CN116286589A - Culture method for reducing acid peak ratio of antibody protein, antibody and cell culture medium - Google Patents
Culture method for reducing acid peak ratio of antibody protein, antibody and cell culture medium Download PDFInfo
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Abstract
The application discloses a culture method for reducing the acid peak ratio of antibody protein, an antibody and a cell culture medium, wherein the culture method comprises the steps of inoculating fusion cells into a basic culture medium for culture, and adding a fed-batch culture medium and a regulator in the culture process; separating antibody protein from the culture product; wherein the modifier comprises Mn 2+ . Mn in the culture system after the addition of the regulator is completed 2+ The molar concentration of (2) is 20-30 mu M. The culture method can effectively reduce the acid peak ratio of the antibody protein, has lower influence on the alkali peak, and compared with the existing culture method, the acid peak ratio of the antibody is reduced by 29.2 percent, and the alkali peak ratio is only increased by 4.5 percent; no need of changing process control conditions, no influence on cell growth and target antibody yield, no cause of other regulations and safetyProblems in terms of.
Description
Technical Field
The application belongs to the technical field of biological pharmacy, and in particular relates to a culture method for reducing the acid peak ratio of antibody protein, an antibody and a cell culture medium.
Background
The charge heterogeneity is due to changes in the surface charge characteristics of the antibody molecule, such as isoelectric points or space charge distribution, due to post-translational modification and degradation, and further, the generation of charge variants. Numerous post-translational modifications and degradation processes in antibodies, such as glycosylation, C-terminal and N-terminal modifications, saccharification, oxidation, multimeric, disulfide bond modifications, and the like, can cause changes in the surface charge characteristics of the antibodies and the generation of charge variants. The occurrence of heterogeneity often causes alterations in antibody structure and function, such as antibody biological activity, pharmacokinetics, thermal stability, immunogenicity, etc., which are a key quality attribute of antibody drugs.
The acid peak of the protein is more likely to cause a change in the functional activity of the protein in terms of charge heterogeneity, while the base peak is typically caused by incomplete cleavage of the C-terminal lysine, which variation generally does not affect the functional activity of the protein, and thus the control of the acid peak is particularly important in antibody production. In the prior art, the means for regulating and controlling the acid peak of the antibody mainly comprises cooling, wherein the cooling can reduce the acid peak, but can reduce the activity of enzyme, influence the protein expression quantity, and meanwhile, the cooling can also lead to the rising of the alkali peak, so that the main peak possibly cannot be increased.
Disclosure of Invention
The purpose of the application is to provide a culture method, an antibody and a cell culture medium for reducing the acid peak ratio of an antibody protein, so as to solve the problems that in the prior art, the activity of enzyme can be reduced, the protein expression quantity is affected, and meanwhile, the temperature reduction can also lead to the rising of an alkali peak, so that a main peak possibly cannot be increased.
In order to achieve the above purpose, a technical scheme adopted in the application is as follows:
a culture method for reducing the acid peak ratio of antibody protein is provided, which comprises the following steps:
inoculating the fusion cells into a basic culture medium for culture, and adding a fed-batch culture medium and a regulator in the culture process;
separating antibody protein from the culture product;
wherein the modifier comprises Mn 2+ 。
In one or more embodiments, mn in the culture system after the addition of the modifier is completed 2+ The molar concentration of (2) is 20-30 mu M.
In one or more embodiments, the modulator is added to the culture system in two portions on days 3, 4 or 5 and 5, 6 or 7 of the culture, respectively.
In one or more embodiments, the modulator is added to the culture system in two portions on days 4 and 6 of the culture, respectively.
In one or more embodiments, the feed medium is added to the culture system three times on days 3,5, and 7 of the culture, respectively.
In one or more embodiments, the modifier is a manganese chloride tetrahydrate solution.
In order to achieve the above purpose, another technical scheme adopted in the application is as follows:
an antibody obtained by culturing the antibody according to any one of the above embodiments is provided.
