CN108531463A - A kind of separation method and composition of enveloped virus particles - Google Patents
A kind of separation method and composition of enveloped virus particles Download PDFInfo
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Abstract
The invention discloses a kind of separation method of enveloped virus particles and compositions comprising by virus liquid into the interchangeable ion-exchange chromatography of row order and hydroxyapatite chromatography.The advantageous effect of the method for the present invention is：It is low to the pre-treating technology requirement of virus liquid, secondary destruction will not be caused to virion, can handle the virus liquid that various culture substrate cultures obtain, treating capacity is big, and processing cost is low in the case where not adjusting process conditions；Target viral particle can be approached with molecular weight by ion-exchange chromatography and hydroxyapatite chromatography but the enveloped virus particles of textural anomaly efficiently separate by the structural homogeneity for improving virion.
Description
Technical field
The invention belongs to viral biology technical fields, and in particular to a kind of purification process of enveloped virus particles, simultaneously
Further relate to the virus composition using isolated enveloped virus particles.
Background technology
Virus can be divided into enveloped virus (enveloped virus) and non-enveloped virus.Non- enveloped virus is only by capsid egg
White and virus gene genome nucleic acid composition, homogeneous simple in structure are easily isolated purifying.And enveloped virus structure is extremely complex, and not
Homogeneous, therefore the enveloped virus difficulty that obtain structure homogeneous is very big.
The basic structure of enveloped virus is：Linear DNA or RNA macromoleculars are located at the inner core of virion, and appearance is
The shell (capsid) formed by multiple nucleoprotein subunits, shell form the nucleocapsid of tight structure with virus gene genome nucleic acid
(nucleocapsid) particle.There is lipid envelope outside nucleocapsid, has 1 outside coating~the several outer albumen of film, the outer albumen of each film
There are multiple copies in each surfaces of viral particles, is clouded in surfaces of viral particles；The outer albumen of film generally comprises three regions：Film
Outskirt, transmembrane region and film inner region, wherein film outskirt accounts for major portion, and transmembrane region and film inner region are very short.The outer egg of the film of enveloped virus
White is all the albumen (glycoprotein) of high glycosylation under normal conditions, and sugar chain portion accounts for the outer protein ratio of film and may be up to
75% or more.Since sugar chain is got on through post-processing connection after virion formation, cause each culture batch interior and training
It is all inhomogeneous in structure to support the enveloped virus particles harvested between criticizing.
In general, coating, with inside points are more stable and homogeneous, the difference of virus particle structure essentially consists in the outer egg of film
In vain, the copy number and its degree of glycosylation of albumen including outside each virion film.This species diversity both may be in virion group
It has just been formed when dress, it is also possible to be destroyed after virion maturation is released in culture solution, including be hydrolyzed by protease, sugar chain
Enzyme degrades or is sheared and caused by mechanical force.
In addition, enveloped virus is the parasitic microbe of stricti jurise, need that (host is thin using other organisms as culture substrate
Born of the same parents).In enveloped virus incubation, cellular matrix will appear the biological effects such as necrosis, apoptosis, and the DNA of cell is cut into
The segment of big low height unevenness is released in culture solution, for example various organelles of intracellular matter, cell fragment, molecular size range
Different carbohydrate, lipid and various protein are also released in culture solution.
Enveloped virus is the main Types in the virus for infect humans and animals, and common pathogenic enveloped virus has rabies
Poison, influenza virus, japanese encephalitis virus, measles virus, rubella virus, varicella virus, mumps virus, dengue fever virus, Chinese mugwort
Grow virus etc..It needs to obtain homogeneous or the higher enveloped virus particles of purity to prepare vaccine or other researchs.
Currently, the separation principle of enveloped virus is mainly detached virion and impurity using molecular size range difference.
Isolation technics route includes mainly：Density gradient ultracentrifugation purification technique and gel filtration chromatography purification technique.But by
The molecular sieve property of part big molecular impurity and active ingredient are very close in virus harvest liquid, rely on molecular sieve principle merely
It is difficult to be effectively separated.This kind of impurity includes：
(1) high molecular weight protein of host cell is derived from.The protein amounts of host cell expression in incubation
At 20,000 kinds or more, molecular size range is uneven, and many protein exist with multimeric forms.Therefore, the host in virus harvest liquid
Cell protein (HCP) is the complicated mixture of a composition, including molecular sieve property and the close component of virion.
(2) DNA of host cell is derived from.The DNA to dissociate in virus harvest liquid is broken, the disease by host cell
What change, necrosis or apoptosis generated, these cell biological processes can all generate genomic DNA and be cut into segment of different sizes
As a result.Therefore, the host cell DNA in virus harvest liquid is also a highly heterogeneous mixture of molecular size range.Some
Processing step can also further increase the inhomogeneity of DNA molecular amount size as the shearing effect generated is concentrated by ultrafiltration.A part
The molecular sieve property and virion of DNA components are very close.
(3) medium additives such as cow's serum is derived from, cow's serum is also a kind of mixture of complicated component.
(4) structural derivative of enveloped virus is derived from.During Virus culture, it will usually it is endless to generate a part of structure
Whole virion, as protein copies number is too low, glycosylation lacks or incomplete virus outside film.The incomplete disease of these structures
The virion of virion and structural integrity has marked difference, but its molecular sieve in biological property and immunological properties etc.
Property is sufficiently close to the normal virion of structure, is not readily separated.
Based on the above reason, existing enveloped virus particles purification process is difficult to have enveloped virus particles and other impurities
Effect separation, it more difficult to accomplish the different or abnormal virion separation of structure.
In addition, existing purification process usually requires that virus liquid is concentrated by ultrafiltration, and the outer albumen of the film of enveloped virus
It is very fragile, it is easily broken during being concentrated by ultrafiltration, causes the heterogeneity of virion even more serious.Existing purifying
Technique is relatively high to the obtained enveloped virus liquid requirement of culture, is only used for the enveloped virus liquid of processing ad hoc approach culture, and one
Denier cultural method changes, then purifying process needs are changed on a large scale.Even if the coating obtained for Same Way culture
Virus liquid is purified, it is also difficult to be ensured the stability between product batches, can not be obtained the highly stable enveloped virus of quality
Grain.
