Cell culture method for efficiently and correctly expressing antibody with asymmetric structure

Abstract

The invention provides a cell perfusion culture method for efficiently and correctly expressing the antibody with the asymmetric structure, a rocking tube model suitable for a perfusion culture process, perfusion culture medium development and the like is established, the asymmetric antibody with a correct structure can be obtained, the daily output can reach 2g / L, the asymmetric antibody with higher antibody purity can be obtained after purification, and the method is suitable for large-scale industrial production. Compared with a traditional fed batch (Fed-Batch) culture mode, the method has a great breakthrough, and has great significance in production of asymmetric antibody products with low expression and poor stability.

Description

Technical Field

[0001] The present disclosure belongs to the field of biotechnology and relates to a cell culture method for an asymmetric structure antibody. Background Art

[0002] Multiple myeloma (MM) (also known as plasma cell myeloma) accounts for 10% of hematological malignancies and approximately 20% of deaths from hematological malignancies. This disease mostly affects middle-aged and elderly people over 45 years old. Although several drugs have been approved by regulatory agencies, including proteasome inhibitors, immunomodulatory drugs, and monoclonal antibodies, MM remains an incurable disease. Among many potential MM treatment targets, B-cell maturation antigen (BCMA) is currently the most successful target, and several approved products are available, including CAR-T, BCMA / CD3 bispecific antibodies, and ADCs. Among them, the drugs with better efficacy are bispecific antibodies targeting BCMA / CD3, and the main products include elranatamab, teclistamab (TEC), etc. In addition, other targets such as G protein-coupled receptor family C group 5 member D (GPRC5D), Fc receptor homolog 5 (FcRH5), SLAM57, CD38, and CD19 are also potential treatment targets for MM. Among them, GPRC5D is expressed at low levels in healthy human tissues but highly expressed in malignant plasma cells, making it a promising immunotherapy target for MM patients. Some studies have found that the expression of BCMA and GPRC5D is not correlated. Based on the BCMA / CD3 bispecific antibody, adding a GPRC5D antibody to obtain a trispecific antibody targeting two tumor-associated antigens (TAAs) of the same antibody can cover both GPRC5D-positive and BCMA-positive tumor patients, and at the same time avoid recurrence caused by the loss of a single antigen, thereby achieving the purpose of improving patient coverage and efficacy.

[0003] Multispecific antibodies (such as trispecific antibodies), due to their high degree of artificial modification and complex structure, are generally asymmetric structures. In the traditional fed-batch culture production mode, when exposed to a culture medium environment with high osmotic pressure, constantly changing pH, high temperature, and a large accumulation of secondary metabolites (such as enzymes) for a long time, multispecific antibodies exhibit more instability and more complex product heterogeneity characteristics, such as aggregation, fragmentation, oxidation, mismatch, etc., increasing the difficulty of product expression and downstream purification. To solve the above problems of trispecific (multispecific) antibodies, experimentalists have developed new cell culture processes and production methods.

[0004] The cultivation method is a perfusion cultivation method. Compared with the traditional fed-batch cultivation process, in this method, cells are inoculated into shake flasks or shake tubes. After the cells grow to a certain density, the old culture medium is discarded (or harvested) by centrifugation every day, and fresh culture medium is continuously perfused (or supplemented) to maintain a high cell density and daily antibody production. Summary of the Invention

[0005] The content of the present disclosure is a cell culture method developed to address the deficiencies in the rapid small-scale production of multispecific antibody products. The method can be used for the rapid and efficient preparation of unstable antibody products, the development of perfusion culture media, and the guidance of the development of large-scale perfusion production processes, etc.

[0006] To achieve the above object, the present disclosure adopts the following technical solutions.

[0007] The present disclosure provides a cell culture method for efficiently and correctly expressing an antibody with an asymmetric structure, and the method includes the following steps:

[0008] (1) Obtain a mammalian cell capable of producing an antibody with an asymmetric structure;

[0009] (2) Cultivate the cells in step (1), obtain a culture solution, and perform separation and purification to obtain an antibody with an asymmetric structure.

[0010] The mammalian cells include CHO cells.

[0011] The cultivation method is a perfusion cultivation method. The culture medium includes a basal medium and a perfusion medium, and the temperature range for perfusion cultivation is 34-37°C.

[0012] In the present disclosure, by adopting the perfusion cultivation method, an antibody with an asymmetric structure is efficiently and correctly expressed.

[0013] Preferably, the antibody with an asymmetric structure is a trispecific antibody that specifically binds to GPRC5D, BCMA, and / or CD3. Preferably, the antibody with an asymmetric structure contains a domain that specifically binds to GPRC5D; preferably, the antibody with an asymmetric structure contains a domain that specifically binds to BCMA; and / or preferably, the antibody with an asymmetric structure contains a domain that specifically binds to CD3.

[0014] Preferably, the trispecific antibody contains the following polypeptides:

[0015] A first polypeptide, comprising: (i) a heavy chain domain of an antigen-binding fragment Fab that can specifically bind to BCMA, (ii) a single-chain antibody (scFv) domain that can specifically bind to CD3, and (iii) a first Fc domain;

[0016] A second polypeptide, comprising: a light chain domain of an antigen-binding fragment Fab capable of specifically binding to BCMA;

[0017] A third polypeptide, comprising: (i) a heavy chain single-domain antibody (VHH) domain capable of specifically binding to GPRC5D and (ii) a second Fc domain.

[0018] The heavy chain domain of the antigen-binding fragment Fab of the first polypeptide and the light chain domain of the antigen-binding fragment Fab of the second polypeptide form a domain that specifically binds to BCMA; the single-chain antibody (scFv) domain forms a domain that specifically binds to CD3; the heavy chain single-domain antibody (VHH) domain forms a domain that specifically binds to GPRC5D; the first Fc domain and the second Fc domain associate with each other.

[0019] Preferably, the domain that specifically binds to BCMA and the domain that specifically binds to CD3 respectively comprise heavy chain CDR1, CDR2, and CDR3 sequences and light chain CDR1, CDR2, and CDR3 sequences, and the domain that specifically binds to GPRC5D comprises heavy chain CDR1, CDR2, and CDR3 sequences.

[0020] Preferably, the domain that specifically binds to GPRC5D comprises HCDR1 having the sequence shown in SEQ ID NO:1, HCDR2 having the sequence shown in SEQ ID NO:2, and HCDR3 having the sequence shown in SEQ ID NO:3. Preferably, the domain that specifically binds to CD3 comprises HCDR1 having the sequence shown in SEQ ID NO:4, HCDR2 having the sequence shown in SEQ ID NO:5, HCDR3 having the sequence shown in SEQ ID NO:6, LCDR1 having the sequence shown in SEQ ID NO:7, LCDR2 having the sequence shown in SEQ ID NO:8, and LCDR3 having the sequence shown in SEQ ID NO:9. Preferably, the domain that specifically binds to BCMA comprises HCDR1 having the sequence shown in SEQ ID NO:10, HCDR2 having the sequence shown in SEQ ID NO:11, and HCDR3 having the sequence shown in SEQ ID NO:12, LCDR1 having the sequence shown in SEQ ID NO:13, LCDR2 having the sequence shown in SEQ ID NO:14, and LCDR3 having the sequence shown in SEQ ID NO:15.

