CN112592948B

CN112592948B - Perfusion culture method of animal cells

Info

Publication number: CN112592948B
Application number: CN202011486772.2A
Authority: CN
Inventors: 陆金康; 胡飞
Original assignee: Foshan Hanteng Biotechnology Co ltd; Foshan Pu Jin Bioisystech Co ltd; Cantonbio Co ltd
Current assignee: Foshan Hanteng Biotechnology Co ltd; Foshan Pu Jin Bioisystech Co ltd; Cantonbio Co ltd
Priority date: 2020-12-16
Filing date: 2020-12-16
Publication date: 2023-05-09
Anticipated expiration: 2040-12-16
Also published as: CN112592948A

Abstract

The invention relates to a perfusion culture method of animal cells, which comprises the following steps: mixing a basic culture medium and a feed medium, and then performing perfusion culture on animal cells; harvesting the biological material; wherein the basal medium is

Growth A, feed medium is

Feed4, the volume ratio of basal medium to Feed medium is (80-90): (10-20), the perfusion culture method of the animal cells can promote proliferation of the animal cells and improve the yield of the therapeutic proteins secreted by the animal cells.

Description

Perfusion culture method of animal cells

Technical Field

The invention relates to the technical field of cell culture, in particular to a perfusion culture method of animal cells.

Background

In recent years, the development of biopharmaceutical technology is faster and the market demand for biopharmaceuticals is larger, and as the demand for biological products (such as proteins, growth factors, vaccines or monoclonal antibodies) produced by using mammalian cells as carriers is increasing, large-scale animal cell culture technology is also attracting more attention.

Common animal cell culture methods include batch culture, fed-batch culture, and perfusion culture. Batch culture is to put cells and culture medium into a reactor at one time for culture, the cells grow continuously, products are formed continuously, and after a period of time, the culture is terminated. The fed-batch culture refers to that a certain amount of culture medium is firstly filled into a reactor, cells are inoculated under proper conditions for culture, the cells are continuously grown, products are continuously formed, in the process, along with continuous consumption of nutrient substances, new nutrient components are continuously supplemented into a reaction container, the cells are further metabolized, and the products are taken out until the whole culture is finished. Fed-batch culture is simply to supplement the reaction vessel with the necessary nutrients to maintain the concentration of nutrients unchanged. Perfusion culture is to continuously take out part of the culture medium during cell growth and product formation, while continuously perfusing new culture medium after the cells and culture medium are added together into the reactor. Perfusion culture is one of the important processes of the current mammalian cell culture, not only can provide a stable environment favorable for cell culture by continuously discharging cell metabolism byproducts, solve the problems of unstable protein quality or low expression quantity and the like, but also can optimize the productivity utilization rate and improve the production efficiency by improving the yield per unit volume.

The current commercial perfusion culture medium in the market has few varieties, has good compatibility with constructed cells to be verified, and lacks perfusion culture medium capable of promoting the growth of cells expressing therapeutic proteins and secreting the therapeutic proteins.

Disclosure of Invention

Based on this, it is necessary to provide a perfusion culture method of animal cells, which can proliferate animal cells rapidly and facilitate secretion of therapeutic proteins by cells when the animal cells are cultured by the perfusion culture method.

A perfusion culture method of animal cells, comprising the steps of:

mixing a basic culture medium and a feed medium, and then performing perfusion culture on animal cells; and

harvesting the biological material;

wherein the basic culture medium is

GrowthA, the feed medium is

Feed4, the volume ratio of the basal medium to the Feed medium is (80-90): (10-20).

In the perfusion culture method of the animal cells, the perfusion culture medium comprises

Growth A and->

Feed4 and the volume ratio of the Feed and the Feed is set to be (80-90): (10-20) by

Growth A and->

The cooperation of Feed4 can promote the proliferation of animal cells, improve the cell density of the animal cells, promote the secretion of therapeutic proteins by the animal cells and improve the yield of the therapeutic proteins. The perfusion culture method of the animal cells has a particularly obvious culture effect on CHO cells secreting anti-CD20 antibodies. />

In one embodiment, the ratio of the volume of basal medium to the volume of feed medium is 90:10.

in one embodiment, from day 15 to day 16 of perfusion culture, the ratio of the volume of the basal medium to the feed medium is (80-85): (20-15).

