A cryopreservation formulation of antibody-producing cells, uses thereof and a method of cryopreservation

Abstract

The application discloses a freezing preparation of antibody production cells, application and freezing method thereof. The freezing preparation comprises 8-10% (v / v) DMSO, 0.1-0.3% (w / v) carboxymethyl cellulose and / or a salt thereof. The freezing preparation has simple components, is easy to realize and has good universality. The recovery activity rate of the antibody production cells subjected to long-term freezing can be greatly improved by using the freezing preparation and the freezing method.

Description

Technical Field

[0001] This invention is applied in the field of biotechnology, specifically relating to a cryopreservation preparation for antibody production cells, its application, and a cryopreservation method. Background Technology

[0002] Monoclonal antibodies (mAbs) and Fc fusion proteins have become mainstream drugs widely used in clinical treatment of autoimmune diseases and tumors. Mammalian cells are the preferred host for the production of therapeutic proteins because they can generate monoclonal antibodies with post-translational modifications, which are essential for bioefficacy and safety. Since the first mammalian-expressed therapeutic protein was produced by Chinese hamster ovary (CHO) cells in 1987, CHO cells have maintained a significant market share. In recent decades, CHO cells have been the most commonly used expression system for various forms of therapeutic proteins and antibodies. Mammalian expression systems are robust, scalable, and relatively inexpensive, and importantly, capable of post-translational modifications, which are crucial for the formation of therapeutic proteins. Over time, different guidelines have been used to regulate the drug development process for CHO cells, including cell line development (CLD) and manufacturing. These guidelines are used to ensure the safety, consistency, and reliability of products used by patients.

[0003] Mammalian cell culture plays a crucial role in the production of biopharmaceuticals, including monoclonal antibodies (mAbs). Over the past few decades, significant work has been done in cell culture technology to increase antibody yield while ensuring quality properties, while reducing production costs and time to market. Research has been conducted on cell engineering, cell culture processes, and culture medium optimization to improve monoclonal antibody yield. Key drivers of an ideal cell culture process are the cell lines used and their specific productivity, the ability to grow and maintain high cell volumes, the maintenance of stable process properties in bioreactors, and the consistent supply of raw materials downstream. Such cell culture processes have fostered close collaboration between biology and engineering to generate continuous innovation.

[0004] A typical mammalian cell culture process begins by thawing a cryopreserved cell line into a cell culture medium, where the cells are suspended and expanded in a shake flask or bag under controlled temperature and carbon dioxide conditions. The cells are then typically diluted and cultured in fresh medium to allow them to grow exponentially and produce a sufficient cell mass to initiate production culture. Cryopreservation or freezing of mammalian cells allows for the long-term preservation of cell lines. This is particularly important when a long time (years) is required to develop and license mammalian cell-derived biopharmaceuticals for commercial use.

[0005] One of the key requirements for the continued commercial success of biotherapeutic drugs is the availability of cell sources to initiate their production. For each target product, which may undergo years of development and then commercialization, a stable cell storage system is crucial. Cryopreservation is commonly used to establish cell banks for producing cell lines. The widely accepted two-tiered cell bank system, including master cell banks (MCBs) and working cell banks (WCBs), was first proposed at the Cell Culture Committee Symposium in 1963. This method of cell storage allows for safety and quality testing of cell sources, which is one of the ways we ensure the safe, reliable, and reproducible delivery of biotherapeutic drugs to patients. Therefore, cryopreservation has developed rapidly over the past two decades.

[0006] The 2015 edition of the Chinese Pharmacopoeia, Part III, "Preparation and Testing Procedures for Animal Cell Substrates for the Production and Testing of Biological Products," stipulates that cell cryopreservation should be performed when most cells are in the logarithmic growth phase, using the optimal cryopreservation method for the cell culture. Furthermore, "the same cooling process should be used for each cryopreservation, and the process should be recorded." Regarding the management of cell banks at all levels, "to ensure good cell viability after cryopreservation, the cell viability before cryopreservation should not be less than 90%. After cryopreservation, a certain amount of cryopreservation tubes representing the entire cryopreservation process should be taken for thawing, and the viability of the thawed cells should not be less than 80%." For the evaluation of cryopreserved cells, "the stability of cells under cryopreservation and cryopreservation conditions can be verified by periodically thawing cells and using data on the viability of thawed cells." The "General Principles for Quality Control Technology Evaluation of Mammalian Cells Used in the Production of Recombinant Products" also states that "when stored cells are thawed and used to produce products, studies and measurements of cell viability and functional activity, which are closely related to cell quality, should be conducted to confirm the stability of the expression capacity of the thawed cells"; and "if production has not been carried out for a long time, the cell bank used for production should be tested for viability at the intervals specified in the marketing application." Multiple regulations emphasize the importance of examining cell viability, and one of the factors affecting the viability of thawed cells is a suitable cryopreservation protocol, including the cryopreservation method and formulation.