[0021] Preferably, the heavy chain single domain antibody (VHH) domain specifically binding to GPRC5D comprises the sequence shown in SEQ ID NO: 16. Preferably, the domain specifically binding to BCMA comprises a heavy chain variable region with a sequence as shown in SEQ ID NO: 17, and / or, the domain specifically binding to BCMA comprises a light chain variable region with a sequence as shown in SEQ ID NO: 18. Preferably, the domain specifically binding to CD3 comprises a heavy chain variable region with a sequence as shown in SEQ ID NO: 19 and a light chain variable region with a sequence as shown in SEQ ID NO: 20.

[0022] In one embodiment, the first polypeptide of the trispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 21, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 23, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO: 22.

[0023] Table 1 Amino acid sequences of the trispecific antibody

[0024]

[0025]

[0026] Preferably, the CHO cells are selected from the group consisting of the following cell lines:

[0027] CHO-K1, DXB-11, DG-44, CHO-S, CHO-K1, CHO-K1 (GS-KO) and other cell lines.

[0028] The basal medium used in the present disclosure is a serum-free, protein-free, chemically defined medium independently developed by the applicant, and its main components include the following substances:

[0029] 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 5 - 7 g / L, glucose 3 - 8 g / L, sodium pyruvate 0.05 - 0.4 g / L, sodium chloride 3 - 5 g / L, potassium chloride 0.2 - 0.4 g / L, sodium selenite 0.0000005 - 0.000060 g / L, manganese sulfate 0.05 - 0.2 g / L, ethanolamine 5 - 25 μg / L, ferric citrate 1 - 10 mg / L, zinc sulfate 0.1 - 0.5 g / L, copper sulfate 0.02 - 0.15 g / L, glutathione 0.05 - 0.3 g / L, magnesium chloride 0.025 - 0.15 g / L, sodium dihydrogen phosphate 0.1 - 0.3 g / L, sodium bicarbonate 1 - 5 g / L, alanine 0.015 - 0.035 g / L, asparagine 0.01 - 0.035 g / L, arginine 0.06 - 0.30 g / L, aspartic acid 0.02 - 0.2 g / L, cystine 0.3 - 0.5 g / L, cysteine 0.05 - 0.2 g / L, glutamic acid 0.001 - 0.09 g / L, glycine 0 - 0.04 g / L, histidine 0.03 - 0.2 g / L, isoleucine 0.05 - 0.25 g / L, leucine 0.05 - 0.25 g / L, lysine 0.02 - 0.25 g / L, methionine 0.02 - 0.2 g / L, phenylalanine 0.055 - 0.075 g / L, proline 0.01 - 0.05 g / L, serine 0.03 - 0.25 g / L, threonine 0.07 - 0.15 g / L, tryptophan 0.005 - 0.025 g / L, tyrosine 0.05 - 0.2 g / L, valine 0.05 - 0.5 g / L, biotin 0.005 - 0.15 mg / L, calcium pantothenate 0.002 - 0.006 g / L, choline chloride 0.002 - 0.09 g / L, folic acid 0.002 - 0.006 g / L, inositol 0.005 - 0.02 g / L, nicotinamide 0.002 - 0.006 g / L, vitamin B6 0.002 - 0.006 g / L, vitamin B2 0.0002 - 0.0006 g / L, vitamin B1 0.002 - 0.006 g / L, vitamin B12 0.000005 - 0.000025 g / L, linoleic acid 0.001 - 0.1 g / L, block copolymer polyether F-68 (Pluronic F-68) 0.2 - 5 g / L.

[0030] The perfusion medium used in the present disclosure is a serum-free and protein-free, chemically defined medium independently developed by the applicant, and the main components include the following substances:

[0031] 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 2 - 7 g / L, glucose 5 - 15 g / L, sodium pyruvate 0.1 - 0.6 g / L, potassium chloride 1 - 5 g / L, sodium selenite 0.0000005 - 0.000060 g / L, manganese sulfate 0.00000005 - 0.0000004 g / L, ethanolamine 5 - 25 μg / L, ferric citrate 0.01 - 1 g / L, zinc sulfate 0.001 - 0.05 g / L, copper sulfate 0.0005 - 0.01 g / L, glutathione 0.005 - 0.03 g / L, magnesium chloride 0.025 - 0.5 g / L, sodium dihydrogen phosphate 0.1 - 5 g / L, sodium bicarbonate 0.2 - 5 g / L, alanine 0.1 - 1.5 g / L, asparagine 0.1 - 3.5 g / L, arginine 0.6 - 6 g / L, aspartic acid 0.2 - 2 g / L, cysteine 0.05 - 0.2 g / L, glutamic acid 0.01 - 0.9 g / L, glycine 0 - 0.4 g / L, histidine 0.03 - 1.5 g / L, isoleucine 0.05 - 2.5 g / L, leucine 0.5 - 2.5 g / L, lysine 0.02 - 0.25 g / L, methionine 0.2 - 1 g / L, phenylalanine 0.55 - 0.75 g / L, proline 0.1 - 0.5 g / L, serine 0.3 - 2.5 g / L, threonine 0.07 - 0.15 g / L, tryptophan 0.05 - 0.25 g / L, tyrosine 0.05 - 1 g / L, valine 0.05 - 0.5 g / L, biotin 0.005 - 0.15 mg / L, calcium pantothenate 0.002 - 0.006 g / L, choline chloride 0.002 - 0.09 g / L, folic acid 0.002 - 0.006 g / L, inositol 0.005 - 0.02 g / L, nicotinamide 0.002 - 0.006 g / L, vitamin B6 0.002 - 0.006 g / L, vitamin B2 0.0002 - 0.0006 g / L, vitamin B1 0.002 - 0.006 g / L, vitamin B12 0.000005 - 0.000025 g / L, linoleic acid 0.001 - 0.1 g / L, block copolymer polyether F-68 (Pluronic F-68) 0.2 - 5 g / L.

[0032] Preferably, the perfusion culture method is carried out in a 50 ml shake tube.

[0033] Preferably, the shake tube can be placed on the tube rack in a shaker for culture, and the culture parameters of the shaker include: the amplitude is 25 - 50 mm, and the rotation speed is 150 - 300 rpm.

[0034] Preferably, the culture volume is 10 - 20 ml, and more preferably, the culture volume is 10 - 15 ml.

[0035] Preferably, the inclination angle of the tube rocking rack is 45-90°, and more preferably, the inclination angle of the tube rocking rack is 45-60°.

[0036] Preferably, the perfusion culture method includes the following operations performed daily: sampling and counting daily, when the cell density > 8×10 6 cells / ml, centrifuging the shaking tube at 800-1500 rpm for 5-6 min, removing the supernatant, resuspending the cells with the basal medium, culturing under the above rotation speed conditions, when the cell density > 30×10 6 cells / ml, centrifuging at 800-1500 rpm for 5-7 min, removing the supernatant, resuspending the cells with the perfusion medium, and the perfusion rate is 1 VVD. The perfusion culture time can be 15-60 days.

