CN101638427A - Method for purifying virus antigens - Google Patents
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- CN101638427A CN101638427A CN 200810043681 CN200810043681A CN101638427A CN 101638427 A CN101638427 A CN 101638427A CN 200810043681 CN200810043681 CN 200810043681 CN 200810043681 A CN200810043681 A CN 200810043681A CN 101638427 A CN101638427 A CN 101638427A
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Abstract
The invention belongs to the technical field of biology and particularly relates to a method for purifying virus antigens, which is characterized in that the purification method comprises the step ofanion exchange chromatographic column purification. The purification method has the advantages of realizing the purification of the virus antigens, greatly reducing the content of impurity protein andresidual DNA of a finished product of a vaccine and producing virus antigens having good immunogenicity and high recovery rate and greatly improving the safety of the finished product of the vaccineunder a production scale, along with mild process condition, good repetitiveness and suitability for large-scale industrial production.
Description
Technical field
The invention belongs to biological technical field, be specifically related to a kind of virus antigen purification process.
Background technology
Because popularity rate height, the consumption of vaccine for man are big, and use object mostly to be infant and children, so all there are strict requirement in the World Health Organization and various countries' medication management department to the amount of residual foreign protein and residual DNA in the vaccine.
Comprise that at the rough vaccine of tradition on the basis of complete malicious vaccine, the purified virus composition prepares purified vaccine, has become the inexorable trend of Future Development.The purifying of virus antigen is a key issue in vaccine research and the production.Vaccine separation and purification research is in the ascendant, both at home and abroad about the also corresponding cumulative year after year with patent of the paper of vaccine and separation and purification thereof.
Should select different separation and purification routes for use at different vaccine products, but generally speaking, all comprise two root phases: just separate and the purifying (see figure 1).Just the main task of separation phase is isolated cell and nutrient solution, smudge cells releasing product (if product is in cell), enriched product and the most of impurity of removal etc., this stage, available separation method comprised cell breaking technology, centrifugal settling and various intermediate processings etc., purification phase is then selected for use variously has high-resolution technology so that product and small amount of interference impurity separate as far as possible, reach required quality standard, ultracentrifugation technology and various chromatographic technique become the current main method that reaches this purpose.
Inactivated vaccine or attenuated vaccine derive from through attenuation or inactivation technology and live pathogen, perhaps from infected pathogenic agent, separate the communicable virion of not tool that obtains.This vaccine is made up of full particle thalline or virus, its pure system process is simply many with respect to the production technique of some subunit vaccines, recombinant vaccine, mainly be to remove viral nucleic acid and foreign protein, generally only relate to simple separation, take physics such as continuously centrifuged, precipitation, filtration or extraction, chemical process to purify in early days mostly., process economics simple to operate and characteristics such as be easy to amplify and certain reasons such as security record are arranged owing to having, continuous-flow centrifugation, precipitation and filtering technique were still generally adopted in the separating of vaccine, but generally all there is purity, vigor and security shortcoming on the low side in the sort of in the past inactivated vaccine and the attenuated vaccine that is obtained by simple separation, is difficult to pass through the approval of medication management mechanism again.
Ripe day by day along with chromatographic technique, though precipitation, centrifugal and filtering technique in the separation and purification process of vaccine, still be widely used, more just as initial step, be used for sepn process just.Chromatographic technique because of its mild condition, good reproducibility, be convenient to industrialization, chromatographic technique substitutes precipitation and conventional art such as centrifugal gradually and also combines with it and become the main flow of vaccine separation and purification.
But the chromatographic separation purifying process of being set up in the existing document also is trapped in laboratory stage mostly, and the purifying process that is adapted to scale operation is still waiting research and development; The complicacy of the route of purification technique often that the highly purified productive target of while novel vaccine brings, this will certainly have a negative impact to the rate of recovery and the production high efficiency of active vaccine.Therefore, need develop be adapted to scale operation, the high purity virus antigen purifying process
Summary of the invention
The purpose of this invention is to provide the good virus antigen purifying novel method of a kind of purification effect, and processing condition gentleness, good reproducibility, be convenient to industrialization.
For this reason, the invention discloses a kind of virus antigen purification process, it is characterized in that described purification process comprises the anion-exchange column purification step.
