阅读说明：本技术 一种白喉毒素无毒突变体crm197蛋白、生产方法以及应用 (Diphtheria toxin non-toxic mutant CRM197 protein, production method and application ) 是由 马伟 曹方 于 2021-02-07 设计创作，主要内容包括：本申请涉及蛋白纯化领域,具体公开了一种白喉毒素无毒突变体CRM197蛋白的生产方法,包括以下步骤：S1、粗纯S1.1离心；S1.2超滤；S1.3一段盐析；S1.4二段盐析；S1.5粗蛋白储存；S2、精纯S2.1粗蛋白复溶；S2.2超滤；S2.3过Q膜；S2.4超滤；S2.5柱层析；S2.6超滤；S3、储存CRM197蛋白S3.1原液配制；S3.2原液储存：储存于-70℃以下。本申请生产方法所制得的CRM197蛋白可用于作为载体蛋白的结合疫苗或联合疫苗的制备。通过本申请的生产方法可制备出高质量、高收率、高稳定性的CRM197蛋白。(The application relates to the field of protein purification, and particularly discloses a production method of diphtheria toxin non-toxic mutant CRM197 protein, which comprises the following steps: s1, centrifuging crude pure S1.1; s1.2, ultrafiltration; s1.3, salting out; s1.4, two-stage salting out; s1.5, storing crude protein; s2, redissolving pure S2.1 crude protein; s2.2, ultrafiltration; s2.3, passing through a Q film; s2.4, ultrafiltration; s2.5, performing column chromatography; s2.6, ultrafiltration; s3, storing CRM197 protein S3.1 stock solution; s3.2 stock solution storage: store at below-70 ℃. The CRM197 protein prepared by the production method can be used for preparing a combined vaccine or a combined vaccine serving as a carrier protein. The CRM197 protein with high quality, high yield and high stability can be prepared by the production method.)

1. A production method of a diphtheria toxin non-toxic mutant CRM197 protein is characterized by comprising the following steps of:

s1 coarse purification

S1.1 centrifugation: collecting supernatant after centrifuging CRM197 fermentation liquor;

s1.2, ultrafiltration: ultrafiltering the supernatant with buffer solution for 10-15 times, and collecting ultrafiltrate;

s1.3 first-stage salting out: adding sulfate according to the volume of the ultrafiltrate being 20-25% (W/V), fully stirring, centrifuging and collecting a first-stage salting-out solution;

s1.4 two-stage salting-out: adding sulfate into the first-stage salting-out solution according to the volume of 20-25% (W/V) of the ultrafiltrate, fully stirring, centrifuging, and collecting precipitate to obtain crude protein;

s1.5 crude protein storage: freezing at-20 deg.C or below;

s2 refined purity

S2.1 redissolving crude protein: fully dissolving the crude protein by using a buffer solution to obtain a crude protein solution;

s2.2, ultrafiltration: filtering the crude protein solution, performing ultrafiltration for 10-15 times by using a buffer solution through a 5-50KD membrane package, and collecting ultrafiltrate I;

s2.3, passing through a Q film: filtering the ultrafiltrate I through a Sartobind Q membrane of the Sartis and collecting the filtrate;

s2.4 ultrafiltration: performing isovolumetric ultrafiltration on the filtrate with buffer solution by a 5-50KD membrane pack for 10-15 times, and collecting ultrafiltrate II;

s2.5, column chromatography: purifying the ultrafiltrate II by anion column chromatography, and collecting the eluent;

s2.6 ultrafiltration: ultrafiltering the eluate with water for injection with 5-50KD membrane for 10-15 times to obtain protein ultrafiltrate, and detecting protein concentration;

s3 storage of CRM197 protein

S3.1, preparing stock solution: adding sucrose and water for injection into the protein ultrafiltrate to enable the final concentration of the sucrose to be 4-7%;

s3.2 stock solution storage: freezing at-70 deg.C.