[0007] The basic principle of cell cryopreservation and thawing is slow freezing and rapid thawing, which maximizes cell viability. Currently, serum-free cell banks often use cell culture media with 5%-15% DMSO as a cryoprotectant, which increases cell membrane permeability to water. Cryopreservation technology uses very low temperatures to preserve the structure and function of living cells. However, for some engineered cells, this concentration of DMSO cannot guarantee that the cell viability after thawing and passage will meet regulatory requirements. The biotechnology industry has developed different commercial cell cryopreservation media or different cryopreservation combination formulations for different cell types, making cryopreservation media more personalized, but greatly reducing their universality. At present, the viability of mammalian cells expressing monoclonal antibodies after thawing is generally around 85%. Three key variables determine how mammalian cells respond to cryopreservation and the process of thawing from the frozen state: (a) the rate of cell cooling; (b) the type and concentration of the protective additives (cryoprotectants) used; and (c) the freezing rate and the speed at which cells are heated to thaw from the frozen state. Since the first successful cryopreservation of cells in 1949, a general consensus has emerged regarding key parameters for cryopreservation, but these parameters can still be optimized for each cell type and cryopreservation scale. Summary of the Invention

[0008] The technical problem to be solved by the present invention is to address the shortcomings of existing cryopreservation preparations for antibody production cells, such as low cell resuscitation viability and poor cell viability after long-term (more than 1 month) cryopreservation of antibody production cells, and to provide a cryopreservation preparation for antibody production cells, its application and cryopreservation method.

[0009] The inventors reduced the DMSO content in traditional cryopreservation formulations and combined them with carboxymethyl cellulose and / or its salts, conventional cell culture medium, and culture medium that had been used to culture cells to be cryopreserved, resulting in a new type of cryopreservation formulation. This formulation can reduce cell damage caused by ice crystal formation during cryopreservation and thawing of antibody-producing cells, and provide a more suitable external environment for the cells, thereby significantly improving the recovery survival rate of long-term cryopreserved antibody-producing cells.

[0010] To address the aforementioned technical problems, one of the technical solutions provided by this invention is: a cryopreservation preparation for antibody production cells, wherein the cryopreservation preparation comprises 8-10% (v / v) DMSO, 0.1-0.3% (w / v) carboxymethyl cellulose and / or its salts, wherein the DMSO content (v / v) is, for example, 8%, 8.2%, 8.5%, 9%, 9.2%, 9.5%, 9.7%, 9.8%, 9.9%, or 10%; and the carboxymethyl cellulose and / or its salts content (v / v) is, for example, 0.1%, 0.15%, 0.2%, 0.25%, or 0.3%.

[0011] In this invention, v / v refers to volume fraction, and w / v refers to mass-volume fraction.

[0012] The antibody-producing cells may be conventional antibody-producing cells in the art.

[0013] Preferably, the cryopreservation preparation further comprises 40-50% (v / v) cell culture medium and 41.7-49.9% (v / v) conditioned medium; the cell culture medium is preferably Hycell CHO cell culture medium, and the conditioned medium is prepared by the following steps: the antibody-producing cells are inoculated into the cell culture medium for culture, and the supernatant is taken after centrifugation as the conditioned medium; the centrifugation is preferably 250g for 5 minutes.

[0014] Those skilled in the art can routinely select a cell culture medium suitable for the antibody-producing cells.

[0015] Those skilled in the art can routinely select culture conditions that are conducive to the survival of the antibody-producing cells in the cell culture medium.

[0016] Preferably, the culture conditions are 37°C, 5% CO2 incubator, shaker culture for 3 days, and / or, the antibody production cells are CHO-K1 cells transfected with antibody expression plasmids.

