CN101638427B - Method for purifying virus antigens - Google Patents
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- CN101638427B CN101638427B CN200810043681.4A CN200810043681A CN101638427B CN 101638427 B CN101638427 B CN 101638427B CN 200810043681 A CN200810043681 A CN 200810043681A CN 101638427 B CN101638427 B CN 101638427B
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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 of anion 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 and residual 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 vaccine under 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 the popularity rate of vaccine for man is high, consumption is large, 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 vaccine.
At the rough vaccine of tradition, comprise that on the basis of complete malicious vaccine, purified virus composition is prepared purified vaccine, has become the inexorable trend of Future Development.The purifying of virus antigen is a key issue in vaccine research and production.Vaccine separation and purification research is in the ascendant, and the domestic and international paper about vaccine and separation and purification thereof and patent be corresponding cumulative year after year also.
For different vaccine products, should select different separation and purification routes, but generally speaking, all comprise two root phases: first separated and purifying (seeing Fig. 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., the available separation method of this one-phase comprises cell breaking technology, centrifugal settling and various intermediate processings etc., purification phase is selected variously has high-resolution technology so that product and a small amount of impurity that disturbs separate as far as possible, reach required quality standard, ultracentrifugation technology and various chromatographic technique become the current main method that reaches this object.
Inactivated vaccine or attenuated vaccine derive from through attenuation or inactivation technology and live pathogen, or from infected pathogenic agent the separated communicable virion of not tool obtaining.This vaccine is comprised of full particle thalline or virus, its pure process processed 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, mostly take in early days the physics such as continuously centrifuged, precipitation, filtration or extraction, chemical process to purify., process economics simple to operate owing to having and be easy to the features such as amplification and have certain reasons such as security record, continuous-flow centrifugation, precipitation and filtering technique are still generally adopted in vaccine separated, but that inactivated vaccine being obtained by simple separation and attenuated vaccine generally all existed purity, vigor and security shortcoming on the low side in the past, be difficult to again by the approval of medication management mechanism.
Ripe day by day along with chromatographic technique, though precipitation, centrifugal and filtering technique in the separation and purification process of vaccine, be still widely used, more just as initial step, for first sepn process.Chromatographic technique because of its mild condition, reproducible, be convenient to industrialization, chromatographic technique substitutes gradually precipitation and the conventional art such as centrifugal also combines with it and becomes the main flow of vaccine separation and purification.
But the chromatographic separation purifying process of setting up in existing document is also trapped in laboratory stage mostly, the purifying process that is adapted to scale operation need research and development; The complicacy of the route of purification technique often that the highly purified productive target of novel vaccine brings simultaneously, this will certainly have a negative impact to the rate of recovery of active vaccine and production high efficiency.Therefore, need to develop and be adapted to scale operation, highly purified virus antigen purifying process
Summary of the invention
The object of this invention is to provide the virus antigen purifying novel method that a kind of purification effect is good, and processing condition gentle, reproducible, 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 anion-exchange column purification step.
Innovative point of the present invention is to have adopted anion-exchange column purification step in purification process, and the anionite-exchange resin in anion-exchange column is as purification media, and the characteristic that it has high absorption carrying capacity, belongs to sepharose ion-exchanger.The advantages such as it is high that this medium has resolving power when isolated protein, simple to operate, reproducible.Its principle is: due to protein molecule to be separated can be respectively under different pH with the electric charge of different attribute, thereby there is reversible exchange between the ion-exchanger as stationary phase and the elutriant of moving phase, by regulating the pH value of elutriant, or change the methods such as ionic concn, the reversibility that makes protein molecule, on stationary phase surface, adsorption and de-adsorption occur changes, and reaches separated different albumen object.Under certain pH environment, viral protein and foreign protein all can be with negative charges, easily be adsorbed onto on anionite, by changing pH and/or the ionic strength of elute soln, viral protein and foreign protein are by wash-out successively, reach further isolated viral stoste and comprise that host cell proteins (Host Cell Protein, HCP) is in interior remaining foreign protein and the object 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 before deactivation or after deactivation, carry out purifying, preferably after 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, adsorb complete with after 1.0mol/L PB (pH6.4～pH7.8) solution drip washing 2-5CV (column volume), with 0.02mol/LPB (pH6.4～pH7.8) solution, carry out linear elution 2-8CV, collect the albumen elution peak containing object virus antigen; Wherein, described drainage column one of is preferably in Phenyl Sepharose, Octyl Sepharose or Butyl Sepharose type organophilic gel post.
