阅读说明：本技术 动物细胞的灌流培养方法 (Perfusion culture method of animal cells ) 是由 陆金康 胡飞 于 2020-12-16 设计创作，主要内容包括：本发明涉及一种动物细胞的灌流培养方法,该动物细胞的灌流培养方法包括以下步骤：将基础培养基和补料培养基混合后灌流培养动物细胞；以及收获生物物质；其中,基础培养基为Growth A,补料培养基为Feed 4,基础培养基与补料培养基的体积之比为(80～90)：(10～20),上述动物细胞的灌流培养方法可以促进动物细胞增殖,提高动物细胞分泌的治疗性蛋白的产量。(The invention relates to a perfusion culture method of animal cells, which comprises the following steps: mixing a basal culture medium and a supplementary culture medium, and performing perfusion culture on animal cells; and harvesting the biological material; wherein the basic culture medium is Growth A, feed medium Feed4, the volume ratio of the basal medium to the Feed medium is (80-90): (10-20), the perfusion culture method of animal cells can promote the proliferation of the animal cells and improve the yield of therapeutic proteins secreted by the animal cells.)

1. A perfusion culture method of animal cells is characterized by comprising the following steps:

mixing a basal culture medium and a supplementary culture medium, and performing perfusion culture on animal cells; and

harvesting the biological material;

wherein the basic culture medium isCHO Growth A, the feed medium isCHO Feed4, the volume ratio of the basic culture medium to the Feed culture medium is (80-90): (10-20).

2. A perfusion culture method of animal cells according to claim 1, wherein the volume ratio of the basal medium to the feed medium is 90: 10.

3. a perfusion culture method of animal cells according to claim 2, wherein the ratio of the volume of the base medium to the volume of the feed medium from day 15 to day 16 of perfusion culture is (80-85): (20-15).

4. A perfusion culture method of animal cells according to claim 2, wherein a volume ratio of the base medium to the feed medium is 80: 20, the volume ratio of the basal medium to the feed medium from day 17 to day 18 of perfusion culture is 85: 15.

5. the perfusion culture method for animal cells according to claim 1, wherein the seeding density of the animal cells is 0.2X 10⁶2X 10 per mL⁶one/mL.

6. A perfusion culture method for animal cells according to claim 1, wherein the perfusion rate of the perfusion culture is 0.5VVD to 2 VVD.

7. A perfusion culture method for animal cells according to claim 1, wherein the perfusion culture temperature is 31 to 37 ℃.

8. A perfusion culture method for animal cells according to any one of claims 1 to 7, wherein the animal cells are animal cells for expressing an anti-CD20 antibody.

9. The perfusion culture method for animal cells according to claim 8, wherein the animal cells are selected from any one of Chinese hamster ovary cells, hybridoma cells, baby hamster kidney cells, and myeloma cells.

10. The perfusion culture method for animal cells according to claim 9, wherein the chinese hamster ovary cells are CHO-K1.

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

Common animal cell culture methods include batch, fed-batch, and perfusion cultures. Batch culture is to put cells and culture medium into a reactor for culture at one time, the cells grow continuously, products are formed continuously, and after a period of culture, the culture is stopped. The fed-batch culture is that a certain amount of culture medium is firstly filled into a reactor, cells are inoculated under proper conditions and cultured, so that the cells continuously grow, and products are continuously formed. Fed-batch culture merely supplements the necessary nutrients to the reaction vessel to maintain the concentration of the nutrients constant. Perfusion culture is to continuously take out part of the culture medium and continuously perfuse new culture medium during the process of cell growth and product formation after cells and the culture medium are added into a reactor. Perfusion culture is one of the important processes of the prior mammalian cell culture, not only can provide a stable environment beneficial to cell culture by continuously discharging cell metabolic 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 of unit volume.

At present, commercial perfusion culture media in the market are few in variety, the matching with constructed cells needs to be verified, and perfusion culture media capable of promoting the growth of cells expressing therapeutic proteins and secreting the therapeutic proteins are lacking.

