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

Abstract

The present invention provides a method for preparing an anti-CD20 antibody rituximab analog, comprising the steps of: (a) culturing cells, wherein the medium used includes a basal medium and a fed-feed medium; and (b) optionally from the culture product The obtained similar drug is isolated; wherein, the fed-adding medium is added with a regulator A, and the regulator A includes: uridine, Mn ²⁺ , galactose and Pluronic; the uridine, Mn ² The molar concentration ratio of ⁺ and galactose is (0.8-1.2):(0.001-0.003):(2-10), and the concentration ratio of uridine and pluronic is 1mmol:(0.02-0.08) g; 45.0%<G1F+G2F<70.0% of the similar drugs. Using the method of the present invention, the Rituxan antibody biosimilar drug meeting the expected glycoform can be stably and highly produced in industrial scale-up production.

Description

A formula for antibody glycoform modification, cell culture method and industrial production applications in

technical field

The present invention relates to the field of biopharmaceuticals, in particular, to a formula for antibody glycoform modification, a cell culture method and its application in industrialized production.

Background technique

With the improvement of production technology, the yield of biological antibody drugs can reach a very high level. However, the glycoform control of antibody molecules has always been a big challenge in the production of antibody drugs. Even for generic drugs, the conventional production process is not enough. It is difficult to meet the quality standards for sugar control. For Rituxan, the original drug of CD20 antibody, glycoform is a very important quality indicator, and the quality standard of N-glycosylation of this antibody is 45.0%<G1F+G2F<70.0%. However, the glycoforms of the Rituxan antibody-like drug produced in the practice of using commercial culture medium are quite different from that of the original drug Rituxan, which is manifested in that the GOF (57.71%) of N-terminal glycosylation is higher than that of the original drug ( The G0F of the original drug is 37.78%); G1F (29.24%) and G2F (7.58%) are lower than the original drug (the G1F and G2F of the original drug are 41.43% and 10.34%, respectively), and the antibody G1F+G2F is 36.82, which is much lower than the antibody Quality standard of N-glycosylation (N-glycosylation quality standard 45.0%<G1F+G2F<70.0%). Therefore, the existing production methods are difficult to meet the national quality control requirements for generic drugs, resulting in the dilemma that the generic drugs cannot be marketed as scheduled even if the patent of the original drug expires. .

Medium is the basic solution to maintain cell survival and growth metabolism, and is one of the most important conditions for cell culture. It provides cells with an easily controlled and standardized living environment. Although cell culture uses chemically synthesized media with standardized production, composition and content, before large-scale production, cell culture media and culture processes often require scale-up validation to achieve large-scale or relatively small scale production. mass production. During the scale-up process, parameter settings such as temperature, pH, dissolved oxygen, and stirring must be considered as a whole to verify whether the cell culture medium and culture process are suitable for commercial production.

Therefore, there is an urgent need in the field to develop a medium that can enable the cultured expression of Rituxan antibody biosimilars to meet the expected glycoforms of the original drug, as well as a stable and high-yield Rituxan antibody biosimilar that meets the expected glycoforms in industrial scale-up production. method.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a medium capable of culturing and expressing Rituxan antibody biosimilars conforming to expected glycoforms, and a method for stably producing Rituxan antibody biosimilars conforming to expected glycoforms in industrial scale-up production.

In a first aspect of the present invention, a method for preparing an anti-CD20 antibody rituximab analog is provided, comprising the steps of:

(a) culturing cells, wherein the medium used includes basal medium and fed-feed medium; and

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

Wherein, the feeding medium is added with a regulator A, and the regulator A includes: uridine, Mn ²⁺ , galactose and Pluronic;

The molar concentration ratio of described uridine, Mn and galactose is ( ^0.8-1.2 ):(0.001-0.003):(2-10), and the concentration ratio of uridine and pluronic is 1mmol:(0.02-0.08 )g;

And, 45.0%<G1F+G2F<70.0% of the obtained similar drugs.

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

(c) monitor the glucose concentration in the culture system, when the glucose concentration is lower than 4g/L, supplement the glucose mother liquor to the culture system, so that the glucose content in the culture system is about 4g/L.

