Method for simultaneously regulating cell expression product aggregates and charge heterogeneity and increasing yield

Abstract

A method for simultaneously regulating cell expression product aggregates and charge heterogeneity, and increasing the yield. The method comprises: adding taurine and glutathione during inoculation. Compared with individually adding taurine or glutathione, adding both taurine and glutathione can synergistically regulate product aggregates, charge heterogeneity and yield.

Description

A method for simultaneously regulating cell expression product aggregates, charge heterogeneity, and increasing yield Technical Field

[0001] The invention relates to a method for simultaneously regulating aggregates and charge heterogeneity of cell expression products and improving yield, and belongs to the field of cells. Background Art

[0002] Most of the therapeutic antibodies currently available and under development on the market, as well as some recombinant therapeutic proteins, are expressed in mammalian cells (primarily Chinese hamster ovary cells, or CHO cells). Protein multimer content is a key quality attribute (CQA) of protein pharmaceuticals and has received increasing attention. The formation of multimers can affect the safety and efficacy of the final product. Protein multimers are generally formed through covalent or non-covalent linkages. Antibody disulfide bonds occur in the endoplasmic reticulum, where redox reactions directly affect antibody disulfide bonds.

[0003] In addition to polymers, therapeutic antibodies or recombinant proteins also exhibit charge heterogeneity. The main causes of charge heterogeneity include various chemical degradation mechanisms (such as isomerization, amidation, cleavage, and oxidation) and addition reactions (covalent addition or glycosylation) occurring at different locations on the protein. Basic peaks are primarily derived from heterogeneity of C-terminal lysine, methionine oxidation, or conversion of aspartic acid, while acidic peaks are primarily derived from N-sugar terminal sialylation and amino acid residue deamidation. These phenomena that cause molecular changes can significantly affect the pH of the molecule and potentially impact product function.

[0004] In addition to quality control, production volume is also a key concern for biopharmaceuticals. As long as quality is met, increasing production volume can significantly reduce costs and increase efficiency. With the continuous development of biopharmaceuticals, the demand for cost control is becoming increasingly stringent, making increasing production volume a goal for every biopharmaceutical company.

[0005] The main approaches to regulating aggregates and charge heterogeneity or increasing yield are through genetic modification, culture medium component modification, and process modification. Genetic modification is complex and time-consuming, and the safety, complexity, and stability of such methods require a long time to verify and confirm, which is not conducive to the approval of biological products. Culture medium component optimization is limited by culture medium components, and the operation is complex, making it difficult to implement aggregates. Improvements through process modification are relatively small. Currently, many studies have found that aggregates or charge can be improved through culture medium additives. For example, adding copper sulfate or β-mercaptoethanol can improve product aggregates, adding flavonoids or adjusting zinc ion concentration can improve protein charge heterogeneity, and increasing feed volume can increase protein yield. However, these methods can only adjust one quality attribute or yield and have little effect on adjusting another quality attribute.

[0006] The purpose of this patent is to develop a method to simultaneously regulate the aggregates, charge heterogeneity and increase the yield of cell expression products. Summary of the Invention

[0007] The present invention aims to provide a method for simultaneously regulating aggregates, charge heterogeneity, and yield. This method allows for the simultaneous regulation of aggregates, charge heterogeneity, and yield, resulting in higher-quality products that can quickly meet demand. The present invention offers a simple process, high cost-effectiveness, and suitability for industrial production.

[0008] The present invention provides a method for simultaneously regulating aggregates, charge heterogeneity, and yield improvement, namely, adding taurine and glutathione during inoculation to simultaneously regulate aggregates, charge heterogeneity, and yield improvement of CHO cell products. This method can reduce antibody aggregates, improve antibody charge heterogeneity, and increase yield at the same time. This method can be applied to improve the quality and increase yield of antibody drugs. Compared with the single effects of adding substances such as copper sulfate and β-mercaptoethanol on charge heterogeneity, aggregates, or yield improvement, the method of the present invention can simultaneously regulate aggregates, charge heterogeneity, and yield improvement.

[0009] In order to address the deficiencies of the prior art, the present invention provides a method for simultaneously regulating aggregates, charge heterogeneity and increasing production, wherein a regulator is added to the cell culture system to regulate aggregates and charge isomers, wherein the regulator is taurine and glutathione; preferably, the regulator can also increase production at the same time.

[0010] In certain specific embodiments, the cell is a CHO-K1 cell; preferably, the CHO-K1 cell comprises an expression vector of a GS gene, and an exogenous gene is inserted downstream of the GS gene.

[0011] In certain specific embodiments, the final concentration of taurine in the cell culture system is 1.34-13.4 mM.

[0012] In certain specific embodiments, the final concentration of glutathione in the cell culture system is 150-300 μM.