[0017] Preferably, the cryopreservation formulation comprises 0.2-0.3% (w / v) carboxymethyl cellulose and / or its salts and 41.7-49.8% (v / v) conditioned medium.

[0018] Preferably, in the antibody-producing cells, the antibody is an anti-VEGF monoclonal antibody or an anti-CD30 monoclonal antibody.

[0019] In a preferred embodiment of the present invention, the anti-VEGF monoclonal antibody is bevacizumab (trade name Avastin), and the CD30 monoclonal antibody is brentuximab.

[0020] In a preferred embodiment of the present invention, the carboxymethyl cellulose salt is sodium carboxymethyl cellulose.

[0021] In a preferred embodiment of the present invention, the cryopreservation preparation comprises 8% (v / v) DMSO, 0.2% (w / v) sodium carboxymethyl cellulose, 41.8% (v / v) conditioned medium and 50% (v / v) cell culture medium.

[0022] To solve the above-mentioned technical problems, the second technical solution provided by the present invention is: the application of the cryopreservation preparation as described in the present invention in the reagent for preparing cryopreserved antibody production cells.

[0023] To solve the above-mentioned technical problems, the third technical solution provided by the present invention is: a method for cryopreserving antibody production cells, wherein the antibody production cells are stored in a cryopreservation preparation as described in the present invention.

[0024] Preferably, the cryopreservation method includes the following steps: mixing the cryopreservation preparation as described above with the antibody-producing cells to obtain a final concentration of the antibody cells of 1–2 × 10⁻⁶. 7 / ml preferably 1.5×10 7 The mixture is prepared at a concentration of / ml. The mixture is placed in a cryovial, which is then placed in a programmed cooling box and placed in a -20°C freezer for 2-3 hours. It is then transferred to a -80°C freezer for 16-30 hours and finally transferred to a liquid nitrogen tank for long-term storage.

[0025] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0026] The reagents and raw materials used in this invention are all commercially available.

[0027] The positive and progressive effects of this invention are as follows:

[0028] The cryopreservation formulation and method for antibody production cells provided by this invention eliminate the need for cryopreservation solutions composed of multiple reagent combinations and personalized cell cryopreservation schemes. This results in a simple and easy-to-implement formulation with good versatility. The cryopreservation solution formulation of this invention can increase the cell recovery viability after one month of cryopreservation from no more than 70% to no less than 87%, preferably no less than 92%, under the original cryopreservation scheme. Furthermore, it can increase the cell recovery viability after long-term cryopreservation (e.g., 6 months) from no more than 72% to over 79%, preferably over 92%. Attached Figure Description

[0029] Figure 1 Comparison of cell viability in Examples 1-8 after cryopreservation of the same cells for 1 week;

[0030] Figure 2 Comparison of cell viability in Examples 1-8 after one month of cryopreservation of the same cells;

[0031] Figure 3 Comparison of cell viability in Examples 1-8 after 6 months of cryopreservation of the same cells;

[0032] Figure 4 This is a schematic diagram of the structure of the expression vector pV69. Detailed Implementation

[0033] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0034] This invention is achieved using the following technical solution:

[0035] All cells 1-6 used in this study were obtained from CHO-K1 host cells using the same transfection method.

[0036] Information on anti-VEGF monoclonal antibodies: amino acid sequence and bevacizumab (trade name) This is consistent with the previous statement. See patent US6884879 B1, "Anti-VEGF antibodies," and the literature, "Humanization of an anti-vascular endothelial growth factor monoclonal antibody for the therapy of solid tumors and other disorders," Cancer Res. 1997; 57(20):4593-9.

[0037] Information on anti-CD30 monoclonal antibody: The amino acid sequence is the same as that of brentuximab, see patent US7090843B1.

