Method for preparing antibodies

Abstract

The present application belongs to the field of biomedicine, and specifically relates to a method for preparing an antibody, comprising adding cobalt chloride, ammonium heptamolybdate, nickel chloride and ammonium metavanadate to a cell culture for producing the antibody.

Description

Technical Field

[0001] The present application belongs to the field of biomedicine, and specifically relates to a method for preparing antibodies. Background Art

[0002] Glycosylation is an important post-translational modification of therapeutic monoclonal antibodies. The two most common forms are O-glycosylation (oligosaccharides are linked to hydroxyl-containing amino acids such as Ser, Thr or Tyr) and N-glycosylation (oligosaccharides are linked to Asn-X-Ser / Thr, where X is any amino acid except Pro). N-glycosylation can affect the physicochemical properties of monoclonal antibodies, including protein folding, solubility, binding, stability, immunogenicity and pharmacokinetics. The efficacy of therapeutic monoclonal antibodies may depend on complement-dependent cytotoxicity (CDC) activity and / or antibody-dependent cell-mediated cytotoxicity (ADCC) activity. Increasing the level of antibody galactosylation can increase the CDC activity of antibodies. In the development of antibody drugs, the distribution of glycoforms is generally regarded as one of the critical quality attributes (CQA) of the product, and the distribution of glycoforms needs to be strictly controlled.

[0003] A common way to regulate antibody galactosylation is to add manganese, galactose or uridine to optimize enzyme activity, glycosylation substrates and sugar carriers, respectively, ultimately increasing the galactosylation level of antibodies. However, there is still a need to further increase the galactosylation level of antibodies. Summary of the invention

[0004] In one aspect, the present application provides a method for preparing an antibody, comprising adding cobalt chloride, ammonium heptamolybdate, nickel chloride and / or ammonium metavanadate to a cell culture producing the antibody.

[0005] In some embodiments, the amount of cobalt chloride added is 1-40 μmol / L. In some embodiments, the amount of cobalt chloride added is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L, 25 μmol / L, 30 μmol / L, 35 μmol / L, 40 μmol / L or the range formed by any of the foregoing values. In some embodiments, the amount of cobalt chloride added is 5-40 μmol / L, 5-35 μmol / L or 6-30 μmol / L. In some embodiments, the amount of cobalt chloride added is 6-30 μmol / L. In some specific embodiments, the amount of cobalt chloride added is 6 μmol / L, 8 μmol / L or 30 μmol / L.

[0006] In some embodiments, the amount of ammonium heptamolybdate added is 0.1-0.15 μmol / L. In some embodiments, the amount of ammonium heptamolybdate added is 0.1 μmol / L, 0.105 μmol / L, 0.11 μmol / L, 0.115 μmol / L, 0.12 μmol / L, 0.125 μmol / L, 0.13 μmol / L, 0.135 μmol / L, 0.14 μmol / L, 0.145 μmol / L, 0.15 μmol / L or the range formed by any of the foregoing values. In some embodiments, the amount of ammonium heptamolybdate added is 0.105-0.15 μmol / L, 0.105-0.145 μmol / L or 0.105-0.14 μmol / L. In some embodiments, the amount of ammonium heptamolybdate added is 0.105-0.14 μmol / L. In some specific embodiments, the added amount of ammonium heptamolybdate is 0.105 μmol / L or 0.14 μmol / L.

[0007] In some embodiments, the nickel chloride is added in an amount of 0.05-0.5 μmol / L. In some embodiments, the nickel chloride is added in an amount of 0.05 μmol / L, 0.1 μmol / L, 0.15 μmol / L, 0.2 μmol / L, 0.25 μmol / L, 0.3 μmol / L, 0.35 μmol / L, 0.4 μmol / L, 0.45 μmol / L, 0.5 μmol / L or the range formed by any of the foregoing values. In some embodiments, the nickel chloride is added in an amount of 0.1-0.4 μmol / L, 0.15-0.3 μmol / L or 0.15-0.2 μmol / L. In some embodiments, the nickel chloride is added in an amount of 0.15-0.2 μmol / L. In some specific embodiments, the nickel chloride is added in an amount of 0.15 μmol / L or 0.2 μmol / L.

[0008] In some embodiments, the amount of ammonium metavanadate added is 25-35 μmol / L. In some embodiments, the amount of ammonium metavanadate added is 25 μmol / L, 25.5 μmol / L, 26 μmol / L, 26.5 μmol / L, 27 μmol / L, 27.5 μmol / L, 28 μmol / L, 28.5 μmol / L, 29 μmol / L, 29.5 μmol / L, 30 μmol / L, 30.5 μmol / L, 31 μmol / L, 31.5 μmol / L, 32 μmol / L, 32.5 μmol / L, 33 μmol / L, 33.5 μmol / L, 34 μmol / L, 34.5 μmol / L, 35 μmol / L or a range formed by any of the foregoing values. In some embodiments, the amount of ammonium metavanadate added is 25-32.5 μmol / L, 27.5-32.5 μmol / L or 30-32.5 μmol / L. In some embodiments, the amount of ammonium metavanadate added is 30-32.5 μmol / L. In some specific embodiments, the amount of ammonium metavanadate added is 30 μmol / L or 32.5 μmol / L.

[0009] In some embodiments, the amount of cobalt chloride added is 6-30 μmol / L, the amount of ammonium heptamolybdate added is 0.105-0.14 μmol / L, the amount of nickel chloride added is 0.15-0.2 μmol / L, and the amount of ammonium metavanadate added is 30-32.5 μmol / L.

[0010] In some embodiments, the amount of cobalt chloride added is 6 μmol / L, 8 μmol / L or 30 μmol / L, the amount of ammonium heptamolybdate added is 0.105 μmol / L or 0.14 μmol / L, the amount of nickel chloride added is 0.15 μmol / L or 0.2 μmol / L, and the amount of ammonium metavanadate added is 30 μmol / L or 32.5 μmol / L.

[0011] In some specific embodiments, the amount of cobalt chloride added is 8 μmol / L, the amount of ammonium heptamolybdate added is 0.14 μmol / L, the amount of nickel chloride added is 0.2 μmol / L, and the amount of ammonium metavanadate added is 30 μmol / L.

[0012] In some specific embodiments, the amount of cobalt chloride added is 6 μmol / L, the amount of ammonium heptamolybdate added is 0.105 μmol / L, the amount of nickel chloride added is 0.15 μmol / L, and the amount of ammonium metavanadate added is 32.5 μmol / L.

