阅读说明：本技术 一种cho细胞表达的重组新型冠状病毒ncp-rbd蛋白的制备方法及其应用 (Preparation method and application of recombinant novel coronavirus NCP-RBD protein expressed by CHO (Chinese hamster ovary) cells ) 是由 孙安徽 孙佳亓 周航 吴常伟 黄恩启 于 2021-08-18 设计创作，主要内容包括：本发明公开了一种CHO细胞表达的重组新型冠状病毒NCP-RBD蛋白的制备方法及其应用,包括以下步骤：(1)细胞复苏培养：取工作细胞株进行复苏培养,得到细胞复苏培养物；(2)细胞扩增培养：将细胞复苏培养物进行扩增,得到细胞扩增培养物；(3)罐培养：将细胞扩增培养物进行罐培养,得到细胞收获液；(4)纯化：对细胞收获液进行纯化,得到所需产物；其中,工作细胞株为表达重组新型冠状病毒NCP-RBD蛋白的重组CHO细胞株。采用本发明所述方法,可以将CHO细胞表达的重组新型冠状病毒NCP-RBD蛋白高效的分离和纯化,适用于规模化生产,同时能有效地用于生产新冠疫苗。(The invention discloses a preparation method and application of a recombinant novel coronavirus NCP-RBD protein expressed by CHO cells, which comprises the following steps: (1) cell recovery culture: taking a working cell strain for recovery culture to obtain a cell recovery culture; (2) and (3) cell amplification culture: amplifying the cell recovery culture to obtain a cell amplification culture; (3) tank culture: performing tank culture on the cell amplification culture to obtain a cell harvest solution; (4) and (3) purification: purifying the cell harvest liquid to obtain a required product; wherein the working cell strain is a recombinant CHO cell strain for expressing recombinant novel coronavirus NCP-RBD protein. The method can efficiently separate and purify the recombinant novel coronavirus NCP-RBD protein expressed by the CHO cells, is suitable for large-scale production, and can be effectively used for producing a novel corona vaccine.)

1. A preparation method of a recombinant novel coronavirus NCP-RBD protein expressed by CHO cells is characterized by comprising the following steps:

(1) cell recovery culture: taking a working cell strain for recovery culture to obtain a cell recovery culture;

(2) and (3) cell amplification culture: amplifying the cell recovery culture to obtain a cell amplification culture;

(3) tank culture: performing tank culture on the cell amplification culture to obtain a cell harvest solution;

(4) and (3) purification: purifying the cell harvest liquid to obtain a required product;

wherein the working cell strain is a recombinant CHO cell strain for expressing recombinant novel coronavirus NCP-RBD protein.

2. The method for preparing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 1, wherein the specific process of cell recovery culture is as follows: inoculating the working cell strain into fresh serum-free culture medium with initial density not lower than 3.0 × 10⁵Each ml of the solution is placed at 37 +/-1 ℃ and 5 +/-1% CO₂Suspension culture is carried out for 2-4 days under the condition of 100-150 rpm, and a cell recovery culture is obtained; wherein the serum-free medium is EX-CELL CD CHO Fusion medium.

3. The method for preparing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 1, wherein the specific process of cell amplification culture is as follows: the cell recovery culture is transferred into a fresh serum-free culture medium, and the initial density is not lower than 3.0 multiplied by 10⁵Each ml is placed at 37 +/-1 ℃ and 5 +/-1% CO₂Suspension culture is carried out for 2-4 days under the condition of 100-150 rpm, and then passage amplification culture is carried out for 2-5 times to obtain a cell amplification culture; wherein the serum-free medium is EX-CELL CD CHO Fusion medium.

4. The method for producing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 1, wherein the specific process of tank culture is as follows:

transferring qualified cell amplification culture to fresh serum-free culture medium, and performing tank culture with initial density not lower than 3.0 × 10⁵Culturing in a tank at a growth temperature of 36.5 + -1 deg.C, dissolved oxygen content of 45% or higher, and pH value of 6.5 or higher;

when the culture is carried out for 3-4 days, the supplementary culture medium is started to be supplemented, the supplementary volume is 10 +/-1% of the initial volume, and the interval of each supplementary culture is 1-2 days; when the culture is carried out for 4-5 days, sugar supplement is started, and the concentration of glucose is not lower than 1g/L during the culture period;

harvesting when the tank culture time reaches 7-13 days and the cell survival rate is 90 +/-10% to obtain cell harvest liquid serving as a production harvest;

wherein the serum-free culture medium is an EX-CELL-Advanced CHO basal culture medium;

the Feed culture medium is EX-CELLAdvanced CHO Feed 1 Feed culture medium;

the qualified cell expansion culture has a cell density of not less than 1.0 × 10⁶Cell expansion culture of individual/ml and cell viability not less than 90%.

5. The method for preparing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 1, wherein the purification comprises the following steps:

clarifying and filtering: centrifuging and filtering the cell harvest liquid to obtain NCP-RBD clarified liquid;

and (3) ultrafiltration concentration: concentrating and ultrafiltering NCP-RBD clarified liquid obtained by clarification and filtration to obtain concentrated liquid;

ion exchange chromatography: carrying out ion exchange chromatography treatment on the concentrated solution to obtain a purified solution;

inactivation: inactivating the purified solution to obtain an inactivated solution;

filtration of viruses and bacteria: and filtering the inactivation solution to remove viruses and bacteria to obtain the required product.