In an embodiment, described sieve chromatography step is for to be splined on molecular sieve gel by the liquid of concentration, and applied sample amount is 1-15%CV (column volume), with PBS solution, carries out wash-out, collects the albumen elution peak that contains object 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 object virus antigen is splined on anion-exchange column, with after the drip washing of 0.01mol/L PB pH7.4 solution, with 0.01mol/L PBpH5.8～7.4 solution containing 0.5～2.0mol/LNaCl, carry out linear elution, collect the albumen elution peak containing object virus antigen.
In an embodiment, described anionresin column packing is one of in Capto Q, Q Sepharose, SOURCE Q, DEAESepharose or 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 hepatitis A viral antigen.
As the art, personnel are known, 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 separated; Purification phase step.
In an embodiment, above-mentioned virus antigen purification process also comprises just separation phase step, and it is selected from isoelectric point precipitation, salting-out process or method of organic solvent extraction, preferably method of organic solvent extraction.
In one embodiment, described method of organic solvent extraction is that the enchylema after 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 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, and this is the technology general knowledge that those skilled in the art should possess.
The advantage of the method disclosed in the present is under industrial scale, realized the purifying to virus antigen, impurity albumen and residual DNA content in vaccine finished product reduce greatly, and the virus antigen immunogenicity of producing is good, the rate of recovery is higher, have improved widely the security of vaccine finished product; Simultaneously present method processing condition gentle, reproducible, be convenient to the production of industrialization on a large scale.
Accompanying drawing explanation
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 thin layer chromatography figure.
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 are only not used in and limit the scope of the invention for the present invention is described.The experimental technique of unreceipted actual conditions in the following example, conventionally 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: ATCC series number is No:CCL-81
YN-5 strain HAV: preserving number is CCTCC NO:V200104 Chinese Typical Representative culture collection center
Major equipment and reagent:
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);
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 purchased 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
With reference to the description in the description in China Patent No. ZL 0210685.9, produce hepatitis A inactivated vaccine (Vero cell) virus antigen stock and finished product.
Virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in working cardial cell storehouse, seed cells in Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition containing 10% calf serum.
B. cell amplification and cultivation: with the amplification of going down to posterity of trypsinase or other suitable Digestive systems digestion Veto cell, put 35 ± 1 ℃ and cultivate and gather in the crops for 21～28 days after recovery Growth of Cells becomes fine and close individual layer.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in to Vero cell.
D. connect results and the merging of poison cell: the cell of cultivating 21～28 days is digested 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 turn, 2～8 ℃ centrifugal 30 minutes, collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, centrifugal 20 minutes of 2～8 ℃ of the speed that turn with per minute 4000, draw upper strata albumen water, to the 0.01mol/L PBS (pH7.4) that supplements equivalent in Centrifuge Cup, repeatedly extract 3～5 times.
Merge all extracts, with the ultra-filtration membrane of MWCO=100KD, concentrate; In ultrafiltration and concentration liquid, add PB buffering salt, and buffering salt is fully dissolved, through Phenyl Sepharose 6 Fast Flow type organophilic gel absorption, adsorb complete with after the drip washing of 1.0mol/L PB (pH7.4) solution, start to carry out linear elution with 0.02mol/L PB (pH7.4) solution, collect electric conductivity value lower than the elutriant of 50mS/cm; 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, general control column volume 5% in) after be splined on Sepharose 4 Fast Flow type molecular sieve gels, with 0.01mol/L PBS (pH7.4) solution, carry out wash-out, collect the second elution peak; The elutriant of collection is replaced as to 0.01mol/L PBS (pH7.4) solution with ultrafiltration and concentration device by damping fluid, completes virus antigen stock preparation.
Hepatitis A inactivated vaccine (Veto cell) method for producing finished product is as follows:
A) formaldehyde treated: it was the formaldehyde of 1: 4000 that above-mentioned virus antigen stock is added to 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
Virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in working cardial cell storehouse, seed cells in Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition containing 10% calf serum.
B. cell amplification and cultivation: with the amplification of going down to posterity of trypsinase or other suitable Digestive systems digestion Vero cell, put 35 ± 1 ℃ and cultivate and gather in the crops for 21～28 days after recovery Growth of Cells becomes fine and close individual layer.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in to Vero cell.
D. connect results and the merging of poison cell: the cell of cultivating 21～28 days is digested 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 turn, 2～8 ℃ centrifugal 30 minutes, collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, centrifugal 20 minutes of 2～8 ℃ of the speed that turn with per minute 4000, draw upper strata albumen water, to the 0.01mol/L PBS (pH7.4) that supplements equivalent in Centrifuge Cup, repeatedly extract 3～5 times.