[0013] In some specific embodiments, the amount of cobalt chloride added is 30 μmol / L, the amount of ammonium heptamolybdate added is 0.14 μmol / L, the amount of nickel chloride added is 0.2 μmol / L, and the amount of ammonium metavanadate added is 30 μmol / L.

[0014] In some specific embodiments, cobalt chloride, ammonium heptamolybdate, nickel chloride and / or ammonium metavanadate are added on the initiation day of the cell culture cycle.

[0015] In some embodiments, the method further comprises adding manganese chloride.

[0016] In some embodiments, the amount of manganese chloride added is 1-20 μmol / L. In some embodiments, the amount of manganese chloride added is 1 μmol / L, 2 μmol / L, 3 μmol / L, 4 μmol / L, 5 μmol / L, 6 μmol / L, 7 μmol / L, 8 μmol / L, 9 μmol / L, 10 μmol / L, 15 μmol / L, 20 μmol / L or the range formed by any of the foregoing values. In some embodiments, the amount of manganese chloride added is 1-15 μmol / L or 1-10 μmol / L. In some embodiments, the amount of manganese chloride added is 1-10 μmol / L. In some specific embodiments, the amount of manganese chloride added is 1 μmol / L, 5 μmol / L or 10 μmol / L.

[0017] In some embodiments, manganese chloride is added on the initiation day of the cell culture cycle.

[0018] In some embodiments, the method further comprises adding cysteine ​​and / or cystine.

[0019] In some embodiments, the amount of cysteine ​​added is 0.1%-5%. In some embodiments, the amount of cysteine ​​added is 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or the range formed by any of the foregoing values. In some embodiments, the amount of cysteine ​​added is 0.1%-2%, 0.5%-2% or 0.5%-1%. In some embodiments, the amount of cysteine ​​added is 0.5%-1%. In some specific embodiments, the amount of cysteine ​​added is 0.5%.

[0020] In some embodiments, cysteine ​​is added during cell culture.

[0021] In some embodiments, the amount of cystine added is 0.1%-5%. In some embodiments, the amount of cystine added is 0.1%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5% or the range formed by any of the foregoing values. In some embodiments, the amount of cystine added is 0.1%-2%, 0.5%-2% or 0.5%-1%. In some embodiments, the amount of cystine added is 0.5%-1%. In some specific embodiments, the amount of cystine added is 0.5%.

[0022] In some embodiments, cystine is added during cell culturing.

[0023] In some embodiments, the method comprises adding cobalt chloride, ammonium heptamolybdate, nickel chloride, ammonium metavanadate, and manganese chloride on the starting day of the culture cycle of the antibody-producing cells, and adding cysteine ​​or cystine during the cell culture.

[0024] In some embodiments, the method comprises adding 6-30 μmol / L cobalt chloride, 0.105-0.14 μmol / L ammonium heptamolybdate, 0.15-0.2 μmol / L nickel chloride, 30-32.5 μmol / L ammonium metavanadate, and 1-10 μmol / L manganese chloride on the starting day of the culture cycle of the antibody-producing cells, and adding 0.5%-1% cysteine ​​or cystine during the cell culture process.

[0025] In some specific embodiments, the method comprises adding 30 μmol / L cobalt chloride, 0.14 μmol / L ammonium heptamolybdate, 0.2 μmol / L nickel chloride, 30 μmol / L ammonium metavanadate, and 10 μmol / L manganese chloride on the starting day of the culture cycle of the antibody-producing cells, and adding 0.5% cystine during the cell culture process.

[0026] In some specific embodiments, said cell culture does not contain ammonium ferric citrate.

[0027] On the other hand, the present application provides a method for culturing antibody-producing cells, the method comprising adding cobalt chloride, ammonium heptamolybdate, nickel chloride, ammonium metavanadate and manganese chloride to the cell culture on the starting day of the cell culture cycle, and optionally adding cysteine ​​or cystine during the cell culture process.

[0028] In some embodiments, the method comprises adding 6-30 μmol / L cobalt chloride, 0.105-0.14 μmol / L ammonium heptamolybdate, 0.15-0.2 μmol / L nickel chloride, and 30-32.5 μmol / L ammonium metavanadate to the cell culture on the starting day of the cell culture cycle, optionally adding 1-10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5%-1% cysteine ​​or cystine during the cell culture process.

[0029] In some embodiments, the method comprises adding 8 μmol / L cobalt chloride, 0.14 μmol / L ammonium heptamolybdate, 0.2 μmol / L nickel chloride, and 30 μmol / L ammonium metavanadate to the cell culture on the starting day of the cell culture cycle, optionally adding 10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5% cysteine ​​or cystine during the cell culture process.

[0030] In some embodiments, the method comprises adding 6 μmol / L cobalt chloride, 0.105 μmol / L ammonium heptamolybdate, 0.15 μmol / L nickel chloride, and 32.5 μmol / L ammonium metavanadate to the cell culture on the starting day of the cell culture cycle, optionally adding 10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5% cysteine ​​or cystine during the cell culture process.

[0031] In some embodiments, the method comprises adding 30 μmol / L cobalt chloride, 0.14 μmol / L ammonium heptamolybdate, 0.2 μmol / L nickel chloride, and 30 μmol / L ammonium metavanadate to the cell culture on the starting day of the cell culture cycle, optionally adding 10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5% cysteine ​​or cystine during the cell culture process.

[0032] In some specific embodiments, the method comprises adding 30 μmol / L cobalt chloride, 0.14 μmol / L ammonium heptamolybdate, 0.2 μmol / L nickel chloride, 30 μmol / L ammonium metavanadate, and 10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5% cystine during the cell culture process.

[0033] The amounts of cobalt chloride, ammonium heptamolybdate, nickel chloride, ammonium metavanadate or manganese chloride added are all amounts of the final concentration after being added to the cell culture.

[0034] In some specific embodiments, said cell culture does not contain ammonium ferric citrate.

[0035] Antibody

[0036] In some embodiments, the antibody is penampalimab, bemosubinib, daratumumab, mepolizumab, ramucirumab, pertuzumab, guselkumab, rituximab, trastuzumab, adalimumab, bevacizumab, epezetuzumab, obinutuzumab, pembrolizumab, lomosozumab, alirocumab, bimegizumab, denosumab, elotuzumab, nivolumab, secukinumab, ustekinumab, belimumab, nemolizumab, lencanezumab, rozelixizumab, faricizumab, or atezolizumab.