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

In another preferred embodiment, the basal medium is selected from commercially available commercial medium, including but not limited to CDFortiCHO media (Thermo Fisher Scientific), Dynamis (Gibco), Balan CD CHO Growth A (Irvine Scientific), Artipro ( GE-Hyclone), CD C1M1 (Life Technology), CD012 (Opman), CD11V (Jianshun Bio).

In another preferred embodiment, the fed-feed medium is selected from commercially available commercial medium, including but not limited to CDEfficientFeed C+AGT Supplement (Thermo Fisher Scientific), Sheff-CHO PLUS PFACF (KERRY), Cell Boost 5 (GE-Hyclone), Cell Boost 4 (GE-Hycone), CD Feed 002 (Opman), PFF05 (Jianshun Bio).

In another preferred embodiment, the temperature of the culture is 30-40°C, preferably 32-38°C, more preferably 37°C.

In another preferred example, after culturing to the 4th-6th day (preferably the 5th day), the culture temperature is adjusted to 34°C; and/or after the culturing to the 7th-9th day (preferably the 8th day), The culture temperature was adjusted to 32°C.

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 cells are selected from the group consisting of CHO cells, NSO cells, HEK293 cells, or a combination thereof.

In another preferred embodiment, the cells are CHO cells.

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

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

In another preferred embodiment, the fed-feed medium is added to the reaction system on the 4th-6th day and the 7th-9th day of the culture, preferably, the reaction is added on the 5th day and the 8th day of the culture. in the system.

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

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

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

In another preferred example, 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 embodiment, in the culture system, the final concentration of uridine, Mn ²⁺ and galactose is 0.3×UMG to 4×UMG, preferably 0.5×UMG to 3×UMG, more preferably 1 ×UMG to 2×UMG;

Wherein, the 1×UMG means:

The uridine concentration was 1 mM, the Mn ²⁺ concentration was 0.002 mM, and the galactose concentration was 5 mM.

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

In another preferred embodiment, the Pluronic is selected from the following group: Pluronic L31, Pluronic L35, Pluronic L38, Pluronic L42, Pluronic L43, Pluronic L44, Pluronic L61, PluronicL62, Pluronic L63, Pluronic L64, Pluronic L65, Pluronic L68 , Pluronic L72, PluronicL75, Pluronic L77, Pluronic L81, Pluronic L84, Pluronic L85, Pluronic L87, PluronicL88, Pluronic L121, Pluronic L122, Pluronic F38, Pluronic F68, Pluronic F108, Pluronic F127, Pluronic P85, Pluronic P94, Pluronic P104, Pluronic P105, Pluronic P123, etc., or a combination 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 example, the regulator A further includes an anti-agglomeration agent that prevents the cells in suspension culture from clumping.

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

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

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

In the second aspect of the present invention, there is provided a culture medium used in the process of preparing anti-CD20 antibody Rituxan-like drugs, the culture medium includes a basal medium and a fed-feed medium,

Wherein, the fed-feed medium comprises regulator A, which comprises: uridine, Mn ²⁺ , galactose and Pluronic;

The molar concentration ratio of described uridine, Mn and galactose is ( ^0.8-1.2 ):(0.001-0.003):(2-10), and the concentration ratio of uridine and pluronic is 1mmol:(0.02-0.08 )g;

And, 45.0%<G1F+G2F<70.0% of the obtained similar drugs.

In another preferred embodiment, the Pluronic is selected from the following group: Pluronic L31, Pluronic L35, Pluronic L38, Pluronic L42, Pluronic L43, Pluronic L44, Pluronic L61, PluronicL62, Pluronic L63, Pluronic L64, Pluronic L65, Pluronic L68 , Pluronic L72, PluronicL75, Pluronic L77, Pluronic L81, Pluronic L84, Pluronic L85, Pluronic L87, PluronicL88, Pluronic L121, Pluronic L122, Pluronic F38, Pluronic F68, Pluronic F108, Pluronic F127, Pluronic P85, Pluronic P94, Pluronic P104, Pluronic P105, Pluronic P123, etc., or a combination 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.

It should be understood that within the scope of the present invention, the above-mentioned technical features of the present invention and the technical features specifically described in the following (eg, the embodiments) can be combined with each other to form new or preferred technical solutions. Due to space limitations, it is not repeated here.

