Cell culture method and application of engineered cysteine antibody expression cell

Abstract

The invention provides a cell culture method and application of an engineered cysteine antibody expression cell. The culture method comprises the following steps: carrying out cell culture on engineered cysteine antibody expression cells under the conditions that the initial temperature is 35-37 DEG C and the initial pH value is 6.7-7.3; in the cell culture process, when cell culture reaches a first condition, the initial temperature is reduced to 30-33 DEG C, and meanwhile, the initial pH is reduced to 6.6-7.2; one or more of the following additives are added in the cell culture process: L-cysteine and / or L-cystine. The method can solve the problem that a method capable of effectively reducing highly acidic charge variants of the engineered cysteine antibody is lacked in the prior art, and is suitable for the field of cell culture of the engineered cysteine antibody.

Description

Technical Field

[0001] The present invention relates to the field of engineered cysteine ​​antibody cell culture, and in particular to a cell culture method for engineered cysteine ​​antibody expressing cells and applications thereof. Background Art

[0002] Engineered cysteine ​​antibodies are a type of antibody that uses genetic engineering technology to precisely insert cysteine ​​residues at specific locations on the antibody, and then use the thiol group on the cysteine ​​to couple with toxin molecules to form a site-specific ADC drug (Antibody-Drug Conjugate). This engineering modification can accurately control the insertion position of the cysteine ​​residue and achieve precise site-specific coupling of the antibody and toxin molecules, thereby significantly improving the uniformity of the ADC drug. In addition, the introduction of cysteine ​​residues will not interfere with the folding and assembly of immunoglobulins, nor will it change the binding mode of antibodies and antigens. Therefore, the obtained ADC drug not only retains its anti-tumor activity in vivo, but also improves tolerance and reduces systemic toxicity.

[0003] During the cell culture stage, the engineered cysteine ​​sites will be cysteine ​​or glutathionylated to form capping. These different forms of capping change the charge distribution on the antibody surface, changing the IEX-HPLC / iCIEF charge heterogeneity analysis from a typical main peak to multiple peaks, resulting in a decrease in the proportion of the main peak and an increase in the proportion of acidic and basic peaks. As a specially modified antibody, the formation of acidic charge heterogeneities is also related to the traditional post-translational modification of antibody proteins, including deamidation, isomerization, oxidation, glycosylation, etc. These modifications may occur in the complementarity determining region (CDR) or other key areas of the antibody, resulting in changes in the charge distribution of the antibody molecule, thereby forming acidic charge heterogeneities.

[0004] The highly acidic charged heterogeneity of engineered cysteine ​​antibodies can have many effects on the quality and performance of the product, such as (1) affecting product quality: the highly acidic peak may represent the presence of unreacted cysteine, degradation products or other acidic impurities in the sample, the presence of which will reduce the purity of the ACD antibody intermediate. If there are a large number of acidic impurities in the antibody intermediate, it may affect its coupling efficiency with the toxin molecule, resulting in a reduced yield of the coupling product and uneven distribution of DAR, thereby affecting the efficacy and safety of the drug; (2) affecting the stability of the product: it may accelerate the hydrolysis or breakage of certain chemical bonds in the antibody intermediate, causing changes in the antibody structure, thereby reducing the chemical stability of the product; (3) affecting the separation and purification of the product: the highly acidic peak may overlap with other peaks or be difficult to distinguish, thereby increasing the difficulty of product separation and purification. In order to remove these impurities, more complex purification processes and more purification steps may be required; (4) affecting the analysis and detection of the product: the highly acidic peak may interfere with the results of chromatographic analysis, making it difficult to qualitatively and quantitatively analyze the product. In addition, in mass spectrometry detection, high acidic peaks may mask or interfere with the signals of target molecules, affecting the accuracy and reliability of the detection results.

[0005] At present, the research on reducing highly acidic charged heterogeneous proteins is all aimed at highly acidic charged heterogeneous proteins caused by traditional protein post-translation modification (PTM). However, for antibodies with engineered cysteine ​​modifications, different forms of capping change the charge distribution on the antibody surface, making charge heterogeneity analysis and optimization more complicated. The existing technology lacks a method that can effectively reduce highly acidic charged heterogeneous proteins of engineered cysteine ​​antibodies. Summary of the invention

[0006] The main purpose of the present invention is to provide a cell culture method for engineered cysteine ​​antibody expressing cells and its application, so as to solve the problem that there is a lack of a method that can effectively reduce the highly acidic charged heterogeneous bodies of engineered cysteine ​​antibodies in the prior art.

[0007] In order to achieve the above object, according to a first aspect of the present invention, a cell culture method for engineered cysteine ​​antibody expressing cells is provided, the cell culture method comprising: culturing the engineered cysteine ​​antibody expressing cells at an initial temperature of 35 to 37° C. and an initial pH of 6.7 to 7.3;

[0008] During the cell culture process, when the cell culture reaches the first condition, the initial temperature is lowered to 30-33° C., and the initial pH is simultaneously lowered to 6.6-7.2;

[0009] One or more of the following additives are added during the cell culture process: L-cysteine ​​and / or L-cystine.

[0010] Furthermore, the first condition includes that the density of live cells during the cell culture process is (10-16)×10 6 cells / mL or on the 6th day of cell culture.

[0011] Furthermore, the seeding density of the engineered cysteine ​​antibody expressing cells in the cell culture is (0.3-0.7)×10 6 cells / mL.

[0012] Furthermore, the total amount of the additive added is 1 to 100 mM.

[0013] Further, the cell culture comprises: culturing the engineered cysteine ​​antibody-expressing cells in a basal medium; adding a feed medium to the basal medium; preferably, the feed medium is added on the 3rd, 5th, 7th, 9th, 11th, and 12th day of the cell culture.

[0014] Further, the basal culture medium includes one or more of Actipro, Vega CHO, AM08, MaxA or Max X.

[0015] Further, the feed medium includes one or more of CB7a, CB7b, AF72V2, CDFS36, Max FA, Max FX or Max FB.

