CN111321188B

CN111321188B - Formula for modifying antibody glycoform, cell culture method and application thereof in industrial production

Info

Publication number: CN111321188B
Application number: CN201811542658.XA
Authority: CN
Inventors: 刘春磊; 王太海; 沈俊; 周新华
Original assignee: GENOR BIOPHARMA CO Ltd
Current assignee: GENOR BIOPHARMA CO Ltd
Priority date: 2018-12-17
Filing date: 2018-12-17
Publication date: 2024-10-29
Anticipated expiration: 2038-12-17
Also published as: CN111321188A

Abstract

The invention provides a method for preparing an anti-CD 20 antibody rituximab analogue, which comprises the following steps: (a) Culturing the cells, wherein the culture medium comprises basal medium and fed-batch medium; and (b) optionally isolating the resulting analog from the culture product; wherein, regulator A is added to the fed-batch culture medium, and the regulator A comprises: uridine, mn ²⁺, galactose, and Pluronic (Pluronic); the mol concentration ratio of uridine, mn ²⁺ and galactose is (0.8-1.2): (0.001-0.003): (2-10), and the concentration ratio of uridine to pramipexole is 1mmol (0.02-0.08) g; and 45.0% < G1F+G2F < 70.0% of the analogue was prepared. The method can stably and highly produce the biological analogue of the rituximab antibody which accords with the expected sugar type in industrial scale-up production.

Description

Formula for modifying antibody glycoform, cell culture method and application thereof in industrial production

Technical Field

The invention relates to the field of biopharmaceuticals, in particular to an antibody glycoform-modified formula, a cell culture method and application in industrial production.

Background

With the improvement of production technology, the yield of biological antibody drugs can reach a high level, however, glycoform control of antibody molecules is always a great challenge in antibody drug production, and even in imitation pharmacy, conventional production processes are difficult to reach quality standards on glycoform control. For rituximab, a CD20 antibody prodrug, glycoform is a very important quality indicator, and the N-glycosylation quality standard of the antibody is 45.0% < g1f+g2f < 70.0%. However, the drug-like glycoforms of rituximab antibodies produced in practical operation using commercial medium are greatly different from the original drug form of rituximab Luo Huatang, and are characterized in that the N-terminal glycosylated G0F (57.71%) is higher than the original drug (original drug G0F is 37.78%); G1F (29.24%), G2F (7.58%) was lower than the original (original G1F, G F41.43%, 10.34% respectively), and the antibody G1F+G2F 36.82 was far lower than the quality standard for N-glycosylation of the antibody (N-glycosylation quality standard 45.0% < G1F+G2F < 70.0%). Therefore, the existing production method is difficult to meet the national quality control requirement of imitation drugs, and the imitation drugs cannot be marketed as expected even if the original medicine-grinding patent is out of date. .

The culture medium is a basic solution for maintaining cell survival and growth metabolism, and is one of the most important conditions for cell culture. It provides a controlled and standardized living environment for cells. Although cell culture uses chemically synthesized media with defined standardized production, composition and content, cell culture media and culture processes typically require extensive process verification prior to mass production to achieve large-scale or larger-scale production. In the amplification process, parameters such as temperature, pH, dissolved oxygen, stirring and the like are required to be integrally considered to verify whether the cell culture medium and the culture process are suitable for commercial production.

Therefore, there is an urgent need in the art to develop a culture medium capable of allowing the cultured expression of a rituximab antibody biosimilar corresponding to the desired glycoform of the original drug, and a method capable of stably producing the rituximab antibody biosimilar corresponding to the desired glycoform in industrial scale-up production.

Disclosure of Invention

The invention aims to provide a culture medium capable of enabling culture and expression of a rituximab antibody biological analogue to meet an expected glycoform, and a method capable of stably and highly producing the rituximab antibody biological analogue meeting the expected glycoform in industrial scale-up production.

In a first aspect of the invention there is provided a method of preparing an anti-CD 20 antibody rituximab analogue comprising the steps of:

(a) Culturing the cells, wherein the culture medium comprises basal medium and fed-batch medium; and

(B) Optionally isolating the resulting analogue from the culture product;

Wherein, regulator A is added to the fed-batch culture medium, and the regulator A comprises: uridine, mn ²⁺, galactose, and Pluronic (Pluronic);

The mol concentration ratio of uridine, mn ²⁺ and galactose is (0.8-1.2): (0.001-0.003): (2-10), and the concentration ratio of uridine to pramipexole is 1mmol (0.02-0.08) g;

And 45.0% < G1F+G2F < 70.0% of the analogue was prepared.