[0037] Preferably, the seeding density is 0.5-20×10 6 cells / ml, and more preferably, 1×10 6 -20×10 6 cells / ml.

[0038] Preferably, the perfusion culture temperature is 34-37 °C, and more preferably, the temperature is 36-37 °C.

[0039] Preferably, during the perfusion culture process, the cell density is regulated by cooling. More preferably, the cooling method is that when the cell density reaches 30×10 6 cells / ml, the culture temperature is reduced from 36-37 °C to 32-34 °C to maintain the cell density. In some specific embodiments of the present disclosure, the peak density is (70-80)×10 6 cells / ml.

[0040] Preferably, the perfusion culture further includes supplementing glucose.

[0041] Preferably, the added amount of glucose is 1-10 g / L, including but not limited to 1 g / L, 2 g / L, 3 g / L, 4 g / L, 5 g / L, 6 g / L, 7 g / L, 8 g / L, 9 g / L, 10 g / L.

[0042] Preferably, the separation and purification in step (2) includes the following steps:

[0043] (1) Filtering the culture solution through a PES membrane to obtain a clarified solution;

[0044] (2) Purifying the clarified solution by affinity chromatography;

[0045] Preferably, the quality analysis index in step (2) is a purity index, including molecular size purity (SEC-HPLC) and charge heterogeneity (iCIEF analysis).

[0046] Compared with the prior art, the present disclosure has the following beneficial effects:

[0047] By adopting the perfusion culture method and optimizing the process parameters, the highest cell density of (70-80)×10 6 cells / ml can be achieved on the shaking tube. The culture time can be up to 30 days, the daily cell viability is above 90%, and the daily antibody concentration is 1.8-2.2 g / L.

[0048] Compared with the fed-batch culture process, the perfusion culture method can obtain a higher cell density, a higher unit yield, and higher purity of the main peak of antibody SEC-HPLC and iCIEF, as well as a lower content of acidic variants.

[0049] The present disclosure provides a cell culture method for efficiently and correctly expressing an antibody with an asymmetric structure, which has the characteristics of low cost, easy operation, simple process, high expression level, and better quality, and provides a new cell culture model for the high yield and high quality of antibodies with an asymmetric structure. BRIEF DESCRIPTION OF THE DRAWINGS

[0050] Figure 1 It shows the influence of the cell loading volume on the viable cell density and viability in the perfusion culture shaking tube model.

[0051] Figure 2 It shows the influence of the cell loading volume on the IgG yield in the perfusion culture shaking tube model.

[0052] Figure 3 It shows the influence of the tilt angle on the viable cell density and viability in the perfusion culture shaking tube model.

[0053] Figure 4 It shows the influence of the tilt angle on the IgG yield in the perfusion culture shaking tube model.

[0054] Figure 5 It shows the influence of the inoculation density on the total cell density and viability in the perfusion culture shaking tube model.

[0055] Figure 6 It shows the influence of the inoculation density on the IgG yield in the perfusion culture shaking tube model.

[0056] Figure 7 It is a comparison chart of the viable cell density and viability between the Fed-Batch culture and the perfusion culture mode.

[0057] Figure 8It is a comparison chart of IgG production between Fed - Batch culture and perfusion culture modes.

[0058] Figure 9 It is a comparison chart of the main peak purity of SEC - HPLC between Fed - Batch culture and perfusion culture modes.

[0059] Figure 10 It is a comparison chart of the main peak purity of iCIEF between Fed - Batch culture and perfusion culture modes.

[0060] Figure 11 It is a comparison chart of the acid region quality of iCIEF between Fed - Batch culture and perfusion culture modes.

[0061] Figure 12 It is a schematic diagram of the structure of the trispecific antibody of the present disclosure. Detailed Description of the Invention

[0063] Terms

[0064] All publications, patents, and patent applications mentioned in this specification are incorporated herein by reference to the extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated herein by reference.

[0065] Before describing the present disclosure in detail below, it should be understood that the present disclosure is not limited to the specific methodologies, protocols, and reagents described herein, as these may vary. It should also be understood that the terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of the present disclosure. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which the present disclosure pertains.

[0066] Certain embodiments disclosed herein include numerical ranges, and certain aspects of the present disclosure may be described in terms of ranges. Unless otherwise stated, it should be understood that the numerical ranges or the description in terms of ranges are only for the purpose of brevity and convenience and should not be considered as a strict limitation on the scope of the present disclosure. Therefore, the description in terms of ranges should be considered to specifically disclose all possible sub - ranges and all possible specific numerical points within that range, just as if these sub - ranges and numerical points were explicitly written herein. The above principles apply equally regardless of the width of the numerical values. When a range is described, the range includes the endpoints of the range.

[0067] When referring to measurable values such as amounts, temporal durations, etc., the term "about" means including a variation of ±20%, or in some cases ±10%, or in some cases ±5%, or in some cases ±1%, or in some cases ±0.1% of the specified value.

[0068] The three-letter and one-letter codes for amino acids used herein are as described in J. Biol. Chem., 243, p3558 (1968).

[0069] As used herein, the term “antibody” can include intact antibodies (e.g., full-length monoclonal antibodies) and any antigen-binding fragments (i.e., antigen-binding portions) or single chains thereof, and can also include products with antigen-specific binding ability formed by modifying (e.g., linking other peptide segments, rearranging functional units, etc.) intact antibodies or their antigen-binding fragments or single chains.

[0070] In one embodiment, an antibody typically refers to a Y-shaped tetrameric protein comprising two heavy (H) polypeptide chains and two light (L) polypeptide chains held together by covalent disulfide bonds and non-covalent interactions. Native IgG antibodies have such a structure. Each light chain consists of a variable domain (VL) and a constant domain (CL). Each heavy chain contains a variable domain (VH) and a constant region.

[0071] Five major classes of antibodies are known in the art: IgA, IgD, IgE, IgG, and IgM, and the corresponding heavy-chain constant domains are designated α, δ, ε, γ, and μ, respectively. IgG and IgA can be further divided into different subclasses. For example, IgG can be divided into IgG1, IgG2, IgG3, and IgG4, and IgA can be divided into IgA1 and IgA2. The light chains of antibodies from any vertebrate species can be assigned to one of two distinct types, called κ and λ, based on the amino acid sequence of their constant domains.

[0072] In the case of IgG, IgA, and IgD antibodies, the constant region contains three domains called CH1, CH2, and CH3 (IgM and IgE have a fourth domain, CH4). In the IgG, IgA, and IgD classes, the CH1 and CH2 domains are separated by a flexible hinge region, which is a variable-length proline- and cysteine-rich segment. Each class of antibody further contains interchain and intrachain disulfide bonds formed by paired cysteine residues.