[0016] Furthermore, the volume of the basal culture medium is 30% to 70% of the volume of the cell culture system; preferably, the feed medium includes a first feed medium and a second feed medium; preferably, the first feed medium includes CB7a, AF72V2, Max FA or Max FX; preferably, the second feed medium includes CB7b, CDFS36 or Max FB; preferably, the total addition amount of the first feed medium is 15% to 35% of the volume of the basal culture medium, and the single addition amount of the second feed medium is 5% to 15% of the volume of the single addition amount of the first feed culture.

[0017] To achieve the above object, according to a second aspect of the present invention, there is provided a method for culturing engineered cysteine ​​antibody-expressing cells for reducing acidic charged heterogeneities in engineered cysteine ​​antibodies or for preparing drugs for engineered cysteine ​​antibodies.

[0018] By applying the technical solution of the present invention, when the engineered cysteine ​​antibody expressing cells are cultured, the engineered cysteine ​​antibody expressing cells are cultured under the conditions of an initial temperature of 35 to 37° C. and an initial pH of 6.7 to 7.3; and during the cell culture process, when the first condition is reached, the initial temperature is lowered to 30 to 33° C., and the initial pH is simultaneously lowered to 6.6 to 7.2; at the same time, one or more of the following additives are added during the cell culture process: L-cysteine ​​and / or L-cystine, which can reduce the highly acidic charge heterogeneity of the engineered cysteine ​​antibody, and is beneficial to improving the quality attributes and stability of the ADC antibody intermediate. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] The drawings constituting a part of the present application are used to provide a further understanding of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0020] Figure 1 The cell culture optimization process and result diagram of the engineered cysteine ​​antibody expressing cells of Examples 1 to 4 of the present invention are shown.

[0021] Figure 2 A curve chart showing the change of live cell density over time during the cell culture process of Example 1 of the present invention.

[0022] Figure 3 A curve chart showing the change of cell viability over time during the cell culture process of Example 1 of the present invention is shown.

[0023] Figure 4 A curve chart showing the change of lactic acid over time during the cell culture process of Example 1 of the present invention.

[0024] Figure 5 A graph showing the change in osmotic pressure over time during the cell culture process of Example 1 of the present invention.

[0025] Figure 6 A curve chart showing the change of live cell density over time during the cell culture process of Example 2 of the present invention.

[0026] Figure 7 A curve chart showing the change of cell viability over time during the cell culture process of Example 2 of the present invention is shown.

[0027] Figure 8 A curve chart showing the change of lactic acid over time during the cell culture process of Example 2 of the present invention.

[0028] Fig. 9 A graph showing the change in osmotic pressure over time during the cell culture process of Example 2 of the present invention.

[0029] Fig.10 A curve chart showing the change of live cell density over time during the cell culture process of Example 3 of the present invention.

[0030] Fig.11 A curve chart showing the change of cell viability over time during the cell culture process of Example 3 of the present invention is shown.

[0031] Fig.12 A curve chart showing the change of lactic acid concentration over time during the cell culture process of Example 3 of the present invention.

[0032] Fig.13 A graph showing the change in osmotic pressure over time during the cell culture process of Example 3 of the present invention.

[0033] Fig.14 A curve chart showing the change of offline pH over time during the cell culture process of Example 3 of the present invention.

[0034] Fig.15 A schematic diagram of the DIM spectrum of the control group antibody obtained by cell culture in Example 3 of the present invention is shown.

[0035] Fig.16 A schematic diagram of the DIM spectrum of the experimental group antibodies obtained by cell culture in Example 3 of the present invention is shown.

[0036] Fig.17 A graph showing the change in live cell density over time during the cell culture process of Example 4 of the present invention.

[0037] Fig.18 A curve chart showing the change of cell viability over time during the cell culture process of Example 4 of the present invention.

[0038] Fig.19 A graph showing the change in lactic acid concentration over time during the cell culture process of Example 4 of the present invention.

[0039] Fig. 20 A graph showing the change in osmotic pressure concentration over time during the cell culture process of Example 4 of the present invention is shown.

[0040] Fig.21 A curve chart showing the change of offline pH over time during the cell culture process of Example 4 of the present invention.

[0041] Fig. 22 A schematic diagram of the antibody DIM spectrum obtained by cell culture in Example 4 of the present invention is shown.

[0042] Fig.23A schematic diagram comparing the acidic peak proportions of antibodies obtained by cell culture in Example 3-C1, Example 3-C2 and Example 4-D1 of the present invention is shown. DETAILED DESCRIPTION

[0043] It should be noted that, in the absence of conflict, the embodiments and features in the embodiments of the present application can be combined with each other. The present invention will be described in detail below in conjunction with the embodiments.

[0044] Terminology explanation:

[0045] Competitive blocking effect of cysteine ​​at engineered sites: Engineered cysteine ​​sites have the ability to bind to thiol-containing groups. By increasing the concentration of the blocking agent cysteine ​​during cell culture, it competes with other blocking agents at the binding site, thereby effectively preventing thiol-containing substances such as GSH from binding to the engineered site.

[0046] As mentioned in the background technology, highly acidic charged heterogeneities of engineered cysteine ​​antibodies may affect the stability, analysis and detection, separation and purification of the product. However, the technical solutions for reducing highly acidic charged heterogeneities in the prior art are all aimed at highly acidic charged heterogeneities caused by post-translational modification of traditional proteins. However, the charge distribution of engineered cysteine ​​modified antibodies is changed due to different forms of capping, making the charge heterogeneity analysis and optimization more complicated. The prior art lacks a method that can effectively reduce the highly acidic peak of engineered cysteine ​​antibodies.

[0047] Therefore, in the present application, the inventors attempted to develop a new cell culture method for engineered cysteine ​​antibody-expressing cells, which can effectively reduce the highly acidic charged heteromers of engineered cysteine ​​antibodies, thereby making the target antibodies secreted by the cells higher in quality. Based on this, a series of protection schemes of the present application are proposed.