Innovative point of the present invention is to have adopted in the purification process anion-exchange column purification step, and the anionite-exchange resin in the anion-exchange column is as purification media, and the characteristic that it has high absorption carrying capacity belongs to the sepharose ion-exchanger.This medium has the resolving power height when isolated protein, simple to operate, advantages such as good reproducibility.Its principle is: because protein molecule to be separated can have the electric charge of different attribute respectively under different pH, thereby between as the elutriant of the ion-exchanger of stationary phase and moving phase reversible exchange takes place, by regulating the pH value of elutriant, or change method such as ionic concn, make protein molecule the attached reversibility of adsorption and desorption take place and change, reach and separate different albumen purposes on the stationary phase surface.Under certain pH environment, viral protein and foreign protein all can have negative charge, easily be adsorbed onto on the anionite, by changing the pH and/or the ionic strength of elute soln, viral protein and foreign protein quilt be wash-out successively, reach and comprise host cell proteins in the further isolated viral stoste (Host Cell Protein is HCP) in the interior remaining foreign protein and the purpose of residual DNA.
In an embodiment, described virus antigen can be comprise hepatitis A virus, encephalitis b virus, poliovirus, rabies virus, epidemic hemorrhagic fever, influenza virus, hepatitis B virus surface antigen, rotavirus or human papillomavirus proteantigen in one of, wherein preferably comprise hepatitis A virus virus antigen;
Virus mentioned above, as hepatitis A virus, encephalitis b virus, poliovirus, rabies virus, epidemic hemorrhagic fever, influenza virus, hepatitis B virus surface antigen, rotavirus or human papillomavirus, can carry out purifying before deactivation or after the deactivation, preferably behind purifying, carry out inactivation of virus.
In an embodiment, a kind of virus antigen purification process is characterized in that described purification phase comprises the following steps:
A) hydrophobic chromatography purifying;
B) concentrate eluant;
C) sieve chromatography;
D) anionresin column purification;
E) fraction collection albumen elution peak;
Wherein, in an embodiment, described hydrophobic chromatography purification step is that virus stock solution used adsorbs through drainage column, absorption finishes with 1.0mol/L PB (behind the solution drip washing 2-5CV (column volume) of pH6.4～pH7.8), (pH6.4～pH7.8) solution carries out linear elution 2-8CV, collects the albumen elution peak that contains the purpose virus antigen with 0.02mol/LPB; Wherein, described drainage column one of is preferably in Phenyl Sepharose, Octyl Sepharose or the Butyl Sepharose type organophilic gel post.
In an embodiment, described sieve chromatography step is splined on molecular sieve gel for the liquid with concentration, and applied sample amount is 1-15%CV (column volume), carries out wash-out with PBS solution, collects the albumen elution peak that contains the purpose virus antigen; Wherein, the preferred Sepharose type of described molecular sieve gel molecular sieve gel.
In an embodiment, described anion-exchange column purification step is that the albumen elutriant that gained contains the purpose virus antigen is splined on anion-exchange column, after the drip washing of 0.01mol/L PB pH7.4 solution, carry out linear elution with the 0.01mol/L PBpH5.8 that contains 0.5～2.0mol/LNaCl～7.4 solution, collect the albumen elution peak that contains the purpose virus antigen.
In an embodiment, described anionresin column packing is one of in Capto Q, Q Sepharose, SOURCE Q, DEAESepharose or the DEAE Sephadex type negatively charged ion gel.
In an embodiment, described virus antigen stock can concentrate with the ultra-filtration membrane of MWCO=100～300KD before consummateization.
In an embodiment, above-mentioned virus antigen purification process is characterized in that described virus antigen is a hepatitis A viral antigen.
Personnel are known as the present technique field, and virus antigen purification process disclosed in this invention also comprises and adopts tissue culture technique or genetic engineering technique to express, gather in the crops virus antigen; Just separate; The purification phase step.
In an embodiment, above-mentioned virus antigen purification process also comprises separation phase step just, and it is selected from isoelectric point precipitation, salting-out process or method of organic solvent extraction, preferred method of organic solvent extraction.
In one embodiment, described method of organic solvent extraction is that the enchylema after the fragmentation adds and isopyknic trichloromethane, and jolting 20 minutes with centrifugal 20 minutes of 2～8 ℃ of the speed of 4000rpm, is drawn upper strata albumen water.