In one or more embodiments, the antibody has an acid peak ratio of less than 15% and a major peak ratio of greater than 67%.
In order to achieve the above object, another technical solution adopted in the present application is:
providing a cell culture medium comprising a basal medium, a feed-through medium and a regulator comprising Mn 2+ 。
In one or more embodiments, the Mn in the cell culture medium 2+ The molar concentration of (2) is 20-30 mu M.
In one or more embodiments, the modifier is a manganese chloride tetrahydrate solution.
The beneficial effect of this application is, in contrast to prior art:
according to the culture method, the regulator comprising manganese ions is added in the cell culture process, so that the acid peak ratio of the antibody protein can be effectively reduced, the influence on the alkali peak is low, the phenomenon that the acid peak is reduced but the alkali peak is increased and the main peak is not increased in the conventional protein acid-alkali peak regulating method is avoided, the change of the protein activity of the antibody is effectively avoided, and compared with the conventional culture method, the acid peak ratio of the antibody obtained by the culture method is reduced by 29.2%, and the alkali peak ratio is only increased by 4.5%;
the culture method does not need to change process control conditions, has no influence on cell growth and target antibody yield, and does not cause other problems in regulation and safety;
when the cell culture medium is applied to the culture of fusion cells, the acid peak ratio of the harvested antibody protein can be obviously reduced, and the protein alkali peak ratio is not influenced, so that the change of the protein activity of the antibody is effectively avoided.
Drawings
FIG. 1 is a schematic flow chart of an embodiment of a method for reducing the acid peak ratio of an antibody protein according to the present application.
Detailed Description
The present application will be described in detail with reference to the embodiments. The embodiments are not intended to be limiting and structural, methodological, or functional changes made by those of ordinary skill in the art in light of the embodiments are intended to be included within the scope of the present application.
The charge heterogeneity is due to changes in the surface charge characteristics of the antibody molecule, such as isoelectric points or space charge distribution, due to post-translational modification and degradation, and further, the generation of charge variants. The antibody charge variants are derived from a large number of sources and the mechanism of generation is quite complex. Studies have shown that many post-translational modifications and degradation processes in antibodies, such as glycosylation, C-terminal and N-terminal modifications, saccharification, oxidation, multimeric, disulfide bond modifications, etc., can lead to changes in the surface charge characteristics of the antibodies and the generation of charge variants. Because antibodies often undergo multiple post-translational modifications and degradations simultaneously during production, storage, and use, the surface charge characteristics of the antibodies are commonly affected, and thus the charge heterogeneity of the antibodies is a combined effect of these post-translational modifications and degradations.
The occurrence of heterogeneity often causes alterations in antibody structure and function, such as antibody biological activity, pharmacokinetics, thermal stability, immunogenicity, etc., which are a key quality attribute of antibody drugs. However, effective control of charge heterogeneity has been a challenge in antibody drug development and production.
As described in the background art, the acid peak of a protein is more likely to cause a change in the functional activity of the protein in terms of charge heterogeneity, whereas the base peak is typically caused by incomplete cleavage of C-terminal lysine, and this variation usually does not affect the function of the protein activity, so that the control of the acid peak is particularly important in antibody production. In the prior art, the means for regulating and controlling the acid peak of the antibody mainly comprises cooling, wherein the cooling can reduce the acid peak, but can reduce the activity of enzyme, influence the protein expression quantity, and meanwhile, the cooling can also lead to the rising of the alkali peak, so that the main peak possibly cannot be increased.
In order to solve the above problems, the applicant developed a culture method for reducing the acid peak ratio of an antibody-like protein, which can effectively reduce the acid peak ratio of an antibody, increase the main peak ratio, and have no influence on cell growth and yield of a target antibody.
Specifically, referring to fig. 1, fig. 1 is a schematic flow chart of an embodiment of a culture method for reducing the acid peak ratio of an antibody protein according to the present application.