Description of drawings

Fig. 1 shows the viable cell density of each experimental group of medium screening in Example 1.

FIG. 2 shows the change curves of cell growth and viability of each experimental group of medium screening in Example 1.

Figure 3 shows the number of viable cells cultured in the 3L reactor in Example 2

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

FIG. 5 shows the antibody expression curve of the 3L reactor culture in Example 2. FIG.

Figure 6 shows a flow chart of the 250L process scaled-up cell culture in Example 3.

Figure 7 shows the cell growth in the seed cultivation stage of the 250L process scaled-up cell culture shake flask in Example 3, and the graph shows the change curve of the number of viable cells and the growth and viability rate in the seed cultivation stage of the shake flask.

Figure 8 shows the cell growth in the seed culture stage of the 15L reactor during the scale-up of the 250L process in Example 3. The figure includes the number of viable cells and the growth and viability curve of seed cells in the seed culture stage of the 15L reactor.

Figure 9 shows the cell growth in the seed culture stage of the 50L reactor during the scale-up of the 250L process in Example 3. The figure includes the number of viable cells and the growth and viability curve of seed cells in the seed culture stage of the 50L reactor.

Figure 10 shows the comparison chart of the cell growth of the R3-01 group (Example 2) in the 250L process scale-up cell culture in Example 3 with the addition of regulator A, the addition of regulator B and the 3L small-scale process cell culture. The arrows in the figure indicate the stage of feeding the fed-batch medium.

Figure 11 shows a comparison chart of the antibody expression of the R3-01 group (Example 2) in the 250L process scale-up cell culture in Example 3 with the addition of regulator A, the addition of regulator B, and the 3L small-scale process cell culture.

Figure 12 shows the types of antibody N-glycosylation.

Detailed ways

After extensive and in-depth research and extensive screening, the inventors unexpectedly developed a method for preparing anti-CD20 antibody rituximab analogs for the first time. Biosimilars with glycoforms similar to the original drug Rituxan can be obtained. Wherein, the regulator A includes: uridine, Mn ²⁺ , galactose and Pluronic, and the molar concentration ratio of the uridine, Mn ²⁺ and galactose is 1:0.002:5, and the The concentration ratio of uridine to pluronic is 1 mmol:0.05 g.

In addition, the inventors carried out a scale-up experiment of a 250L reactor on the basis of the 3L reactor sugar type pilot process. In the culture product obtained by this culture system, the isolated antibody product, on the premise of ensuring cell viability and antibody yield, has an N-glycosylation level G1F+G2F value of 51.44, which is significantly higher than that in the prior art. It can reach 36.82, which is very close to the G1F+G2F value of the original research drug Rituxan of 51.77. The present invention has been completed on this basis.

the term

As used herein, the terms "Rituxan-like drug", "Rituxan generic drug", "anti-CD20 antibody Rituxan-like drug" and "anti-CD20 antibody Rituxan-Generic drug" are used interchangeably and refer to the original drug that has been approved for registration (reference drug). ) The anticancer drug-Rituxan (rituximab injection) is similar to therapeutic biological products in terms of quality, safety and efficacy. Specifically, it can reach the N-glycosylation quality standard of 45.0%<G1F+G2F<70.0%.

culture medium

Including commercial basal medium and fed-feed medium, since the commercial medium does not disclose specific formula information, the following table 1 is the commonly used commercial medium:

Table 1 Commonly used commercial media

The configuration method of the above-mentioned medium needs to be carried out according to the requirements of the medium specification.

Preparation

Key ingredients and addition ratio of Regulator A:

Molecular composition of regulator A used in Table 2 Examples 1-3

substance Preparation method (1L) moles/L uridine 6.11g 25mmol MnCl2 6.34g 0.05mmol Galactose 22.52g 125mmol Planfack F68 1.25g 1.25g

In other embodiments, Pluronic may be selected from the group consisting of Pluronic L31, Pluronic L35, PluronicL38, Pluronic L42, Pluronic L43, Pluronic L44, Pluronic L61, Pluronic L62, PluronicL63, Pluronic L64, Pluronic L65, Pluronic L68, Pluronic L72, Pluronic L75, PluronicL77, Pluronic L81, Pluronic L84, Pluronic L85, Pluronic L87, Pluronic L88, PluronicL121, Pluronic L122, Pluronic F38, Pluronic F68, Pluronic F108, Pluronic F127, Pluronic P85, Pluronic P94, Pluronic P104, Pluronic P1025, Pluronic P123 etc., or a combination thereof. The addition ratio is 0.03-0.06 g of Planfac per 1 mmol of uridine.

