolase substrate that mimics polysorbate. If a hydrolase or a compound with hydrolase activity is present in the sample to be analyzed, the artificial hydrolase substrate is cleaved by the hydrolase / hydrolase activity and the released alcohol residue of the artificial hydrolase substrate is detected. The detection of the released alcohol residue of the artificial hydrolase substrate is by a label attached thereto. The label can be freely selected. Thus, the detection can be performed by any known method, such as, for example, Simoa®, Gyrolab®, Elecsys®, ELISA, etc. In a preferred embodiment, the detection is by electrochemiluminescence immunoassay.

[0066] The present invention is based, at least in part, on the discovery that the presence of bovine serum albumin with a high monomer content is advantageous in terms of assay sensitivity, which can increase the hydrolase activity of the hydrolases as well as the sensitivity of the assay.

[0067] overview Methods and techniques useful for carrying out the invention are described, for example, in Ausubel, FM (ed.), Current Protocols in Molecular Biology, Volumes I-III (1997); Glover, ND, and Hames, BD, eds., DNA Cloning: A Practical Approach, Volumes I and II (1985), Oxford University Press; Freshney, RI (ed.), Animal Cell Culture-a practical approach, IRL Press Limited (1986); Watson, JD et al., Recombinant DNA, 2nd ed., CHSL Press (1992); Winnacker, EL, From Genes to Clones; NY, VCH Publishers (1987); Celis, J., ed., Cell Biology, 2nd ed., Academic Press (1998); Freshney, RI, Culture of Animal Cells: A Manual of Basic Technique, 2nd ed., Alan R. Liss, Inc., NY (1987).

[0068] The use of recombinant DNA technology allows the creation of derivatives of nucleic acids. Such derivatives can be modified, for example, by substitution, alteration, replacement, deletion or insertion, at individual or several nucleotide positions. Modification or derivatization can be performed, for example, by site-directed mutagenesis. Such modifications can be easily carried out by those skilled in the art (see, for example, Sambrook, J. et al., Molecular Cloning: A laboratory manual (1999) Cold Spring Harbor Laboratory Press, New York, USA; Hames, BD and Higgins, SG, Nucleic acid hybridization-a practical approach (1985) IRL Press, Oxford, England).

[0069] It should be noted that, as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, a reference to "a cell" includes a plurality of such cells, and equivalents thereof known to those of skill in the art, and so forth. Similarly, the terms "a" (or "an"), "one or more," and "at least one" may be used interchangeably herein. It should also be noted that the terms "comprising," "including," and "having" may be used interchangeably.

[0070] The term "about" refers to a range of ±20% of the numerical value that follows it. In certain embodiments, the term "about" refers to a range of ±10% of the numerical value that follows it. In certain embodiments, the term "about" refers to a range of ±5% of the numerical value that follows it. The term "comprising" also includes the term "consisting of."

[0071] cell As used herein, the term "cell clone" refers to a mammalian cell that contains an exogenous nucleotide sequence capable of expressing a polypeptide, i.e., a recombinant mammalian cell. Such a recombinant mammalian cell is a cell into which one or more exogenous nucleic acid(s) have been introduced, including the progeny of such a cell. In certain embodiments, the cell clone is a mammalian cell that contains a nucleic acid encoding a heterologous polypeptide. Thus, the term "cell clone containing a nucleic acid encoding a heterologous polypeptide" refers to a recombinant mammalian cell that contains an exogenous nucleotide sequence integrated into the genome of the mammalian cell and is capable of expressing a heterologous polypeptide. In certain embodiments, the cell clone is a mammalian cell that contains an exogenous nucleotide sequence integrated into a single site within a locus of the cell's genome. In a preferred embodiment, the cell clone is a mammalian cell that contains an exogenous nucleotide sequence integrated into a single site within a locus of the cell's genome, the exogenous nucleotide sequence comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least one first selection marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and the recombination recognition sequences are all different.

[0072] The term "recombinant cell" as used herein refers to a cell after genetic modification, such as a cell that expresses a heterologous polypeptide of interest and can be used for the production of said heterologous polypeptide on any scale. For example, a "cell clone" refers to a cell in which the coding sequence of the heterologous polypeptide of interest is stably introduced into the genome. For example, a "recombinant mammalian cell containing an exogenous nucleotide sequence" that has been subjected to recombinase-mediated cassette exchange (RMCE), in which the coding sequence of the polypeptide of interest is stably introduced into the genome of the host cell, is a particular "cell clone".

[0073] As used herein, a "cell clone" includes the primary transformed cell as well as progeny derived therefrom regardless of the number of passages. The progeny may not be completely identical to the parent cell, for example in nucleic acid content, but may contain mutations. Mutant progeny that have the same function or biological activity as screened or selected for in the originally transformed cell are included.

[0074] An "isolated cell clone" refers to a cell clone that has been separated from a component of its natural environment.

[0075] An "isolated nucleic acid" refers to a nucleic acid molecule that is separated from a component of its natural environment.

[0076] An "isolated polypeptide" or an "isolated antibody" or an "isolated by-product" refers to a polypeptide molecule or an antibody molecule that has been separated from a component of its natural environment, respectively. In certain embodiments, an isolated polypeptide or an isolated antibody is purified to greater than 95% or greater than 99% purity, for example, as determined by electrophoresis (e.g., SDS-PAGE, isoelectric focusing (IEF), capillary electrophoresis, CE-SDS) or chromatography (e.g., size exclusion chromatography or ion exchange or reverse phase HPLC). For a review of methods for assessing antibody purity, see, for example, Flatman, S. et al., J. Chrom. B848 (2007) 79-87.

[0077] Assay The detection of the released alcohol residue of the artificial hydrolase substrate in the method according to the invention is by means of a label attached thereto. The label can be freely selected. Thus, the detection can be carried out by any known method, for example Simoa®, Gyrolab®, Elecsys®, ELISA, etc.

[0078] The principles of various different immunoassays are described, for example, in Hage, DS (Anal. Chem. 71 (1999) 294R-304R). Lu, B. et al. (Analyst 121 (1996) 29R-32R) report oriented immobilization of antibodies for use in immunoassays. Avidin-biotin mediated immunoassays are reported, for example, in Wilchek, M., and Bayer, EA, in Methods Enzymol. 184 (1990) 467-469.

[0079] The term "immunoassay" refers to any technique that utilizes a specific binding molecule, such as an antibody, to capture and / or detect a specific target for qualitative or quantitative analysis. In general, an immunoassay is characterized by the following steps: (1) immobilization or capture of the analyte, and (2) detection and measurement of the analyte. The analyte can be captured, i.e., bound, to any solid surface, such as a membrane, a plastic plate, or any other solid surface.

[0080] In general, immunoassays can be performed in three different formats: one with direct detection, one with indirect detection, or one with a sandwich assay.

[0081] Direct detection immunoassays use a detection (or tracer) antibody that can be measured directly. Enzymes or other molecules or radiation allow the production of a signal that produces color, fluorescence, or electrochemiluminescence that allows the signal to be visualized or measured.

[0082] In an indirect assay, a primary antibody that binds to the analyte is used to provide a defined target for a secondary antibody that specifically binds to the target provided by the primary antibody (called the detector or tracer antibody). The secondary antibody produces a measurable signal.

[0083] Sandwich assays use two reagents: a capture reagent and a tracer (detector) reagent. The capture reagent is used to bind (immobilize) the analyte from solution, so that it can be specifically removed from the sample. The tracer (detector) reagent is used in a second step to generate a signal (directly or indirectly, as described above). The sandwich format requires two reagents, each with a distinct binding site on the analyte. Furthermore, since both reagents must bind to the target simultaneously, they must not interfere with each other.

[0084] Amino acids contain a variety of reactive side chains for conjugation to members of binding pairs, such as polypeptides / proteins, polymers (e.g., PEG, cellulose or polystyrol), or enzymes. Chemically reactive groups of amino acids are, for example, amino groups (lysine, alpha-amino group), thiol groups (cystine, cysteine ​​and methionine), carboxylic acid groups (aspartic acid, glutamic acid), and sugar alcohol groups. Such methods are described, for example, in "Bioconjugation", MacMillan Ref. Ltd., 1999, pages 50-100.

[0085] One of the most common reactive groups of amino acid side chains is the aliphatic ε-amine of the amino acid lysine. Lysine amines are reasonably good nucleophiles above pH 8.0 (pKa=9.18) and therefore react easily and cleanly with a variety of reagents to form stable bonds. Amine-reactive reagents react primarily with α-amino groups of lysine and proteins. Reactive esters, especially N-hydroxy-succinimide (NHS) esters, are among the most commonly used reagents for the modification of amine groups. The optimal pH for reactions in aqueous environments is pH 8.0-9.0. Isothiocyanates are amine-modifying reagents that form thiourea bonds with proteins. Isothiocyanates react with proteinaceous amines in aqueous solutions (optimally at pH 9.0-9.5). Aldehydes react with aliphatic and aromatic amines, hydrazines, and hydrazides under mild aqueous conditions to form imine intermediates (Schiff bases). Schiff bases can be selectively reduced with mild or strong reducing agents (such as sodium borohydride or sodium cyanoborohydride) to induce stable alkylamine linkages. Other reagents that have been used to modify amines are acid anhydrides. For example, diethylenetriaminepentaacetic anhydride (DTPA) is a bifunctional chelating agent that contains two amine-reactive anhydride groups. It can react with the termini and ε-amine groups of amino acids to form amide bonds. The anhydride ring opens to generate multivalent metal chelating arms that can strongly bind to metals in coordination complexes.

[0086] Another common reactive group is the thiol residue from the sulfur-containing amino acid cystine and its reduction product cysteine ​​(or hemi-cystine). Cysteine ​​is more nucleophilic than amines and contains a free thiol group, which is generally the most reactive functional group in proteins. Thiols are generally reactive at neutral pH, so they can selectively bind to other molecules in the presence of amines. Because free sulfhydryl groups are relatively reactive, proteins with these groups often exist in their oxidized form as disulfide groups or disulfide bonds. In such proteins, reduction of the disulfide bonds with reagents such as dithiothreitol (DTT) is necessary to generate reactive free thiols. Thiol-reactive reagents are reagents that bind to thiol groups on polypeptides to form thioether-linked products. These reagents react rapidly at slightly acidic to neutral pH, and therefore can react selectively in the presence of amine groups. The literature reports the use of several thiolated cross-linking reagents, such as Traut's reagent (2-iminothiolane), succinimidyl (acetylthio)acetate (SATA) and sulfosuccinimidyl 6-[3-(2-pyridyldithio)propionamido]hexanoate (Sulfo-LC-SPDP), to provide an efficient method to introduce multiple sulfhydryl groups via reactive amino groups. Haloacetyl derivatives, such as iodoacetamide, form thioether bonds and are also reagents for thiol modification. Further useful reagents are maleimides. The reaction of maleimides with thiol-reactive reagents is essentially the same as with iodoacetamide. Maleimides react rapidly at slightly acidic to neutral pH.

[0087] Another common reactive group is the carboxylic acid. Polypeptides contain carboxylic acid groups at the C-terminal position and in the side chains of aspartic and glutamic acids. The relatively low reactivity of carboxylic acids in water usually makes it difficult to use these groups to selectively modify polypeptides. In this case, the carboxylic acid group is usually converted to a reactive ester using a water-soluble carbodiimide and reacted with a nucleophile such as an amine, hydrazide, or hydrazine. The amine-containing reagent must be weakly basic to selectively react with the activated carboxylic acid in the presence of the more highly basic ε-amine of lysine to form a stable amide bond. When the pH is increased above 8.0, protein cross-linking can occur.

[0088] Sodium periodate can be used to oxidize the alcohol moiety of the sugar in the carbohydrate moiety to an aldehyde. Each aldehyde group can be reacted with an amine, hydrazide, or hydrazine as described for carboxylic acids. A Schiff base intermediate is formed which can be reduced to an alkylamine by reduction of the intermediate with sodium cyanoborohydride (mild and selective) or sodium borohydride (strong) water-soluble reducing agents.

[0089] The binding of a polypeptide or amino acid to its binding partner can be performed by various methods, such as chemical binding or binding via a binding pair. The term "binding partner" as used herein refers to, for example, a solid support, a polypeptide, a detectable label, a member of a specific binding pair. In certain embodiments, the binding of a polypeptide or amino acid to its binding partner is performed by chemical binding via the N-terminus and / or ε-amino group (lysine), the ε-amino groups of various lysines, the carboxy-, sulfhydryl-, hydroxyl-, and / or phenolic functional groups of the amino acid backbone of the antibody, and / or the sugar alcohol group of the carbohydrate structure. In one preferred embodiment, the carboxylic acid residue is bound to biotin, and the immobilization to the solid support is performed via avidin or streptavidin-immobilized solid support. In one preferred embodiment, the alcohol residue is bound to digoxigenin by a covalent bond.

[0090] "Solid phase" refers to non-fluid substances, including particles (including microparticles and beads) made from materials such as polymers, metals (paramagnetic, ferromagnetic particles), glass, and ceramics; gel substances such as silica, alumina, and polymer gels; capillaries, which may be made from polymers, metals, glass, and / or ceramics; zeolites and other porous materials; electrodes; microtiter plates; solid strips; and cuvettes, tubes, or other spectrometer sample containers. The solid phase components of the assay are distinguished from inert solid surfaces in that a "solid support" contains at least one moiety on its surface that is intended to chemically interact with the molecule. The solid phase may be a stationary component, such as a chip, tube, strip, cuvette, or microtiter plate, or a non-stationary component, such as beads and microparticles. A variety of microparticles may be used that allow for either non-covalent or covalent attachment of substances. Such particles include polymer particles, such as polystyrene and poly(methyl methacrylate); gold particles, such as gold nanoparticles, gold colloids; and ceramic particles, such as silica, glass, metal oxide particles, and the like. See, for example, Martin, CR et al., Analytical Chemistry-News & Features, 70 (1998) 322A-327A, or Butler, JE, Methods 22 (2000) 4-23.

[0091] Chromogens (fluorescent or luminescent groups and dyes), enzymes, NMR-active groups or metal particles are examples of "detectable labels". Metal chelates that can be detected by electrochemiluminescence are also signal-emitting groups, such as ruthenium chelates, e.g., ruthenium(bispyridyl)3 2+Chelates are particularly preferred. Suitable ruthenium labeling groups are described, for example, in EP 0580979, WO 90 / 05301, WO 90 / 11511 and WO 92 / 14138. For direct detection, the labeling group may be selected from any known detectable marker group, such as dyes, luminescent labeling groups, such as chemiluminescent groups, for example acridinium esters or dioxetanes, or fluorescent dyes, such as fluoresceins, coumarins, rhodamines, oxazines, resorufins, cyanines and their derivatives. Other examples of labeling groups are luminescent metal complexes, such as ruthenium or europium complexes, enzymes, such as those used in ELISA or CEDIA (cloned enzyme donor immunoassays, for example EP-A-0061888), and radioisotopes.