[0073] The terms "variable region" or "variable domain" show significant variation in amino acid composition from one antibody to another and are mainly responsible for antigen recognition and binding. The variable regions of each light chain / heavy chain pair form the antibody binding site, such that a complete IgG antibody has two binding sites (i.e., it is bivalent). The variable domain of the heavy chain (VH) and the variable domain of the light chain (VL) each contain three regions with extreme variability, called hypervariable regions (HVRs), or more commonly, complementarity-determining regions (CDRs). VH and VL each have 4 framework regions FR, denoted as FR1, FR2, FR3, and FR4 respectively. Thus, the CDR and FR sequences typically occur in the following sequence in the heavy chain variable domain (or light chain variable domain): FR1-HCDR1(LCDR1)-FR2-HCDR2(LCDR2)-FR3-HCDR3(LCDR3)-FR4.

[0074] The term "antibody fragment" encompasses at least a portion of a complete antibody. As used herein, a "fragment" of an antibody molecule includes an "antigen-binding fragment" of the antibody, and the term "antigen-binding fragment" refers to a polypeptide fragment in an immunoglobulin or antibody that specifically binds or reacts with a selected antigen or an immunogenic determinant portion thereof, or a fusion protein product further derived from such a fragment, such as a single-chain antibody, the extracellular binding region in a chimeric antigen receptor, etc. Exemplary antibody fragments or their antigen-binding fragments include, but are not limited to: variable light chain fragments, variable heavy chain fragments, Fab fragments, F(ab’)2 fragments, Fd fragments, Fv fragments, single-domain antibodies, linear antibodies, single-chain antibodies (scFv), and bispecific or multispecific antibodies formed from antibody fragments, etc.

[0075] The term "Fab" or "Fab fragment" refers to a monovalent antibody fragment consisting of the VH and CH1 domains of the heavy chain and the VL and CL domains of the light chain. The term "F(ab’)2" or "F(ab’)2 fragment" contains 2 Fab fragments and the hinge region and is a bivalent antibody fragment.

[0076] The term "single-chain antibody" or "scFv" refers to a fusion protein containing at least one antibody fragment including the variable region of the light chain and at least one antibody fragment including the variable region of the heavy chain, wherein the light chain and heavy chain variable regions are adjacent (e.g., via a synthetic linker such as a short flexible polypeptide linker), and can be expressed as a single-chain polypeptide, and wherein the scFv retains the specificity of the complete antibody from which it is derived. Unless specified, the scFv can have the VL and VH variable regions in any order (e.g., with respect to the N-terminus and C-terminus of the polypeptide), and the scFv can include VL-linker-VH or can include VH-linker-VL.

[0077] "VHH domain", also known as heavy chain single domain antibody, VHH, VHH antibody fragment, VHH antibody, nanobody, is the variable domain of an antigen-binding immunoglobulin called "heavy chain antibody" (i.e., "antibody lacking light chain") (Hamers-Casterman C, Atarhouch T, Muyldermans S, Robinson G, Hamers C, Songa EB, Bendahman N, Hamers R.: "Naturally occurring antibodies devoid of light chains"; Nature 363, 446-448 (1993)). The term "VHH domain" is used to distinguish the variable domain from the heavy chain variable domain (referred to as "VH domain" in the present disclosure) and the light chain variable domain (referred to as "VL domain") present in antibodies with a conventional four-peptide chain structure. The VHH domain specifically binds to an epitope without the need for other antigen-binding domains (in contrast to the VH or VL domains in antibodies with a conventional four-peptide chain structure, where the epitope is recognized by the VL domain together with the VH domain). The VHH domain is a small, stable and efficient antigen recognition unit formed by a single immunoglobulin domain. The terms "heavy chain single domain antibody", "VHH domain", "VHH", "VHH domain", "VHH antibody fragment", "VHH antibody" and "heavy chain antibody variable region" are used interchangeably. "VHH domain" includes but is not limited to natural antibodies produced by camelids, antibodies produced by camelids and then humanized, or antibodies obtained by screening using phage display technology.

[0078] In this article, "antibody" can be used in the broadest sense and can include, for example, polyclonal antibodies, monoclonal antibodies, chimeric antibodies, humanized antibodies and primatized antibodies, CDR-grafted antibodies, human antibodies (including recombinantly produced human antibodies), recombinantly produced antibodies, intracellular antibodies, multispecific antibodies, bispecific antibodies, monovalent antibodies, multivalent antibodies, anti-idiotypic antibodies, synthetic antibodies (including mutant proteins and their variants), and so on.

[0079] The term "monoclonal antibody" (or "mAb") refers to an antibody that is substantially homogeneous and specific for a particular antigen epitope, produced by a single cell clone. Monoclonal antibodies can be prepared using a variety of techniques known in the art, including hybridoma technology, recombinant technology, phage display technology, transgenic animals, synthetic technology, or combinations of the above techniques.

[0080] The term "antigen" refers to a substance that is recognized and specifically bound by an antibody or an antibody-binding fragment. Broadly speaking, an antigen can include any immunogenic fragment or determinant of a selected target, including a single epitope, a multi-epitope, a single domain, a multi-domain, a complete extracellular domain (ECD) or a protein. Peptides, proteins, glycoproteins, polysaccharides and lipids, their parts and combinations can all constitute antigens. Non-limiting exemplary antigens include tumor antigens or pathogen antigens, etc. "Antigen" can also refer to a molecule that elicits an immune response. Any form of antigen or cells or preparations containing the antigen can be used to generate antibodies specific for the antigenic determinant.

[0081] The term "multispecific" means that an antigen-binding molecule is capable of specifically binding to multiple different antigenic determinants. The term "trispecific antibody" refers to an antibody that is specific for three different antigens (or epitopes).

[0082] The term "specific binding" means that the binding is selective for the antigen and can be distinguished from unwanted or non-specific interactions. The ability of an antibody to specifically bind to an antigenic determinant can be determined by enzyme-linked immunosorbent assay (ELISA) or other techniques familiar to those skilled in the art.

[0083] When making antibodies or multispecific antibodies with the variable regions in the present disclosure, the constant region is not particularly limited. Constant regions known to those skilled in the art or self-obtained constant regions can be used. Amino acid mutations can also be introduced into the constant region part (for example, mutations that increase or decrease the binding of Fc to receptors or FcRn).

[0084] The term "fed-batch culture", also known as semi-continuous culture or fed culture, in English is Fed-batch Culture, which refers to adding a certain amount of materials to the culture system in a certain way during the cell batch culture process to meet the cell growth requirements until the end of the culture to take out the product.

[0085] The term "perfusion culture", also known as continuous culture, in English is Perfusion Culture, which refers to a cell culture technique of continuously perfusing fresh culture medium, while the culture solution flows out at the same flow rate for cell retention, and the product is harvested as the culture solution is taken out.

[0086] The term "perfusion rate" means continuously providing fresh culture medium to the cells at the same speed while removing the depleted waste culture medium, usually expressed by VVD, that is, the perfusion volume per day. For example, perfusion of 10 ml of culture medium into a system with a working volume of 10 ml per day is expressed as 1 VVD.

[0087] The term "affinity chromatography" refers to protein A affinity chromatography. Protein A is a cell wall protein of Staphylococcus aureus that can specifically bind to the Fc segment of antibodies, and purify the target protein from a complex culture medium.

[0088] The term "SEC-HPLC" refers to size exclusion chromatography, which is a chromatographic technique based on the separation principle of molecular size or hydrodynamic radius differences.