The culture method comprises the following steps:
s100, inoculating the fusion cells into a basic culture medium for culture, and adding a fed-batch culture medium and a regulator in the culture process.
Wherein the modifier comprises Mn 2+ 。
The selection of the basal medium and the fed-batch medium is not limited in this embodiment, and any suitable commercial medium can be selected according to the type of the fused cells, so that stable culture and proliferation of the cells can be ensured, and the effect of this embodiment can be achieved.
At present, mn in the common fusion cell culture process 2+ The ion as a metal ion has application only in regulating the glycoform of protein, and has no application in regulating the acid-base peak of proteinIs used.
In the present embodiment, mn is added to cell culture 2+ The ion is used for adjusting the acid-base peak of the protein, the process control condition is not required to be changed, and the cell growth and the target antibody yield are not affected; meanwhile, the acid peak ratio is reduced, the alkali peak is not increased, and the phenomenon that the common protein acid-base peak adjusting method reduces the acid peak but increases the alkali peak and the main peak is not increased is avoided.
Mn 2+ Ions are important as metal ions required for cell growth, and the addition of ions at a certain limiting concentration has no effect on cell growth and does not cause other regulatory and safety problems.
Specifically, in one application scenario, mn of the present embodiment 2+ The ion may be manganese chloride tetrahydrate, and in other application scenarios, other manganese compounds may be used, which can achieve the effects of the present embodiment.
In one application scenario, mn in the culture system after the addition of the regulator is completed 2+ The molar concentration of the manganese ion can be 20-30 mu M, so that the influence of the too low manganese ion concentration on the peak regulation effect of the protein acid and the base is avoided, and the influence of the too high manganese ion concentration on the cell metabolism is avoided.
In the process of cell culture, the first 7 days are mainly the process of cell proliferation, and the 7 th day is mainly the process of expressing antibodies, so that the condition that a single addition of a regulator generates larger stirring on culture is avoided, and in one application scene, the antibody can be selectively added into a reaction system in 3 rd, 4 th or 5 th days and 5 th, 6 th or 7 th days of culture in two times.
Preferably, in one application scenario, the reaction system may be added in two portions on days 4 and 6 of the culture.
In one application scenario, the fed-batch medium may be added to the reaction system three times on days 3,5, and 7 of the culture.
S200, separating antibody protein from the culture product.
After cell culture, antibodies can be isolated from the culture product. The method for separating and harvesting the antibody in the present embodiment is not limited, and any conventional harvesting method can achieve the effects of the present embodiment, for example, centrifugation harvesting and the like.
The application also provides an antibody obtained by culturing by the culture method, in one embodiment, the acid peak proportion of the antibody can be less than 15%, and the main peak proportion can be more than 67%, so that the change of the protein activity of the antibody is effectively avoided.
The application also provides a cell culture medium comprising a basal medium, a fed-batch medium and a regulator comprising Mn 2+ 。
In one embodiment, mn in the cell culture medium 2+ The molar concentration of (2) may be 20 to 30. Mu.M.
In one embodiment, mn 2+ The ion may be manganese chloride tetrahydrate, and in other embodiments, other manganese compounds may be used, which can achieve the effects of the present embodiment.
When the cell culture medium is applied to the culture of fusion cells, the acid peak ratio of the harvested antibody protein can be obviously reduced, and the protein alkali peak ratio is not influenced, so that the change of the protein activity of the antibody is effectively avoided.
The present application is further described in detail below with reference to specific embodiments, which are not intended to be limiting, but rather structural, methodological, or functional transformations of one of ordinary skill in the art based on the present embodiments are included within the scope of the present application.
Cell strain information of the fused cells: the fusion cells used in the following embodiments of the present application were CHO-K1 cells from ATCC (American Type Culture Collection) expressing an antibody of the IgG1 type.
Medium information: the basic culture medium is Driving
Eden B400S with feed medium of Driving +.>
Eden F400aS and Driving->
Eden F200bS, all from the open-air-fold-based biotechnology company.
