CN113980103B - Method for purifying mSEB antigen protein - Google Patents

Abstract

The invention discloses a method for purifying mSEB antigen protein, which solves the technical problems that in the prior art, the purity of mSEB protein stock solution obtained by an mSEB protein purification process is not high, the storage stability is poor, and the purity is reduced. It comprises the following steps: s1, crushing thalli; s2, clarifying and filtering; s3, SPFF column chromatography; s4, SPHP column chromatography; s5, phenyl HP column chromatography; s6, G25 column chromatography; s7, Q HP column chromatography; s8, preparing a stock solution. The mSEB protein stock solution obtained by the purification method has high purity and good storage stability, and the condition of purity reduction can not occur.

Description

Method for purifying mSEB antigen protein

Technical Field

The invention relates to the field of protein purification, in particular to a method for purifying mSEB antigen protein.

Background

The staphylococcus aureus is an important pathogenic bacteria causing nosocomial infection and community infection, develops into a difficulty of clinical treatment due to multi-drug resistance, and needs to develop vaccines to control infection and epidemic.

The staphylococcus aureus is called 'mesophilic bacteria', is taken as the representative of gram-positive bacteria, and is an important pathogenic bacteria for causing nosocomial infection and community infection. Infection is characterized by acute and suppurative infection, and can cause suppurative infection of skin, soft tissues and the like locally, and the infection is not healed for a long time; the whole body can cause severe infection and complications such as acute pneumonia, sepsis, endocarditis, septic arthritis, osteomyelitis and the like, and the death rate is up to 20 percent. Meanwhile, the exotoxin of the staphylococcus aureus can also cause systemic lethal infection such as food poisoning, scald-like skin syndrome, toxic shock syndrome and the like.

With the long-term and wide-spread use of antibiotics, bacterial resistance has become increasingly prominent, and methicillin-resistant staphylococcus aureus (MRSA), which is a typical representative, has been discovered for the first time in 1961 until now, and has become one of the most frequent nosocomial infectious pathogens in the global ICU ward, postoperative infection, burns, war wounds and the like. Meanwhile, the drug has strong pathogenicity, wide transmission path and easy outbreak and epidemic, and develops into a difficulty of clinical treatment due to multi-drug resistance, and is called as a first super bacterium.

MRSA is classified into hospital-associated MRSA (HA-MRSA) and community-associated MRSA (CA-MRSA).

In 2009, CDC in the united states was reported in the journal of new england medicine to have about 9 tens of thousands of severe MRSA infections and about 2 tens of thousands of fatal cases in the united states each year. Meeting data of "new progress of MRSA nosocomial infection diagnosis and treatment strategy" in China in 12 months 2010 shows that the incidence rate of MRSA infection in hospital in China is about 8%, the average hospitalization time of each nosocomial infected patient is prolonged by 14 days, the cost is increased by 6542 yuan, and the direct loss caused by nosocomial infection in China is over 150 hundred billion yuan each year. Meanwhile, due to abuse of antibiotics, the isolation and detection rate of clinical highly drug-resistant staphylococcus aureus, especially MRSA, rises year by year. The national bacterial drug resistance monitoring report in 2011 shows that the staphylococcus aureus position list clinical gram positive bacteria separation amount is first in 273808 strains of bacteria detected in 129 national trimethyl hospitals, the MRSA detection rate accounts for 60% of the detected staphylococcus aureus, and the wide drug resistance rate exceeds 40%. Based on this severe situation, china has classified MRSA as one of 12 pathogenic microorganisms that may have a significant impact on national health in the 21 st century. Currently, MRSA and hepatitis B and AIDS are listed as three most refractory infectious diseases in the world, and are in the first place. Currently, vancomycin is the last line of defense for treating MRSA infection, but with the sequential appearance of vancomycin-resistant staphylococcus aureus (VRSA) in 2002, MRSA and VRSA with highly developed drug resistance are in a global spreading trend, so that clinical staphylococcus aureus infection faces a serious challenge of "drug free treatability".

The development speed of antibiotics is far from the development speed of bacterial drug resistance, and safe and effective staphylococcus aureus vaccine can become an effective means for preventing staphylococcus aureus infection. The WHO has proposed a "six-point policy package plan" for "resistance to drug-resistant bacteria" in 2011, and emphasized that development of immune prevention and control products such as innovative vaccines and the like are supported in the future. Therefore, the research on immune control of staphylococcus aureus infection is enhanced, and the development of a safe and effective novel staphylococcus aureus vaccine has important practical and strategic significance in the aspects of effectively controlling the wide infection and the large-scale outbreak of staphylococcus aureus, greatly reducing the incidence of infection in staphylococcus aureus hospital, spreading drug resistance and the like.

Staphylococcus aureus vaccine mSEB antigen protein profile:

SEB is a superantigen exotoxin, usually produced by pathogenic staphylococcus aureus, especially MRSA, and is a major cause of septic shock, systemic inflammatory response, and food poisoning in humans. As SEB is a super antigen, is not limited by MHC molecules, and is directly combined with T cell antigen epitope under the condition of independent antigen recognition, thereby activating T cells, promoting the T cells to release a large amount of cytokines to cause clinical symptoms such as toxin shock syndrome and the like.

The Blast comparison proves that the amino acid sequence of SEB is highly conserved in each strain of staphylococcus aureus. Studies in a mouse model prove that SEB has better humoral response advantage than other antigens, and the prepared human SEB monoclonal antibody can effectively treat toxic shock syndrome common to staphylococcus aureus infection. The National Institute for Allergy and Infectious Disease (NIAID) in 2010 has used wild-type SEB to prepare vaccines, which have entered phase I clinical studies in humans.

The applicant refers to related research literature construction to obtain SEB three-site (L45R, Y89A, Y94A) mutant mSEB as one of antigen components of recombinant staphylococcus aureus vaccine. In vivo and in vitro experiments prove that: mSEB no longer has toxicity and superantigen activity of wild SEB; animal safety evaluation and immune protection results also showed: mSEB eliminates enterotoxin activity, ensures good safety and retains good immunogenicity. The BALB/c mice generated by single-component immunization have the immune toxicity attack protection rate of more than 25 percent. In addition, no other biological function changes were found.

The existing purification technology of mSEB protein is to directly carry out protein capture on the bacterial liquid through SPHP chromatography so as to achieve the purpose of primarily purifying target protein. But this step is subject to a higher back pressure during the process amplification.

The recombinant mSEB protein is present in soluble form in the E.coli cytoplasm after translation. Because 2 cysteines exist in the sequence, the thiol groups of the cysteines exist in active reduced form in the reducing environment in the cytoplasm of the escherichia coli, and because the prior mSEB purification process has no special renaturation step, disulfide bond formation is not complete enough, and partial cysteines exist in free thiol form. During stock storage, these free sulfhydryl groups spontaneously form disulfide bonds, which if formed by cysteines between two protein molecules, produce mSEB dimers or multi-aggregates, resulting in a decrease in purity.

The applicant found that the prior art has at least the following technical problems:

1. the existing purification technology of mSEB protein is to directly carry out protein capturing on the bacterial breaking liquid through SPHP chromatography, and the situation of higher back pressure can occur in the process of technological amplification;

2. in the prior art, the mSEB protein stock solution obtained by the mSEB protein purification process has low purity, poor storage stability and reduced purity.

Disclosure of Invention

The invention aims to provide a method for purifying mSEB antigen protein, which aims to solve the technical problems that in the prior art, the purity of mSEB protein stock solution obtained by an mSEB protein purification process is not high, the storage stability is poor, and the purity is reduced.