Therefore, develop that a kind of purification technique route is succinct, separative efficiency is high, separation selectivity is good, to enveloped virus structure
Destructive small enveloped virus particles separation method has very important significance.
Invention content
The purpose of the present invention is to provide a kind of method for separate coating virion, this method has treatment conditions
Mildly, it is concisely and efficiently advantage.
It is another object of the present invention to provide a kind of diseases of the enveloped virus particles isolated containing the above method
Malicious composition.
The technical solution used in the present invention is：
A method of it being used for separate coating virion, including virus liquid is subjected to ion-exchange chromatography and hydroxy-apatite
Stone chromatographic runs, wherein ion-exchange chromatography carries out before hydroxyapatite chromatography；Or ion-exchange chromatography is in hydroxy-apatite
It is carried out after rock layers analysis.
As being further improved for the above method, ion-exchange chromatography includes：
A) optional to pre-equilibrate operation：Pre- put down is carried out to ion exchange column including using ion exchange to pre-equilibrate liquid
Weighing apparatus；
B) loading operates：By virus liquid or sample liquid loading after hydroxyapatite chromatography to ion exchange column；
C) optional balancing run：Including using ion-exchange equilibrium liquid to be balanced ion exchange column；
D) optional pre- elution action：Including using the pre- eluent of ion exchange to carry out prewashing to ion exchange column
It is de-；
E) elution action：Ion exchange column is eluted using ion-exchanging eluent.
As being further improved for the above method, further comprise using the second ion-exchanging eluent to ion exchange layer
Analysis column is eluted.
As being further improved for the above method, the first ion-exchanging eluent and the second ion of collecting wash-off respectively are handed over
Eluent is changed, the virion of different structure, composition or purity is obtained.
As being further improved for the above method, hydroxyapatite chromatography includes：
A) optional to pre-equilibrate operation：Hydroxyapatite chromatography column is carried out in advance including using hydroxyapatite to pre-equilibrate liquid
Balance：
B) loading operates：By virus liquid or sample liquid loading after ion-exchange chromatography to hydroxyapatite chromatography column；
C) optional balancing run：Including using hydroxyapatite equilibrium liquid to be balanced hydroxyapatite chromatography column；
D) optional pre- elution action：It is pre- including using the pre- eluent of hydroxyapatite to carry out hydroxyapatite chromatography column
Elution；
E) elution action：Hydroxyapatite chromatography column is eluted using hydroxyapatite eluent.
As being further improved for the above method, further comprise using the second hydroxyapatite eluent to hydroxy-apatite
Stone chromatographic column is eluted.
As being further improved for the above method, the first hydroxyapatite eluent and the second hydroxyl of wash-off are collected respectively
Apatite eluent obtains the virion of different structure, composition or purity.
As being further improved for the above method, without intermediate chromatography between ion-exchange chromatography and hydroxyapatite chromatography
Operation.
As being further improved for the above method, without intermediate behaviour between ion-exchange chromatography and hydroxyapatite chromatography
Make.
As being further improved for the above method, ion-exchange chromatography is anion-exchange chromatography.
As being further improved for the above method, ion exchange pre-equilibrates liquid, ion-exchange equilibrium liquid, ion exchange prewashing
De- liquid, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, the pre- eluent of hydroxyapatite, hydroxyl
The pH value of base apatite eluent stands alone as 7.0~9.5.
As being further improved for the above method, ion exchange pre-equilibrates liquid, ion-exchange equilibrium liquid, ion exchange prewashing
De- liquid, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, the pre- eluent of hydroxyapatite, hydroxyl
Base apatite eluent stands alone as phosphate buffer, Tris-HCl buffer solutions, Tricine buffer solutions, Hepes buffer solutions, sweet ammonia
Acid buffer, TEA buffer solutions, veronal buffer.
As being further improved for the above method, ion exchange pre-equilibrates liquid, ion-exchange equilibrium liquid, ion exchange prewashing
De- liquid, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, the pre- eluent of hydroxyapatite, hydroxyl
Base apatite eluent is phosphate buffer.
As being further improved for the above method, ion exchange pre-equilibrates liquid, ion-exchange equilibrium liquid, ion exchange prewashing
De- liquid, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, the pre- eluent of hydroxyapatite, hydroxyl
Base apatite eluent is independently added with water soluble salt.
As being further improved for the above method, enveloped virus include rabies viruses, influenza virus, japanese encephalitis virus,
Measles virus, rubella virus, varicella virus, mumps virus, dengue fever virus or AIDS virus.
A kind of enveloped virus composition, including virion isolated as stated above.
Further include stabilizer as being further improved for above-mentioned enveloped virus composition.
As being further improved for above-mentioned enveloped virus composition, stabilizer includes sucrose and albumin.
As being further improved for above-mentioned enveloped virus composition, the mass percentage of sucrose is 0.5~10%.
As being further improved for above-mentioned enveloped virus composition, the mass percentage of albumin is 1~20%.
The beneficial effects of the invention are as follows：
(1) in the method for the present invention, the pre-treating technology requirement of virus liquid is low, and treatment conditions are mild, will not be to virion
Secondary destruction is caused, while having that treating capacity is big, the low feature of processing cost.
(2) the method for the present invention improves the structural homogeneity of virion, passes through ion-exchange chromatography and hydroxyapatite
Target viral particle can be approached with molecular weight but the enveloped virus particles of textural anomaly efficiently separate by chromatography.
(3) the method for the present invention can handle what various culture substrate cultures obtained in the case where not adjusting process conditions
Virus liquid has better adaptability.
(4) the method for the present invention further reduced impurity residual quantity.
(5) the method for the present invention improves the tolerance (Process robustness) of purifying process, even if virus liquid matter
There is notable difference in amount, also can carry out calibration by purification process, the consistency between ensureing harvest group in batches.