[0037] In some embodiments, the antibody is an anti-ROR1 antibody, an anti-Claudin6 antibody, a GIPR antibody / GLP-1 fusion protein, or an anti-EpCAM×CD3 bispecific antibody.

[0038] In some embodiments, the antibody is an anti-TIM-3 antibody or an anti-LAG-3 antibody.

[0039] Table S1. CDR sequences of anti-TIM-3 antibodies

[0040]

[0041] In some embodiments, the anti-TIM-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 1, HCDR2 shown in the amino acid sequence of SEQ ID NO: 2, HCDR3 shown in the amino acid sequence of SEQ ID NO: 3, LCDR1 shown in the amino acid sequence of SEQ ID NO: 4, LCDR2 shown in the amino acid sequence of SEQ ID NO: 5, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 6.

[0042] In some embodiments, the anti-TIM-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 7, HCDR2 shown in the amino acid sequence of SEQ ID NO: 8, HCDR3 shown in the amino acid sequence of SEQ ID NO: 9, LCDR1 shown in the amino acid sequence of SEQ ID NO: 10, LCDR2 shown in the amino acid sequence of SEQ ID NO: 11, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 12.

[0043] In some embodiments, the anti-TIM-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 13 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 14.

[0044] In some embodiments, the anti-TIM-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 15 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 16.

[0045] In some embodiments, the anti-TIM-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 17 and a light chain shown in the amino acid sequence of SEQ ID NO: 18.

[0046] In some embodiments, the anti-TIM-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 19 and a light chain shown in the amino acid sequence of SEQ ID NO: 20.

[0047] Table S2. CDR sequences of anti-LAG-3 antibodies

[0048]

[0049] In some embodiments, the anti-LAG-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 21, HCDR2 shown in the amino acid sequence of SEQ ID NO: 22, HCDR3 shown in the amino acid sequence of SEQ ID NO: 23, LCDR1 shown in the amino acid sequence of SEQ ID NO: 24, LCDR2 shown in the amino acid sequence of SEQ ID NO: 25, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 26.

[0050] In some embodiments, the anti-LAG-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 27, HCDR2 shown in the amino acid sequence of SEQ ID NO: 28, HCDR3 shown in the amino acid sequence of SEQ ID NO: 29, LCDR1 shown in the amino acid sequence of SEQ ID NO: 30, LCDR2 shown in the amino acid sequence of SEQ ID NO: 31, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 32.

[0051] In some embodiments, the anti-LAG-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 33 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 34.

[0052] In some embodiments, the anti-LAG-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 35 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 36.

[0053] In some embodiments, the anti-LAG-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 37 and a light chain shown in the amino acid sequence of SEQ ID NO: 38.

[0054] In some embodiments, the anti-LAG-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 39 and a light chain shown in the amino acid sequence of SEQ ID NO: 40.

[0055] It will be understood by those skilled in the art that, unless otherwise specified, the term "CDR" or "complementarity determining region" of a given antibody or region thereof (e.g., variable region) should be understood to encompass complementarity determining regions defined by any known scheme. Although Tables S1-S2 have shown CDR sequences, however, when referring to antibodies defined by specific CDR sequences disclosed herein, the scope of the antibodies also encompasses antibodies defined by CDR sequences converted to other arbitrary numbering system definitions (e.g., a combination of one or more of the definitions of Kabat, Chothia, CCG, IMGT or Contact, etc., which are well known in the art).

[0056] cell

[0057] The method of the present application can be used to cultivate a variety of cells. In some embodiments, the cultured cells are eukaryotic cells, such as plant and / or animal cells. The animal cells can be mammalian cells, fish cells, insect cells, amphibian cells or bird cells. A variety of mammalian cell lines suitable for cultivation can be obtained from depository institutions and commercial suppliers. Cells that can be used in the method of the present application include but are not limited to Chinese hamster ovary cells (CHO), CHO-S cells, CHO-DG44 cells, or any other cell type known to those skilled in the art.

[0058] Culture medium

[0059] In some embodiments, the basal medium for cell culture may include serum-free and / or animal-free products or components. As will be appreciated by those skilled in the art, animal or mammalian cells may be cultured in a defined medium without undue experimentation, provided that it is suitable for the specific cells being cultured. Commercial culture media may be used, including but not limited to: CD OptiCHO, Hycell, CD CHO, GrowthA, Dynamis, Iscov's Modified Dulbecco's Medium, RPMI 1640, Minimum Essential Medium-α (MEM-α), Dulbecco's Modified Eagle's Medium (DMEM), DME / F12, Earle's Basal Medium with Earle's BSS, High Glucose DMEM with Glutamine, High Glucose DMEM without Glutamine, Low Glucose DMEM without Glutamine, Glasgow's MEM, GMEM with Glutamine, Ham's F-10 with Glutamine, Ham's with Glutamine F-12, IMDM with HEPES and glutamine, IMDM with HEPES and without glutamine, McCoy's Modified Medium, Medium 199, Eagle's MEM-Earle's BSS with glutamine, Eagle's MEM-Earle's BSS without glutamine, Eagle's MEM-Hanks BSS without glutamine, NCTC-109 with glutamine, Richter's CM with glutamine, RPMI 1640 with HEPES, glutamine and / or penicillin-streptomycin, RPMI 1640 with glutamine, RPMI 1640 without glutamine, or any other medium known to those of skill in the art formulated for a particular cell type. Supplemental components or ingredients, including optional components, may be added to the above exemplary media at appropriate concentrations or amounts as needed or as desired and as known and practiced by those of skill in the art using routine skills.

[0060] In some embodiments, the feed medium of the specific nutrient that is difficult to prepare or consumes rapidly in the cell culture can also be supplemented in the cell culture. Such nutrients can be amino acids, such as tyrosine, cysteine ​​and / or cystine. For example, concentrated tyrosine solution can be fed independently to the cell culture grown in the cell culture medium containing tyrosine. Concentrated solutions of tyrosine and cystine can also be fed independently to the cell culture grown in the cell culture medium lacking tyrosine, cystine and / or cysteine.