In addition, an anti-caking agent can be added according to experimental needs, and the anti-caking agent is selected from dextran sulfate, dextran 40, dextran 70, or a combination thereof. In Examples 1-3, the anti-caking agent used is dextran sulfate, preferably, the molecular weight of dextran sulfate is 5000 Daltons. In 1L of regulator A, add 0.25g of molecular weight 5000 Dalton's Dextran Sulfate. Generally, the mass ratio of pluronic and anticaking agent is 1:(0.2-0.8), preferably 1:(0.4-0.7), more preferably 1:(0.5-0.6).

Conditioner B does not contain F68, dextran sulfate two components.

conventional cell culture methods

The cells used to prepare the antibody are selected from CHO cells, NSO cells and HEK293 cells, and these cells comply with conventional cell culture methods, including cell recovery, basal medium resuspending, cell expansion, feeding culture and other stages, wherein, The initial cell seeding density for cell expansion is 1x 10 ⁶ cells/ml; during the culture process, daily sampling is required to detect viable cell density, cell viability, and cell fluid physicochemical indicators, including glucose, glutamine, lactate, and NH4+. level; the supplementation of the fed-batch medium is generally controlled when the glucose is lower than 2g/L and the glutamine is lower than 0.5mM. In addition, when the glucose content is lower than 4g/L, the glucose solution is added to 4g/L. Generally, when the cell viability is lower than 70% or when cultured for 14±2 days, the culture medium can be harvested for detection of antibody expression and detection of N-glycoform.

Antibody glycoforms

Antibody glycosylation is a key quality attribute of therapeutic antibody drugs, and the main form of glycosylation is N-glycosylation at the Fc terminal. See Figure 12 for several forms of N-glycosylation.

N-glycosylation analysis method

Dilute the sample to the concentration to be tested, add 2.5 μL PNGase F (glycosidase), mix well, centrifuge for 5s, and take a water bath at 50°C for 1 hour. Add 40 μL of HILIC labeling reagent, then add 60 μL of methanol, close the cap of the centrifuge tube, and place it in a water bath at 80 °C for 75 min. The samples were taken out and centrifuged at 15,000 rpm for 20 minutes at 10°C to precipitate proteins. Take the supernatant into a 1.5mL centrifuge tube, freeze-dried in vacuum, and set the temperature to 4°C. After the sample was dried, 40 μL of 50% ACN was added and mixed evenly; after centrifugation at 12000 rpm for 5 min, 18 μL of the supernatant was taken into the inner tube, and the bottom air bubbles were removed.

The samples were run on Wastes UPLC and analyzed using a HILIC column with an injection volume of 2 μL, an excitation wavelength of 360 nm and an emission wavelength of 425 nm, and a fluorescence detector was used for detection. Gradient elution was used, and the elution gradient was set as Table 3 below.

Table 3 Elution gradient settings

Advantages of the present invention include:

1) Through the exploration of ingredients and dosage, an effective glycoform regulator was identified, which not only controlled the glycoform specification to meet the needs of industrial production, but also ensured a higher yield/amount of cells and antibodies.

2) Compared with commercial sugar-type regulators, the composition is simple, and the production cost is greatly saved.

3) Since the formulations of commercial glycoform modifiers are all unpublished information, the present invention further breaks the technical monopoly of commercial glycoform modifiers.

4) Breaking through the technical barriers for the production of imported generic antibody drugs, greatly accelerating the process of listing domestic generic antibody drugs.

The present invention will be further described below in conjunction with specific embodiments. It should be understood that these examples are only used to illustrate the present invention and not to limit the scope of the present invention. The experimental method of unreceipted specific conditions in the following examples, usually according to normal conditions, such as people such as Sambrook, molecular cloning: conditions described in laboratory manual (New York:Cold Spring Harbor Laboratory Press, 1989), or according to manufacturer the proposed conditions. Percentages and parts are weight percentages and parts unless otherwise specified.