Disclosure of Invention

Accordingly, there is a need for a perfusion culture method for animal cells, which can rapidly proliferate animal cells and facilitate the cells to secrete therapeutic proteins when the animal cells are cultured by the perfusion culture method.

A perfusion culture method of animal cells comprises the following steps:

mixing a basal culture medium and a supplementary culture medium, and performing perfusion culture on animal cells; and

harvesting the biological material;

wherein the basic culture medium isGrowth A, the feed medium isFeed4, wherein the volume ratio of the basic culture medium to the Feed culture medium is (80-90): (10-20).

The perfusion culture medium used in the perfusion culture method of the animal cells comprisesGrowth A andfeed4 and the volume ratio of the two is set to (80-90): (10 to 20) byGrowth A andthe 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 effect on culturing CHO cells secreting anti-CD20 antibodies.

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

in one embodiment, the volume ratio of the base medium to the feed medium is (80-85): (20-15).

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

in one embodiment, the animal cells are seeded at a density of 0.2X 10⁶2X 10 per mL⁶one/mL.

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

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 antibody concentration for example 2;

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

FIG. 5 is a graph of antibody concentration for comparative example 1;

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

fig. 7 is an antibody concentration curve of comparative example 2.

Detailed Description

The present invention will now be described more fully hereinafter for purposes of facilitating an understanding thereof, and may be embodied in many different forms and are 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 in the description of the invention herein is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention.

Unless otherwise specified, during perfusion culture herein, perfusion of fresh medium or withdrawal of spent medium may be continuous, gradual, intermittent or any combination of these or all.

The invention provides a perfusion culture medium, which comprises a basal culture medium and a supplementary culture medium, wherein the basal culture medium isGrowth A, feed mediumFeed4, the volume ratio of the basal medium to the Feed medium is (80-90): (10-20). After verification, will containGrowth A andfeed4 and the volume ratio of the two is set to (80-90): the perfusion culture medium (10-20) is used for perfusion culture of anti-CD20 antibody-secreting CHO cells, and can obviously improve the cell density of anti-CD20 antibody (anti-CD20) -secreting CHO cells and improve the yield of anti-CD20 antibodies.

In an alternative embodiment, the ratio of the volumes of the basal medium to the feed medium is 90: 10. 88: 12. 85: 15. 83: 17 or 80: 20. in one embodiment, the volume ratio of the basal medium to the 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 the basal medium to the feed medium is as described above. Of course, in other embodiments, the perfusion medium 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 capable of binding to the resistance marker and viability marker in the host cell line, including, but not limited to, geneticin (G4118), neomycin, hygromycin B, puromycin, zeocin, methionine sulfimide or methotrexate.

An embodiment of the present invention further provides a method for preparing the perfusion medium, including: the perfusion medium was prepared by mixing the components of 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 mix the components uniformly.

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

An embodiment of the present invention also provides a perfusion culture method of animal cells, including steps a to c. Specifically, the method comprises the following steps:

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

Specifically, the step of culturing a cell culture comprising a cell culture medium and animal cells is to shorten the time until a desired active density is reached after the animal cells are 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 that favors cell proliferation is more beneficial to shorten the culture time to achieve a cell mass 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) was a transformed cell line, obtained from chinese hamster ovary cells in 1957. CHO cells belong to the fibroblast group and are secretory cells commonly used for expressing proteins required by the human body, such as antibodies. Further, the animal cell was a Chinese hamster ovary cell K1 (abbreviated as CHO-K1) 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 an immunoglobulin heavy chain nor synthesize an immunoglobulin light chain by itself), perc.6 cells (engineered cells developed by the Crucell company in cooperation with Royal DSM company, which is genetically engineered from human embryonic retinoblastocytes), and the like.

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

Step b: and mixing the basic culture medium and the supplementary culture medium, and performing perfusion culture on the cell culture.