[0061] The antibody preparation method and the antibody-producing cell culture method provided in the present application have high process controllability, significantly improve the expression amount and galactosylation level of the antibody, and can improve production efficiency, reduce production costs, and optimize product quality. DETAILED DESCRIPTION

[0062] the term

[0063] Unless otherwise specified, the following terms used in this application have the following meanings. A particular term should not be considered to be uncertain or unclear in the absence of a special definition, but should be understood according to the common meaning in the art. When a trade name appears in this application, it is intended to refer to the corresponding commodity or its active ingredient.

[0064] The term "antibody" is used in the broadest sense to cover natural antibodies and artificial antibodies of various structures, including but not limited to monoclonal antibodies, polyclonal antibodies, monospecific antibodies, multispecific antibodies (e.g., bispecific antibodies, trispecific antibodies), and single-chain antibodies, as long as they show the desired antigen-binding activity.

[0065] The term "variable domain" or "variable region" refers to the domain of an antibody that is involved in binding the antibody to an antigen. For example, a natural four-chain antibody (e.g., derived from humans, mice, etc.) has a heavy chain variable region (also referred to as a heavy chain variable domain, VH or VH domain) and a light chain variable region (also referred to as a light chain variable domain, VL or VL domain). In most cases, each variable domain of a natural antibody is essentially composed of four "framework regions (FR)" and three "complementarity determining regions (CDR)". The four framework regions are respectively referred to as framework region 1 (or FR1), framework region 2 (or FR2), framework region 3 (or FR3), and framework region 4 (or FR4); the framework region is separated by three complementary determining regions, which are respectively referred to as complementary determining region 1 (or CDR1), complementary determining region 2 (or CDR2), and complementary determining region 3 (or CDR3) in the art and hereinafter. Therefore, the general structure of the variable domain can be expressed as follows: FR1-CDR1-FR2-CDR2-FR3-CDR3-FR4. The variable domain confers specificity to the antibody for the antigen by providing the antigen binding site.

[0066] The term "complementarity determining region" (CDR), also known as "hypervariable region" (HVR). A natural four-chain antibody usually contains six CDRs, three in the heavy chain variable region, namely heavy chain CDR1 (HCDR1), heavy chain CDR2 (HCDR2) and heavy chain CDR3 (HCDR3); three in the light chain variable region, namely light chain CDR1 (LCDR1), light chain CDR2 (LCDR2) and light chain CDR3 (LCDR3).

[0067] There are many methods to divide and define CDR at present. Among them, Kabat definition divides CDR based on sequence variability, and is the most commonly used (Elvin A.Kabat, et al, Sequences of Proteins of Immunological Interest, 5th edition, Public Health Service, National Institutes of Health, Bethesda, Md. (1991)); and Chothia definition is based on the position of structural loop (Cyrus Chothia, et al, Canonical Structures for the Hypervariable Regions of Immunoglobulins, J.Mol. Biol. 196:901-917 (1987)). AbM definition is a compromise between Kabat definition and Chothia definition, and is used by AbM antibody modeling software of Oxford Molecular. The basis of "contact" definition CDR is the analysis of available complex crystal structure. In addition, there are definitions such as IMGT and CCG. However, it should be noted that the boundaries of the CDRs of the same antibody variable region obtained by different division definitions may be different, that is, the CDR sequences of the same antibody variable region divided and defined by different methods may be different. Therefore, when it comes to defining antibodies by specific CDR sequences, the scope of the antibodies also covers antibodies defined by CDR sequences of any other definitions (e.g., a combination of one or more of the definitions of Kabat, IMGT, Chothia, Contact, AbM, CCG, etc.).

[0068] The term "culture cycle" refers to the cycle from cell inoculation to shake flask culture, "D0" refers to the starting day of the culture cycle, which is day 0 of the culture cycle, "D5" refers to day 5 of the culture cycle, and so on.

[0069] The terms "include" or "comprising" should be construed in an open, non-exclusive sense, ie, "including, but not limited to."

[0070] As used herein, "about" means within the acceptable error range for a particular value as determined by one of ordinary skill in the art, which depends in part on how the value is measured or determined, i.e., the limitations of the measurement system. For example, "about" may mean within 1 or more than 1 standard deviation according to the practice in the art. Alternatively, "about" may mean a range of up to ±5%, such as fluctuations within ±2%, within ±1%, or within ±0.5% of a given specific numerical range. When a specific value is given in the application or claims, unless otherwise stated, the meaning of "about" should be considered to be within the acceptable error range for that specific value. In this article, unless otherwise stated, all values ​​involving dosages, times, step parameters or conditions are modified by "about" by default.

[0071] Example

[0072] Although the foregoing disclosure has been described in detail by way of illustration and example for purposes of clear understanding, it will be apparent to one of ordinary skill in the art, based on the teachings of the present disclosure, that certain changes and modifications may be made to the disclosure without departing from the spirit and scope of the appended claims. The following examples are provided by way of illustration only and are not intended to be limiting. One of ordinary skill in the art will readily recognize a variety of non-critical parameters that may be changed or modified to produce substantially similar results.

[0073] Unless otherwise specified, all reagents used in the examples can be purchased from commercial sources.

[0074] The present application also provides the following specific implementation schemes, but is not limited thereto:

[0075] Embodiment 1. A method for preparing an antibody, characterized in that the method comprises adding cobalt chloride, ammonium heptamolybdate, nickel chloride and ammonium metavanadate to a cell culture for producing the antibody.

[0076] Embodiment 2. The method according to Embodiment 1 is characterized in that the amount of cobalt chloride added is 6-30 μmol / L.

[0077] Embodiment 3. The method according to any one of Embodiments 1-2 is characterized in that the amount of cobalt chloride added is 6 μmol / L, 8 μmol / L or 30 μmol / L.

[0078] Embodiment 4. The method according to any one of Embodiments 1-3 is characterized in that the addition amount of ammonium heptamolybdate is 0.105-0.14 μmol / L.

[0079] Embodiment 5. The method according to any one of Embodiments 1 to 4 is characterized in that the addition amount of ammonium heptamolybdate is 0.105 μmol / L or 0.14 μmol / L.

[0080] Embodiment 6. The method according to any one of Embodiments 1-5 is characterized in that the amount of nickel chloride added is 0.15-0.2 μmol / L.

[0081] Embodiment 7. The method according to any one of Embodiments 1-6 is characterized in that the amount of nickel chloride added is 0.15 μmol / L or 0.2 μmol / L.

[0082] Embodiment 8. The method according to any one of Embodiments 1-7 is characterized in that the amount of ammonium metavanadate added is 30-32.5 μmol / L.