Merge all extracts, with the ultra-filtration membrane of MWCO=100KD, concentrate; In ultrafiltration and concentration liquid, add PB buffering salt, and buffering salt is fully dissolved, again through Phenyl Sepharose 6 Fast Flow type organophilic gel absorption, adsorb complete with after the drip washing of 1.0mol/L PB (pH7.4) solution, start to carry out linear elution with 0.02mol/L PB (pH7.4) solution, collect electric conductivity value lower than the elutriant (referring to Fig. 2) of 50mS/cm; 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, general control column volume 5% in) after be splined on Sepharose 4 Fast Flow type molecular sieve gels, with 0.01mol/L PBS (pH7.4) solution, carry out wash-out, collect the second elution peak (referring to Fig. 3); This collection liquid is splined on to Q Sepharose Fast Flow type anion column, with after the drip washing of 0.01mol/LPB (pH7.4) solution, start to carry out linear elution with 0.01mol/L PB (pH 7.4) solution containing 0.6mol/L NaCl, collect the first elution peak (referring to Fig. 4).The elutriant of collection is replaced as to 0.01mol/LPBS (pH7.4) solution with ultrafiltration and concentration device by damping fluid, obtains virus antigen stock.
Hepatitis A inactivated vaccine (Vero cell) method for producing finished product is as follows:
A) formaldehyde treated: it was the formaldehyde of 1: 4000 that above-mentioned virus antigen stock is added to 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
Virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in working cardial cell storehouse, seed cells in Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition containing 10% calf serum.
B. cell amplification and cultivation: with the amplification of going down to posterity of trypsinase or other suitable Digestive systems digestion Vero cell, put 35 ± 1 ℃ and cultivate and gather in the crops for 21～28 days after recovery Growth of Cells becomes fine and close individual layer.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in to Vero cell.
D. connect results and the merging of poison cell: the cell of cultivating 21～28 days is digested 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 turn, 2～8 ℃ centrifugal 30 minutes, collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, centrifugal 20 minutes of 2～8 ℃ of the speed that turn with per minute 4000, draw upper strata albumen water, to the 0.01mol/L PBS (pH7.4) that supplements equivalent in Centrifuge Cup, repeatedly extract 3～5 times.
Merge all extracts, with the ultra-filtration membrane of MWCO=100KD, concentrate; In ultrafiltration and concentration liquid, add PB buffering salt, and buffering salt is fully dissolved, again through Octyl Sepharose 4 Fast Flow type organophilic gel absorption, adsorb complete with after the drip washing of 1.0mol/L PB (pH7.4) solution, start to carry out linear elution with 0.02mol/L PB (pH7.4) solution, collect electric conductivity value lower than the elutriant of 58mS/cm; 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, general control column volume 5% in) after be splined on Sepharose 4 Fast Flow type molecular sieve gels, with 0.01mol/L PBS (pH7.4) solution, carry out wash-out, collect the second elution peak; This collection liquid is splined on to SOURCE 30Q type anion column, with after the drip washing of 0.01mol/L PB (pH7.4) solution, starts to carry out linear elution with 0.01mol/L PB (pH7.4) solution containing 0.6mol/LNaCl, collect the first elution peak.The elutriant of collection is replaced as to 0.01mol/L PBS (pH7.4) solution with ultrafiltration and concentration device by damping fluid, obtains virus antigen stock.
Embodiment 4: the preparation of hepatitis A inactivated vaccine (Vero cell) virus antigen stock
Virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in working cardial cell storehouse, seed cells in Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition containing 10% calf serum.
B. cell amplification and cultivation: with the amplification of going down to posterity of trypsinase or other suitable Digestive systems digestion Vero cell, put 35 ± 1 ℃ and cultivate and gather in the crops for 21～28 days after recovery Growth of Cells becomes fine and close individual layer.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in to Vero cell.
D. connect results and the merging of poison cell: the cell of cultivating 21～28 days is digested 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 turn, 2～8 ℃ centrifugal 30 minutes, collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, centrifugal 20 minutes of 2～8 ℃ of the speed that turn with per minute 4000, draw upper strata albumen water, to the 0.01mol/L PBS (pH7.4) that supplements equivalent in Centrifuge Cup, repeatedly extract 3～5 times.