Description of the drawings
Fig. 1 is the SDS-PAGE protein electrophoresis Coomassie brilliant blues dye for the target viral component that 1 purifying of the experiment of embodiment 1 obtains
(band 1-3 passes through electrophoresis result after purification to color result for the virus liquid that Vero cell flask cultures obtain；Band 4 is albumen
Marker；Band 5-7 passes through electrophoresis result after purification for the virus liquid that Vero cell spinner bottle cultures obtain；Band 8-10 is
The virus liquid that Vero bioreactor cultures obtain is by electrophoresis result after purification).
Fig. 2 is the virus liquid difference purification phase SDS- that embodiment 1 tests that 1 Vero bioreactor cultures obtain
PAGE electrophoresis Coomassie brilliant blue analysis results (Marker：Protein molecular weight standard (Mr：Molecular weight, unit KD)；1 is disease
Malicious harvest liquid；2 be ion-exchange chromatography elution fraction；3-4 is CHT elution fractions, and 3 be the sample before desalination, and 4 is after desalinations
Sample).
Fig. 3 is the electricity that embodiment 1 tests that 1 Vero bioreactor cultures harvest virus liquid virion after purification
Mirror photo.
Fig. 4 be 2 Vero cell flask cultures of embodiment harvest japanese encephalitis virus through different purification process after purification
SDS-PAGE electrophoresis silver staining analysis results (Marker：Protein molecular weight standard (Mr：Molecular weight, unit KD)；1 this method
Purification of samples；2 gel filtration method purification of samples；3 Ultracentrifugation Method purification of samples, 4 virus harvest liquids).
Fig. 5 is that the purified restrovirus particle Electronic Speculum of japanese encephalitis virus of 2 Vero cell flask cultures of embodiment harvest is shone
Piece.
Fig. 6 is the influenza virus of 3 chick embryo culture of embodiment harvest through different purification process SDS-PAGE Gel Electrophoresis Silvers after purification
Contaminate analysis result (Marker：Protein molecular weight standard (Mr：Molecular weight, unit KD)；1 virus harvest liquid；2 ultracentrifugations
Method purification of samples；3 gel filtration method purification of samples, 4 this method purification of samples).
Fig. 7 is the influenza virus virion electromicroscopic photograph after purification of 3 chick embryo culture of embodiment harvest.
Specific implementation mode
A method of it being used for separate coating virion, including virus liquid is subjected to ion-exchange chromatography and hydroxy-apatite
Stone chromatographic runs, wherein ion-exchange chromatography carries out before hydroxyapatite chromatography；Or ion-exchange chromatography is in hydroxy-apatite
It is carried out after rock layers analysis.
Ion exchange column used in the technology of the present invention can be various existing ion exchange columns.It is exemplary
Anion exchange chromatography include but not limited to：DEAE cellulose, the Poros PI of Applied Biosystems
20, PI 50, HQ 10, HQ 20, HQ 50, D 50, MonoQ, MiniQ, Source 15Q and 30Q, Q, DEAE and ANX
The QAE SEPHADEX of Sepharose Fast Flow, Q Sepharose High Performance, GE HealthcareTM
With FAST Q SEPHAROSETM, the WP PEI of J.T.Baker, WP DEAM, WP QUAT, Biochrom Labs Inc.
UNOsphere Q, Macro-Prep DEAE and the Macro- of Hydrocell DEAE and Hydrocell QA, BioRad
The Ceramic HyperD Q of Prep High Q, Pall Technologies, Ceramic HyperD DEAE, Q HyperZ,
Trisacryl M and LS DEAE, Spherodex LS DEAE, QMA Spherosil LS, QMA Spherosil M, DOWEX
Fine Mesh Strong Base Type I and Type π Anion Resins and DOWEX MONOSPHERE 77, Dow
The weakly-basic anion displacement chromatography column of Liquid Separationd, Matrex Cellufme A200 of Millipore,
A500, Q500 and Q800, EMD'sEMD TMAE、EMD DEAE andEMD DMAE,
The Amberlite of Sigma-Aldrich is weak and strong anion displacement chromatography column type I and II, DOWEX weak and
strong anion exchangers type I and II、Diaion weak and strong anion exchangers
Type I and II, Duolite, TSK gel Q and the DEAE 5PW and 5PW-HR of Tosoh,SuperQ-
650S, 650M and 650C3 QAE-550C and 650S, DEAE-650M and 650C, QA52, DE23 of Whatman, DE32, DE51,
DE52, DE53, Express-Ion D and Express-Ion Q.Commercial high power capacity resin includes but not limited to：GigaCap
Q-650M (Tosoh), Capto Q (GE Healthcare), EshmunoTMQ (EMD) and NuviaTMQ(Bio-rad).One
In a little examples, anion-exchange chromatography includes DEAE chromatographies.In some instances, DEAE chromatographies are selected from following any：In some instances with DEAE cellulose chromatographic columns, DEAE chromatographic columns areChromatographic column.In some instances, in some instances, anion-exchange chromatography includes quaternary ammonium salt (Q) chromatography
Column.In some instances, Q chromatographs column chromatography selected from following any：Q With
Illustratively cation-exchange chromatography post includes but not limited to：MonoS, MiniS, Source 15S and 30S, SP
Sepharose Fast FlowTM, SP Sepharose High Performance of GE Healthcare, TosohThe Macro-Prep High S of SP-650S and SP-650M, BioRad, Pall Technologies
Fractogel EMD SE of Ceramic HyperD S, Trisacryl M and LS SP and Spherodex LS SP, EMD,
The TSK Gel SP 5PW and SP-5PW-HR of the Poros S-10 and S-20 of Applied Biosystems, Tosoh, Applied
The Poros HS-20 and HS 50 of Biosystems, EMD'sSE52, SE53 of EMD S03, Whatman and
CM Sepharose Fast Flow, the Biochrom Labs Inc.'s of Express-Ion S, GE Healthcare
Ceramic HyperD CM of Macro-Prep CM, the Pall Technologies of Hydrocell CM, BioRad,
The Matrex Cellufme C500 and C200 of Trisacryl M CM, Trisacryl LS CM, Millipore, Whatman
CM52, CM32, CM23 and Express-Ion C, Tosoh'sCM-650S, CM-650M and CM-650C,
WP CBX from, the Dow Liquid of BAKERBOND Carboxy-Sulfon, the J.T Baker of J.T.Baker
Amberlite Weak Cation Exchangers of the DOWEX MAC-3, Sigma-Aldrich of Separations,
The Fractogel EMD of DOWEX Weak Cation Exchanger and Diaion Weak Cation Exchangers, EMD
The DOWEX Fine Mesh of Hydrocell SP, the Dow Liquid Separations of COO-, Biochrom Labs Inc.