[0083] Embodiment 9. The method according to any one of Embodiments 1 to 8 is characterized in that the addition amount of ammonium metavanadate is 30 μmol / L or 32.5 μmol / L.

[0084] Embodiment 10. The method according to any one of Embodiments 1-9 is characterized in that the amount of cobalt chloride added is 6-30 μmol / L, the amount of ammonium heptamolybdate added is 0.105-0.14 μmol / L, the amount of nickel chloride added is 0.15-0.2 μmol / L, and the amount of ammonium metavanadate added is 30-32.5 μmol / L.

[0085] Embodiment 11. The method according to any one of Embodiments 1-10 is characterized in that the amount of cobalt chloride added is 6 μmol / L, 8 μmol / L or 30 μmol / L, the amount of ammonium heptamolybdate added is 0.105 μmol / L or 0.14 μmol / L, the amount of nickel chloride added is 0.15 μmol / L or 0.2 μmol / L, and the amount of ammonium metavanadate added is 30 μmol / L or 32.5 μmol / L.

[0086] Embodiment 12. The method according to any one of Embodiments 1-11 is characterized in that the amount of cobalt chloride added is 8 μmol / L, the amount of ammonium heptamolybdate added is 0.14 μmol / L, the amount of nickel chloride added is 0.2 μmol / L, and the amount of ammonium metavanadate added is 30 μmol / L.

[0087] Embodiment 13. The method according to any one of Embodiments 1-11 is characterized in that the amount of cobalt chloride added is 6 μmol / L, the amount of ammonium heptamolybdate added is 0.105 μmol / L, the amount of nickel chloride added is 0.15 μmol / L, and the amount of ammonium metavanadate added is 32.5 μmol / L.

[0088] Embodiment 14. The method according to any one of Embodiments 1-11 is characterized in that the amount of cobalt chloride added is 30 μmol / L, the amount of ammonium heptamolybdate added is 0.14 μmol / L, the amount of nickel chloride added is 0.2 μmol / L, and the amount of ammonium metavanadate added is 30 μmol / L.

[0089] Embodiment 15. The method according to any one of Embodiments 1 to 14, characterized in that the cobalt chloride, ammonium heptamolybdate, nickel chloride and ammonium metavanadate are added on the starting day of the cell culture cycle.

[0090] Embodiment 16. The method according to any one of Embodiments 1-15, characterized in that the method further comprises adding manganese chloride.

[0091] Embodiment 17. The method according to Embodiment 16 is characterized in that the amount of manganese chloride added is 1-10 μmol / L.

[0092] Embodiment 18. The method according to Embodiment 16 or 17 is characterized in that the added amount of manganese chloride is 1 μmol / L, 5 μmol / L or 10 μmol / L.

[0093] Embodiment 19. The method according to any one of Embodiments 16-18 is characterized in that the manganese chloride is added on the starting day of the cell culture cycle.

[0094] Embodiment 20. The method according to any one of embodiments 1-19, characterized in that the method further comprises adding cysteine ​​and / or cystine.

[0095] Embodiment 21. The method according to Embodiment 20 is characterized in that the added amount of cysteine ​​or cystine is 0.5%-1%.

[0096] Embodiment 22. The method according to Embodiment 20 or 21 is characterized in that the added amount of cysteine ​​or cystine is 0.5%.

[0097] Embodiment 23. The method according to any one of Embodiments 20-22 is characterized in that the cysteine ​​or cystine is added during the cell culture process.

[0098] Embodiment 24. The method according to any one of Embodiments 1-23 is characterized in that the method includes adding 6-30 μmol / L cobalt chloride, 0.105-0.14 μmol / L ammonium heptamolybdate, 0.15-0.2 μmol / L nickel chloride, 30-32.5 μmol / L ammonium metavanadate and 1-10 μmol / L manganese chloride on the starting day of the culture cycle of the antibody-producing cells, and adding 0.5%-1% cysteine ​​or cystine during the cell culture process.

[0099] Embodiment 25. The method according to Embodiment 24 is characterized in that the method includes adding 30 μmol / L cobalt chloride, 0.14 μmol / L ammonium heptamolybdate, 0.2 μmol / L nickel chloride, 30 μmol / L ammonium metavanadate and 10 μmol / L manganese chloride on the starting day of the culture cycle of the antibody-producing cells, and adding 0.5% cystine during the cell culture process.

[0100] Embodiment 26. A method for culturing antibody-producing cells, the method comprising adding 6-30 μmol / L cobalt chloride, 0.105-0.14 μmol / L ammonium heptamolybdate, 0.15-0.2 μmol / L nickel chloride and 30-32.5 μmol / L ammonium metavanadate to the cell culture on the starting day of the cell culture cycle, optionally adding 1-10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5%-1% cystine during the cell culture process.

[0101] Embodiment 27. The method according to Embodiment 26 is characterized in that the method includes adding 8 μmol / L cobalt chloride, 0.14 μmol / L ammonium heptamolybdate, 0.2 μmol / L nickel chloride and 30 μmol / L ammonium metavanadate to the cell culture on the starting day of the cell culture cycle, optionally adding 10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5% cystine during the cell culture process.

[0102] Embodiment 28. The method according to Embodiment 26 is characterized in that the method includes adding 6 μmol / L cobalt chloride, 0.105 μmol / L ammonium heptamolybdate, 0.15 μmol / L nickel chloride and 32.5 μmol / L ammonium metavanadate to the cell culture on the starting day of the cell culture cycle, optionally adding 10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5% cystine during the cell culture process.

[0103] Embodiment 29. The method according to Embodiment 26 is characterized in that the method includes adding 30 μmol / L cobalt chloride, 0.14 μmol / L ammonium heptamolybdate, 0.2 μmol / L nickel chloride and 30 μmol / L ammonium metavanadate to the cell culture on the starting day of the cell culture cycle, optionally adding 10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5% cystine during the cell culture process.

[0104] Embodiment 30. The method according to Embodiment 26 is characterized in that the method includes adding 30 μmol / L cobalt chloride, 0.14 μmol / L ammonium heptamolybdate, 0.2 μmol / L nickel chloride, 30 μmol / L ammonium metavanadate and 10 μmol / L manganese chloride to the cell culture on the starting day of the cell culture cycle, and adding 0.5% cystine during the cell culture process.

[0105] Embodiment 31. The method according to any one of Embodiments 1-30 is characterized in that the cell culture does not contain ammonium ferric citrate.