[0092] Examples of binding pairs are antigen / antibody, biotin or biotin analogues such as aminobiotin, iminobiotin or desthiobiotin / avidin or streptavidin, sugar / lectin, nucleic acid or nucleic acid analogue / complementary nucleic acid, and receptor / ligand such as steroid hormone receptor / steroid hormone. In one preferred embodiment, the first binding pair member comprises a hapten, an antigen and a hormone. In a particular embodiment, the hapten is selected from the group consisting of digoxin, digoxigenin and biotin and their analogues. The second partner of such binding pairs, such as antibodies, streptavidin, etc., is usually labeled to allow direct detection, for example by labeling as described above.

[0093] antibody General information relating to the nucleotide sequences of human immunoglobulin light and heavy chains is given in Kabat EA et al., Sequences of Proteins of Immunological Interest, 5th Ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991).

[0094] The term "antibody" herein is used in the broadest sense and encompasses a variety of antibody structures including, but not limited to, full length antibodies, monoclonal antibodies, multispecific antibodies (e.g., bispecific antibodies), and antibody-antibody fragment-fusions and combinations thereof.

[0095] The term "full-length antibody" refers to an antibody that has a structure substantially similar to that of a natural antibody. A full-length antibody comprises two full-length antibody light chains, each of which comprises, from N-terminus to C-terminus, a light chain variable region and a light chain constant domain, and two full-length antibody heavy chains, each of which comprises, from N-terminus to C-terminus, a heavy chain variable region, a first heavy chain constant domain, a hinge region, a second heavy chain constant domain, and a third heavy chain constant domain. In contrast to a natural antibody, a full-length antibody may comprise additional immunoglobulin domains, such as one or more additional scFvs, or heavy or light chain Fab fragments, or scFabs that are conjugated to one or more ends of different chains of a full-length antibody, but only one fragment at each end. These complexes are also encompassed by the term full-length antibody.

[0096] The term "antibody binding site" refers to a pair of heavy and light chain variable domains. To ensure proper binding to the antigen, these variable domains are cognate variable domains, i.e. belong together. An antibody binding site comprises at least three HVRs (e.g., in the case of a VHH) or three to six HVRs (e.g., in the case of a conventional antibody with a naturally occurring, i.e., VH / VL pair). In general, the amino acid residues of an antibody involved in antigen binding form the binding site. These residues are usually contained in a pair of an antibody heavy chain variable domain and a corresponding antibody light chain variable domain. An antibody antigen binding site comprises amino acid residues from "hypervariable regions" or "HVRs". "Framework" or "FR" regions are variable domain regions other than the hypervariable region residues as defined herein. Thus, the light and heavy chain variable domains of an antibody comprise, from the N- to C-terminus, the regions FR1, HVR1, FR2, HVR2, FR3, HVR3, and FR4. In particular, the HVR3 region of the heavy chain variable domain is the region that contributes most to antigen binding and defines the binding specificity of the antibody. A "functional binding site" can bind to its target. The term "bind" refers to the binding of a binding site to its target in an in vitro assay, and in a specific embodiment, in a binding assay. Such a binding assay can be any assay, as long as a binding event can be detected. "Binding" can be determined, for example, using an ELISA assay.

[0097] The term "hypervariable region" or "HVR" as used herein refers to each of the regions of an antibody variable domain that comprise stretches of amino acid residues that are hypervariable sequences ("complementarity determining regions" or "CDRs") and / or form structurally defined loops ("hypervariable loops") and / or contain residues that contact the antigen ("antigen contacts"). Typically, antibodies contain six HVRs, three in the heavy chain variable domain VH (H1, H2, H3) and three in the light chain variable domain VL (L1, L2, L3).

[0098] HVR includes: (a) hypervariable loops occurring at amino acid residues 26-32 (L1), 50-52 (L2), 91-96 (L3), 26-32 (H1), 53-55 (H2), and 96-101 (H3) (Chothia, C. and Lesk, A. M., J. Mol. Biol. 196 (1987) 901-917); (b) CDRs occurring at amino acid residues 24-34 (L1), 50-56 (L2), 89-97 (L3), 31-35b (H1), 50-65 (H2), and 95-102 (H3) (Kabat, E. A. et al., Sequences of Proteins of Immunological Interest, 5th ed., Public Health Service, National Institutes of Health, Bethesda, MD (1991), NIH Publication 91-3242); (c) antigen contacts occurring at amino acid residues 27c-36 (L1), 46-55 (L2), 89-96 (L3), 30-35b (H1), 47-58 (H2), and 93-101 (H3) (MacCallum et al., J. Mol. Biol. 262:732-745 (1996)); and (d) A combination of (a), (b), and / or (c), comprising amino acid residues 46-56 (L2), 47-56 (L2), 48-56 (L2), 49-56 (L2), 26-35 (H1), 26-35b (H1), 49-65 (H2), 93-102 (H3), and 94-102 (H3).

[0099] Unless otherwise indicated, HVR residues and other residues in the variable domain (eg, FR residues) are numbered herein according to Kabat et al., supra.

[0100] The "class" of an antibody refers to the type of constant domain or region, preferably the Fc region, possessed by the heavy chain. There are five major classes of antibodies: IgA, IgD, IgE, IgG, and IgM, some of which can be further divided into subclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA1, and IgA2. The heavy chain constant domains corresponding to the different classes of immunoglobulins are called α, δ, ε, γ, and μ, respectively.

[0101] The term "heavy chain constant region" refers to the region of an immunoglobulin heavy chain that includes the constant domains, i.e., the CH1 domain, the hinge region, the CH2 domain, and the CH3 domain. In one embodiment, the human IgG constant region extends from Ala118 to the carboxyl terminus of the heavy chain (numbering according to Kabat EU index). However, the C-terminal lysine (Lys447) of the constant region may or may not be present (numbering according to Kabat EU index). The term "constant region" refers to a dimer that includes two heavy chain constant regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0102] The term "heavy chain Fc region" refers to the C-terminal region of an immunoglobulin heavy chain, including at least a portion of the hinge region (middle and lower hinge regions), the CH2 domain, and the CH3 domain. In one embodiment, the human IgG heavy chain Fc region extends from Asp221 or Cys226 or Pro230 to the carboxyl terminus of the heavy chain (numbering according to Kabat EU index). Thus, the Fc region is smaller than the constant region, but the C-terminal portion is identical to it. However, the C-terminal lysine (Lys447) of the heavy chain Fc region may or may not be present (numbering according to Kabat EU index). The term "Fc region" refers to a dimer comprising two heavy chain Fc regions that can be covalently linked to each other via hinge region cysteine ​​residues that form interchain disulfide bonds.

[0103] The term "valency" as used within this application refers to the presence of a particular number of binding sites within an antibody. Thus, the terms "bivalent," "tetravalent," and "hexavalent" refer to the presence of two binding sites, four binding sites, and six binding sites, respectively, within an antibody.

[0104] "Monospecific antibody" refers to an antibody that has a single binding specificity, i.e., that specifically binds to one antigen. Monospecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., F(ab')2) or combinations thereof (e.g., full-length antibodies with additional scFv or Fab fragments). Monospecific antibodies need not be monovalent. That is, monospecific antibodies may contain more than one binding site that specifically binds to one antigen. For example, natural antibodies are monospecific but bivalent.

[0105] "Multispecific antibodies" refer to antibodies that have binding specificities for at least two different epitopes on the same antigen or two different antigens. Multispecific antibodies can be prepared as full-length antibodies or antibody fragments (e.g., Fab bispecific antibodies) or combinations thereof (antibody-antibody fragment-fusions, e.g., full-length antibodies conjugated to additional scFv or Fab fragments). Multispecific antibodies are at least bivalent, i.e., contain two antigen-binding sites. Furthermore, multispecific antibodies are at least bispecific. Thus, bivalent bispecific antibodies are the simplest form of multispecific antibodies. Engineered antibodies with two, three or more (e.g., four) functional antigen-binding sites have been reported (see, e.g., U.S. Patent Application Publication No. 2002 / 0004587).

[0106] In certain embodiments of all aspects and other embodiments of the present invention, the cell clone produces a multispecific antibody. In certain embodiments, one of the binding specificities is for a first antigen and the other is for a different second antigen. In certain embodiments, the multispecific antibody binds to two different epitopes of the same antigen. In certain embodiments, the second epitopes on the same antigen are non-overlapping epitopes. In certain embodiments, the antibody is a bispecific antibody. In a preferred embodiment, the bispecific antibody is a trivalent bispecific antibody or a bivalent bispecific antibody.

[0107] Techniques for producing multispecific antibodies include, but are not limited to, recombinant co-expression of two immunoglobulin heavy-light chain pairs with different specificities (see Milstein, C. and Cuello, A.C., Nature 305 (1983) 537-540, WO 93 / 08829, and Traunecker, A. et al., EMBO J. 10 (1991) 3655-3659), and "knob-in-hole" engineering (see, e.g., U.S. Pat. No. 5,731,168). Multispecific antibodies can also be produced by a number of techniques, including the manipulation of electrostatic steering effects to create antibody Fc heterodimeric molecules (WO 2009 / 089004); cross-linking of two or more antibodies or fragments (see, e.g., U.S. Pat. No. 4,676,980, and Brennan, M., et al., Science 229 (1985) 81-83); the production of bispecific antibodies using leucine zippers (see, e.g., Kostelny, SA et al., J. Immunol. 148 (1992) 1547-1553); the use of general light chain technology to circumvent light chain mispairing problems (see, e.g., WO 98 / 50431); the use of "diabody" technology to create bispecific antibody fragments (see, e.g., Holliger, P. et al., Proc. Natl. Acad. Sci. USA 90 (1993) 6444-6448); and by the preparation of trispecific antibodies as described, for example, in Tutt, A. et al., J. Immunol. 147 (1991) 60-69.

[0108] Engineered antibodies with three or more antigen binding sites, including, for example, "Octopus antibodies," or DVD-Igs, are also included herein (see, for example, WO 2001 / 77342 and WO 2008 / 024715). Other examples of multispecific antibodies with three or more antigen binding sites can be found in WO 2010 / 115589, WO 2010 / 112193, WO 2010 / 136172, WO 2010 / 145792, and WO 2013 / 026831. Bispecific antibodies or antigen-binding fragments thereof also include "dual-acting Fabs" or "DAFs" (see, for example, U.S. Patent Application Publication Nos. 2008 / 0069820 and WO 2015 / 095539).

[0109] Multispecific antibodies can also be provided in an asymmetric manner with domain crossover in one or more binding arms of the same antigen specificity, i.e. by exchanging VH / VL domains (see, e.g., WO 2009 / 080252 and WO 2015 / 150447), CH1 / CL domains (see WO 2009 / 080253) or complete Fab arms (see WO 2009 / 080251, WO 2016 / 016299; see also Schaefer et al., Proc. Natl. Acad. Sci. USA 108 (2011) 1187-1191, and Klein et al., MAbs 8 (2016) 1010-1020).

[0110] In one preferred embodiment of all aspects and other embodiments of the invention, the multispecific antibody comprises Fab fragments in which either the heavy and light chain variable or constant regions have been exchanged, i.e. in one chain the heavy chain VH variable domain is directly connected via a peptide linker to the light chain CL constant domain and in the respective other chain the light chain VL variable domain is directly connected via a peptide linker to the heavy chain CH1 constant domain.

[0111] Thus, a domain-swapped Fab fragment comprises a polypeptide chain composed of a light chain variable region (VL) and a heavy chain constant region 1 (CH1), and a polypeptide chain composed of a heavy chain variable region (VH) and a light chain constant region (CL).

[0112] Asymmetric Fab arms can also be engineered by introducing charged or uncharged amino acid mutations at the domain interface to direct correct Fab pairing. See, e.g., WO 2016 / 172485.

[0113] The antibody or fragment may also be a multispecific antibody as described in WO 2009 / 080254, WO 2010 / 112193, WO 2010 / 115589, WO 2010 / 136172, WO 2010 / 145792 or WO 2010 / 145793.

[0114] The antibody or fragment thereof may also be a multispecific antibody as disclosed in WO 2012 / 163520.

[0115] A variety of additional molecular formats of multispecific antibodies are known in the art and are included herein (see, e.g., Spiess et al., Mol. Immunol. 67 (2015) 95-106).

[0116] Bispecific antibodies are generally antibody molecules that specifically bind to two different, non-overlapping epitopes on the same antigen or to two epitopes on different antigens.

