Quality regulation and control method for improving acidic charge heteroplasmon level of antibody protein

Abstract

The invention discloses a quality regulation and control method for improving the level of an acid charge variant of an antibody protein, which comprises the following steps: in a cell culture process, adding beta-nicotinamide adenine dinucleotide into a culture system, and finally obtaining an antibody with an increased proportion of the acid charge variant. The method provided by the invention enriches a method library for regulating and controlling the quality of an antibody protein product, is high in universality, low in cost, simple and convenient to operate and good in safety, and can be used for remarkably increasing the proportion of acidic charge variants for a plurality of monoclonal cell strains and a plurality of protein molecules; various adding modes in the method can achieve the technical effect of remarkably increasing the level of acidic charge variants, negative effects on cell growth and protein yield are avoided, lactic acid metabolism of cells can be remarkably improved, osmotic pressure can be reduced, a healthier cell culture environment can be provided, meanwhile, the protein amount is remarkably increased, and the cell culture cost is reduced. And the cIEF acid peak proportion of the protein product is increased by 8.7%-27.4%.

Description

Technical Field

[0001] The present invention belongs to the field of biotechnology, and in particular relates to a quality control method for improving the level of acidic charged heterogeneous forms of antibody proteins. Background Art

[0002] Protein drugs are an important component of current biopharmaceuticals. Among them, glycoproteins in the form of antibodies, antibody-based fusion proteins, and antibody-drug conjugates are the three most common antibody protein drugs. The quality attributes of antibody protein drugs mainly include protein purity, charge heterogeneity distribution, glycosylation modification, etc. Good quality attributes play a vital role in the normal function of such drugs. Among them, protein charge heterogeneity, that is, the uneven distribution of charge on the protein surface, is one of the most common key quality attributes of antibody drugs. The charge heterogeneity of antibody drugs may affect the three-dimensional structure, solubility, and interaction with other molecules of the antibody, thereby affecting its biological activity, stability, bioavailability, immunogenicity, etc. Therefore, close attention should be paid to the charge heterogeneity distribution of antibody proteins in the biopharmaceutical process.

[0003] The mechanisms of protein charge heterogeneity are complex and often associated with post-translational modifications. Studies have shown that the formation of basic charge variants is associated with glutamine cyclization, succinimidation, and carbon-terminal lysine or carbon-terminal amidation at the protein's N-terminus, while the formation of acidic charge variants is associated with sialylation, glycation, oxidation, or deamidation. Given the potential impact of charge variant distribution on drug efficacy and the complexity of the mechanisms of charge variant formation, researchers need to develop diverse regulatory strategies to regulate or control the distribution of charge variants at all stages of biopharmaceutical R&D, including cell line development, upstream cell culture process optimization, downstream protein purification process optimization, and formulation optimization. Acidic charge variants (ACVs) are a key quality control indicator due to their impact on protein function and stability. Therefore, developing effective quality control methods to improve the level of acidic charge variants in antibody proteins is of great theoretical and practical value.

[0004] In the prior art, changes in the upstream cell culture process can effectively regulate the distribution of charged heterogeneous species. For example, adding carboxypeptidase B at the end of cell culture can reduce the proportion of basic charged heterogeneous species by increasing the cleavage of lysine at the carbon terminus of the protein; while improving fermentation conditions such as lowering the culture temperature and adjusting the pH can reduce the proportion of acidic charged heterogeneous species by reducing deamination and reducing the reduction of disulfide bonds; in addition, light can trigger the oxidation of vitamins or amino acids in the culture medium, thereby increasing the proportion of acidic charged heterogeneous species; extending the culture time and increasing the culture temperature can also increase the proportion of acidic charged heterogeneous species. However, these methods will lead to a decrease in cell harvest viability and changes in other quality attributes such as glycoforms, so the scope of application is limited and the application prospects are poor. In the prior art, heterogeneous species are separated and removed through downstream processing steps. However, this method increases production costs and reduces yields, and has significant deficiencies in comprehensiveness and efficiency.