In order to achieve the above purpose, the present invention provides the following technical solutions:

a method for purifying an mSEB antigen protein comprising the steps of:

s1, thallus disruption

Dissolving and crushing mSEB thalli to obtain a bacterial liquid A;

s2, clarifying and filtering

Centrifuging the bacterial liquid A in the step S1, and collecting a supernatant; clarifying and filtering the supernatant to obtain bacterial liquid B;

s3, SPFF column chromatography

Performing primary purification on the bacterial liquid B obtained in the step S2 by adopting GE SPFF filling chromatographic columns to obtain SPFF eluent;

s4, SPHP column chromatography

Performing moderate purification on the SPFF eluent obtained in the step S3 by adopting GE SPHP packing to fill a chromatographic column to obtain the SPHP eluent;

s5, phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; the SPHP eluent comprises the following components in percentage by volume: ammonium sulfate solution = 1:0.5 to 5.0; the ammonium sulfate solution was 20mM PB+3M (NH) ₄ ） ₂ SO ₄ The pH is 6.0; obtaining the dilution of the SPHP eluent to be purified in the next step;

e2, filling a chromatographic column with GE phenyl HP filler, and carrying out fine purification on the dilution of the SPHP eluent to obtain phenyl HP eluent;

s6, G25 column chromatography

Desalting and changing the phenyl HP eluent by adopting a G25 chromatographic column to obtain a G25 chromatographic liquid;

S7, Q HP column chromatography

Carrying out chromatography on the G25 chromatographic liquid by adopting a Q HP chromatographic column to obtain a Q HP flow-through liquid;

s8, preparing a stock solution

Sterilizing and filtering the Q HP flow penetrating fluid in a biosafety cabinet or a sterile isolator to obtain mSEB protein stock solution;

the method also comprises protein renaturation before the step S5, wherein the protein renaturation method comprises the steps of carrying out SPFF column chromatography column renaturation in the step S3 or carrying out protease cofactor method renaturation in the step S4;

when the PFF column chromatography column renaturation is performed in step S3,

(1) the step S3 specifically comprises the following steps:

chromatography column: column height 18-20 cm, column diameter 200mm, column volume 5.5-6.5L;

mobile phase a:20mM PB (pH 6.0);

mobile phase B:20mM PB+5mM GSH+0.5mM GSSG (pH 6.0);

mobile phase C:20mM PB+1M NaCl (pH 6.0);

loading parameters:

sample loading flow rate: 36-72L/h; ultraviolet detection wavelength: UV 280nm; after loading, the column is equilibrated by using a mobile phase A;

complex balance 1 parameters:

flow rate: performing on-column renaturation by using a mobile phase B after the baseline is leveled by re-balancing 2-3 column volumes at 36-72L/h;

flow rate: 3.6-7.2L/h, and carrying out on-column renaturation for 4-20h; after the next day renaturation was completed, the column was equilibrated with mobile phase a;

Complex equilibrium 2 parameters:

flow rate: 36-72L/h, re-balancing 1-2 column volumes, and starting to elute when the base line is leveled;

elution parameters: the flow rate is 36-72L/h; the gradient is: a: c=0-50%, 10 column volumes;

detecting a UV280 absorption spectrum in the elution process, wherein the initial collection UV value (mAU) is more than 600mAU; ending the collection of the UV value (mAU) less than 700mAU to obtain the SPFF eluent;

(2) the step S4 specifically comprises the following steps:

d1, diluting the SPFF eluent obtained in the step S3 by 2-5 times to obtain an SPFF eluent diluent; controlling the conductivity of the dilution liquid of the SPFF eluent to be 2.0-8.0 ms/cm, centrifuging the dilution liquid of the SPFF eluent by using a high-speed refrigerated centrifuge, and centrifuging parameters: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

d2, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d3, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent to obtain the SPHP eluent;

when the protease cofactor method renaturation is performed in step S4,

(1) the step S3 specifically comprises the following steps:

when primary purification is performed by SPFF column chromatography:

chromatography column: column height 18-20 cm, column diameter 200mm, column volume 5.5-6.5L;

buffer solution:

SPFF balance liquid: PB having a pH of 6.0 to 6.5 and a pH of 20 to 50 mM;

SPFF eluent: 20mM PB+1M NaCl, pH 6.0;

sample loading flow rate: 36-72L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 36-72L/h, balance volume: 5-8 CV to UV value and stable electric conduction;

eluting: the elution flow rate is 36-72L/h, the elution gradient is 0-50% B,10CV;

collection criteria: collecting peaks when UV280 nm=200-600 mAU, and stopping collecting when UV280 nm=700-200 mAU to obtain SPFF eluent;

(2) the step S4 specifically comprises the following steps:

d1, diluting the SPFF eluent obtained in the step S3 by 2-5 times to obtain an SPFF eluent diluent; controlling the conductivity of the dilution of the SPFF eluent to be 2.0-8.0 ms/cm, adding CuSO with the final concentration of 1-50 mu mM ₄ Stirring the solution at a rotating speed of 50-100 RPM, and preserving heat for 4-20 hours at a temperature of 2-8 ℃;

d2, centrifuging the dilution of the SPFF eluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

d3, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d4, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent,

chromatography column: column height 18-20 cm, column diameter 200mm, column volume 5.5-6.5L;

Buffer solution:

SPHP balancing solution: PB having a pH of 6.0 to 6.5 and a pH of 20 to 50 mM;

SPHP eluate: 20mM PB+1M NaCl, the pH of which is between 6.0 and 6.5;

sample loading flow rate: 12-24L/h;

complex balance: using a balance buffer solution to re-balance the chromatographic column, wherein the flow rate is 12-24L/h; balance volume: 2-5 CV to UV value and stable electric conduction;

eluting: the elution flow rate is 12-24L/h, the elution gradient is 0-50% B,10CV;

collection criteria: peak collection was started when UV280 nm=200-600 mAU and stopped when UV280 nm=1000-200 mAU, resulting in SPHP eluate.

Further, in the step S1, the cell disruption is specifically:

a1, dissolving mSEB thallus, wherein the mass volume ratio of the mSEB thallus is as follows: cell lysis solution = 1: 15-25, dissolving mSEB bacteria at a temperature not exceeding 20 ℃;

crushing the mSEB thalli, and crushing the mSEB thalli for 2 to 4 times by using a homogenizer at the temperature of not more than 20 ℃ and the pressure of 700 to 800bar after the mSEB thalli are dissolved to obtain bacterial liquid A.

Further, in the step A1, the cell lysis solution is PB in an amount of 20 to 50mM, and the pH thereof is 6.0 to 6.5.

Further, in the step S2, the clarification and filtration specifically includes:

b1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

B2, clarifying and filtering the supernatant collected in the step B1 by using a Cobetter 0.6-0.8 mu m or Pall PDH4 deep layer filter plate.

Further, in the step S5, when fine purification is performed by Phenyl HP column chromatography,

chromatography column: the column height is 9-11 cm, the diameter of the column is 140mm, and the column volume is 1-2L;

buffer solution:

phenyl HP equilibration: 20-50 mM PB+0.25-2.5M (NH 4) 2SO4, the pH value of which is 6.0-6.5;

phenyl HP eluate: PB having a pH of 6.0 to 6.5 and a pH of 20 to 50 mM;

sample loading flow rate: 12-24L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 12-24L/h, balance volume: 3-5 CV to UV value, pH value and stable electric conductivity;

eluting: elution flow rate: 12-24L/h, elution gradient: 0-100% B,10CV;

sample collection: collecting the eluent, wherein the UV280 nm=500-1500 mAU starts to be collected, and stopping collecting when the UV280 nm=1000-200 mAU is used for obtaining the phenyl HP eluent.