[0048] In a first typical embodiment of the present application, a cell culture method for engineered cysteine ​​antibody-expressing cells is provided, the cell culture method comprising: culturing the engineered cysteine ​​antibody-expressing cells at an initial temperature of 35 to 37°C (including but not limited to 35°C, 35.5°C, 36°C, 36.5°C or 37°C) and an initial pH of 6.7 to 7.3 (including but not limited to 6.7, 6.8, 6.9, 7.0, 7.1, 7.2 or 7.3); during the cell culture process, when the cell culture reaches the first condition, lowering the initial temperature to 30 to 33°C (including but not limited to 30, 31, 32 or 33°C), and simultaneously lowering the initial pH to 6.6 to 7.2 (including but not limited to 6.6, 6.7, 6.8, 6.9, 7.0, 7.1 or 7.2); adding one or more of the following additives during the cell culture process: L-cysteine ​​and / or L-cystine. The additive may also be a derivative of L-cysteine ​​or a derivative of L-cystine. The above-mentioned derivatives of L-cysteine ​​or derivatives of L-cystine are all sulfur-containing metabolites, which are derived from cysteine ​​during their synthesis. The above-mentioned derivatives include but are not limited to methionine, biotin, thiazolidine, thiamine, lipoic acid and coenzyme A.

[0049] Regarding the expression of engineered cysteine ​​modified antibodies, the different forms of capping formed during the cell culture process will change the charge distribution on the antibody surface, and the relationship between the difference in engineering site capping and the change in acidic charge heterogeneity is still unclear, making the charge heterogeneity analysis and optimization more complicated. It is difficult for existing technologies to solve the problem of excessive acidic charge heterogeneity in engineered antibodies.

[0050] Compared with traditional antibodies, engineered cysteine ​​antibodies have a higher proportion of acidic charge isomers. In addition to the acidic isomers formed by traditional PTMs, it may also be related to the end-capping modification of the engineering site. Deamidation, as a common PTM modification method, is considered to be one of the key factors causing the formation of acidic isomers. During the deamidation process, the amide group in the protein is converted into a carboxyl group. This chemical change directly leads to a change in the charge state of the protein, increasing its acidity, and thereby increasing the proportion of acidic charge isomers. In addition to traditional PTM modifications, the reason why engineered cysteine ​​antibodies have a higher proportion of acidic charge isomers may also be because the cysteine ​​at the engineering site is easily combined with thiol-containing acidic groups such as glutathione (GSH), which further increases its acidic charge isomers.

[0051] Generally speaking, lower temperatures slow down the rate of deamidation reaction, thereby slowing down the modification process. In addition, acidic environment (i.e., lower pH conditions) is not conducive to the generation of deamidation reaction intermediate succinimide, thereby delaying the process of deamidation reaction. Therefore, by finely regulating the temperature and pH value during the culture process, the deamidation reaction can be effectively controlled, thereby reducing the acidic isomer level of traditional antibodies. In the process of cell culture of engineered cysteine ​​antibody expression cells, the present application adopts a culture method controlled by lower temperature and pH when the temperature is lowered, so as to reduce the proportion of highly acidic charge heterogeneous bodies in engineered cysteine ​​antibodies. Although the method shows certain applicability, the effect is not obvious, proving that only by lowering the temperature and pH to interfere with traditional PTM modification, the formation of acidic charge heterogeneous bodies is reduced, and the effect of reducing the acidic charge heterogeneous bodies formed during the culture of engineered cysteine ​​antibody expression cells is not obvious.

[0052] As mentioned above, the formation of acidic charge isomers of engineered cysteine ​​is not only related to traditional PTMs, but also closely related to the end-capping modification of the engineering site, which together determine the acidic peak proportion of engineered cysteine.

[0053] In the process of reducing the excessively high acidic peak of antibody-expressing cells caused by traditional PTM, although the strategy of lowering the temperature is often adopted, the temperature range of the reduction is often small. Compared with the prior art, the temperature reduction strategy of the present application has a larger temperature reduction range and does not affect the growth and metabolism of the cells. Combined with the pH control strategy and the additive addition strategy, a significant effect has been achieved in reducing the acidic peak of engineered cysteine ​​antibody-expressing cells.

[0054] This application can reduce the acidic charge isomer ratio of antibodies by reducing the deamidation modification level of traditional PTMs through precise regulation of the temperature and pH value during cell culture. The specific implementation methods of this application have verified that the low pH environment combined with the lower temperature is controlled within the above range of variation, and the addition of the above additives during cell culture can significantly reduce the acidic charge isomer ratio.

[0055] The screening and use of additives in the present application mainly consider the influence of four aspects of the mechanism of action: first, by introducing different forms of cysteine ​​and utilizing the competitive capping effect of cysteine ​​at the engineered sites, the capping state of these sites can be precisely regulated, thereby affecting the antibody charge heterogeneity; second, based on the recognition that the TCA (Tricarboxylic Acid Cycle) cycle is the central link of energy metabolism in organisms, by using specific additives to promote the TCA cycle, the metabolic rate of glutathione (GSH) is indirectly accelerated, thereby reducing the potential interference caused by GSH capping; third, the thiomethyl group of neutral methionine can undergo certain chemical reactions under specific conditions and be converted into a thiol or other forms of sulfur atoms, thereby forming a disulfide bond with the thiol group of cysteine; fourth, in order to verify the effect of GSH capping on the acidic peak, an experimental group with the addition of GSH is set in the specific embodiments of the present application, and the effect of GSH capping on acidic charge heterogeneity is evaluated by observing the GSH capping and acidic peak changes at the engineered sites after the addition of GSH.

[0056] The inventors of the present application have found through a large number of experimental screenings and analyses that the competitive blocking effect of different forms of cysteine ​​at the engineered site can effectively increase the Cys (cysteine) blocking ratio; the Cys content in the culture medium can change the GSH blocking ratio of the engineered site, and the addition of GSH can increase the GSH blocking ratio of the engineered site, resulting in an increase in the acidic peak. The additive of the present application is preferably one or more of L-cysteine, L-cystine, a derivative of L-cysteine, or a derivative of L-cystine. However, the addition of the above additives during the cell culture process, although it can reduce GSH at the engineered site, has no obvious effect on the improvement of the formation of acidic charge heteromers.