[0089] The term "iCIEF" refers to imaging capillary isoelectric focusing electrophoresis, and the principle is that proteins have different isoelectric point charge states under different pH conditions, thereby realizing protein separation. Detailed implementation manners

[0090] To further elaborate on the technical means and implementation effects adopted by the present disclosure, the present disclosure will be further described below in combination with embodiments and the accompanying drawings. It can be understood that the following described embodiments are part of the embodiments of the present disclosure, rather than all of the embodiments, and are only used to explain the present disclosure, rather than limiting the present disclosure.

[0091] For those not specifying specific techniques or conditions in the embodiments, they shall be carried out according to the techniques or conditions described in the literature in this field or according to the product specifications. For reagents or instruments not indicating the manufacturer, they are all conventional products that can be obtained through commercial purchase.

[0092] Some experimental material information adopted in the embodiments of the present disclosure is as follows:

[0093] 50 ml shaking tubes: purchased from Corning, product number: 431720

[0094] Carbon dioxide incubator shaker: purchased from Infors, model: Multitron Pro

[0095] Shaking tube rack: purchased from Infors, model: Celtron

[0096] CountStar cell counter: purchased from Alit International Trading Co., Ltd., model: IC1000

[0097] Haier refrigerator: purchased from Haier, model: BCD-321WDJ

[0098] High-speed refrigerated centrifuge: purchased from ThermoFisher Scientific, model: Sorvall ST16RProtein A packing material: purchased from Cytiva, product number: 10298838

[0099] PES filter membrane: purchased from Merck, product number: SLGPR33RB

[0100] Biosensor analyzer: purchased from Sillman Technology Co., Ltd., model: M-100

[0101] Macromolecular interaction instrument: purchased from fortebio company, model: Octet Qke system

[0102] SEC-HPLC analyzer: purchased from Waters Corporation, model: e2695-2489 HPLC

[0103] Chromatographic column: XBridge Protein BEH SEC Purchased from Waters Corporation, product number: PN.186007640

[0105] Guard column: XBridge Protein BEH SEC Purchased from Waters Corporation, product number: PN.186007638)

[0107] Imaging capillary isoelectric focusing electrophoresis instrument: purchased from protein Sample company, model: Maurice C Example 1

[0108] Construction and screening of cell lines expressing anti-GPRC5D, BCMA and CD3 multispecific antibodies

[0109] (1) Construction of monoclonal antibody recombinant plasmid

[0110] The gene sequences encoding three polypeptides (sequences are SEQ ID NO:21, SEQ ID NO:23 and SEQ ID NO:22 respectively) of the trispecific antibody of the present disclosure were synthesized by a gene synthesis company and respectively ligated to the multiple cloning insertion site (MCS) of the vector. After construction, the correctness of the recombinant expression vector was confirmed by sequencing verification. Then, the plasmid was extracted using a plasmid midiprep kit, quantified by an ultra-micro spectrophotometer and used, or stored at -80 °C for later use.

[0111] (2) Host cell resuscitation and shake flask culture

[0112] The host cells used in this study were CHOZN ZFN-Modified GS- / -CHO cell line (hereinafter referred to as "CHOZN cells"), which were purchased from Merck. The CHOZN cells used in the present invention were taken out of liquid nitrogen, quickly placed in a 37°C water bath for rapid thawing, and added to the 37°C preheated EX-CELL CD CHO Fusion medium in an ultra-clean workbench. After mixing, the cells were counted using a CountStar cell counter, and the inoculation density was controlled at 1×10 6 cells / ml, at 36.5°C, 80% humidity, 5% CO 2 , shake flask culture was carried out in a shaker at 110-150 rpm.

[0113] (3) Cell transfection

[0114] Cells were seeded 24 hours before transfection at a density of 0.3-0.7×10 6 cells / ml, culture conditions were 36.5°C, 5% CO 2 , 80% humidity, 110-150rpm. On the day of transfection, use EX-CELL CD CHO Fusion medium preheated at 37°C to prepare a density of 4-8×10 6 cells / ml of cell suspension, and take 800μl and mix with 20-100μg plasmid. Use BioRad electroporator for transfection, and the transfection conditions are set as follows: voltage 300V, Capacitance 950μF, electric shock mode Exponential Decay, electric shock number 1, and then start the electric shock. Immediately after the electric shock, mix the cells with preheated culture medium and transfer them to a carbon dioxide incubator for static culture. The incubator setting parameters are: 36.5℃, 5% CO 2 , 80% humidity.

[0115] (4) Cell pool screening

[0116] After 24 hours of static culture, cell pool screening was performed. The culture medium used was 80% EX-CELL CHO Cloning medium + 20% EX-CELL CD CHO Fusion medium. The initial inoculation density was 3000-6000 cells / well. After inoculation, the 96-well plate was placed in a carbon dioxide incubator for static culture. The culture conditions were: 36.5°C, 5% CO 2 , 80% humidity. Select 2-3 96-well plates and observe the cell growth under a microscope every 3-4 days.

[0117] After the cells cover the wells, use a macromolecular interaction instrument to evaluate the expression level, and select the cell pools with higher expression levels for expansion to a 24-well plate. Culture conditions: 36.5 °C, 5% CO 2 , 80% humidity, 110 - 150 rpm. Observe the cell growth every 2 - 5 days. After the cell density is high and the cell state is good, perform cell counting and yield evaluation. Based on the combined results of cell number and yield, screen for high-yield cell pools for the screening of monoclonal cell lines.

[0118] (5) Screening of monoclonal cell lines

[0119] Using the limited dilution method, inoculate the cell Pools with higher expression levels into a 96-well plate at a density of <0.5 cell / well. The culture medium used is 80% EX-CELL CHO Cloning medium + 20% EX-CELL CD CHO Fusion medium. After inoculation, place the 96-well plate in a carbon dioxide incubator for static culture. Culture conditions: 36.5 °C, 5% CO 2 , 80% humidity. Take images on the 0th day, 1st day, 2nd day, 7th day, and 14th day respectively. At the same time, select 2 - 3 96-well plates and observe the cell growth with a microscope every 3 - 4 days.

[0120] After the cells cover the wells, use a macromolecular interaction instrument to evaluate the expression level, and select the cell pools with higher expression levels for expansion to a 24-well plate. Culture conditions: 36.5 °C, 5% CO 2 , 80% humidity, 110 - 150 rpm. Observe the cell growth every 2 - 5 days. After the cell density is high and the cell state is good, perform cell counting and yield evaluation. Based on the combined results of cell number and yield, screen for high-expressing monoclonal cell lines for shake flask Fed-Batch culture evaluation, collect information such as expression level and quality, and finally screen for candidate monoclonal cell lines according to the expression level and quality results.

[0121] Example 2

[0122] In this example, perfusion culture is carried out. The culture model is a 50 ml shake tube. The basal medium and perfusion medium are both independently developed by the applicant. Inoculate at 1×10 6 cells / ml. The liquid volumes of cells in the shake tubes are 10 ml, 15 ml, and 20 ml respectively. Adjust the tilt angle of the shake tube to 60° (the angle between the shake tube rack and the horizontal plane of the shaker is 60°). The culture parameters of the shake tube are set as follows: temperature 36.5 °C, rotation radius 50 mm, rotation speed 160 rpm, humidity 80%, 5% CO 2 , and inoculate and culture for 4 days.