[0005] Therefore, this field needs to develop a quality control method for improving the level of acidic charged heterogeneous forms of antibody proteins that is highly universal, low-cost, simple to operate, safe, and has a wide range of applications, so as to enrich the method library for quality control of antibody protein products. Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a quality control method for improving the level of acidic charged heterogeneous forms of antibody proteins. During the cell culture process, β-nicotinamide adenine dinucleotide is added to the culture system, and finally an antibody with an increased proportion of acidic charged heterogeneous forms is obtained.

[0007] In a preferred embodiment, the concentration of β-nicotinamide adenine dinucleotide is 1-10 mM, more preferably, the concentration of β-nicotinamide adenine dinucleotide is 3-6 mM, and most preferably, the concentration of β-nicotinamide adenine dinucleotide is 3 mM, 4 mM, 5 mM or 6 mM.

[0008] In a preferred embodiment, the cells are cultured in a fed-batch format, and the culture is terminated after 14 days of culture or when the cell viability falls below 60%. More preferably, the culture is terminated after 14 days of culture or when the cell viability falls below 60%.

[0009] In a preferred embodiment, the initial culture volume is 50 mL, and the initial culture temperature is 36.5±1° C. More preferably, the initial culture temperature is 35.5° C., 36.5° C., or 37.5° C.

[0010] In a preferred embodiment, when the viable cell density reaches 10.00×10 6 cells / mL to 14.00×10 6cells / mL or on the sixth day of culture, the culture temperature was lowered to 33±1°C. More preferably, the viable cell density reached 10.00×10 6 cells / mL、11.00×10 6 cells / mL、12.00×10 6 cells / mL、13.00×10 6 cells / mL or 14.00×10 6 cells / mL, the culture temperature was lowered to 33±1°C. More preferably, the viable cell density reached 10.00×10 6 cells / mL to 14.00×10 6 cells / mL or when the culture reaches the sixth day, the culture temperature can be lowered to 33±1° C. More preferably, the culture temperature is lowered to 32° C., 33° C. or 34° C.

[0011] In a preferred embodiment, β-nicotinamide adenine dinucleotide is added to the culture system as follows: on the 8th day of culture, β-nicotinamide adenine dinucleotide is added based on the initial volume to a final concentration of 6 mM.

[0012] In a preferred embodiment, β-nicotinamide adenine dinucleotide is added to the culture system as follows: on the 7th day of culture, β-nicotinamide adenine dinucleotide is added to the initial volume at a concentration of 3 mM; the culture is continued until the 10th day, and 3 mM β-nicotinamide adenine dinucleotide is added based on the initial volume.

[0013] In a preferred embodiment, β-nicotinamide adenine dinucleotide is added to the culture system as follows: on the 9th day of culture, β-nicotinamide adenine dinucleotide is added based on the initial volume to a final concentration of 6 mM.

[0014] In a preferred embodiment, the cells are mammalian cells, including CHO cells.

[0015] In a second aspect, the present application provides the use of the aforementioned quality control method for increasing the level of acidic charged heterogeneous forms of antibody proteins in the preparation of antibody drugs and regulators, wherein the regulator is used to increase the level of acidic charged heterogeneous forms of antibody proteins.

[0016] The sample pretreatment method of the present invention has the following beneficial effects:

[0017] 1. The present invention provides a quality control method for improving the level of acidic charged heterogeneous variants of antibody proteins. During the cell culture process, a specific amount of β-nicotinamide adenine dinucleotide (NAD) is added at a specific time to improve the level of acidic charged heterogeneous variants of antibody proteins. At the same time, it has the advantages of low cost, simple operation and good universality, enriching the existing method library for quality control of antibody protein products.

[0018] 2. The present invention provides a quality control method for improving the level of acidic charged variants in antibody proteins. This method not only does not negatively impact cell growth and protein yield, but also significantly improves cellular lactate metabolism and reduces lactate levels. Furthermore, the protein quality is significantly improved, and the cIEF acidic peak ratio of the protein product is significantly increased. These advantages are conducive to enhancing the robustness of the cell culture process, thereby reducing the risk of scale-up.