[0057] The present application uses specific additives to effectively reduce the end-capping modification of acidic groups such as GSH at the engineering site, and combines the low temperature and low pH culture process during cell culture to reduce the deamidation modification of traditional PTM, thereby greatly reducing the content of acidic charge variants in cysteine ​​antibodies and solving the problem of excessive acidic charge heterogeneities in engineered antibodies.

[0058] In a preferred embodiment, the first condition includes that the density of viable cells during cell culture is (10-16)×10 6 cells / mL or on the 6th day of cell culture.

[0059] In the cell culture process of the present application, if the viable cell density (i.e., the viable cells of the engineered cysteine ​​antibody-expressing cells) reaches 10×10 6 cells / mL, and when it reaches 10×10 6cells / mL, lower the initial temperature and initial pH of the cell culture; however, if the viable cell density does not reach 10×10 cells / mL during the 1-6 day culture, 6 cells / mL, that is, the initial temperature and initial pH of the cell culture are lowered on the 6th day of culture. The above two conditions of lowering temperature and pH can be flexibly selected by those skilled in the art according to the actual growth conditions of the cell culture.

[0060] In a preferred embodiment, the seeding density of engineered cysteine ​​antibody expressing cells in cell culture is (0.3-0.7)×10 6 cells / mL.

[0061] In a preferred embodiment, the total amount of additives added is 1 to 100 mM, including but not limited to 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85, 90, 95 or 100 mM, preferably 2.5 to 100 mM; preferably, the amount of additives added in each batch is ≥ 2.5 mM.

[0062] The additive of the present application is preferably one or more of L-cysteine, L-cystine, L-cysteine ​​derivatives or L-cysteine ​​derivatives, by increasing the engineering site Cys (cysteine) end-capping modification, thereby reducing GSH end-capping modification, reducing the acidic peak proportion. The amount of the additive supplemented by the present application and the time of supplementation will not affect the growth and metabolism of the engineered cysteine ​​antibody expressing cells, so the additive addition period of the present application can be added in batches during the process of cell culture, and only needs to meet the period of antibody expression in the middle and late stages of cell culture, and the cell culture system contains the above-mentioned additive to achieve the increase of the engineering site Cys end-capping modification. In the specific embodiment of the present application, additives are added in batches on the 5th, 8th and 11th days of cell culture, and the increase of the engineering site Cys end-capping modification can be achieved. In the previous research of the present application, additives were added in batches on the 0th, 3rd, 5th, 7th, 9th, 11th days of cell culture, and the effect of adding additives in batches on the 5th, 8th and 11th days of cell culture was consistent, that is, the increase of the engineering site Cys end-capping modification was achieved, but the metabolism of the cell was not affected. Therefore, those skilled in the art can add the additives in batches during the cell culture process according to the actual cell culture conditions and conventional additive addition strategies.

[0063] In a preferred embodiment, the cell culture comprises: culturing engineered cysteine ​​antibody-expressing cells in a basal medium; during the cell culture process, adding a feed medium to the basal medium; preferably, the feed medium is added on the 3rd, 5th, 7th, 9th, 11th, and 12th days of cell culture.

[0064] In a preferred embodiment, the basal medium comprises one or more of Actipro, Vega CHO, AM08, Max A, and Max X; preferably, the feed medium comprises one or more of CB7a, CB7b, AF72V2, CDFS36, Max FA, Max FX, or Max FB. Preferably, the basal medium is Actipro, and the feed medium is CB7a and CB7b.

[0065] In a preferred embodiment, the volume of the basal culture medium is 30% to 70% of the volume of the cell culture system; preferably, the feed medium includes a first feed medium and a second feed medium; preferably, the first feed medium includes CB7a, AF72V2, Max FA or Max FX; preferably, the second feed medium includes CB7b, CDFS36 or MaxFB; preferably, the total addition amount of the first feed medium is 15% to 35% of the volume of the basal culture medium; preferably, the single addition amount of the second feed medium is 5% to 15% of the volume of the single addition amount of the first feed culture.

[0066] The culture medium for the above-mentioned cell culture of the present application includes but is not limited to the types of the above-mentioned basal culture medium and feed culture medium. Those skilled in the art can screen and use the culture medium according to actual needs. The culture method of the engineered cysteine ​​antibody-expressing cells of the present application can reduce the production of highly acidic charged heterogeneous bodies and reduce the proportion of their acidic peaks, thereby improving the quality attributes and stability of ADC antibody intermediates. The volume of the basal culture medium and the amount of feed culture medium added include but are not limited to the above ranges, and those skilled in the art can adjust them according to the actual cell growth conditions.

[0067] The volume of the above-mentioned cell culture system is the volume of the culture container for cell culture. Those skilled in the art can flexibly select a suitable culture container according to actual production needs, and adjust the two according to the range of the addition amount of the basal culture medium and the feed culture medium recorded above, and the effect of the present application can be achieved.

[0068] In a preferred embodiment, the cells are harvested on the 14th day of culture or when the cell viability is lower than 70%.

[0069] The method of cell culture of engineered cysteine ​​antibody-expressing cells in this application aims to significantly reduce the proportion of highly acidic charged heterogeneous bodies by adjusting the culture process (such as using lower temperature and pH strategies after cooling) and using additives. Using the cell culture method of this application, the acidic peak proportion of engineered cysteine ​​antibodies was reduced to 32.12%, which is much lower than the unoptimized level. At the same time, the cell culture method of this application also increases the proportion of Cys capping modifications at the engineering site, thereby helping to enhance the structural stability of ADC antibody intermediates and is conducive to the development of the field of engineered cysteine ​​antibodies.