[0038] Preparation of linearized recombinant plasmids: Based on the amino acid sequences of the light and heavy chains of the monoclonal antibody, Shanghai Meiji and Suzhou Genewise designed and synthesized the nucleotide sequences of the light and heavy chain genes of the monoclonal antibody. These sequences were then cloned into a self-made PV69 vector to obtain the expression recombinant plasmids of the light and heavy chains. *E. coli* carrying the monoclonal antibody light and heavy chain recombinant plasmids were inoculated into LB medium and cultured overnight for plasmid preparation. The extracted plasmids were digested with Sal I, and the digested plasmids were extracted with phenol / chloroform and precipitated with anhydrous ethanol. The obtained plasmid DNA was washed with 75% ethanol, sterilely air-dried, dissolved in sterile water, and 1 μl of plasmid was diluted 100-fold to detect OD. 260nm and OD 280nm The value is calculated, and the plasmid concentration is determined.

[0039] Construction of the pV69 vector: Using glutamine synthetase (GS) as a selection marker, the specific structure of the vector is as follows:

[0040] Expression vector: pV69 (see structural diagram) Figure 4 )

[0041] Sources of carrier components:

[0042] Promoter: pCMV-IE; poly A: SV40 poly A; IRES: internal ribosome entry site; IVS (intervening sequence).

[0043] Selective markers: Prokaryotes - Ampr (Ampicillin); Eukaryotes - Neo (Neomycin)

[0044] Functional genes: glutamine synthase gene (gsE); anti-apoptotic gene bcl-2

[0045] Replicants: Prokaryote - Col E1 ori; Eukaryote - SV40 ori;

[0046] Multiple cloning sites: Sma I-Xma I-EcoRI-Bcl I

[0047] Preparation method of antibody production cells: The cells used are CHO-K1 (ATCC CCL-61). Preparation method of antibody production cells: CHO-K1 cells are prepared at 3.5 × 10⁻⁶ cells / year. 5 At a density of 0.5 ml, cells were seeded into 24-well plates. 24 hours after seeding, PEI transfection was used to introduce linearized recombinant plasmid light and heavy strands at a 3:1 ratio, total plasmid DNA at 0.5 μg / well, and DNA:PEI at a 1:2 ratio into CHO-K1 cells. 24 hours after transfection, the cells in the 24-well plates were digested, and 100 μl was seeded into 96-well plates at 1000 cells / well. The seeding medium was GMEM-S (SAFC) + 8% dFBS (PAA) + 1×GS Supplement (SAFC). 48 hours after seeding in the 96-well plates, MSX (Sigma) at a final concentration of 25 μM was added as a selection pressure, and the cells were cultured for 2–4 weeks until colony formation. High-expression clones were amplified into 24-well plates. After confluence, 1×10⁻⁶ cells were cultured. 5 For density quantification, clones with expression levels greater than 4000 ng / ml after 48 hours were selected and seeded into 96-well plates for subcloning using limiting dilution. The resulting monoclonal cells were also analyzed for expression levels in 96-well and 24-well plates to obtain adherent monoclonal cell lines. These monoclonal cells were then acclimated to serum-free culture to obtain suspension culture antibody production cell lines. Among them, anti-VEGF monoclonal antibody-1#, 2#, and 3# cells were three replicates of anti-VEGF monoclonal antibody cells, and anti-CD30 monoclonal antibody-4#, 5#, and 6# cells were three replicates of anti-CD30 monoclonal antibody cells.

[0048] Preparation and Counting of Cells for Cryopreservation: Count the antibody-producing cells prepared using the aforementioned method, ensuring a cell viability of at least 90% before cryopreservation. Cell viability (%) = (Number of viable cells / Total number of cells) * 100, where total number of cells = number of viable cells + number of dead cells. Countstar IC1000 cell counter. Counting Procedure: 1) Aseptically aspirate 1 ml of well-mixed cell suspension. 2) Stain 50 μl of the cell suspension with 50 μl of 0.2% trypan blue solution, and mix thoroughly by pipetting. 3) Aspirate 20 μl of the stained cell solution and spot it onto a cell counting chamber. 4) Insert the cell counting chamber into the cell counter slot and count the cells. 5) Record the viable cell density value and calculate the total number of cells required for cryopreservation.

[0049] Preparation of conditioned medium: After cell counting, the antibody production cell solution, which was cultured at 37°C and 5% CO2 in a shaker for 3 days and suspended in HycellCHO medium, was centrifuged (250g, centrifuged for 5 minutes). The supernatant after centrifugation is the conditioned medium for cryopreservation. The cells obtained by centrifugation are used for cryopreservation.