Unless otherwise specified, the materials and reagents used in the examples are all commercially available products.

Example 1: Cell Culture Media Screening and Determination

1.1 Cell culture medium screening

In the ultra-clean workbench, take about 12 mL of cell fluid from each 250 mL shake flask, take a total of 7 bottles, and add 38 mL of 8 kinds of basal medium in different proportions. The composition of basal medium is shown in Table 4, and 7 experimental groups are set up. They are F1, F2, F3, F4, F5, F6, F7 respectively, and the control group is F8. The culture volume was 50 mL, and the flasks were placed in a carbon dioxide shaker at 37.0 °C, 120 rpm, 5.0% CO ₂ for culture, inoculated at an initial culture density of 1 x 10 ⁶ cells/mL, and co-cultured for 14 days. Among them, GE001 (GE-Hyclone) is a commercially available medium, which can be used for glycoform regulation. For details, please refer to the product manual.

Table 4 Basic culture composition of each experimental group

The temperature of the carbon dioxide shaker was adjusted to 34.0°C until the 5th day of cultivation, and the temperature of the carbon dioxide shaker was adjusted to 32.0°C until the 8th day of cultivation. During the culturing process, the fed-feed medium needs to be supplemented to provide the nutrients needed for cell growth during the culture process. When the glucose content in the shake flask is lower than 4g/L, supplement the glucose solution with a concentration of 30% (containing 300g/L of glucose) into the shake flask to make the glucose content in the medium to about 4g/L.

Table 5 The composition and supplementation strategy of fed-feed medium in each experimental group

On the 5th, 8th, 11th, 12th, 13th, and 14th days, samples were taken to detect the cell density, and 1mL of the cell supernatant was collected on D5, D8, D11 and D14 days to measure the antibody content. Clear for glycoform detection.

Table 6 Final concentration of key ingredients in Regulator A

Table 7 Final concentration of key ingredients of regulator B

1.2 Cell culture medium determination

In terms of cell growth, it can be found from the cell growth of each experimental group in Figure 1 and Figure 2 that although the cell viability of the F5, F6, F7, and F8 groups during the culture process (Figure 2) is equivalent, the actual number of viable cells There are great differences in the levels of the cells. The F7 group did not add any regulators, and only used the commercial fed-feed medium. The highest cell density was only about 10×10 ⁶ cells/mL. The other six experimental groups had the highest cell density. 20×10 ⁶ cells/mL or more. Compared with the other 5 experimental groups, the F5 and F6 experimental groups could maintain higher cell density and viability in the later stage. The F8 group was the regulator control group, which did not contain F68 and anti-aggregation agent, and its highest viable cell density was only 10×10 ⁶ cells/mL.

In terms of antibody expression, it can be seen from the antibody expression level (Figure 3) that the antibody expression levels of shake flasks F5 and F6 are significantly better than 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 Rituxan standard, and the expected glycoform value G1F+G2F was closer to the standard. Therefore, the overall performance of the cell culture process of the F5 and F6 experimental groups is the best, which can be used as the basis for the small-scale test process of the 3L reactor sugar.

Table 8 Summary of N-glycoform results of each experimental group

Example 2: 3L cell culture process

2.1 Cell recovery

Take out the cell cryopreservation tube from the liquid nitrogen tank and immediately put it into a 37°C water bath to quickly thaw the cell fluid across the freezing point, preventing the formation of ice crystals in the cell fluid to pierce the cell membrane and affect the cells.

After the cells were completely thawed, they were removed from the water bath and disinfected with 75% alcohol. Then, open the cryopreservation tube in the ultra-clean workbench, aspirate the cell fluid with a sterile pipette, put it into a 50 mL shake flask filled with 10 mL of culture medium, and take out about 0.5 mL of cell suspension after shaking. count. The flasks were incubated in a carbon dioxide shaker (37.0°C, 120 rpm, 5.0% CO2).