[0070] In a second typical embodiment of the present application, a method for culturing the above-mentioned engineered cysteine ​​antibody cells is provided for reducing acidic charge heterogeneities in engineered cysteine ​​antibodies or preparing drugs for engineered cysteine ​​antibodies. When the method for culturing the above-mentioned engineered cysteine ​​antibody cells of the present application is applied to the preparation of ADC drugs, the quality attributes and stability of the engineered cysteine ​​antibodies can be improved.

[0071] The beneficial effects of the present application will be further explained in detail below in conjunction with specific embodiments.

[0072] Unless otherwise specified, all reagents in the examples of this application are conventional commercially available products. The equipment information is shown in Table 1, and the reagent information is shown in Table 2.

[0073] Table 1

[0074]

[0075]

[0076] Table 2

[0077] Reagent name level brand Part Number batch number CD02 Cell culture Quacell A11002 16G02 Actipro Cell culture Hyclone SH31037.01 MJ30641099C 10% ADCF Antifoam Solution Cell culture Hyclone SH30897.02 AH3001456 Cell Boost 7a Cell culture Hyclone SH31026.01 MJ30646393B Cell Boost 7b Cell culture Hyclone SH31027.01CN MH29971903B Vega CHO Cell culture OPM P121662 17080803A AM08 Cell culture OPM C210838 16121702A Max A Cell culture Maibang MB1101.102 MB231228A01 Max X Cell culture Maibang MB1113.102 MB230726A AF72V2 Cell culture OPM C230802 17080303A Max FA Cell culture Maibang MB1201.102 MB20230913A01 Max FX Cell culture Maibang MB1220.102 MB230705A01 CDFS36 Cell culture OPM C672069 16111002C Max FB Cell culture Maibang MB1202.102 MB231011A01 Glucose Cell culture Sigma 1.04074.0500 K51565174106 <![CDATA[NaHCO3]]> AR Sinopharm 10018960 20210913 <![CDATA[Na2CO3]]> AR Sinopharm 10019260 20190409 NaOH AR Sinopharm 10019718 20190402 L-Cysteine Cell culture Aladdin C108238-100g K2307438 L-Cystine Cell culture Aladdin C118619-25g H1231042 Zinc sulfate heptahydrate Cell culture Aladdin Z118879-500g L1926011 L-Methionine Cell culture Aladdin M101131-100g J2317037 Reduced Glutathione Cell culture Aladdin G105427-5g J1915128

[0078] The host cell CHO-K1Q used in the examples of the present application was purchased from Zhongshan Kangtian Shenghe Biotechnology Co., Ltd. (referred to as Kangsheng Bio). By constructing the cell line to express an engineered cysteine ​​antibody with two mutation sites, it is finally possible to achieve site-specific conjugation of DAR4 (4 drug molecules are conjugated to each antibody molecule). Figure 1 A schematic diagram of the process flow of the engineered cysteine ​​antibody cell culture scheme of Examples 1 to 4 of the present application is shown.

[0079] Example 1

[0080] This example uses different culture media and combinations to screen a culture medium combination with a relatively low acidic peak ratio, and evaluates its effect on antibody expression. The specific experimental steps are as follows:

[0081] Cell recovery and expansion: The cells were recovered and passaged in CD02 medium containing 25 μM MSX.

[0082] Process detection and analysis: Samples were taken regularly during the culture process to detect cell density, cell viability, and nutrient and metabolite concentrations. The protein expression of the samples on the harvest day was analyzed (HPLC-Protein A), and the antibody charge heterogeneity was detected after affinity purification (IEX).

[0083] Experimental design: There are 5 groups in total in the experiment, and the corresponding groups are shown in Table 3.

[0084] In this example, culture media suitable for culturing engineered cysteine ​​antibody-expressing cells were screened, and 5 culture media and combinations were selected. The basal culture media were Actipro, Vega CHO, AM08, Max A and Max X, and the feed culture media were CB7a&CB7b, AF72V2&CDFS36, Max FA&Max FB and Max FX&Max FB. The culture media were screened in a shake tube.

[0085] Cell culture: (0.4~0.6)×10 6 The initial density of cells / mL was inoculated into the basal culture medium, the culture system was 50mL shaking tube, the initial culture volume was 20mL, the initial temperature was 36.5℃, the rotation speed was 225rpm, the humidity was 80%, and the CO2 concentration was 6%.

[0086] Cooling strategy: When the density of viable cells reaches 12×10 6 cells / mL and cooled to 33.0℃.

[0087] Feed strategy: feeding started on the 3rd day. The first feed medium and the second feed medium were added at 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, and 3.0% + 0.3% of the initial culture volume on the 3rd, 5th, 7th, 9th, 11th, and 12th days, respectively. The feed amount can be adjusted appropriately according to the actual cell growth conditions. The actual feed addition amount is shown in Table 3.

[0088] Sugar supplementation strategy: Use a sugar acid analyzer to detect the residual sugar content. When the glucose concentration in the culture system is lower than 6g / L, add glucose to the culture system to 8g / L.

[0089] Harvest standard: Harvest on the 14th day of culture.

[0090] Table 3

[0091]

[0092] The antibody expression and IEX test results are shown in Table 4. Different culture medium combinations had an expression level of 4.58-6.24 g / L at harvest, among which the acidic peaks of groups A1, A3, and A4 (Actipro+CB7a&CB7b, AM08+AF72V2&CDFS36, and MaxA+Max FA&MaxFB) accounted for relatively low proportions, 65.16%, 68.12%, and 67.82%, respectively, and the expression levels were 6.24 g / L, 5.45 g / L, and 4.32 g / L, respectively. The acidic peak of the Actipro+CB7a&CB7b culture medium combination was relatively low and the expression level was high, and this culture medium combination was selected for subsequent experimental exploration.

[0093] Table 4

[0094]

[0095] In Table 4, Peak 1 to Peak 7 refer to being uniformly divided into 7 different peaks according to the peak elution time. Among them, based on the fact that acidic substances will elute before other substances during the ion exchange process, the ratio of the sum of the peak areas of Peak 1, Peak 2, and Peak 3 to the total area of ​​the peaks is divided into the acidic peak ratio. Peak 1 to Peak 7 and the acidic peak ratio in the following table have the above meanings.