6. The method for preparing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 5, wherein the clarification filtration comprises the following steps: performing low-speed centrifugation and deep filtration to obtain cell-harvested clarified liquid and obtain NCP-RBD clarified liquid;

wherein the low-speed centrifugation condition is 200-1000 g for 5-20 min;

the deep filtration adopts a Supracap 100 bag filter, the nominal precision is 0.5-15 mu m, and the buffer solution is 10-30 mM PB buffer solution.

7. The method for preparing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 5, wherein the ultrafiltration concentration comprises the following steps:

and (3) concentrating and ultrafiltering the NCP-RBD clarified liquid by adopting a 10-50 kDa ultrafiltration membrane package, wherein the concentration multiple is 2-10 times, the ultrafiltration buffer solution is 10-30 mM PB buffer solution, and the washing and filtering multiple is 2-10 times, so as to obtain a concentrated solution.

8. The method for preparing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 5, wherein the ion exchange chromatography comprises the following steps:

anion exchange chromatography: passing the concentrated solution through an anion exchange chromatography column, enabling the concentrated solution to interact with anion exchange filler, and collecting flow-through solution in a flow-through mode, namely the first-step column purified solution;

cation exchange chromatography: and (3) passing the first-step column purified liquid through a cation exchange chromatography column again, enabling the first-step column purified liquid to interact with cation exchange filler, eluting, and collecting target protein eluent, namely the second-step column purified liquid.

9. The method for producing the recombinant novel coronavirus NCP-RBD protein according to claim 8, wherein said anion exchange chromatography comprises anion exchange filler with-CH₂N+(CH₃)₃Anion exchange packing of quaternary ammonium functional groups;

the anion exchange chromatography specifically comprises the following steps:

using 10-30 mM PB buffer solution to balance the chromatographic column until the baseline is stable;

and (3) passing the concentrated solution through an anion exchange chromatography column, enabling the concentrated solution to interact with anion exchange filler, and collecting flow-through solution, namely the first-step column purification solution.

10. The method for producing the recombinant novel coronavirus NCP-RBD protein according to claim 8, wherein the cation exchange filler in the cation exchange chromatography comprises-CH₂CH₂CHSO₃A cation exchange filler of a hydroxypropyl functional group;

the cation exchange chromatography specifically comprises the following steps:

using 10-30 mM PB buffer solution to balance the chromatographic column until the baseline is stable;

passing the first-step column purified liquid through a cation exchange chromatographic column to enable the first-step column purified liquid to interact with cation exchange filler;

after the sample loading is finished, the chromatographic column is balanced again by using 10-30 mM PB buffer solution to make the baseline stable;

eluting with 10-30 mM PB buffer solution containing NaCl, and collecting target protein eluate, namely the second-step column purification solution, wherein the concentration of NaCl is not lower than 0.1M.

11. The method for preparing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 8, wherein the inactivation is performed by the following steps: diluting the second-step column purification solution to 400-800 mu g/ml of protein content, adjusting the pH value of the second-step column purification solution to 3-4 by using a phosphoric acid solution, uniformly mixing, and inactivating at 15-26 ℃ for 2-4 h; and (4) adjusting the pH value back to 6-8 by using a NaOH solution to obtain the inactivation solution.

12. The method for preparing the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells according to claim 5, wherein the specific process for filtering viruses and bacteria is as follows: the inactivated solution is sequentially subjected to virus removal by an SV4 virus removal filter and filtration sterilization by a Supor EKV membrane Acropak20 filter.

13. A recombinant novel coronavirus NCP-RBD protein expressed by CHO cells produced by the method of any one of claims 1-12.

14. The application of a recombinant novel coronavirus NCP-RBD protein expressed by CHO cells is characterized in that: the recombinant novel coronavirus NCP-RBD protein expressed by CHO cells for preparing a novel corona vaccine, which is prepared by the method of any one of claims 1-12.

Technical Field

The invention relates to the technical field of biological engineering, in particular to a preparation method and application of a recombinant novel coronavirus NCP-RBD protein expressed by CHO cells.

Background

The CHO expression system is a eukaryotic expression system widely applied to preparation of macromolecular protein medicaments at present. The CHO cell belongs to mammalian cells, has high evolution degree and a complete protein translation and modification system, so that the expressed protein is similar to the protein in a human body from structure to function, and has smaller risk of side effect when being used as medicinal protein. The RBD domain of the novel coronavirus is known to be a glycoprotein, and CHO cells in eukaryotic cells are the first choice for recombinant glycosyl protein production. Has accurate post-transcriptional modification function, and the expressed glycosylated drug protein is closest to natural protein molecules in the aspects of molecular structure, physicochemical property and biological function; the culture medium can achieve high-density culture in a serum-free suspension culture medium, has higher shear force and osmotic pressure resistance, can achieve a culture volume of more than 1000L, and can be produced in a large scale; CHO has a mature commercialized technical platform to ensure the high-efficiency and stable expression of foreign protein; the recombinant protein can be efficiently expressed in extracellular secretion mode, endogenous protein of the recombinant protein is rarely secreted, and separation and purification of downstream products are facilitated; at present, more and more medicinal proteins are efficiently expressed in CHO cells, and partial vaccines such as hepatitis B vaccines, herpes zoster virus vaccines and the like are put on the market and have better safety.