Reagent information and preparation method: mnCl 2 ·4H 2 O(Sigma，M5005-100G)。
4mM MnCl 2 ·4H 2 The preparation method of O comprises the following steps: weigh 0.0289g MnCl 2 ·4H 2 O (Sigma, M5005-100G) powder is dissolved in 36mL of ultrapure water, stirred and mixed for more than 20min, filtered and sterilized by a 0.22 μm filter membrane, and stored at 4 ℃ for later use.
Example 1:
an antibody protein is obtained by culturing by the following culture method:
CHO-K1 fused cell seeds were prepared according to the method of (0.8.+ -. 0.2). Times.10 6 Inoculating cells/mL into 250mL flat bottom shake flask, and performing Fed-batch culture with initial culture volume of 80mL and basal medium of Driving
Eden B400S. The shake culture conditions are as follows: 36.5 ℃ and 6.0% CO 2 Concentration, wheelbase 50mm, rotational speed 120rpm; when the culture was carried out to day 5, the temperature was switched to 33.0 ℃.
When cultured to day 3,5,7, driving was added at an initial culture volume of 5% (v/v) each time
Eden F400aS and Driving +.1 in initial culture volume of 0.5% (v/v)>
EdenF200bS。
On days 4 and 6 of the culture, 40. Mu.L of 4mM MnCl was added, respectively 2 ·4H 2 O。
During the culture, the glucose concentration was measured with a Nova biochemical analyzer (BioProfile 100) and the glucose concentration was controlled at 8.0g/L on days 3,5,7 with 300g/kg of the glucose concentrate.
The harvest was centrifuged at 4000rpm for 15min when cultured to day 9.
Example 2:
the method of culturing the antibody protein of this example is basically the same as that of example 1, except that:
in this example, 100. Mu.L of 4mM MnCl was added to each of the culture days 4 and 6 2 ·4H 2 O。
Example 3:
the method of culturing the antibody protein of this example is basically the same as that of example 1, except that:
in this example, 200. Mu.L of 4mM MnCl was added to each of the culture plates on days 4 and 6 of the culture 2 ·4H 2 O。
Example 4:
the method of culturing the antibody protein of this example is basically the same as that of example 1, except that:
in this example, 300. Mu.L of 4mM MnCl was added to each of the culture plates at days 4 and 6 2 ·4H 2 O。
Comparative example 1:
the method of culturing the antibody protein of this comparative example was substantially the same as in example 1, except that:
this comparative example was carried out on days 4,6 of culture without any addition of reagents.
Comparative example 2:
the method of culturing the antibody protein of this comparative example was substantially the same as in example 1, except that:
in this comparative example, 20. Mu.L of 4mM MnCl was added to each of the culture plates at days 4 and 6 of the culture 2 ·4H 2 O。
Specific addition schemes of examples 1 to 4 and comparative examples 1 to 2 and the concentration of manganese ions in the culture system after completion of addition are shown in the following table:
effect example 1: analysis of cell growth influence
For the culture procedures of comparative examples 1 to 2 and examples 1 to 4, 1mL was sampled for viable CELL density (VCC) and CELL Viability (VIA) assays on day 0,3,5,7,9 of culture, respectively, using a Beckman CELL viability analyzer (Vi-CELL XR), giving the following table data:
from the above table data, it is understood that the growth of cells in the culture process of examples 1 to 4 and comparative example 2 was substantially the same as that of the cells in the culture process of comparative example 1, and that the addition of the regulator including manganese ions in batches during the culture process had no effect on the growth of cells.
Effect example 2: antibody expression level influence analysis
For the culture procedures of comparative examples 1 to 2 and examples 1 to 4, at the time of centrifugation harvest at the 9 th day of culture, supernatants were collected, filtered through a 0.22 μm filter membrane, and then sub-packed with 2X 1mL, and the antibody expression amount (Titer) in the supernatants was detected by the Titer-HPLC method, to obtain the following table data.