In another preferred embodiment, the method further comprises the steps of:

(c) Monitoring the glucose concentration in the culture system, and supplementing the glucose mother liquor into the culture system when the glucose concentration is lower than 4g/L so as to ensure that the glucose content in the culture system is about 4g/L.

In another preferred embodiment, the concentration of glucose in the glucose mother liquor is 200-400g/L, preferably 250-350g/L, more preferably 300g/L.

In another preferred embodiment, the basal medium is selected from commercial media, including but not limited to CD FortiCHO media(Thermo Fisher Scientific)、Dynamis(Gibco)、Balan CD CHO Growth A(IrvineScientific)、Artipro(GE-Hyclone)、CD C1M1(Life Technology)、CD012( oltman, CD11V (healthy organism).

In another preferred embodiment, the feed medium is selected from commercial media, including but not limited to CD EfficientFeed C+AGT Supplement(Thermo Fisher Scientific)、Sheff-CHO PLUS PF ACF(KERRY)、Cell Boost 5(GE-Hyclone)、Cell Boost 4(GE-Hycone)、CD Feed 002( oltman, PFF05 (healthy organism).

In another preferred embodiment, the temperature of the cultivation is 30-40 ℃, preferably 32-38 ℃, more preferably 37 ℃.

In another preferred embodiment, the culture temperature is adjusted to 34℃on days 4-6 (preferably day 5) of culture; and/or the culture temperature is adjusted to 32℃on days 7-9 (preferably 8).

In another preferred embodiment, the volume of the culture system is 0.1 to 500L, preferably 3 to 300L, more preferably 100 to 250L.

In another preferred embodiment, the cell is selected from the group consisting of: CHO cells, NS0 cells, HEK293 cells, or a combination thereof.

In another preferred embodiment, the cell is a CHO cell.

In another preferred embodiment, the CHO cells are selected from the group consisting of CHO-S cells, CHO-K1 cells, CHO-DG44 cells.

In another preferred embodiment, the CHO cells are CHO-S cells.

In another preferred embodiment, the fed-batch medium is added to the reaction system at the time of cultivation up to the 4 th to 6 th and the 7 th to 9 th days, respectively, and preferably, is added to the reaction system at the time of cultivation up to the 5 th and the 8 th days, respectively.

In another preferred embodiment, the total amount of the fed-batch medium is 10 to 30v/v%, preferably 15 to 25v/v%, more preferably 20v/v% of the total reaction system.

In another preferred embodiment, the amount of the fed-batch medium added each time is 3 to 20v/v%, preferably 5 to 15v/v%, more preferably 10v/v% of the total reaction system.

In another preferred embodiment, the proportion of regulator A in the feed medium is from 0 to 100v/v%, preferably from 0 to 75v/v%, more preferably from 25 to 50v/v%.

In another preferred embodiment, the molar concentration ratio of uridine, mn ²⁺ and galactose is (0.9-1.1): (0.0015-0.0025): (3-8), preferably 1:0.002:5.

In another preferred example, the final concentrations of uridine, mn ²⁺ and galactose in the culture system are 0.3×umg to 4×umg, preferably 0.5×umg to 3×umg, more preferably 1×umg to 2×umg;

wherein the 1×umg represents:

Uridine concentration was 1mM, mn ²⁺ concentration was 0.002mM, galactose concentration was 5mM.

In another preferred embodiment, the concentration ratio of uridine to pramipexole is 1mmol (0.03-0.06) g, preferably 1mmol:0.05g.

In another preferred embodiment, the pramipexole is selected from the following group ：Pluronic L31、Pluronic L35、Pluronic L38、Pluronic L42、Pluronic L43、Pluronic L44、Pluronic L61、Pluronic L62、Pluronic L63、Pluronic L64、Pluronic L65、Pluronic L68、Pluronic L72、Pluronic L75、Pluronic L77、Pluronic L81、Pluronic L84、Pluronic L85、Pluronic L87、Pluronic L88、Pluronic L121、Pluronic L122、Pluronic F38、Pluronic F68、Pluronic F108、Pluronic F127、Pluronic P85、Pluronic P94、Pluronic P104、Pluronic P105、Pluronic P123, and the like, or a combination thereof.

In another preferred embodiment, the pramipexole is selected from the group consisting of: pluronic F68, pluronic F77, pluronic F88, pluronic F87, pluronic L65, pluronic L38, or combinations thereof.

In another preferred embodiment, the pramipexole is Pluronic F68.

In another preferred embodiment, the regulator A further comprises an anti-clumping agent that prevents clumping of the suspension culture cells.