[0106] Implementation Plan 32. The method according to any one of embodiments 1 to 31 is characterized in that the antibody is an anti-TIM-3 antibody, an anti-LAG-3 antibody, an anti-ROR1 antibody, an anti-Claudin6 antibody, a GIPR antibody / GLP-1 fusion protein, an anti-EpCAM×CD3 bispecific antibody, penamprimab, bemosubizumab, daratumumab, mepolizumab, ramucirumab, pertuzumab, guselkumab, rituximab, trastuzumab, adalimumab, bevacizumab, epezetuzumab, obinutuzumab, pembrolizumab, lomosotuzumab, alirocumab, bimegilide, denosumab, evolocumab, nivolumab, secukinumab, ustekinumab, belimumab, nemolimumab, lencanezumab, rozelixizumab, faricizumab or atezolizumab.

[0107] Embodiment 33. The method according to Embodiment 32, characterized in that the anti-TIM-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 1, HCDR2 shown in the amino acid sequence of SEQ ID NO: 2, HCDR3 shown in the amino acid sequence of SEQ ID NO: 3, LCDR1 shown in the amino acid sequence of SEQ ID NO: 4, LCDR2 shown in the amino acid sequence of SEQ ID NO: 5, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 6; or

[0108] The anti-TIM-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 7, HCDR2 shown in the amino acid sequence of SEQ ID NO: 8, HCDR3 shown in the amino acid sequence of SEQ ID NO: 9, LCDR1 shown in the amino acid sequence of SEQ ID NO: 10, LCDR2 shown in the amino acid sequence of SEQ ID NO: 11, and LCDR3 shown in the amino acid sequence of SEQ ID NO: 12.

[0109] Embodiment 34. The method according to Embodiment 32 or 33, characterized in that the anti-TIM-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 13 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 14; or

[0110] The anti-TIM-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 15 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 16.

[0111] Embodiment 35. The method according to any one of Embodiments 32-34, characterized in that the anti-TIM-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 17 and a light chain shown in the amino acid sequence of SEQ ID NO: 18; or

[0112] The anti-TIM-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 19 and a light chain shown in the amino acid sequence of SEQ ID NO: 20.

[0113] Embodiment 36. The method according to Embodiment 32 is characterized in that the anti-LAG-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 21, HCDR2 shown in the amino acid sequence of SEQ ID NO: 22, HCDR3 shown in the amino acid sequence of SEQ ID NO: 23, LCDR1 shown in the amino acid sequence of SEQ ID NO: 24, LCDR2 shown in the amino acid sequence of SEQ ID NO: 25 and LCDR3 shown in the amino acid sequence of SEQ ID NO: 26; or

[0114] The anti-LAG-3 monoclonal antibody comprises HCDR1 shown in the amino acid sequence of SEQ ID NO: 27, HCDR2 shown in the amino acid sequence of SEQ ID NO: 28, HCDR3 shown in the amino acid sequence of SEQ ID NO: 29, LCDR1 shown in the amino acid sequence of SEQ ID NO: 30, LCDR2 shown in the amino acid sequence of SEQ ID NO: 31 and LCDR3 shown in the amino acid sequence of SEQ ID NO: 32.

[0115] Embodiment 37. The method according to Embodiment 32 or 36, characterized in that the anti-LAG-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 33 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 34; or

[0116] The anti-LAG-3 monoclonal antibody comprises a heavy chain variable region shown in the amino acid sequence of SEQ ID NO: 35 and a light chain variable region shown in the amino acid sequence of SEQ ID NO: 36.

[0117] Embodiment 38. The method according to any one of Embodiments 32, 36-37, characterized in that the anti-LAG-3 monoclonal antibody comprises a heavy chain shown in the amino acid sequence of SEQ ID NO: 37 and a light chain shown in the amino acid sequence of SEQ ID NO: 38; or

[0118] The anti-LAG-3 monoclonal antibody comprises a heavy chain represented by the amino acid sequence of SEQ ID NO: 39 and a light chain represented by the amino acid sequence of SEQ ID NO: 40.

[0119] Example 1 Preparation method of anti-ROR1 monoclonal antibody

[0120] Take a cell of the anti-ROR1 monoclonal antibody frozen working cell bank (CHO cells, 1 mL) from the liquid nitrogen tank, thaw it in a 37°C water bath, transfer it to a cell culture shake flask containing basal medium or basal medium supplemented with a final concentration of 8 μmol / L cobalt chloride hexahydrate, 0.14 μmol / L ammonium heptamolybdate tetrahydrate, 0.2 μmol / L nickel chloride hexahydrate, and 30 μmol / L ammonium metavanadate, and place it in a carbon dioxide constant temperature incubator for culture. The cell density is about 1.5-5.0×10 6 cells / mL, and the subculture density is about 0.4-0.9×10 6 cells / ml, and the shaking flask culture conditions were: 37.0℃, 5% CO2, 130 rpm. The culture medium with cells in logarithmic growth phase and good condition was used as seed medium.

[0121] The seed solution was inoculated into the bioreactor (Sartorius, Ambr ® 15) were cultured in basal medium or basal medium supplemented with a final concentration of 6 μmol / L cobalt chloride hexahydrate, 0.105 μmol / L ammonium heptamolybdate tetrahydrate, 0.15 μmol / L nickel chloride hexahydrate, and 32.5 μmol / L ammonium metavanadate; the temperature was initially set at 37.0°C, and when the cell density reached 10×10 6 cells / mL, the temperature was adjusted to 33.0℃; the speed was set to 900 rpm; the DO-related O2 automatic control was set to 40%; the pH value-related CO2 automatic control was initially set to 7.15±0.35, and the pH was adjusted to 7.00±0.30 after cooling; the air sparger was continuously turned on, and the ventilation volume was 0.10 mL / min; the culture period was 16 days. After 16 days of culture, the cells were harvested, and the antibodies were purified after centrifugation, and the protein expression and glycoforms were determined.

[0122] High performance liquid chromatography was used to detect the protein at a wavelength of 280 nm and calculate the protein expression.

[0123] The samples were digested with IdeS enzyme and separated by a reverse phase chromatography hydrophobic interaction chromatography column. The molecular weights of different glycoform modifications were detected by liquid chromatography-mass spectrometry, and the content of each glycoform was calculated.