In the above-mentioned purge process, drip washing or eluting liquid volume can be according to different virus or different in addition variation and the adjustment of expressing culture systems, this be those skilled in the art the technology general knowledge that should possess.
The advantage of the method disclosed in the present is under industrial scale, realized purifying to virus antigen, impurity albumen and residual DNA content in the vaccine finished product reduce greatly, and the virus antigen immunogenicity of being produced is good, the rate of recovery is higher, have improved the security of vaccine finished product widely; Simultaneously present method processing condition gentleness, good reproducibility, be convenient to industrialization production on a large scale.
Description of drawings
The purifying schema of Fig. 1 virus antigen.
Fig. 2 Phenyl Sepharose 6 Fast Flow organophilic gel purifying color atlass.
Fig. 3 Sepharose 4 Fast Flow molecular sieve gel chromatography color atlass.
Fig. 4 Q Sepharose Fast Flow type ion column purifying color atlas.
Fig. 5 SDS-PAGE electrophorogram.
Embodiment
Below in conjunction with specific embodiment, further illustrate the present invention.Should be understood that these embodiment only to be used to the present invention is described and be not used in and limit the scope of the invention.The experimental technique of unreceipted actual conditions in the following example, usually according to normal condition, or the condition of advising according to manufacturer.Ratio and per-cent are based on weight, unless stated otherwise.
Material source:
Vero cell work seed cell: the ATCC series number is No:CCL-81
YN-5 strain HAV: preserving number is CCTCC NO:V200104 China typical culture collection center
Major equipment and reagent:
The cell Ultrasonic Cell Disruptor: SONICS VCF1500, peak power output is 1500W
Ultra-fine filter: Pellicon 2 Cassette Filter; Biomax-100A (MWCO=100KD) (Millipore company);
Ultrafiltration and concentration device, (MWCO=100KD) (Millipore company);
The chromatogram purification instrument:
Pilot (GE Healthcare company)
Chromatographic column: BPG 140/950; INdEX 140/500 (GE Healthcare company)
Purification media: Phenyl Sepharose, Octyl Sepharose, Butyl Sepharose type organophilic gel (GE Healthcare company);
Sepharose 4 Fast Flow type molecular sieve gels; Capto Q, Q Sepharose, SOURCE Q, DEAE Sepharose or DEAE Sephadex type negatively charged ion gel; (GE Healthcare company)
Calf serum is available from Lanzhou people's marine life technology company limited; Cell culture fluid is 199 substratum (GIBCO);
Embodiment 1: hepatitis A inactivated vaccine (Vero cell) virus antigen stock and finished product preparation
Produce hepatitis A inactivated vaccine (Vero cell) virus antigen stock and finished product with reference to the description in the description among the China Patent No. ZL 0210685.9.
The virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in the working cardial cell storehouse, seed cells in the Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition that contain 10% calf serum.
B. cell amplification and cultivation: treat that the recovery cell grows into behind the fine and close individual layer with the amplification of going down to posterity of trypsinase or other suitable Digestive system digestion Veto cell, put 35 ± 1 ℃ and cultivate and gathered in the crops in 21～28 days.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in the Vero cell.
D. connect the results and the merging of poison cell: the cell that will cultivate 21～28 days digests with trypsinase or other suitable Digestive systems, the many bottles of cell harvesting liquid that same cell dissociation is criticized, with per minute 3000～4000 change, 2～8 ℃ centrifugal 30 minutes, the collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With the cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after the fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, and centrifugal 20 minutes of 2～8 ℃ of speed of changeing with per minute 4000 are drawn upper strata albumen water, and the 0.01mol/L PBS (pH7.4) that replenishes equivalent in Centrifuge Cup extracts 3～5 times repeatedly.
