CN111321188A

CN111321188A - Formula for modifying antibody glycoform, cell culture method and application in industrial production

Info

Publication number: CN111321188A
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: 2020-06-23

Abstract

The invention provides a method for preparing anti-CD 20 antibody rituximab, which comprises the following steps: (a) culturing the cells, wherein the culture medium used comprises a basal medium and a feed medium; and (b) optionally isolating the resulting analog from the culture product; wherein, the feeding culture medium is added with a regulator A, and the regulator A comprises: uridine, Mn²⁺Galactose and Pluronic; the uridine and Mn²⁺And galactose in a molar concentration ratio of (0.8-1.2) to (0.001-0.003) to (2-10), and uridine to pluronic in a concentration ratio of 1mmol to (0.02-0.08) g; and 45.0% < G1F + G2F < 70.0% of the similar drug prepared. Using the method of the present invention, rituximab organisms conforming to the desired glycoform can be stably produced in high yield in an industrial scale-upSimilar to the medicine.

Description

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

Technical Field

The invention relates to the field of biological pharmacy, in particular to an antibody glycoform modification 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 drugs, the conventional production process has difficulty in reaching the quality standard of glycoform control. For the CD20 antibody original research drug, rituximab, the glycoform is a very important quality index, and the N-glycosylation quality standard of the antibody is 45.0% < G1F + G2F < 70.0%. However, the rituximab-like drug glycoform produced in practical procedures using commercial culture media differs greatly from the original research drug rituximab form, in particular in that G0F (57.71%) glycosylated at the N-terminus is higher than that of the original research drug (37.78% for G0F); G1F (29.24%) and G2F (7.58%) are lower than those of the original medicines (41.43% and 10.34% for G1F and G2F respectively), and the quality standard of the antibody G1F + G2F is 36.82 which is far lower than that of the antibody N-glycosylation (45.0% < G1F + G2F < 70.0% for the quality standard of the N-glycosylation). Therefore, the existing production method is difficult to meet the national quality control requirements of the counterfeit drugs, and the counterfeit drugs cannot be listed as soon as possible even if the original drug patents are overdue. .

The culture medium is a basic solution for maintaining the survival and growth metabolism of cells and is one of the most important conditions for cell culture. It provides a convenient controlled and standardized living environment for cells. Although cell culture uses chemically synthesized media that have been standardized for production, composition, and content, cell culture media and culture processes often require scale-up procedures to be validated for large-scale or larger-scale production before large-scale production can occur. In the amplification process, parameter settings such as temperature, pH, dissolved oxygen, stirring and the like need to be considered integrally 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 culture expression of rituximab biosimilar drugs to conform to the expected glycoform of the original research drug, and a method capable of stably producing the rituximab biosimilar drugs conforming to the expected glycoform at a high yield in an industrial scale-up production.

Disclosure of Invention

The invention aims to provide a culture medium which can ensure that the culture expression of the rituximab biosimilar drug conforms to the expected glycoform, and a method for stably producing the rituximab biosimilar drug conforming to the expected glycoform in high yield 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 used comprises a basal medium and a feed medium; and

(b) optionally isolating the resulting analog from the culture product;

wherein, the feeding culture medium is added with a regulator A, and the regulator A comprises: uridine, Mn²⁺Galactose and Pluronic;

the uridine and Mn²⁺And galactose in a molar concentration ratio of (0.8-1.2) to (0.001-0.003) to (2-10), and uridine to pluronic in a concentration ratio of 1mmol to (0.02-0.08) g;

and 45.0% < G1F + G2F < 70.0% of the similar drug prepared.

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

(c) monitoring the glucose concentration in the culture system, and when the glucose concentration is lower than 4g/L, supplementing a glucose mother liquor into the culture system to enable the glucose content in the culture system to be about 4 g/L.

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

In another preferred embodiment, the basal medium is selected from commercially available commercial media including, but not limited to, CDFortiCHO media (Thermo Fisher Scientific), Dynamis (Gibco), Balan CD CHO Growth A (Irvine Scientific), Artipro (GE-Hyclone), CD 1M1(Life Technology), CD012 (Ompman), CD11V (Jianshun).