The WP Sulfonic, Sigma- of the UNOsphere S, J.T.Baker of Strong Acid Cation Resin, BioRad
The Amberlite Strong Cation Exchangers of Aldrich, the Diaion of DOWEX Strong Cation
The PI 1 of Strong Cation Exchanger, Whatman.Commercial high power capacity resin includes but not limited to：GigaCap S-
650M (Tosoh), EshmunoTMS (EMD), NuviaTMS (BioRad),XS (Applied Biosystems) and
Capto S(GE Healthcare)。
Hydroxyapatite chromatography column includes but not limited to：(model has ceramic hydroxyapatite chromatographic column：CHT Type I and
Type II, Bio-Rad Laboratories, Hercules, Calif.), the super gel hydroxyapatite chromatography column (models of HA
Have：Pall Corp., East Hills, N.Y.) and ceramic fluor-apatite chromatographic column (model has：CFT Type I and Type
II、Bio-Rad Laboratories、Hercules、Calif.)。
As being further improved for the above method, ion-exchange chromatography includes：
It is optional to pre-equilibrate operation：Ion exchange column is pre-equilibrated including using ion exchange to pre-equilibrate liquid；
Loading operates：By virus liquid or sample liquid loading after hydroxyapatite chromatography to ion exchange column；
Optional balancing run：Including using ion-exchange equilibrium liquid to be balanced ion exchange column；
Optional pre- elution action：Including using the pre- eluent of ion exchange to elute ion exchange column in advance；
Elution action：Ion exchange column is eluted using ion-exchanging eluent.
Optional operating procedure can be according to the type of different ions displacement chromatography column, concrete model, virus liquid or sample liquid
The case where etc. be adjusted correspondingly.
As being further improved for the above method, further comprise using the second ion-exchanging eluent to ion exchange layer
Analysis column is eluted.
As being further improved for the above method, the first ion-exchanging eluent and the second ion of collecting wash-off respectively are handed over
Eluent is changed, the virion of different structure, composition or purity is obtained.
As being further improved for the above method, hydroxyapatite chromatography includes：
It is optional to pre-equilibrate operation：Pre- put down is carried out to hydroxyapatite chromatography column including using hydroxyapatite to pre-equilibrate liquid
Weighing apparatus；
Loading operates：By virus liquid or sample liquid loading after ion-exchange chromatography to hydroxyapatite chromatography column；
Optional balancing run：Including using hydroxyapatite equilibrium liquid to be balanced hydroxyapatite chromatography column；
Optional pre- elution action：Including using the pre- eluent of hydroxyapatite to carry out prewashing to hydroxyapatite chromatography column
It is de-；
Elution action：Hydroxyapatite chromatography column is eluted using hydroxyapatite eluent.
The case where optional operating procedure can be according to the concrete model of different hydroxyapatite chromatographies, virus liquid or sample liquid
Etc. being adjusted correspondingly.
Further comprise eluting hydroxyapatite chromatography column using the second hydroxyapatite eluent.
As being further improved for the above method, the first hydroxyapatite eluent and the second hydroxyl of wash-off are collected respectively
Apatite eluent obtains the virion of different structure, composition or purity.
To avoid reducing virion integrality, as being further improved for the above method, ion-exchange chromatography and hydroxyl
Without intermediate chromatographic runs between apatite chromatography.
To be further simplified operation, there is no intermediary operation between ion-exchange chromatography and hydroxyapatite chromatography.
As being further improved for the above method, ion-exchange chromatography is anion-exchange chromatography.
As being further improved for the above method, ion exchange pre-equilibrates liquid, ion-exchange equilibrium liquid, ion exchange prewashing
De- liquid, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, the pre- eluent of hydroxyapatite, hydroxyl
The pH value of base apatite eluent both can be identical, can also different or part it is identical, part is different.According to specific
Chromatography condition, the pH value of each solution can stand alone as 7.0~9.5,7.0~9.0,7.0~8.5,7.2~8.0,7.2~
7.8,7.3~7.8,7.3~7.6,7.6.The pH value of each solution can be adjusted correspondingly according to specific chromatography condition, this
A little specific chromatography conditions include but not limited to the type, concrete model, hydroxyapatite chromatography column of ion exchange column
Copy number, amino acid composition, degree of glycosylation, the phosphoric acid of concrete model, the type of virus, the one or more outer membrane proteins of virus
Change degree, the dosage etc. of each solution.By the adjusting to each solution ph, different solution properties are obtained, are met different
It needs.