[0117] In certain embodiments of all of the aspects and other embodiments of the invention, the bispecific antibody comprises: Domain-swapped 1+1 bispecific antibodies (CrossMab) (a) a bispecific full-length IgG antibody comprising a first light chain and a first heavy chain pair comprising a first Fab fragment, and a second light chain and a second heavy chain pair comprising a second Fab fragment, In the first Fab fragment, (a) only the CH1 and CL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises the VL and CH1 domains, and the heavy chain of the first Fab fragment comprises the VH and CL domains); (b) only the VH and VL domains are replaced by each other (i.e. the light chain of the first Fab fragment comprises the VH and CL domains and the heavy chain of the first Fab fragment comprises the VL and CH1 domains); or (c) the CH1 and CL domains and the VH and VL domains are replaced by each other (i.e., the light chain of the first Fab fragment comprises the VH and CH1 domains, and the heavy chain of the first Fab fragment comprises the VL and CL domains); the second Fab fragment comprises a light chain comprising a VL and a CL domain, and a heavy chain comprising a VH and a CH1 domain; the first heavy chain and the second heavy chain both comprise a CH3 domain, and both CH3 domains are complementarily engineered by respective amino acid substitutions to support heterodimerization of the first heavy chain and the second heavy chain, (in one preferred embodiment, one CH3 domain comprises a knob mutation and the respective other CH3 domain comprises a hole mutation); C-terminal Fab domain fused 2+1 bispecific antibody (BS) Bispecific full-length IgG antibodies, including: (a) a full-length antibody comprising two pairs of full-length antibody light chains and full-length antibody heavy chains, wherein a binding site formed by each pair of full-length heavy chains specifically binds to a first antigen; and (b) one additional Fab fragment fused to the C-terminus of one heavy chain of the full-length antibody, wherein the binding site of the additional Fab fragment specifically binds to a second antigen; an additional Fab fragment that specifically binds to a second antigen, which contains a domain crossover such that (a) the light chain variable domain (VL) and the heavy chain variable domain (VH) are substituted for each other, or (b) the light chain constant domain (CL) and the heavy chain constant domain (CH1) are substituted for each other; Bispecific one-arm single chain antibody (A bispecific one-arm single chain antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: - a light chain (comprising a variable light domain and a light chain constant domain); - a light / heavy chain combination, which comprises, from N-terminus to C-terminus, a variable light domain, a light chain constant domain, a peptide linker, a variable heavy domain, a CH1 domain, a hinge region, a CH2 domain, and a CH3 domain with a knob or hole mutation. - a heavy chain (comprising, from N-terminus to C-terminus, the variable heavy domain, the CH1 domain, the hinge region, the CH2 domain, the CH3 domain with a hole or knob mutation); Bispecific two-arm single chain antibody A bispecific two-arm single chain antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: - light chain / heavy chain combination 1 (comprising, from N-terminus to C-terminus, variable light chain domain 1, light chain constant domain, peptide linker, variable heavy chain domain 1, CH1 domain, hinge region, CH2 domain, CH3 domain with knob or hole mutation); - light chain / heavy chain combination 2 (comprising, from N-terminus to C-terminus, variable light chain domain 2, light chain constant domain, peptide linker, variable heavy chain domain 2, CH1 domain, hinge region, CH2 domain, CH3 domain with hole or knob mutations); Common light chain bispecific antibodies A common light chain bispecific antibody comprising a first binding site that specifically binds to a first epitope or antigen and a second binding site that specifically binds to a second epitope or antigen, wherein the individual chains are as follows: - a light chain (comprising, from N-terminus to C-terminus, a variable light domain and a light chain constant domain); - heavy chain 1 (comprising, from N-terminus to C-terminus, variable heavy domain 1, CH1 domain, hinge region, CH2 domain, CH3 domain with hole or knob mutation); heavy chain 2 (comprising, from N-terminus to C-terminus, variable heavy domain 2, CH1 domain, hinge region, CH2 domain, CH3 domain with a knob or hole mutation); 2+1 Bispecific Antibody (TCB) with N-Terminal Fab Domain Insertion (A bispecific full-length antibody having an additional heavy chain N-terminal binding site with domain swapping, a first Fab fragment and a second Fab fragment, the respective binding sites of which specifically bind to a first antigen, - a third Fab fragment, the binding site of the third Fab fragment specifically binds to a second antigen, the third Fab fragment comprising a domain crossover such that the variable light domain (VL) and the variable heavy domain (VH) are replaced by one another; and - comprising an Fc region, the Fc region comprising a first Fc region polypeptide and a second Fc region polypeptide; the first Fab fragment and the second Fab fragment comprise a heavy chain fragment and a full-length light chain, respectively; the C-terminus of the heavy chain fragment of the first Fab fragment is fused to the N-terminus of the first Fc region polypeptide; the C-terminus of the heavy chain fragment of the second Fab fragment is fused to the N-terminus of the variable light chain domain of a third Fab fragment, and the C-terminus of the CH1 domain of the third Fab fragment is fused to the N-terminus of a second Fc region polypeptide; Antibody-multimer fusion (A fusion polypeptide, (a) an antibody heavy chain and an antibody light chain, (b) a first fusion polypeptide comprising, from N-terminus to C-terminus, a first portion of a non-antibody multimeric polypeptide, an antibody heavy chain CH1 domain or an antibody light chain constant domain, an antibody hinge region, an antibody heavy chain CH2 domain, and an antibody heavy chain CH3 domain, and a second fusion polypeptide comprising, from N-terminus to C-terminus, a second portion of a non-antibody multimeric polypeptide, and an antibody light chain constant domain if the first polypeptide comprises the antibody heavy chain CH1 domain, or an antibody heavy chain CH1 domain if the first polypeptide comprises the antibody light chain constant domain; (In the formula, (i) the antibody heavy chain of (a) and the first fusion polypeptide of (b), (ii) the antibody heavy chain of (a) and the antibody light chain of (a), and (iii) the first fusion polypeptide of (b) and the second fusion polypeptide of (b) are each independently covalently linked to each other by at least one disulfide bond; (In the formula, The variable domains of the antibody heavy chain and the antibody light chain form a binding site that specifically binds to an antigen.

[0118] The CH3 domains of the heavy chains of the antibody may be modified by the "knob-into-hole" technique. This is described in detail with some examples in, for example, WO 96 / 027011, Ridgway, JB et al., Protein Eng. 9 (1996) 617-621; and Merchant, AM et al., Nat. Biotechnol. 16 (1998) 677-681. In this method, the interaction surfaces of the two CH3 domains are modified to increase the heterodimerization of these two CH3 domains, and thereby the heterodimerization of the polypeptides containing them. Each of the two CH3 domains (of the two heavy chains) may be a "knob" and each other a "hole". The introduction of disulfide bridges further stabilizes the heterodimers (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681; Atwell, S. et al., J. Mol. Biol. 270 (1997) 26-35) and increases the yield.

[0119] The mutation T366W in the CH3 domain (of the antibody heavy chain) is designated as a "knob mutation" and the mutations T366S, L368A, Y407V in the CH3 domain (of the antibody heavy chain) are designated as "hole mutations" (numbering according to the EU index of Kabat). Additional interchain disulfide bridges between CH3 domains (Merchant, AM et al., Nature Biotech. 16 (1998) 677-681) can also be used, for example, by introducing a S354C mutation in the CH3 domain of the heavy chain carrying a "knob mutation" (designated as a "knob-cys-mutation") and a Y349C mutation in the CH3 domain of the heavy chain carrying a "hole mutation" (designated as a "hole-cys-mutation") (numbering according to the EU index of Kabat).

[0120] The term "domain crossover" as used herein refers to the deviation of the domain sequences from that of the native antibody in that in a pair of an antibody heavy chain VH-CH1 fragment and its corresponding cognate antibody light chain, i.e., in the antibody Fab (fragment-antigen binding), at least one heavy chain domain is replaced by its corresponding light chain domain, and vice versa. There are three general types of domain crossovers: (i) crossovers of CH1 and CL domains, where the domain crossover in the light chain results in a VL-CH1 domain sequence and the domain crossover in the heavy chain fragment results in a VH-CL domain sequence (or a full-length antibody heavy chain having a VH-CL-hinge-CH2-CH3 domain sequence); (ii) domain crossovers of VH and VL domains, where the domain crossover in the light chain results in a VH-CL domain sequence and the domain crossover in the heavy chain fragment results in a VL-CH1 domain sequence; and (iii) domain crossovers of a complete light chain (VL-CL) and a complete VH-CH1 heavy chain fragment ("Fab crossover"), where the domain crossover results in a light chain with a VH-CH1 domain sequence and the domain crossover results in a heavy chain fragment with a VL-CL domain sequence (all domain sequences listed above are in the N-terminal to C-terminal direction).

[0121] As used herein, the term "replaced with each other" with respect to corresponding heavy and light chain domains refers to the domain crossover described above. Thus, when CH1 and CL domains are "replaced with each other", this term refers to the domain crossover described under item (i) and the resulting heavy and light chain domain sequences. Thus, when VH and VL are "replaced with each other", this term refers to the domain crossover described under item (ii), and when CH1 and CL domains are "replaced with each other" and VH and VL domains are "replaced with each other", this term refers to the domain crossover described under item (iii). Bispecific antibodies comprising domain crossovers have been reported, for example, in WO2009 / 080251, WO2009 / 080252, WO2009 / 080253, WO2009 / 080254, and Schaefer, W. et al., Proc. Natl. Acad. Sci USA 108 (2011) 11187-11192. Such antibodies are generally referred to as CrossMab.

[0122] In certain embodiments of all aspects and other embodiments of the present invention, the multispecific antibody also comprises at least one Fab fragment comprising either a domain crossover between the CH1 domain and the CL domain as described in item (i) above, or a domain crossover between the VH domain and the VL domain as described in item (ii) above, or a domain crossover between the VH-CH1 domain and the VL-VL domain as described in item (iii) above. In the case of a multispecific antibody with domain crossover, the Fabs that specifically bind to the same antigen are constructed to have the same domain sequence in certain embodiments. Therefore, when more than one Fab with domain crossover is included in a multispecific antibody, the Fabs specifically bind to the same antigen.

[0123] As used herein, the term "recombinant antibody" refers to all antibodies (chimeric, humanized and human) that are prepared, expressed, produced or isolated by recombinant means, such as recombinant cells. This includes antibodies isolated from recombinant cells, such as NS0, HEK, BHK, amniotic cells, CHO cells, etc.

[0124] As used herein, the term "antibody fragment" refers to a molecule other than an intact antibody that contains a portion of an intact antibody that binds to the antigen to which the intact antibody binds, i.e., it is a functional fragment. Examples of antibody fragments include, but are not limited to, Fv, Fab, Fab', Fab'-SH, F(ab')2, bispecific Fab, diabody, linear antibody, single chain antibody molecule (e.g., scFv or scFab).

[0125] Recombinant methods Antibodies can be produced using recombinant methods and compositions, for example, as described in U.S. Patent No. 4,816,567. For these methods, one or more isolated nucleic acids encoding the antibody are provided.

[0126] In one aspect of the invention, there is provided a method for producing an antibody, comprising culturing a cell clone comprising nucleic acid encoding the antibody under conditions suitable for expression of the antibody, and optionally recovering the antibody from the host cell (or host cell culture medium), wherein the cell clone has been selected by a method according to the invention.

[0127] For recombinant production of an antibody, a nucleic acid encoding the antibody is generated / designed / synthesized and inserted into one or more vectors for further cloning and / or expression in a cell. Such nucleic acid can be readily isolated and sequenced using conventional procedures (e.g., by using oligonucleotide probes capable of specifically binding to genes encoding the heavy and light chains of the antibody), or can be produced by recombinant methods, or can be obtained by chemical synthesis.

[0128] Typically, for recombinant mass production of a polypeptide of interest, such as a therapeutic antibody, a cell clone that stably expresses and secretes the polypeptide is required. This cell clone is called a "recombinant cell clone" or a "recombinant production cell clone," and the overall process used to generate such cells is called "cell line development." In the first step of the cell line development process, a suitable host cell, such as, for example, in certain embodiments, a CHO cell, is transfected with one or more nucleic acid sequences suitable for expressing the polypeptide of interest. In the second step, cell clones that stably express the polypeptide of interest are selected based on the co-expression of a selectable marker that has been co-transfected with the nucleic acid encoding the polypeptide of interest.

[0129] The nucleic acid that codes for a polypeptide, i.e., the coding sequence, is called a structural gene. Such a structural gene is pure coding information. Therefore, its expression requires additional regulatory elements. Therefore, the structural gene is usually incorporated into a so-called expression cassette. The minimum control elements required for an expression cassette to be functional in a mammalian cell are a promoter that is functional in said mammalian cell, located upstream, i.e., 5', of the structural gene, and a polyadenylation signal sequence that is functional in said mammalian cell, located downstream, i.e., 3', of the structural gene. The promoter sequence, the structural gene sequence, and the polyadenylation signal sequence are arranged in an operably linked form.

[0130] If the polypeptide of interest is a heteromultimeric polypeptide composed of different polypeptides, such as an antibody or complex antibody format, not only a single expression cassette is required, but multiple expression cassettes, each containing different structural genes, i.e., at least one expression cassette for each of the different polypeptides (chains) of the heteromultimeric polypeptide (heteromultimeric antibody). For example, a full-length antibody is a heteromultimeric polypeptide that contains two copies of a light chain and two copies of a heavy chain. Thus, a full-length antibody is composed of two different polypeptides. Thus, two expression cassettes are required for the expression of a full-length antibody, one for the light chain and one for the heavy chain. For example, if the full-length antibody is a bispecific antibody, i.e., the antibody contains two different binding sites that specifically bind to two different antigens / epitopes on the same antigen, the two light chains as well as the two heavy chains are also different from each other. Thus, such a bispecific full-length antibody is composed of four different polypeptides, and therefore four expression cassettes are required.

[0131] The expression cassette for the polypeptide of interest is then incorporated into one or more so-called "expression vectors". An "expression vector" is a nucleic acid that provides all the elements required to amplify the vector in bacterial cells and to express the contained structural gene in mammalian cells. Typically, an expression vector contains a prokaryotic plasmid propagation unit, which contains, for example in the case of E. coli, an origin of replication and a prokaryotic selection marker, as well as a eukaryotic selection marker, and an expression cassette required for the expression of the structural gene of interest. An "expression vector" is a transport vehicle for introducing the expression cassette into a mammalian host cell to generate a polypeptide-expressing cell clone.

[0132] As outlined in the previous paragraph, the more complex the polypeptide to be expressed, the more number of different expression cassettes is required. Essentially, the number of expression cassettes increases with the size of the nucleic acid to be integrated into the genome of the host cell. At the same time, the size of the expression vector also increases. However, the practical upper limit of the vector size is in the range of about 15 kbp, above which the efficiency of manipulation and processing decreases significantly. This problem can be addressed by using two or more expression vectors. Thereby, the expression cassette can be divided between different expression vectors, each of which contains only a part of the expression cassette, resulting in a reduction in size.

[0133] Cell line development (CLD) for generating recombinant cells expressing heterologous polypeptides, such as multispecific antibodies, uses either random integration (RI) or targeted integration (TI) of nucleic acids containing the respective expression cassettes required for the expression and production of the heterologous polypeptides of interest.

[0134] Using RI, typically multiple vectors or fragments thereof are integrated into the genome of a cell at the same or different loci.

[0135] Using TI, typically a single copy of a transgene containing different expression cassettes is integrated into a defined "hot spot" in the genome of the host cell.

[0136] Suitable host cells for the generation of cell clones for the expression of (glycosylated) antibodies are generally derived from multicellular organisms, such as vertebrates.

[0137] host cell Any mammalian host cell line adapted to grow in suspension may be used to generate recombinant cell clones that can be treated with the methods according to the invention. Moreover, regardless of the integration method, i.e., in the case of RI and TI, any mammalian host cell may be used.