Specifically, when animal cells are cultured to a certain cell amount (i.e., a cell amount satisfying the density of perfusion culture), the animal cells are transferred to a perfusion apparatus for perfusion culture. The inoculation density of the animal cells is 0.2X 10⁶2X 10 per mL⁶one/mL. At this seeding density, the animal cells can be made to adapt to the perfusion culture environment more quickly. Alternatively, the seeding density of the animal cells is 0.2X 10⁶0.5X 10 units/mL⁶1X 10 units/mL⁶1.5X 10 units/mL⁶2/mL or 2X 10⁶one/mL. Further, the inoculation density of the animal cells was 0.5X 10⁶1X 10 to one/mL⁶one/mL.

In the present embodiment, the perfusion rate of the perfusion culture is 0.5VVD to 2 VVD. "VVD" means the volume of the container per day. Optionally, the perfusion rate of the perfusion culture is 0.5VVD, 0.8VVD, 1VVD, 1.5VVD, or 2 VVD.

Specifically, perfusion culture is alternate tangential flow perfusion culture or tangential flow perfusion culture. The alternate tangential flow perfusion culture refers to perfusion culture in which cells are intercepted by adopting an Alternate Tangential Flow (ATF) filtration technology in cell culture; tangential flow perfusion culture refers to perfusion culture in which cells are retained during the cell culture process using a tangential flow (TFF) filtration technique. In the perfusion culture of adopting tangential flow (TFF) filtration technology to intercept cells, cell sap forms a continuous annular flow direction through the action of a peristaltic pump, after entering a fiber membrane, waste liquid is discharged out of a system through the membrane, and the cells return to the culture system along with a loop. In perfusion culture for intercepting cells by adopting an Alternate Tangential Flow (ATF) filtration technology, reciprocating flow of a culture medium in a tank in an interception device is realized by reciprocating blowing and sucking action of a diaphragm pump, and meanwhile metabolic waste is discharged through the diaphragm along with the culture medium and the cells are intercepted in a reactor. When ATF is used, the alternating motion creates a scouring action in the filter membrane, which helps to prevent clogging of the fibre membrane.

Specifically, the reaction vessel for perfusion culture is not particularly limited, and may be, for example, a bioreactor or a culture shake tube. "bioreactor" herein refers primarily to a reaction vessel with a capacity above 2L for culturing cells to produce a product of interest.

In one embodiment, the reaction vessel for perfusion culture is a bioreactor, and perfusion culture using the bioreactor facilitates control of fresh medium perfusion and spent medium withdrawal. In one specific example, the bioreactor is a N-stage bioreactor (production bioreactor), and when a general culture (e.g., batch culture) is used in the N-stage bioreactor, the amount of cells can quickly reach the upper limit of the system capacity due to nutrient or equipment limitations, and the viability of the cells is quickly reduced. When the N-stage bioreactor uses perfusion culture, the cell density can be effectively controlled, which can be prevented, and the cell density is usually controlled by semi-continuous or continuous cell rows, and the cell amount can be stably maintained for a long production period. Of course, the bioreactor is typically fitted with a cell retention device so that the cells remain in the bioreactor. Further, the cell retention device comprises a hollow fiber filter. Optionally, 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, a 50mL culture shake tube or a 100mL culture shake tube. Optionally, in the process of culturing the cells by using the culture shake tube, a shaking table with 200-300 rpm and 50mm of amplitude is adopted for culturing; the used medium can be removed from the culture shake tube by centrifugation. For example, centrifugation is carried out for 5min to 10min at 200g to 300 g. Of course, fresh medium is added after the spent medium is removed. It will be appreciated that in other embodiments, the manner of removing the spent media is not limited to centrifugation, but may be otherwise.