The specific detection method comprises the following steps: the expression level of antibodies in the cell supernatants was assessed using a Waters e2695 high performance liquid chromatography system and a MabPac Protein a affinity chromatography column (Thermo, 35mm x 4mm,13 μm) (Titer-HPLC), using mobile phase a (50mM PB,150mM NaCl,5%ACN,pH =7.5) and mobile phase B (50mM PB,150mM NaCl,5%ACN,pH =2.5). The HPLC system and column were equilibrated with 100% mobile phase A, then eluted with 100% mobile phase B for 3 minutes at a flow rate of 2.0mL/min, column temperature: 25 ℃. Antibody solutions of different concentrations (0.25 mg/mL,0.5mg/mL,1.0mg/mL,2mg/mL and 4 mg/mL) were prepared as external standard solutions, 10. Mu.L were injected, and a standard curve was fitted.
And (3) taking cell supernatant, centrifuging, and directly injecting sample, wherein the injection amount is 10 mu L, and the detection wavelength is 280nm.
| Comparative example 1 | Comparative example 2 | Example 1 | Example 2 | Example 3 | Example 4 | |
| Titer(g/L) | 3.02 | 2.97 | 3.06 | 2.88 | 3.18 | 3.10 |
As is clear from the above table data, the amounts of antibody expression in examples 1 to 4 were not greatly different from those in comparative examples 1 to 2, and the addition of a regulator including manganese ions in batches during the culture had no effect on the expression of the antibodies.
Effect example 3: CEX mass analysis
For the culture procedures of comparative examples 1 to 2 and examples 1 to 4, at the time of centrifugation harvest at the 9 th day of culture, supernatants were collected, filtered through a 0.22 μm filter membrane and split into 2X 10mL, affinity purified by Protein A, and then subjected to N-Glycan mass analysis to obtain the data of the following table.
The Protein A affinity purification method specifically comprises the following steps: protein a affinity chromatography was performed using the MabSelect SuRe packing from Cytiva to isolate the antibody of interest in the cell culture supernatant. First, the protein A column was equilibrated with 50mM Tris-HAC,150mM NaCl equilibration solution at pH 7.2 in an equilibration volume of 5 column volumes, then the cell culture supernatant was applied to the column, and after the end of the sample application, the column was rinsed again with equilibration solution to remove excess cell supernatant in an rinse volume of 5 column volumes. Finally, the target antibody was eluted with 50mM NaAC-HAC buffer pH 3.7, with an elution volume of 3 column volumes. The eluate was neutralized with 1M Tris buffer, pH adjusted to 5.5 and the final antibody concentration was determined by spectrophotometry NannoDrosone.
The CEX detection method specifically comprises the following steps: the charge heterogeneity (CEX-HPLC) of monoclonal antibodies was tested by a Waters e2695 high performance liquid chromatography system and a weak cation column ProPac WCX-10 (Thermo, 250 x 4.0mm I.D.,10 μm) using mobile phase A (6 mmol/L NaH) 2 PO 4 ,4mmol/L Na 2 HPO 4 ) And mobile phase B (6 mmol/L NaH) 2 PO 4 ,4mmol/L Na 2 HPO 4 0.4mol/L NaCl). The HPLC system and column were equilibrated with 95% mobile phase A at a flow rate of 1.0mL/min until the absorbance baseline stabilized. A gradient elution procedure was then performed from 5% b to 25% b for 30 minutes. The reference and sample were diluted to 2mg/mL with ultrapure water and the sample loading was 50. Mu.L. Column temperature: the detection wavelength was 280nm at 35 ℃.
| Sequence number | Acid peak (%) | Main peak (%) | Alkali peak (%) |
| Comparative example 1 | 20.08 | 62.23 | 17.70 |
| Comparative example 2 | 21.25 | 61.15 | 17.61 |
| Example 1 | 18.16 | 63.63 | 18.21 |
| Example 2 | 17.58 | 63.92 | 18.50 |
| Example 3 | 14.30 | 67.69 | 18.01 |
| Example 4 | 14.21 | 67.46 | 18.34 |
From the above data, the acid peak ratio of the antibodies of examples 1 to 4 was significantly reduced as compared with comparative example 1 without the modifier, wherein the acid peak ratio of the antibody of example 4 was 14.21%, which was reduced by 29.2% as compared with comparative example 1.