Because of the long culture period of mammalian cell expression, various protein polymers exist in the supernatant of cell culture solution, and cell host proteins are also released, thereby influencing the purity and uniformity of target proteins. For protein drugs (e.g., antibodies) for therapeutic use, higher purity is often required to ensure that toxicity or immunogenicity due to impurities is avoided.

Disclosure of Invention

One of the objects of the present invention is: aiming at the technical problems to be solved, the preparation method of the recombinant novel coronavirus NCP-RBD protein expressed by the CHO cell is provided, the NCP-RBD protein can be effectively expressed, the method is suitable for subsequent large-scale production, and the recombinant novel coronavirus NCP-RBD protein with higher purity and activity can be effectively prepared.

In order to achieve the purpose, the invention provides the following technical scheme:

a preparation method of a recombinant novel coronavirus NCP-RBD protein expressed by CHO cells comprises the following steps:

(1) cell recovery culture: taking a working cell strain for recovery culture to obtain a cell recovery culture;

(2) and (3) cell amplification culture: amplifying the cell recovery culture to obtain a cell amplification culture;

(3) tank culture: performing tank culture on the cell amplification culture to obtain a cell harvest solution;

(4) and (3) purification: purifying the cell harvest liquid to obtain a required product;

wherein the working cell strain is a recombinant CHO cell strain for expressing recombinant novel coronavirus NCP-RBD protein.

Preferably, the specific process of the cell recovery culture is as follows: inoculating the working cell strain into fresh serum-free culture medium with initial density not lower than 3.0 × 10⁵Each ml of the solution is placed at 37 +/-1 ℃ and 5 +/-1% CO₂Suspension culture is carried out for 2-4 days under the condition of 100-150 rpm, and a cell recovery culture is obtained; wherein the serum-free medium is EX-CELL CD CHO Fusion medium.

Preferably, the specific process of the cell amplification culture is as follows: the cell recovery culture is transferred into a fresh serum-free culture medium, and the initial density is not lower than 3.0 multiplied by 10⁵Each ml of the solution is placed at 37 +/-1 ℃ and 5 +/-1% CO₂Suspension culture is carried out for 2-4 days under the condition of 100-150 rpm, and then passage amplification culture is carried out for 2-5 times to obtain a cell amplification culture; wherein the serum-free medium is EX-CELL CD CHO Fusion medium.

Preferably, the specific process of the tank culture is as follows:

transferring qualified cell amplification culture to fresh serum-free culture medium, and performing tank culture with initial density not lower than 3.0 × 10⁵Culturing in a tank at a growth temperature of 36.5 + -1 deg.C, dissolved oxygen content of 45% or higher, and pH value of 6.5 or higher;

when the culture is carried out for 3-4 days, the supplementary culture medium is started to be supplemented, the supplementary volume is 10 +/-1% of the initial volume, and the interval of each supplementary culture is 1-2 days; when the culture is carried out for 4-5 days, sugar supplement is started, and the concentration of glucose is not lower than 1g/L during the culture period;

harvesting when the tank culture time reaches 7-13 days and the cell survival rate is 90 +/-10% to obtain cell harvesting solution as a production harvest;

wherein the serum-free culture medium is an EX-CELL-Advanced CHO basal culture medium;

the Feed culture medium is EX-CELLAdvanced CHO Feed 1 Feed culture medium;

the qualified cell expansion culture has a cell density of not less than 1.0 × 10⁶Cell expansion culture of individual/ml and cell viability not less than 90%.

Preferably, the purification comprises the following steps:

clarifying and filtering: centrifuging and filtering the cell harvest liquid to obtain NCP-RBD clarified liquid;

and (3) ultrafiltration concentration: concentrating and ultrafiltering NCP-RBD clarified liquid obtained by clarification and filtration to obtain concentrated liquid;

ion exchange chromatography: carrying out ion exchange chromatography treatment on the concentrated solution to obtain a purified solution;

inactivation: inactivating the purified solution to obtain an inactivated solution;

filtration of viruses and bacteria: and filtering the inactivation solution to remove viruses and bacteria to obtain the required product.

Preferably, the specific process of clarifying filtration is as follows: performing low-speed centrifugation and deep filtration to obtain cell-harvested clarified liquid and obtain NCP-RBD clarified liquid;

wherein the low-speed centrifugation condition is 200-1000 g for 5-20 min;

the deep filtration adopts a Supracap 100 bag filter, the nominal precision is 0.5-15 mu m, and the buffer solution is 10-30 mM PB buffer solution.

Preferably, the specific process of ultrafiltration concentration is as follows:

and (3) concentrating and ultrafiltering the NCP-RBD clarified liquid by adopting a 10-50 kDa ultrafiltration membrane package, wherein the concentration multiple is 2-10 times, the ultrafiltration buffer solution is 10-30 mM PB buffer solution, and the washing and filtering multiple is 2-10 times, so as to obtain a concentrated solution.