In one embodiment, the perfusion medium is in the early stage of perfusion cultureGrowth A andthe Feed4 volume ratio is (80-90): (10-20); in the late stage of perfusion culture, in perfusion culture mediumGrowth A andthe Feed4 volume ratio is (80-85): (20-15). In the early stage of perfusion culture,growth A accounts for a large proportion, so that animal cells can rapidly proliferate, and the Growth A is reduced in the late stage of perfusion cultureGrowth A and corresponding increaseThe proportion of Feed4 is beneficial to the accumulation of the antibody and the increase of the yield of the antibody.

Specifically, the perfusion culture medium is cultured in the perfusion culture medium from day 15 to day 16Growth A andthe Feed4 volume ratio is (80-85): (20-15). In an alternative embodiment, the perfusion culture medium is a perfusion culture medium beginning on days 15 to 16Growth A andfeed4 volume ratio 80: 20 to 15 parts. Further, the perfusion culture medium is added from the 17 th to the 18 th days of the perfusion cultureGrowth A andthe Feed4 volume ratio was adjusted to 85: 15.

in one embodiment, the perfusion culture medium is in the perfusion culture medium for the first 14 days of the perfusion cultureGrowth A andfeed4 volume ratio 90: 10, starting from day 15 to day 16 of perfusion culture, in perfusion mediumGrowth A andthe Feed4 volume ratio was adjusted to 80: 20; in perfusion medium from day 17 to day 18 of perfusion cultureGrowth A andthe Feed4 volume ratio was adjusted to 85: 15.

step c: harvesting the biological material.

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

It should be noted that the harvesting of the biological material is performed during the continuous perfusion culture, not after the perfusion culture is finished, 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 viable 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 late stage of perfusion culture is further optimized, so that the amount of the biological substances produced by the perfusion culture method of the animal cells is higher.

DETAILED DESCRIPTION OF EMBODIMENT (S) OF INVENTION

The following detailed description is given with reference to specific examples. The following examples are not specifically described, and other components except inevitable impurities are not included. Reagents and instruments used in the examples are all conventional in the art and are not specifically described. The experimental procedures, in which specific conditions are not indicated in the examples, were carried out according to conventional conditions, such as those described in the literature, in books, or as recommended by the manufacturer. The following examplesThe cell line used was CHO-K1 (from ATCC, American type culture collection) for expressing anti-CD20 antibody;feed4 andgrowth A was from Irvine Scientific; EX-Advanced HD Perfusion Medium is from Sigma, cat number 24370C.

EXAMPLE 1 Medium proportioning Screen

11 culture shake tubes of 50mL are taken as reaction vessels, and are numbered A to K, and 20mL of culture medium is respectively filled into each culture shake tube (the composition of the culture medium corresponding to each culture shake tube is shown in Table 1); then at 0.3 × 10⁶Inoculum Density of individuals/mL CHO-K1 for expression of anti-CD20 antibody and at 85% humidity, 5.0% CO₂In the conditions of (1), shaking culture at 225rpm and an amplitude of 50mm was carried out, and a sample was taken every 2 to 3 days to count the cell density and to plot a growth curve. The growth curves of the respective groups are shown in FIG. 1, in which FIG. 1, the abscissa is time (unit: day) and the ordinate is viable cell density (VC, unit 10)⁶one/mL).

TABLE 1

As can be seen from FIG. 1, the group F medium was most effective in culturing CHO-K1 expressing anti-CD20 antibody, and the highest cell density reached 1.67X 10⁷The cell density of the other groups increased or decreased slowly, and the cell density of the F group increased sharply, particularly after 5 days of culture, indicating that the cells were present in the other groupsIn accordance with the culture mediumFeed4 andgrowth in a volume ratio of 10: 90, the culture medium of group F was more suitable for the proliferation of CHO-K1 expressing anti-CD20 antibody.