Further, in the step S6, when the G25 column chromatography is used for desalting and changing the liquid,

chromatography column: the column height is 28-30 cm, the column diameter is 300mm, and the column volume is 20-22L;

balancing solution: 10-50 mML-His+0.9-1.5% NaCl, and the pH value is 6.0-6.5;

sample loading flow rate: 50-80L/h, rt=15 min;

The sample loading amount is less than or equal to 30% of the volume of the column bed;

detecting the absorption of the eluent UV280, starting to collect when the UV280nm is more than or equal to 50mAU, and stopping collecting when the UV280nm is less than or equal to 50mAU to obtain G25 chromatographic liquid; if the chromatography is performed in separate steps, the eluates are combined each time.

Further, in the step S7, when the QHP column chromatography is performed,

chromatography column: column height 9-11 cm, column diameter 140mm, column volume 1-2L;

balancing solution: 10-50 mM L-His+0.9% -1.5% NaCl, the pH is 6.0-6.5;

sample loading flow rate: 12-24L/h;

collecting fluid penetration, and collecting standard: collecting is started when the UV280nm is more than or equal to 50mAU, and collecting is stopped when the UV280nm is less than or equal to 50mAU, so that the Q HP flow-through liquid is obtained.

Further, in the step S8, a 0.22 μm filter is used for the sterilization filtration.

Based on the technical scheme, the embodiment of the invention at least has the following technical effects:

the mSEB protein stock solution obtained by the purification method of the mSEB antigen protein provided by the invention has high purity, good storage stability and no purity drop, and particularly has the following advantages:

(1) According to the invention, a clarification and filtration step is added, fine particles, a large amount of nucleic acid and other impurities in the bacterial liquid A are removed, the clarity of the bacterial liquid B is improved, the high column pressure of the subsequent chromatographic packing is reduced, and the service life of the packing is prolonged;

(2) According to the invention, SPFF column chromatography (SPFF cation chromatography) is added as a primary purification step, and target proteins are rapidly captured, so that the target proteins are concentrated and stabilized from the bacterial-breaking supernatant with complex components; setting SPHP column chromatography (SPHP cation chromatography) as a moderate purification step, removing a large amount of impurities such as HCP, nucleic acid, enzyme and possibly aggregates which are not removed in the SPFF step contained in mSEB cells; in addition, a protein renaturation step is added in the primary purification or moderate purification step, 2 cysteines of mSEB are oxidized under the influence of oxidation environment, active sulfhydryl groups are changed into inert disulfide bonds, disulfide bonds are formed, the stability of the protein is improved, dimers and multimers formed by mismatch of disulfide bonds of mSEB protein during renaturation are removed during SPHP column chromatography and Phenyl HP column chromatography, and therefore the purity and stability of stock solution are improved;

(4) According to the invention, phenyl HP column chromatography (PHP hydrophobic chromatography) is set as a fine purification step, and meanwhile, gradient elution peak collection standard is optimized, trace impurities are removed, so that various indexes of target proteins meet the quality standard requirements;

(5) The invention adopts G25 chromatography to replace buffer solution, adopts Q HP chromatography to effectively remove endotoxin, effectively links with the previous steps, and finally adopts a 0.22 mu m filter membrane to sterilize and filter a final sample, and then carries out split charging operation to obtain protein stock solution for storage.

Drawings

FIG. 1 is an electrophoretogram of mSEB protein stock solution obtained in comparative example 1;

FIG. 2 is an electrophoretogram of mSEB protein stock solution obtained in example 1;

FIG. 3 is an electrophoretogram of the intermediate process sample of comparative example 1;

FIG. 4 is an electrophoretogram of the intermediate process sample of example 1.

Detailed Description

Example 1:

the invention provides a method for purifying mSEB antigen protein, which comprises the following steps:

s1, thallus disruption

A1, dissolving mSEB thallus, wherein the mass volume ratio of the mSEB thallus is as follows: cell lysis solution = 1:20, dissolving mSEB bacteria at a temperature not exceeding 20 ℃; the thallus dissolving solution is PB with pH of 6.0 and 20 mM;

crushing the mSEB thalli, namely crushing the mSEB thalli for 3 times by using a homogenizer at the temperature of not more than 20 ℃ and the pressure of 750bar after the mSEB thalli are dissolved to obtain a bacterial liquid A; sampling 1 ml/branch×20 branches (1) of the supernatant of the bacterial liquid A to obtain a sample (1);

s2, clarifying and filtering

B1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000g, centrifuging for 20min at 2-8 ℃, and collecting supernatant;

b2, clarifying and filtering the supernatant collected in the step B1 by using a cowetter of 0.6-0.8 mu m to obtain bacterial liquid B; detecting the comprehensive turbidity values before and after filtration, and recording turbidity data; sampling 1 ml/branch X20 branch (2) of bacterial liquid B to obtain a sample (2);

S3, SPFF column chromatography

And (2) carrying out primary purification on the bacterial liquid B by adopting GE SPFF filler to fill a chromatographic column, wherein when the SPFF column chromatography is used for primary purification:

chromatography column: the column height was about 19cm, the column diameter was 200mm, and the column volume was about 6.0L.

Buffer solution:

SPFF balance liquid: PB at pH6.0, 20 mM;

SPFF eluent: 20mM PB+1M NaCl, pH 6.0;

sample loading flow rate: 36-72L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 54L/h, equilibrium volume: 6CV, reaching UV value and stable electric conduction;

eluting: the elution flow rate is 54L/h, the elution gradient is 0-50% B,10CV;

collection criteria: collecting peaks when UV280 nm=400 mAU, stopping collecting when UV280 nm=300 mAU, obtaining SPFF eluent;

recording the volume of the eluted SPFF eluent, and sampling the SPFF eluent to obtain a sample (3) with the volume of 1 ml/branch multiplied by 20 branch (3);

regeneration: regeneration was performed by washing the column 1-2CV with 20mM PB+1M NaCl (pH 6.0);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 3 times to obtain the SPFF eluent diluent; controlling the conductivity of the SPFF eluent to be 5.0 ms/cm, adding a CuSO4 solution with the final concentration of 25 mu mM, stirring at a speed of 75RPM, and preserving heat at 5 ℃ for 12 hours;

D2, centrifuging the dilution of the SPFF eluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000g, centrifuging for 20min at 2-8 ℃, and collecting supernatant;

d3, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d4, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent,

chromatography column: the column height is about 19cm, the column diameter is 200mm, and the column volume is about 6.0L;

buffer solution:

SPHP balancing solution: PB at pH6.0, 20 mM;

SPHP eluate: 20mM PB+1M NaCl, pH 6.0;

sample loading flow rate: 18L/h;

complex balance: re-balancing the chromatographic column with balanced buffer solution at flow rate of 18L/h; balance volume: 3CV to UV value, smooth conductance;

eluting: the elution flow rate is 18L/h, the elution gradient is 0-50% B,10CV;

collection criteria: collecting peaks when UV280 nm=400 mAU, and stopping collecting when UV280 nm=800 mAU, to obtain an SPHP eluent;

the volume of SPHP eluate was recorded, and the SPHP eluate was sampled at 1ml per branch×20 branches (4) as sample (4);

regeneration: regeneration was performed by washing the column 1-2CV with 20mM PB+1M NaCl (pH 6.0);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