2. The production method according to claim 1, wherein the S1.1 centrifugation is performed at 7500rpm for 20-40 min at 2-8 ℃.

3. The production method according to claim 1, wherein the first-stage salting-out of S1.3 and the second-stage salting-out of S1.4 are performed by centrifugation at 7500rpm for 15-30 min at 2-8 ℃.

4. The method of claim 1, wherein the buffer is one of 10mM PB and 10mM PBS.

5. The method of claim 1, wherein the sulfate is a combination of one or more of ammonium sulfate, sodium sulfate, and magnesium sulfate.

6. The process according to claim 1, wherein in S2.2 ultrafiltration, the crude protein solution is clarified and filtered using a 0.45 μm filter.

7. The method of claim 1, wherein the stock solution S3.1 is formulated to have a final sucrose concentration of 5%.

8. The process according to claim 1, wherein the S2.5 column chromatography is a DEAE SFF column or a Q column.

9. A diphtheria toxin non-toxic mutant CRM197 protein produced by the process of any one of claims 1 to 8.

10. Use of the nontoxic mutant CRM197 protein of diphtheria toxin according to claim 9 as a carrier protein for the preparation of a conjugate or combination vaccine.

Technical Field

The application relates to the field of protein purification, in particular to diphtheria toxin non-toxic mutant CRM197 protein, a production method and application.

Background

CRM197 is a non-toxic variant of diphtheria toxin, and compared to the native diphtheria toxin, CRM197 protein is structurally such that only the glycine residue at position 52 of the a fragment is substituted with a glutamic acid residue, so that its enzymatic activity is lost and thus it cannot exert a toxic effect on cells; but because the protein sequence and the structure of the B fragment of the protein are not changed, the CRM197 protein without toxicity can still be combined with the receptor of a sensitive cell. The CRM197 protein has the advantages of no toxicity, highest integrity, least mutation, closest structure and most powerful receptor blocking capacity compared with other diphtheria toxins and variants. CRM197 therefore becomes an ideal polysaccharide conjugate vaccine carrier protein.

At present, the purity of the protein obtained by purifying the CRM197 fermentation liquor by adopting the existing purification method is not high, so a new purification method of the CRM197 protein is urgently needed.

Disclosure of Invention

In order to improve the purity of the protein purified by the CRM197 fermentation liquor, the application provides a diphtheria toxin non-toxic mutant CRM197 protein, a production method and application.

The production method of the diphtheria toxin non-toxic mutant CRM197 protein provided by the application adopts the following technical scheme:

in a first aspect, the present application provides a method for producing a diphtheria toxin non-toxic mutant CRM197 protein, comprising the steps of:

s1 coarse purification

S1.1 centrifugation: collecting supernatant after centrifuging CRM197 fermentation liquor;

s1.2, ultrafiltration: ultrafiltering the supernatant with buffer solution for 10-15 times, and collecting ultrafiltrate;

s1.3 first-stage salting out: adding sulfate according to the volume ratio of 20-25% (W/V) of the ultrafiltrate, fully stirring, centrifuging and collecting a first-stage salting-out solution;

s1.4 two-stage salting-out: adding sulfate into the first-stage salting-out solution according to the volume of 20-25% (W/V) of the ultrafiltrate, fully stirring, centrifuging, and collecting precipitate to obtain crude protein;

s1.5 crude protein storage: freezing at-20 deg.C or below;

s2 refined purity

S2.1 redissolving crude protein: fully dissolving the crude protein by using a buffer solution to obtain a crude protein solution;

s2.2, ultrafiltration: filtering the crude protein solution, performing ultrafiltration for 10-15 times by using a buffer solution through a 5-50KD membrane package, and collecting ultrafiltrate I;

s2.3, passing through a Q film: filtering the ultrafiltrate I through a Sartobind Q membrane of the Sartis and collecting the filtrate;