Preferably, the ion exchange chromatography comprises the following steps:

anion exchange chromatography: passing the concentrated solution through an anion exchange chromatography column, allowing the concentrated solution to interact with anion exchange filler, and collecting flow-through solution in a flow-through mode, namely the first-step column purified solution;

cation exchange chromatography: and (3) passing the first-step column purified liquid through a cation exchange chromatography column again, enabling the first-step column purified liquid to interact with cation exchange filler, eluting, and collecting target protein eluent, namely the second-step column purified liquid.

Preferably, the anion exchange packing in the anion exchange chromatography is a belthas-CH₂N+(CH₃)₃Anion exchange packing of quaternary ammonium functional groups;

the anion exchange chromatography specifically comprises the following steps:

using 10-30 mM PB buffer solution to balance the chromatographic column until the baseline is stable;

and (3) passing the concentrated solution through an anion exchange chromatography column, enabling the concentrated solution to interact with anion exchange filler, and collecting flow-through solution, namely the first-step column purification solution.

Preferably, the cation exchange packing in the cation exchange chromatography is provided with-CH₂CH₂CHSO₃A cation exchange filler of a yellow propyl functional group;

the cation exchange chromatography specifically comprises the following steps:

using 10-30 mM PB buffer solution to balance the chromatographic column until the baseline is stable;

passing the first-step column purified liquid through a cation exchange chromatographic column to enable the first-step column purified liquid to interact with cation exchange filler;

after the sample loading is finished, the chromatographic column is balanced again by using 10-30 mM PB buffer solution to make the baseline stable;

eluting with 10-30 mM PB buffer solution containing NaCl, and collecting target protein eluent, namely the second-step column purification solution, wherein the concentration of NaCl is not lower than 0.1M.

Preferably, the specific process of inactivation is as follows: diluting the second-step column purification solution to 400-800 mu g/ml of protein content, adjusting the pH value of the second-step column purification solution to 3-4 by using a phosphoric acid solution, uniformly mixing, and inactivating at 15-26 ℃ for 2-4 h; and (4) adjusting the pH value back to 6-8 by using a NaOH solution to obtain the inactivation solution.

Preferably, the specific process for filtering viruses and bacteria is as follows: the inactivated solution is sequentially subjected to virus removal by an SV4 virus removal filter and filtration sterilization by a Supor EKV membrane Acropak20 filter.

The second purpose of the invention is that: provides a recombinant novel coronavirus NCP-RBD protein expressed by CHO cells, and the protein is prepared by the method.

The third purpose of the invention is: provides an application of a recombinant novel coronavirus NCP-RBD protein expressed by CHO cells for preparing a novel corona vaccine.

The invention has the beneficial effects that:

the method can efficiently separate and purify the recombinant novel coronavirus NCP-RBD protein expressed by the CHO cells, is suitable for large-scale production, and can be effectively used for producing a novel corona vaccine.

Drawings

FIG. 1 shows the purity (electrophoresis) of a Q column chromatography sample obtained by screening recombinant coronavirus protein packing, wherein M is Marker 26616; wherein each electrophoresis strip represents a sample collected in each step of the column chromatography step, and the specific meanings are as follows: q flow through; Q-0.2M NaCl-1; Q-0.2M NaCl-2; Q-0.2M NaCl-3; Q-0.2M NaCl-4; Q-0.4M NaCl;

FIG. 2 shows the DEAE column chromatography sample purity (electrophoresis) detection map for recombinant coronavirus protein filler screening, M: Marker 26616; wherein each electrophoresis strip represents a sample collected in each step of the column chromatography step, and the specific meanings are as follows: DEAE flow through; DEAE-0.2M NaCl-1; DEAE-0.2M NaCl-2; DEAE-0.2M NaCl-3.

FIG. 3 shows the purity (electrophoresis) of a sample obtained by screening CM column chromatography using recombinant coronavirus protein packing, wherein M is Marker 26616; wherein each electrophoresis strip represents a sample collected in each step of the column chromatography step, and the specific meanings are as follows: 1. clarifying the solution; CM flow through.

FIG. 4 shows the POROS column chromatography sample purity (electrophoresis) detection spectrum for recombinant coronavirus protein packing screening, wherein M is Marker 26616; wherein each electrophoresis strip represents a sample collected in each step of the column chromatography step, and the specific meanings are as follows: stock solution Q; POROS flow through 1; POROS flow through 2; POROS flow through 3; POROS flow through 4; POROS-0.5 MNaCl; POROS-0.5M NaOH.

FIG. 5 shows the purity (electrophoresis) of the sample obtained from the inactivation process of the recombinant coronavirus protein, M: marker 26616; 1. blank control; 2. beta-propiolactone inactivation (24h) control group; 3. beta-propiolactone inactivation (24h) experimental group; 4. low pH inactivation (4h) control group; 5. low pH inactivation (4h) experimental group; 6. formaldehyde inactivation (24h) control group; 7. formaldehyde inactivation (24h) experimental group.