In another preferred embodiment, the anti-caking agent comprises: dextran sulfate, dextran 40, dextran 70, or a combination thereof.

In another preferred embodiment, the anti-caking agent is dextran sulfate.

In another preferred embodiment, the mass ratio of the pramipexole to the anti-caking agent is 1 (0.2-0.8), preferably 1 (0.4-0.7), more preferably 1 (0.5-0.6).

In a second aspect of the present invention, there is provided a medium for use in the preparation of an anti-CD 20 antibody rituximab analogue, the medium comprising a basal medium and a fed-batch medium,

Wherein, the feeding culture medium comprises a regulator A, and the regulator A comprises: uridine, mn ²⁺, galactose, and Pluronic (Pluronic);

And 45.0% < G1F+G2F < 70.0% of the analogue was prepared.

In another preferred embodiment, the pramipexole is Pluronic F68.

It is understood that within the scope of the present invention, the above-described technical features of the present invention and technical features specifically described below (e.g., in the examples) may be combined with each other to constitute new or preferred technical solutions. And are limited to a space, and are not described in detail herein.

Drawings

FIG. 1 shows the viable cell density of each experimental group screened with the medium of example 1.

FIG. 2 shows the cell growth and activity change curves for each experimental group of the medium screening in example 1.

FIG. 3 shows the number of living cells cultured in the 3L reactor of example 2

FIG. 4 shows the cell growth and activity change curves of the 3L reactor culture in example 2.

FIG. 5 shows the expression profile of the 3L reactor cultured antibodies in example 2.

FIG. 6 shows a flow chart of a 250L process scale-up cell culture in example 3.

FIG. 7 shows the growth of cells during the shake flask seed culture stage of the 250L process of example 3, showing the number of viable cells and the change in growth rate during the shake flask seed culture stage.

FIG. 8 shows cell growth during the 15L reactor seed culture stage of the 250L process scale-up in example 3. The figure includes a 15L reactor seed culture stage of viable cell count and seed cell growth activity curve.

FIG. 9 shows cell growth during the 50L reactor seed culture stage of the 250L process scale-up in example 3. The figure includes a 50L reactor seed culture stage of viable cell count and seed cell growth activity curve.

FIG. 10 is a graph showing the comparison of cell growth in the 250L process-expanded cell culture of example 3 with the addition of modifier A, modifier B, and the R3-01 group (example 2) of 3L pilot cell culture. The arrows in the figure show the phase of feeding the feed medium.

FIG. 11 shows a comparison of the expression of antibodies from group R3-01 (example 2) in a 250L process-expanded cell culture of example 3 with the addition of regulator A, regulator B and 3L pilot cell culture.

FIG. 12 shows the type of antibody N-glycosylation.

Detailed Description

The inventors have conducted extensive and intensive studies to develop, for the first time, a method for preparing an anti-CD 20 antibody rituximab-like drug by mass screening, which can obtain a bio-mimetic drug having a sugar-like nature similar to rituximab as an original drug by using a fed-batch medium to which a regulator A is added in a cell culture system. Wherein, the regulator A comprises: uridine, mn ²⁺, galactose and pramipexole (Pluronic), and the molar concentration ratio of uridine, mn ²⁺ and galactose was 1:0.002:5, and the concentration ratio of uridine to pramipexole was 1mmol:0.05g.

Furthermore, the present inventors performed a 250L reactor scale-up experiment on the basis of a 3L reactor sugar bench scale-up process. In the culture product obtained by the culture system, the N-glycosylation level G1F+G2F value of the isolated antibody product can reach 51.44 under the premise of ensuring the cell activity rate and the antibody yield, is obviously higher than 36.82 which can be achieved in the prior art, and is very close to the G1F+G2F value 51.77 of the original medicine rituximab. The present invention has been completed on the basis of this finding.

Terminology

As used herein, the terms "rituximab analog," "anti-CD 20 antibody rituximab analog," and "anti-CD 20 antibody rituximab" are used interchangeably to refer to therapeutic biologicals that are similar in quality, safety, and efficacy to rituximab (rituximab injection), which is an approved registered, as an anticancer drug. In particular, it can reach N-glycosylation quality standard 45.0% < G1F+G2F < 70.0%.

Culture medium

Including commercial basal medium and fed-batch medium, table 1 below is a common commercial medium since commercial medium does not disclose specific formulation information:

Table 1 commercial Medium commonly used

The above-described method for preparing the medium is required to be carried out according to the requirements of the medium specification.