In another preferred embodiment, the Feed medium is selected from commercially available commercial media including, but not limited to, CDEffectintFeed C + AGT Supplement (Thermo Fisher Scientific), Sheff-CHO PLUS PFACF (KERRY), Cell Boost 5(GE-Hyclone), Cell Boost 4 (GE-Hycon), CD Feed 002 (Omegan), PFF05 (Jianjian Probiotics).

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

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

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

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 cell is 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 feeding medium is added to the reaction system at the 4 th to 6 th day and the 7 th to 9 th day, respectively, preferably at the 5 th and 8 th day, respectively.

In another preferred embodiment, the feeding medium is added in a total amount of 10 to 30 v/v%, preferably 15 to 25 v/v%, more preferably 20 v/v% of the total reaction system.

In another preferred embodiment, the feeding medium is added in an amount of 3 to 20 v/v%, preferably 5 to 15 v/v%, more preferably 10 v/v% per time of the total reaction system.

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

In another preferred embodiment, said uridine, Mn²⁺The molar concentration ratio of galactose to galactose is (0.9-1.1): (0.0015-0.0025): 3-8), preferably 1:0.002: 5.

In another preferred embodiment, uridine and Mn are contained in the culture system²⁺And galactose at a final concentration of 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 1mM, Mn²⁺The concentration was 0.002mM and the galactose concentration was 5 mM.

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

In another preferred embodiment, the pluronic is selected from the group consisting of: 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 P68653, Pluronic P94, Pluronic P86104, Pluronic F84105, Pluronic P84123, or combinations thereof.

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

In another preferred embodiment, the Pluronic is Pluronic F68.

In another preferred embodiment, said modulator a further comprises an anti-caking agent which 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 pluronic 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 culture medium for use in the preparation of anti-CD 20 antibody rituximab, said culture medium comprising a basal medium and a feed medium,

wherein the feeding medium comprises a regulator A, and the regulator A comprises: uridine, Mn²⁺Galactose and Pluronic;

and 45.0% < G1F + G2F < 70.0% of the similar drug prepared.

In another preferred embodiment, the Pluronic is Pluronic F68.

It is to be understood that within the scope of the present invention, the above-described features of the present invention and those specifically described below (e.g., in the examples) may be combined with each other to form new or preferred embodiments. Not to be reiterated herein, but to the extent of space.

Drawings

FIG. 1 shows viable cell densities of the culture medium screening of each experimental group in example 1.

FIG. 2 shows the cell growth and viability profiles of the experimental groups screened with medium in example 1.

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

FIG. 4 shows the cell growth and viability profiles of the 3L reactor cultures of example 2.

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

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

FIG. 7 shows the cell growth in the 250L process scale-up shake flask seed cultivation stage of example 3, showing the viable cell number and growth viability curves for the shake flask seed cultivation stage.

FIG. 8 shows the growth of cells during the seed culture phase of a 15L reactor during the scale-up of the 250L process in example 3. The figure includes the viable cell number and seed cell growth viability curves for the 15L reactor seed culture stage.

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

FIG. 10 is a graph showing a comparison of the growth of cells in the group R3-01 (example 2) in the 250L process scale-up cell culture in example 3 with the addition of regulator A, regulator B and the 3L pilot process cell culture. The arrows in the figure indicate the stage of supplementing the fed medium.

FIG. 11 is a graph showing a comparison of the expression of the antibody in the group R3-01 (example 2) in the 250L process scale-up cell culture in example 3 with the addition of regulator A, regulator B and the 3L pilot scale cell culture.

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

Detailed Description

The present inventors have made extensive and intensive studies and, as a result of extensive screening, have unexpectedly developed for the first time a method for producing an anti-CD 20 antibody rituximab analogue by using it in a cell culture systemAdding a fed-batch culture medium of the regulator A to obtain the biosimilar drug with the sugar type similar to that of the original drug rituximab. Wherein, the regulator A comprises: uridine, Mn²⁺Galactose and Pluronic (Pluronic), and said uridine, Mn²⁺And galactose at a molar concentration ratio of 1:0.002:5, and said uridine to pluronic at a concentration ratio of 1mmol:0.05 g.