S5, phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; the SPHP eluent comprises the following components in percentage by volume: ammonium sulfate solution = 1:3.0; the ammonium sulfate solution is 20mM PB+3M (NH 4) 2SO4, and the pH value of the ammonium sulfate solution is 6.0; obtaining the dilution of the SPHP eluent to be purified in the next step;

e2, filling a chromatographic column with GE Phenyl HP filler, finely purifying the dilution of the SPHP eluent, and when finely purifying Phenyl HP column chromatography,

chromatography column: the height of the column is about 10cm, the diameter of the column is 140mm, and the volume of the column is about 1.5L;

buffer solution:

phenyl HP equilibration: 20mM PB+1.5M (NH 4) 2SO4, pH 6.0;

phenyl HP eluate: PB at pH6.0, 20 mM;

sample loading flow rate: 18L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 18L/h, equilibrium volume: 4CV to UV value, pH, stable electric conductance;

eluting: elution flow rate: 18L/h, elution gradient: 0-100% B,10CV;

sample collection: collecting the eluent, wherein the UV280 nm=1000 mAU starts to be collected, and when the UV280 nm=200 mAU stops to be collected, the phenyl HP eluent is obtained;

recording the volume of phenyl HP eluate, and sampling the phenyl HP eluate by 1 ml/branch X20 branch (5) to obtain a sample (5);

regeneration: regeneration was performed by washing the column 1-2CV with 20mM PB (pH 6.0);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s6, G25 column chromatography

Desalting and changing the phenyl HP eluent by adopting a G25 chromatographic column, and when desalting and changing the liquid by adopting G25 chromatographic column chromatography,

chromatography column: the column height is about 29cm, the column diameter is 300mm, and the column volume is about 20L;

balancing solution: 10mM L-His+0.9% NaCl, pH 6.0;

sample loading flow rate: 50-80L/h;

the sample loading amount is less than or equal to 30% of the volume of the column bed;

detecting the absorption of the eluent UV280, starting to collect when the UV280 nm=50mAU, and stopping collecting when the UV280nm is less than or equal to 50mAU (if the eluent is analyzed in a fractional manner, combining each eluent) to obtain a G25 chromatographic liquid;

mixing the eluates, and sampling 1 ml/branch×20 branch (6) to obtain sample (6);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s7, Q HP column chromatography

The chromatography is carried out on the G25 chromatographic liquid by adopting a Q HP chromatographic column, when the chromatography is carried out by adopting the Q HP column chromatography,

chromatography column: the column height is about 10cm, the column diameter is 140mm, and the column volume is about 1.5L;

balancing solution: 10mM L-His+0.9% NaCl, pH 6.0;

sample loading flow rate: 18L/h;

Collecting fluid penetration, and collecting standard: collecting when UV280 nm=50 mAU, and stopping collecting when UV280 nm=40 mAU to obtain a Q HP stream penetrating fluid;

sampling 1 ml/branch X20 branch (7) of the Q HP flow through liquid, which is the sample (7);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s8, preparing a stock solution

And (3) sterilizing and filtering the Q HP flow-through liquid in a biosafety cabinet or a sterile isolator by adopting a 0.22 mu m filter to obtain mSEB protein stock solution.

Example 2:

the invention provides a method for purifying mSEB antigen protein, which comprises the following steps:

s1, thallus disruption

A1, dissolving mSEB thallus, wherein the mass volume ratio of the mSEB thallus is as follows: cell lysis solution = 1:25, dissolving mSEB bacteria at a temperature of not more than 20 ℃; the thallus dissolving solution is PB with pH of 6.5 and 50 mM;

crushing the mSEB thalli, namely crushing the mSEB thalli for 2 times by using a homogenizer at the temperature of not more than 20 ℃ and the pressure of 800bar after the mSEB thalli are dissolved to obtain a bacterial liquid A; sampling 1 ml/branch×20 branches (1) of the supernatant of the bacterial liquid A to obtain a sample (1);

s2, clarifying and filtering

B1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: centrifuging at 15000g at 2deg.C for 30min, and collecting supernatant;

B2, clarifying and filtering the supernatant collected in the step B1 by using a cowetter of 0.6-0.8 mu m to obtain bacterial liquid B; detecting the comprehensive turbidity values before and after filtration, and recording turbidity data; sampling 1 ml/branch X20 branch (2) of bacterial liquid B to obtain a sample (2);

s3, SPFF column chromatography

And (2) carrying out primary purification on the bacterial liquid B by adopting GE SPFF filler to fill a chromatographic column, wherein when the SPFF column chromatography is used for primary purification:

chromatography column: the column height was about 19cm, the column diameter was 200mm, and the column volume was about 6.0L.

Buffer solution:

SPFF balance liquid: PB at pH6.5, 50 mM;

SPFF eluent: 20mM PB+1M NaCl, pH 6.5;

sample loading flow rate: 72L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 72L/h, equilibrium volume: 8CV to UV value, smooth conductance;

eluting: the elution flow rate is 72L/h, the elution gradient is 0-50% B,10CV;

collection criteria: collecting peaks when UV280 nm=600 mAU, stopping collecting when UV280 nm=300 mAU, obtaining SPFF eluent;

recording the volume of the eluted SPFF eluent, and sampling the SPFF eluent to obtain a sample (3) with the volume of 1 ml/branch multiplied by 20 branch (3);

regeneration: regeneration was performed by washing the column 1-2CV with 50mM PB+1M NaCl (pH 6.5);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

And (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 for 5 times to obtain the SPFF eluent diluent; controlling the conductivity of the dilution of the SPFF eluent to be 8.0 ms/cm, adding a CuSO4 solution with the final concentration of 50 mu mM, stirring at a rotating speed of 100 RPM, and preserving the temperature at 8 ℃ for 4 hours;

d2, centrifuging the dilution of the SPFF eluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: centrifuging at 2-8 ℃ for 30min at 15000g, and collecting supernatant;

d3, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d4, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent,

chromatography column: the column height is about 19cm, the column diameter is 200mm, and the column volume is about 6.0L;

buffer solution:

SPHP balancing solution: PB at pH6.5, 50 mM;

SPHP eluate: 20mM PB+1M NaCl, pH 6.5;

sample loading flow rate: 24L/h;

complex balance: using a balance buffer solution to re-balance the chromatographic column, wherein the flow rate is 24L/h; balance volume: 5CV to UV value, smooth conductance;

eluting: the elution flow rate is 24L/h, the elution gradient is 0-50% B,10CV;

collection criteria: peak collection was started when UV280 nm=600 mAU, and collection was stopped when UV280 nm=200 mAU, yielding an SPHP eluate;

The volume of SPHP eluate was recorded, and the SPHP eluate was sampled at 1ml per branch×20 branches (4) as sample (4);

regeneration: regeneration was performed by washing the column 1-2CV with 50mM PB+1M NaCl (pH 6.5);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s5, phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; the SPHP eluent comprises the following components in percentage by volume: ammonium sulfate solution = 1:5.0; the ammonium sulfate solution is 20mM PB+3M (NH 4) 2SO4, the pH value of the ammonium sulfate solution is 6.0, and the SPHP eluent diluent is obtained to be subjected to the next purification;

e2, filling a chromatographic column with GE Phenyl HP filler, finely purifying the dilution of the SPHP eluent, and when finely purifying Phenyl HP column chromatography,

chromatography column: the height of the column is about 10cm, the diameter of the column is 140mm, and the volume of the column is about 1.5L;

buffer solution:

phenyl HP equilibration: 50mM PB+2.5M (NH 4) 2SO4, pH 6.5;

phenyl HP eluate: PB at pH6.5, 50 mM;

sample loading flow rate: 24L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 24L/h, equilibrium volume: 5CV to UV value, pH, stable electric conductance;

eluting: elution flow rate: 24L/h, elution gradient: 0-100% B,10CV;