Merge all extracts, with the ultra-filtration membrane of MWCO=100KD, concentrate; In ultrafiltration and concentration liquid, add PB buffering salt, and buffering salt is fully dissolved, through Butyl Sepharose Fast Flow type organophilic gel, adsorb again, adsorb complete with after the drip washing of 1.0mol/L PB (pH7.2) solution, start to carry out linear elution with 0.02mol/L PB (pH7.2) solution, collect electric conductivity value lower than the elutriant of 61mS/cm; 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, general control column volume 5% in) after be splined on Sepharose 4 Fast Flow type molecular sieve gels, with 0.01mol/L PBS (pH7.4) solution, carry out wash-out, collect the second elution peak; This collection liquid is splined on to DEAE Sephadex A-50 type anion column, with after the drip washing of 0.01mol/L PB (pH7.4) solution, start to carry out linear elution with 0.01mol/L PB (pH6.0) solution containing 0.8mol/LNaCl, collect the first elution peak.The elutriant of collection is replaced as to 0.01mol/L PBS (pH7.4) solution with ultrafiltration and concentration device by damping fluid, obtains virus antigen stock.
Embodiment 5: the preparation of hepatitis A inactivated vaccine (Vero cell) virus antigen stock
Virus antigen stock production method is as follows:
A. cell recovery; Get 1 or several cell pipes in working cardial cell storehouse, seed cells in Tissue Culture Flask, add 37 ± 1 ℃ of cultivations of 199 substratum postposition containing 10% calf serum.
B. cell amplification and cultivation: with the amplification of going down to posterity of trypsinase or other suitable Digestive systems digestion Vero cell, put 35 ± 1 ℃ and cultivate and gather in the crops for 21～28 days after recovery Growth of Cells becomes fine and close individual layer.
C. inoculate HAV: YN-5 strain hepatitis A virus (HAV) is inoculated in to Vero cell.
D. connect results and the merging of poison cell: the cell of cultivating 21～28 days is digested 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 turn, 2～8 ℃ centrifugal 30 minutes, collecting cell precipitation is merged into 1 batch under aseptic condition.
E. purifying:
With cell supersonic wave crusher with 1200W～1600W output rating smudge cells.Enchylema after fragmentation adds and isopyknic trichloromethane, jolting 20 minutes, centrifugal 20 minutes of 2～8 ℃ of the speed that turn with per minute 4000, draw upper strata albumen water, to the 0.01mol/L PBS (pH7.4) that supplements equivalent in Centrifuge Cup, repeatedly extract 3～5 times.
Merge all extracts, with the ultra-filtration membrane of MWCO=100KD, concentrate; Ultrafiltration and concentration liquid adsorbs through Capto Q type negatively charged ion glue, adsorb complete with after the drip washing of 0.02mol/L PB (pH7.4) solution, with 0.01mol/LPB (pH7.4) solution containing 2.0mol/LNaCl, carry out linear elution, collect the elutriant containing HAV-Ag albumen; The elutriant of collecting adds PB buffering salt, and buffering salt is fully dissolved, through Phenyl Sepharose 6 Fast Flow type organophilic gel absorption, adsorb complete with after the drip washing of 1.0mol/L PB (pH7.4) solution, start to carry out linear elution with 0.02mol/L PB (pH7.4) solution, collect electric conductivity value lower than the elutriant of 50mS/cm; The elutriant of collecting is concentrated into proper volume, and (this volume is according to the viscosity situation of concentrated solution, general control column volume 5% in) after be splined on Sepharose 4Fast Flow type molecular sieve gel, with 0.01mol/L PBS (pH7.4) solution, carry out wash-out, collect the second elution peak; The elutriant of collection is replaced as to 0.01mol/L PBS (pH7.4) solution with ultrafiltration and concentration device by damping fluid, obtains virus antigen stock.
Embodiment 6
Method routinely and commercially available reagent, carry out respectively total protein concentration, antigen titre, residual DNA content, residual bovine serum albumin content, HCP content detection to the virus antigen stock of embodiment 1～5 gained (abbreviation antigen stock) and finished product;
Result is as table 1.
Table 1. related substances content detection result
Table 2: antigen relative recovery cartogram
From table 1 and table 2, 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 greatly to reduce, and antigen titre does not reduce, its antigen rate of recovery does not reduce significantly yet.
Embodiment 7SDS-PAGE electrophoresis
The hepatitis A inactivated vaccine of embodiment 1 and 2 prepared purifying (Vero cell) virus antigen is carried out to SDS-PAGE electrophoresis, and then silver dyes.
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.Result shows: with conventional chromatography purification comparison, in hepatitis A inactivated vaccine (Vero cell) virus antigen obtaining through purification process of the present invention, component and the content of residual protein reduce greatly.