In addition, the present inventors conducted a 250L reactor scale-up experiment based on a 3L reactor sugar type bench test process. In the culture product obtained by the culture system, the value of the N-glycosylation level G1F + G2F of the separated antibody product can reach 51.44 on the premise of ensuring the cell viability and the antibody yield, is obviously higher than 36.82 which can be reached in the prior art, and is very close to the value 51.77 of G1F + G2F of the original research medicine rituximab. The present invention has been completed based on this finding.

Term(s) for

As used herein, the terms "rituximab-like drug", "anti-CD 20 antibody rituximab-like drug", and "anti-CD 20 antibody rituximab-like drug" are used interchangeably to refer to a therapeutic biological product that has similarities in quality, safety, and effectiveness with the original drug (reference drug) anticancer drug, rituximab injection, that has been approved for registration. Specifically, the N-glycosylation quality standard can reach 45.0% < G1F + G2F < 70.0%.

Culture medium

Including commercial basal media and feed media, since commercial media do not disclose specific formulation information, table 1 below is a commonly used commercial medium:

table 1 general commercial culture Medium

The method of disposing the medium is required to be performed according to the requirements of the medium specification.

Preparation method

Key components and addition proportion of the regulator A are as follows:

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

Substance(s)	Preparation method (1L)	Mole number/L
			Urine sweet	6.11g	25mmol
MnCl2	6.34g	0.05mmol
			Galactose	22.52g	125mmol
Polang F68	1.25g	1.25g

In other embodiments, the Pluronic 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 F94, Pluronic L85, Pluronic P104, Pluronic P123, Pluronic P combinations thereof, or the like. The addition ratio is 0.03-0.06 g of Plumbago every 1mmol of uridine.

In addition, an anti-caking agent can be added according to the experimental requirements, and the anti-caking agent is selected from dextran sulfate, dextran 40, dextran 70 or the combination thereof. In examples 1-3, the anti-caking agent used was dextran sulfate, preferably with a molecular weight of 5000 daltons, to which 0.25g of dextran sulfate with a molecular weight of 5000 daltons was added in 1L of regulator a. Generally, the mass ratio of the pluronic to the anti-caking agent is 1 (0.2-0.8), preferably 1 (0.4-0.7), more preferably 1 (0.5-0.6).

Regulator B does not contain F68, a dextran sulfate component.

Conventional cell culture methods

The cells for preparing the antibody are selected from CHO cells, NS0 cells and HEK293 cells, and the cells follow the conventional cell culture method and comprise cell recovery, basic medium resuspension, cell amplification, feed culture and other stages, wherein the initial cell inoculation density of the cell amplification is 1x 10⁶Number of cells/ml; during the culture process, daily sampling is needed to detect the density and the vitality of living cells and physical and chemical indexes of cell liquid, including the levels of glucose, glutamine, lactic acid and NH4 +; the supplement of the fed-batch culture solution is generally controlled to be less than 2g/L of glucose and less than 0.5mM of glutamine, and the fed-batch culture medium is added into the reaction system respectively at the 4 th to 6 th day and the 7 th to 9 th day of culture according to empirical research, preferably at the 5 th day and the 8 th day of culture; in addition, when the glucose content is lower than 4g/L, the glucose solution is supplemented to 4 g/L. Generally, the culture broth can be harvested when the cell viability is less than 70% or at 14. + -.2 days of culture for the detection of the expression amount of the antibody and the detection of the N-glycoform.

Antibody glycoforms

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

N-glycosylation assay

The sample is diluted to the concentration to be detected, 2.5 mu L of PNGase F (glycosidase) is added, the mixture is mixed evenly, and the mixture is instantaneously separated for 5 seconds and is bathed in water at 50 ℃ for 1 hour. Adding 40 μ L HILIC labeling reagent, adding 60 μ L methanol, covering the centrifugal tube, and water-bathing at 80 deg.C for 75 min. The sample was removed, centrifuged at 15000rpm for 20 minutes at 10 ℃ to precipitate the protein. The supernatant was taken in a 1.5mL centrifuge tube, freeze dried under vacuum, set at 4 ℃. After the sample is dried, 40 mu L of 50% ACN is added and mixed evenly; after centrifugation at 12000rpm for 5min, 18. mu.L of the supernatant was placed in the inner cannula, and air bubbles at the bottom were removed.