FIG. 6 shows the purity (electrophoresis) of the purified intermediate of the recombinant novel coronavirus protein, M: marker 26616;

1. clear liquid with purity of 96.3%; 2. the purity of the concentrated solution is 85.7%; 3. the first step of column purification liquid has the purity of 95.8 percent; 4. the second step column purified liquid, the purity 97.8%.

FIG. 7 high performance liquid chromatography (size exclusion) detection profile of the final product of the purification of recombinant novel coronavirus protein.

FIG. 8 high performance liquid chromatography (reversed phase chromatography) detection pattern of the final product of the purification of the recombinant novel coronavirus protein.

FIG. 9 shows the cell density and antigen expression curve of the recombinant novel coronavirus NCP-RBD protein tank culture.

Detailed Description

The following examples further describe the invention in detail. It is to be understood, however, that these examples are set forth for purposes of illustration only and are not intended to limit the invention.

Example 1: large-scale culture of recombinant novel coronavirus protein NCP-RBD in 5L bioreactor

The large-scale culture production process of the recombinant protein is divided into two parts, namely CELL recovery and amplification culture and tank culture, wherein the CELL recovery and amplification culture adopts an EX-CELL CD CHO Fusion culture medium, the tank culture adopts an EX-CELL-Advanced CHO Fed-batch basal medium as initial culture, and the EX-CELL-Advanced CHO Fed-1 Fed-batch culture medium is Fed-batch cultured to increase the expression of the recombinant protein, and the process parameters are as follows:

(1) cell resuscitation

Taking a working cell strain (the cell strain is a recombinant CHO cell strain expressing recombinant novel coronavirus NCP-RBD protein and purchased from Chineman bioscience, Beijing, the following operation can be directly carried out on the purchased cell strain, and preferably, the following operation is carried out after the purchased cell strain is established in a three-level cell bank), and taking the working cell strain from a liquid nitrogen tank according to the initial density of not less than 3.0 x 10⁵Is inoculated toEX-CELL CD CHO Fusion medium, at 37 + -1 deg.C and 5 + -1% CO₂Culturing at 100-150 rpm for 2-4 days to obtain cell recovery culture.

(2) Cell expansion culture

Recovering the cells from the culture at an initial density of not less than 3.0X 10⁵Inoculating to EX-CELL CD CHO Fusion culture medium at 37 + -1 deg.C and 5 + -1% CO₂Culturing for 2-4 days under the condition of 100-150 rpm, and carrying out subculture for 2-5 times to obtain a cell amplification culture.

(3) Tank culture

Subjecting the qualified cell amplification culture (the qualified cell amplification culture has a cell density of not less than 1.0 × 10)⁶Cell expansion culture of individual/ml and cell viability not less than 90%. ) Inoculating to bioreactor for tank culture with initial density not lower than 3.0 × 10⁵And (2) per ml, using an EX-CELL-Advanced CHO basic culture medium as a tank culture initiation culture medium, wherein the initiation working volume is 3L, the set temperature is 36.5 +/-1 ℃, the rotating speed is not higher than 350rpm, the pH is not lower than 6.5, and the dissolved oxygen is not lower than 45%: when the culture is carried out for 3-4 days, feeding and supplementing an EX-CELLAdvanced CHO Feed 1 Feed culture medium, wherein the Feed amount accounts for 10 +/-1% of the initial working volume, and the interval of each Feed is 1-2 days; when the culture is carried out for 4-5 days, sugar supplement is started, and the glucose content in the culture solution is controlled to be more than or equal to 1g/L by glucose supplement. And when the culture time reaches 7-13 days and the cell survival rate is 90 +/-10%, discharging the culture tank to obtain a cell harvest liquid, namely the production harvest.

(4) During the culture process, sampling every day to detect the cell density, the antigen content and the protein purity, and finding that the cell density, the antigen content and the antigen purity all increase along with the increase of the number of days of culture, and the cell density reaches 1.0 multiplied by 10 when the culture is carried out for 7 days⁷The antigen content is more than 600 mug/ml, the antigen purity is 71.7%, and the result is shown in figure 9.

Example 2: three batches of continuous large-scale culture expression recombinant novel coronavirus protein NCP-RBD

(1) The pilot scale culture process of the recombinant novel coronavirus protein NCP-RBD was verified by performing a continuous three-batch tank culture by the method of example 1.

(2) During the culture process, sampling every day to detect the cell density, the antigen content and the protein purity, and finding that the cell density, the antigen content and the antigen purity all increase along with the increase of the number of days of culture, and the cell density reaches 1.0 multiplied by 10 when the culture is carried out for 8 days⁷More than one antigen per ml, the antigen content reaches more than 500 mu g/ml, the antigen purity (protein purity) of the final product reaches more than 50 percent, and the specific results are shown in the following table 1.

TABLE 1 determination of cell density and antigen content in the product of three consecutive tank cultures

Example 3: purification process of recombinant novel coronavirus protein NCP-RBD

The harvest (cell harvest) for producing the recombinant novel coronavirus protein NCP-RBD produced in example 1 is used for separation and purification of the recombinant protein, and the specific steps are as follows:

(1) clarifying and filtering, and performing low-speed centrifugation and deep filtration to obtain clarified liquid of cells to obtain NCP-RBD clarified liquid.