In one embodiment, from day 15 to day 16 of perfusion culture, the ratio of the volume of basal medium to the volume of feed medium is 80:20, starting from day 17 to day 18 of perfusion culture, the volume ratio of the basal medium to the feed medium is 85:15.

in one embodiment, the animal cells are seeded at a density of 0.2X10 ⁶ individual/mL ultra2×10 ⁶ And each mL.

In one embodiment, the perfusion rate of the perfusion culture is 0.5VVD to 2VVD.

In one embodiment, the perfusion culture temperature is 31℃to 37 ℃.

In one embodiment, the animal cell is an animal cell expressing an anti-CD20 antibody.

In one embodiment, the animal cell is selected from any one of chinese hamster ovary cells, hybridoma cells, baby hamster kidney cells, and myeloma cells.

In one embodiment, the chinese hamster ovary cell is CHO-K1.

Drawings

FIG. 1 is a growth curve of example 1;

FIG. 2 is a growth curve of example 2;

FIG. 3 is a graph of the concentration of antibodies of example 2;

FIG. 4 is a growth curve of comparative example 1;

FIG. 5 is a graph of the concentration of antibodies of comparative example 1;

FIG. 6 is a growth curve of comparative example 2;

FIG. 7 is a graph showing the concentration of the antibody of comparative example 2.

Detailed Description

The present invention will be described more fully hereinafter in order to facilitate an understanding of the invention, which may be embodied in many different forms and is not limited to the embodiments described herein. Rather, these embodiments are provided so that this disclosure will be thorough and complete.

Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. The terminology used herein in the description of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

The perfusion of fresh medium or the removal of spent medium during perfusion culture herein may be continuous, stepwise, intermittent or a combination of any or all of these, unless specifically indicated.

An embodiment of the invention provides a perfusion culture medium, which comprises a basal culture medium and a feed culture medium, wherein the basal culture medium is

Growth A, feed medium +.>

Feed4, the volume ratio of basal medium to Feed medium is (80-90): (10-20). Verified to contain->

Growth A and->

Feed4 and the volume ratio of the Feed and the Feed is set to be (80-90): the perfusion medium of (10-20) can obviously improve the cell density of CHO cells secreting anti-CD20 antibody (anti-CD 20) and improve the yield of the anti-CD20 antibody when used for perfusion culture of the CHO cells secreting the anti-CD20 antibody. />

In an alternative specific example, the ratio of the volume of basal medium to the volume of feed medium is 90:10. 88: 12. 85:15. 83:17 or 80:20. in one embodiment, the volume ratio of basal medium to feed medium is (85-90): (10-15).

In one embodiment, the perfusion medium is composed of a basal medium and a feed medium, and the ratio of basal medium and feed medium is as described above. Of course, in other embodiments, the perfusion medium described above includes other components in addition to the basal medium and the feed medium. Such as an antifoaming agent, and, for example, one or more selection agents that can bind to resistance markers and viability markers in the host cell line, including, but not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfimide, or methotrexate.

The invention also provides a preparation method of the perfusion culture medium, which comprises the following steps: the components of the perfusion medium are mixed to prepare the perfusion medium. Of course, the manner of mixing the components of the perfusion medium is not particularly limited, and it is only necessary to uniformly mix the components.

The preparation method of the perfusion culture medium is simple and convenient to operate.

The invention further provides a perfusion culture method of the animal cells, which comprises the steps a to c. Specifically:

step a: culturing a cell culture comprising a cell culture medium and animal cells.

Specifically, the step of culturing a cell culture comprising a cell culture medium and animal cells is to shorten the time for the animal cells to reach a desired activity density after being inoculated into a bioreactor, and to increase the initial cell amount. It is to be understood that in the step of culturing a cell culture comprising a cell culture medium and animal cells, the cell culture medium is not particularly limited and may be any medium that can be used for culturing animal cells. Of course, selection of a medium favorable for cell proliferation is more favorable for shortening the culture time to reach a cell amount suitable for perfusion culture inoculation density.