Merge all extracts, concentrate with the ultra-filtration membrane of MWCO=100KD; Add the PB buffering salt in the ultrafiltration and concentration liquid, and buffering salt is fully dissolved, adsorb through Phenyl Sepharose 6 Fast Flow type organophilic gels, absorption finishes with after the drip washing of 1.0mol/L PB (pH7.4) solution, begin to carry out linear elution, collect the elutriant that electric conductivity value is lower than 50mS/cm with 0.02mol/L PB (pH7.4) solution; The elutriant of collecting concentrates with the ultra-filtration membrane of MWCO=100KD, (this volume is according to the viscosity situation of concentrated solution to be concentrated into proper volume, generally be controlled at column volume 5% in) after be splined on Sepharose 4 Fast Flow type molecular sieve gels, carry out wash-out with 0.01mol/L PBS (pH7.4) solution, collect second elution peak; The elutriant of collecting is replaced as 0.01mol/L PBS (pH7.4) solution with the ultrafiltration and concentration device with damping fluid, finishes the virus antigen stock preparation.
Hepatitis A inactivated vaccine (Veto cell) finished product production method is as follows:
A) formaldehyde treated: it was 1: 4000 formaldehyde that above-mentioned virus antigen stock is added final concentration, through deactivation in 37 ± 1 ℃, 12 days.
B) ultrafiltration and concentration gets product.
Embodiment 2: hepatitis A inactivated vaccine (Vero cell) virus antigen stock and finished product preparation
The virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in the working cardial cell storehouse, seed cells in the Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition that contain 10% calf serum.
B. cell amplification and cultivation: treat that the recovery cell grows into behind the fine and close individual layer with the amplification of going down to posterity of trypsinase or other suitable Digestive system digestion Vero cell, put 35 ± 1 ℃ and cultivate and gathered in the crops in 21～28 days.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in the Vero cell.
D. connect the results and the merging of poison cell: the cell that will cultivate 21～28 days digests with trypsinase or other suitable Digestive systems, the many bottles of cell harvesting liquid that same cell dissociation is criticized, with per minute 3000～4000 change, 2～8 ℃ centrifugal 30 minutes, the collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With the cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after the fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, and centrifugal 20 minutes of 2～8 ℃ of speed of changeing with per minute 4000 are drawn upper strata albumen water, and the 0.01mol/L PBS (pH7.4) that replenishes equivalent in Centrifuge Cup extracts 3～5 times repeatedly.
Merge all extracts, concentrate with the ultra-filtration membrane of MWCO=100KD; Add the PB buffering salt in the ultrafiltration and concentration liquid, and buffering salt is fully dissolved, adsorb through Phenyl Sepharose 6 Fast Flow type organophilic gels again, absorption finishes with after the drip washing of 1.0mol/L PB (pH7.4) solution, begin to carry out linear elution, collect the elutriant (referring to Fig. 2) that electric conductivity value is lower than 50mS/cm with 0.02mol/L PB (pH7.4) solution; The elutriant of collecting concentrates with the ultra-filtration membrane of MWCO=100KD, (this volume is according to the viscosity situation of concentrated solution to be concentrated into proper volume, generally be controlled at column volume 5% in) after be splined on Sepharose 4 Fast Flow type molecular sieve gels, carry out wash-out with 0.01mol/L PBS (pH7.4) solution, collect second elution peak (referring to Fig. 3); Should collect liquid and be splined on Q Sepharose Fast Flow type anion column, after the drip washing of 0.01mol/LPB (pH7.4) solution, begin to carry out linear elution, collect first elution peak (referring to Fig. 4) with 0.01mol/L PB (pH 7.4) solution that contains 0.6mol/L NaCl.The elutriant of collecting is replaced as 0.01mol/LPBS (pH7.4) solution with the ultrafiltration and concentration device with damping fluid, promptly gets virus antigen stock.
Hepatitis A inactivated vaccine (Vero cell) finished product production method is as follows:
A) formaldehyde treated: it was 1: 4000 formaldehyde that above-mentioned virus antigen stock is added final concentration, through deactivation in 37 ± 1 ℃, 12 days;
B) ultrafiltration and concentration gets product.
Embodiment 3: the preparation of hepatitis A inactivated vaccine (Vero cell) virus antigen stock
The virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in the working cardial cell storehouse, seed cells in the Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition that contain 10% calf serum.
B. cell amplification and cultivation: treat that the recovery cell grows into behind the fine and close individual layer with the amplification of going down to posterity of trypsinase or other suitable Digestive system digestion Vero cell, put 35 ± 1 ℃ and cultivate and gathered in the crops in 21～28 days.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in the Vero cell.