[0019] 3. β-Nicotinamide adenine dinucleotide (NAD) is an oxidized β-nicotinamide adenine dinucleotide coenzyme naturally present in cells. It is non-animal derived, molecularly stable, and easy to use. At the same time, NAD is currently widely used in scientific research, food, health products, anti-aging products, medicine, and beauty, among other industries. This application uses NAD as a regulator to increase the level of acidic charged heteromers in antibody proteins, which has strong safety and applicability, and has broad prospects in the fields of antibody drug preparation. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 This is a graph showing the changes in viable cell density over time in each group in clone 1;

[0021] Figure 2 This is a curve diagram of the cell viability of each group in clone 1 over time;

[0022] Figure 3 This is a graph showing the changes in viable cell density over time in each group in clone 2;

[0023] Figure 4 This is a curve diagram of the cell viability of each group changing with time in clone 2;

[0024] Figure 5 This is a graph showing the changes in viable cell density over time in each group in clone 3;

[0025] Figure 6 This is a curve diagram of the cell viability of each group in clone 3 over time;

[0026] Figure 7 This is a graph showing the change of glucose concentration in the culture medium of each group over time in clone 1;

[0027] Figure 8This is a graph showing the change of lactic acid concentration in the culture medium of each group over time in clone 1;

[0028] Figure 9 This is a graph showing the osmotic pressure changes over time in the culture medium of each group in clone 1;

[0029] Figure 10 This is a graph showing the change of glucose concentration in the culture medium of each group over time in clone 2;

[0030] Figure 11 This is a graph showing the change of lactic acid concentration in the culture medium of each group over time in clone 2;

[0031] Figure 12 This is a graph showing the osmotic pressure changes over time in the culture medium of each group in clone 2;

[0032] Figure 13 This is a graph showing the change of glucose concentration in the culture medium of each group over time in clone 3;

[0033] Figure 14 This is a graph showing the change in lactic acid concentration in the culture medium of each group over time in clone 3;

[0034] Figure 15 This is a graph showing the osmotic pressure changes over time in the culture medium of each group in clone 3;

[0035] Figure 16 The bar graph shows the acidic peak ratio in capillary isoelectric focusing (cIEF) of each group in clone 1;

[0036] Figure 17 This is a bar graph showing the proportion of acidic peaks in capillary isoelectric focusing (cIEF) of each group in clone 2;

[0037] Figure 18 The figure is a bar graph showing the ratio of acidic peaks in capillary isoelectric focusing (cIEF) of each group in clone 3. DETAILED DESCRIPTION

[0038] The following is a clear and complete description of the technical solution of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts are within the scope of protection of the present invention.

[0039] In the following examples, all reagents used unless otherwise specified are commercially available. β-Nicotinamide adenine dinucleotide (NAD) was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd., catalog number N111610. Culture medium information: The basal medium was ActiPro medium (HyClone, SH31037); the feed medium was Cell Boost 7a (HyClone, SH31026) and Cell Boost 7b (HyClone, SH31027). If specific techniques or conditions are not described in the examples, it should be understood that the techniques or conditions described in the prior art or the instructions for commercial products were used.

[0040] Example 1

[0041] This example illustrates the operational process of using the quality control method provided in this application to increase the level of acidic charged variants of antibody proteins.

[0042] 1. Preparation of NAD solution

[0043] Prepare 10 mL of a 125 mM NAD solution: First, weigh 9.17 g of purified water into a clean beaker. Then, weigh 0.83 g of NAD powder and add it to the purified water. Mix using a magnetic stirrer until completely dissolved. Filter-sterilize the solution using a certified 0.22 μm filter in a biosafety cabinet. The prepared NAD solution can be stored in a -60°C refrigerator, protected from light, for up to 1 month. Before use, thaw the solution in a refrigerator at 2-8°C, protected from light, to avoid repeated freeze-thaw cycles.

[0044] 2. Initial cell culture

[0045] Batch fed-batch culture of a single clone of CHO cells was performed in 250 mL shake flasks. Cell culture conditions were: shaker speed 125 rpm, humidity 80%, and carbon dioxide concentration 8%. The basal medium was ActiPro, and the feed medium was Cell Boost 7a / 7b. The ratio of Cell Boost 7a:Cell Boost 7b was 9:1 to 11:1. Feeds were administered daily from day 3 until the day before culture ended, at a feed ratio of 0% to 4% of the initial culture volume.