Specifically, the animal cell is selected from any one of chinese hamster ovary cells, hybridoma cells, baby hamster kidney cells, and myeloma cells.

In one embodiment, the animal cell is a CHO cell. The chinese hamster ovary cell line (CHO cell) is a transformed cell line obtained from chinese hamster ovary cells in 1957. CHO cells, which are fibroblasts, are a type of secretory cell that is commonly used to express proteins, such as antibodies, required by the human body. Further, the animal cell is a chinese hamster ovary cell K1 (CHO-K1 for short) for expressing an anti-CD20 antibody. Of course, in other embodiments, the animal cells are not limited to CHO cells, but may be other mammalian cell lines suitable for industrial production, such as NSO cells (derived from the mouse myeloma cell line NS-1, which does not secrete immunoglobulin heavy chains nor synthesize immunoglobulin light chains by themselves), perc.6 cells (engineered cells developed by the company cruell and company Royal DSM, which are genetically engineered from human embryonic retinoblasts), and the like.

It will be appreciated that in some embodiments, step a may be omitted. For example, step a is not necessary when the amount of cells used for perfusion culture can satisfy the inoculation density at the time of perfusion culture.

Step b: the cell culture is perfusion-cultured after mixing the basal medium and the feed medium.

Specifically, when the animal cells are cultured to a certain cell amount (i.e., cell amount satisfying the density of perfusion culture), the animal cells are transferred to a perfusion apparatus for perfusion culture. The inoculation density of animal cells was 0.2X10 ⁶ Per mL-2×10 ⁶ And each mL. At this seeding density, animal cells can be made to adapt more quickly to the perfusion culture environment. Alternatively, the inoculation density of animal cells is 0.2X10 ⁶ 0.5X10 g/mL ⁶ Per mL, 1X 10 ⁶ individual/mL, 1.5X10) ⁶ individual/mL or 2X 10 ⁶ And each mL. Further, the inoculation density of the animal cells was 0.5X10 ⁶ Per mL-1×10 ⁶ And each mL.

In this embodiment, the perfusion rate of the perfusion culture is 0.5 to 2VVD. "VVD" refers to the volume of the container per day. Alternatively, the perfusion rate of the perfusion culture is 0.5VVD, 0.8VVD, 1VVD, 1.5VVD or 2VVD.

Specifically, the perfusion culture is an alternate tangential perfusion culture or a tangential perfusion culture. Alternate tangential flow perfusion culture refers to perfusion culture in which cells are trapped by an Alternate Tangential Flow (ATF) filtration technique in cell culture; tangential flow perfusion culture refers to perfusion culture in which cells are trapped during cell culture using tangential flow (TFF) filtration techniques. In perfusion culture where tangential flow (TFF) filtration is used to retain cells, the cell fluid is pumped by peristaltic pump to form a continuous annular flow direction, and after entering the fiber membrane, the waste fluid is discharged out of the culture system through the membrane, and the cells are returned to the culture system along with the loop. In perfusion culture of cells trapped by Alternate Tangential Flow (ATF) filtration, the reciprocating flow of the medium in the tank in the trapping device is realized by the reciprocating blowing and sucking action of a diaphragm pump, and meanwhile, metabolic wastes are trapped in the reactor as the medium is discharged through the membrane. When ATF is used, the alternating motion creates a scouring action in the filter membrane, helping to prevent clogging of the fibrous membrane.

Specifically, the reaction vessel for perfusion culture is not particularly limited, and may be, for example, a bioreactor or a culture shaking tube. "bioreactor" as used herein refers primarily to a reaction vessel having a capacity of 2L or more for culturing cells to produce a desired product.

In one embodiment, the reaction vessel for perfusion culture is a bioreactor, and perfusion culture by using the bioreactor is more convenient for controlling the perfusion of fresh culture medium and the removal of used culture medium. In a specific example, the bioreactor is an N-stage bioreactor (production bioreactor), and when the N-stage bioreactor is used in general culture (e.g., batch culture), the cell viability is rapidly decreased because the cell volume can rapidly reach the upper limit of the system capacity due to nutrition or equipment limitations. In the case of the perfusion culture in the N-stage bioreactor, the effective control of the cell density can be realized, the above situation can be prevented, the cell density is usually controlled by using semi-continuous or continuous cell discharge, and the cell quantity can be stably maintained for a long production period. Of course, bioreactors are typically fitted with a cell retention device to retain cells in the bioreactor. Further, the cell retention device comprises a hollow fiber filter. Alternatively, the hollow fiber filter has a pore size of 0.1 μm to 0.5 μm.