Preparation method

Key components and addition proportion of the regulator A:

TABLE 2 molecular composition of modifier A used in examples 1-3

Substance (B)	Preparation method (1L)	Mole number/L
			Niaogangan (sweet urine)	6.11g	25mmol
MnCl2	6.34g	0.05mmol
			Galactose	22.52g	125mmol
Pramipexole F68	1.25g	1.25g

In other embodiments, pramipexole may be selected from Pluronic L31、Pluronic L35、Pluronic L38、Pluronic L42、Pluronic L43、Pluronic L44、Pluronic L61、Pluronic L62、Pluronic L63、Pluronic L64、Pluronic L65、Pluronic L68、Pluronic L72、Pluronic L75、Pluronic L77、Pluronic L81、Pluronic L84、Pluronic L85、Pluronic L87、Pluronic L88、Pluronic L121、Pluronic L122、Pluronic F38、Pluronic F68、Pluronic F108、Pluronic F127、Pluronic P85、Pluronic P94、Pluronic P104、Pluronic P105、Pluronic P123, and the like, or a combination thereof. The adding proportion is that 0.03-0.06 g of pramipexole is configured for each 1mmol of uridine.

In addition, an anti-caking agent selected from dextran sulfate, dextran 40, dextran 70, or combinations thereof may be added as desired. In examples 1-3, the anti-caking agent used was dextran sulfate, preferably having a molecular weight of 5000 daltons, and 0.25g of dextran sulfate having a molecular weight of 5000 daltons was added to 1L of modifier A. Typically, the mass ratio of pramipexole to anti-caking agent is 1 (0.2-0.8), preferably 1 (0.4-0.7), more preferably 1 (0.5-0.6).

Modifier B contains no two components, F68, dextran sulfate.

Conventional cell culture methods

Cells for antibody production selected from CHO cells, NS0 cells and HEK293 cells following conventional cell culture methods including various stages of cell resuscitation, basal medium resuspension, cell expansion, feed culture, etc., wherein the initial cell seeding density of cell expansion is 1x 10 ⁶ cells/ml; in the culture process, sampling and detecting the density, the activity and the physicochemical indexes of the cell fluid, including the levels of glucose, glutamine, lactic acid and NH4+, every day is needed; the feeding of the fed-batch culture solution is generally controlled to be carried out when the glucose is lower than 2g/L and the glutamine is lower than 0.5mM, and according to experience, the fed-batch culture medium is respectively added into the reaction system from the 4 th day to the 6 th day and from the 7 th day to the 9 th day of the culture, and preferably is respectively added into the reaction system from the 5 th day and the 8 th day of the culture; in addition, when the glucose content was less than 4g/L, the glucose solution was supplemented to 4g/L. In general, the culture broth can be harvested when the cell viability is below 70% or at 14.+ -. 2 days of culture for detection of the amount of antibody expression and detection of N-glycoform.

Antibody glycoform

Glycosylation modification of antibodies is a key quality attribute of therapeutic antibody drugs, with the primary glycosylation form being the N-glycosylation modification of the Fc terminus. Several forms of N-glycosylation are shown in FIG. 12.

N-glycosylation assay methods

The sample was diluted to the concentration to be examined, 2.5. Mu.L PNGase F (glycosidase) was added, mixed well, transiently separated for 5s, and water-bath was performed at 50℃for 1 hour. 40. Mu.L of HILIC labeling reagent was added, 60. Mu.L of methanol was further added, the centrifuge tube was capped, and the mixture was then placed in a water bath at 80℃for 75 minutes. Samples were removed and centrifuged at 15000rpm at 10℃for 20 minutes to precipitate proteins. The supernatant was taken in a 1.5mL centrifuge tube and lyophilized in vacuo at a temperature of 4 ℃. After the sample is dried, 40 mu L of 50% ACN is added and mixed uniformly; after centrifugation at 12000rpm for 5min, 18. Mu.L of the supernatant was placed in the inner tube to remove the bottom air bubbles.

Samples were run on Wasters UPLC and analyzed using a HILIC column with a sample loading of 2. Mu.L, excitation wavelength of 360nm, emission wavelength of 425nm, and detection using a fluorescence detector. Gradient elution was used and the elution gradient was set to table 3 below.

TABLE 3 elution gradient set-up

The advantages of the invention include:

1) Through the exploration of components and dosages, an effective glycoform regulator is determined, so that the glycoform specification is controlled to meet the industrial production requirement, and the yield/quantity of cells and antibodies is ensured to be higher.

2) Compared with the commercialized sugar-type regulator, the sugar-type regulator has simple components and greatly saves the production cost.

3) The present invention also further breaks the technical monopoly of commercial glycomodulators, since the formulation of commercial glycomodulators is not disclosed.