Buffer solution can preferably stablize condition when chromatography, advantageously ensure that the stability of tomographic results.As above-mentioned
Further improvements in methods, ion exchange pre-equilibrate liquid, ion-exchange equilibrium liquid, the pre- eluent of ion exchange, ion exchange and wash
De- liquid, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, the pre- eluent of hydroxyapatite, hydroxyapatite eluent
It is slow to stand alone as phosphate buffer, Tris-HCl buffer solutions, Tricine buffer solutions, Hepes buffer solutions, glycine buffer, TEA
The buffer solution commonly used in the art such as fliud flushing, veronal buffer.Further, it is flat to pre-equilibrate liquid, ion exchange for ion exchange
Weigh liquid, the pre- eluent of ion exchange, ion-exchanging eluent, hydroxyapatite pre-equilibration liquid, hydroxyapatite equilibrium liquid, hydroxyl
The pre- eluent of apatite, hydroxyapatite eluent are the identical buffer solution of of the same race or anion.Particularly, ion exchange is pre- flat
Weigh liquid, ion-exchange equilibrium liquid, the pre- eluent of ion exchange, ion-exchanging eluent, hydroxyapatite pre-equilibration liquid, hydroxyl phosphorus
The pre- eluent of lime stone equilibrium liquid, hydroxyapatite, hydroxyapatite eluent are phosphate buffer.The pH value of each solution can be only
Stand is 7.0~9.5,7.0~9.0,7.0~8.5,7.2~8.0,7.2~7.8,7.3~7.8,7.3~7.6,7.6.Each solution
PH value can be adjusted correspondingly according to specific chromatography condition, these specific chromatography conditions include but not limited to ion
Type, concrete model, the type of virus, the copy number of the one or more outer membrane proteins of virus, the amino acid group of displacement chromatography column
At, the dosage etc. of degree of glycosylation, phosphorylation degree, each solution.By the adjusting to each solution ph, meet different need
It wants.In some instances, the concentration of buffer solution intermediate ion (such as anion, phosphate anion) is about 1-50mM, 1-40mM, 5-
30mM, 10-30mM, 15-25mM, 18-22mM or 20mM.In some instances, buffer solution intermediate ion (such as anion, phosphate radical
Ion) concentration be about 1-20mM, 1-10mM, 2-8mM, 4-6mM or 5mM.
In some instances, the pH value that ion exchange pre-equilibrates liquid is about 6.0-10.0,6.5-9.5,7.0-9.5,7.0-
9.0,7.0-8.5,7.0-8.0,7.2-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, ion exchange pre-equilibrates
The concentration of liquid intermediate ion (such as anion, phosphate anion) is about 1-80mM, 1-50mM, 3-40mM, 5-30mM, 5-30mM,
10-30mM, 15-25mM, 18-22mM or 20mM.In some instances, ion exchange pre-equilibrate liquid in salt (such as NaCl) it is dense
Degree is about 1-500mM, 10-400mM, 50-300mM, 100-200mM, 125-175mM or 150mM.In some instances, ion
It is phosphate buffer, the phosphate concentration with about 10-30mM or 20mM to exchange and pre-equilibrate liquid.
In some instances, the pH value of ion-exchange equilibrium liquid is about 6.0-10.0,6.5-9.5,7.0-9.5,7.0-
9.0,7.0-8.5,7.0-8.0,7.2-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, ion-exchange equilibrium liquid
The concentration of intermediate ion (such as anion, phosphate anion) is about 1-50mM, 1-40mM, 5-30mM, 10-30mM, 15-25mM,
18-22mM or 20mM.In some instances, ion-exchange equilibrium liquid is further added with salt, such as sodium salt, NaCl.In some realities
Example in, in ion-exchange equilibrium liquid the concentration of salt (such as NaCl) be about 1-500mM, 10-400mM, 50-300mM, 100-200mM,
125-175mM or 150mM.In some instances, ion-exchange equilibrium liquid be phosphate buffer, have about 10-30mM or
The phosphate concentration of 20mM.In some instances, the further NaCl containing 100-200mM, 150mM of phosphate buffer.
In some instances, the pH value of the pre- eluent of ion exchange is about 6.0-10.0,6.5-9.5,7.0-9.5,7.0-
9.0,7.0-8.5,7.0-8.0,7.2-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, ion exchange elutes in advance
The pH value of liquid and ion exchange pre-equilibrate the pH differences of liquid and/or ion-exchange equilibrium liquid less than 2,1,1.5,1,0.8,0.5,
0.2、0.1.In some instances, the concentration of the pre- eluent intermediate ion of ion exchange (such as anion, phosphate anion) is about
1-50mM, 1-40mM, 5-30mM, 10-30mM, 15-25mM, 18-22mM or 20mM.In some instances, ion exchange prewashing
Added with concentration be about 1-700mM, 10-600mM in de- liquid, 50-500mM, 100-350mM, 150-300mM, 175-275mM,
Or the NaCl of 250-300mM.In some instances, it is about added with concentration in the pre- eluent of ion exchange (such as phosphate buffer)
The salt of 1-700mM, 10-600mM, 50-500mM, 100-350mM, 150-300mM, 175-275mM or 250-300mM are (such as
NaCl).In some instances, the pre- eluent of ion exchange is that the phosphoric acid of 10-30mM or 20mM that phosphorus acid ion concentration is are slow
Fliud flushing.In some instances, it is about 200-300mM (such as 250mM), 20-100mM that phosphate buffer, which is further added with concentration,
The NaCl of (such as 50mM) or 50-180mM (such as 120mM).
In some instances, the pH value of ion-exchanging eluent is about 6.0-10.0,6.5-9.5,7.0-9.5,7.0-
9.0,7.0-8.5,7.0-8.0,7.2-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, ion exchange elutes in advance
The pH value of liquid and the pH value difference of any solution of previous utilization are less than 2,1,1.5,1,0.8,0.5,0.2,0.1.At some
In example, middle salt (such as NaCl) concentration of ion-exchanging eluent (such as phosphate buffer) be about 50-1000mM, 100-800mM,
Or 200-700mM.In some instances, middle salt (such as NaCl) concentration of ion-exchanging eluent (such as phosphate buffer) is about
250-750mM, 300-700mM, 350-650mM, 400-600mM, 450-600mM or 500-550mM.In some instances, from
Middle salt (such as NaCl) concentration that son exchanges eluent (such as phosphate buffer) is about 100-500mM, 150-450mM, 200-
400mM, 250-350mM, 275-325mM or 300mM.