2.2 Shake flask cell expansion

Use Dynamis (Gibco) + MTX (add 0.5 mL of 1mM MTX solution per liter of basal medium) medium, sample and count every three days and then passage once, at a cell density of 1.0×10 ⁶ cells/mL, passage to 4 cells The amount of cells required in the 3L reactor was transferred to four 3L reactors to continue the culture.

2.3 3L reactor inoculation

About 1.6L of Dynamis (Gibco) medium was pumped into 4 3L reactors respectively. After sterility check, about 220mL of seed cells were inoculated into 4 3L reactors respectively. After mixing evenly, about 220mL of cell fluid was removed. Make each 3L reactor an initial culture volume of approximately 1.6L. The reactor temperature was adjusted to 34.0°C until the 5th day of cultivation, and the reactor temperature was adjusted to 32.0°C until the 8th day of cultivation. Four 3L reactors were fed with medium supplementation according to the fed-feed medium supplementation strategy in Table 9. When the glucose content in the shake flask is lower than 4g/L, supplement the glucose solution with a concentration of 30% (containing 300g/L of glucose) into the reactor to make the glucose content in the medium to about 4g/L. The culture conditions of R3-01 and R3-02 are the same, and the culture conditions of R3-03 and R3-04 are the same. The culture parameters of the four 3L reactors are shown in Table 9.

Table 9 3L reactor process parameter settings

2.4 Sampling and retaining samples

During the cell culture process of the 3L reactor, the 3L reactor was sampled and counted every day except the 6th and 7th day, and the cell fluid was tested for biochemical parameters. After each sampling, 1 mL × 2 tubes of cell supernatant were kept at -20°C for protein content detection; 50 mL × 1 tube of cell supernatant was reserved for each reactor on the 11th and 14th days. The solution was stored at -20°C for glycoform detection.

2.5 Results and Discussion

In terms of cell growth, it can be seen from Figure 4 that the two groups of parallel reactors have similar cell growth, the four 3L reactors have similar growth in the early stage, and the highest density is about 27 × 10 ⁶ cells/mL. In the later stage, R3-03 and R3- The cell density and cell viability of 04 decreased significantly, while R3-01 and R3-02 maintained higher cell density and viability.

In terms of antibody expression, it can be seen from the antibody expression level (Figure 5) that the two groups of parallel reactor cells have similar antibody expression levels, and the antibody expression levels of R3-01 and R3-02 are better than that of R3-03 and R3-04. From the antibody glycoform detection results (Table 10), it can be seen that the glycoform detection results of the 4 reactors are similar. To sum up, the overall performance of the R3-01 and R3-02 process conditions is more suitable as the basis for the scale-up production of the 250L reactor process.

Table 10 Detection results of antibody N-glycoform in 3L reactor

Example 3: 250L process scale-up culture

3.1 Cell culture process

As shown in Figure 6.

3.2 Cell recovery

Take out the cell cryopreservation tube from the liquid nitrogen tank and immediately put it into a 37°C water bath. After the cells are completely thawed, take them out from the water bath and disinfect them with 75% alcohol. Then, open the cryopreservation tube in the ultra-clean workbench, aspirate the cell fluid with a sterile pipette, put it into a 50 mL shake flask containing the culture, and take out about 0.5 mL of the cell suspension for cell counting after shaking. The flasks were incubated in a carbon dioxide shaker (37.0°C, 120 rpm, 5.0% CO ₂ ).

3.3 Shake flask seed culture

The next day, after the cells were sampled and counted, the cells were transferred to a sterile centrifuge tube and centrifuged at 700 rpm for 5 min. The supernatant was discarded, and the cells were resuspended in fresh Dynamis (Gibco) (containing MTX) into a culture flask to make the final density about 1.0×10 ⁶ cells/mL. After that, the cells were sampled and counted every three days, and the cells were passaged at a density of 1.0×10 ⁶ cells/mL, and the passage medium was MTX-containing basal medium. Passage to the required amount of cells in a 15L reactor, and then transfer to a 15L reactor to continue culturing.