Running the sample on a Waster UPLC, analyzing by using a HILIC column, carrying out sample injection with 2 mu L, excitation wavelength of 360nm and emission wavelength of 425nm, and detecting by using a fluorescence detector. Gradient elution was used, with the elution gradient set to table 3 below.

TABLE 3 elution gradient setup

The advantages of the invention include:

1) through component and dosage exploration, an effective glycoform regulator is determined, the glycoform specification is controlled to meet the requirement of industrial production, and higher yield/quantity of cells and antibodies is ensured.

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

3) The invention further breaks the technical monopoly of the commercial sugar-type regulator because the formula of the commercial sugar-type regulator is public information.

4) Breaks through the technical barrier of the imitation drug production of imported antibody drugs and greatly accelerates the process of marketing domestic imitation antibody drugs.

The invention will be further illustrated with reference to the following specific examples. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention. Experimental procedures without specific conditions noted in the following examples, generally followed by conventional conditions, such as Sambrook et al, molecular cloning: the conditions described in the laboratory Manual (New York: Cold Spring harbor laboratory Press,1989), or according to the manufacturer's recommendations. Unless otherwise indicated, percentages and parts are percentages and parts by weight.

The materials and reagents used in the examples were all commercially available products unless otherwise specified.

Example 1: cell culture medium screening and determination

1.1 cell culture Medium screening

In an ultra-clean bench, about 12mL of cell fluid is taken from each 250mL shake flask, 7 flasks are taken, 38mL8 basal media with different proportions are respectively added, the composition of the basal media is shown in Table 4, 7 experimental groups are set, and the numbers of the experimental groups are respectively F1, F2, F3, F4, F5, F6 and F7, and the number of the control group is F8. The culture volume was 50mL, and the shake flask was placed at 37.0 ℃ and 120rpm with 5.0% CO₂The cultivation was carried out in a carbon dioxide shaker at 1 × 10⁶cells/mL of initial culture density were inoculated for a total of 14 days. Wherein GE001(GE-Hyclone) is a commercial culture medium and can be used for sugar type adjustment, and the specific using method is described in the product specification.

TABLE 4 basal culture composition of each experimental group

The temperature of the carbon dioxide shaker was adjusted to 34.0 ℃ by day 5 and to 32.0 ℃ by day 8. The fed-batch culture medium is required to be supplemented in the culture process so as to provide nutrients required by cell growth in the culture process, and the composition and the supplement strategy of the fed-batch culture medium of each experimental group are shown in a table 5. When the glucose content in the shake flask is lower than 4g/L, a 30% glucose solution (containing 300g/L glucose) is supplemented into the shake flask to make the glucose content in the culture medium to be about 4 g/L.

TABLE 5 feeding culture Medium composition and supplement strategy for each experimental group

Samples were taken on

days

5, 8, 11, 12, 13, 14 to determine cell density, and 1mL of cell supernatant was retained for antibody content on days D5, D8, D11, and D14, and about 8mL of cell supernatant was retained for glycoform determination on days D11 and D14.

TABLE 6 Final concentration of key ingredients of Modulator A

TABLE 7 Final concentration of key ingredients of Modulator B

1.2 cell culture Medium determination

In terms of cell growth, it can be seen from the cell growth of each experimental group in fig. 1 and 2 that, although the cell viability rates (fig. 2) of the F5, F6, F7 and F8 groups during the culture process are equivalent, the level of the actual viable cell number is greatly different, the F7 group is not added with any regulator, only the commercialized fed-batch culture medium is used, and the highest cell density is only 10 × 10⁶About one cell/mL, and the highest cell density of the other six experimental groups is 20 × 10⁶F5 and F6 compared with other 5 experimental groups can maintain higher cell density and survival rate in the later period, while F8 is a regulator control group, does not contain F68 and anti-agglomeration agent, and the highest viable cell density is only 10 × 10⁶Individual cells/mL.