D. connect the results and the merging of poison cell: the cell that will cultivate 21～28 days digests with trypsinase or other suitable Digestive systems, the many bottles of cell harvesting liquid that same cell dissociation is criticized, with per minute 3000～4000 change, 2～8 ℃ centrifugal 30 minutes, the collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With the cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after the fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, and centrifugal 20 minutes of 2～8 ℃ of speed of changeing with per minute 4000 are drawn upper strata albumen water, and the 0.01mol/L PBS (pH7.4) that replenishes equivalent in Centrifuge Cup extracts 3～5 times repeatedly.
Merge all extracts, concentrate with the ultra-filtration membrane of MWCO=100KD; Add the PB buffering salt in the ultrafiltration and concentration liquid, and buffering salt is fully dissolved, adsorb through Octyl Sepharose 4 Fast Flow type organophilic gels again, absorption finishes with after the drip washing of 1.0mol/L PB (pH7.4) solution, begin to carry out linear elution, collect the elutriant that electric conductivity value is lower than 58mS/cm with 0.02mol/L PB (pH7.4) solution; The elutriant of collecting concentrates with the ultra-filtration membrane of MWCO=100KD, (this volume is according to the viscosity situation of concentrated solution to be concentrated into proper volume, generally be controlled at column volume 5% in) after be splined on Sepharose 4 Fast Flow type molecular sieve gels, carry out wash-out with 0.01mol/L PBS (pH7.4) solution, collect second elution peak; Should collect liquid and be splined on SOURCE 30Q type anion column, and after the drip washing of 0.01mol/L PB (pH7.4) solution, begin to carry out linear elution, collect first elution peak with 0.01mol/L PB (pH7.4) solution that contains 0.6mol/LNaCl.The elutriant of collecting is replaced as 0.01mol/L PBS (pH7.4) solution with the ultrafiltration and concentration device with damping fluid, promptly gets virus antigen stock.
Embodiment 4: the preparation of hepatitis A inactivated vaccine (Vero cell) virus antigen stock
The virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in the working cardial cell storehouse, seed cells in the Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition that contain 10% calf serum.
B. cell amplification and cultivation: treat that the recovery cell grows into behind the fine and close individual layer with the amplification of going down to posterity of trypsinase or other suitable Digestive system digestion Vero cell, put 35 ± 1 ℃ and cultivate and gathered in the crops in 21～28 days.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in the Vero cell.
D. connect the results and the merging of poison cell: the cell that will cultivate 21～28 days digests with trypsinase or other suitable Digestive systems, the many bottles of cell harvesting liquid that same cell dissociation is criticized, with per minute 3000～4000 change, 2～8 ℃ centrifugal 30 minutes, the collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With the cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after the fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, and centrifugal 20 minutes of 2～8 ℃ of speed of changeing with per minute 4000 are drawn upper strata albumen water, and the 0.01mol/L PBS (pH7.4) that replenishes equivalent in Centrifuge Cup extracts 3～5 times repeatedly.
Merge all extracts, concentrate with the ultra-filtration membrane of MWCO=100KD; Add the PB buffering salt in the ultrafiltration and concentration liquid, and buffering salt is fully dissolved, adsorb through Butyl Sepharose Fast Flow type organophilic gel again, absorption finishes with after the drip washing of 1.0mol/L PB (pH7.2) solution, begin to carry out linear elution, collect the elutriant that electric conductivity value is lower than 61mS/cm with 0.02mol/L PB (pH7.2) solution; The elutriant of collecting concentrates with the ultra-filtration membrane of MWCO=100KD, (this volume is according to the viscosity situation of concentrated solution to be concentrated into proper volume, generally be controlled at column volume 5% in) after be splined on Sepharose 4 Fast Flow type molecular sieve gels, carry out wash-out with 0.01mol/L PBS (pH7.4) solution, collect second elution peak; Should collect liquid and be splined on DEAE Sephadex A-50 type anion column, and after the drip washing of 0.01mol/L PB (pH7.4) solution, begin to carry out linear elution, collect first elution peak with 0.01mol/L PB (pH6.0) solution that contains 0.8mol/LNaCl.The elutriant of collecting is replaced as 0.01mol/L PBS (pH7.4) solution with the ultrafiltration and concentration device with damping fluid, promptly gets virus antigen stock.