[0046] The seeding density is (0.30-0.50)×10 6 cells / mL, the initial culture volume was 50 mL, and the initial culture temperature was set to 36.5°C. During the culture process, samples were taken daily starting from the third day for offline testing of viable cell density, cell viability, glucose concentration, lactate concentration, and osmotic pressure.

[0047] Culture until the sixth day, or the viable cell density reaches (10.00-14.00)×106 cells / mL (whichever comes first), cool to 33°C.

[0048] 3. Add NAD

[0049] There are three ways to add NAD, you can choose any one of them:

[0050] (1) On the 8th day of culture, NAD was added once based on the initial volume to a final concentration of 6 mM;

[0051] (2) On the 7th day of culture, 3 mM NAD was added based on the initial volume, and then on the 10th day of culture, 3 mM NAD was added again based on the initial volume;

[0052] (3) On the 9th day of culture, NAD was added once based on the initial volume to a final concentration of 6 mM.

[0053] 4. Cell harvesting and result detection

[0054] Fermentation was terminated and the cell culture fluid was harvested on day 14, or when cell viability fell below 60%, whichever occurred first. On the day of harvest, samples were taken and protein concentration was measured offline using a cedex instrument. The cell harvest fluid was centrifuged and subjected to a one-step Protein A affinity purification before being analyzed by capillary isoelectric focusing (cIEF) to determine charge variant distribution.

[0055] Comparative Example 1

[0056] Based on the culture conditions and operations in Example 1, in this Comparative Example 1, three groups of experiments were designed for three clones, with a total of 8 culture conditions.

[0057] 1. Culture conditions

[0058] Clone 1:

[0059] Control group 1a, no NAD was added;

[0060] Experimental group 1b, on day 8 of culture, 6 mM NAD was added once based on the initial volume;

[0061] In experimental group 1c, 3 mM NAD was added on the 7th and 10th days of culture based on the initial volume (total 6 mM).

[0062] Clone 2:

[0063] Control group 2a, no NAD was added;

[0064] In experimental group 2b, 6 mM NAD was added once based on the initial volume on day 8 of culture;

[0065] In experimental group 2c, 3 mM NAD was added on the 7th and 10th days of culture based on the initial volume (total 6 mM).

[0066] Clone 3:

[0067] Control group 3a, no NAD was added;

[0068] In experimental group 3b, 6 mM NAD was added once based on the initial volume on day 9 of culture.

[0069] For the eight groups described above, the remaining experimental procedures were the same as those described in Example 1. During the culture process, cell growth (viable cell density, cell viability) and metabolism (glucose, lactate, osmotic pressure) were monitored. Protein production and quality were measured on day 14 of culture.

[0070] 2. Results and Discussion

[0071] The cell growth curves of the above groups are shown in Figure 2. Figure 1-6 As shown (see the accompanying drawings and accompanying descriptions for the corresponding relationship), it can be seen that in terms of cell growth: after adding NAD, the live cell density and cell viability are comparable to those of the control group, indicating that adding NAD does not affect cell growth.

[0072] The cell metabolism curves of the above groups are as follows: Figure 7-15 As shown (see the accompanying figures and figure captions for corresponding relationships), it can be seen that in terms of cellular metabolism: after adding NAD, the glucose level in the culture medium increased slightly, and the lactate level immediately decreased after the addition of NAD, and the downward trend was maintained for several days, indicating that cellular lactate metabolism was significantly improved. After adding NAD, the osmotic pressure temporarily decreased compared to the control group, indicating a temporary increase in cellular metabolism, which may be related to the increase in lactate consumption.

[0073] The protein yield results of the above groups are shown in Table 1 below.

[0074] Table 1 Protein yield results

[0075]

[0076] As shown in Table 1, protein production remained unchanged in clones 1 and 3 after NAD addition. For clone 2, a single addition of 6 mM NAD on day 8 increased overall protein production by 11.3%, while the addition of 3 mM NAD on days 7 and 10 had no effect. These results indicate that NAD supplementation has no negative effect on overall protein production and, under certain conditions, may even have a positive effect.