s2.4 ultrafiltration: performing isovolumetric ultrafiltration on the filtrate with buffer solution by a 5-50KD membrane pack for 10-15 times, and collecting ultrafiltrate II;

s2.5, column chromatography: purifying the ultrafiltrate II by anion column chromatography, and collecting the eluent;

s2.6 ultrafiltration: ultrafiltering the eluate with water for injection with 5-50KD membrane for 10-15 times to obtain protein ultrafiltrate, and detecting protein concentration;

s3 storage of CRM197 protein

S3.1, preparing stock solution: adding sucrose and water for injection into the protein ultrafiltrate to enable the final concentration of the sucrose to be 4-7%;

s3.2 stock solution storage: freezing at-70 deg.C.

By adopting the technical scheme, firstly, bacterial thalli, metabolites, culture components and the like are removed through centrifugation and ultrafiltration in the rough purification, part of foreign proteins can be removed through further one-stage salting-out, the target protein is precipitated through two-stage salting-out, and the protein yield and the protein purity can be greatly improved through the two-stage salting-out.

Secondly, ultrafiltration in fine purification can remove salt and displace a buffer system at the same time; and further removing impurities such as partial impurity protein, pigment and the like through a Q membrane, and further purifying through column chromatography to obtain the target protein.

In addition, CRM197 protein stock solution prepared from the purified CRM197 protein can be directly frozen and stored, so that the process is shortened, the energy consumption and the production cost are reduced, and the CRM197 protein stock solution is easy to store and use in later period compared with a vacuum drying storage method; and a vacuum freeze-drying process is not adopted, so that the loss of protein freeze-drying is avoided, and the protein yield is improved.

Therefore, the production method can greatly improve the purity of the protein purified by the CRM197 fermentation liquor, the protein yield and the long-term stability of protein storage.

Preferably, in S1.1 centrifugation, the centrifugation is carried out at 7500rpm for 20-40 min at 2-8 ℃.

By adopting the technical scheme, the purity of the CRM197 protein and the yield of the CRM197 protein can be further improved.

Preferably, the first-stage salting-out of S1.3 and the second-stage salting-out of S1.4 are performed at 7500rpm for 15-30 min and at 2-8 ℃.

By adopting the technical scheme, the purity of the CRM197 protein and the yield of the CRM197 protein can be further improved.

Preferably, the buffer is one of 10mM PB and 10mM PBs.

Preferably, the sulfate is one or more of ammonium sulfate, sodium sulfate and magnesium sulfate.

Preferably, in S2.2 ultrafiltration, the crude protein solution is clarified and filtered using a 0.45 μm filter.

By adopting the technical scheme, the purity of the CRM197 protein and the yield of the CRM197 protein can be further improved.

Preferably, the final concentration of sucrose in the S3.1 stock solution preparation is 5%.

By adopting the technical scheme, the protein stock solution can be directly frozen and stored, and the long-term stability of CRM197 protein is further improved.

Preferably, the S2.5 column chromatography is performed using a DEAE SFF column Q column.

By adopting the technical scheme, the adsorption effect is better, and the protein purity is improved.

In a second aspect, the present application provides a diphtheria toxin non-toxic mutant, CRM197 protein, produced using the above production process.

In a third aspect, the application provides the use of the diphtheria toxin non-toxic mutant CRM197 protein as described above as a carrier protein for the preparation of a conjugate vaccine or combination vaccine.

In summary, the present application has the following beneficial effects:

1. because the method adopts twice salting out and ultrafiltration in the coarse purification and adopts Q membrane, ultrafiltration and anion column chromatography purification in the fine purification, the CRM197 protein fine purification method can obtain CRM197 protein stock solution with high yield and high quality.

2. In the application, the CRM197 protein stock solution with higher stability can be obtained by preferably adopting the preparation and storage method disclosed by the invention.

3. The method shortens the process, reduces the energy consumption and the production cost, is easy to store and use at the later stage, avoids the loss of freeze-drying of the protein and improves the yield of the protein by direct freezing storage.