[0138] Examples of useful mammalian host cell lines are human amniotic cells (e.g., CAP-T cells as described in Woelfel, J. et al., BMC Proc. 5 (2011) p. 133); monkey kidney CV1 line transformed with SV40 (COS-7); human embryonic kidney lines (e.g., HEK293 cells or HEK293T cells as described in Graham, FL et al., J. Gen Virol. 36 (1977) 59-74); baby hamster kidney cells (BHK); mouse Sertoli cells (e.g., TM4 cells as described in Mather, JP, Biol. Reprod. 23 (1980) 243-252); monkey kidney cells (CV1); African green monkey kidney cells (VERO-76); human cervical carcinoma cells (HELA); canine kidney cells (MDCK; buffalo rat liver cells (BRL 3A); human lung cells (W138); human hepatocytes (HepG2); mouse mammary tumor (MMT060562); TRI cells, e.g., as described in Mather, JP et al., Annals NYAcad. Sci. 383 (1982) 44-68; MRC5 cells; and FS4 cells. Other useful mammalian host cell lines include Chinese hamster ovary (CHO) cells, including DHFR-CHO cells (Urlaub, G. et al., Proc. Natl. Acad. Sci. USA 77 (1980) 4216-4220), and myeloma cell lines, e.g., Y0, NS0, and Sp2 / 0. For a review of certain mammalian host cell lines suitable for antibody production, see, e.g., Yazaki, P. and Wu, AM, Methods in Molecular Biology, Vol. 248, in Lo, BKC (ed.), Humana Press, Totowa, NJ (2004), pp. 255-268.

[0139] In certain embodiments of all aspects and other embodiments of the present invention, the mammalian host cell is, for example, a Chinese Hamster Ovary (CHO) cell (e.g., CHO K1, CHO DG44, etc.), a human embryonic kidney (HEK) cell, a lymphoid cell (e.g., Y0, NS0, Sp2 / 0 cell), or a human amniotic cell (e.g., CAP-T, etc.). In one preferred embodiment, the mammalian (host) cell is a CHO cell. Thus, similarly, the cell clone is a CHO cell.

[0140] For TI, any known or future mammalian host cell suitable for TI that includes the landing site described herein integrated at a single site within a genomic locus may be used in the present invention. Such cells are referred to as mammalian TI host cells. In certain embodiments, the mammalian TI host cell is a hamster cell, a human cell, a rat cell, or a mouse cell that includes a landing site as described herein. In a preferred embodiment, the mammalian TI host cell is a CHO cell. In certain embodiments, the mammalian TI host cell is a Chinese hamster ovary (CHO) cell, a CHO K1 cell, a CHO K1SV cell, a CHO DG44 cell, a CHO DUKXB-11 cell, a CHO K1S cell, or a CHO K1M cell that includes the landing site described herein integrated at a single site within a genomic locus.

[0141] In certain embodiments of all aspects and other embodiments of the invention, the mammalian TI host cell comprises an integrated landing site, the landing site comprising one or more recombination recognition sequences (RRS). The RRS can be recognized by a recombinase, for example, Cre recombinase, FLP recombinase, Bxb1 integrase, or φC31 integrase. The RRS can be selected, independently of each other, from the group consisting of LoxP sequence, LoxP L3 sequence, LoxP 2L sequence, LoxFas sequence, Lox511 sequence, Lox2272 sequence, Lox2372 sequence, Lox5171 sequence, Loxm2 sequence, Lox71 sequence, Lox66 sequence, FRT sequence, Bxb1 attP sequence, Bxb1 attB sequence, φC31 attP sequence, and φC31 attB sequence. If multiple RRSs must be present, the selection of each sequence is dependent on the other, to the extent that non-identical RRSs are selected.

[0142] The present invention is exemplified below in CHO cells, which are presented only to illustrate the invention and should not be construed as limiting, the true scope of the invention being set forth in the following claims.

[0143] Targeted embedding One method for producing recombinant mammalian cell clones to be treated in the methods according to the invention is recombinant cell clones produced by using targeted integration (TI) for the introduction of coding nucleic acid.

[0144] In targeted integration, site-specific recombination is used to introduce exogenous nucleic acid at a specific locus in the genome of a mammalian TI host cell to generate a recombinant cell clone. It is an enzymatic process in which the sequence of the integration site in the genome is exchanged with the exogenous nucleic acid. One system used to perform such a nucleic acid exchange is the Cre-lox system. The enzyme that catalyzes the exchange is Cre recombinase. The sequence to be exchanged is defined by the location of two lox(P) sites in the genome as well as in the exogenous nucleic acid. These lox(P) sites are recognized by Cre recombinase. Nothing more is needed, i.e. no ATP etc. Originally, the Cre-lox system was found in bacteriophage P1.

[0145] The Cre-lox system functions in a variety of cell types, including mammalian, plant, bacterial, and yeast cells.

[0146] In certain embodiments of all aspects and other embodiments of the invention, exogenous nucleic acids encoding heterologous polypeptides are integrated into mammalian TI host cells by single or double recombinase-mediated cassette exchange (RMCE), resulting in recombinant mammalian cell clones, such as recombinant CHO cell clones, in which a defined, specific expression cassette sequence is integrated into the genome at a single locus.

[0147] The Cre-LoxP site-specific recombination system is widely used in many biological experimental systems. Cre recombinase is a 38 kDa site-specific DNA recombinase that recognizes 34 bp LoxP sequences. Cre recombinase is derived from bacteriophage P1 and belongs to the tyrosine family of site-specific recombinases. Cre recombinase can mediate both intra- and intermolecular recombination between LoxP sequences. The LoxP sequence consists of an 8 bp non-palindromic core region flanked by two 13 bp inverted repeats. Cre recombinase binds to the 13 bp repeats, thereby mediating recombination within the 8 bp core region. Cre-LoxP-mediated recombination occurs with high efficiency and does not require any other host factors. If two LoxP sequences are positioned in the same orientation on the same nucleotide sequence, Cre recombinase-mediated recombination will excise the DNA sequence located between the two LoxP sequences as a covalently closed circle. If two LoxP sequences are located in opposite orientations on the same nucleotide sequence, Cre recombinase-mediated recombination will invert the orientation of the DNA sequence located between the two sequences. If two LoxP sequences are on two different DNA molecules and one DNA molecule is circular, Cre recombinase-mediated recombination will result in the integration of the circular DNA sequence.

[0148] A "recombination recognition sequence" (RRS) is a nucleotide sequence that is recognized by a recombinase and is necessary and sufficient for a recombinase-mediated recombination event. An RRS can be used to define the location at which a recombination event occurs within a nucleotide sequence.

[0149] The term "concordant RRS" indicates that recombination occurs between two RRSs.

[0150] In certain embodiments, the two matching RRSs are the same.

[0151] In certain embodiments of all aspects and other embodiments of the present invention, the RRS can be recognized by Cre recombinase. In certain embodiments, the RRS can be recognized by FLP recombinase. In certain embodiments, the RRS can be recognized by Bxb1 integrase.

[0152] In certain embodiments, the RRS can be recognized by φC31 integrase.

[0153] In certain embodiments of all aspects and other embodiments of the present invention, both RRSs are wild-type LoxP sequences. In certain embodiments, both RRSs are mutant LoxP sequences. In certain embodiments, both RRSs are wild-type FRT sequences. In certain embodiments, both RRSs are mutant FRT sequences. In certain embodiments, the two matching RRSs are different sequences but can be recognized by the same recombinase. In certain embodiments, the first matching RRS is a Bxb1 attP sequence and the second matching RRS is a Bxb1 attB sequence. In certain embodiments, the first matching RRS is a φC31 attB sequence and the second matching RRS is a φC31 attB sequence.

[0154] A "two-plasmid RMCE" strategy or "double RMCE" is used in the method according to the present invention when a combination of two vectors is used. For example, but not limited to, the integrated landing site may comprise three RRSs, such as an arrangement in which a third RRS ("RRS3") is present between a first RRS ("RRS1") and a second RRS ("RRS2"), the first vector comprises two RRSs that match the first and third RRSs on the integrated exogenous nucleotide sequence, and the second vector comprises two RRSs that match the third and second RRSs on the integrated exogenous nucleotide sequence.

[0155] The two-plasmid RMCE strategy involves using three RRS sites to carry out two independent RMCEs simultaneously. Thus, the landing site of a mammalian TI host cell using the two-plasmid RMCE strategy contains a third RRS site (RRS3) that has no cross activity towards either the first RRS site (RRS1) or the second RRS site (RRS2). The two plasmids to be targeted require the same flanking RRS sites for efficient targeting, with one plasmid (front) flanked by RRS1 and RRS3 and the other (back) flanked by RRS3 and RRS2. Furthermore, in two-plasmid RMCE, two selection markers are also required. One selection marker expression cassette was split into two parts. The front plasmid contains a promoter followed by a start codon and an RRS3 sequence. The back plasmid lacks a start codon (ATG) and has the RRS3 sequence fused to the N-terminus of the selection marker coding region. It may be necessary to insert additional nucleotides between the RRS3 site and the selection marker sequence to ensure in-frame translation of the fusion protein, i.e., operative linkage. Only when both plasmids are correctly inserted will the complete expression cassette of the selection marker be assembled, thus conferring resistance to the respective selection agent to the cells.

[0156] Two-plasmid RMCE involves a double recombination crossover event between two heterospecific RRSs in a target genomic locus and a donor DNA molecule, catalyzed by a recombinase. Two-plasmid RMCE is designed to combine and introduce copies of DNA sequences from a front vector and a back vector into a predetermined locus in the genome of a mammalian TI host cell. RMCE can be performed such that sequences of the prokaryotic vector are not introduced into the mammalian TI host cell genome, thus reducing and / or preventing unwanted triggering of host immune or defense mechanisms. The RMCE procedure can be repeated with multiple DNA sequences.

[0157] In certain embodiments of all aspects and other embodiments of the present invention, targeted integration is achieved by two rounds of RMCE, where two different DNA sequences are both integrated into a predetermined site in the genome of a corresponding RRS of a mammalian TI host cell, and each DNA sequence includes at least one expression cassette encoding a portion of a heteromultimeric polypeptide and / or at least one selection marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, targeted integration is achieved by multiple rounds of RMCE, where DNA sequences from multiple vectors are all integrated into a predetermined site in the genome of a mammalian TI host cell, and each DNA sequence includes at least one expression cassette encoding a portion of a heteromultimeric polypeptide and / or at least one selection marker or portion thereof flanked by two heterospecific RRSs. In certain embodiments, the selection marker can be partially encoded on a first vector and partially encoded on a second vector, such that only correct integration of both by double RMCE allows expression of the selection marker.

[0158] In certain embodiments of all aspects and other embodiments of the invention, targeted integration by recombinase-mediated recombination integrates various expression cassettes for selection markers and / or multimeric polypeptides into one or more predefined integration sites of the host cell genome without including sequences derived from the prokaryotic vector.

[0159] An exemplary mammalian TI host cell suitable for use in the methods according to the invention is a CHO cell with a landing site integrated at a single site within a locus in its genome, the landing site containing three heterospecific loxP sites for DNA recombination mediated by Cre recombinase.

[0160] In this example, the heterospecific loxP sites are L3, LoxFas, and 2L (see, for example, Lanza et al., Biotechnol. J. 7 (2012) 898-908; Wong et al., Nucleic Acids Res. 33 (2005) e147), where L3 and 2L are adjacent to the 5' and 3' ends of the landing site, respectively, and LoxFas is located between the L3 and 2L sites. The landing site further contains a bicistronic unit that couples the expression of a selection marker via an IRES to the expression of a fluorescent GFP protein, allowing the landing site to be stabilized by positive selection, and the absence of the site to be selected after transfection and Cre recombination (negative selection). The green fluorescent protein (GFP) is useful for monitoring the RMCE reaction.

[0161] This organization of the landing sites as outlined in the previous paragraph allows the simultaneous integration of two vectors, for example the so-called front vector with L3 and LoxFas sites and the back vector with LoxFas and 2L sites inside. The functional elements of the selection marker gene, different from the one present in the landing sites, can be distributed between both vectors: the promoter and the start codon can be located on the front vector, whereas the coding region and the polyadenylation signal sequence are located on the back vector. Only the correct recombinase-mediated integration of the nucleic acid from both vectors induces resistance to the respective selection agents.

[0162] Typically, a mammalian TI host cell is a mammalian cell that comprises a landing site that integrates at a single site within a locus in the genome of the mammalian cell, the landing site comprising a first recombination recognition sequence and a second recombination recognition sequence adjacent to at least a first selectable marker, and a third recombination recognition sequence located between the first recombination recognition sequence and the second recombination recognition sequence, and wherein the recombination recognition sequences are all different.

[0163] The selection marker may be selected from the group consisting of genes encoding aminoglycoside phosphotransferases (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418 APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid. Alternatively, the selection marker may be a fluorescent protein selected from the group consisting of green fluorescent protein (GFP), enhanced GFP (eGFP), synthetic GFP, yellow fluorescent protein (YFP), enhanced YFP (eYFP), cyan fluorescent protein (CFP), mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald6, CyPet, mCFPm, Cerulean, and T-Sapphire.

[0164] An exogenous nucleotide sequence is a nucleotide sequence that is not native to a particular cell, but can be introduced into the cell by a DNA delivery method, such as transfection, electroporation, or transformation. In certain embodiments, the mammalian TI host cell comprises at least one landing site that is integrated into one or more integration sites in the genome of the mammalian cell. In certain embodiments, the landing site is integrated into one or more integration sites within a specific locus of the genome of the mammalian cell.

[0165] In certain embodiments of all aspects and other embodiments of the present invention, the integrated landing site comprises at least one selection marker. In certain embodiments, the integrated landing site comprises a first RRS, a second RRS and a third RRS, and at least one selection marker. In certain embodiments, the selection marker is located between the first RRS and the second RRS. In certain embodiments, the two RRSs are adjacent to at least one selection marker. That is, the first RRS is located 5' (upstream) of the selection marker, and the second RRS is located 3' (downstream) of the selection marker. In certain embodiments, the first RRS is adjacent to the 5' end of the selection marker, and the second RRS is adjacent to the 3' end of the selection marker. In certain embodiments, the landing site comprises a first RRS, a second RRS and a third RRS, and at least one selection marker is located between the first RRS and the third RRS.

[0166] In certain embodiments of all aspects and other embodiments of the invention, the selection marker is located between the first RRS and the second RRS, and these two adjacent RRSs are different from each other. In certain preferred embodiments, the first adjacent RRS is a LoxP L3 sequence and the second adjacent RRS is a LoxP 2L sequence. In certain embodiments, the LoxP L3 sequence is located 5' of the selection marker and the LoxP 2L sequence is located 3' of the selection marker. In certain embodiments, the first adjacent RRS is a wild-type FRT sequence and the second adjacent RRS is a mutant FRT sequence. In certain embodiments, the first adjacent RRS is a Bxb1 attP sequence and the second adjacent RRS is a Bxb1 attB sequence. In certain embodiments, the first adjacent RRS is a φC31 attP sequence and the second adjacent RRS is a φC31 attB sequence. In certain embodiments, the two RRSs are arranged in the same direction.