In another embodiment, the reaction vessel for perfusion culture is a culture shake tube. For example, 50mL of culture shake tube or 100mL of culture shake tube. Optionally, in the process of culturing cells by using a culture shaking tube, a shaking table with the amplitude of 50mm and the speed of 200 rpm-300 rpm is adopted for culturing; centrifugation may be used to remove the spent medium from the culture shake tube. For example, 200g to 300g, for 5min to 10min. Of course, fresh medium is added after the spent medium is removed. It will be appreciated that in other embodiments, the manner in which the spent media is removed is not limited to centrifugation, but may be otherwise.

In one embodiment, the perfusion medium is in a pre-perfusion phase

Growth A and->

The volume ratio of Feed4 is (80-90): (10-20); in the late stage of perfusion culture, the perfusion medium is +.>

Growth A and->

The volume ratio of Feed4 is (80-85): (20-15). In the early stage of perfusion culture, +.>

Growth A occupies a relatively large area, so that animal cells proliferate rapidly, and +.>

The duty ratio of Growth A and increase accordingly +.>

The duty ratio of Feed4 is favorable for accumulation of antibodies and increases the yield of the antibodies.

Specifically, from day 15 to day 16 of perfusion culture, the medium is perfused

Growth A and->

The volume ratio of Feed4 is (80-85): (20-15). In an alternative specific example, from day 15 to day 16 of perfusion culture +.>

Growth A and

feed4 volume ratio is 80: 20-15. Further, from the 17 th day to the 18 th day of the perfusion culture, the perfusion culture medium is +.>

Growth A and->

The Feed4 volume ratio was adjusted to 85:15./>

In one embodiment, the perfusion medium is in the first 14 days of perfusion culture

Growth A and->

Feed4 volume ratio is 90:10, starting from day 15 to day 16 of perfusion culture, in the perfusion medium +.>

Growth A and->

The Feed4 volume ratio was adjusted to 80:20, a step of; from day 17 to day 18 of perfusion culture, the perfusion culture medium is +.>

Growth A and

the Feed4 volume ratio was adjusted to 85:15.

step c: harvesting the biological material.

Specifically, biological material produced by cells is continuously harvested during perfusion culture. In this embodiment, the cultured cells are CHO-K1 for the expression of the anti-CD20 antibody, and thus, the anti-CD20 antibody produced by the animal cells is harvested. Of course, in other embodiments, depending on the animal cells being cultured, the corresponding products may be harvested.

It should be noted that harvesting the biomass is performed during the continuous perfusion culture process, not after the perfusion culture is completed, i.e., step c is not performed after step b.

The perfusion culture method of the animal cells is simple and convenient, is beneficial to industrial production, and has high living cell density, high yield, short production period and high production efficiency due to the adoption of the perfusion culture medium. In addition, the culture medium in the later stage of perfusion culture is further optimized, so that the amount of biological substances produced by the method for culturing the animal cells by perfusion culture is higher.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following is a detailed description of specific embodiments. The following examples are not specifically described but do not include other components than the unavoidable impurities. Reagents and apparatus used in the examples, unless otherwise specified, are all routine choices in the art. The experimental methods without specific conditions noted in the examples were carried out according to conventional conditions, such as those described in the literature, books, or recommended by the manufacturer. The cell line used in the examples below was CHO-K1 (from ATCC, american type culture collection) for expression of anti-CD20 antibodies;

feed4 and->

Growth A is from Irvine Scientific; EX- & lt- & gt>

Advanced HD Perfusion Medium from Sigma, cat number 24370C.