Detailed Description

The present application will be described in further detail with reference to examples.

Raw materials and sources

The CRM197 fermentation broth is produced by Aimeiweixin biopharmaceutical industry (Zhejiang) Co., Ltd;

PB is produced by almimes weixin biopharmaceuticals (zhejiang) limited;

PBS is produced by eimeria weixin biopharmaceutical (zhejiang) limited;

sucrose is manufactured by chemical reagents of national drug group limited;

ammonium sulfate, sodium sulfate, magnesium sulfate are produced by national pharmaceutical group chemical reagents, ltd;

DEAE SFF columns and Q columns are produced by bersee borsch;

water for injection is produced by aimeiweixin biopharmaceuticals (zhejiang) limited.

Examples

Example 1

A method for producing a diphtheria toxin non-toxic mutant CRM197 protein, comprising the steps of:

s1 coarse purification

S1.1 centrifugation: subpackaging the CRM197 fermentation liquor into centrifuge cups, centrifuging at 7500rpm for 20min at 2 ℃, and collecting supernatant;

s1.2, ultrafiltration: after clarifying and filtering the supernatant, concentrating the supernatant to 1/10 volume by using a 30KD membrane package, performing ultrafiltration for 15 times by using a 10mM PB solution, and collecting ultrafiltrate;

s1.3 first-stage salting out: adding ammonium sulfate into ultrafiltrate at a volume of 25% (W/V), stirring, packaging into centrifuge cup, centrifuging at 7500rpm for 15min at 2 deg.C, and collecting a salting-out solution.

S1.4 two-stage salting-out: adding ammonium sulfate into the first-stage salting-out solution according to the volume ratio of 20% (W/V) of the ultrafiltrate, stirring fully, subpackaging into centrifuge cups, centrifuging at 7500rpm for 15min and 2 ℃, and collecting precipitate to obtain crude protein;

s1.5 crude protein storage: freezing at-20 deg.C or below;

s2 refined purity

S2.1 redissolving crude protein: fully dissolving the crude protein by using 10mM PBS to obtain a crude protein solution;

s2.2, ultrafiltration: clarifying and filtering the crude protein solution with a 0.45-micrometer filter membrane, performing isovolumetric ultrafiltration on the crude protein solution with 10mM PBS through a 30KD membrane for 15 times, and collecting ultrafiltrate I;

s2.3, passing through a Q film: filtering the ultrafiltrate I through a Sartobind Q membrane of the Sartis and collecting the filtrate;

s2.4 ultrafiltration: performing equal-volume ultrafiltration on the filtrate by using 10mM PB through a 30KD membrane package for 15 times, and collecting ultrafiltrate II;

s2.5, column chromatography: purifying the ultrafiltrate II by DEAE SFF anion column chromatography, collecting the eluate, and specifically washing the column with 0.5M sodium hydroxide solution at 40mL/min for 2 CVs; then washing the column with physiological saline at 40mL/min for 3 CVs; finally, the balance was stabilized to baseline with 6 CV of 10 mMPB.

Loading the sample at a flow rate of 40mL/min, balancing with 10mM PB, washing with a pre-washing solution until the baseline is stable, eluting with an eluent, collecting the sample when the light absorption value at 280nm is greater than 20mAU, and stopping collection when the light absorption value is less than 20 mAU;

s2.6 ultrafiltration: ultrafiltering the eluate with water for injection with 30KD membrane for 10 times to obtain protein ultrafiltrate, and detecting protein concentration.

S3, storing CRM197 protein

S3.1, preparing stock solution: calculating the protein content of the ultrafiltrate according to the detection result, and calculating the protein content according to the protein content: the sucrose is 1: 9 adding sucrose, and supplementing water for injection to make the final concentration of sucrose be 4%, so as to obtain CRM197 protein stock solution.

S3.2 stock solution storage: freezing at-70 deg.C.