4) Breaks through the technical barriers of the simulated pharmaceutical production of imported antibody drugs, and greatly accelerates the process of marketing the domestic simulated antibody drugs.

The invention will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present invention and are not intended to limit the scope of the present invention. The experimental procedure, which does not address the specific conditions in the examples below, is generally followed by routine conditions, such as, for example, sambrook et al, molecular cloning: conditions described in the laboratory Manual (New York: cold Spring Harbor Laboratory Press, 1989) or as recommended by the manufacturer. Percentages and parts are weight percentages and parts unless otherwise indicated.

Unless otherwise indicated, the materials and reagents used in the examples were all commercially available products.

Example 1: cell culture media selection and determination

1.1 Cell culture Medium Screen

In an ultra-clean bench, about 12mL of cell liquid is taken from each 250mL of shaking flask, 7 bottles are taken, 38mL of 8 basic culture mediums with different proportions are respectively added, the basic culture mediums are shown in a table 4, 7 experimental groups are arranged, the numbers of the experimental groups are F1, F2, F3, F4, F5, F6 and F7, and the reference group is F8. The culture volume was 50mL, and the flask was placed in a carbon dioxide shaker at 37.0℃and 120rpm with 5.0% CO ₂, and inoculated at an initial culture density of 1X 10 ⁶ cells/mL and co-cultured for 14 days. Wherein GE001 (GE-Hyclone) is a commercial medium and can be used for sugar type regulation, and the specific application method is shown in the specification of the product.

TABLE 4 basal culture composition for each experimental group

The temperature of the carbon dioxide shaker was adjusted to 34.0℃by the 5 th day of cultivation and to 32.0℃by the 8 th day of cultivation. The fed-batch culture medium is added during the culture process to provide nutrient substances required by the growth of cells during the culture process, and the composition and the feeding strategy of the fed-batch culture medium of each experimental group are shown in Table 5. When the glucose content in the shake flask was lower than 4g/L, a 30% glucose solution (containing 300g/L of glucose) was supplemented to the shake flask to bring the glucose content in the medium to about 4 g/L.

Table 5 composition of the fed-batch medium and feeding strategy for each experimental group

Samples were taken on days 5,8, 11, 12, 13, 14, cell densities were measured, and 1mL of cell supernatants were left for days D5, D8, D11, and D14 for antibody content, and about 8mL of cell supernatants were left for glycoform detection on days D11 and D14.

TABLE 6 final concentrations of key components of regulator A

TABLE 7 final concentrations of key ingredients of regulator B

1.2 Cell culture Medium determination

In terms of cell growth, it was found from the cell growth of each experimental group in FIGS. 1 and 2 that although the cell viability of the F5, F6, F7 and F8 groups during the culture (FIG. 2) was comparable, there was a large difference in the level of the actual viable cell count, and that the F7 group was not added with any regulator, and only a commercial fed-batch medium was used, and the maximum cell density was only about 10X 10 ⁶ cells/mL, and the maximum cell density of the other six experimental groups was 20X 10 ⁶ cells/mL or more. F5 and F6 experimental groups were able to maintain higher cell densities and viability later than the other 5 experimental groups. And the F8 group is a regulator control group, does not contain F68 and anti-caking agent, and has the highest living cell density of only 10 multiplied by 10 ⁶ cells/mL.

In terms of antibody expression, as can be seen from the condition of the antibody expression level (FIG. 3), the antibody expression levels of shake flasks F5 and F6 are significantly better than those of other experimental groups, which are 1.729g/L and 1.674g/L, respectively. And from the antibody glycoform detection results (table 8), the F5 and F6 experimental groups were closer to the glycoform expected values g1f+g2f of the rituximab standard and to the standard. Therefore, the whole cell culture process of the F5 and F6 experimental groups has optimal performance, and can be used as the basis of the 3L reactor sugar type small test process.

Table 8 summary of N-glycoform results for each experimental group

Example 2:3L cell culture process

2.1 Cell resuscitation

Taking out the cell freezing tube from the liquid nitrogen tank, immediately putting the cell freezing tube into a water bath at 37 ℃ to enable the cell liquid to quickly defrost and cross the freezing point, and preventing the cell liquid from forming ice crystals to puncture cell membranes to influence the cells.

After the cells were completely thawed, they were removed from the water bath and sterilized with 75% alcohol. Then, the frozen tube was opened in an ultra clean bench, the cell fluid was aspirated by a sterile pipette, placed in a 50mL shake flask containing 10mL of medium, shaken well, and then taken out about 0.5mL of cell suspension for cell counting. Shake flasks were incubated in a carbon dioxide shaker (37.0 ℃,120rpm,5.0% CO 2).