Sample collection: collecting the eluent, wherein the collection is started when the UV280 nm=1500mAU, and the collection is stopped when the UV280 nm=500mAU, so as to obtain phenyl HP eluent;

recording the volume of phenyl HP eluate, and sampling the phenyl HP eluate by 1 ml/branch X20 branch (5) to obtain a sample (5);

regeneration: regeneration was performed by washing the column 1-2CV with 50mM PB (pH 6.5);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s6, G25 column chromatography

Desalting and changing the phenyl HP eluent by adopting a G25 chromatographic column, and when desalting and changing the liquid by adopting G25 chromatographic column chromatography,

chromatography column: the column height is about 29cm, the column diameter is 300mm, and the column volume is about 20L;

balancing solution: 50mM L-His+1.5% NaCl, pH 6.5;

sample loading flow rate: 50-80L/h;

the sample loading amount is less than or equal to 30% of the volume of the column bed;

detecting the absorption of the eluent UV280, starting to collect when UV280 nm=50mAU, and stopping to collect when UV280 nm=50mAU (if the eluent is analyzed in a fractional manner, combining each eluent) to obtain a G25 chromatographic liquid;

mixing the eluates, and sampling 1 ml/branch×20 branch (6) to obtain sample (6);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

S7, Q HP column chromatography

The chromatography is carried out on the G25 chromatographic liquid by adopting a Q HP chromatographic column, when the chromatography is carried out by adopting the Q HP column chromatography,

chromatography column: the column height is about 10cm, the column diameter is 140mm, and the column volume is about 1.5L;

balancing solution: 50mM L-His+1.5% NaCl, pH 6.5;

sample loading flow rate: 12-24L/h;

collecting fluid penetration, and collecting standard: collecting when UV280 nm=60 mAU, and stopping collecting when UV280 nm=50 mAU to obtain a Q HP stream penetrating fluid;

sampling 1 ml/branch X20 branch (7) of the Q HP flow through liquid, which is the sample (7);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s8, preparing a stock solution

And (3) sterilizing and filtering the Q HP flow-through liquid in a biosafety cabinet or a sterile isolator by adopting a 0.22 mu m filter to obtain mSEB protein stock solution.

Example 3:

the invention provides a method for purifying mSEB antigen protein, which comprises the following steps:

s1, thallus disruption

A1, dissolving mSEB thallus, wherein the mass volume ratio of the mSEB thallus is as follows: cell lysis solution = 1:15, dissolving mSEB bacteria at a temperature of not more than 20 ℃; the thallus dissolving solution is PB with pH of 6.2 and 35 mM;

crushing the mSEB thalli, namely crushing the mSEB thalli for 4 times by using a homogenizer at the temperature of not more than 20 ℃ and the pressure of 700bar after the mSEB thalli are dissolved to obtain a bacterial liquid A; sampling 1 ml/branch×20 branches (1) of the supernatant of the bacterial liquid A to obtain a sample (1);

S2, clarifying and filtering

B1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 13500g, centrifuging at 2 ℃ for 25min, and collecting supernatant;

b2, clarifying and filtering the supernatant collected in the step B1 by using a Pall PDH4 deep filter plate to obtain bacterial liquid B; detecting the comprehensive turbidity values before and after filtration, and recording turbidity data; sampling 1 ml/branch X20 branch (2) of bacterial liquid B to obtain a sample (2);

s3, SPFF column chromatography

And (2) carrying out primary purification on the bacterial liquid B by adopting GE SPFF filler to fill a chromatographic column, wherein when the SPFF column chromatography is used for primary purification:

chromatography column: the column height was about 19cm, the column diameter was 200mm, and the column volume was about 6.0L.

Buffer solution:

SPFF balance liquid: PB at pH6.2, 35 mM;

SPFF eluent: 20mM PB+1M NaCl, pH 6.2;

sample loading flow rate: 36L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 36L/h, equilibrium volume: 5CV to UV value, smooth conductance;

eluting: the elution flow rate is 36L/h, the elution gradient is 0-50% B,10CV;

collection criteria: collecting peaks when UV280 nm=200 mAU, stopping collecting when UV280 nm=700 mAU, obtaining SPFF eluent;

recording the volume of the eluted SPFF eluent, and sampling the SPFF eluent to obtain a sample (3) with the volume of 1 ml/branch multiplied by 20 branch (3);

Regeneration: regeneration was performed by washing the column 1-2CV with 35mM PB+1M NaCl (pH 6.2);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 2 times to obtain an SPFF eluent diluent; controlling the conductivity of the dilution of the SPFF eluent to be 2.0 ms/cm, adding a CuSO4 solution with the final concentration of 1 mu mM, stirring at a speed of 50RPM, and preserving heat at 2 ℃ for 20 hours;

d2, centrifuging the dilution of the SPFF eluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12500g, centrifuging at 2deg.C for 25min, and collecting supernatant;

d3, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d4, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent,

chromatography column: the column height is about 19cm, the column diameter is 200mm, and the column volume is about 6.0L;

buffer solution:

SPHP balancing solution: PB at pH6.2, 35 mM;

SPHP eluate: 20mM PB+1M NaCl, pH 6.2;

sample loading flow rate: 12L/h;

complex balance: using a balance buffer solution to re-balance the chromatographic column, wherein the flow rate is 12L/h; balance volume: 2CV to UV value, smooth conductance;

Eluting: the elution flow rate is 12L/h, the elution gradient is 0-50% B,10CV;

collection criteria: peak collection was started when UV280 nm=200 mAU, and collection was stopped when UV280 nm=1000 mAU, yielding an SPHP eluate;

the volume of SPHP eluate was recorded, and the SPHP eluate was sampled at 1ml per branch×20 branches (4) as sample (4);

regeneration: regeneration was performed by washing the column 1-2CV with 35mM PB+1M NaCl (pH 6.2);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s5, phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; the SPHP eluent comprises the following components in percentage by volume: ammonium sulfate solution = 1:0.5; the ammonium sulfate solution is 20mM PB+3M (NH 4) 2SO4, and the pH value of the ammonium sulfate solution is 6.0; obtaining the dilution of the SPHP eluent to be purified in the next step;

e2, filling a chromatographic column with GE Phenyl HP filler, finely purifying the dilution of the SPHP eluent, and when finely purifying Phenyl HP column chromatography,

chromatography column: the height of the column is about 10cm, the diameter of the column is 140mm, and the volume of the column is about 1.5L;

buffer solution:

phenyl HP equilibration: 35mM PB+1.5M (NH 4) 2SO4, pH 6.2;

phenyl HP eluate: PB at pH6.2, 35 mM;

Sample loading flow rate: 12L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 12L/h, equilibrium volume: 3CV to UV value, pH, stable conductivity;

eluting: elution flow rate: 12L/h, elution gradient: 0-100% B,10CV;

sample collection: collecting the eluent, wherein the UV280 nm=500 mAU starts to be collected, and stopping collecting when the UV280 nm=1000 mAU is collected to obtain phenyl HP eluent;

recording the volume of phenyl HP eluate, and sampling the phenyl HP eluate by 1 ml/branch X20 branch (5) to obtain a sample (5);

regeneration: regeneration was performed by washing the column 1-2CV with 35mM PB (pH 6.2);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s6, G25 column chromatography

Desalting and changing the phenyl HP eluent by adopting a G25 chromatographic column, and when desalting and changing the liquid by adopting G25 chromatographic column chromatography,

chromatography column: the column height is about 29cm, the column diameter is 300mm, and the column volume is about 20L;

balancing solution: 35mM L-His+1.2% NaCl, pH 6.2;

sample loading flow rate: 50-80L/h;

the sample loading amount is less than or equal to 30% of the volume of the column bed;

detecting the absorption of the eluent UV280, starting to collect when the UV280 nm=55mAU, and stopping to collect when the UV280 nm=45mAU (if the eluent is analyzed in a fractional manner, combining each eluent) to obtain a G25 chromatographic liquid;

Mixing the eluates, and sampling 1 ml/branch×20 branch (6) to obtain sample (6);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s7, Q HP column chromatography

The chromatography is carried out on the G25 chromatographic liquid by adopting a Q HP chromatographic column, when the chromatography is carried out by adopting the Q HP column chromatography,

chromatography column: the column height is about 10cm, the column diameter is 140mm, and the column volume is about 1.5L;

balancing solution: 35mM L-His+1.2% NaCl, pH 6.2;

sample loading flow rate: 12L/h;

collecting fluid penetration, and collecting standard: collecting when UV280 nm=55mau, and stopping collecting when UV280 nm=45mau to obtain a Q HP stream penetrating fluid;

sampling 1 ml/branch X20 branch (7) of the Q HP flow through liquid, which is the sample (7);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s8, preparing a stock solution

And (3) sterilizing and filtering the Q HP flow-through liquid in a biosafety cabinet or a sterile isolator by adopting a 0.22 mu m filter to obtain mSEB protein stock solution.