Example 1 of the present application also provides a diphtheria toxin non-toxic mutant CRM197 protein prepared by the production method described in example 1.

The application example 1 also provides the application of the diphtheria toxin non-toxic mutant CRM197 protein described in example 1 as a carrier protein in preparing a combined vaccine or a combined vaccine.

Example 2

A method for producing a diphtheria toxin non-toxic mutant CRM197 protein, comprising the steps of:

s1 coarse purification

S1.1 centrifugation: subpackaging the CRM197 fermentation liquor into centrifuge cups, centrifuging at 7500rpm for 30min at 5 ℃, and collecting supernatant;

s1.2, ultrafiltration: after clarifying the filtered supernatant, the supernatant was concentrated to 1/10 volumes using a 5KD membrane pack, and then ultrafiltrated 12 times using 10mM PBS solution, and the ultrafiltrate was collected.

S1.3 first-stage salting out: adding sodium sulfate into ultrafiltrate at a volume of 22% (W/V), stirring for 40min, packaging into centrifuge cup, centrifuging at 7500rpm for 20min at 5 deg.C, and collecting a salting-out solution.

S1.4 two-stage salting-out: adding sodium sulfate into the first-stage salting-out solution according to the volume ratio of the ultrafiltrate to 22% (W/V), stirring for 40min, subpackaging into centrifuge cups, centrifuging at 7500rpm for 20min and 5 deg.C, and collecting precipitate to obtain crude protein;

s1.5 crude protein storage: freezing at-20 deg.C or below;

s2 refining

S2.1 redissolving crude protein: fully dissolving the crude protein by using 10mM PB to obtain a crude protein solution;

s2.2, ultrafiltration: clarifying and filtering the crude protein solution with a 0.45-micrometer filter membrane, performing isovolumetric ultrafiltration with a 5KD membrane by using 10mM PBS for 12 times, and collecting ultrafiltrate I;

s2.3, passing through a Q film: filtering the ultrafiltrate I through a Sartobind Q membrane of the Sartis and collecting the filtrate;

s2.4 ultrafiltration: performing equal-volume ultrafiltration on the filtrate for 12 times by using 10mM PBS through a 5KD membrane package, and collecting ultrafiltrate II;

s2.5, column chromatography: purifying the ultrafiltrate II by DEAE SFF anion column chromatography, and collecting the eluent;

specifically, the column is washed with 0.5M sodium hydroxide solution at 40mL/min for 2 CVs; then washing the column with physiological saline at 40mL/min for 3 CVs; finally, the balance was stabilized to baseline with 6 CV of 10 mMPB.

Loading the sample at a flow rate of 40mL/min, balancing with 10mM PB, washing with a pre-washing solution until the baseline is stable, eluting with an eluent, collecting the sample when the light absorption value at 280nm is greater than 20mAU, and stopping collection when the light absorption value is less than 20 mAU;

s2.6 ultrafiltration: ultrafiltering the eluate with water for injection with 5KD membrane for 12 times to obtain protein ultrafiltrate;

s3, storing CRM197 protein

S3.1, preparing stock solution: calculating the protein content of the protein ultrafiltrate according to the detection result, and calculating the protein content according to the protein content: the sucrose is 1: 9 adding sucrose, and supplementing water for injection to make the final concentration of sucrose be 5% to obtain CRM197 protein stock solution;

s3.2 stock solution storage: freezing at-70 deg.C.

The application example 2 also provides a diphtheria toxin non-toxic mutant CRM197 protein prepared by the production method described in example 2.

The application example 2 also provides the application of the diphtheria toxin non-toxic mutant CRM197 protein described in example 2 as a carrier protein in preparing a combined vaccine or a combined vaccine.