[0013] In certain specific embodiments, the final concentration of the regulator in the cell culture system is 13.4 mM taurine and 150 μM glutathione.

[0014] In certain embodiments, the method comprises adding a modulator to the cells at the time of seeding.

[0015] The above method can be carried out by feeding cells in a chemically defined medium commonly used in the art, with taurine and glutathione added before inoculation. For example, a chemically defined CHO medium is used and cultured in a fed-batch manner.

[0016] In certain specific embodiments, the culture conditions of the method are 36.5° C., 5-8% CO 2 , 110-130 rpm, and 80% humidity.

[0017] In some specific protocols, the temperature is lowered to 31-34° C. on the fifth day of culture.

[0018] The present invention also provides a regulator, which comprises taurine and glutathione.

[0019] The present invention also provides the use of the regulator in regulating cell expression product aggregates, charge heterogeneity and increasing yield in cell culture.

[0020] The raw materials and reagents described in the present invention are all commercially available. Beneficial effects:

[0021] The present invention simultaneously adds taurine and glutathione to the CHO cell culture system, which can reduce the expression of antibody aggregates and the content of antibody acidic isomers, while also increasing antibody production. The effects produced are as follows:

[0022] (1) Effect on aggregates: aggregate expression decreased by 6.5%, and the main peak increased by 6.4%;

[0023] (2) Effect on charge isomers: the acid peak decreased by 6.4%, the base peak decreased by 4.8%, and the main peak increased by 11.2%;

[0024] (3) Impact on yield: yield increased by 0.79g / L.

[0025] Compared with the addition of taurine or glutathione alone, the simultaneous addition of taurine and glutathione has a synergistic effect on aggregates, charge isomers and yield, increasing the reduction of aggregates by 2.2 times and the increase of the main peak by 2.6 times; in terms of charge isomers, the reduction of the acidic peak content increased by 1.3 times and the increase of the main peak increased by 3.6 times; at the same time, the yield increased by 1.2 times.

[0026] In general, the present invention is simple to operate, low in cost, and suitable for the commercial production of various therapeutic antibodies or biosimilar drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 shows the effect of taurine on the growth and viability of CHO cells;

[0028] Figure 2 shows the effect of taurine on charge heterogeneity of CHO cell products;

[0029] Figure 3 shows the effect of taurine on CHO cell product yield;

[0030] Figure 4 shows the effect of glutathione on CHO cell product aggregates;

[0031] Figure 5 shows the effect of glutathione on CHO cell product yield;

[0032] Figure 6 shows the effect of taurine + glutathione on CHO cell product aggregates;

[0033] Figure 7 shows the effect of taurine + glutathione on the charge heterogeneity of CHO cell products;

[0034] FIG8 shows the effect of taurine + glutathione on the product yield of CHO cells. DETAILED DESCRIPTION

[0035] The present invention is further described and illustrated below with reference to specific examples, but the present invention is not limited to the scope of the examples. Experimental methods without specific conditions in the following examples are performed according to conventional methods and conditions, or selected according to the instructions.

[0036] The experimental materials used in the following examples are as follows:

[0037] The cells used in the following examples are CHO-K1 cells, which are from Merck and have the product number CHOGS-1VL.

[0038] Taurine and glutathione used in the following examples were purchased from Sigma, with product numbers SMB00177-1MG and PHR1359-500MG, respectively.

[0039] Example 1: Effect of taurine on CHO cell expression products

[0040] The specific implementation methods are as follows:

[0041] 1.1 Cell culture

[0042] After cell recovery and 3 to 4 passage expansion, the cells were cultured at 0.5 × 10 6 Cells were seeded at a density of 10 cells / mL in chemically defined basal medium (CHO MaxX, Catalog No. MB1113.102) with an initial culture volume of 30 mL and a shake flask volume of 125 mL. Culture conditions were: 36.5°C, 125 rpm, 5% CO2, and 80% humidity. The temperature was lowered to 33°C on day 5, and the culture was terminated on day 14 when cell viability dropped below 80%.

[0043] Taurine experimental group: taurine was added during cell inoculation; the final concentrations were 6.7 mM, 13.4 mM, and 19.1 mM, respectively.

[0044] Control group: no additives were added.

[0045] 1.2 Detection methods and instruments

[0046] The expression level of monoclonal antibodies in the supernatant was determined by ProA-HPLC (Protein A-High Performance Liquid Chromatography). To detect aggregates and charge heterogeneity, the supernatant sample was first purified by Protein A affinity purification. Aggregates were analyzed by gel exclusion chromatography (SEC) in the ProA-purified sample. Charge heterogeneity was analyzed by IEC-HPLC (ion exchange-high performance liquid chromatography) in the purified sample.

[0047] In this embodiment, the cell viability and density were detected using a Beckman cell counter, and nutrients such as glucose and lactic acid were detected using a Cedex biochemical analyzer.