[0167] In certain embodiments, the two RRSs are both oriented in the forward or reverse direction. In certain embodiments, the two RRSs are positioned in opposite directions.

[0168] In certain embodiments of all aspects and other embodiments of the invention, the integrated landing site comprises a first and a second selection marker flanked by two RRSs, the first selection marker being different from the second selection marker. In certain embodiments, both of the two selection markers, independently of each other, are selected from the group consisting of a glutamine synthetase selection marker, a thymidine kinase selection marker, a HYG selection marker, and a puromycin resistance selection marker. In certain embodiments, the integrated landing site comprises a thymidine kinase selection marker and a HYG selection marker. In certain embodiments, the first selection marker is an aminoglycoside phosphotransferase (APH) (e.g., hygromycin phosphotransferase (HYG), neomycin, and G418). APH), dihydrofolate reductase (DHFR), thymidine kinase (TK), glutamine synthetase (GS), asparagine synthetase, tryptophan synthase (indole), histidinol dehydrogenase (histidinol D), and genes encoding resistance to puromycin, blasticidin, bleomycin, phleomycin, chloramphenicol, zeocin, and mycophenolic acid, and the second selection marker is selected from the group consisting of GFP, eGFP, synthetic GFP, YFP, eYFP, CFP, mPlum, mCherry, tdTomato, mStrawberry, J-red, DsRed monomer, mOrange, mKO, mCitrine, Venus, YPet, Emerald, CyPet, mCFPm, Cerulean, and T-Sapphire fluorescent protein. In a particular embodiment, the first selection marker is a glutamine synthetase selection marker and the second selection marker is a GFP fluorescent protein, hi a particular embodiment, the two RRS flanking both selection markers are different.

[0169] In certain embodiments of all aspects and other embodiments of the present invention, the selection marker is operably linked to a promoter sequence. In certain embodiments, the selection marker is operably linked to an SV40 promoter. In certain embodiments, the selection marker is operably linked to a human cytomegalovirus (CMV) promoter.

[0170] Lipase Lipases (EC 3.1.1.x) are found in animals, plants, and microorganisms and belong to a family of hydrolases that cleave carboxylic acid esters. Their natural function is to hydrolyze triglycerides to diglycerides, monoglycerides, fatty acids, and glycerol. This is called lipolysis. Lipases are not limited to the hydrolysis of triglycerides (this is only true for triacylglycerol lipases, 3.1.1.3). There are also other families of lipases that hydrolyze other substrates (e.g. phospholipids), such as phospholipases (3.1.1.32). All of these are considered to be related to polysorbate degradation.

[0171] Lipolysis is an enzymatic reaction and therefore an equilibrium reaction.

[0172] Lipases act at the lipid / water interface and are only slightly active in the absence of this boundary layer.[7,8] The activity of most lipases increases significantly upon contact with nonpolar lipid droplets or micelles in aqueous media, a process called interfacial activation,[9] and they are specifically linked to these interfaces.

[0173] An important feature of some lipases is the presence of a mobile structural element, a lid or flap, located above the enzyme active site. When the so-called "lid" is closed, the active site is protected from the environment and inaccessible to the substrate, and the lipase is inactive. In the open conformation, the substrate has access to the active site and can undergo enzymatic transformation.

[0174] Lipases have a much lower catalytic activity in aqueous media and a higher catalytic activity in organic media.

[10]

[0175] The term "hydrolase" is used herein in the broadest sense to refer to a molecule / compound with hydrolytic activity. Thus, in general, a "hydrolase" is an enzyme that hydrolyzes a chemical bond. For example, a "hydrolase" is an ester-cleaving enzyme that derives a carboxylic acid, with the derived free carboxylic acid and the corresponding alcohol as the final product. Alternatively, a hydrolase is an enzyme that cleaves a covalent bond by using hydrolase activity, i.e., a water molecule. Non-limiting examples of "hydrolases" are lipases, esterases, thioesterases, phospholipases, or ceramidases.

[0176] "Hydrolytic activity" as used herein as known in the art refers to the ability to hydrolyze chemical bonds. Hydrolysis refers to the cleavage of chemical bonds with water molecules acting as nucleophiles. Preferably, the molecule / compound with hydrolytic activity is a protein or polypeptide with hydrolytic activity. Thus, the protein or polypeptide with hydrolytic activity is an enzyme with hydrolytic activity. Thus, the hydrolase is an enzyme with hydrolytic activity. The hydrolase may be an esterase or an amidase.

[0177] The term "esterase" is well known in the art and in the context of the present invention refers to an enzyme that catalyzes the hydrolysis of ester bonds to produce acids and alcohols. In other words, esterases can act on ester bonds. Esterases are a diverse category of enzymes that include acetyl esterases, phosphatases, nucleases, thioesterases, and carboxylic ester hydrolases.

[0178] Carboxylic ester hydrolases hydrolyze carboxylic esters to alcohols and carboxylates using a water molecule. Most preferably, the hydrolase / esterase (such as carboxylic ester hydrolases) is a lipase. Lipases catalyze the hydrolysis of fatty acid esters / lipids, including triglycerides, fats and oils, to fatty acid and alcohol head groups.

[0179] In particular embodiments of all aspects and other embodiments of the invention, the hydrolase(s) of the method according to the invention is lipoprotein lipase (accession number: G3H6V7), palmitoyl protein thioesterase 1 (accession number: G3HN89), acid ceramidase (accession number: G3GZB2), C-terminal domain of fatty acid synthase (accession number: G3GXD7), putative phospholipase b-like 2 (accession number: G316T1), lysosomal acid lipase (accession number: G3HQY6), lysosomal phospholipase, phospholipase A2, group VII (PLA2G7) and group XV (PLA2G15), sialate O-acetyl esterase.

[0180] In certain embodiments of all aspects and other embodiments of the invention, the hydrolase enzyme of the method according to the invention is lipoprotein lipase, putative phospholipase b-like 2, and / or lysosomal phospholipase, due to their demonstrated hydrolase activity towards detergents such as polysorbates, which results in the formation of (visible) particles in the composition and / or reduced stability of the composition.

[0181] In a particular embodiment of all aspects and other embodiments of the invention, the hydrolase of the method according to the invention is a lysosomal phospholipase, in particular lysosomal phospholipase A2 (LPLA2) (Accession Number: G3HKV9).

[0182] In certain embodiments of all aspects and other embodiments of the invention, the hydrolase / hydrolase activity, i.e., hydrolytic activity, is based on the presence of one or more hydrolases selected from the group consisting of lipoprotein lipase, palmitoyl protein thioesterase, acid ceramidase, the C-terminal domain of fatty acid synthase, putative phospholipase b-like 2, lysosomal acid lipase, lysosomal phospholipase and sialate O-acetylesterase.

[0183] In certain embodiments of all aspects and other embodiments of the invention, the hydrolase is selected from the group consisting of lipoprotein lipase, palmitoyl protein thioesterase, acid ceramidase, the C-terminal domain of fatty acid synthase, putative phospholipase b-like 2, lysosomal acid lipase, lysosomal phospholipase, and sialate O-acetyl esterase.

[0184] Nucleic Acid Tags An "oligonucleotide" is a short, single-stranded nucleic acid, usually consisting of up to about 15 nucleotide monomers linked by a phosphodiester bond between the 3' carbon atom of one sugar molecule and the 5' carbon atom of the other sugar molecule. The monomers (in the general sense) contained in an oligonucleotide may be naturally occurring monomers as well as non-naturally occurring monomers, also called nucleotide analogs. In a non-limiting manner, exemplary analogs include sugar moieties other than ribose or deoxyribose, particularly ribose in which the sugar ring is "locked" by a methylene bridge connecting the 2'-O atom and the 4'-C atom. For the purposes of this disclosure, the term nucleotide encompasses natural and non-natural nucleotides as monomers in an oligonucleotide. Thus, an oligonucleotide according to this definition may be composed exclusively of natural or non-natural monomers or may be composed of a mixture thereof. Furthermore, unless otherwise stated, it is understood that various classes of non-natural monomers (e.g., PNA, D-LNA, L-LNA, homoDNA (containing hexose sugars), HNA (containing hexitol sugars, hexitol nucleic acids), L-DNA, etc.) may be included in an oligonucleotide.

[0185] In certain embodiments of all of the aspects and other embodiments of the invention, the immobilized tag is a nucleic acid tag.

[0186] A nucleic acid tag is one member of a binding pair of two distinct compatible nucleic acid partners, ie, complementary nucleic acid sequences.

[0187] In certain embodiments of all aspects and other embodiments of the invention, the immobilization tag is a nucleic acid tag comprised of an LNA oligonucleotide.

[0188] Several such single-stranded all-LNA oligonucleotides have been found, which are exemplary embodiments of the nucleic acid sequence tag, i.e., non-limiting examples of oligonucleotides that can be used as immobilization tags, since they can be bound to other complementary sequences by hybridization and duplex formation under non-denaturing conditions.The following list provides a non-limiting compilation thereof.It is understood that the listed sequences represent oligonucleotides that contain all LNA nucleosides, i.e., only LNA monomers.The sequences are given in the conventional orientation, i.e., from the 5' end to the 3' end.The sequences are paired, i.e., two consecutive sequences can be used as hybridization pairs, one of which is a nucleic acid tag in an artificial substrate, and the other is bound to a solid phase for capture.

[0189] In certain embodiments of all of the aspects and other embodiments of the invention, the nucleic acid sequence tag comprises: SEQ ID NO:01: tgctcctg; SEQ ID NO:02: caggagca; SEQ ID NO: 09: tgctcctgt; SEQ ID NO: 10: acaggagca; SEQ ID NO:11: gtgcgtct; SEQ ID NO:12: agacgcac; SEQ ID NO:13: gttggtgt; SEQ ID NO:14: acaccaac; SEQ ID NO:33: cttcc; SEQ ID NO:34: ggaag; SEQ ID NO:42: gctcc; SEQ ID NO:43: ggagc; SEQ ID NO:16: gttggt; SEQ ID NO:46: ccaac; SEQ ID NO:16: gttggt; SEQ ID NO:19: caccaac; SEQ ID NO:16: gttggt; SEQ ID NO:17: caacacaccaac; SEQ ID NO:16: gttggt; SEQ ID NO:18: acacaccaac; SEQ ID NO:16: gttggt; SEQ ID NO:14: acaccaac; SEQ ID NO:16: gttggt; SEQ ID NO:20: accaac; SEQ ID NO:40: ctgtca; SEQ ID NO:41: tgacag; SEQ ID NO:44: tgctcc; SEQ ID NO:45: ggagca; SEQ ID NO:35: tcttcc; SEQ ID NO:36: ggaaga; SEQ ID NO:21: gttggtg; SEQ ID NO:17: caacacaccaac; SEQ ID NO:21: gttggtg; SEQ ID NO:18: acacaccaac; SEQ ID NO:21: gttggtg; SEQ ID NO:14: acaccaac; SEQ ID NO:21: gttggtg; SEQ ID NO:19: caccaac; SEQ ID NO:21: gttggtg; SEQ ID NO:20: accaac; SEQ ID NO:01: tgctcctg; SEQ ID NO:02: caggagca; SEQ ID NO:11: gtgcgtct; SEQ ID NO:12: agacgcac; SEQ ID NO:13: gttggtgt; SEQ ID NO:17: caacacaccaac; SEQ ID NO:13: gttggtgt; SEQ ID NO:18: acacaccaac; SEQ ID NO:13: gttggtgt; SEQ ID NO:14: acaccaac; SEQ ID NO:13: gttggtgt; SEQ ID NO:19: caccaac; SEQ ID NO:13: gttggtgt; SEQ ID NO:20: accaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:17: caacacaccaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:18: acacaccaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:14: acaccaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:19: caccaac; SEQ ID NO:22: gttggtgtg; SEQ ID NO:20: accaac; SEQ ID NO:09: tgctcctgt; SEQ ID NO:15: caggagc; SEQ ID NO: 09: tgctcctgt; SEQ ID NO: 10: acaggagca; SEQ ID NO:09: tgctcctgt; SEQ ID NO:02: caggagca; SEQ ID NO:22: gttggtgtg; SEQ ID NO:30: cacaccaac; SEQ ID NO:37: ttctcttcc; SEQ ID NO:38: ggaagagaa; SEQ ID NO:23: gttggtgtgttg; SEQ ID NO:17: caacacaccaac; SEQ ID NO:23: gttggtgtgttg; SEQ ID NO:18: acacaccaac; SEQ ID NO:23: gttggtgtgttg; SEQ ID NO:14: acaccaac; SEQ ID NO:23: gttggtgtgttg; SEQ ID NO:19: caccaac; SEQ ID NO:23: gttggtgtgttg: SEQ ID NO:20: accaac; SEQ ID NO:31: gttggtgtgttggtg; SEQ ID NO:32: caccaacacaccaac; SEQ ID NO:28: aaaaaaaaa; SEQ ID NO:24: ttttttttt; SEQ ID NO:28: aaaaaaaaa; SEQ ID NO:25: tttttttt; SEQ ID NO:28: aaaaaaaaa; SEQ ID NO:26: tttttt; SEQ ID NO:28: aaaaaaaaa; SEQ ID NO:27: tttttt; SEQ ID NO:39: aaaaaa; SEQ ID NO:27: tttttt is selected from the group consisting of:

[0190] In a particular embodiment of all aspects and other embodiments of the present invention, a pair of two distinct compatible binding partners is used in the method according to the present invention. In a particular embodiment, the pair is a pair of all-LNA single-stranded oligonucleotides, one of which is used as a nucleic acid tag in the alcohol residue of the artificial substrate according to the present invention, and the other of which is used to capture / immobilize the free alcohol residue in the method according to the present invention. In a particular embodiment, the pair of all-LNA single-stranded oligonucleotides is (SEQ ID NO:01):(SEQ ID NO:02); (SEQ ID NO:09):(SEQ ID NO:10); (SEQ ID NO:11):(SEQ ID NO:12); (SEQ ID NO:13):(SEQ ID NO:14); (SEQ ID NO:09):(SEQ ID NO:15); (SEQ ID NO:16):(SEQ ID NO:20); (SEQ ID NO:21):(SEQ ID NO:18); (SEQ ID NO:21): (SEQ ID NO:20); (SEQ ID NO:21):(SEQ ID NO:19); (SEQ ID NO:23): (SEQ ID NO:17); and SEQ ID NO:25: SEQ ID NO:28 is selected from the group consisting of:

[0191] In one preferred embodiment of all aspects and other embodiments of the invention, the pair of two distinct compatible binding partners is a pair of all-LNA single-stranded oligonucleotides of SEQ ID NO: 16 and SEQ ID NO: 20. The binding pair is therefore gttggt:accaac.