In terms of antibody expression, as can be seen from the antibody expression level (FIG. 3), the antibody expression levels of shake flasks F5 and F6 are obviously superior to those of other experimental groups, namely 1.729g/L and 1.674g/L respectively. And from the results of antibody glycoform detection (table 8), the glycoform expected values G1F + G2F of the F5 and F6 experimental groups closer to rituximab standard are closer to the standard. Therefore, the cell culture process of the F5 and F6 experimental groups is optimal as a whole, and can be used as the basis of a sugar type lab process of a 3L reactor.

TABLE 8 summary of the results of the N-glycoforms of the experimental groups

Example 2: 3L cell culture process

2.1 cell Resuscitation

Taking out the cell freezing tube from the liquid nitrogen tank, immediately placing the tube into a water bath at 37 ℃ to ensure that the cell sap is quickly thawed and crosses over the freezing point, and preventing the cell sap 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% ethanol. Then, the cryopreserved tube was opened in a clean bench, the cell fluid was aspirated by a sterile pipette, and the cell fluid was put into a 50mL shake flask containing 10mL of a medium, shaken, and then about 0.5mL of a cell suspension was taken out to count the cells. The shake flask was 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 per liter of basal medium) medium was used, and the cells were passaged every three days after sampling and counting at a cell density of 1.0 × 10⁶And (4) carrying out passage on each cell/mL, carrying out passage to the cell amount required by 4 3L reactors, and then transferring to 4 3L reactors for continuous culture.

2.33L reactor inoculation

About 1.6L of Dynamis (Gibco) medium was pumped into 4 3L reactors, and about 220mL of seed cells were inoculated into 4 3L reactors, respectively, after sterility testing, and about 220mL of cell sap was removed after mixing, respectively, so that the initial culture volume of each 3L reactor was about 1.6L. The reactor temperature was adjusted to 34.0 ℃ by day 5 and 32.0 ℃ by day 8. Fed-batch media supplementation was performed on 4 3L reactors according to the fed-batch media supplementation strategy of table 9. When the glucose content in the shake flask is lower than 4g/L, a 30% glucose solution (containing 300g/L glucose) is supplemented into the reactor to make the glucose content in the culture medium to be about 4 g/L. The culture conditions of R3-01 and R3-02 are the same, the culture conditions of R3-03 and R3-04 are the same, and the culture parameters of 4 3L reactors are shown in Table 9.

TABLE 93L reactor Process parameter settings

2.4 sampling and Retention

In the process of cell culture in the 3L reactor, the 3L reactor is sampled and counted every day except for the 6 th day and the 7 th day, biochemical parameter detection is carried out on cell sap, 1mL × 2 tube cell supernatant is reserved and stored at the temperature of 20 ℃ below zero after each sampling for detecting protein content, and 50mL × 1 tube cell supernatant is reserved and stored at the temperature of 20 ℃ below zero for detecting sugar type in each reactor on the 11 th day and the 14 th day.

2.5 results and discussion

In terms of cell growth, it can be seen from FIG. 4 that the cells in the two parallel reactors grow similarly, the cells in the first stage of the 4 3L reactors grow similarly, and the highest density is about 27 × 10⁶The cell density and the cell survival rate of each cell per mL and later R3-03 and R3-04 are obviously reduced, while R3-01 and R3-02 maintain higher cell density and survival rate.

In terms of antibody expression, it can be seen from the antibody expression level (FIG. 5) that the antibody expression levels of two parallel reactor cells are similar, and the R3-01 and R3-02 antibody expression levels are better than those of R3-03 and R3-04. As can be seen from the results of the antibody glycoform detection (Table 10), the glycoform detection results were similar in the 4 reactors. In conclusion, the overall process conditions of R3-01 and R3-02 are more suitable for the basis of the process scale-up production of the 250L reactor.

TABLE 103L reactor antibody N-glycoform assay results

Example 3: 250L process scale-up culture

3.1 cell culture procedure

As shown in fig. 6.