[0123] After 4 days of inoculation and culture, samples were taken to detect cell density and cell viability. On Day 4, perfusion was carried out with the basal medium, centrifuged at 800 - 1500 rpm, the supernatant was removed, and perfusion was carried out at a perfusion rate of 1 VVD, that is, the cells were resuspended with 10 ml, 15 ml, and 20 ml of the basal medium respectively; the operation was repeated once every 24 hours. On Day 6, perfusion was started with the perfusion medium, centrifuged at 800 - 1500 rpm, the supernatant was removed, and the cells were resuspended with 10 ml, 15 ml, and 20 ml of the perfusion medium respectively; the operation was repeated once every 24 hours. On Day 7, the temperature was lowered to 34°C. After perfusion with the perfusion medium, in addition to detecting cell density and cell viability, biochemical parameters and IgG content were also detected daily, with the glucose content not less than 1 g / L, and the culture ended after 20 days.

[0124] Example 3

[0125] In this example, perfusion culture in a shake flask model was carried out. Both the basal medium and the perfusion medium were independently developed by the applicant. Inoculation was carried out at 1×10 6 cells / ml, the cell loading volume was 10 ml, and the tilt angle of the shake flask was adjusted to 45° and 90°. When the tilt angle was 45°, the shaker speed was 160 rpm, and when the tilt angle was 90°, the shaker speed was 300 rpm. Other shaker parameter settings: temperature 36.5°C, rotation radius 50 mm, humidity 80%, 5% CO 2 , and inoculation and culture were carried out for 4 days.

[0126] After 4 days of inoculation and culture, samples were taken to detect cell density and cell viability. On Day 4, perfusion was carried out with the basal medium, centrifuged at 800 - 1500 rpm, the supernatant was removed, and perfusion was carried out at a perfusion rate of 1 VVD, that is, the cells were resuspended with 10 ml of the basal medium; the operation was repeated once every 24 hours. On Day 6, perfusion was started with the perfusion medium, centrifuged at 800 - 1500 rpm, the supernatant was removed, and the cells were resuspended with 10 ml of the perfusion medium; the operation was repeated once every 24 hours. On Day 7, the temperature was lowered to 34°C. After perfusion with the perfusion medium, in addition to detecting cell density and cell viability, biochemical parameters and IgG content were also detected daily, with the glucose content not less than 1 g / L, and the culture ended after 20 days.

[0127] Example 4

[0128] In this example, perfusion culture in a shake flask model was carried out. Both the basal medium and the perfusion medium were independently developed by the applicant. Inoculation was carried out at 20×10 6 cells / ml, the cell loading volume was 10 ml, the tilt angle of the shake flask was 60°, the shaker speed was 160 rpm, and the shaker culture parameter settings: temperature 36.5°C, rotation radius 50 mm, humidity 80%, 5% CO 2 .

[0129] One day after inoculation and cultivation, samples were taken to detect cell density, cell viability, biochemical parameters, and IgG content. On Day 1, perfusion culture medium was used for perfusion, centrifuged at 800 - 1500 rpm, the supernatant was removed, and the cells were resuspended with 10 ml of perfusion culture medium; this operation was repeated once every 24 hours. On Day 2, the temperature was lowered to 34°C. The glucose content was not less than 1 g / L, and the cultivation ended after 20 days.

[0130] Example 5

[0131] In this example, perfusion culture was carried out. The culture model was a 50-ml shake flask. Both the basal medium and the perfusion culture medium were self-developed media. Inoculation was carried out at 1×10 6 cells / ml, the cell loading volume was 10 ml, the tilt angle of the shake flask was adjusted to 45° (the angle between the shake flask holder and the horizontal plane of the shaker was 45°). The culture parameters of the shake flask were set as follows: temperature 36.5°C, rotation radius 50 mm, rotation speed 160 rpm, humidity 80%, 5% CO 2 , and the inoculation and cultivation lasted for 4 days. Four days after inoculation and cultivation, samples were taken to detect cell density and cell viability. On Day 4, perfusion was carried out with the basal medium, centrifuged at 800 - 1500 rpm, the supernatant was removed, and perfusion was carried out at a perfusion rate of 1 VVD, that is, the cells were resuspended with 10 ml of basal medium; the operation was repeated after 24 h. On Day 6, perfusion was started with the perfusion culture medium, centrifuged at 800 - 1500 rpm, the supernatant was removed, and the cells were resuspended with 10 ml of perfusion culture medium; this operation was repeated once every 24 hours. On Day 7, the temperature was lowered to 34°C. When using the perfusion culture medium for perfusion, in addition to detecting cell density and cell viability, biochemical parameters and IgG content also needed to be detected every day. The glucose content was not less than 1 g / L, and the cultivation ended after 30 days. The cell supernatant removed daily needed to be clarified and filtered and stored at -20°C. After the cultivation ended, the clarified solution was subjected to affinity chromatography purification for quality research.

[0132] Example 6

[0133] In this example, shake flask Fed-batch culture was carried out. Both the basal medium and the feeding medium were self-developed by the applicant. The inoculation density was 0.5×10 6 cells / ml, the culture volume was 20 ml, the culture time was 16 days, the temperature was lowered to 32°C on Day 6. After the cultivation ended, a clarified solution was obtained, subjected to affinity chromatography, and the target protein was obtained for quality research.

[0134] In order to comparatively illustrate the influence of the culture process parameters of each example of the present disclosure on perfusion culture on the culture effect, the cell density, cell viability, IgG yield, etc. under different conditions were tested and compared.

[0135] As Figure 1 and Figure 2 shown, they are respectively the effects of the volume of cell fluid in the shaking tube on cell density, viability and IgG production in the culture method of Example 2 of the present disclosure. It can be seen from Figure 1 that although the volume of cell loading has little effect on the cell peak value, the cell viability is maintained well under the 10 ml culture condition, which is better than the 15 ml and 20 ml condition groups. It can be seen from Figure 2 that when the volume of cell loading is 10 ml, the IgG concentration is relatively high, and the IgG content is in the range of (1.0 - 2.0) g / L / day, which is better than the culture condition groups with the volume of cell loading of 15 ml or 20 ml.

[0136] As Figure 3 and Figure 4 shown, they are respectively the effects of the inclination angle between the shaking tube and the horizontal plane of the shaker on cell density, cell viability and IgG production in the culture methods of Example 2 and Example 3 of the present disclosure. It can be seen from Figure 3 that when the inclination angle between the shaking tube and the horizontal plane of the shaker is 60° or 45°, the cell peak value and viability are relatively high, and the peak density is (70 - 80)×10 6 cells / ml. It can be seen from Figure 4 that when the inclination angle between the shaking tube and the horizontal plane of the shaker is 45°, in the later stage of the culture time, the IgG content is in the range of (2.0 - 2.2) g / L / day, which is better than the culture condition groups with 60° and 90° inclinations.