[0192] Description of Specific Embodiments of the Invention Herein, we report a new assay using a tripartite artificial hydrolase substrate that mimics polysorbate. If a hydrolase, e.g., esterase or lipase, is present in the sample being analyzed, the artificial hydrolase substrate is cleaved by the hydrolase and the production of the enzymatic cleavage product, i.e., the free alcohol residue of the artificial substrate, is detected.

[0193] The detection of the released alcohol residue of the artificial hydrolase substrate in the method according to the invention is by means of a label attached thereto. The label can be freely selected. Thus, the detection can be carried out by any known method, for example Simoa®, Gyrolab®, Elecsys®, ELISA, etc.

[0194] In one preferred embodiment, the hydrolytic enzyme is an esterase and detection is by electrochemiluminescence immunoassay.

[0195] The present invention is based, at least in part, on the discovery that the presence of highly monomeric bovine serum albumin is advantageous with respect to assay sensitivity. This allows for increasing the hydrolytic activity of the hydrolytic enzyme. At the same time, it allows for increased sensitivity of the assay. This allows for the establishment of a detection limit in the low single picomolar range (1 pg / mL) and a quantification limit in the low double picomolar range (10 pg / mL).

[0196] The following provides an exemplary description of the method according to the present invention. This is done merely to outline the method according to the present invention. It should not be construed as limiting. The true scope of the present invention is set forth in the appended claims.

[0197] For the production of pharmaceuticals, CHO cells are preferred. During the production process, i.e., during cultivation, cell lysis occurs, which in particular releases proteolytic and hydrolytic enzymes. When therapeutic proteins are secreted into the extracellular medium, they come into contact with released host cell protein (HCP) impurities. Some HCPs have physicochemical properties similar to those of the pharmaceutical and are co-purified as a result. Other HCPs are co-purified as hitchhikers with the pharmaceutical due to direct protein-protein interactions [1].

[0198] In downstream processing (DSP), HCP contamination must be reduced to an acceptable level, since even small amounts of HCP contamination can affect product quality and stability and / or cause adverse effects in patients, such as immune reactions. This is achieved by using purification steps. However, some HCPs are difficult to remove in DSP, as they are involved in non-covalent interactions with the product and have chromatographic behavior similar to that of therapeutic proteins [2].

[0199] More than 90% of HCPs are typically removed by Protein A chromatography in DSPs [3]. Further purification (polishing) steps such as hydrophobic interaction chromatography (HIC) help to significantly reduce residual HCP concentrations [4]. However, traces of HCPs remain in the preparation. Health agencies take this into account by stipulating that the presence of HCPs is minimized to an acceptable residual amount, i.e., below a defined threshold for each product [5]. Most biotechnology products approved by the Food and Drug Administration (FDA) have less than 100 ppm of HCP contaminants.

[0200] HCP contaminants are usually detected using quantitative immunological tests such as ELISA. However, multianalyte ELISAs detect only the total amount of HCPs and therefore cannot estimate the identity or amount of individual HCPs present. Liquid chromatography combined with tandem mass spectrometry (LC-MS / MS) and protein-specific ELISAs are suitable for identifying individual HCPs. The amount of HCPs relative to an active pharmaceutical ingredient (API) is usually very low, so that identification and quantification using LC-MS / MS is hindered even after concentrating those HCPs [6].

[0201] Protein aggregation in biotherapeutics is associated with immune reactions and loss of active drug components. A common method to reduce non-specific interactions and stabilize proteins is to add surface active compounds to protein formulations.

[0202] Surface active agents, or surfactants, consist of molecules that have a polar hydrophilic part and a non-polar hydrophobic part. They are called amphiphiles. They reduce interfacial stresses and increase the solubility of products.

[11]

[0203] Surfactants can be subdivided into four main groups: anionic, cationic, zwitterionic and non-ionic surfactants. Anionic surfactants have a negative charge on the polar part of the molecule and cationic surfactants have a positive charge on the polar end. Zwitterionic surfactants have positive and negative charges that depend on the environment and non-ionic surfactants have no charge on the hydrophilic part of the molecule.

[0204] Polysorbates are nonionic surfactants that are widely used in the pharmaceutical industry due to their stabilizing properties, low toxicity, and high biocompatibility.

[12] The most frequently used surfactants in pharmaceutical products are polysorbate 20 (PS20), polysorbate 80 (PS80), and poloxamer 188, which are usually added to products at mass concentrations of 0.001%–0.1%.

[13]

[0205] Polysorbates belong to the group of poly(ethylene glycol) sorbitan fatty acid esters. They consist of 20 ethylene oxide units, a sorbitan ring, and fatty acids of 12 to 18 carbon atoms. The fatty acid ester of PS20 is formed mainly from lauric acid and is therefore called polyoxyethylene (POE)-(20)-sorbitan monolaurate. PS80 contains mainly oleic acid and is therefore called polyoxyethylene-(80)-sorbitan monooleate [14-21]. Figure 1 shows an idealized polysorbate structure.

[0206] As is well known, technical polysorbates are primarily heterogeneous mixtures with different attached fatty acids and varying degrees of esterification (see Table 1).

[0207] Table 1: Percentage composition of fatty acids contained in heterogeneous mixtures of polysorbates 20 and 80 according to the European Pharmacopoeia (Ph.Eur.) and the United States Pharmacopoeia (USP). The bolded fatty acids are the most common fatty acids in each polysorbate. TIFF2025500334000003.tif55128

[0208] The advantage of polysorbates is that they are readily biodegradable

[22] . However, this advantage is also their biggest drawback in terms of stability in protein formulations, which has been a major challenge in the pharmaceutical industry for several years. In some cases, polysorbates can undergo significant degradation and therapeutic proteins can no longer be adequately protected from interfacial interactions

[23] .

[0209] Furthermore, if free fatty acids accumulate, they may exceed their solubility limit and precipitate, forming visible particles that may affect the stability and quality of the product

[24] . Due to the complex interrelationships between surfactants, active ingredients, and free fatty acids, the basic mechanisms of action that promote precipitation via specific nucleation events are not yet fully understood and may vary from formulation to formulation

[25] . Particles in parenteral products are undesirable as they may cause harm to patients [23, 26].

[0210] Ultra-purified and high-purity grades are two types of PS20 used in the formulation of biopharmaceuticals. Compared with high-purity grade PS20, ultra-purified PS20 contains a higher proportion of secondary and higher-order esters, which play an important role in the solubilization of free fatty acids. In addition, ultra-purified PS20 is characterized by lower levels of process-related impurities, such as free fatty acids, and thus reduces or delays the occurrence of particle formation

[27] .

[0211] Polysorbate degradation can occur either by oxidation or hydrolysis (see Figure 2)

[28] .

[0212] The hydrolysis of fatty acid esters can be carried out enzymatically or via an acid / base catalyzed mechanism, the latter being negligible under normal storage and formulation conditions, as the acid / base catalyzed mechanism has been found to play a key role mainly in experiments carried out at high pH and high temperature

[29] .

[0213] Enzymatic degradation of polysorbates is less well characterized than chemical degradation, and only a small proportion of the responsible enzymes have been identified [30,31,73]. Hall et al. identified lysosomal phospholipase A2 (LPLA2) in purified antibodies that caused polysorbate degradation, but antibodies in the absence of LPLA2 showed no polysorbate degradation

[30] . Dixit et al. found that phospholipase B-like 2 (PLBL2) is involved in polysorbate degradation

[32] . However, this study used a recombinant variant with a purity of about 90% for this protein. Therefore, possible enzyme contamination cannot be completely excluded. A study by Zhang et al. examined the purity of recombinant PLBL2 and was able to identify several different lipases. Thus, the increase in hydrolytic activity in Dixit et al. could possibly be attributed to contamination with lipases. Furthermore, Zhang et al. showed that PLBL2-depleted samples did not result in a decrease in lipase activity. Similarly, no correlation could be established between the concentration of PLBL2 in samples and polysorbate degradation.

[33] A study by Chiu et al. showed that removal of lipoprotein lipase (LPL) using LPL knockout CHO cells helped to reduce the degradation of PS20 and PS80.

[31]

[0214] Hydrolysis results in the release of free fatty acids and non-esterified sorbitan / isosorbide forms. The solubility of fatty acids decreases with increasing chain length. This can promote particle formation [34,35,36]. A risk factor for product quality in pharmaceutical solutions is the appearance of fatty acid particles, which may be either visible or invisible to the human eye, derived from the cleavage of polysorbates [37,38]. The observed fatty acid particles can be clearly distinguished from protein particles using analytical methods. Fatty acid particles are composed mostly of cleaved long-chain free fatty acids, which are poorly soluble in aqueous solutions, especially at low temperatures

[24] .

[0215] There are various analytical methods for the degradation of polysorbates. One method is to measure the hydrolase activity of the in-process sample. Another is to carry out the determination of free fatty acids. A further method is mixed-mode liquid chromatography with a light scattering detector (HPLC-ELSD) to determine the remaining PS20 content

[17] . Furthermore, another possibility is the fluorescent micellar assay, in which the fluorescent dye N-phenyl-1-naphthylamine is absorbed into the hydrophobic core of PS micelles, allowing the determination of the polysorbate content

[39] .

[0216] The Lipase Enzyme Activity Against Polysorbates (LEAP) assay can be used to determine the hydrolase / hydrolase activity, i.e., hydrolytic activity, of in-process samples

[40] . As shown graphically in Figure 3, hydrolytically active components present in the sample cleave the ester bond of the non-fluorescent substrate, 4-methylumbelliferyl fatty acid ester. This results in an increase in the fluorescent alcohol residue, 4-methylumbelliferone (MU). The increase in the fluorescent signal with an absorbance of 355 nm and an emission of 460 nm is continuously monitored while the assay is incubated for 2 hours at 37 °C in a plate reader.

[0217] The rate of MU production was derived from the slope of the fluorescence time curve and the initial rate of the reaction (k raw ) This method expresses the rate of self-cleavage of a substrate in a given sample matrix (k self開裂The lipase activity of the sample is determined according to equation (1) by subtracting the autocleavage rate from the initial rate and converting the fluorescent signal to μMMU by plotting a standard curve of MU on the same plate. TIFF2025500334000004.tif9128k raw = reaction rate of the sample in RFU / h, k self開裂 = reaction rate of the enzyme blank in RFU / h, a = calculated conversion factor of fluorescence signal to MU concentration in RFU / μM.

[0218] The limit of quantification (LOQ) of this assay is 0.1 μMMU / h and the limit of detection is 0.4 μMMU / h

[40] .

[0219] The Free Fatty Acid Mass Spectroscopy (FAMS) method for the quantification of FFAs (free fatty acids)

[41] is carried out using, for example, ultra-performance liquid chromatography coupled to a mass spectrometer (UPLC-MS). The separation of molecules for FFA analysis is based on reversed-phase liquid chromatography (RPLC). Among all RPLC columns, the octadecyl (C18) column is the most common. Since hydrophobic molecules have a high affinity for the stationary phase, while hydrophilic molecules have a low affinity for the column, hydrophobic molecules pass through the column faster and are detected first. Thus, the shorter lauric acid with a C12 chain is eluted first, followed by the longer myristic acid with a C14 chain

[42] .

[0220] Mass spectrometry is used as an analytical technique to determine the mass of molecules. An ion source, in this case electrospray ionization (ESI), produces gas ions from molecules in the liquid phase. A mass analyzer separates the ions according to their mass-to-charge ratio (m / z), and a detector, QDa, counts the ions for each m / z ratio

[43] .

[0221] The concentration of free fatty acids is calculated according to the following formula (2): TIFF2025500334000005.tif7128

[0222] (Table 2) LOQ and operating range of the FAMS method. TIFF2025500334000006.tif32128

[0223] Small amounts of the enzyme are involved in the cleavage of polysorbates in pharmaceutical products and are difficult or impossible to detect using existing analytical methods such as LEAP or FAMS.

[0224] Both LEAP and FAMS have limiting factors: the LEAP assay is a robust but not very sensitive assay; residual hydrolases / hydrolase activity, i.e. hydrolytic activity, cannot be detected at low levels, for example at the final process step; FAMS is a very robust and sensitive method, but is very time consuming to use; other methods require sample pretreatment or chromatographic pre-separation before carrying out the determination of hydrolases / hydrolase activity (hydrolytic activity).

[0225] As described herein, previously used methods for both HCP identification / quantification and determination of hydrolytic activity are hampered by the small amounts of HCP involved in polysorbate degradation in biopharmaceutical formulations.

[0226] For these reasons, suitable methods are needed for the rapid, sensitive and specific detection of hydrolases / hydrolase activity (hydrolytic activity), i.e. hydrolytically active HCPs.

[0227] The present invention is based, at least in part, on the discovery that in certain embodiments, a Polysol-Based Assay (EPA) based electroluminescence-based immunoassay can overcome at least all of these limiting factors, as outlined above.

[0228] The EPA according to the invention is based on the hydrolysis of a tripartite artificial esterase substrate. For detection, the electrochemiluminescence immunoassay (ECLIA) method can be used. ECLIA is an analytical, highly selective and sensitive technique that can be used to determine the enzyme activity using appropriate (artificial) substrates.

[0229] The method according to the invention provides more rapid and sensitive enzyme activity measurements than the above-mentioned assays.

[0230] Detection of polysorbate degradation caused by hydrolases / hydrolase activity (hydrolytic activity) of HCPs (still) present in the sample is achieved by combining hydrolytic cleavage with immunoassay-based quantification of the resulting enzymatic cleavage products.

[0231] For this purpose, we used an artificial substrate that mimics polysorbate. The functional diagram of the artificial substrate is shown in Figure 4. The essential feature of the substrate is that different functional groups are bonded to the carboxylic acid residue and the alcohol residue, respectively.

[0232] More specifically, due to the low catalytic activity, the determination of hydrolases / hydrolase activity (hydrolytic activity) benefits from the removal of one of the cleavage products as well as the uncleaved artificial substrate from the reaction mixture.