3.2 cell Resuscitation

Taking out the cell freezing tube from the liquid nitrogen tank, immediately placing the tube into a water bath at 37 ℃, taking out the tube from the water bath after the cells are completely thawed, and performing disinfection treatment by using 75% alcohol. Then, the cryopreservation tube was opened in a clean bench, the cell fluid was aspirated by a sterile pipette, and the cell fluid was put into a 50mL shake flask containing the culture, shaken well, and then taken out about 0.5mL of the cell suspension for cell counting. The shake flask was placed in a carbon dioxide shaker (37.0 ℃, 120rpm, 5.0% CO)₂) Culturing in medium.

3.3 Shake flask seed culture

The following day, after counting the cells, the cells were transferred to a sterile centrifuge tube and centrifuged at 700rpm for 5min, the supernatant discarded, and the cells were resuspended in culture flasks using fresh Dynamis (Gibco) (containing MTX) to a final density of 1.0 × 10⁶cells/mL, and passage after every three days sampling and counting, the cell density of the passage is 1.0 × 10⁶cells/mL, and the subculture medium is a basal medium containing MTX. Passage is carried out until the cell amount is required by a 15L reactor, and then the cell amount is transferred to the 15L reactor for continuous culture.

3.415L reactor seed culture

Before 15L reactor inoculation, the tank is assembled (pH electrode needs to be calibrated in advance) to ensure correct pipeline connection, 5L WFI is injected into the tank, and then the tank and related accessories are subjected to wet heat sterilization treatment at 121 ℃ for 120 mins. After the tank is sterilized, the WFI in the tank is fully cooled, about 7.9L Dynamis (Gibco) culture medium is added, the reactor parameters are set to 37.0 ℃, 120rpm is used for carrying out culture medium sterility test, the test time is 18-24 hours, and after sterility verification is correct, the dissolved oxygen electrode and the pH correction electrode are calibrated.

3.550L reactor seed culture

50L reactor seed culture A50L Hyclone disposable reactor was used. Prior to use, the reaction bag is mounted, sterilized electrodes (pH electrodes calibrated) are inserted into the reaction bag, and about 42.5L Dynamis (Gibco) medium is added. Stirring speed 90rpm, temperature 37.0 deg.C, culturing for 18-24 hr, sampling and examining sterility. And after sterile verification, calibrating the dissolved oxygen electrode and correcting the pH electrode.

50L reactor at a density of about 1.0 × 10⁶One cell/mL was inoculated and about 5.3L of seed solution was added to the 15L reactor. The culture parameters are DO: 50%, pH: 7.00 ± 0.20, temperature: stirring at 37.0 ℃: 90 rpm. The culture is continued until day 3, and the reactor is transferred to a 250L reactor for further culture. Daily sampling is carried out to detect cell density, cell viability and metabolic parameters.

3.6250L reactor seed culture

250L reactor A250L Hyclone disposable reactor was used for seed culture. Prior to use, the reaction bag is mounted, sterilized electrodes (pH electrodes calibrated) are inserted into the reaction bag, and about 150L Dynamis (Gibco) medium is added. Stirring speed 60rpm, temperature 37.0 deg.C, culturing for 18-24 hr, sampling and examining sterility. And after sterile verification, calibrating the dissolved oxygen electrode and correcting the pH electrode.

At a density of about 1.0 × 10⁶One cell/mL was inoculated and about 20L of seed solution was added to a 50L reactor. The culture parameters are DO: 50%, pH: 7.00 ± 0.20, temperature: stirring at 37.0 ℃: 80 rpm.

The set temperature of the reactor culture was adjusted to 34.0 ℃ until day 5, and to 32.0 ℃ until day 5. During the culture, pH was controlled with 7.5% sodium bicarbonate solution and CO2 gas, and defoaming was performed with 5% defoamer solution. The culture was supplemented with feeding medium on

days

5 and 8, and when the glucose content in the reactor was less than 4.0g/L, a 30% glucose solution (containing 300g/L glucose) was fed to the reactor to bring the glucose content in the medium to about 4.0 g/L. Daily sampling is carried out to detect cell density, cell viability and metabolic parameters.

3.7 Retention of sample

From the 0 th day to the end of the culture, 1mL of cell supernatant in 2 tubes is reserved every day and is stored at the temperature of-20 ℃ for detecting the protein content; and (3) reserving 40mL of cell supernatant in 2 tubes on the 11 th day and the 14 th day, and preserving the cell supernatant at the temperature of-20 ℃ for detecting the physicochemical property of the protein.