[0124] The results are shown in Table 1. In the cell culture producing the antibody, the addition of cobalt chloride hexahydrate, ammonium heptamolybdate tetrahydrate, nickel chloride hexahydrate, and ammonium metavanadate increased the protein expression by 213.94%, and the protein FA2G1 (monogalactoglycan) and FA2G2 (digalactoglycan) by 53.86% and 121.74%, respectively.

[0125] Table 1 Glycoforms of anti-ROR1 monoclonal antibodies

[0126] Culture medium Expression (g / L) FA2G1(%) FA2G2(%) Basic culture medium 2.08 8.04 0.46 Basic culture medium + cobalt chloride hexahydrate + ammonium heptamolybdate tetrahydrate + nickel chloride hexahydrate + ammonium metavanadate 6.53 12.37 1.02

[0127] Example 2 Preparation method of anti-Claudin6 monoclonal antibody

[0128] Take a cell of the anti-Claudin6 monoclonal antibody frozen working cell bank (CHO cells, 1 mL) from the liquid nitrogen tank, thaw it in a 37°C water bath, transfer it to a cell culture shake flask containing basal medium or basal medium supplemented with a final concentration of 8 μmol / L cobalt chloride hexahydrate, 0.14 μmol / L ammonium heptamolybdate tetrahydrate, 0.2 μmol / L nickel chloride hexahydrate, and 30 μmol / L ammonium metavanadate, and place it in a carbon dioxide constant temperature incubator for culture. The cell density is about 1.5-6.0×10 6cells / mL, and the subculture density is about 0.2-0.8×10 6 cells / ml, and the shaking flask culture conditions were: 37.0℃, 5% CO2, 130rpm. The culture medium with cells in logarithmic growth phase and good condition was used as seed medium.

[0129] The seed solution was inoculated into a shake flask and cultured in a basal medium or a basal medium supplemented with a final concentration of 8 μmol / L cobalt chloride hexahydrate, 0.14 μmol / L ammonium heptamolybdate tetrahydrate, 0.2 μmol / L nickel chloride hexahydrate, and 30 μmol / L ammonium metavanadate. The temperature was initially set at 37.0°C. When the cell density reached 9-12×10 6 cells / mL, the temperature was adjusted to 33.0℃; the rotation speed was set to 130 rpm; the culture period was 13 days. After 13 days of culture, the cells were harvested, centrifuged and antibody purification was performed, and the protein expression and glycoforms were determined.

[0130] High performance liquid chromatography was used to detect the protein at a wavelength of 280 nm and calculate the protein expression.

[0131] The samples were digested with IdeS enzyme and separated by a reverse phase chromatography hydrophobic interaction chromatography column. The molecular weights of different glycoform modifications were detected by liquid chromatography-mass spectrometry, and the content of each glycoform was calculated.

[0132] The results are shown in Table 2. In the cell culture producing the antibody, the addition of cobalt chloride hexahydrate, ammonium heptamolybdate tetrahydrate, nickel chloride hexahydrate and ammonium metavanadate increased the protein expression by 93.66%, and the proteins FA2G1 and FA2G2 increased by 35.57% and 69.44%, respectively.

[0133] Table 2 Glycoforms of anti-Claudin6 monoclonal antibodies

[0134] Culture medium Expression level (g / L) FA2G1(%) FA2G2(%) Basic culture medium 3.31 23.98 1.80 Basic culture medium + cobalt chloride hexahydrate + ammonium heptamolybdate tetrahydrate + nickel chloride hexahydrate + ammonium metavanadate 6.41 32.51 3.05

[0135] Example 3 Preparation method of guselkumab

[0136] Take a cell of the Guselkumab cryopreserved working cell bank (CHO cells, 1 mL) from the liquid nitrogen tank, thaw it in a 37°C water bath, transfer it to a cell culture shake flask containing basal culture medium, and culture it in a carbon dioxide constant temperature incubator. The cell density is about 1.5-5.0×10 6 cells / mL, and the subculture density is about 0.4-0.9×10 6 cells / ml, and the shaking flask culture conditions were: 37.0℃, 5% CO2, 130 rpm. The culture medium with cells in logarithmic growth phase and good condition was used as seed medium.

[0137] The seed solution was inoculated into the bioreactor (Sysbio, BioTop ® D) were cultured in the medium shown in Table 3; the temperature was initially set at 37.0°C. When the cell density reached 10×10 6 cells / mL, the temperature was adjusted to 33.0℃; the speed was set to 200 rpm; the DO-related O2 automatic control was set to 40%; the pH value-related CO2 automatic control was initially set to 7.15±0.35, and the pH was adjusted to 7.00±0.30 after cooling; the air sparger was continuously turned on, and the ventilation volume was 20 mL / min; the culture period was 14 days; cysteine ​​or cystine was added during the culture process, and the amount of cysteine ​​or cystine added (g) = cell culture volume (mL) × 0.5%. The cells were harvested after 14 days of culture, and the antibodies were purified after centrifugation to determine the protein expression and glycoforms.

[0138] High performance liquid chromatography was used to detect the protein at a wavelength of 280 nm and calculate the protein expression.

[0139] The samples were digested with IdeS enzyme and separated by a reverse phase chromatography hydrophobic interaction chromatography column. The molecular weights of different glycoform modifications were detected by liquid chromatography-mass spectrometry, and the content of each glycoform was calculated.

[0140] Table 3 Culture medium

[0141]

[0142] The results are shown in Table 4. When manganese chloride was added to the cell culture for antibody production, the levels of antibody FA2G1 and FA2G2 increased, but with the increase of manganese chloride concentration, the levels of antibody FA2G1 and FA2G2 did not increase significantly. Compared with the addition of manganese chloride alone, the levels of antibody FA2G1 and FA2G2 did not increase further after adding manganese chloride, galactose and uridine at the same time. The addition of ammonium ferric citrate resulted in a decrease in the galactosylation level of the antibody. In the cell culture for antibody production, the addition of cobalt chloride hexahydrate, ammonium heptamolybdate tetrahydrate, nickel chloride hexahydrate and ammonium metavanadate, and the addition of cystine can break through the plateau period of manganese chloride affecting the galactosylation level, and prepare antibodies with high expression and high galactosylation levels.