2.2 Shake flask cell expansion

Dynamis (Gibco) +MTX (0.5 mL of 1mM MTX solution is added to each liter of basal medium) is used for culture, sampling and counting are carried out every three days, and then passage is carried out, the culture is carried out according to the cell density of 1.0X10. 10 ⁶ cells/mL passage, the culture is carried out to the cell quantity required by 4 3L reactors, and then the culture is carried out to 4 3L reactors for continuous culture.

2.3 3L reactor inoculation

About 1.6L Dynamis (Gibco) medium was pumped into the 4 3L reactors, respectively, and after sterile inspection, about 220mL of seed cells were inoculated into the 4 3L reactors, respectively, and after uniform mixing, about 220mL of cell fluid was removed, respectively, to give an initial culture volume of about 1.6L per 3L reactor. The reactor temperature was adjusted to 34.0℃by culturing to 5 days and to 32.0℃by culturing to 8 days. The 4 3L reactors were fed-batch medium fed according to the fed-batch medium fed-batch strategy of Table 9. When the glucose content in the shake flask was lower than 4g/L, a 30% glucose solution (containing 300g/L of glucose) was fed into the reactor to make the glucose content in the medium about 4 g/L. The culture parameters of the 4 3L reactors are shown in Table 9, wherein R3-01 and R3-02 are the same, and R3-03 and R3-04 are the same.

TABLE 9 3L reactor Process parameter settings

2.4 Sampling and sample leaving

During the cell culture in the 3L reactor, the 3L reactor was sampled and counted every day except for day 6 and day 7, and the cell sap was subjected to biochemical parameter detection. After each sampling, reserving 1mL multiplied by 2 tube cell supernatant, and preserving at-20 ℃ for detecting protein content; 50mL x 1 tube cell supernatants were left for each reactor on days 11 and 14 and stored at-20℃for glycoform detection.

2.5 Results and discussion

In terms of cell growth, it can be seen from FIG. 4 that the two parallel sets of reactors have similar cell growth, the 4 3L reactors have similar early growth, the highest density is about 27X 10 ⁶ cells/mL, the later stages of R3-03 and R3-04 show a relatively significant decrease in cell density and cell viability, and R3-01 and R3-02 maintain higher cell densities and viability.

In terms of antibody expression, it can be seen from the case of antibody expression levels (FIG. 5) that the two parallel sets of reactor cells had similar antibody expression levels, with the R3-01 and R3-02 antibody expression levels being superior to those of R3-03 and R3-04. From the antibody glycoform detection results (Table 10), it can be seen that the 4 reactor glycoform detection results are similar. In conclusion, the overall performance of the process conditions of R3-01 and R3-02 is more suitable for being used as the basis of the enlarged production of the 250L reactor process.

TABLE 10 3N-sugar type detection results for reactor antibody of 3L

Example 3:250L process amplification culture

3.1 Cell culture procedure

As shown in fig. 6.

3.2 Cell resuscitation

Taking out the cell cryopreservation tube from the liquid nitrogen tank, immediately placing the cell cryopreservation tube into a water bath at 37 ℃, taking out the cell from the water bath after the cell is completely thawed, and sterilizing the cell by using 75% alcohol. Then, the frozen tube was opened in an ultra clean bench, the cell fluid was aspirated by a sterile pipette, placed in a 50mL shake flask which had been filled with culture, and after shaking, about 0.5mL of cell suspension was taken out for cell counting. Shake flasks were incubated in a carbon dioxide shaker (37.0 ℃,120rpm,5.0% CO ₂).

3.3 Seed culture in shake flask

The next day, after cell sampling and counting, the cells were transferred into a sterile centrifuge tube and centrifuged at 700rpm for 5min. The supernatant was discarded, and the cells were resuspended in a culture flask with fresh Dynamis (Gibco) (containing MTX) to a final density of about 1.0X10 ⁶ cells/mL. After every three days sampling and counting, the passage is carried out, the density of the passage cells is 1.0X10 ⁶ cells/mL, and the passage culture medium is a basal culture medium containing MTX. The cells were passaged to the desired cell volume in the 15L reactor and transferred to the 15L reactor for further culture.