In some instances, hydroxyapatite pre-equilibrate liquid pH value be about 6.0-10.0,6.5-9.5,7.0-9.5,
7.0-9.0,7.0-8.5,7.0-8.0,7.2-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, hydroxyapatite
The concentration for pre-equilibrating the ion (such as anion, phosphate anion) of liquid (such as phosphate buffer) is about 1-50mM, 1-40mM, 5-
30mM, 10-30mM, 15-25mM, 18-22mM or 20mM.In some instances, (such as phosphoric acid is slow for hydroxyapatite pre-equilibration liquid
Fliud flushing) the concentration of ion (such as anion, phosphate anion) be about 1-20mM, 1-10mM, 2-8mM, 4-6mM or 5mM.
In some examples, hydroxyapatite pre-equilibrates the concentration of the ion (such as anion, phosphate anion) of liquid (such as phosphate buffer)
About 50~220mM.In some instances, hydroxyapatite pre-equilibrates liquid (such as phosphate buffer) and is further added with concentration about
For the salt (such as NaCl) of 1-500mM, 10-400mM, 50-300mM, 100-200mM, 125-175mM or 150mM.In some realities
In example, it is about 100-1000mM, 300- that hydroxyapatite, which pre-equilibrates liquid (such as phosphate buffer) and is further added with concentration,
The salt (such as NaCl) of 800mM, 400-700mM, 500-600mM or 550mM.In some instances, hydroxyapatite pre-equilibrates liquid
(such as phosphate buffer) is not added with salt.
In some instances, the pH value of hydroxyapatite equilibrium liquid is about 6.0-10.0,6.5-9.5,7.0-9.5,7.0-
9.0,7.0-8.5,7.0-8.0,7.2-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, hydroxyapatite balances
The concentration of the ion (such as anion, phosphate anion) of liquid (such as phosphate buffer) is about 1-50mM, 1-40mM, 5-30mM, 10-
30mM, 15-25mM, 18-22mM or 20mM.In some instances, the ion of hydroxyapatite equilibrium liquid (such as phosphate buffer)
The concentration of (such as anion, phosphate anion) is about 1-20mM, 1-10mM, 2-8mM, 4-6mM or 5mM.In some instances,
Hydroxyapatite equilibrium liquid (such as phosphate buffer) be further added with concentration be about 1-500mM, 10-400mM, 50-300mM,
The salt (such as NaCl) of 100-200mM, 125-175mM or 150mM.In some instances, hydroxyapatite equilibrium liquid (such as phosphoric acid
Buffer solution) to be further added with concentration be about 100-1000mM, 300-800mM, 400-700mM, 500-600mM or 550mM
Salt (such as NaCl).
In some instances, the pH value of the pre- eluent of hydroxyapatite be about 6.0-10.0,6.5-9.5,7.0-9.5,
7.0-9.0,7.0-8.5,7.0-8.0,7.2-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, hydroxyapatite
The concentration of the ion (such as anion, phosphate anion) of pre- eluent (such as phosphate buffer) is about 1-50mM, 1-40mM, 5-
30mM, 10-30mM, 15-25mM, 18-22mM or 20mM.In some instances, (such as phosphoric acid is slow for the pre- eluent of hydroxyapatite
Fliud flushing) the concentration of ion (such as anion, phosphate anion) be about 1-20mM, 1-10mM, 2-8mM, 4-6mM or 5mM.
In some examples, the concentration of the ion (such as anion, phosphate anion) of the pre- eluent of hydroxyapatite (such as phosphate buffer)
About 1-200mM, 20-180mM, 30-150mM, 40-120mM, 50-100mM, 40-60mM, 80-120mM, 50mM or
100mM.In some instances, it is about 1- that the pre- eluent of hydroxyapatite (such as phosphate buffer), which is further added with concentration,
The salt (such as NaCl) of 500mM, 10-400mM, 50-300mM, 100-200mM, 125-175mM or 150mM.In some instances,
It is about 100-1000mM, 300-800mM, 400- that the pre- eluent of hydroxyapatite (such as phosphate buffer), which is further added with concentration,
The salt (such as NaCl) of 700mM, 500-600mM or 550mM.
In some instances, the pH value of hydroxyapatite eluent is about 6.0-10.0,6.5-9.5,7.0-9.5,7.0-
9.0,7.0-8.5,7.0-8.0,7.2-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, hydroxyapatite elutes
The concentration of the ion (such as anion, phosphate anion) of liquid (such as phosphate buffer) is about 50-500mM, 75-350mM, 80-
320mM、90-310mM、100-300mM、80-120mM、150-230mM、180-220mM、250-330mM、280-380mM、73-
223mM, 100mM, 200mM or 300mM.In some instances, the ion of hydroxyapatite eluent (such as phosphate buffer)
The concentration of (such as anion, phosphate anion) is about 200mM.In some instances, hydroxyapatite eluent (such as phosphoric acid buffer
Liquid) the concentration of ion (such as anion, phosphate anion) be about 100-200mM, 125-175mM or 150mM.In some realities
In example, the concentration of the ion (such as anion, phosphate anion) of hydroxyapatite eluent (such as phosphate buffer) is about 1-
200mM, 20-180mM, 30-150mM, 40-120mM, 50-100mM, 40-60mM, 80-120mM, 50mM or 100mM.One
In a little examples, hydroxyapatite eluent (such as phosphate buffer) be further added with concentration be about 1-500mM, 10-400mM,
The salt (such as NaCl) of 50-300mM, 100-200mM, 125-175mM or 150mM.In some instances, hydroxyapatite elutes
It is about 100-1000mM, 300-800mM, 400-700mM, 500-600mM that liquid (such as phosphate buffer), which is further added with concentration,
Or the salt (such as NaCl) of 550mM.