Example 4:

this embodiment differs from embodiment 1 in that:

s3, SPFF column chromatography

And (2) carrying out primary purification on the bacterial liquid B by adopting GE SPFF filler to fill a chromatographic column, wherein when the SPFF column chromatography is used for primary purification:

Chromatography column: the column height is about 19cm, the column diameter is 200mm, and the column volume is about 6.0L;

mobile phase a:20mM PB (pH 6.0);

mobile phase B:20mM PB+5mM GSH+0.5mM GSSG (pH 6.0);

mobile phase C:20mM PB+1M NaCl (pH 6.0);

loading parameters:

sample loading flow rate: 54L/h; ultraviolet detection wavelength: UV280 nm; after loading, the column is equilibrated by using a mobile phase A;

complex balance 1 parameters:

flow rate: 54L/h, re-balancing 3 column volumes, and carrying out on-column renaturation by using a mobile phase B for 20CV when a base line is leveled;

flow rate: 5.4L/h, and carrying out on-column renaturation overnight for 12h; after the next day renaturation was completed, the column was equilibrated with mobile phase a;

complex equilibrium 2 parameters:

flow rate: 54L/h, re-balancing 2 column volumes, and starting to elute when the base line is leveled;

elution parameters: the flow rate is 54L/h; the gradient is: a: c=0-50%, 10 column volumes;

the elution process detects UV280 absorbance spectrum, initial collection UV value (mAU) =620 mAU; ending the collection of UV values (mAU) =680 mAU, resulting in SPFF eluate;

recording the volume of the eluted SPFF eluent, and sampling the SPFF eluent to obtain a sample (3) with the volume of 1 ml/branch multiplied by 20 branch (3);

regeneration: regeneration was performed by washing the column 1-2CV with 20mM PB+1M NaCl (pH 6.0);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

And (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 3 times to obtain the SPFF eluent diluent; controlling the conductivity of the dilution of the SPFF eluent to be 5.0 ms/cm, and centrifuging the dilution of the SPFF eluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000g, centrifuging at 5 ℃ for 20min, and collecting a supernatant;

d2, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d3, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent,

chromatography column: the column height is about 19cm, the column diameter is 200mm, and the column volume is about 6.0L;

buffer solution:

SPHP balancing solution: PB at pH6.0, 20 mM;

SPHP eluate: 20mM PB+1M NaCl, pH 6.0;

sample loading flow rate: 18L/h;

complex balance: re-balancing the chromatographic column with balanced buffer solution at flow rate of 18L/h; balance volume: 3CV to UV value, smooth conductance;

eluting: the elution flow rate is 18L/h, the elution gradient is 0-50% B,10CV;

collection criteria: peak collection was started when UV280 nm=400 mAU and stopped when UV280 nm=200 mAU, yielding an SPHP eluate;

The volume of SPHP eluate was recorded, and the SPHP eluate was sampled at 1ml per branch×20 branches (4) as sample (4);

regeneration: regeneration was performed by washing the column 1-2CV with 20mM PB+1M NaCl (pH 6.0);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the column was stored using 0.01M NaOH or 20% ethanol solution.

The same procedure as in example 1 was repeated to obtain mSEB protein stock solution.

Example 5:

this embodiment differs from embodiment 1 in that:

s3, SPFF column chromatography

And (2) carrying out primary purification on the bacterial liquid B by adopting GE SPFF filler to fill a chromatographic column, wherein when the SPFF column chromatography is used for primary purification:

chromatography column: column height about 19cm, column diameter 200mm, column volume 6.0L;

mobile phase a:20mM PB (pH 6.0);

mobile phase B:20mM PB+5mM GSH+0.5mM GSSG (pH 6.0);

mobile phase C:20mM PB+1M NaCl (pH 6.0);

loading parameters:

sample loading flow rate: 72L/h; ultraviolet detection wavelength: UV 280nm; after loading, the column is equilibrated by using a mobile phase A;

complex balance 1 parameters:

flow rate: 72L/h, re-balancing 2-3 column volumes, and carrying out on-column renaturation by using the mobile phase B when the base line is leveled;

flow rate: 7.2L/h, and carrying out on-column renaturation overnight for 4h; after the next day renaturation was completed, the column was equilibrated with mobile phase a;

Complex equilibrium 2 parameters:

flow rate: 72L/h, re-balancing 2 column volumes, and starting to elute when the base line is leveled;

elution parameters: the flow rate is 72L/h; the gradient is: a: c=0-50%, 10 column volumes;

detecting a UV280 absorption spectrum in the elution process, wherein the initial collection UV value (mAU) is more than 600mAU; ending the collection of the UV value (mAU) less than 700mAU to obtain the SPFF eluent; recording the volume of the eluted SPFF eluent, and sampling the SPFF eluent to obtain a sample (3) with the volume of 1 ml/branch multiplied by 20 branch (3);

regeneration: regeneration was performed by washing the column 1-2CV with 50mM PB+1M NaCl (pH 6.5); 10

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 for 5 times to obtain the SPFF eluent diluent; controlling the conductivity of the dilution of the SPFF eluent to be 8.0 ms/cm, and centrifuging the dilution of the SPFF eluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: centrifuging at 15000g at 2deg.C for 30min, and collecting supernatant;

d2, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d3, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent,

Chromatography column: the column height is about 19cm, the column diameter is 200mm, and the column volume is about 6.0L;

buffer solution:

SPHP balancing solution: PB at pH6.5, 50 mM;

SPHP eluate: 20mM PB+1M NaCl, pH 6.5;

sample loading flow rate: 24L/h; complex balance: using a balance buffer solution to re-balance the chromatographic column, wherein the flow rate is 24L/h; balance volume: 5CV to UV value, smooth conductance;

eluting: the elution flow rate is 24L/h, the elution gradient is 0-50% B,10CV;

collection criteria: peak collection was started when UV280 nm=600 mAU, and collection was stopped when UV280 nm=200 mAU, yielding an SPHP eluate;

the volume of SPHP eluate was recorded, and the SPHP eluate was sampled at 1ml per branch×20 branches (4) as sample (4);

regeneration: regeneration was performed by washing the column 1-2CV with 50mM PB+1M NaCl (pH 6.5);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the column was stored using 0.01M NaOH or 20% ethanol solution.

The same procedure as in example 1 was repeated to obtain mSEB protein stock solution.