Example 3

A method for producing a diphtheria toxin non-toxic mutant CRM197 protein, comprising the steps of:

s1 coarse purification

S1.1 centrifugation: subpackaging the CRM197 fermentation liquor into centrifuge cups, centrifuging at 7500rpm for 40min at 8 ℃, and collecting supernatant;

s1.2, ultrafiltration: after clarifying the filtered supernatant, it was concentrated to 1/10 volumes with 50KD and then ultrafiltered 15 times with 10mM PB solution;

s1.3 first-stage salting out: adding magnesium sulfate according to the volume ratio of 25% (W/V) of ultrafiltrate, stirring thoroughly for 30min, subpackaging into centrifuge cup, centrifuging at 7500rpm for 30min and 8 deg.C, and collecting the first-stage salting-out solution.

S1.4 two-stage salting-out: adding magnesium sulfate into the first-stage salting-out solution according to the volume ratio of 25% (W/V) of the ultrafiltrate, fully stirring for 40min, subpackaging into centrifuge cups, and centrifuging at 7500rpm for 30min and 8 ℃ to collect precipitate to obtain crude protein. Centrifuging and collecting the precipitate to obtain crude protein;

s1.5 crude protein storage: freezing at-20 deg.C or below;

s2 refining

S2.1 redissolving crude protein: fully dissolving the crude protein by using 10mM PBS to obtain a crude protein solution;

s2.2, ultrafiltration: clarifying and filtering the crude protein solution with a 0.45-micrometer filter membrane, performing equal-volume ultrafiltration with 10mM PBS through a 50KD membrane for 10 times, and collecting ultrafiltrate I;

s2.3, passing through a Q film: filtering the ultrafiltrate I through a Sartobind Q membrane of the Sartis and collecting the filtrate;

s2.4 ultrafiltration: performing equal-volume ultrafiltration on the filtrate by using 10mM PB through a 50KD membrane package for 10 times, and collecting ultrafiltrate II;

s2.5, column chromatography: purifying the ultrafiltrate II by DEAE SFF anion column chromatography, and collecting the eluent;

specifically, the column is washed with 0.5M sodium hydroxide solution at 40mL/min for 2 CVs; then washing the column with physiological saline at 40mL/min for 3 CVs; finally, the balance was stabilized to baseline with 6 CV of 10 mMPB.

Loading the sample at a flow rate of 40mL/min, balancing with 10mM PB, washing with a pre-washing solution until the baseline is stable, eluting with an eluent, collecting the sample when the light absorption value at 280nm is greater than 20mAU, and stopping collection when the light absorption value is less than 20 mAU;

s2.6 ultrafiltration: ultrafiltering the eluate with water for injection with 50KD membrane for 15 times to obtain protein ultrafiltrate.

S3, storing CRM197 protein

S3.1, preparing stock solution: calculating the protein content of the protein ultrafiltrate according to the detection result, and calculating the protein content according to the protein content: the sucrose is 1: 9 adding cane sugar, and supplementing water for injection to make the final concentration of the cane sugar be 7% to obtain CRM197 protein stock solution.

S3.2 stock solution storage: freezing at-70 deg.C.

Example 4

The difference from example 1 is that S2.5 column chromatography: purifying the ultrafiltrate II by Q anion column chromatography, and collecting the eluent.

Example 5

The difference from example 1 is that in the preparation of S3.1 stock solution, water for injection is added to make the final concentration of sucrose 10%.

Example 6

The difference from example 1 is that in the preparation of S3.1 stock solution, water for injection is added to make the final concentration of sucrose 3%.

Example 7

The difference from example 1 is that in S1.1 centrifugation, 7500rpm, 10min, 1 ℃ centrifugation was used.

Example 8

The difference from example 1 is that in S1.1 centrifugation, 7500rpm, 45min, 9 ℃ centrifugation was used.

Example 9

The difference from example 1 is that the first salting-out in S1.3 and the second salting-out in S1.4 were performed by centrifugation at 7500rpm for 10min at 1 ℃.