[0096] The cell growth and metabolism results of different groups in this example are as follows: Figure 2 to Figure 5 As shown, Figure 2 is a curve graph showing changes in cell density over time; Figure 3 The curve of cell viability changing with time; Figure 4 is a curve of lactic acid concentration changing with time; Figure 5 This is a graph showing the change of osmotic pressure over time.

[0097] Example 2

[0098] Based on Example 1, this example uses a combination of Actipro+CB7a&CB7b culture medium and additives with different mechanisms of action to explore the effects of additives on engineering site capping and acidic charge heterogeneity. The specific experimental steps are as follows:

[0099] Cell recovery and expansion: The cells were recovered and passaged in CD02 medium containing 25 μM MSX.

[0100] Process detection and analysis: Samples are taken regularly during the culture process to detect cell density, cell viability, and nutrient and metabolite concentrations. The protein expression of the samples on the day of harvest is analyzed (HPLC-Protein A), and the antibody charge heterogeneity (IEX) and mass spectrometry (DIM) are detected after affinity purification. Combined with the DIM results, the modification of the engineering site is quantitatively and qualitatively analyzed.

[0101] Experimental design: There are 6 groups in total in the experiment, and the specific groups are shown in Table 5.

[0102] In this example, additives with different mechanisms of action were screened, and the five selected additives were L-cysteine, L-cystine, zinc sulfate, methionine, and reduced glutathione. The screening of the additives was performed in a shake tube.

[0103] Cell culture: (0.4~0.6)×10 6 The initial density of cells / mL was inoculated into the basal culture medium, the culture system was 50mL shaking tube, the initial culture volume was 20mL, the initial temperature was 36.5℃, the rotation speed was 225rpm, the humidity was 80%, and the CO2 concentration was 6%.

[0104] Cooling strategy: When the density of viable cells reaches 12×10 6 When the concentration of cells / mL is above 33.0℃, cool down to 33.0℃.

[0105] Feeding strategy: feeding started on the 3rd day, and CB7a and CB7b were fed with 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, and 3.0% + 0.3% of the initial culture volume on the 3rd, 5th, 7th, 9th, 11th, and 12th days, respectively. The feed amount can be adjusted appropriately according to the actual cell growth conditions. The actual feed addition amount is shown in Table 5.

[0106] Sugar supplementation strategy: When the glucose concentration in the culture system was lower than 6 g / L, glucose was added to the culture system to reach 8 g / L.

[0107] Use of additives: L-cysteine, L-cystine, and reduced glutathione were added at 5 mM on days 5, 8, and 11, respectively; zinc sulfate was added at 40 μM on D3; and methionine was added at 5 mM in the basal medium.

[0108] Harvest standard: Harvest on the 14th day of culture.

[0109] Table 5

[0110]

[0111] The results of antibody expression and IEX quality are shown in Table 6. According to the expression results, different additives have little effect on antibody expression (the expression of group B3 is lower, which may be due to the high pH of alkaline-soluble L-cystine, which leads to a decrease in cell viability when added later). The IEX results show that the proportion of acidic peaks has not changed significantly. Further combined with the DIM test results, the changes in the end-capping form of the engineering site are analyzed. The antibody DIM test results are detailed in Table 7.

[0112] According to the results of DIM, L-cysteine ​​and L-cystine added to groups B2 and B3 effectively increased the blocking ratio of Cys at the engineered site and competitively inhibited the blocking ratio of GSH; while zinc sulfate and methionine added to groups B4 and B5 had no obvious effect on the blocking ratio of Cys; on the contrary, the addition of reduced glutathione significantly increased the blocking ratio of GSH. This result reversely verified the correlation between the blocking ratio of GSH at the engineered site and the GSH content in the culture medium. Combined with the analysis of IEX results, the proportion of acidic peaks showed an increasing trend with the increase of GSH blocking ratio, which also revealed that GSH blocking has an important influence on acidic isomers.

[0113] The cell growth and metabolism results of different groups in this example are as follows: Figure 6 to Figure 9 As shown. Among them, Figure 6 is a curve graph showing changes in cell density over time; Figure 7 The curve of cell viability changing with time; Figure 8 is a curve of lactic acid concentration changing with time; Fig. 9 This is a graph showing the change of osmotic pressure over time.

[0114] Table 6

[0115]

[0116] Table 7

[0117]

[0118] Note: The DIM analysis results are the end-capping forms of the engineered sites. In Table 7, "Cys (1), GSH (3)" is taken as an example, which means that there is 1 Cys end-capping and 3 GSH end-capping among the 4 end-cappings of the engineered sites. The specific interpretations of the other expressions in Table 7 in this form are similar.

[0119] Example 3

[0120] To explore the effect of traditional PTM reduction strategy (low temperature & low pH) on the acidic peak of engineered cysteine ​​antibodies, the specific experimental steps are as follows:

[0121] Cell recovery and expansion: Cells were recovered and passaged in CD02 medium containing 25 μM MSX and expanded for 2 generations in CD02 medium without MSX before inoculation.

[0122] Process detection and analysis: Samples were taken every day during the culture process to detect cell density, cell viability, and nutrient and metabolite concentrations. The protein expression of the samples on the harvest day was analyzed (HPLC-Protein A), and the antibody charge heterogeneity was detected after one-step affinity purification (IEX). Combined with DIM detection, the modification of the engineering site was quantitatively and qualitatively analyzed. Finally, PTM detection was used to verify the relationship between PTM changes and acidic charge isomers.

[0123] Experimental design: There are 2 groups in the experiment, and the specific groups are shown in Table 8.

[0124] Cell culture: (0.4~0.6)×10 6 The initial density of cells / mL was inoculated into Actipro basal medium, the culture system was a 3L reactor, the initial culture volume was 1.5L, the initial temperature was 36.5℃, the rotation speed was 250rpm, and the pH was controlled at 7.00+0.20. The pH was controlled by a single CO2 mode, and CO2 was automatically introduced when it exceeded the upper control limit, and 10% Na2CO3 was automatically added when it was lower than the lower control limit.