Example 6:

this embodiment differs from embodiment 1 in that:

s3, SPFF column chromatography

Chromatography column: column height about 19cm, column diameter 200mm, column volume 6.0L;

mobile phase a:20mM PB (pH 6.0);

mobile phase B:20mM PB+5mM GSH+0.5mM GSSG (pH 6.0);

Mobile phase C:20mM PB+1M NaCl (pH 6.0);

loading parameters:

sample loading flow rate: 36L/h; ultraviolet detection wavelength: UV280 nm; after loading, the column is equilibrated by using a mobile phase A;

complex balance 1 parameters:

flow rate: re-balancing 2 column volumes at 36L/h, and carrying out on-column renaturation by using the mobile phase B for 20CV when the base line is leveled;

flow rate: 3.6L/h, and carrying out on-column renaturation overnight for 20h; after the next day renaturation was completed, the column was equilibrated with mobile phase a;

complex equilibrium 2 parameters:

flow rate: 36L/h, re-balancing 1 column volume, and starting to elute when the base line is leveled;

elution parameters: the flow rate is 36L/h; the gradient is: a: c=0-50%, 10 column volumes;

detecting a UV280 absorption spectrum in the elution process, wherein the initial collection UV value (mAU) is more than 600mAU; ending the collection of the UV value (mAU) less than 700mAU to obtain the SPFF eluent; recording the volume of the eluted SPFF eluent, and sampling the SPFF eluent to obtain a sample (3) with the volume of 1 ml/branch multiplied by 20 branch (3);

regeneration: regeneration was performed by washing the column 1-2CV with 35mM PB+1M NaCl (pH 6.2);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV;

and (3) preserving: the chromatographic column is preserved with 0.01M NaOH or 20% ethanol solution;

s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 2 times to obtain an SPFF eluent diluent; controlling the conductivity of the dilution of the SPFF eluent to be 2.0 ms/cm, and centrifuging the dilution of the SPFF eluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000g, centrifuging at 8 ℃ for 30min, and collecting a supernatant;

D2, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d3, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent,

chromatography column: the column height is about 19cm, the column diameter is 200mm, and the column volume is about 6.0L;

buffer solution:

SPHP balancing solution: PB at pH6.2, 35 mM;

SPHP eluate: 20mM PB+1M NaCl, pH 6.2;

sample loading flow rate: 12L/h;

complex balance: using a balance buffer solution to re-balance the chromatographic column, wherein the flow rate is 12L/h; balance volume: 2CV to UV value, smooth conductance;

eluting: the elution flow rate is 12L/h, the elution gradient is 0-50% B,10CV;

collection criteria: peak collection was started when UV280 nm=200 mAU, and collection was stopped when UV280 nm=1000 mAU, yielding an SPHP eluate;

the volume of SPHP eluate was recorded, and the SPHP eluate was sampled at 1ml per branch×20 branches (4) as sample (4);

regeneration: regeneration was performed by washing the column 1-2CV with 35mM PB+1M NaCl (pH 6.2);

CIP: CIP was performed using 1M NaOH solution to flush column 1-2 CV; 5

And (3) preserving: the column was stored using 0.01M NaOH or 20% ethanol solution.

The same procedure as in example 1 was repeated to obtain mSEB protein stock solution.

2. Comparative example

Comparative example 1:

unlike example 1, the following is: s4, SPHP column chromatography

D1, diluting the SPFF eluent obtained in the step S3 by 2-5 times to obtain an SPFF eluent diluent; controlling the conductivity of the dilution liquid of the SPFF eluent to be 2.0-8.0 ms/cm (without adding CuSO4 solution);

with the rest of example 1, mSEB protein stock solution was obtained.

3. Experimental example

1. The resulting mSEB protein stocks of examples 1 to 6 and comparative example 1 and the intermediate process samples were subjected to electrophoretic purity comparison and were detected by Image Lab analysis software of BioRad corporation, and the results are shown in fig. 1 to 4 and table 1 below.

As can be seen from fig. 1-4 and table 1, after renaturation of mSEB, the sample sampled after the SPHP column chromatography can meet the 95% purity requirement, i.e. can meet the quality standard, while comparative example 1 cannot meet 97% after Phenyl HP, while the purity of mSEB protein stock solution obtained in examples 1-6 can reach 100.0% and meet the stock solution purity standard, and is far higher than that of comparative example 1; in comparative example 1, the data of sample (4), sample (5), sample (6), sample (7) and mSEB protein stock were suddenly changed since comparative example 1 was not subjected to renaturation, and the process sample was not subjected to such a change, because the non-renaturation produced aggregates, resulting in poor purity.

2. The mSEB protein stock solutions obtained in examples 1 to 6 and comparative example 1 were stored at an ultralow temperature of-60 to-80℃for 12 months, and purity was measured after 1 month, 3 months, 6 months, 9 months and 12 months, respectively, and the results are shown in Table 2 below.

As can be seen from Table 2, the mSEB protein stock solutions obtained in examples 1 to 6 can meet the requirement of no significant decrease in purity for 12 months; whereas comparative example 1 was below the quality standard already at 6 months, stability could only be maintained for 3 months. Since the detection time is already lower than the standard in 6 months and 9 months, the detection is not needed in 12 months, and the product is failed. The mSEB protein stocks obtained in examples 1-6 did not have aggregates produced, and had a purity close to zero. The method is sufficient for explaining that the renaturation effect of the embodiment of the invention is good, the problem of mSEB polymer can be well solved, and the purity of mSEB protein stock solution and the storage stability of the mSEB protein stock solution are improved.

Claims (8)

1. A method for purifying an mSEB antigen protein, comprising the steps of:

s1, thallus disruption

Dissolving and crushing mSEB thalli to obtain a bacterial liquid A;

s2, clarifying and filtering

Centrifuging the bacterial liquid A in the step S1, and collecting a supernatant; clarifying and filtering the supernatant to obtain bacterial liquid B;

S3, SPFF column chromatography

Performing primary purification on the bacterial liquid B obtained in the step S2 by adopting GE SPFF filling chromatographic columns to obtain SPFF eluent;

s4, SPHP column chromatography

Performing moderate purification on the SPFF eluent obtained in the step S3 by adopting GE SPHP packing to fill a chromatographic column to obtain the SPHP eluent;

s5, phenyl HP column chromatography

E1, diluting the SPHP eluent with an ammonium sulfate solution; the SPHP eluent comprises the following components in percentage by volume: ammonium sulfate solution = 1:0.5 to 5.0; the ammonium sulfate solution was 20mM PB+3M (NH) ₄ ） ₂ SO ₄ The pH is 6.0; obtaining the dilution of the SPHP eluentPurifying the mixture in the next step;

e2, filling a chromatographic column with GE phenyl HP filler, and carrying out fine purification on the dilution of the SPHP eluent to obtain phenyl HP eluent;

s6, G25 column chromatography

Desalting and changing the phenyl HP eluent by adopting a G25 chromatographic column to obtain a G25 chromatographic liquid;

s7, Q HP column chromatography

Carrying out chromatography on the G25 chromatographic liquid by adopting a Q HP chromatographic column to obtain a Q HP flow-through liquid;

s8, preparing a stock solution

Sterilizing and filtering the Q HP flow penetrating fluid in a biosafety cabinet or a sterile isolator to obtain mSEB protein stock solution;

the method also comprises protein renaturation before the step S5, wherein the protein renaturation method comprises the steps of carrying out SPFF column chromatography column renaturation in the step S3 or carrying out protease cofactor method renaturation in the step S4;