As being further improved for the above method, ion exchange pre-equilibrates liquid, ion-exchange equilibrium liquid, ion exchange prewashing
De- liquid, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, the pre- eluent of hydroxyapatite, hydroxyl
Base apatite eluent is independently added with water soluble salt.The addition of water soluble salt, thus it is possible to vary the parameter of solution makes it meet not
With the needs of chromatography condition, these chromatography conditions include but not limited to the type, concrete model, hydroxyl phosphorus of ion exchange column
The concrete model of lime stone chromatographic column, the type of virus, the copy number of the one or more outer membrane proteins of virus, amino acid composition, sugar
Base degree, phosphorylation degree etc..In some instances, the concentration of salt (such as sodium salt, specific as NaCl) be about 1-500mM,
10-400mM, 50-300mM, 100-200mM, 125-175mM or 150mM.In some instances, salt (such as sodium salt, it is specific such as
NaCl concentration) is about 100-1000mM, 300-800mM, 400-700mM, 500-600mM or 550mM.
As being further improved for the above method, before carrying out ion-exchange chromatography and hydroxyapatite chromatography, to disease
Venom is pre-processed.
As being further improved for the above method, enveloped virus includes but not limited to rabies viruses, influenza virus, B-mode brain
The enveloped virus such as scorching virus, measles virus, rubella virus, varicella virus, mumps virus, dengue fever virus or AIDS virus.
A kind of enveloped virus composition, including isolated virion as stated above.
Further include stabilizer as being further improved for above-mentioned enveloped virus composition.
As being further improved for above-mentioned enveloped virus composition, stabilizer includes sucrose and albumin.
As being further improved for above-mentioned enveloped virus composition, the mass percentage of sucrose is 0.5~10%.
As being further improved for above-mentioned enveloped virus composition, the mass percentage of albumin is 1~20%.
In some examples, the condition of ion-exchange chromatography is (such as specific according to the copy numbers of one or more outer membrane proteins
The combination of the copy number of outer membrane protein, one or more outer membrane proteins) and/or one or more outer membrane proteins glycosylation journey
Degree, phosphorylation degree determine.In some instances, the condition of ion-exchange chromatography includes at least one in the following conditions：Column
Type (such as anion or cation), the model of column, it is pre- that ion exchange pre-equilibrates liquid, ion-exchange equilibrium liquid, ion exchange
Eluent, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, the pre- eluent of hydroxyapatite,
The type, concentration of salt and/or volume is used in the ion concentration of hydroxyapatite eluent, pH, specific buffer solution；Virus liquid
Volume or applied sample amount.
In some instances, virus includes one or more outer membrane proteins.Ion-exchange chromatography or hydroxyapatite chromatography
Condition can according to one or more of the following conditions determine：A) the amino acid composition of one or more outer membrane proteins；b)
The copy number of one or more outer membrane proteins；C) degree of glycosylation of one or more outer membrane proteins；D) one or more outer membranes
The phosphorylation degree of albumen.
In some instances, the ion concentration of eluent and the copy number of one or more outer membrane proteins are proportional.One
In a little examples, the degree of glycosylation of the ion concentration of eluent and one or more outer membrane proteins is in inverse ratio.
In some instances, only there are one outer membrane proteins for enveloped virus.The ion concentration of eluent and unique outer membrane
The copy number of albumen is proportional, is in inverse ratio with degree of glycosylation.In some instances, unique outer membrane protein has and preferably copies
Shellfish number range and/or preferred degree of glycosylation range.Such as with the disease without preferred copy number range or degree of glycosylation
Poison is compared, and preferred copy number range and/or preferred degree of glycosylation range make virus composition have higher be immunized
Originality.
With reference to embodiment, the present invention is further illustrated, and however, it is not limited to this.
Embodiment 1
The present embodiment is by by taking the purifying of rabies viruses particle as an example, and the present invention is further illustrated.
In the present embodiment, viral purity and virus protein proportion grading method are：The virus component of purifying harvest is passed through
It is denaturalized SDS- polyacrylamide gel electrophoresises (SDS-PAGE) to analyze, after Silver stain or coomassie brilliant blue staining, gel is placed in
It is scanned under gel imaging system, the ratio that each virus protein accounts for total protein is calculated in peak area normalization method, with all virus proteins
The sum of ratio is calculated as viral purity.
Vero cell DNAs residual quantity and NIH potency are pressed《Pharmacopoeia of People's Republic of China》Three institute's support methods in 2015 into
Row.
Experiment 1
This experiment is with Vero cells (spinner culture, flask culture, bioreactor culture), human diploid cell, chicken embryo
For the CTN-1V strain rabies venom of culture, the purification process of the present invention is further described.
1, virus-culturing fluid pre-processes
The virus-culturing fluid of harvest is filtered clarification with 0.45 μm of miillpore filter, obtains viral pretreatment fluid.
2, anion-exchange chromatography
(1) column equilibration：Capto-DEAE chromatographic columns are balanced, equilibrium liquid is：The 20mMol/L phosphoric acid buffers of pH7.6
Liquid (being 150mMol/L containing sodium chloride).
(2) viruses adsorption：Virus liquid after filtering is clarified flows through the Capto-DEAE chromatographic columns after balance, and sample-adding terminates
Afterwards, continue to balance chromatographic column with the equilibrium liquid of 2-5 times of column volume.
(3) pre- elution：The chromatographic column for having adsorbed virus is eluted in advance with pre- eluent, pre- eluent is：PH7.6's
20mMol/L phosphate buffers (being 200mMol/L containing sodium chloride).
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.6's
20mMol/L phosphate buffers (being 500mMol/L containing sodium chloride), are received according to the optical absorption peak of tomographic system detector instruction
Collect eluent, obtains product A.
3, hydroxyapatite chromatography
(1) column equilibration：CHT chromatographic columns are balanced, equilibrium liquid is：The 20mMol/L phosphate buffers of pH7.6.
(2) viruses adsorption：Product A will be crossed and flow through the CHT chromatographic columns after balance, after sample-adding, with 2-5 times of column volume
Equilibrium liquid continues to balance chromatographic column.
(3) pre- elution：The chromatographic column for having adsorbed virus is eluted in advance with pre- eluent, pre- eluent is：PH7.6's
100mMol/L phosphate buffers.
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.6's
200mMol/L phosphate buffers collect eluent according to the optical absorption peak of tomographic system detector instruction, obtain target viral
Grain component.