3.8 results and discussion

3.8.1 Shake flask seed data

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

3.8.215L seed culture data

The growth curve of the seed cells in the 15L reactor is shown in FIG. 8.

3.8.350L seed culture data

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

3.8.4250L seed culture data

A comparison of the cell growth and viability curves for the 250L addition of regulator A, the 250L addition of regulator B, and the 3L pilot plant described in example 2 in the culture of R3-01, according to the fed-batch culture addition strategy of example 2R3-01, is shown in FIG. 10. In terms of cell growth, the sugar type process of adding the regulator A into the 250L reactor is superior to the culture conditions of 3L pilot culture and adding the regulator B into the 250L reactor in terms of cell growth and cell viability.

The comparison of the antibody expression in the 250L reactor with regulator A, in the 250L reactor with regulator B and in the 3L pilot culture process is shown in FIG. 11; the results of the detection of the N-glycoform of the antibody are shown in Table 11. In the aspect of antibody expression, the antibody yield of the regulator A added into the 250L reactor is higher than that of the culture conditions of the 3L small test and the regulator B added into the 250L reactor, and the N-glycoform of the antibody is better than that of the regulator B added into the 3L small test and the 250L reactor, and has higher similarity compared with the original medicine.

TABLE 11 comparison of results for N-glycoforms of antibodies

Example 4 investigation of other Medium combinations

On the basis of examples 1-3, the present inventors also tried development strategies using other commercial media in combination with regulator A, such as CD FortiCHO media (Thermo Fisher Scientific) as basal medium, Sheff-CHOPLUS PF ACF (KERRY) with regulator A according to a 50: preparing a fed-batch culture medium according to the proportion of 50; or Balan CD CHOGrowth A (IrvineScientific) as a basal medium, CD Feed 002 (Omoman) and regulator A were mixed according to a ratio of 75: 25 preparing a fed-batch culture medium; or CD ClM1(Life Technology) as basal medium, Cel l Boost 5(GE-Hyclone) with regulator A according to 25: 75, the fed-batch culture medium can achieve better effects of regulating the sugar type and improving the antibody yield.

All documents referred to herein are incorporated by reference into this application as if each were individually incorporated by reference. Furthermore, it should be understood that various changes and modifications of the present invention can be made by those skilled in the art after reading the above teachings of the present invention, and these equivalents also fall within the scope of the present invention as defined by the appended claims.

Claims

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

(b) optionally isolating the resulting analog from the culture product;

and 45.0% < G1F + G2F < 70.0% of the similar drug prepared.

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

3. The method according to claim 1, wherein the volume of the culture system is 0.1-500L, preferably 3-300L, more preferably 100-250L.

4. The method according to claim 1, wherein the feeding medium is added to the reaction system at the 4 th to 6 th day and the 7 th to 9 th day, respectively, preferably at the 5 th day and the 8 th day, respectively.

5. The method according to claim 1, wherein the feeding medium is added in a total amount of 10 to 30 v/v%, preferably 15 to 25 v/v%, more preferably 20 v/v% of the total reaction system.

6. The method of claim 1, wherein said uridine, Mn²⁺The molar concentration ratio of galactose to galactose is (0.9-1.1): (0.0015-0.0025): 3-8), preferably 1:0.002: 5.

7. The method of claim 1, wherein uridine, Mn, are present in the culture system²⁺And galactose at a final concentration of 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:

8. The method according to claim 1, wherein the concentration ratio of uridine to pluronic is 1mmol (0.03-0.06) g, preferably 1mmol:0.05 g.

9. The method of claim 1, wherein said Pluronic is Pluronic F68.

10. A culture medium used in the process of preparing anti-CD 20 antibody rituximab, which is characterized in that the culture medium comprises a basal culture medium and a feeding culture medium,

the molar concentration ratio of the uridine, Mn2+ and galactose is (0.8-1.2): 0.001-0.003: (2-10), and the concentration ratio of the uridine to the pluronic is 1mmol: (0.02-0.08) g;

and 45.0% < G1F + G2F < 70.0% of the similar drug prepared.