Embodiment 5: the preparation of hepatitis A inactivated vaccine (Vero cell) virus antigen stock
The virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in the working cardial cell storehouse, seed cells in the Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition that contain 10% calf serum.
B. cell amplification and cultivation: treat that the recovery cell grows into behind the fine and close individual layer with the amplification of going down to posterity of trypsinase or other suitable Digestive system digestion Vero cell, put 35 ± 1 ℃ and cultivate and gathered in the crops in 21～28 days.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in the Vero cell.
D. connect the results and the merging of poison cell: the cell that will cultivate 21～28 days digests with trypsinase or other suitable Digestive systems, the many bottles of cell harvesting liquid that same cell dissociation is criticized, with per minute 3000～4000 change, 2～8 ℃ centrifugal 30 minutes, the collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With the cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after the fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, and centrifugal 20 minutes of 2～8 ℃ of speed of changeing with per minute 4000 are drawn upper strata albumen water, and the 0.01mol/L PBS (pH7.4) that replenishes equivalent in Centrifuge Cup extracts 3～5 times repeatedly.
Merge all extracts, concentrate with the ultra-filtration membrane of MWCO=100KD; Ultrafiltration and concentration liquid adsorbs through Capto Q type negatively charged ion glue, absorption finishes with after the drip washing of 0.02mol/L PB (pH7.4) solution, carry out linear elution with the 0.01mol/LPB that contains 2.0mol/LNaCl (pH7.4) solution, collect the elutriant that contains the hepatitis A virus (HAV) antigen protein; The elutriant of collecting adds the PB buffering salt, and buffering salt is fully dissolved, adsorb through Phenyl Sepharose 6 Fast Flow type organophilic gels, absorption finishes with after the drip washing of 1.0mol/L PB (pH7.4) solution, begin to carry out linear elution, collect the elutriant that electric conductivity value is lower than 50mS/cm with 0.02mol/L PB (pH7.4) solution; The elutriant of collecting is concentrated into proper volume, and (this volume is according to the viscosity situation of concentrated solution, generally be controlled at column volume 5% in) after be splined on Sepharose 4Fast Flow type molecular sieve gel, carry out wash-out with 0.01mol/L PBS (pH7.4) solution, collect second elution peak; The elutriant of collecting is replaced as 0.01mol/L PBS (pH7.4) solution with the ultrafiltration and concentration device with damping fluid, promptly gets virus antigen stock.
Embodiment 6
Method routinely and commercially available reagent carry out total protein concentration, antigen titre, residual DNA content, residual bovine serum albumin content, HCP content detection respectively to the virus antigen stock (abbreviation antigen stock) and the finished product of embodiment 1～5 gained;
Result such as table 1.
Table 1. related substances content detection result
Table 2: antigen relative recovery cartogram
From table 1 and table 2 as can be known, compare with existing method (embodiment 1), adopt the residual DNA content of hepatitis A inactivated vaccine (Vero cell) finished product of the method for the invention production, residual bovine serum albumin content, HCP content all to reduce greatly, and antigen titre does not reduce, and its antigen rate of recovery does not reduce significantly yet.
Embodiment 7SDS-PAGE electrophoresis
Hepatitis A inactivated vaccine (Vero cell) virus antigen of embodiment 1 and 2 prepared purifying is carried out the SDS-PAGE electrophoresis, and silver dyes then.
The result is referring to Fig. 4, and 1: standard molecular weight albumen; 2: the hepatitis A inactivated vaccine of embodiment 1 prepared purifying (Vero cell) virus antigen; 3: the hepatitis A inactivated vaccine of embodiment 2 prepared purifying (Vero cell) virus antigen.The result shows: compare with the chromatography purification of routine, the component and the content of residual protein reduce greatly in hepatitis A inactivated vaccine (Vero cell) virus antigen that process purification process of the present invention obtains.
Embodiment 8 rabies virus antigen purifying
The rabies virus antigen purification process is as follows:
A) Shou Huo inactivation of virus liquid is concentrated into proper volume with the ultra-filtration membrane of MWCO=100KD, with 4 ℃ of deactivations of 1: 1000 beta-propiolactone 24 hours, and 37 ℃ of hydrolysis 2 hours.
B) inactivation of virus liquid is splined on Sepharose 4Fast Flow type molecular sieve gel, and (go up the sample volume be generally column volume 3%) carry out wash-out with 0.01mol/L PBS (pH7.4) solution, collect first elution peak.