[0050] Preparation of sodium carboxymethyl cellulose solution: Add 98g of water for injection to a mixing bottle equipped with a stirrer. While stirring, slowly and evenly add 2g of sodium carboxymethyl cellulose to the mixing bottle, continuing to stir until the sodium carboxymethyl cellulose and water are completely mixed, thus preparing a sodium carboxymethyl cellulose solution with a concentration of 20g / L. Then, autoclave at 121℃ for 30 minutes and cool before use.

[0051] Preparation of methylcellulose solution: Add 98g of water for injection to a mixing bottle equipped with a stirrer. While stirring, slowly and evenly add 2g of methylcellulose to the mixing bottle, continuing to stir until the methylcellulose and water are completely mixed, preparing a methylcellulose solution with a concentration of 20g / L. Then, autoclave at 121℃ for 30 minutes and cool before use.

[0052] In the following examples, all cells used were antibody-producing cells obtained by the above method, all conditioned media were conditioned media prepared according to the above method, and all sodium carboxymethyl cellulose in the formulation were sodium carboxymethyl cellulose solutions prepared by the above method.

[0053] Example 1: Experiment on the cryopreservation effect of cell culture medium + 10 v / v % DMSO

[0054]

[0055] Prepare the cell cryopreservation solution according to the table above: 10% of the total cryopreservation volume of DMSO + 45% of the total cryopreservation volume of Hycell CHO medium + 45% of the total cryopreservation volume of conditioned medium.

[0056] The cells were centrifuged at 250g (Eppendorf 5430R) for 5 minutes. The cell pellet was mixed with cryopreservation buffer by pipetting and then aliquoted into cryovials (Thermo nunc 377267, 1.8ml container, cell cryopreservation specification 1.5*10⁻⁶) according to the predetermined cryopreservation specifications. 7 Cells / ml). The cryovials were placed in a programmed cooling box (Thermo Nalgene 5100-0001) and kept at -20°C for 2-3 hours, then transferred to -80°C for 16-30 hours, and finally transferred to liquid nitrogen for long-term storage. At 1 week, 1 month, and 6 months after entering the liquid nitrogen tank, three different numbered cell lines for each antibody were removed from the tank and thawed at 37°C for 2 minutes. The cell suspension was then transferred to 9 ml of Hycell CHO medium, centrifuged at 250g for 5 minutes, and resuspended in 30 ml of fresh Hycell CHO medium to dilute the cell pellet. The resuspended cell suspension was used for viability testing, and the results are shown in the table below.

[0057]

[0058] Example 2: Experiment on the cryopreservation effect of cell culture medium + 8v / v% DMSO

[0059]

[0060] Prepare the cell cryopreservation solution according to the table above: 8% of the total cryopreservation volume of DMSO + 46% of the total cryopreservation volume of Hycell CHO medium + 46% of the total cryopreservation volume of conditioned medium.

[0061] The cells were centrifuged at 250g for 5 minutes. The cell pellet was mixed with cryopreservation buffer and then aliquoted into cryovials according to the predetermined cryopreservation specifications. The cryovials were placed in a programmed cooling box and stored at -20°C for 2-3 hours, then transferred to -80°C for 16-30 hours, and finally transferred to liquid nitrogen for long-term storage. At 1 week, 1 month, and 6 months after storage in liquid nitrogen, three different numbered cell lines for each antibody were removed from the liquid nitrogen tank and thawed at 37°C for 2 minutes. The cell suspension was then transferred to 9 ml of Hycell CHO medium, centrifuged at 250g for 5 minutes, and resuspended in 30 ml of fresh Hycell CHO medium to dilute the cell pellet. The resuspended cell suspension was used for viability testing, and the results are shown in the table below.

[0062]

[0063] Example 3: Experiment on the cryopreservation effect of cell culture medium + 10 v / v% DMSO + 0.1 w / v% sodium carboxymethyl cellulose

[0064]

[0065] Prepare the cell cryopreservation solution according to the table above: 10% of the total cryopreservation volume of DMSO + 0.1% sodium carboxymethyl cellulose + 49.9% of the total cryopreservation volume of Hycell CHO medium + 40% of the total cryopreservation volume of conditioned medium.