1) Centrifuging at low speed for 5-20 min at 200-1000 g, preferably at 220g for 10 min;

2) the deep filtration filter is a Supracap 100 bag filter, and the nominal precision is 0.5-15 mu m;

3) and (3) membrane package cleaning: washing 5-10 membrane volumes with purified water, and rinsing 1-5 membrane volumes with 10-30 mM PB buffer (pH 6.0-7.0);

4) centrifuging, and filtering the supernatant with a deep-layer filtering bag filter; after filtration, the supernatant is obtained by top washing at least 1 membrane volume with 10-30 mM PB buffer (pH6.0-7.0).

(2) And (3) ultrafiltration concentration: and concentrating and washing and filtering the clear solution by adopting an ultrafiltration membrane package to obtain a concentrated solution.

1) The ultrafiltration membrane package is a 10 kD-50 kD ultrafiltration membrane package (Pellicon 2mini cassette);

2) and (3) membrane package cleaning: washing 5-10 membrane volumes with purified water, washing 1-5 membrane volumes with 0.5M NaOH, and circulating for at least 30 min; finally, cleaning 5-10 membrane volumes by using purified water;

3) film moistening and washing: rinsing with 10-30 mM PB buffer (pH6.0-7.0) for 1-5 membrane volumes;

4) concentrating the clear solution by 2-10 times by using a 10 kD-50 kD ultrafiltration membrane; and (3) washing and filtering the concentrated sample by 2-10 times by using 10-30 mM PB buffer solution (pH 6.0-7.0) to obtain a concentrated solution.

(3) Anion column chromatography

1) Name of chromatography packing: with-CH being selected₂N+(CH₃)₃Anion exchange fillers of quaternary ammonium functional groups, such as Q sepharose HP, Q Betarose HP, and the like;

2) column balancing: equilibrating 8-15 column volumes with 10-30 mM PB buffer (pH6.0-7.0);

3) loading: directly loading the concentrated solution, and performing UV₂₈₀Collecting flow-through peaks when the flow-through peaks reach 100-300 mAU;

4) and (3) re-balancing: after the sample loading is finished, re-balancing 5-10 column volumes by using 10-30 mM PB buffer solution (pH6.0-7.0), and stopping collecting flow crossing peaks when UV280 is reduced to 100-300 mAU;

5) and (3) carrying out anion column chromatography on the concentrated solution, combining the hybrid protein with the filler, collecting the target protein in a flow-through mode, and carrying out electrophoresis detection on a purity map shown in figure 6, wherein the purity of the first-step column purified solution is more than 70%.

(4) Cation column chromatography

1) Name of chromatography packing: by the presence of-CH₂CH₂CHSO₃Cationic exchange fillers of the hydroxypropyl functional group, such as POROS 50HS, Diamond SPMustang;

2) column balancing: equilibrating 5-10 column volumes with 10-30 mM PB buffer (pH6.0-7.0);

3) loading: directly loading the first-step column purification solution;

4) and (3) re-balancing: after the sample loading is finished, re-balancing 5-10 cylinder volumes by using 10-30 mM PB buffer solution (pH6.0-7.0);

5) and (3) elution: buffering with 10-30 mM PB containing not less than 0.1M NaClWashing with a washing solution (pH6.0-7.0), eluting with UV₂₈₀Collecting the elution peak when the peak reaches 100-300 mAU, and stopping collecting the peak when the UV280 is reduced to 100-300 mAU;

6) and (3) passing the Q flow through cation column chromatography, collecting the target protein in an elution mode, and detecting a purity map by electrophoresis as shown in figure 6, wherein the purity of the column purified liquid in the second step is more than 95%.

(5) And inactivating, namely inactivating viruses in the diluent of the column purified liquid in the second step by adopting a low pH method.

1) Diluting: adding a proper amount of 10-30 mM PB buffer solution (pH6.0-7.0) into the second-step column purification solution to dilute the second-step column purification solution until the protein content is 400-800 mu g/ml;

2) inactivation: adjusting the pH value of the diluted protein solution to 3-4 by using a phosphoric acid solution, uniformly mixing, and inactivating at 15-26 ℃ for 2-4 h; adjusting the pH value to 6-8 by using NaOH solution to obtain an inactivation solution;

(6) and (3) removing viruses by nanofiltration and sterilizing by filtration, and sequentially carrying out nanofiltration to the inactivated solution to remove the viruses and filtering and sterilizing by a 0.2-micron filter membrane.

1) The virus removal prefilter was a Fluorodyne II Filter, the nanofiltration Filter was an SV4 virus removal Filter, the Filter supplier: PALL (PALL of Party testing)

2) Cleaning a filter membrane: washing 5-10 film volumes with sterile water;

3) rinsing a filter membrane: rinsing with 10-30 mM PB buffer (pH6.0-7.0) for 1-5 membrane volumes;

4) and (3) nanofiltration virus removal: and removing the virus from the inactivation solution by nanofiltration.