When the PFF column chromatography column renaturation is performed in step S3,

(1) the step S3 specifically comprises the following steps:

chromatography column: column height 18-20 cm, column diameter 200mm, column volume 5.5-6.5L;

mobile phase a:20mM PB, pH6.0;

mobile phase B:20mM PB+5mM GSH+0.5mM GSSG,pH6.0;

mobile phase C:20mM PB+1M NaCl,pH6.0;

loading parameters:

sample loading flow rate: 36-72L/h; ultraviolet detection wavelength: UV280 nm; after loading, the column is equilibrated by using a mobile phase A;

complex balance 1 parameters:

flow rate: performing on-column renaturation by using a mobile phase B after the baseline is leveled by re-balancing 2-3 column volumes at 36-72L/h;

flow rate: 3.6-7.2L/h, and carrying out on-column renaturation for 4-20h; after the next day renaturation was completed, the column was equilibrated with mobile phase a;

complex equilibrium 2 parameters:

flow rate: 36-72L/h, re-balancing 1-2 column volumes, and starting to elute when the base line is leveled;

elution parameters: the flow rate is 36-72L/h; the gradient is: a: c=0-50%, 10 column volumes;

detecting a UV280 absorption spectrum in the elution process, wherein the initial collection UV value (mAU) is more than 600mAU; ending the collection of the UV value (mAU) less than 700mAU to obtain the SPFF eluent;

(2) the step S4 specifically comprises the following steps:

d1, diluting the SPFF eluent obtained in the step S3 by 2-5 times to obtain an SPFF eluent diluent; controlling the conductivity of the dilution liquid of the SPFF eluent to be 2.0-8.0 ms/cm, centrifuging the dilution liquid of the SPFF eluent by using a high-speed refrigerated centrifuge, and centrifuging parameters: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

D2, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d3, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent to obtain the SPHP eluent;

when the protease cofactor method renaturation is performed in step S4,

(1) the step S3 specifically comprises the following steps:

when primary purification is performed by SPFF column chromatography:

chromatography column: column height 18-20 cm, column diameter 200mm, column volume 5.5-6.5L;

buffer solution:

SPFF balance liquid: PB having a pH of 6.0 to 6.5 and a pH of 20 to 50 mM;

SPFF eluent: 20mM PB+1M NaCl, pH 6.0;

sample loading flow rate: 36-72L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 36-72L/h, balance volume: 5-8 CV to UV value and stable electric conduction;

eluting: the elution flow rate is 36-72L/h, the elution gradient is 0-50% B,10CV;

collection criteria: collecting peaks when UV280 nm=200-600 mAU, and stopping collecting when UV280 nm=700-200 mAU to obtain SPFF eluent;

(2) the step S4 specifically comprises the following steps:

d1, step S3 is performed to obtainDiluting the SPFF eluent by 2 to 5 times to obtain the SPFF eluent diluent; controlling the conductivity of the dilution of the SPFF eluent to be 2.0-8.0 ms/cm, adding CuSO with the final concentration of 1-50 mu mM ₄ Stirring the solution at a rotating speed of 50-100 RPM, and preserving heat for 4-20 hours at a temperature of 2-8 ℃;

d2, centrifuging the dilution of the SPFF eluent by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

d3, performing bacteria reduction filtration on the supernatant by adopting a filter membrane of 0.22 mu m, and purifying by a column;

d4, filling a chromatographic column with GE SPHP filler, and performing moderate purification on the dilution of the SPFF eluent,

chromatography column: column height 18-20 cm, column diameter 200mm, column volume 5.5-6.5L;

buffer solution:

SPHP balancing solution: PB having a pH of 6.0 to 6.5 and a pH of 20 to 50 mM;

SPHP eluate: 20mM PB+1M NaCl, the pH of which is between 6.0 and 6.5;

sample loading flow rate: 12-24L/h;

complex balance: using a balance buffer solution to re-balance the chromatographic column, wherein the flow rate is 12-24L/h; balance volume: 2-5 CV to UV value and stable electric conduction;

eluting: the elution flow rate is 12-24L/h, the elution gradient is 0-50% B,10CV;

collection criteria: peak collection was started when UV280 nm=200-600 mAU and stopped when UV280 nm=1000-200 mAU, resulting in SPHP eluate.

2. The method for purifying mSEB antigen protein as claimed in claim 1, wherein in the step S1, the cell disruption is specifically:

A1, dissolving mSEB thallus, wherein the mass volume ratio of the mSEB thallus is as follows: cell lysis solution = 1: 15-25, dissolving mSEB bacteria at a temperature not exceeding 20 ℃;

crushing the mSEB thalli, and crushing the mSEB thalli for 2 to 4 times by using a homogenizer at the temperature of not more than 20 ℃ and the pressure of 700 to 800bar after the mSEB thalli are dissolved to obtain bacterial liquid A.

3. The method for purifying an mSEB antigen protein as claimed in claim 2, wherein in the step A1, the cell lysis solution is 20 to 50mM PB and has a pH of 6.0 to 6.5.

4. The method for purifying mSEB antigen protein as claimed in claim 1, wherein in step S2, the clarification filtration is specifically:

b1, centrifuging the bacterial liquid A in the step S1 by using a high-speed refrigerated centrifuge, wherein the centrifugation parameters are as follows: 12000-15000 g, centrifuging for 20-30 min at 2-8 ℃, and collecting supernatant;

b2, clarifying and filtering the supernatant collected in the step B1 by using a Cobetter 0.6-0.8 mu m or Pall PDH4 deep layer filter plate.

5. The method for purifying mSEB antigen protein according to claim 1, wherein in the step S5, when fine purification is performed by Phenyl HP column chromatography,

chromatography column: the column height is 9-11 cm, the diameter of the column is 140mm, and the column volume is 1-2L;

Buffer solution:

phenyl HP equilibration: 20 to 50mM PB+0.25 to 2.5M (NH) ₄ ） ₂ SO ₄ The pH value is 6.0-6.5;

phenyl HP eluate: PB having a pH of 6.0 to 6.5 and a pH of 20 to 50 mM;

sample loading flow rate: 12-24L/h;

complex balance: the column was equilibrated again with equilibration buffer, equilibration flow rate: 12-24L/h, balance volume: 3-5 CV to UV value, pH value and stable electric conductivity;

eluting: elution flow rate: 12-24L/h, elution gradient: 0-100% B,10CV;

sample collection: collecting the eluent, wherein the UV280 nm=500-1500 mAU starts to be collected, and stopping collecting when the UV280 nm=1000-200 mAU is used for obtaining the phenyl HP eluent.

6. The method for purifying an mSEB antigen protein according to claim 1, wherein in the step S6, when desalting and liquid exchange are performed by G25 column chromatography,

chromatography column: the column height is 28-30 cm, the column diameter is 300mm, and the column volume is 20-22L;

balancing solution: 10-50 mML-His+0.9-1.5% NaCl, and the pH value is 6.0-6.5;

sample loading flow rate: 50-80L/h, rt=15 min;

the sample loading amount is less than or equal to 30% of the volume of the column bed;

detecting the absorption of the eluent UV280, starting to collect when the UV280nm is more than or equal to 50mAU, and stopping collecting when the UV280nm is less than or equal to 50mAU to obtain G25 chromatographic liquid; if the chromatography is performed in separate steps, the eluates are combined each time.

7. The method for purifying an mSEB antigen protein according to claim 1, wherein in the step S7, when the Q HP column chromatography is performed,

chromatography column: column height 9-11 cm, column diameter 140mm, column volume 1-2L;

balancing solution: 10-50 mM L-His+0.9% -1.5% NaCl, the pH is 6.0-6.5;

sample loading flow rate: 12-24L/h;

collecting fluid penetration, and collecting standard: collecting is started when the UV280nm is more than or equal to 50mAU, and collecting is stopped when the UV280nm is less than or equal to 50mAU, so that the Q HP flow-through liquid is obtained.

8. The method for purifying an mSEB antigen protein as claimed in any one of claims 1 to 7, wherein in step S8, a 0.22 μm filter is used for the sterilization filtration.

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