[0066] The cells were centrifuged at 250g for 5 minutes. The cell pellet was mixed with cryopreservation buffer and then aliquoted into cryovials according to the predetermined cryopreservation specifications. The cryovials were placed in a programmed cooling box and stored at -20°C for 2-3 hours, then transferred to -80°C for 16-30 hours, and finally transferred to liquid nitrogen for long-term storage. At 1 week, 1 month, and 6 months after storage in liquid nitrogen, three different numbered cell lines for each antibody were removed from the liquid nitrogen tank and thawed at 37°C for 2 minutes. The cell suspension was then transferred to 9 ml of Hycell CHO medium, centrifuged at 250g for 5 minutes, and resuspended in 30 ml of fresh Hycell CHO medium to dilute the cell pellet. The resuspended cell suspension was used for viability testing, and the results are shown in the table below.

[0067]

[0068] Example 4: Experiment on the cryopreservation effect of cell culture medium + 8 v / v% DMSO + 0.2 w / v% sodium carboxymethyl cellulose

[0069]

[0070] Prepare the cell cryopreservation solution according to the table above: 8% of the total cryopreservation volume of DMSO + 0.2% sodium carboxymethyl cellulose + 41.8% of the total cryopreservation volume of Hycell CHO medium + 50% of the total cryopreservation volume of conditioned medium.

[0071] The cells were centrifuged at 250g for 5 minutes. The cell pellet was mixed with cryopreservation buffer and then aliquoted into cryovials according to the predetermined cryopreservation specifications. The cryovials were placed in a programmed cooling box and stored at -20°C for 2-3 hours, then transferred to -80°C for 16-30 hours, and finally transferred to liquid nitrogen for long-term storage. At 1 week, 1 month, and 6 months after storage in liquid nitrogen, three different numbered cell lines for each antibody were removed from the liquid nitrogen tank and thawed at 37°C for 2 minutes. The cell suspension was then transferred to 9 ml of Hycell CHO medium, centrifuged at 250g for 5 minutes, and resuspended in 30 ml of fresh Hycell CHO medium to dilute the cell pellet. The resuspended cell suspension was used for viability testing, and the results are shown in the table below.

[0072]

[0073] Example 5: Experiment on the cryopreservation effect of cell culture medium + 10 v / v% DMSO + 0.2 w / v% sodium carboxymethyl cellulose

[0074]

[0075] Prepare the cell cryopreservation solution according to the table above: 10% of the total cryopreservation volume of DMSO + 0.2% sodium carboxymethyl cellulose + 49.8% of the total cryopreservation volume of Hycell CHO medium + 40% of the total cryopreservation volume of conditioned medium.

[0076] The cells were centrifuged at 250g for 5 minutes. The cell pellet was mixed with cryopreservation buffer and then aliquoted into cryovials according to the predetermined cryopreservation specifications. The cryovials were placed in a programmed cooling box and stored at -20°C for 2-3 hours, then transferred to -80°C for 16-30 hours, and finally transferred to liquid nitrogen for long-term storage. At 1 week, 1 month, and 6 months after storage in liquid nitrogen, three different numbered cell lines for each antibody were removed from the liquid nitrogen tank and thawed at 37°C for 2 minutes. The cell suspension was then transferred to 9 ml of Hycell CHO medium, centrifuged at 250g for 5 minutes, and resuspended in 30 ml of fresh Hycell CHO medium to dilute the cell pellet. The resuspended cell suspension was used for viability testing, and the results are shown in the table below.

[0077]

[0078] Example 6: Experiment on the cryopreservation effect of cell culture medium + 8 v / v% DMSO + 0.3 w / v% sodium carboxymethyl cellulose

[0079]

[0080] Prepare the cell cryopreservation solution according to the table above: 8% of the total cryopreservation volume of DMSO + 0.3% sodium carboxymethyl cellulose + 41.7% of the total cryopreservation volume of Hycell CHO medium + 50% of the total cryopreservation volume of conditioned medium.