3.4 15L reactor seed culture

Before inoculating the 15L reactor, assemble the tank (the pH electrode needs to be calibrated in advance), ensure that the pipeline is connected correctly, inject 5L WFI into the tank, and then sterilize the tank and related accessories at 121°C for 120mins. After the tank is sterilized, wait for it to be fully cooled, empty the WFI in the tank, add about 7.9L Dynamis (Gibco) medium, set the reactor parameters as temperature 37.0 ° C, 120 rpm to conduct aseptic investigation of the medium, investigate Time 18-24 hours, after sterility verification is correct, calibrate the dissolved oxygen electrode and calibrate the pH electrode.

3.5 50L reactor seed culture

A 50L Hyclone disposable reactor was used for seed culture in the 50L reactor. Before use, install the reaction bag, then insert the sterilized electrode (the pH electrode has been calibrated) into the reaction bag, and then add about 42.5L of Dynamis (Gibco) medium. The stirring speed was 90 rpm, the temperature was 37.0 °C, and the culture was carried out for 18-24 hours. After sterility verification is correct, calibrate the DO electrode and calibrate the pH electrode.

A 50L reactor was inoculated at a density of about 1.0×10 ⁶ cells/mL, and about 5.3L of the 15L reactor seed solution was added. The culture parameters were DO: 50%, PH: 7.00±0.20, temperature: 37.0°C, stirring: 90 rpm. Culture to the third day, transfer to a 250L reactor to continue the culture. Daily sampling was performed to detect cell density, cell viability and metabolic parameters.

3.6 250L reactor seed culture

The 250L reactor seed culture uses a 250L Hyclone disposable reactor. Before use, install the reaction bag, then insert the sterilized electrode (the pH electrode has been calibrated) into the reaction bag, and then add about 150L of Dynamis (Gibco) medium. The stirring speed was 60 rpm, the temperature was 37.0 °C, and the culture was carried out for 18-24 hours. After sterility verification is correct, calibrate the DO electrode and calibrate the pH electrode.

Inoculate at a density of about 1.0×10 ⁶ cells/mL, and add about 20 L of the 50 L reactor seed solution. The culture parameters were DO: 50%, PH: 7.00±0.20, temperature: 37.0°C, stirring: 80 rpm.

On the 5th day, the set temperature of the reactor culture was adjusted to 34.0°C, and until the 5th day, the set temperature of the reactor culture was adjusted to 32.0°C. During the incubation, pH was controlled with 7.5% sodium bicarbonate solution and CO2 gas, and defoaming was performed with 5% defoamer solution. On the 5th and 8th days of culture, the fed-feed medium was added. When the glucose content in the reactor was lower than 4.0 g/L, a glucose solution with a concentration of 30% (containing 300 g/L of glucose) was added to the reactor to make the solution. The glucose content in the medium was about 4.0 g/L. Daily sampling was performed to detect cell density, cell viability and metabolic parameters.

3.7 Reserve samples

From the 0th day of culture to the end of the culture, 1mL*2 tubes of cell supernatant were kept at -20°C every day for the detection of protein content; 40mL*2 tubes of cells were kept on the 11th and 14th days The supernatant was stored at -20°C for the detection of protein physicochemical properties.

3.8 Results and Discussion

3.8.1 Shake Flask Seed Data

Seed cell growth curves in shake flasks (Figure 7).

3.8.2 15L Seed Culture Data

Seed cell growth curve in 15L reactor (Figure 8).

3.8.3 50L Seed Culture Data

The cell growth curve of the 50L reactor is shown in Figure 9.

3.8.4 250L Seed Culture Data

According to the feeding strategy of R3-01 in Example 2, the cell growth in R3-01 culture in the case of adding 250L of Regulator A, 250L of Regulator B, and the 3L pilot process described in Example 2 were compared. And the comparison of the change curve of the activity rate is shown in Figure 10. In terms of cell growth, the cell growth and cell viability of the glycoform process cell culture with Regulator A added to the 250L reactor were better than those of the 3L pilot culture and the 250L reactor with Regulator B added.

Figure 11 shows the comparison of antibody expression in 250L reactor adding regulator A, 250L reactor adding regulator B and 3L small-scale culture process; the comparison of antibody N-glycoform detection results is shown in Table 11. In terms of antibody expression, the antibody yield of the 250L reactor with Regulator A was higher than that of the 3L test and the 250L reactor with Regulator B, and the antibody N-glycoforms were better than the 3L test and the 250L reactor. The batch of agent B is more similar to the original research drug.