5) The sterile filtration filter was a Supor EKV membrane Acropak20 filter, filter supplier: PALL;

6) and (3) degerming and filtering: after the virus is removed by nanofiltration, the sample is sterilized by a 0.2 mu m filter, thus obtaining the stock solution.

Finally, 1062mg of protein can be obtained from 4L of cell harvest, the purity of the purified final product is 100% by high performance liquid chromatography (molecular exclusion chromatography and reverse phase chromatography), and the detection maps are respectively shown in FIG. 7 and FIG. 8; the protein recovery and antigen recovery of key intermediates of each step are shown in table 2 below, with a final purified protein recovery of 13.0% and an antigen recovery of 44.2%.

TABLE 2 protein recovery and antigen recovery in Key steps of the purification Process

Name of intermediate product	Protein recovery (%)	Recovery ratio of antigen (%)
			Clear liquid	/	/
Concentrated solution	22.1	60.5
			First step column purification solution	16.9	60.1
Second step column purification solution	13.6	40.8
			Stock solution	13.0	44.2

The recombinant novel coronavirus NCP-RBD protein expressed by the CHO cells obtained in the embodiment can be used for preparing a novel corona vaccine, preferably, the recombinant novel coronavirus NCP-RBD protein obtained after purification is selected and added with an aluminum hydroxide adjuvant to prepare the novel corona vaccine, and the protein concentration in the novel corona vaccine is 50 mu g/ml.

Example 4: three batches of continuous pilot scale purification of recombinant novel coronavirus protein NCP-RBD

(1) The pilot scale purification process of the recombinant novel coronavirus protein NCP-RBD was verified by three consecutive pilot scale purifications by the purification method of example 3.

(2) In the purification process, the intermediate products of the key steps, namely clarified liquid, concentrated liquid, first-step column purified liquid and second-step column purified liquid, are sampled to detect the protein content, the antigen content and the protein purity so as to investigate the rationality of the control range of key process parameters and the effectiveness of impurity removal in the purification process.

The purity of the intermediate and the stock solution of the 3 batches of key steps is detected by an electrophoresis method, the purities of the clarified solution, the concentrated solution, the first-step column purification solution, the second-step column purification solution and the stock solution are respectively more than 50%, 60%, 70%, 95% and 95%, the recovery rate of the finally purified protein can reach more than 10%, the recovery rate of the antigen can reach more than 30%, and the results are shown in the following table 3. The method proves that each purification step can improve the purity of the protein and can obtain higher protein or antigen recovery rate, so that the setting of relevant parameters of each key step of the purification process in the preparation process of the stock solution is reasonable, the production process can ensure that the purity and the recovery rate of the antigen meet the requirements, the repeatability of the result is good, and the process is stable and controllable.

TABLE 3 purity and recovery test results for three consecutive batches of purified intermediates

Comparison experiment of column chromatography process filler of recombinant novel coronavirus protein NCP-RBD

According to the properties of NCP-RBD protein of the novel recombinant coronavirus and the characteristics of different chromatographic fillers, fillers such as strong cation exchange resin (POROS 50HS), weak cation exchange resin (CM Sepharose FF), strong anion exchange resin (Q Sepharose HP), weak anion exchange resin (DEAE Sepharose FF) and the like are respectively selected for carrying out a comparison experiment.

Comparative example 1: q column chromatography

Adopting Q Sepharose HP filler, and balancing the filler by using 10-30 mM PB buffer solution (pH7.0-8.0); taking the cell harvest obtained in example 1, clarifying to obtain a clarified liquid, loading the clarified liquid into a sample with 4 column volumes, collecting the flow-through peak if the flow-through peak is out of the peak, and sampling; eluting with 10-30 mM PB buffer solution (pH7.0-8.0) containing different NaCl concentrations (0.2-1.0M), collecting elution peaks, mixing uniformly, and sampling; the electrophoresis detection result of the Q column chromatography sample is shown in figure 1, when the pH value of the sample is 8.0, the target antigen is captured in a flow-through mode, and macromolecular hybrid protein is removed after being combined with the filler.

Comparative example 2: DEAE column chromatography

Adopting DEAE Sepharose FF filler, and balancing the filler by using 10-30 mM PB buffer solution (pH7.0-8.0); taking the cell harvest liquid prepared in the example 1, clarifying to obtain clarified liquid, loading the clarified liquid into a sample with 4 column volumes, collecting the flow-through peak if the flow-through peak passes through the peak, and uniformly mixing and sampling; eluting with 10-30 mM PB (pH7.0-8.0) buffer solutions containing different NaCl concentrations (0.2-1.0M), collecting elution peaks, mixing uniformly, and sampling; the result of the electrophoresis detection of the DEAE column chromatography sample is shown in figure 2, and because a small part of antigen is combined with DEAE Sepharose FF filler, the elution contains target antigen, which causes antigen loss.