EXAMPLE 1 Medium proportion screening

11 culture shaking tubes of 50mL were used as reaction vessels and were numbered A to K, and 20mL of medium was filled into each culture shaking tube (the composition of the medium corresponding to each culture shaking tube is shown in Table 1); then at 0.3X10 ⁶ Inoculation Density of individuals/mL CHO-K1 for expression of anti-CD20 antibody was inoculated at 85% humidity, 5.0% CO ₂ Under the conditions of 225rpm and 50mm amplitude, the cell density was sampled and counted every 2 to 3 days, and a growth curve was drawn. The growth curves of the groups are shown in FIG. 1, in which the abscissa in FIG. 1 represents time (in days) and the ordinate in viable cell density (VC, in 10 ⁶ and/mL).

TABLE 1

As can be seen from FIG. 1, the culture medium of group F has the best effect on culturing CHO-K1 expressing anti-CD20 antibody, and the highest cell density can reach 1.67×10 ⁷ The cell density of the other group increased or decreased slowly per mL, especially after 5 days of culture, while the cell density of group F increased sharply, thus demonstrating that the following is true for the other group medium

Feed4 and->

The Growth volume ratio is 10:90, more suitable for proliferation of CHO-K1 expressing anti-CD20 antibodies.

Example 2

In example 1, the screening resulted in

Feed4 and->

The optimum mixing ratio of Growth A was found during the culture process to increase the cell density after a period of time by comparison of

Feed4 and->

Further optimization of the mixing ratio of Growth A can achieve a better culture effect. Specifically, during perfusion culture the ∈Di->

Feed4

The culture method for further optimizing the mixing ratio of Growth A comprises the following steps:

10mL of perfusion medium was added to a 50mL culture rocker to inoculate a 1X 10 density ⁶ Inoculating CHO-K1 for expressing anti-CD20 antibody at 85% humidity and 5.0% CO ₂ Under the conditions of 300rpm and 50mm amplitude, followed by the following operations:

the cell density was counted by sampling and centrifuged to change the liquid, 1VVD per day. The supernatant after centrifugation was used for biochemical, metabolic, yield index and the like detection, and the pellet after centrifugation was resuspended to 10mL with fresh perfusion medium (specific composition is shown in Table 2) and cultured continuously. That is, in the first 15 days of perfusion culture, the medium was perfused

Growth A and->

The ratio of the volumes of Feed4 is 90:10; from day 16 on, perfusion medium +.>

Growth A and->

The ratio of the volumes of Feed4 was adjusted to 80:20, a step of; from day 18, perfusion medium +.>

Growth A and->

The ratio of the volumes of Feed4 was adjusted to 85:15.

The growth curve was plotted against the recorded cell density and the antibody concentration curve was plotted against the daily detected antibody content in the centrifuged supernatant, the results are shown in fig. 2 and 3.

TABLE 2

As can be seen from FIGS. 2 and 3, according to the culture process of example 2, the highest Viable Cell Density (VCD) of CHO-K1 can be up to 7X 10 when CHO-K1 expressing an anti-CD20 antibody is cultured ⁷ The expression level of the anti-CD20 antibody (anti-CD 20) per day is stabilized at 3.5 g/L-4.3 g/L in the plateau phase.

Comparative example 1

The perfusion culture method of comparative example 1 was different from that of example 2 except that the culture conditions were the same, and in comparative example 1, the perfusion culture medium used was 100% in 19 days of the perfusion culture

Growth A。

The growth curve and antibody concentration curve of comparative example 1 are shown in fig. 4 and 5. In FIG. 4, the Growth curve represented by BalanCD Growth A is the Growth curve obtained by the perfusion culture method of comparative example 1, medium A (from

Growth A and->

Feed4 was 90 by volume: 10) is a growth curve obtained by the perfusion culture method of example 2. In FIG. 5, the antibody concentration curve represented by BalanCD Growth A is the antibody concentration curve obtained by the perfusion culture method of comparative example 1, and the antibody concentration curve represented by the culture medium A is the antibody concentration curve obtained by the perfusion culture method of example 2.