[0125] Protocol design: C1 is the control group, and C2 is the experimental group controlled by low temperature and low pH. The control group was cooled to 33.0℃, and the pH was not adjusted; the experimental group was cooled to 32.0℃, and the pH was adjusted to 6.9±0.15, and the rest of the culture process remained the same.

[0126] Process control: When the viable cell density reaches 12×10 6 When the concentration is above cells / mL, adjust to the designed temperature and pH.

[0127] Feeding strategy: feeding started on the 3rd day, and CB7a and CB7b were fed with 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, and 3.0% + 0.3% of the initial culture volume on the 3rd, 5th, 7th, 9th, 11th, and 12th days, respectively. The feed amount can be adjusted appropriately according to the actual cell growth conditions. The actual feed addition amount is shown in Table 8.

[0128] Sugar supplementation strategy: When the glucose concentration in the culture medium is lower than 4.5g / L, add glucose solution to 5.5g / L.

[0129] Harvest standard: Harvest on the 14th day of culture.

[0130] Table 8

[0131]

[0132] The cell growth and metabolism results were as follows Figure 10 to Figure 14 As shown, Fig.10is a curve graph showing changes in cell density over time; Fig.11 The curve of cell viability changing with time; Fig.12 is a curve of lactic acid concentration changing with time; Fig.13 is a graph showing changes in osmotic pressure over time; Fig.14 The offline pH curve is a time-dependent curve. The cell growth and metabolism under different culture conditions were normal, and the highest viable cell density was 20×10 6 cells / mL, the viability at harvest was above 90.0%, the lactic acid concentration at harvest was lower than 0.8g / L, the osmotic pressure was below 390mOsm / kg, and the pH fluctuated normally within the parameter control range during the culture process.

[0133] The results of antibody expression, IEX quality, and PTM detection are shown in Table 9. The antibody expression results showed that under this low pH and low temperature culture environment, the antibody expression level decreased slightly, from 6.53 g / L to 5.14 g / L. However, given that the cell viability was well maintained under this culture environment, there is still a lot of room for optimization in the later stage to increase the antibody expression level. The IEX quality results showed that compared with the control group, the low pH and low temperature control strategy adopted by the experimental group effectively reduced the acidic charge heterogeneity, specifically, the acidic peak ratio decreased from 63.57% to 50.13%.

[0134] The results of PTM showed that the low temperature and low pH control strategy adopted by the experimental group effectively reduced the deamidation modification in PTM, specifically the proportion of deamidation decreased from 9.0% to 7.5%. This result further proved that low temperature and low pH did reduce the production of acidic variants by reducing traditional PTM modifications (especially deamidation modifications). Although this control strategy to reduce traditional PTM is effective for engineered cysteine ​​antibodies, the effect is not significant, and the proportion of acidic charge heterogeneous bodies still needs to be further reduced.

[0135] The antibody DIM test results and graphs are shown in Table 10. Fig.15 and Fig.16 As shown, Fig.15 This is the DIM spectrum of the control antibody; Fig.16 This is the DIM spectrum of the antibody in the experimental group. According to the DIM results, there is a high proportion of GSH modification in the engineering sites of both the control group and the experimental group, which may also be the reason for the high proportion of acidic charge isomers. Therefore, it is still necessary to reduce or eliminate the acidic charge isomers caused by the end-capping of acidic groups such as GSH at the engineering sites.

[0136] Table 9

[0137]

[0138] Table 10

[0139]

[0140] Example 4

[0141] The effects of the combined use of additives and low temperature & low pH control strategies on acidic charge heterogeneity were investigated. The specific experimental steps are as follows:

[0142] Cell recovery and expansion: Cells were recovered and passaged in CD02 medium containing 25 μM MSX and expanded for 2 generations in CD02 medium without MSX before inoculation.

[0143] Process detection and analysis: Samples were taken every day during the culture process to detect cell density, cell viability, and nutrient and metabolite concentrations. The protein expression of the samples on the harvest day was analyzed (HPLC-Protein A), and the antibody charge heterogeneity (IEX), post-translational modification (PTM), and mass spectrometry (DIM) were detected after one-step affinity purification.

[0144] Experimental design: There is 1 group in the experiment, as shown in Table 11 below.

[0145] Cell culture: (0.4~0.6)×10 6 The initial density of cells / mL was inoculated into Actipro basal medium, the culture system was a 3L reactor, the initial culture volume was 1.5L, the initial temperature was 36.5℃, the rotation speed was 250rpm, and the pH was controlled at 7.00+0.20. The pH was controlled by a single CO2 mode, and CO2 was automatically introduced when it exceeded the upper control limit, and 10% Na2CO3 was automatically added when it was lower than the lower control limit.

[0146] Protocol design: The same culture process as that of Group C2 in Example 3 was adopted, and L-cysteine ​​was added during the culture process.

[0147] Process control: When the viable cell density reaches 12×10 6 cells / mL, cool to 32.0°C and adjust pH to 6.90+0.15.

[0148] Additives: L-cysteine ​​was added at 5 mM on days 5, 8, and 11.

[0149] Fed addition strategy: feeding started on the 3rd day, and CB7a and CB7b were fed with 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, 4.0% + 0.4%, and 3.0% + 0.3% of the initial culture volume on the 3rd, 5th, 7th, 9th, 11th, and 12th days, respectively. The feed amount can be adjusted appropriately according to the actual cell growth conditions. The actual feed addition amount is shown in Table 11.

[0150] Sugar supplementation strategy: When the glucose concentration in the culture medium is lower than 4.5g / L, add glucose solution to 5.5g / L.

[0151] Harvest standard: Harvest on the 14th day of culture.