Example 2

In example 1, screening gaveFeed4 andthe optimal mixing ratio of Growth A is found in the culture process, and after a period of culture, the cell density is increased by the pairFeed4 andthe Growth A mixture ratio is further optimized to obtain better culture effect. Specifically, during perfusion culture processFeed4 andthe 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 shake tube to inoculate a density of 1X 10⁶individuals/mL were inoculated with CHO-K1 for expression of anti-CD20 antibody at 85% humidity, 5.0% CO₂In the condition (1), shaking cultivation at 300rpm and an amplitude of 50mm is carried out, and then the following operations are carried out:

samplingCell density was counted and centrifuged for fluid changes, 1VVD per day. The centrifuged supernatant was used for biochemical, metabolic, yield index detection, and the like, and the centrifuged pellet was resuspended to 10mL with fresh perfusion medium (specific composition shown in table 2), and cultured. That is, in the perfusion medium within the first 15 days of the perfusion cultureGrowth A andthe volume ratio of Feed4 is 90: 10; perfusion in culture medium from day 16Growth A andthe volume ratio of Feed4 was adjusted to 80: 20; perfusion in culture medium from day 18 onwardsGrowth A andthe volume ratio of Feed4 was adjusted to 85: 15.

Growth curves were plotted against the recorded cell densities and antibody concentration curves were plotted against the daily measured antibody content in the centrifuged supernatants, the results being shown in FIGS. 2 and 3.

TABLE 2

As can be seen from FIGS. 2 and 3, according to the cultivation process in example 2, the highest Viable Cell Density (VCD) of CHO-K1 could reach 7X 10 when CHO-K1 expressing anti-CD20 antibody was cultivated⁷individuals/mL, in the plateau phase, the expression level of anti-CD20 antibody (anti-CD20) per dayThe stability is 3.5 g/L-4.3 g/L.

Comparative example 1

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

The growth curve and the 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 (consisting ofGrowth A andfeed4 is 90: 10 ratio) was 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 medium A is the antibody concentration curve obtained by the perfusion culture method of example 2.

As can be seen from FIGS. 4 and 5, the method of usingGrowth A andfeed4 was mixed at a volume ratio of 90: the highest viable cell density of the perfusion culture method of perfusion culture medium (hereinafter referred to as culture medium A) prepared at a ratio of 10 reaches 7 x 10⁷one/mL, higher than that of the single useGrowth A As perfusion Medium (hereinafter referred to as Medium B) for perfusion culture method, highest viable cell density (5X 10)⁷one/mL). The perfusion culture method using medium A has a higher plateau antibody expression level than the perfusion culture method using medium B, and the perfusion culture method using medium A has a plateau antibody expression level of 3.5-4.3 g/L per day, while the perfusion culture method using medium B has a plateau antibody expression level of 0.6-0.7 g/L per day.

Comparative example 2

The perfusion culture method of comparative example 2 is different from that of example 2 except that the culture medium used in comparative example 1 is EX-Advanced HD Perfusion Medium。

The growth curve and the antibody concentration curve of comparative example 2 are shown in fig. 6 and 7. In FIG. 6, the growth curve represented by excel HD perfusion medium is the growth curve obtained by the perfusion culture method of comparative example 3, and the growth curve represented by Medium A is the growth curve obtained by the perfusion culture method of example 2. In FIG. 7, the antibody concentration curve represented by excel 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 is clear from FIGS. 6 and 7, the highest viable cell density of the perfusion culture method using the medium A reached 7X 10⁷one/mL higher than that of EX-Advanced HD Perfusion Medium (hereinafter referred to as Medium C) as a Perfusion Medium mainly used for Perfusion culture of CHO cells secreting anti-CD20 antibody, and having high cell density and high anti-CD20 antibody yield when CHO cells secreting anti-CD20 antibody are perfused and cultured using the MediumHighest viable cell density of the flow culture method (about 5.5X 10)⁷one/mL) and the plateau-phase antibody expression amount is higher in the perfusion culture method using the medium A than in the perfusion culture method using the medium C, the plateau-phase antibody expression amount is stabilized at 3.5g/L to 4.3g/L per day in the perfusion culture method using the medium A, and the plateau-phase antibody expression amount 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 showed an earlier antibody expression and a shorter production cycle than the perfusion culture method using medium C.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.