[0077] The capillary isoelectric focusing (cIEF) test results of the above groups are shown in Table 2 below. The acidic peak ratio bar graph of each group is shown in Figure 16-18 shown.

[0078] Table 2cIEF results

[0079]

[0080] As shown in Table 2 and Figure 16-18 As shown, in terms of protein quality:

[0081] For clone 1 (expressing protein 1), the cIEF acidic peak ratio of the protein product increased by 8.7% after a single addition of 6 mM NAD on day 8 of culture. Furthermore, the acidic peak ratio increased by 9.4% when 3 mM NAD was added on days 7 and 9 of culture, respectively.

[0082] For clone 2 (expressing protein 2), the cIEF acidic peak ratio of the protein product increased by 12.0% after a single addition of 6 mM NAD on day 8 of culture. Furthermore, the acidic peak ratio increased by 13.5% when 3 mM NAD was added on days 7 and 9 of culture, respectively.

[0083] For clone 3 (expressing protein 3), when 6 mM NAD was added once on day 9 of culture, the cIEF acidic peak ratio of the protein product increased by 27.4%.

[0084] It can be seen that under this method, the cIEF acidic peak ratio of protein products can be increased by 8.7%-27.4%.

[0085] In summary, this method can significantly increase the proportion of acidic charged heterogeneous species for multiple monoclonal cell lines and multiple protein molecules, and the various addition methods in this method can all achieve the technical effect of significantly increasing the level of acidic charged heterogeneous species, fully demonstrating the effectiveness and universality of this method.

[0086] In summary, the above embodiments are merely preferred embodiments of the present invention and are not intended to limit the scope of protection of the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the scope of protection of the present invention.

Claims

1. A quality control method for improving the level of acidic charged heterogeneous forms of antibody proteins, characterized in that: During the cell culture process, β-nicotinamide adenine dinucleotide is added to the culture system, and an antibody with an increased proportion of acidic charged heteromers is finally obtained.

2. The quality control method for improving the level of acidic charged variants of antibody proteins according to claim 1, characterized in that: The added concentration of the β-nicotinamide adenine dinucleotide is 1-10 mM.

3. The quality control method for improving the level of acidic charged variants of antibody proteins according to claim 1, characterized in that: The cell culture is carried out in a fed-batch culture mode, and the culture is terminated after 14 days or when the cell viability is lower than 60%.

4. The quality control method for improving the level of acidic charged variants of antibody proteins according to claim 1, characterized in that: The initial culture volume was 50 mL, and the starting culture temperature was 36.5 ± 1 °C.

5. The quality control method for improving the level of acidic charged variants of antibody proteins according to claim 1, characterized in that: When the viable cell density reached 10.00 × 10 6 cells / mL to 14.00×10 6 cells / mL or on the sixth day of culture, lower the culture temperature to 33±1℃.

6. The quality control method for improving the level of acidic charged variants of antibody proteins according to claim 1, characterized in that: The method of adding β-nicotinamide adenine dinucleotide to the culture system is as follows: on the 8th day of culture, β-nicotinamide adenine dinucleotide is added based on the initial volume to a final concentration of 6 mM.

7. The quality control method for improving the level of acidic charged variants of antibody proteins according to claim 1, characterized in that: β-nicotinamide adenine dinucleotide was added to the culture system as follows: on the 7th day of culture, β-nicotinamide adenine dinucleotide was added based on the initial volume to a concentration of 3 mM; culture was continued until the 10th day, and 3 mM β-nicotinamide adenine dinucleotide was added based on the initial volume.

8. The quality control method for improving the level of acidic charged variants of antibody proteins according to claim 1, characterized in that: The method of adding β-nicotinamide adenine dinucleotide to the culture system is as follows: on the 9th day of culture, β-nicotinamide adenine dinucleotide is added based on the initial volume to a final concentration of 6 mM.

9. The quality control method for improving the level of acidic charged variants of antibody proteins according to claim 1, characterized in that: The cells are mammalian cells, including CHO cells.

10. Use of the quality control method for improving the level of acidic charged heteromorphs of antibody proteins according to any one of claims 1 to 9 in the preparation of antibody drugs and regulators, characterized in that: The regulator is used to increase the level of acidic charged heteromorphs of antibody proteins.

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