Comparative example 3: CM column chromatography

Adopting CM Sepharose FF filler, and balancing the filler by using 10-30 mM PB buffer solution (pH7.0-8.0); taking the cell harvest obtained in example 1, clarifying to obtain a clarified liquid, loading the clarified liquid into a sample with 4 column volumes, collecting the flow-through peak if the flow-through peak is out of the peak, and sampling; eluting with 10-30 mM PB buffer solution (pH7.0-8.0) containing different NaCl concentrations (0.2-1.0M), collecting elution peaks, and sampling; the electrophoresis detection result of the CM column chromatography sample is shown in figure 3, when the pH value of the sample is 7.0, CM Sepharose FF chromatography is adopted, the target antigen and macromolecular heteroprotein are collected in a flow-through mode, the purity of the target antigen cannot be improved, and the aim of removing impurities cannot be achieved.

Comparative example 4: POROS column chromatography

Adopting POROS 50HS filler and using 10-30 mM PB buffer solution (pH7.0-8.0) to balance the filler; taking the cell harvest liquid prepared in the example 1, clarifying to obtain a clarified liquid, loading the clarified liquid into a sample with the volume of 15-25 columns, adjusting the pH value to 7.0, loading the sample, collecting the flow-through peak if the flow-through peak passes through the peak, and sampling; eluting with 10-30 mM PB buffer solution (pH7.0-8.0) containing 0.5M NaCl, collecting elution peaks, mixing uniformly, and sampling; the electrophoresis detection result of the POROS column chromatography sample is shown in figure 4, no target antigen exists in POROS flow-through, so that the target antigen is almost completely combined with POROS 50HS filler, 10-30 mM PB buffer solution (pH 7.0-8.0) containing 0.5M NaCl is used for elution, and the antigen can be completely eluted by eluent after electrophoresis detection.

According to comparative examples 1, 2, 3, 4 it can be seen that: selecting Q Sepharose HP as an anion exchange chromatography filler, and POROS 50HS as a cation exchange chromatography filler, wherein the Q Sepharose HP chromatography captures a target antigen in a flow-through mode; POROS 50HS chromatography captures the antigen of interest in an elution mode.

Second, the inactivation process contrast experiment of the recombinant novel coronavirus protein NCP-RBD

According to Chinese pharmacopoeia (current edition), three existing inactivation processes of beta-propiolactone inactivation, low pH inactivation and formaldehyde inactivation of the current virus vaccine are studied, the antigen purity and the antigen content are detected by sampling, and the difference between the antigen purity and the antigen specific activity of a control group and an experimental group is compared, so that whether the inactivation process has influence on the antigen is analyzed, and the inactivation mode with the minimum influence on the antigen is selected.

And (3) taking the diluent of the column purified liquid in the second step according to the inactivation process of the following table 4 to respectively perform three inactivation modes of beta-propiolactone inactivation, low-pH inactivation and formaldehyde inactivation.

(1) The beta-propiolactone inactivation process comprises the following steps: taking the diluent of the column purification liquid in the second step, adding beta-propiolactone according to the ratio of 1:4000, inactivating for 24 hours at the temperature of 2-8 ℃, and hydrolyzing for 2 hours at the temperature of 37 ℃; and (3) additionally arranging a control group without adding beta-propiolactone, inactivating at 2-8 ℃ for 24 hours, and hydrolyzing at 37 ℃ for 2 hours.

(2) And (3) a low pH inactivation process: taking the second step column purified liquid diluent, adjusting the pH to 3.5, standing for 4h at room temperature, and adjusting the pH to 7.0; the control group was prepared without pH adjustment and left at room temperature for 4 hours.

(3) The formaldehyde inactivation process comprises the following steps: adding formaldehyde with final concentration of 100 μ g/ml into the second-step column purified solution, and standing at 37 deg.C for 24 hr; a control group without formaldehyde was placed at 37 ℃ for 24 hours.

TABLE 4 comparative table of inactivation process

TABLE 5 inactivation test results for antigen purity and specific activity

Serial number	Sample name	Purity (HPLC)	Specific activity of antigen
				1	Blank control group	100.0％	1.18
2	Beta-propiolactone inactivation (24h) control group	100.0％	1.18
				3	Beta-propiolactone inactivation (24h) experimental group	100.0％	1.05
4	Low pH inactivation (4h) control group	100.0％	1.21
				5	Low pH inactivation (4h) panel	100.0％	1.18
6	Formaldehyde inactivation (24h) control group	/	/
				7	Formaldehyde inactivation (24h) experimental group	/	/

After formaldehyde is inactivated at 37 ℃ for 24h, the electrophoresis detects that a degradation zone appears in a control group, and the electrophoresis result is shown in figure 5, so that the formaldehyde inactivation method is excluded. Inactivation of beta-propiolactone and low pH inactivation, no degradation zone appears in the experimental group electrophoresis detection, the purity detection is 100%, and the specific activities of the antigens are basically consistent with those before inactivation (as shown in the table 5); compared with the two methods, the low-pH method has higher safety, convenient operation and no residue of other substances, and the method has wide application in the current antibody preparation; therefore, a low pH inactivation method was chosen as the inactivation process for recombinant novel coronavirus vaccine (CHO cell) stock solution and β -propiolactone inactivation was chosen as the alternative method.

Although the present invention has been described in detail with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some technical features may be equivalently replaced; and such modifications or substitutions do not depart from the spirit and scope of the corresponding technical solutions of the embodiments of the present invention.