[0233] Thus, one of the residues of the artificial substrate is bound to the first label. The type of binding and the type of the first label are not limited as long as the binding is stable under the assay conditions and the first label can be specifically bound by the binding partner in the presence of the nucleic acid tag and the second label. The first label is used to bind each binding residue and the intact artificial substrate to a solid phase that immobilizes these compounds, respectively. This allows for easy removal. The remaining residues of each remain in solution and can be separated from the solid phase by standard techniques. In one preferred embodiment, the carboxylic acid residue of the artificial substrate is bound to the first label.

[0234] For detection, each other residue is bound to a second label. This residue is the cleavage product detected / quantified. The second label is different from the first label so that both labels can be selectively bound in the presence of each other. In addition, there is no need to place any further requirements on the second label. In one preferred embodiment, the cleavage product detected / quantified is the alcohol residue of the artificial substrate.

[0235] In order to improve the determination and quantification of the residue that is bound to the second label, immobilization tag can be further bound to the detected / quantified residue.Therefore, detection / quantification can be carried out as solid-phase reaction, for example, ELISA or ECLIA.In one preferred embodiment, immobilization tag is nucleic acid tag.

[0236] In addition to the functional characteristics outlined above, no restrictions are required regarding the structure of the artificial substrate. Thus, based on their technical knowledge of using scaffolds, linkers, labels, binding methods and immobilization tags, those skilled in the art can easily design an artificial substrate suitable for carrying out the method according to the invention without undue burden.

[0237] The hydrolytically active HCP cleaves the ester bond, releasing the alcohol and carboxylic acid residues from the artificial substrate (see Figure 4). R1 ​​on the carboxylic acid residue as well as R1 on the uncleaved artificial substrate enter into a non-covalent biological bond with its binding partner and are then removed. The remaining alcohol containing R2 and R3 can be determined by a specific antibody that binds to R3, e.g., labeled with a ruthenium complex for electrochemiluminescence-based detection. A class of high affinity RNA analogs (R2), e.g., LNA, is used to capture the alcohol residue on magnetic beads coated with nucleic acids with complementary sequences.

[0238] For example, when antibodies labeled with ruthenium complexes are used for electrochemiluminescence-based detection, the incubation / reaction mixture is transferred to the measuring cell of the Cobas e411 system (Roche Diagnostics GmbH, Mannheim, Germany) where the magnetic microparticles are retained on the electrode surface. A washing step removes anything that is not attached to the electrode surface. A voltage is applied to the electrode to induce chemiluminescence, which is measured by a photomultiplier tube. The results are then determined using an external calibration curve.

[0239] The present invention is based, at least in part, on the finding that among possible influencing factors such as, for example, type of buffer salt, divalent salt, temperature, and concentration of buffer, NaCl, presence of methionine, concentration and type of BSA, the factors of type and concentration of BSA, presence of NaCl, and BSA*NaCl interaction have a statistically significant effect on enzyme activity in the pH range of pH 7 to pH 8 (see FIG. 5 and Example 1). Non-significant factors are shown in a half-normal probability plot on a line with a normal distribution. Significant factors deviate from this line. The further an element is from the line, the greater its influence.

[0240] The present invention is based, at least in part, on the discovery that among possible influencing factors such as type of buffer salt, divalent salt, temperature, and concentrations of buffer, NaCl, methionine, BSA and divalent salt at acidic pH values ​​in the range of pH 6-7, the factor concentrations of buffer, BSA as well as the BSA*NaCl interaction have a statistically significant effect on enzyme activity (see FIG. 6 and Example 1).

[0241] The present invention is based, at least in part, on the discovery that the addition of intermediate concentrations of detergents has a positive effect on enzyme activity, especially when the sample contains only small amounts of hydrolases / hydrolase activity (hydrolytic activity).

[0242] The effect of the presence or absence and concentration of a detergent is shown in FIG. 7. As an exemplary sample, a purified monoclonal antibody preparation was used. This sample had already been purified using two chromatographic steps and therefore contains only traces of residual hydrolases / hydrolase activity (hydrolysis activity). As an exemplary detergent, Triton® X-100 is used. It can be seen that in the absence of detergent, the initiation of cleavage of the artificial substrate is delayed. In contrast, in the presence of 0.25% (w / v) detergent, the enzymatic reaction is inhibited. At intermediate concentrations, neither delay nor inhibition of the reaction is observed. Thus, in one preferred embodiment, the concentration of the detergent is in the range including 0.05% (w / v) to 0.15% (w / v).

[0243] In certain embodiments of all of the aspects and other embodiments of the invention, the buffer solution or incubation mixture further comprises a surfactant.

[0244] In certain embodiments, the surfactant is a non-ionic surfactant.

[0245] In a particular embodiment, the surfactant is an aromatic hydrocarbon lipophilic or hydrophobic group with a hydrophilic polyethylene oxide chain.In a particular embodiment, the surfactant has an average of 9.5 ethylene oxide units, and the hydrocarbon is a 4-(1,1,3,3-tetramethylbutyl)-phenyl group.In a particular embodiment, the surfactant is octoxynol-9.Octoxynol-9 is commercially available under the trade names Triton X-100 and Nonidet P40.

[0246] In certain embodiments, the surfactant is an alkyl polyglucoside. In certain embodiments, the surfactant is a mixture of 58.0-62.0 (w / v)% D-glucopyranose, oligomeric, decyl octyl glycoside and 38.0-42.0 (w / v)% water.

[0247] The effect of temperature on the assay according to the invention is shown in Figure 8. Protein A chromatography purified monoclonal monospecific antibodies were used as samples. The samples were treated with the method according to the invention for 22 hours at different temperatures: 34°C, 37°C and 40°C. Blank corrected values ​​are plotted as a bar graph in Figure 8.

[0248] The present invention is based, at least in part, on the discovery that the use of (3-(N-morpholino)propanesulfonic acid) (MOPS) as a buffer in the methods according to the invention results in better assay sensitivity.

[0249] As an example, a MOPS-based buffer was compared to a TRIS-based buffer. The composition of the 10x stock solution is shown in Table 3 below.

[0250] (Table 3) Buffer composition TIFF2025500334000007.tif44128

[0251] The concentrations that can be used to prepare the buffer solutions depend on the solubility of the respective buffer salts in water. Thus, to prepare a 10x concentrated stock solution, a 1.5M concentration of MOPS was used. Similarly, a 3M concentration of TRIS was used. Both are close to the solubility of the respective buffer in water at a pH value of 7. This provides the highest buffering capacity in both cases.

[0252] The amount of hydrolysate produced was determined, and the results are shown in a bar graph in Table 4 below and in Figure 12 (TRIS-based buffer on the left, MOPS-based buffer on the right).

[0253] (Table 4) Amount of hydrolysate produced depending on the buffer used. TIFF2025500334000008.tif41143

[0254] It has been found that the use of MOPS-based buffers increases hydrolase / hydrolase activity (hydrolysis activity). Without wishing to be bound by theory, it is hypothesized that the pKa value of MOPS of 7.02 at 37° C. provides better buffering capacity at a pH value of 7.0.

[0255] The present invention is based, at least in part, on the discovery that BSA significantly affects enzyme activity. In particular, the present invention is based, at least in part, on the discovery that the use of inactive bovine serum albumin results in an increased signal-to-noise ratio, thereby improving sensitivity.

[0256] The presence of BSA in the buffer results in improved enzyme activity and therefore also increases the sensitivity of the assay.

[0257] BSA from various sources or vendors was compared as shown in Table 5 below.

[0258] Table 5: Various BSA sources. TIFF2025500334000009.tif32143

[0259] Relative results, ie blank corrected and normalized to the minimum overall value, are shown in Table 6 below and in FIG.

[0260] Table 6: Relative amounts of hydrolysate produced depending on BSA used. TIFF2025500334000010.tif32141 * : Negative value after blank correction

[0261] It is found that the use of Cys34 oxidized BSA results in the best hydrolase / hydrolase activity (hydrolysis activity). Without being bound by this theory, it is believed that the use of inactive bovine serum albumin may increase the hydrolase activity in the sample, thereby increasing the sensitivity of the assay. In a preferred embodiment, the inactive bovine serum albumin is bovine serum albumin in which more than 80% of the cysteine ​​residues at position 34 are oxidized / in oxidized form. In a particular embodiment, more than 90% of the cysteine ​​residues at position 34 are oxidized / in oxidized form. In a particular embodiment, more than 95% of the cysteine ​​residues at position 34 are oxidized / in oxidized form. In a preferred embodiment, more than 80% of the bovine serum albumin is present in monomeric form (not bound to a second polypeptide at cysteine ​​residue 34). In a particular embodiment, more than 90% of the bovine serum albumin is present in monomeric form. In a particular embodiment, more than 95% of the bovine serum albumin is present in monomeric form.

[0262] A scheme of an exemplary assay according to the invention is shown in FIG.

[0263] In one preferred embodiment of all aspects and other embodiments of the invention, the incubation mixture of the assay according to the invention comprises 20-30 mM sodium chloride, 3-10 μM BSA / oxidized Cys34 with a monomer content of 95% or more, 100-200 mM MOPS, 3-10 mM magnesium chloride, 0.05-0.15% (w / v) surfactant, the therapeutic polypeptide has a concentration of 0.1-250 mg / mL and a pH value of 6.8-7.2, and the incubation is carried out at 35-40° C. for 8-36 hours.

[0264] In certain embodiments of all of the aspects and other embodiments of the invention, steps (a) and (b) are performed in parallel with two different aliquots of the sample, and the aliquots are mixed prior to performing step (c).

[0265] Below is comparative data for the assay according to the invention and the FAMS assay.

[0266] The readout of the assay according to the invention is a rate with units of pg / mL / mg / h, whereas the readout of the FAMS assay is a rate with units of ng / mg / d. Therefore, the results of these two assays are not directly comparable. As outlined above, regulatory authorities require the reduction of HCP below a certain threshold limit. Similarly, the reduction of hydrolases / hydrolase activity (hydrolysis activity) must be below a threshold so that particle formation is prevented. Therefore, the comparison of the assays was performed using the determination of the relative rate, i.e. the relative remaining hydrolases / hydrolase activity (hydrolysis activity) in the test sample compared to the starting value.

[0267] Samples containing different therapeutic polypeptides and at different purification stages were analyzed. As these therapeutic polypeptides are different, different orders of chromatographic steps were also used for their purification. As the assay according to the invention is for determining the residual hydrolases / hydrolase activity (hydrolytic activity), this difference does not affect the assay readout, as the same sample aliquots are used for both assays. Similarly, the specific sequence or binding specificity of the therapeutic polypeptides does not affect the assay.

[0268] Therapeutic polypeptide 1 is a full-length bivalent bispecific antibody of the IgG1 subclass in CrossMab format.

[0269] Therapeutic polypeptide 2 is a full-length bivalent monospecific antibody of the IgG1 subclass.

[0270] Therapeutic polypeptide 3 is a trivalent bispecific antibody in the TCB format.

[0271] Therapeutic polypeptide 4 is a full-length bivalent monospecific antibody.

[0272] Therapeutic polypeptide 5 is a bivalent, bispecific Fab.

[0273] Residual hydrolase / hydrolase activity (hydrolytic activity) below a threshold in a therapeutic polypeptide preparation will not result in appreciable polysorbate degradation during storage. Therefore, it is necessary to determine whether a therapeutic polypeptide preparation has hydrolase / hydrolase activity (hydrolytic activity) below a threshold to ensure an adequate storage period.

[0274] To compare the results of the assay according to the invention and the FAMS assay, the difference between the respective measurements was used, therefore the average of the results of the two assays is assumed to be the correct value.

[0275] As outlined above, it is necessary to determine the residual hydrolase / hydrolase activity (hydrolysis activity). Therefore, for the methods to be comparable, the residual hydrolase / hydrolase activity (hydrolysis activity) determined by the different methods should be within 100% of each other. This percentage difference represents the difference between the measured value and the assumed exact value in percent. It is used to report the difference between the measured value and the exact value, i.e. to measure exactly how close the measured value is to the true value.

[0276] The data are shown in Table 7.

[0277] (Table 7) Comparison of FAMS and EPA experimental results. TIFF2025500334000011.tif219143TIFF2025500334000012.tif73143n.d.=Undecided nc = not determined and cannot be calculated EPA-1 = assay according to the invention with 100 ng / mL of therapeutic polypeptide in incubation mixture; no pooling of samples before determination; 1:3 dilution before electrochemiluminescence measurement EPA-2 = Assay according to the invention with 200 ng / mL of therapeutic polypeptide in the incubation mixture; pooling of two parallel incubated aliquots before determination; no further dilution before electrochemiluminescence measurement

[0278] The data from the above tables are shown as bar graphs in Figures 10 and 11 for the exemplary therapeutic polypeptide 3. The assay according to the invention and the FAMS assay, the most sensitive assay for determining hydrolases / hydrolase activity (hydrolytic activity), provide comparative data, however it can be seen that the assay according to the invention can be performed within 48 hours instead of the two weeks required for the FAMS assay. ***

[0279] The following examples and figures are provided to aid the understanding of the present invention, the true scope of which is set forth in the appended claims. It is understood that modifications can be made in the procedures set forth without departing from the spirit of the invention.

[0280] References TIFF2025500334000013.tif30133TIFF2025500334000014.tif233142TIFF2025500334000015.tif55142 [Brief description of the drawings]

[0281] [Figure 1] An ideal polysorbate structure with a non-polar fatty acid at one end and polar sorbitol and its anhydrides at the other end, where w+x+y+z represents the total number of moles of ethylene oxide per mole of sorbitol and ethoxylated sorbitol anhydrides, and the sum must not exceed 20. [Diagram 2] Oxidative and hydrolytic degradation pathways of polysorbate 20

[44] . Long-chain fatty acids are released by hydrolytic cleavage of the fatty acid ester bond (red bolt icon). Oxidative cleavage (blue bolt icon) results in a mixture of degradation products. [Diagram 3] Ester cleavage of 4-methylumbelliferyl fatty acid esters leads to the fluorescent head group 4-methylumbelliferone and fatty acids. [Figure 4] Schematic diagram of the chemical structure of an artificial substrate according to the invention with three different residues. R3 is an antigen of certain cardenolides, e.g. digoxigenin. R2 is a class of high affinity RNA analogues and R1 is part of a non-covalent biological binding pair, e.g. biotin (biotin / avidin pair). The lightning arrow indicates an ester bond. A hydrolytically active HCP cleaves this ester bond, resulting in the release of an alcohol and an acid from the substrate. [Diagram 5] Graphical representation of the factors tested for their effect on enzyme activity. (A) Semi-normal probability plot. (B) Pareto plot. [Figure 6] Graphical representation of factors tested for their effect on enzyme activity over the pH range of 6 to 7. (A) Semi-normal probability plot. (B) Pareto plot. [Figure 7] Graphical representation of surfactant effects. [Figure 8] Effect of temperature on the method according to the invention. [Figure 9] Schematic of an exemplary assay according to the present invention. [Figure 10] 4 is a bar graph of percent error of the FAMS assay for an exemplary in-process sample of therapeutic polypeptide 3. [Figure 11] 4 is a bar graph of the percent error of a method according to the invention for an exemplary in-process sample of therapeutic polypeptide 3. [Figure 12] Bar graph showing the amount of hydrolysate produced by the method according to the invention depending on the buffer used: TRIS-based buffers on the left, MOPS-based buffers on the right [Figure 13] The effect of BSA and different grades of BSA on the method according to the invention. EXAMPLES

[0282] Description of the embodiment Example 1 Designing an experiment using a Placket-Burman design The buffer screening process identifies factors that affect enzyme activity.