[0048] 1.3 Results

[0049] 1A and 1B show that there is basically no effect. Figure 2 shows that the addition of different taurine concentrations results in changes in both the acid peak and the main peak compared to the control group. However, the addition of 13.4 mM taurine results in the largest change, with the acid peak decreasing by 4.9% and the main peak increasing by 3.1%, indicating that taurine can improve the charge heterogeneity of monoclonal antibodies. Figure 3 shows that the addition of taurine results in basically no change in the expression level.

[0050] Example 2: Effect of glutathione on CHO cell expression products

[0051] The specific method is the same as that of Example 1, except that glutathione is added to the culture medium during inoculation; the final concentrations are 100 μM, 150 μM and 200 μM, respectively.

[0052] Analysis of the effect of glutathione on aggregates shows that after adding glutathione, different concentrations have an effect on reducing aggregates, but the final concentration of 150 μM has the best effect. The aggregate expression of the antibody decreased by 3%, and the main peak (monomer) increased by 2.5%. Analysis of the effect of adding glutathione on the expression level shows that adding different concentrations of glutathione has little effect on the expression level, as shown in Figure 5, indicating that adding glutathione can improve the aggregates of the CHO cell expression product without affecting its expression level.

[0053] Example 3: Effect of Taurine + Glutathione on CHO Cell Expression Products

[0054] The specific method is the same as Example 1, except that taurine and glutathione are added to the culture medium at the same time during inoculation; the final concentrations are 13.4 mM and 150 μM, respectively.

[0055] Analysis of the effect of simultaneous addition of taurine and glutathione on aggregates showed that the aggregate expression decreased by 6.5% and the main peak (monomer) increased by 6.4% as shown in Figure 6. Analysis of its effect on charge isomers showed that simultaneous addition of taurine and glutathione reduced the acid peak by 6.4%, reduced the base peak by 4.8%, and increased the main peak by 11.2% as shown in Figure 7. Analysis of the effect of simultaneous addition of taurine and glutathione on yield showed that the yield increased by 1.2 times as shown in Figure 8.

[0056] In summary, Examples 1 to 3 show that the addition of 13.4 mM taurine can reduce the acidic peak content and increase the main peak ratio without affecting the yield. The addition of 150 μM glutathione alone can reduce the aggregate content and increase the main peak ratio. The simultaneous addition of 13.4 mM taurine and 150 μM glutathione can reduce aggregates, reduce the acidic peak, and increase the yield. Compared with the addition of taurine alone, the simultaneous addition of taurine and glutathione increased the reduction in acidic peak content from 4.9% to 6.4%, a 1.3-fold increase; the increase in the main peak content from 3.1% to 11.2%, a 3.6-fold increase; and the yield increased by 1.2-fold. Compared with the addition of glutathione alone, the simultaneous addition of taurine and glutathione increased the reduction in aggregate content from 3% to 6.5%, a 2.2-fold increase; the increase in the main peak content from 2.5% to 6.4%, a 2.6-fold increase; and the yield increased by 1.2-fold. The above results indicate that the simultaneous addition of taurine and glutathione has a synergistic effect on charge heterogeneity, changes in aggregate content, and yield.

[0057] Table 1 Effects of additives on charge heterogeneity, aggregates and yield

[0058] -Represents no impact

[0059] Although the present invention has been disclosed above in terms of preferred embodiments, it is not intended to limit the present invention. Anyone familiar with this technology can make various changes and modifications without departing from the spirit and scope of the present invention. Therefore, the scope of protection of the present invention should be based on the definition of the claims.

Claims

1. A method for simultaneously regulating the polymers, charge heterogeneity and yield of cell expression products, characterized in that: A regulator is added into the cell culture system, wherein the regulator is taurine and glutathione.

2. The method according to claim 1, wherein The final concentration of taurine is 1.34-13.4 mM.

3. The method according to claim 1, wherein The final concentration of glutathione is 150-300 μM.

4. The method according to claim 1, wherein The regulators were added to the culture medium at the time of cell seeding.

5. The method according to claim 1, wherein The products include but are not limited to monoclonal antibodies, bispecific antibodies, and recombinant proteins.

6. The method according to claim 1, wherein The cells include but are not limited to CHO-K1 cells.

7. The method according to claim 1, wherein The culture conditions of the cells are: 36.5° C., 110-130 rpm, 5-8% CO 2 , and 80% humidity.

8. The method according to claim 7, wherein On the fifth day of cultivation, the temperature was lowered to 31-34°C.

9. A regulator, characterized in that The regulator contains taurine and glutathione.

10. Use of the regulator according to claim 9 in regulating aggregates, charge heterogeneity and increasing yield of cell expression products in cell culture.

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