[0137] As Figure 5 and Figure 6 shown, they are respectively the effects of different inoculation densities on cell density and IgG production in the culture methods of Example 2 and Example 4 of the present disclosure. It can be seen from Figure 5 and Figure 6 that when the inoculation density is as high as 20×10 6 cells / ml and when the inoculation density is 1.0×10 6 cells / ml, the IgG content shows basically the same performance under the same cell density and viability conditions. The cell peak value is (70 - 80)×10 6 cells / ml, and the IgG content is in the range of (1.0 - 2.0) g / L / day. High-density inoculation can reduce the perfusion time of the basal medium in the early stage, and perfusion culture can be directly carried out using the perfusion medium after inoculation.

[0138] According to the above conditions, preferably, for the cell culture method of a trispecific antibody targeting GPRC5D, BCMA, and CD3, the perfusion culture parameters based on the rocking tube model are as follows: the culture volume is 10 ml, the tilt angle is 45°, the rotation radius of the shaker is 50 mm, the shaker speed is 160 rpm, the culture temperature is 36.5 °C, and after the viable cell density (VCD) > 30×10 6 cells / ml, the temperature is lowered to 34 °C, and the seeding density is 1×10 6 cells / ml, and the glucose content is not less than 1 g / L.

[0139] As Figure 7 and Figure 8 shown, they are respectively the comparisons of cell density and IgG production under the Fed-batch culture and perfusion culture modes in the culture methods of Examples 5 and 6 of the present disclosure. As can be seen from Figure 7 , the perfusion culture mode can increase the peak cell density. As can be seen from Figure 8 , the total protein production under the perfusion culture mode is significantly higher than that under the Fed-batch culture mode.

[0140] As Figure 9 shown, they are respectively the comparisons of SEC-HPLC quality results under the Fed-batch culture and perfusion culture modes in the culture methods of Examples 5 and 6 of the present disclosure. As can be seen from Figure 9 , the perfusion culture mode can improve the purity of the SEC-HPLC main peak.

[0141] As Figure 10 shown, they are respectively the comparisons of iCIEF quality results under the Fed-batch culture and perfusion culture modes in the culture methods of Examples 5 and 6 of the present disclosure. As can be seen from Figure 10 , the perfusion culture mode can improve the purity of the iCIEF main peak.

[0142] As Figure 11 shown, they are respectively the comparisons of iCIEF acid region quality results under the Fed-batch culture and perfusion culture modes in the culture methods of Examples 5 and 6 of the present disclosure. As can be seen from Figure 11 , the perfusion culture mode can reduce the proportion of the iCIEF acid region, thereby improving the main peak purity.

[0143] The applicant declares that the present disclosure illustrates the detailed method of the present disclosure through the above embodiments, but the present disclosure is not limited to the above methods, that is, it does not mean that the present disclosure must rely on the above detailed methods to be implemented. Those skilled in the art should understand that they can still modify the technical solutions recorded in the foregoing embodiments, or perform equivalent replacements on some or all of the technical features; and these modifications or replacements do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present disclosure.

Claims

1. A cell culture method for efficiently and correctly expressing an antibody with an asymmetric structure, characterized in that, the method comprises the following steps: (1) Obtaining a mammalian cell capable of producing an antibody with an asymmetric structure; (2) Culturing the cell in step (1), obtaining a culture solution and performing separation and purification to obtain an antibody with an asymmetric structure, and the culture method is a perfusion culture method.

2. The method according to claim 1, wherein the culture medium used in the perfusion culture method comprises a basal medium and a perfusion medium.

3. The method according to claim 2, wherein the basal medium is an animal-free, protein-free, chemically defined medium, and the main components comprise the following substances: 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 5 - 7 g / L, glucose 3 - 8 g / L, sodium pyruvate 0.05 - 0.4 g / L, sodium chloride 3 - 5 g / L, potassium chloride 0.2 - 0.4 g / L, sodium selenite 0.0000005 - 0.000060 g / L, manganese sulfate 0.05 - 0.2 g / L, ethanolamine 5 - 25 μg / L, ferric citrate 1 - 10 mg / L, zinc sulfate 0.1 - 0.5 g / L, copper sulfate 0.02 - 0.15 g / L, glutathione 0.05 - 0.3 g / L, magnesium chloride 0.025 - 0.15 g / L, sodium dihydrogen phosphate 0.1 - 0.3 g / L, sodium bicarbonate 1 - 5 g / L, alanine 0.015 - 0.035 g / L, asparagine 0.01 - 0.035 g / L, arginine 0.06 - 0.30 g / L, aspartic acid 0.02 - 0.2 g / L, cystine 0.3 - 0.5 g / L, cysteine 0.05 - 0.2 g / L, glutamic acid 0.001 - 0.09 g / L, glycine 0 - 0.04 g / L, histidine 0.03 - 0.2 g / L, isoleucine 0.05 - 0.25 g / L, leucine 0.05 - 0.25 g / L, lysine 0.02 - 0.25 g / L, methionine 0.02 - 0.2 g / L, phenylalanine 0.055 - 0.075 g / L, proline 0.01 - 0.05 g / L, serine 0.03 - 0.25 g / L, threonine 0.07 - 0.15 g / L, tryptophan 0.005 - 0.025 g / L, tyrosine 0.05 - 0.2 g / L, valine 0.05 - 0.5 g / L, biotin 0.005 - 0.15 mg / L, calcium pantothenate 0.002 - 0.006 g / L, choline chloride 0.002 - 0.09 g / L, folic acid 0.002 - 0.006 g / L, inositol 0.005 - 0.02 g / L, nicotinamide 0.002 - 0.006 g / L, vitamin B6 0.002 - 0.006 g / L, vitamin B2 0.0002 - 0.0006 g / L, vitamin B1 0.002 - 0.006 g / L, vitamin B12 0.000005 - 0.000025 g / L, linoleic acid 0.001 - 0.1 g / L, block copolymer polyether F-68 (Pluronic F-68) 0.2 - 5 g / L.

4. The method according to claim 2 or 3, wherein the perfusion medium is an animal-free, protein-free, chemically defined medium, and the main components include the following substances: 4-(2-Hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES) 2 - 7 g / L, glucose 5 - 15 g / L, sodium pyruvate 0.1 - 0.6 g / L, potassium chloride 1 - 5 g / L, sodium selenite 0.0000005 - 0.000060 g / L, manganese sulfate 0.00000005 - 0.0000004 g / L, ethanolamine 5 - 25 μg / L, ferric citrate 0.01 - 1 g / L, zinc sulfate 0.001 - 0.05 g / L, copper sulfate 0.0005 - 0.01 g / L, glutathione 0.005 - 0.03 g / L, magnesium chloride 0.025 - 0.5 g / L, sodium dihydrogen phosphate 0.1 - 5 g / L, sodium bicarbonate 0.2 - 5 g / L, alanine 0.1 - 1.5 g / L, asparagine 0.1 - 3.5 g / L, arginine 0.6 - 6 g / L, aspartic acid 0.2 - 2 g / L, cysteine 0.05 - 0.2 g / L, glutamic acid 0.01 - 0.9 g / L, glycine 0 - 0.4 g / L, histidine 0.03 - 1.5 g / L, isoleucine 0.05 - 2.5 g / L, leucine 0.5 - 2.5 g / L, lysine 0.02 - 0.25 g / L, methionine 0.2 - 1 g / L, phenylalanine 0.55 - 0.75 g / L, proline 0.1 - 0.5 g / L, serine 0.3 - 2.5 g / L, threonine 0.07 - 0.15 g / L, tryptophan 0.05 - 0.25 g / L, tyrosine 0.05 - 1 g / L, valine 0.05 - 0.5 g / L, biotin 0.005 - 0.15 mg / L, calcium pantothenate 0.002 - 0.006 g / L, choline chloride 0.002 - 0.09 g / L, folic acid 0.002 - 0.006 g / L, inositol 0.005 - 0.02 g / L, nicotinamide 0.002 - 0.006 g / L, vitamin B6 0.002 - 0.006 g / L, vitamin B2 0.0002 - 0.0006 g / L, vitamin B1 0.002 - 0.006 g / L, vitamin B12 0.000005 - 0.000025 g / L, linoleic acid 0.001 - 0.1 g / L, block copolymer polyether F-68 (Pluronic F-68) 0.2 - 5 g / L.