[0283] For this experiment, host cell culture fluid (HCCF) was used.

[0284] Two categorical factors were used: type of buffer salt and divalent cation, and a series of seven factors - pH, temperature, buffer, NaCl, methionine, BSA concentration and divalent salt concentration. The results are reported in Table 8 below.

[0285] Table 8: DoE conditions; E=acid conditions; F=basic conditions. TIFF2025500334000016.tif87135

[0286] The experimental conditions were selected using the Placket-Burman Design, which is a two-level fractional factorial design in which all factors have only two values. The low (-) and high (+) values ​​of these factors to be tested are listed in Table 8 above.

[0287] Based on these factors, two plans of the Placket-Burman design were created (see Table 9 below): one plan served as the acidic buffer template and the other plan served as the alkaline buffer template.

[0288] To be able to exclude chemical cleavage of the substrate by the buffer composition, a sample in the absence of hydrolytic enzyme, the so-called buffer blank, was also measured, which was then subtracted from each sample value to obtain the value of the enzyme activity alone.

[0289] (Table 9) Plans for the Placket-Burman design. TIFF2025500334000017.tif81140

[0290] Each pattern was replicated on a 96-well plate, and the experiment was repeated twice under the same conditions.

[0291] The results under alkaline conditions are shown in Table 10, and those under acidic conditions in Table 11.

[0292] Table 10: Alkaline DoE results. TIFF2025500334000018.tif100128

[0293] Table 11: Acidity DoE results. TIFF2025500334000019.tif98128

[0294] Example 2 - Comparative Example LEAP assay A non-limiting example for measuring, determining or quantifying hydrolase / hydrolase activity (hydrolytic activity) is an assay that detects lipolytic activity, or in other words, a lipase activity assay (e.g., as described by Jahn et al.), also referred to herein as the LEAP (Lipase Enzyme Assay for Polysorbates) assay (Jahn (2020) Pharm. Res. 37:118).

[0295] This assay allows to measure, determine or quantify hydrolase / hydrolase activity (hydrolytic activity) and is a lipase activity assay (or in other words an assay to detect lipolytic activity) that measures the conversion of 4-methylumbelliferone caprylate (4-MUCA) to 4-methylumbelliferone (4-MU). The assay using 4-MUCA as substrate can be carried out as described below.

[0296] 10 μL of a composition containing the protein to be determined hydrolase / hydrolase activity (hydrolase activity) can be mixed with 80 μL of reaction buffer (150 mM Tris-HCl pH 8.0, 0.25% (w / v) TritonX-100 and 0.125% (w / v) gum arabic) and 10 μL of 4-MUCA substrate (1 mM in DMSO). Reactions can be set up in 96-well half-area polystyrene plates (black clear flat bottom with lid, Corning Incorporated) and the increase in fluorescent signal (excitation at 355 nm, emission at 460 nm) can be monitored every 10 minutes, for example by incubating the reaction plate at 37° C. for 2 hours in an Infinite 200Pro plate reader (Tecan Life Sciences) to derive the 4-MU production rate. The 4-MU production rate of the composition containing the protein can be compared to the starting composition containing the protein.

[0297] Example 3 - Comparative Example FAMS assay The following paragraphs describe exemplary sample preparation and analysis procedures for performing a FAMS assay.

[0298] The FAMS assay measures lipase activity by quantifying the accumulation of free fatty acids from the hydrolysis of polysorbate 20.

[0299] All samples (antibody solutions and buffer controls) may be supplemented with stock solutions of 1% (w / v) ultra-purified polysorbate 20 (Croda Health Care) and 0.25ML of methionine (Sigma Aldrich, Art No. M5308) to obtain a final concentration of 0.04% (w / v) ultra-purified polysorbate 20 and 0.01ML-methionine per sample. For each spiked sample, aliquots of 190 μL may be transferred into five capped glass vials (called t0, t1, t2, t3, t4) that may be used for sample incubation. Glass vial t0 may be frozen at -70°C immediately after their preparation until analysis. Glass vials t1, t2, t3 and t4 may be incubated upright at 25°C and protected from light in an incubator. Incubation of the glass vials may be stopped one at a time over the next 5 to 14 days, after which the glass vials may be frozen at -70°C until analysis.

[0300] For analysis, the frozen samples can be allowed to come to ambient temperature for 1 hour. 50 mg of each fatty acid, lauric acid 2 d 23 (Sigma Aldrich, Cat. No. 451401) and myristic acid 13 C 14 Stock solutions of stable isotope-labeled fatty acids may be prepared by dissolving 1 μg / mL of 100 μL of precipitation reagent (Sigma Aldrich, Cat. No. 605689) in 50 mL of 80% acetone / 20% methanol to obtain a precipitation reagent with a final concentration of 1 μg / mL for each investigated fatty acid. 50 μL of sample solution may be added to 200 μL of precipitation reagent in a reaction tube, mixed by vortexing, and kept at room temperature for 1 h to precipitate the proteins. The precipitate may be centrifuged by centrifugation at 15,000 × g for 15 min at 20 °C, and 100 μL of the supernatant may be transferred to a new reaction tube and mixed with 100 μL of mobile phase A (20 mM ammonium acetate). After spinning down at 15,000 × g for 15 min at 20 °C, 50-100 μL of the solution may be transferred to an LC-MS vial (300 μL fixed insert vial (clear, screw top), Thermo Scientific, Cat. No. 03-FISV).

[0301] Separation of free fatty acids (FFAs) can be achieved on an ACQUITY UPLCH-Class system (Waters Corp. Milford, MA, USA) equipped with a temperature-controllable autosampler and column compartment by using a Jupiter® C4 RP column (300 Å, 2×50 mm, 5 μm) (Phenomenex, Cat. No. 00B-4167-B0). Mobile phase A can be 20 mM ammonium acetate (Sigma Aldrich, Cat. No. 73594) and mobile phase B can be 100% methanol (Merck, Cat. No. 1.06007.2500), which can be performed for 4 min at a flow rate of 0.4 mL / min by applying the following gradient: initial condition: 70% mobile phase B, 0.5 min to 3.4 min: gradient can be changed linearly from 70% to 85% mobile phase B, 3.5 min to 4.0 min: 70% mobile phase B. The autosampler may be maintained at 20°C and the column compartment at 60°C. The injection volume may be set at 8 μL. Detection may be performed on a connected QDa Performance mass spectrometer (Waters) with an external backing pump in negative ion mode. MS settings may be cone voltage 15 V, source temperature 120 °C capillary voltage 800 V, probe temperature 600 °C, mass range 50-1000 m / z and sampling frequency 2 Hz. All samples may be analyzed in triplicate.

[0302] Data evaluation may be performed using Target Lynx as part of Mass Lynx Software Version 4.1 (SCN781) (Waters Corp., Milford, Massachusetts, USA). The lauric and myristic acid contents may be determined by comparing the peak areas of the fatty acids with their respective internally labeled standards using the following equation (3): TIFF2025500334000020.tif10132 where the Σ peak area of ​​the FFA and the Σ peak area of ​​the internal standard refer to the sum of the monoisotopic and isotopic peaks at +1 / +2 or -1 / -2, respectively.

[0303] To determine the rate of fatty acid release, the free fatty acid concentration of each sample can be plotted against incubation time and the degradation can be extracted from the slope of the linear regression.

[0304] Example 4 Assays according to the invention An exemplary method, including sample preparation and its measurement, for performing an Elecsys-based Polysorbase Activity (EPA) assay according to the present invention is provided in this Example.

[0305] The EPA assay determines the hydrolase / hydrolase activity (hydrolytic activity) in a sample. The source or matrix of the sample does not affect EPA. Therefore, samples from a variety of different purification stages (from HCCF to UFDF pools) can be analyzed.

[0306] The artificial hydrolase substrate according to the invention (FIG. 4) was used in this example, (1) a biotin moiety as R1 attached via an ester bond, the biotin moiety being susceptible to cleavage by hydrolytic enzymes present in the sample; (2) L-locked nucleic acid (LNA) as R2, used to immobilize the cleaved substrate on a solid surface; (3) Digoxigenin as R3, used for detection of immobilized cleavage substrate with ruthenium-labeled anti-digoxigenin antibody. Includes.

[0307] Mix the sample and analysis buffer to obtain a mixture containing final concentrations of 150 mM MOPS, 5 mM MgCl, 6 µM BSA, 25 mM NaCl, 0.1% (w / v) Triton X-100 and 0.1-250 mg / mL protein at a pH value of 7. Incubate this reaction mixture at 37 °C for the respective time, i.e., 1, 3, 5, 7, 22 or 48 h.

[0308] Standard EPA reactions are performed in duplicate incubating the substrate with the samples to be measured (antibody solution and buffer control) at various time points (up to 48 hours).

[0309] In the next step, the digested biotin moieties together with the undigested substrate are removed from the reaction mixture using magnetic streptavidin beads (Cytiva, art. no. 28985799) according to the manufacturer's instructions.

[0310] The corresponding replicates are mixed (pooled) and the content of released alcohol moieties of the artificial hydrolase substrate is determined using L-LNA capture and Digoxygenin determination (as readout) on a Cobas e411 analyzer according to the manufacturer's instructions. The readout is the amount of hydrolysate detected (pg) per ml of sample.

[0311] The amount of hydrolysate (pg / ml) detected in the corresponding sample is corrected for the amount of hydrolysate (pg / ml) detected in a blank sample (e.g., using water instead of a protein sample) according to equation (3). Blank correction value = hydrolysate detected in sample (pg / ml) - hydrolysate detected in blank (pg / ml) (3)

[0312] The blank-corrected values ​​for each sample are normalized to its protein concentration to give a final readout expressed in pg of hydrolysate detected per mg of protein according to equation (4). Normalized hydrolytic activity (pg / mg) = blank-corrected value (pg / mL) * 1000 mL / µg protein per well (4)

[0313] To calculate the rate at which the hydrolases present in the sample digest the artificial hydrolase substrate, the hydrolysis rate is determined and expressed as pg of hydrolysate detected per mg of protein per hour according to formula (5): Hydrolysis rate (pg / mg / h) = normalized activity (pg / mg) / incubation time (h) (5)

Claims

1. 1. A method for determining hydrolytic enzyme activity in a sample, comprising: (a) incubating the sample or an aliquot thereof with a buffer solution to form an incubated sample; the buffer solution comprises a buffer substance, an artificial hydrolase substrate, a salt, and bovine serum albumin; the sample comprises a therapeutic polypeptide; The artificial hydrolase substrate is a carboxylic acid residue attached to a first label, and an alcohol residue attached to the nucleic acid tag and to a second label different from the first label; containing an ester bond covalently linking the step of cleaving the ester bond in the artificial hydrolase substrate into a free alcohol residue and a free carboxylic acid residue if hydrolase activity and / or hydrolase is present in the sample; (b) removing free carboxylic acid residues and artificial hydrolase substrates from the incubated sample to obtain a depleted sample, the step of removing by contacting the incubated sample with a first solid phase that specifically binds the first label, and then separating the incubation mixture, depleted of free carboxylic acids and artificial hydrolase substrates, from the first solid phase; (c) determining the hydrolase activity and / or the presence of hydrolases in the sample by determining the second label in the depleted sample, the step of determining by capturing the free alcohol residue on a second solid phase via the nucleic acid tag and subsequently detecting the free alcohol residue captured on the solid phase with an antibody that specifically binds to the second label, the antibody being bound to one or more detectable labels; The method comprising:

2. The method of claim 1 , wherein the hydrolytic enzyme is an esterase.

3. The method of any one of claims 1 to 2, wherein the detection is by electrochemiluminescence immunoassay and the second label is a ruthenium label.

4. 3. The method of claim 1, wherein the detection is by an antibody that specifically binds to the second label, the antibody being bound to one or more ruthenium labels.

5. The method of claim 3, wherein the ruthenium label is a detectable ruthenium label.

6. The method of any one of claims 1 to 2, wherein the first label and the second label are independently selected from a group of specific binding pairs.

7. The method of any one of claims 1 to 2, wherein the nucleic acid tag is a locked nucleic acid.

8. The method according to any one of claims 1 to 2, wherein the nucleic acid tag has a sequence of TGGTTG in the 3' to 5' direction.

9. The alcohol of the artificial hydrolase substrate and the carboxylic acid of the artificial hydrolase substrate are each independently one or more ethylene oxide (CH ２ -CH ２ 3. The method of claim 1, wherein the hydroxyl group comprises a hydroxyl group, ...

10. The method according to any one of claims 1 to 2, wherein the buffer solution has a pH value of about 7.

11. The method of any one of claims 1 to 2, wherein the incubation is carried out at a temperature of about 37°C.

12. The method of any one of claims 1 to 2, wherein the incubation is for up to 22 hours.

13. The method of any one of claims 1 to 2, wherein the incubation mixture comprises bovine serum albumin at a final concentration of 3 μM to 10 μM.

14. The incubation mixture contained a final concentration of about 150 mM MOPS, about 5 mM MgCl ２ 3. The method according to claim 1, wherein the solution contains about 25 mM NaCl and about 6 μM Cys34 oxidized bovine serum albumin.

15. The method of any one of claims 1 to 2, wherein the first label is biotin or a variant thereof, and the second label is digoxigenin or a variant thereof.

16. The method of any one of claims 1 to 2, wherein the incubation mixture further comprises a surfactant at a final concentration of about 0.05% to 0.15% (w / v).

17. 17. The method of claim 16, wherein the surfactant is octoxynol-9.

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