C) should collect liquid and be splined on Q Sepharose Fast Flow type anion column, after the drip washing of 0.01mol/L PB (pH7.4) solution, begin to carry out the stage gradient wash-out with 0.01mol/L PB (pH5.8) solution that contains 1.0mol/LNaCl, collection contains the proteic elution peak of virus antigen.
Scope of the present invention is not subjected to the restriction of described specific embodiments, and described embodiment is only desired also to comprise the method and the component of functional equivalent in the scope of the invention as the single example of illustrating all respects of the present invention.In fact, except content as herein described, those skilled in the art can easily grasp multiple improvement of the present invention with reference to above description and accompanying drawing.Described improvement also falls within the scope of appended claims.
Claims (15)
1. a virus antigen purification process is characterized in that, comprises the anion-exchange column purification step in the described purification process.
2. virus antigen purification process according to claim 1, it is characterized in that described virus antigen can be comprise hepatitis A virus, encephalitis b virus, poliovirus, rabies virus, epidemic hemorrhagic fever, influenza virus, hepatitis B virus surface antigen, rotavirus or human papillomavirus proteantigen in one of.
3. virus antigen purification process according to claim 1, the filler that it is characterized in that described anion-exchange column are one of in Capto Q, Q Sepharose, SOURCE Q, DEAE Sepharose or the DEAE Sephadex type negatively charged ion gel.
4. virus antigen purification process according to claim 1 is characterized in that described purification process comprises the following steps:
A) hydrophobic chromatography purifying;
B) concentrate eluant;
C) sieve chromatography;
D) anionresin column purification;
E) fraction collection albumen elution peak.
5. virus antigen purification process according to claim 4 is characterized in that, described hydrophobic chromatography purification step is that virus antigen adsorbs through drainage column, and absorption finishes with pH6.4～7.81.0mol/LPB solution drip washing; PH6.4～7.80.01mol/LPB solution carries out linear elution then, collects the albumen elution peak that contains the purpose virus antigen.
6. virus antigen purification process according to claim 5 is characterized in that, described drainage column is selected from PhenylSepharose, Octyl Sepharose or Butyl Sepharose type organophilic gel post.
7. virus antigen purification process according to claim 4 is characterized in that, described concentrate eluant step adopts the ultra-filtration membrane of MWCO=100～300KD to concentrate.
8. virus antigen purification process according to claim 4, it is characterized in that, described sieve chromatography step is splined on molecular sieve gel for the liquid that the hydrophobic chromatography purifying is collected, applied sample amount is 1-5%CV, carry out wash-out with 0.01mol/L PBS pH6.4～7.4 solution, collect the albumen elution peak that contains the purpose virus antigen.
9. virus antigen purification process according to claim 8 is characterized in that, the preferred Sepharose type of described molecular sieve gel molecular sieve gel.
10. virus antigen purification process according to claim 4, it is characterized in that, described anion-exchange column purification step is that the albumen elutriant that gained contains the purpose virus antigen is splined on anion-exchange column, after 0.01mol/L PB pH 7.4 solution drip washing, carry out linear elution with the pH 5.8～7.40.01mol/L PB solution that contains 0.5～2.0mol/LNaCl, collect the albumen elution peak of purpose virus antigen.
11. virus antigen purification process according to claim 10 is characterized in that, the filler of described anion-exchange column is one of in Capto Q, Q Sepharose, SOURCE Q, DEAE Sepharose or the DEAE Sephadex type negatively charged ion gel.
12. virus antigen purification process according to claim 4 is characterized in that described purification process also comprises separating step just, it is selected from isoelectric point precipitation, salting-out process or method of organic solvent extraction.
13. virus antigen purification process according to claim 12 is characterized in that the described method of separation phase just is a method of organic solvent extraction.
14. virus antigen purification process according to claim 13, it is characterized in that described method of organic solvent extraction is the enchylema adding and isopyknic trichloromethane after the fragmentation, jolting 20 minutes with centrifugal 20 minutes of 2～8 ℃ of the speed of 4000rpm, is drawn upper strata albumen water.
15., it is characterized in that described virus antigen is a hepatitis A viral antigen according to the described virus antigen purification process of claim 4-14.
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