3.4 Seed culture in 15L reactor

Before the 15L reactor is inoculated, the tank body is assembled (the pH electrode needs to be calibrated in advance) to ensure that the pipeline connection is correct, 5L of WFI is injected into the tank body, and then the tank body and related accessories are subjected to heat sterilization treatment at 121 ℃ for 120 min. After the tank is sterilized, the WFI in the tank is emptied after the tank is sufficiently cooled, about 7.9L Dynamis (Gibco) medium is added, the parameters of the reactor are set to be the temperature of 37.0 ℃ and 120rpm for the sterility inspection of the medium, the inspection time is 18-24 hours, and after the sterility inspection is correct, the dissolved oxygen electrode and the pH correction electrode are calibrated.

3.5 Seed culture in 50L reactor

50L reactor seed culture was performed using a 50L Hyclone disposable reactor. Before use, the reaction bag was installed, the sterilized electrode (pH electrode calibrated) was inserted into the reaction bag, and about 42.5L Dynamis (Gibco) medium was added. The mixture was incubated at a stirring speed of 90rpm and a temperature of 37.0℃for 18 to 24 hours, and examined for sterility by a sampling microscope. After sterility verification, the dissolved oxygen electrode and the corrected pH electrode were calibrated.

The 50L reactor was seeded at a density of about 1.0X10 ⁶ cells/mL and 15L reactor seed solution was added to about 5.3L. The culture parameters are DO:50%, pH: 7.00+/-0.20, temperature: stirring at 37.0 ℃): 90rpm. Culturing was continued by transferring to a 250L reactor until day 3. Daily sampling was performed to measure cell density, cell viability and metabolic parameters.

3.6 Seed culture in 250L reactor

250L reactor seed culture was performed using a 250L Hyclone disposable reactor. Before use, the reaction bag was installed, the sterilized electrode (pH electrode calibrated) was inserted into the reaction bag, and then about 150L Dynamis (Gibco) medium was added. The culture was carried out at a stirring speed of 60rpm and a temperature of 37.0℃for 18 to 24 hours, and the sterility was examined by a sampling microscopic examination. After sterility verification, the dissolved oxygen electrode and the corrected pH electrode were calibrated.

The inoculation was performed at a density of about 1.0X10 ⁶ cells/mL and about 20L of the 50L reactor seed solution was added. The culture parameters are DO:50%, pH: 7.00+/-0.20, temperature: stirring at 37.0 ℃): 80rpm.

Culturing until 5 days, adjusting the set temperature of the reactor culture to 34.0 ℃, culturing until 5 days, and adjusting the set temperature of the reactor culture to 32.0 ℃. During the incubation, the pH was controlled with 7.5% sodium bicarbonate solution and CO2 gas, and defoamed with 5% defoamer solution. The fed-batch medium was fed to the reactor at 5 th and 8 th days of the culture, and when the glucose content in the reactor was lower than 4.0g/L, a 30% glucose solution (containing 300g/L of glucose) was fed to the reactor so that the glucose content in the medium became about 4.0g/L. Daily sampling was performed to measure cell density, cell viability and metabolic parameters.

3.7 Sample preparation

From day 0 to the end of the culture, 1mL of the supernatant of the 2-tube cells was left every day and kept at-20℃for protein content detection; 40mL of the supernatant of the x 2 tube cell was left at day 11 and day 14 and stored at-20℃for the detection of the physicochemical properties of the protein.

3.8 Results and discussion

3.8.1 Shake flask seed data

The growth curve of the shake flask seed cells is shown (FIG. 7).

3.8.2 15L seed culture data

The 15L reactor seed cell growth curve is shown (FIG. 8).

3.8.3 50L seed culture data

The 50L reactor cell growth curve is shown (FIG. 9).

3.8.4 250L seed culture data

The comparison of the cell growth and activity change curves in R3-01 culture in the 3L pilot scale process described in example 2 with 250L of regulator A, 250L of regulator B and the 3L pilot scale process described in example 2 was performed according to the fed-batch culture addition strategy of example 2R3-01, respectively, as shown in FIG. 10. In terms of cell growth, both cell growth and cell viability for sugar-type process cell culture with regulator A added to the 250L reactor were better than the 3L pilot culture and the 250L reactor with regulator B added.

The comparison of antibody expression for 250L reactor addition modifier A, 250L reactor addition modifier B and 3L pilot culture process is shown in FIG. 11; the results of the N-glycoform detection of the antibodies are shown in Table 11. In terms of antibody expression, the antibody yield of the regulator A added in the 250L reactor is higher than that of the culture conditions of the regulator B added in the 3L pilot cell and the regulator B added in the 250L reactor, and the N-glycoform of the antibody is superior to that of the 3L pilot cell and the regulator B added in the 250L reactor in batches, so that the antibody has higher similarity with the original ground medicament.