The alkali peak ratio of the antibodies of examples 1 to 4 was less different from that of comparative example 1 without the addition of the regulator, wherein the alkali peak ratio of the antibody of example 2, in which the alkali peak ratio was the largest, was increased by only 4.5% as compared with comparative example 1.
Therefore, the method reduces the acid peak ratio and has low influence on the alkali peak, and avoids the phenomenon that the common protein acid-base peak adjusting method reduces the acid peak but increases the alkali peak, and the main peak is not increased.
Comparative examples 1 to 4 and comparative example 2 show that when the manganese ion addition concentration is too low, the peak shaving effect on the acid peak is poor.
Comparative examples 1 to 4, in which the acid peak ratio was gradually decreased as the manganese ion addition concentration was increased, it was found that the manganese ion and the regulation of the acid peak ratio were in an inverse relationship, but in order to avoid the influence of excessively high manganese ions on the cell metabolism, the manganese ion concentration in the culture system should be controlled to 30 μm or less.
The previous description of the disclosure is provided to enable any person skilled in the art to make or use the disclosure. Various modifications to the disclosure will be readily apparent to those skilled in the art, and the generic principles defined herein may be applied to other variations without departing from the scope of the disclosure. Thus, the disclosure is not limited to the examples and designs described herein but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.
Claims (10)
1. A culture method for reducing the acid peak ratio of an antibody protein, comprising:
inoculating the fusion cells into a basic culture medium for culture, and adding a fed-batch culture medium and a regulator in the culture process;
separating antibody protein from the culture product;
wherein the modifier comprises Mn 2+ 。
2. The method according to claim 1, wherein Mn is added to the culture system after completion of the addition of the regulator 2+ The molar concentration of (2) is 20-30 mu M.
3. The culture method according to claim 1, wherein the regulator is added to the culture system in two portions on days 3, 4 or 5 and 5, 6 or 7 of the culture, respectively.
4. A culture method according to claim 3, wherein the regulator is added to the culture system in two portions on days 4 and 6 of the culture, respectively.
5. The method according to claim 4, wherein the fed-batch medium is added to the culture system three times on days 3,5 and 7 of the culture, respectively.
6. The method according to claim 1, wherein the regulator is a manganese chloride tetrahydrate solution.
7. An antibody obtained by culturing by the culture method according to any one of claims 1 to 6.
8. The antibody of claim 7, wherein the antibody has an acid peak ratio of less than 15% and a major peak ratio of greater than 67%.
9. A cell culture medium is characterized by comprising a basal medium, a fed-batch medium and a regulator, wherein the regulator comprises Mn 2+ 。
10. The culture method according to claim 1, wherein Mn in the cell culture medium 2+ The molar concentration of (2) is 20-30 mu M; and/or the number of the groups of groups,
the regulator is a tetrahydrate manganese chloride solution.
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| CN114990049A (en) * | 2022-04-26 | 2022-09-02 | 鼎康(武汉)生物医药有限公司 | Method for simultaneously regulating glycoform and charge heterogeneity of cell expression product |
| CN115651953A (en) * | 2022-11-17 | 2023-01-31 | 康日百奥生物科技(苏州)有限公司 | Antibody medicine, preparation method and cell culture medium |
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| CN114990049A (en) * | 2022-04-26 | 2022-09-02 | 鼎康(武汉)生物医药有限公司 | Method for simultaneously regulating glycoform and charge heterogeneity of cell expression product |
| CN115651953A (en) * | 2022-11-17 | 2023-01-31 | 康日百奥生物科技(苏州)有限公司 | Antibody medicine, preparation method and cell culture medium |
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