[0081] The cells were centrifuged at 250g for 5 minutes. The cell pellet was mixed with cryopreservation buffer and then aliquoted into cryovials according to the predetermined cryopreservation specifications. The cryovials were placed in a programmed cooling box and stored at -20°C for 2-3 hours, then transferred to -80°C for 16-30 hours, and finally transferred to liquid nitrogen for long-term storage. At 1 week, 1 month, and 6 months after storage in liquid nitrogen, three different numbered cell lines for each antibody were removed from the liquid nitrogen tank and thawed at 37°C for 2 minutes. The cell suspension was then transferred to 9 ml of Hycell CHO medium, centrifuged at 250g for 5 minutes, and resuspended in 30 ml of fresh Hycell CHO medium to dilute the cell pellet. The resuspended cell suspension was used for viability testing, and the results are shown in the table below.

[0082]

[0083] Example 7: Experiment on the cryopreservation effect of cell culture medium + 8 v / v% DMSO + 0.2 w / v% methylcellulose

[0084]

[0085] Prepare the cell cryopreservation solution according to the table above: 8% of the total cryopreservation volume of DMSO + 0.2% methylcellulose + 41.8% of the total cryopreservation volume of Hycell CHO medium + 50% of the total cryopreservation volume of conditioned medium.

[0086] The cells were centrifuged at 250g for 5 minutes. The cell pellet was mixed with cryopreservation buffer and then aliquoted into cryovials according to the predetermined cryopreservation specifications. The cryovials were placed in a programmed cooling box and stored at -20°C for 2-3 hours, then transferred to -80°C for 16-30 hours, and finally transferred to liquid nitrogen for long-term storage. At 1 week, 1 month, and 6 months after storage in liquid nitrogen, three different numbered cell lines for each antibody were removed from the liquid nitrogen tank and thawed at 37°C for 2 minutes. The cell suspension was then transferred to 9 ml of Hycell CHO medium, centrifuged at 250g for 5 minutes, and resuspended in 30 ml of fresh Hycell CHO medium to dilute the cell pellet. The resuspended cell suspension was used for viability testing, and the results are shown in the table below.

[0087]

[0088] Example 8: Experiment on the cryopreservation effect of cell culture medium + 10 v / v% DMSO + 0.2 w / v% methylcellulose

[0089]

[0090] Prepare the cell cryopreservation solution according to the table above: 10% of the total cryopreservation volume of DMSO + 0.2% methylcellulose + 49.8% of the total cryopreservation volume of Hycell CHO medium + 40% of the total cryopreservation volume of conditioned medium.

[0091] The cells were centrifuged at 250g for 5 minutes. The cell pellet was mixed with cryopreservation buffer and then aliquoted into cryovials according to the predetermined cryopreservation specifications. The cryovials were placed in a programmed cooling box and stored at -20°C for 2-3 hours, then transferred to -80°C for 16-30 hours, and finally transferred to liquid nitrogen for long-term storage. At 1 week, 1 month, and 6 months after storage in liquid nitrogen, three different numbered cell lines for each antibody were removed from the liquid nitrogen tank and thawed at 37°C for 2 minutes. The cell suspension was then transferred to 9 ml of Hycell CHO medium, centrifuged at 250g for 5 minutes, and resuspended in 30 ml of fresh Hycell CHO medium to dilute the cell pellet. The resuspended cell suspension was used for viability testing, and the results are shown in the table below.

[0092]

[0093] Summary: Data Statistics and Analysis of Examples 2-8

[0094] The comparison of the effects of the cryopreservation solutions prepared in the above embodiments on the cryopreservation of the aforementioned six types of cells for 1 week, 1 month, and 6 months is shown in the figures below. Figure 1 , Figure 2 , Figure 3 .

[0095] The p-values ​​of cell cryopreservation survival rates in Examples 3-6 (for anti-VEGF monoclonal antibody production) relative to the survival rate in Example 2 are shown in the table below.

[0096]

[0097] The p-values ​​of cell cryopreservation survival rates in Examples 3-6 (anti-CD30 monoclonal antibody production) relative to the survival rate in Example 2 are shown in the table below.

[0098]

[0099] The p-values ​​of cell cryopreservation survival rates from anti-VEGF monoclonal antibody production in Examples 4-6 relative to Example 3 are shown in the table below.

[0100]

[0101] The p-values ​​of cell cryopreservation survival rates from anti-CD30 monoclonal antibody production in Examples 4-6 relative to Example 3 are shown in the table below.