Embodiment 8 rabies virus antigen purifying
Rabies virus antigen purification process is as follows:
A) the inactivation of virus liquid of results is concentrated into proper volume with the ultra-filtration membrane of MWCO=100KD, and with 4 ℃ of deactivations of the beta-propiolactone of 1: 1,000 24 hours, 37 ℃ were hydrolyzed 2 hours.
B) inactivation of virus liquid is splined on Sepharose 4Fast Flow type molecular sieve gel, and (loading volume be generally column volume 3%) carries out wash-out with 0.01mol/L PBS (pH7.4) solution, collect the first elution peak.
C) this collection liquid is splined on to Q Sepharose Fast Flow type anion column, with after the drip washing of 0.01mol/L PB (pH7.4) solution, start to carry out stage gradient wash-out with 0.01mol/L PB (pH5.8) solution containing 1.0mol/LNaCl, collect the elution peak containing virus antigen albumen.
Scope of the present invention is not subject to the restriction of described specific embodiments, and described embodiment is only wanted, as the single example of illustrating all respects of the present invention, also to comprise method and the component of functional equivalent in the scope of the 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 description and accompanying drawing above.Within described improvement also falls into the scope of appended claims.
Claims (1)
1. a virus antigen purification process, is characterized in that, described purification process comprises the following steps:
A) the vero extraction liquid of cell that hepatitis A virus infects concentrates with the ultra-filtration membrane of MWCO=100KD;
B) step a) prepared ultrafiltration and concentration liquid through Phenyl Sepharose 6 Fast Flow type organophilic gels absorption, adsorb complete with after the drip washing of 1.0mol/L PB pH7.4 solution, with 0.02mol/L PB pH7.4 solution, carry out linear elution again, collect electric conductivity value lower than the elutriant of 50mS/cm;
C) elutriant of step b) collecting concentrates with the ultra-filtration membrane of MWCO=100KD, is splined on Sepharose 4 Fast Flow type molecular sieve gels, with 0.01mol/L PBS pH7.4 solution, carries out wash-out, collects the second elution peak;
D) by step c) the collection liquid that obtains is splined on Q Sepharose Fast Flow type anion column, with after the drip washing of 0.01mol/L PB pH7.4 solution, with the 0.01mol/L PB pH7.4 solution containing 0.6mol/L NaCl, carry out linear elution, collect the first elution peak;
E) by steps d) elutriant collected is replaced as 0.01mol/L PBS pH7.4 solution with the ultrafiltration and concentration device of MWCO=100KD by damping fluid, obtains virus antigen stock;
Described virus antigen is hepatitis A viral antigen.
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| CN102552898B (en) * | 2011-12-21 | 2013-08-07 | 中国医学科学院医学生物学研究所 | Purification preparation method of human rotavirus inactivated vaccines by utilizing ion exchange chromatography |
| CN102628032A (en) * | 2012-03-30 | 2012-08-08 | 中国农业大学 | Purifying process of animal viruses |
| CN103571800B (en) * | 2012-07-27 | 2018-04-24 | 江苏康润生物科技有限公司 | A kind of method of host DNA in removal vaccine |
| CN104558166B (en) * | 2015-01-30 | 2017-12-26 | 肇庆大华农生物药品有限公司 | The method for reducing non-specific antibody in anti-GCRV polyclonal antibody |
| CN106668854A (en) * | 2016-12-23 | 2017-05-17 | 江苏中慧元通生物科技有限公司 | Quadrivalent subunit influenza vaccine and preparation method thereof |
| CN108524929B (en) * | 2017-03-06 | 2019-05-07 | 广州瑞贝斯药业有限公司 | A kind of production method of rabies vacciness |
| CN107365362B (en) * | 2017-07-04 | 2021-02-05 | 武汉科前生物股份有限公司 | Method for large-scale production of high-purity porcine circovirus ORF2 protein |
| CN110274982B (en) * | 2019-06-27 | 2021-09-14 | 中牧实业股份有限公司 | Quantitative detection method for rabies virus inactivated antigen |
| CN111249456A (en) * | 2020-03-13 | 2020-06-09 | 武汉生物制品研究所有限责任公司 | Purification method of rabies virus inactivated vaccine |
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| CN1434056A (en) * | 2002-01-25 | 2003-08-06 | 杭州泰士生物科技有限公司 | Method for producing recombination human hepatitis B virus core antigen |
| CN1415628A (en) * | 2002-08-14 | 2003-05-07 | 上海天伟生物制药有限公司 | Method for extracting luteinizing hormone from human urine |
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