Example 10

The difference from example 1 is that the first salting-out in S1.3 and the second salting-out in S1.4 were performed by centrifugation at 7500rpm for 10min at 1 ℃.

Comparative example

Comparative example 1

The difference from example 1 is that the Q-coated film of step S2.3 is removed.

Comparative example 2

The difference from example 1 is that step S1.3 is eliminated for salting out.

Comparative example 3

The difference from example 1 is that CRM197 protein stock was stored using lyophilization.

Performance test

First, yield detection

The yield was evaluated in mg of protein content harvested after the final purification/volume L of crude pure fermentation broth, and the specific results are shown in Table 1;

wherein, the protein content detection: the detection is carried out according to the second method of appendix VIB in the third part of the Chinese pharmacopoeia.

Second, quality detection

1) Identification test: the method is carried out by adopting a method 3403 in the current edition of Chinese pharmacopoeia;

the detection standard is as follows: should form a clear precipitate line with diphtheria toxoid immune serum;

2) protein purity: using a liquid chromatograph;

the detection standard is as follows: more than 90 percent;

3) bacterial endotoxin: adopting the method of the current edition general rule 1143 of Chinese pharmacopoeia;

the detection standard is as follows: the protein content is less than or equal to 5 EU/mug;

4) and (3) sterile test: the method is carried out by adopting a method of the current edition general rule 1101 of Chinese pharmacopoeia;

the detection standard is as follows: qualified;

the results are shown in Table 2.

Third, stability testing

1) Repeated freeze-thaw test: taking stock solution stored at below-70 ℃, standing at room temperature, slowly and automatically dissolving until all the stock solution is dissolved, then placing the melted stock solution at below-70 ℃, and repeatedly freezing and thawing for three times.

The detection standard is as follows: the protein purity after repeated freeze thawing is more than 90 percent;

the detection results are shown in tables 3-4

2) Long-term stability investigation experimental results: the CRM197 protein stock solution stored at the temperature of below 70 ℃ is subjected to long-term stability test investigation, and the change trend of the purity of the main quality standard protein is observed.

The results are shown in Table 5

Analysis of detection results

TABLE 1 table of the results of the yield test

TABLE 2 quality test results table

Table 3 examples 1-4 test table of repeated freeze-thaw test results

Table 4 examples 5-8 repeated freeze-thaw test results test table

TABLE 5 test tables for results of repeated freeze-thaw tests of examples 9-10 and comparative examples 1-2

TABLE 6 Long-term stability investigation experiment results table

From the above data, it can be seen that the protein purity of the protein stock solutions obtained in examples 1-10 is higher than 92%, and the protein yield is higher than 69%. The protein stock solution obtained in the comparative example 1-2 contains impurity protein, so that the protein yield is high, but the protein purity is greatly reduced.

Comparative example 3 the protein stock solution obtained by the freeze-drying storage method has a protein yield close to that of the protein stock solutions obtained in examples 1-10, but has a low protein purity due to the loss of protein in the freeze-drying process. Therefore, by adopting the storage method of the protein stock solution, the process flow can be greatly saved, and the energy consumption and the production cost can be reduced while the higher protein yield is kept and the protein purity is improved.

After the protein stock solutions of examples 1-10 are examined for 24 months long-term stability, the purity of the main quality standard protein is slightly reduced but still higher than 90%, and the quality standard (the standard established by the enterprise registration application) is met; in each long-term examination time point, the protein stock solutions of examples 1-10 were repeatedly frozen and thawed 3 times, and the protein purity of the main quality standard was slightly reduced, but was higher than 90%, which met the quality standard (the standard established by the enterprise registration application). Therefore, the validity period of the CRM197 protein stock solution prepared by the method can be basically determined to be not less than 24 months.

The present embodiment is only for explaining the present application, and it is not limited to the present application, and those skilled in the art can make modifications of the present embodiment without inventive contribution as needed after reading the present specification, but all of them are protected by patent law within the scope of the claims of the present application.