[0152] Table 11

[0153]

[0154]

[0155] The cell growth and metabolism results of the low temperature & low pH + additives in this example and the low temperature & low pH group (C2 group) in Example 3 are compared as follows: Figures 17 to 21 As shown, Fig.17 is a curve graph showing the change of live cell density over time; Fig.18 The curve of cell viability changing with time; Fig.19 is a curve of lactic acid concentration changing with time; Fig. 20 is a curve of lactic acid concentration changing with time; Fig.21 The curve of offline pH changing with time. The highest living cell density was 22.8×10 6 cells / mL, and the viability was above 90.0% at harvest. The lactic acid concentration was 0.826g / L and the osmotic pressure was 359mOsm / kg at harvest. During the culture process, the pH fluctuated normally within the parameter control range.

[0156] The results of antibody expression, IEX quality and PTM detection are shown in Table 12. According to the expression results, the antibody expression was 5.16 g / L. Compared with the C2 experimental group in Example 3, the addition of L-cysteine ​​had no effect on the antibody expression. According to the IEX results, the addition of L-cysteine ​​to the C2 experimental group further reduced the proportion of acidic charged heterogeneous forms, and the data showed that it was reduced from 50.13% of the C2 group in Example 3 to 32.12%. According to the PTM results, the addition of L-cysteine ​​to the C2 experimental group had little effect on traditional PTM, specifically, it was reduced from 7.5% of the C2 group in Example 3 to 7.0%.

[0157] The antibody DIM test results and spectra are shown in Table 13 and Fig. 22 According to the results of DIM, the addition of L-cysteine ​​to the C2 experimental group successfully reduced the proportion of GSH capping at the engineered site, which indicates that L-cysteine ​​competes with glutathione at the engineered site, thereby increasing the proportion of Cys capping at the engineered site and correspondingly reducing the proportion of GSH acidic group capping.

[0158] Combined with the results of IEX (ion exchange chromatography), PTM (post-translational modification) and DIM (mass spectrometry), IEX showed that the acidic peak ratio was significantly reduced, while the reduction of deamidation in PTM was relatively small. DIM showed that the GSH (glutathione) acidic group at the engineered site was competitively replaced by a large number of Cys (cysteine). This further confirmed that the acidic charge isomers of engineered cysteine ​​are not only closely related to traditional PTMs, but also closely related to the end-capping modification of the engineered site, and the two together determine the acidic peak ratio of engineered cysteine.

[0159] Table 12

[0160]

[0161] Table 13

[0162]

[0163] The schematic diagram of the comparison of the antibody acidic peak after the cell culture in Example 3 and Example 4 of the present application is as follows Fig.23 As shown. Based on the above results, it can be seen that the selection of culture medium Actipro+CB7a&CB7b, combined with the use of additives (L-cysteine) and low temperature & low pH culture process, can significantly reduce the acidic charge heterogeneity of engineered cysteine ​​antibodies.

[0164] From the above description, it can be seen that the above embodiments of the present invention achieve the following technical effects: the present application optimizes the cell culture method of engineered cysteine ​​antibody-expressing cells, strictly controls the temperature and pH during the cell culture process, and controls the lower temperature and pH after cooling to reduce the acidic charge isomers caused by traditional PTM modification. At the same time, the additive L-cysteine ​​is used in combination to reduce the end-capping ratio of acidic groups such as GSH at the engineering site, and increase the ratio of specific Cys end-capping, thereby greatly reducing the acidic charge isomer ratio of engineered cysteine, solving the problem of high acidic charge isomers of engineered cysteine ​​antibodies in the prior art, and further improving the product quality and stability of ADC antibody intermediates.

[0165] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. For those skilled in the art, the present invention may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present invention shall be included in the protection scope of the present invention.

Claims

1. A cell culture method for engineered cysteine ​​antibody expressing cells, characterized in that: The cell culture method comprises: The engineered cysteine ​​antibody expressing cells are cultured at an initial temperature of 35 to 37° C. and an initial pH of 6.7 to 7.3; During the cell culture, when the cell culture reaches the first condition, lowering the initial temperature to 30-33° C., and simultaneously lowering the initial pH to 6.6-7.2; During the cell culture process, one or more of the following additives are added: L-cysteine ​​and / or L-cystine.

2. The cell culture method according to claim 1, characterized in that The first condition includes that the density of live cells during the cell culture process is (10-16)×10 6 cells / mL or on the 6th day of the cell culture.

3. The cell culture method according to claim 1, characterized in that The seeding density of the engineered cysteine ​​antibody expressing cells in the cell culture is (0.3-0.7)×10 6 cells / mL.

4. The cell culture method according to claim 1, characterized in that The total amount of the additive added is 1-100 mM.

5. The cell culture method according to claim 1, characterized in that: The cell culture comprises: culturing the engineered cysteine ​​antibody-expressing cells in a basic culture medium; During the cell culturing, adding a feed medium to the basal medium; Preferably, the feed medium is added on day 3, 5, 7, 9, 11 or 12 of the cell culture.

6. The cell culture method according to claim 5, characterized in that The basal culture medium includes one or more of Actipro, Vega CHO, AM08, Max A or Max X.

7. The cell culture method according to claim 6, characterized in that The feed medium includes one or more of CB7a, CB7b, AF72V2, CDFS36, Max FA, Max FX or Max FB.

8. The cell culture method according to claim 6, characterized in that: The volume of the basic culture medium is 30% to 70% of the volume of the cell culture system; Preferably, the feed medium comprises a first feed medium and a second feed medium; Preferably, the first feed medium comprises one or more of CB7a, AF72V2, Max FA or Max FX; Preferably, the second feed medium comprises one or more of CB7b, CDFS36 or Max FB.

9. The cell culture method according to claim 8, characterized in that: The total addition amount of the first feed culture medium is 15% to 35% of the volume of the basal culture medium, and the single addition amount of the second feed culture medium is 5% to 15% of the volume of the single addition amount of the first feed culture medium.

10. Use of the cell culture method for engineered cysteine ​​antibody expressing cells according to any one of claims 1 to 9 in reducing acidic charge heterogeneities in engineered cysteine ​​antibodies or preparing drugs for engineered cysteine ​​antibodies.

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