As can be seen from fig. 4 and 5, the method of using the following

Growth A and->

Feed4 was 90 by volume: 10 (hereinafter referred to as medium A) to achieve a maximum viable cell density of 7X 10 ⁷ individual/mL, higher than just +.>

Growth A was used as the highest viable cell density (5X 10) of the perfusion culture method of the perfusion medium (hereinafter referred to as medium B) ⁷ and/mL). In addition, the expression level of the antibody in the platform phase of the perfusion culture method adopting the culture medium A is higher than that of the perfusion culture method adopting the culture medium B, the expression level of the antibody in the platform phase is stabilized at 3.5 g/L-4.3 g/L in the perfusion culture method adopting the culture medium A, and the expression level of the antibody in the platform phase is stabilized at 0.6 g/L-0.7 g/L in the perfusion culture method adopting the culture medium B.

Comparative example 2

The perfusion culture method of comparative example 2 was different from that of example 2 except that in comparative example 1, in 19 days of the perfusion culture, EX-

Advanced HD Perfusion Medium。

The growth curve and antibody concentration curve of comparative example 2 are shown in fig. 6 and 7. In FIG. 6, excell HD perfusion medium shows a growth curve obtained by the perfusion culture method of comparative example 3, and culture medium A shows a growth curve obtained by the perfusion culture method of example 2. In fig. 7, the antibody concentration curve represented by Excell HD perfusion medium is the antibody concentration curve obtained by the perfusion culture method of comparative example 2, and the antibody concentration curve represented by medium a is the antibody concentration curve obtained by the perfusion culture method of example 2.

As can be seen from FIGS. 6 and 7, the highest viable cell density of the perfusion culture method using Medium A reached 7X 10 ⁷ The volume per mL is higher than that of EX-

Advanced HD Perfusion Medium As a perfusion medium (hereinafter referred to simply as medium C, which is a medium mainly used in perfusion culture of CHO cells secreting anti-CD20 antibody), when CHO cells secreting anti-CD20 antibody are perfusion-cultured using this medium, the highest viable cell density (about 5.5X10 ⁷ And the amount of antibody expression in the plateau phase of the perfusion culture method using the medium A is higher than that of the perfusion culture method using the medium C, wherein the amount of antibody expression in the plateau phase is stabilized at 3.5g/L to 4.3g/L per day in the perfusion culture method using the medium A, and the amount of antibody expression in the plateau phase is stabilized at 1.8g/L to 2.1g/L per day in the perfusion culture method using the medium C. In addition, the perfusion culture method using medium a has earlier antibody expression and shorter production cycle than the perfusion culture method using medium C.

The technical features of the above-described embodiments may be arbitrarily combined, and all possible combinations of the technical features in the above-described embodiments are not described for brevity of description, however, as long as there is no contradiction between the combinations of the technical features, they should be considered as the scope of the description.

The above examples illustrate only a few embodiments of the invention, which are described in detail and are not to be construed as limiting the scope of the invention. It should be noted that it will be apparent to those skilled in the art that several variations and modifications can be made without departing from the spirit of the invention, which are all within the scope of the invention. Accordingly, the scope of protection of the present invention is to be determined by the appended claims.

Claims

1. A perfusion culture method of animal cells, comprising the steps of:

harvesting the biological material;

the basic culture medium is BalanCD CHO Growth A, the Feed supplement culture medium is BalanCD CHO Feed4, and the volume ratio of BalanCD CHO Growth A to BalanCD CHO Feed4 is 90 in the first 15 days of perfusion culture: 10; starting from day 16 of perfusion culture, the volume ratio of BalanCD [ CHO Growth ] A to BalanCD [ CHO Feed4 ] was adjusted to 80:20, starting from day 18 of perfusion culture, the volume ratio of the BalanCD cube CHO Growth A to the BalanCD cube CHO Feed4 is adjusted to be 85:15;

the animal cell is CHO-K1, and the CHO-K1 is used for expressing anti-CD20 antibody.

2. The perfusion culture method of animal cells according to claim 1, wherein the inoculation density of the animal cells is 0.2 x 10 ⁶ Per mL to 2X 10 ⁶ And each mL.

3. The perfusion culture method of animal cells according to claim 1, wherein the perfusion rate of the perfusion culture is 0.5VVD to 2VVD.

4. The perfusion culture method of animal cells according to claim 1, wherein the perfusion culture temperature is 31-37 ℃.