[0102]

[0103] Based on the above statistical results, we can conclude that:

[0104] Using Example 2 as a comparison, it can be seen that all results of Example 3 (including after 1 week, 1 month, and 6 months) are significantly different from those of Example 2, with p-values ​​after 1 week being less than 0.5 and p-values ​​after 1 month and 6 months being less than 0.005.

[0105] If Example 3 is used as the comparison object, the data of Examples 4-6 after 1 month and 6 months are significantly different from those of Example 2, with the p-values ​​after 1 month being less than 0.5 and the p-values ​​after 6 months being less than 0.001.

[0106] In summary, the cryopreservation formulation with 8-10% (v / v) DMSO, 0.1-0.3% (w / v) carboxymethyl cellulose and / or its salts, 41.7-49.9% (v / v) conditioned medium, and 40-50% (v / v) cell culture medium significantly improved the cryopreservation survival rate of antibody production cells compared to existing technologies. In this invention, the viability after 1 week of cryopreservation increased from 83.39% to no less than 91.74%, the viability after 1 month of cryopreservation increased from 74.09% to no less than 89.06%, and the viability after 6 months of cryopreservation increased from 61.85% to no less than 82.17%.

[0107] A preferred cryopreservation formulation consists of 8-10% (v / v) DMSO, 0.2-0.3% (w / v) sodium carboxymethyl cellulose, 41.7-49.8% (v / v) conditioned medium, and 40-50% (v / v) cell culture medium. In this invention, compared to a cryopreservation formulation with 0.1% (w / v) sodium carboxymethyl cellulose (as in Example 2), the cryopreservation survival rate for antibody production cells is further improved in medium- to long-term (1-6 months) cryopreservation. The survival rate after 1 month of cryopreservation is further increased to no less than 92.92%, and the survival rate after 6 months of cryopreservation is further increased to no less than 92.63%.

Claims

1. A cryopreservation preparation for antibody production cells, characterized in that, The cryopreservation formulation consists of 8 v / v% DMSO, 0.2-0.3 w / v% sodium carboxymethyl cellulose, 41.7-41.8 v / v% Hycell CHO cell culture medium, and 50 v / v% conditioned medium. The conditioned medium is prepared by the following steps: the antibody-producing cells are seeded in the Hycell CHO cell culture medium and cultured. After centrifugation, the supernatant is taken as the conditioned medium. The antibody-producing cells are CHO-K1 cells transfected with antibody expression plasmids.

2. The cryopreservation formulation as described in claim 1, characterized in that, The centrifugation was performed at 250g for 5 minutes.

3. The cryopreservation formulation as described in claim 2, characterized in that, The culture conditions were 37°C, 5% carbon dioxide incubator, and shaker culture for 3 days.

4. The cryopreservation formulation according to any one of claims 1-3, characterized in that, In the antibody-producing cells, the antibody is an anti-VEGF monoclonal antibody or an anti-CD30 monoclonal antibody.

5. The cryopreservation formulation as described in claim 4, characterized in that, The anti-VEGF monoclonal antibody is bevacizumab, and the CD30 monoclonal antibody is butuximab.

6. The cryopreservation formulation as described in claim 1, characterized in that, The cryopreservation formulation consists of 8 v / v% DMSO, 0.2 w / v% sodium carboxymethyl cellulose, 41.8 v / v% Hycell CHO cell culture medium and 50 v / v% conditioned medium.

7. The use of the cryopreservation formulation according to any one of claims 1-6 in the preparation of cryopreserved antibody production cells.

8. A method for cryopreserving antibody-producing cells, characterized in that, The antibody-producing cells are stored in the cryopreservation formulation as described in any one of claims 1-6.

9. The cryopreservation method as described in claim 8, characterized in that, The cryopreservation method includes the following steps: mixing the cryopreservation preparation and the antibody-producing cells to obtain a final concentration of the antibody-producing cells of 1~2×10⁻⁶. 7 The mixture is prepared by placing the mixture in cryovials, then placing the cryovials in a programmed cooling box, placing them in a -20°C freezer for 2-3 hours, transferring them to a -80°C freezer for 16-30 hours, and finally transferring them to a liquid nitrogen tank for long-term storage.

10. The cryopreservation method as described in claim 9, characterized in that, The final concentration of the antibody-producing cells obtained in the cryopreservation method is 1.5 × 10⁻⁶. 7 A mixture of particles per ml.

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