5. The method according to any one of claims 1 - 4, wherein the antibody with an asymmetric structure comprises a domain specifically binding to GPRC5D.

6. The method according to any one of claims 1 - 5, wherein the antibody with an asymmetric structure is a trispecific antibody that can specifically bind to GPRC5D, BCMA, and CD3, and comprises the following polypeptides: The first polypeptide, comprising: (i) a heavy chain domain of an antigen-binding fragment Fab that can specifically bind to BCMA, (ii) a single-chain antibody (scFv) domain that can specifically bind to CD3, and (iii) a first Fc domain; The second polypeptide, comprising: a light chain domain of an antigen-binding fragment Fab that can specifically bind to BCMA; A third polypeptide, comprising: (i) a heavy chain single domain antibody (VHH) domain capable of specifically binding to GPRC5D and (ii) a second Fc domain; The heavy chain domain of the antigen-binding fragment Fab of the first polypeptide and the light chain domain of the antigen-binding fragment Fab of the second polypeptide form a domain that specifically binds to BCMA; the single-chain antibody (scFv) domain forms a domain that specifically binds to CD3; the heavy chain single domain antibody (VHH) domain forms a domain that specifically binds to GPRC5D; the first Fc domain and the second Fc domain associate with each other.

7. The method according to claim 5 or 6, wherein the domain specifically binding to GPRC5D comprises HCDR1 having the sequence shown in SEQ ID NO: 1, HCDR2 having the sequence shown in SEQ ID NO: 2, and HCDR3 having the sequence shown in SEQ ID NO:

3. Preferably, the domain specifically binding to GPRC5D comprises the sequence shown in SEQ ID NO:

16.

8. The method according to claim 6 or 7, wherein the domain specifically binding to CD3 comprises HCDR1 having the sequence shown in SEQ ID NO: 4, HCDR2 having the sequence shown in SEQ ID NO: 5, HCDR3 having the sequence shown in SEQ ID NO: 6, LCDR1 having the sequence shown in SEQ ID NO: 7, LCDR2 having the sequence shown in SEQ ID NO: 8, and LCDR3 having the sequence shown in SEQ ID NO: 9; and / or the domain specifically binding to BCMA comprises HCDR1 having the sequence shown in SEQ ID NO: 10, HCDR2 having the sequence shown in SEQ ID NO: 11, and HCDR3 having the sequence shown in SEQ ID NO: 12, LCDR1 having the sequence shown in SEQ ID NO: 13, LCDR2 having the sequence shown in SEQ ID NO: 14, and LCDR3 having the sequence shown in SEQ ID NO:

15.

9. The method according to any one of claims 6-8, wherein the domain specifically binding to BCMA comprises a heavy chain variable region shown in SEQ ID NO: 17 and a light chain variable region shown in SEQ ID NO: 18; and / or the single-chain antibody (scFv) domain specifically binding to CD3 comprises a heavy chain variable region shown in SEQ ID NO: 19 and a light chain variable region shown in SEQ ID NO:

20.

10. The method according to any one of claims 6-9, wherein the first polypeptide of the trispecific antibody comprises the amino acid sequence shown in SEQ ID NO: 21, the second polypeptide comprises the amino acid sequence shown in SEQ ID NO: 23, and the third polypeptide comprises the amino acid sequence shown in SEQ ID NO:

22.

11. The method according to any one of the preceding claims, wherein the mammalian cell for producing an asymmetric structure antibody is a CHO cell. Preferably, the CHO cell is selected from the group consisting of the following cell lines: DXB-11, DG-44, CHO-S, CHO-K1, CHO-K1(GS-KO) and other cell lines.

12. The method according to any one of the preceding claims, wherein the culture model is a shake tube or a shake flask; Preferably, the culture model is a tube shaking model, characterized in that: the culture volume accounts for 20-40% of the marked volume of the shaking tube, the inclination angle of the shaking tube is 45°-90°, the amplitude of the shaker is 25-50 mm, the rotation speed is 150-300 rpm, the inoculation density is 0.5-20×10 6 cells / ml, and the culture temperature is 34-37°C.

13. The perfusion culture process parameter control according to any one of the preceding claims includes: Samples are counted daily. When the cell density > 8×10 6 cells / ml, centrifuge at 800 - 1500 rpm, remove the supernatant, and resuspend the cells in the basal medium; repeat the above operation after 24 h; when the cell density > 30×10 6 cells / ml, centrifuge at 800 - 1500 rpm, remove the supernatant, and resuspend the cells in the perfusion medium; repeat the above operation after 24 h, and the culture time is 15 - 60 days.

14. The method according to any one of claims 12-13, preferably, the shaker parameters include an amplitude of 50 mm and a rotation speed of 160-300 rpm.

15. The method according to any one of claims 12-14, preferably, the culture volume is 10-20 ml.

16. The method according to claim 15, preferably, the culture volume is 10-15 ml.

17. The method according to any one of claims 12-16, preferably, the inclination angle of the shake tube is 45-60°.

18. The method according to any one of claims 12-17, preferably, the culture temperature of the shaker is 36-37 °C.

19. According to the method described in any one of claims 12-18, preferably, the inoculation density is 1×10 6 -20×10 6 cells / ml.

20. The method according to any one of claims 12-19, preferably, the cell density is regulated by cooling during the perfusion culture process.

21. According to the method of claim 20, preferably, the temperature reduction method is that when the cell density reaches 30×10 6 cells / ml, the culture temperature is reduced from 36 - 37°C to 32 - 34°C.

22. The method according to any one of claims 12-21, characterized in that the perfusion culture method further includes adding glucose, and the addition amount of glucose is 1-10 g / L.

23. The method according to any one of claims 12-22, with a culture volume of 10 ml, an inclination angle of 45°, a shaker rotation radius of 50 mm, a shaker rotation speed of 160 rpm, a culture temperature of 36.5 °C, VCD > 30×10 6 After cooling to 34 °C at 30×10 6 cells / ml, the inoculation density is 1×10 cells / ml, and the glucose content is not less than 1 g / L.

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