Table 11 Comparison table of antibody N-glycoform results

The exploration of other medium combinations of embodiment 4

On the basis of Examples 1-3, the present invention also attempts to use other commercial media in combination with the development strategy of Regulator A, such as CD FortiCHO media (Thermo Fisher Scientific) as the basal medium, Sheff-CHOPLUS PF ACF (KERRY ) and Conditioner A in a ratio of 50:50 to prepare a fed-feed medium; or Balan CD CHOGrowth A (IrvineScientific) as a basal medium, CD Feed 002 (Opman) and Conditioner A in a ratio of 75:25 to prepare a flow-feeding medium Add medium; or CD ClM1 (Life Technology) as the basal medium, Cell Boost 5 (GE-Hyclone) and Regulator A according to the ratio of 25:75 to prepare the fed medium, all of which can play a better role in glycoforms regulation and the effect of antibody yield enhancement.

All documents mentioned herein are incorporated by reference in this application as if each document were individually incorporated by reference. In addition, it should be understood that after reading the above teaching content of the present invention, those skilled in the art can make various changes or modifications to the present invention, and these equivalent forms also fall within the scope defined by the appended claims of the present application.

Claims (10)

1. a method for preparing anti-CD20 antibody Rituxan analogs, is characterized in that, comprises the steps: (a) culturing cells, wherein the medium used includes basal medium and fed-feed medium; and (b) optionally isolating the resulting analog from the culture product; Wherein, the feeding medium is added with a regulator A, and the regulator A includes: uridine, Mn ²⁺ , galactose and Pluronic; The molar concentration ratio of described uridine, Mn and galactose is ( ^0.8-1.2 ):(0.001-0.003):(2-10), and the concentration ratio of uridine and pluronic is 1mmol:(0.02-0.08 )g; And, 45.0%<G1F+G2F<70.0% of the obtained similar drugs. 2. The method of claim 1, wherein the method further comprises the steps of: (c) monitor the glucose concentration in the culture system, when the glucose concentration is lower than 4g/L, supplement the glucose mother liquor to the culture system, so that the glucose content in the culture system is about 4g/L. 3. The method of claim 1, wherein the volume of the culture system is 0.1-500L, preferably 3-300L, more preferably 100-250L. 4. method as claimed in claim 1, is characterized in that, described feeding medium is cultivated to the 4th-6th day and the 7th-9th day respectively and adds in the reaction system, preferably, is cultivated to the 4th day respectively. 5 and 8 days were added to the reaction system. 5. method as claimed in claim 1 is characterized in that, the total amount that described feeding medium adds is 10-30v/v% of total reaction system, preferably 15-25v/v%, more preferably 20v/v%. 6. method as claimed in claim 1 is characterized in that, the molar concentration ratio of described uridine, Mn ²⁺ and galactose is (0.9-1.1): (0.0015-0.0025): (3-8), more Good place 1:0.002:5. 7. The method of claim 1, wherein in the culture system, the final concentration of uridine, Mn ²⁺ and galactose is 0.3×UMG to 4×UMG, preferably 0.5×UMG to 3 ×UMG, more preferably 1×UMG to 2×UMG; Wherein, the 1×UMG means: The uridine concentration was 1 mM, the Mn ²⁺ concentration was 0.002 mM, and the galactose concentration was 5 mM. 8. method as claimed in claim 1 is characterized in that, the concentration ratio of described uridine and pluronic is 1mmol:(0.03-0.06)g, preferably 1mmol:0.05g. 9. The method of claim 1, wherein the Pluronic is Pluronic F68. 10. A substratum used in the process of preparing anti-CD20 antibody Rituxan analogs, wherein the substratum comprises a basal medium and a fed-feed medium, Wherein, the fed-feed medium comprises regulator A, which comprises: uridine, Mn ²⁺ , galactose and Pluronic; The molar concentration ratio of described uridine, Mn and galactose is (0.8-1.2):(0.001-0.003):(2-10), and the concentration ratio of uridine and pluronic is 1mmol:(0.02-0.08)g ; And, 45.0%<G1F+G2F<70.0% of the obtained similar drugs.

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