[0143] Table 4 Glucose forms of guselkumab

[0144] serial number Expression level (g / L) FA2G1(%) FA2G2(%) 1 5.72 13.28 1.12 2 5.56 17.66 1.75 3 5.68 18.14 1.81 4 4.57 15.36 1.44 5 4.43 22.33 3.02 6 5.53 34.88 5.81

[0145] By adding cobalt chloride, ammonium heptamolybdate, nickel chloride and ammonium metavanadate to the cell culture for producing antibodies, anti-TIM-3 antibodies (the heavy chain is shown in SEQ ID NO: 17 and the light chain is shown in SEQ ID NO: 18, the heavy chain is shown in SEQ ID NO: 19 and the light chain is shown in SEQ ID NO: 20) with high expression level and high galactosylation level, anti-LAG-3 antibodies (the heavy chain is shown in SEQ ID NO: 37 and the light chain is shown in SEQ ID NO: 38, the heavy chain is shown in SEQ ID NO: 39 and the light chain is shown in SEQ ID NO: 40), anti-ROR1 antibody, anti-Claudin6 antibody, GIPR antibody / GLP-1 fusion protein, anti-EpCAM×CD3 bispecific antibody, penamprimab, bemosubizumab, daratumumab, mepolizumab, ramucirumab, pertuzumab, guselkumab, rituximab, trastuzumab, adalimumab, bevacizumab, epezetuzumab, obinutuzumab, pembrolizumab, lomosozumab, alirocumab, bimegizumab, denosumab, eloumab, nivolumab, secukinumab, ustekinumab, belimumab, nemolimumab, lencanezumab, rozelixizumab, faricizumab, atezolizumab and other antibodies.

Claims

1. A method for preparing an antibody, characterized in that: Cultivating antibody-producing cells in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 6-30 μmol / L, ammonium heptamolybdate at a final concentration of 0.105-0.14 μmol / L, nickel chloride at a final concentration of 0.15-0.2 μmol / L, and ammonium metavanadate at a final concentration of 30-32.5 μmol / L; or Antibody-producing cells are cultured in a culture consisting of a basal medium, cobalt chloride at a final concentration of 6-30 μmol / L, ammonium heptamolybdate at a final concentration of 0.105-0.14 μmol / L, nickel chloride at a final concentration of 0.15-0.2 μmol / L, ammonium metavanadate at a final concentration of 30-32.5 μmol / L, and manganese chloride at a final concentration of 1-10 μmol / L, wherein the antibody is guselkumab.

2. The method according to claim 1, characterized in that preparing a seed solution in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 8 μmol / L, ammonium heptamolybdate at a final concentration of 0.14 μmol / L, nickel chloride at a final concentration of 0.2 μmol / L, and ammonium metavanadate at a final concentration of 30 μmol / L, and culturing antibody-producing cells in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 6 μmol / L, ammonium heptamolybdate at a final concentration of 0.105 μmol / L, ammonium heptamolybdate at a final concentration of 0.15 μmol / L, nickel chloride, and ammonium metavanadate at a final concentration of 32.5 μmol / L, wherein the antibody is an anti-ROR1 monoclonal antibody; preparing a seed solution in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 8 μmol / L, ammonium heptamolybdate at a final concentration of 0.14 μmol / L, nickel chloride at a final concentration of 0.2 μmol / L, and ammonium metavanadate at a final concentration of 30 μmol / L, and culturing an antibody-producing cell in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 8 μmol / L, ammonium heptamolybdate at a final concentration of 0.14 μmol / L, nickel chloride at a final concentration of 0.2 μmol / L, and ammonium metavanadate at a final concentration of 30 μmol / L; or A seed solution was prepared in a basal medium, and antibody-producing cells were cultured in a culture consisting of a basal medium, cobalt chloride at a final concentration of 30 μmol / L, ammonium heptamolybdate at a final concentration of 0.14 μmol / L, nickel chloride at a final concentration of 0.2 μmol / L, ammonium metavanadate at a final concentration of 30 μmol / L, and manganese chloride at a final concentration of 10 μmol / L, wherein the antibody is guselkumab.

3. The method according to claim 1 or 2, characterized in that 0.5% cystine was added during the culture of guselkumab-producing cells.

4. A method for culturing cells producing antibodies, characterized in that: Cultivating antibody-producing cells in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 6-30 μmol / L, ammonium heptamolybdate at a final concentration of 0.105-0.14 μmol / L, nickel chloride at a final concentration of 0.15-0.2 μmol / L, and ammonium metavanadate at a final concentration of 30-32.5 μmol / L; or Antibody-producing cells are cultured in a culture consisting of a basal medium, cobalt chloride at a final concentration of 6-30 μmol / L, ammonium heptamolybdate at a final concentration of 0.105-0.14 μmol / L, nickel chloride at a final concentration of 0.15-0.2 μmol / L, ammonium metavanadate at a final concentration of 30-32.5 μmol / L, and manganese chloride at a final concentration of 1-10 μmol / L, and 0.5% cystine is added during the cell culture process, and the antibody is guselkumab.

5. The method according to claim 4, characterized in that preparing a seed solution in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 8 μmol / L, ammonium heptamolybdate at a final concentration of 0.14 μmol / L, nickel chloride at a final concentration of 0.2 μmol / L, and ammonium metavanadate at a final concentration of 30 μmol / L, and culturing antibody-producing cells in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 6 μmol / L, ammonium heptamolybdate at a final concentration of 0.105 μmol / L, ammonium heptamolybdate at a final concentration of 0.15 μmol / L, nickel chloride, and ammonium metavanadate at a final concentration of 32.5 μmol / L, wherein the antibody is an anti-ROR1 monoclonal antibody; preparing a seed solution in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 8 μmol / L, ammonium heptamolybdate at a final concentration of 0.14 μmol / L, nickel chloride at a final concentration of 0.2 μmol / L, and ammonium metavanadate at a final concentration of 30 μmol / L, and culturing an antibody-producing cell in a culture medium consisting of a basal medium, cobalt chloride at a final concentration of 8 μmol / L, ammonium heptamolybdate at a final concentration of 0.14 μmol / L, nickel chloride at a final concentration of 0.2 μmol / L, and ammonium metavanadate at a final concentration of 30 μmol / L; or A seed solution was prepared in a basal medium, and antibody-producing cells were cultured in a culture consisting of a basal medium, cobalt chloride at a final concentration of 30 μmol / L, ammonium heptamolybdate at a final concentration of 0.14 μmol / L, nickel chloride at a final concentration of 0.2 μmol / L, ammonium metavanadate at a final concentration of 30 μmol / L, and manganese chloride at a final concentration of 10 μmol / L, and 0.5% cystine was added during the cell culture process. The antibody was guselkumab.

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