Table 11 comparison table of N-glycoform results of antibodies

EXAMPLE 4 fumbling of other Medium combinations

Based on examples 1-3, the present invention also attempted to use other commercial media in combination with the development strategy of regulator A, such as CD FortiCHO media (Thermo FISHER SCIENTIFIC) as basal media, sheff-CHO PLUS PF ACF (KERRY) with regulator A according to 50:50, preparing a fed-batch culture medium; or Balan CD CHO Growth A (IRVINESCIENTIFIC) as basal medium, CD Feed 002 (oldman) with regulator A according to 75:25, preparing a fed-batch culture medium in proportion; or CD ClM (Life Technology) as basal medium, cel Boost 5 (GE-Hyclone) with regulator A according to 25:75, can play a role in better glycoform regulation and antibody yield improvement.

All documents mentioned in this disclosure are incorporated by reference in this disclosure as if each were individually incorporated by reference. Further, it will be appreciated that various changes and modifications may be made by those skilled in the art after reading the above teachings, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.

Claims

1. A method of preparing an anti-CD 20 antibody rituximab analog comprising the steps of:

(B) Optionally isolating the resulting analogue from the culture product;

Wherein, regulator A is added to the fed-batch culture medium, and the regulator A comprises: uridine, mn ²⁺, galactose, pluronic (Pluronic) and dextran sulfate; the pramipexole is Pluronic F68;

the mol concentration ratio of uridine, mn ²⁺ and galactose is (0.9-1.1): 0.0015-0.0025): 3-8, and the concentration ratio of uridine to pramipexole is 1mmol (0.03-0.06) g; the mass ratio of the pramipexole to the dextran sulfate is 1 (0.4-0.7);

and 45.0% < G1F+G2F < 70.0% of the analogue was prepared.

2. The method of claim 1, wherein the method further comprises the step of:

3. The method of claim 2, wherein the glucose concentration in the glucose mother liquor is from 200 to 400g/L.

4. A method according to claim 3, wherein the concentration of glucose in the glucose mother liquor is 250-350g/L.

5. The method of claim 4, wherein the glucose concentration in the glucose mother liquor is 300g/L.

6. The method according to any one of claims 2 to 5, wherein the volume of the culture system is 0.1 to 500L.

7. The method of claim 6, wherein the culture system has a volume of 3 to 300L.

8. The method of claim 7, wherein the culture system has a volume of 100 to 250L.

9. The method of any one of claims 1-5, wherein the cell is selected from the group consisting of: CHO cells, NS0 cells, HEK293 cells, or a combination thereof.

10. The method according to any one of claims 1 to 5, wherein the fed-batch medium is added to the reaction system at the time of cultivation to the 4 th to 6 th days and 7 th to 9 th days, respectively.

11. The method of claim 10, wherein the feed medium is added to the reaction system at day 5 and day 8, respectively.

12. The method according to any one of claims 1 to 5, wherein the total amount of the fed-batch medium is 10 to 30v/v% of the total reaction system.

13. The method of claim 12, wherein the total amount of feed medium added is 15-25v/v% of the total reaction system.

14. The method of claim 12, wherein the total amount of feed medium added is 20v/v% of the total reaction system.

15. The method of any one of claims 1-5, wherein the molar concentration ratio of uridine, mn ²⁺, and galactose is 1:0.002:5.

16. The method of any one of claims 1-5, wherein the final concentration of uridine, mn ²⁺ and galactose in the culture system is 0.3 x UMG to 4 x UMG;

wherein the 1×umg represents:

17. The method of claim 16, wherein the final concentration of uridine, mn ²⁺, and galactose in the culture system is 0.5 x UMG to 3 x UMG.

18. The method of claim 17, wherein the final concentration of uridine, mn ²⁺, and galactose in the culture system is 1 x UMG to 2 x UMG.

19. The method of any one of claims 1-5, wherein the concentration ratio of uridine to pramipexole is 1 mmol/0.05 g.

20. A culture medium used in the process of preparing the anti-CD 20 antibody merozoite analogue is characterized in that the culture medium comprises a basal culture medium and a fed-batch culture medium,

Wherein, the feeding culture medium comprises a regulator A, and the regulator A comprises: uridine, mn ²⁺, galactose, pluronic (Pluronic) and dextran sulfate; the pramipexole is Pluronic F68;

the mol concentration ratio of uridine, mn2+ and galactose is (0.9-1.1): (0.0015-0.0025): (3-8), and the concentration ratio of uridine and pramipexole is 1mmol (0.03-0.06) g; the mass ratio of the pramipexole to the dextran sulfate is 1 (0.4-0.7);

and 45.0% < G1F+G2F < 70.0% of the analogue was prepared.