The analysis for the target viral grain fraction that the virus liquid of different culture media matter culture obtains after purification is shown in Table 1 and Fig. 1-
Fig. 2.
The characteristic for the target viral grain fraction that the virus liquid of 1 different culture media matter culture of table obtains after purification
Fig. 1 is the SDS-PAGE protein electrophoresis Coomassie brilliant blues dye for the target viral component that 1 purifying of the experiment of embodiment 1 obtains
(band 1-3 passes through electrophoresis result after purification to color result for the virus liquid that Vero cell flask cultures obtain；Band 4 is albumen
Marker；Band 5-7 passes through electrophoresis result after purification for the virus liquid that Vero cell spinner bottle cultures obtain；Band 8-10 is
The virus liquid that Vero bioreactor cultures obtain is by electrophoresis result after purification).It can be seen from the figure that different trainings
The rabies viruses particle that foster matrix culture obtains is after identical method isolated or purified, and protein band position is almost
It overlaps.Absolutely prove has high purity by the method for the present invention rabies viruses particle after purification, and the method for the present invention can be with
The rabies viruses obtained suitable for purifying or detaching different culture media matter culture, substantially increases technique adaptability.
Fig. 2 is the virus liquid difference purification phase SDS- that embodiment 1 tests that 1 Vero bioreactor cultures obtain
PAGE electrophoresis Coomassie brilliant blue analysis results (Marker：Protein molecular weight standard (Mr：Molecular weight, unit KD)；1 is disease
Malicious harvest liquid；2 be ion-exchange chromatography elution fraction；3-4 is CHT elution fractions, and 3 be the sample before desalination, and 4 is after desalinations
Sample).
Test electromicroscopic photograph such as Fig. 3 institutes of 1 Vero bioreactor cultures harvest virus liquid virion after purification
Show.It can be seen from the figure that virus particle structure is complete, there is typical rabies viruses particle shape.
Experiment 2：
This experiment makees into one the purification process of the present invention by taking the CTN-1V of Vero cell culture strain rabies venom as an example
The explanation of step.
1, virus-culturing fluid pre-processes
The virus-culturing fluid of harvest is filtered clarification with 0.45 μm of miillpore filter, obtains viral pretreatment fluid.
2, anion-exchange chromatography
(1) column equilibration：Capto-DEAE chromatographic columns are balanced, equilibrium liquid is：The 20mmol/L phosphoric acid buffers of pH7.6
Liquid (being 150mmol/L containing sodium chloride).
(2) viruses adsorption：Virus liquid after filtering is clarified flows through the Capto-DEAE chromatographic columns after balance, and sample-adding terminates
Afterwards, continue to balance chromatographic column with the equilibrium liquid of 2-5 times of column volume.
(3) pre- elution：The chromatographic column for having adsorbed virus is eluted in advance with pre- eluent, pre- eluent is：PH7.6's
20mmol/L phosphate buffers (being 200mmol/L containing sodium chloride).
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.6's
20mmol/L phosphate buffers (being 500mmol/L containing sodium chloride), are received according to the optical absorption peak of tomographic system detector instruction
Collect eluent, obtains product A.
3, hydroxyapatite chromatography
(1) column equilibration：CHT chromatographic columns are balanced, equilibrium liquid is：The 20mmol/L phosphate buffers of pH7.6.
(2) viruses adsorption：Product A will be crossed and flow through the CHT chromatographic columns after balance, after sample-adding, with 2-5 times of column volume
Equilibrium liquid continues to balance chromatographic column.
(3) pre- elution：The chromatographic column for having adsorbed virus is eluted in advance with pre- eluent, pre- eluent is：PH7.6's
50mmol/L phosphate buffers.
(4) product A is eluted：Product A elutions are carried out to the chromatographic column for having adsorbed virus with eluent, eluent A is：
The 100mmol/L phosphate buffers of pH7.6.
(5) product B is eluted：Product B elutions are carried out to the chromatographic column for having adsorbed virus with eluent, eluent B is：
The 200mmol/L phosphate buffers of pH7.6.
(6) product C is eluted：Product C elutions are carried out to the chromatographic column for having adsorbed virus with eluent, eluent C is：
The 300mmol/L phosphate buffers of pH7.6.
4, the analysis for the target viral grain fraction that the virus liquid of different culture media matter culture obtains after purification
The characteristic of the different elution fractions of table 2
The experiment 1 of embodiment 1 and experiment 2 the result shows that, the method have the advantages that：
(1) pre-treating technology of virus liquid requires low, and secondary destruction will not be caused to virion, and treating capacity is big, processing
It is at low cost.
(2) structural homogeneity for improving virion can be by mesh by ion-exchange chromatography and hydroxyapatite chromatography
Mark virion is approached with molecular weight but the enveloped virus particles of textural anomaly efficiently separate.
(3) virus liquid that various culture substrate cultures obtain can be handled in the case where not adjusting process conditions.
(4) it further reduced impurity residual quantity.
(5) tolerance (Process robustness) of purifying process is improved, even if virus liquid quality occurs obviously
Difference also can carry out calibration, the consistency between ensureing harvest group in batches by purification process.
Embodiment 2
The present embodiment is by by taking the purifying of japanese encephalitis virus particle as an example, and the present invention is further illustrated.
This experiment makees into one the purification process of the present invention by taking the P3 of Vero cell culture strain japanese encephalitis virus liquid as an example
The explanation of step.
1, virus-culturing fluid pre-processes
The virus-culturing fluid of harvest is filtered clarification with 0.45 μm of miillpore filter, obtains viral pretreatment fluid.
2, anion-exchange chromatography
(1) column equilibration：Capto-DEAE chromatographic columns are balanced, equilibrium liquid is：The 20mMol/L phosphoric acid buffers of pH8.0
Liquid.
(2) viruses adsorption：Virus liquid after filtering is clarified flows through the Capto-DEAE chromatographic columns after balance, and sample-adding terminates
Afterwards, continue to balance chromatographic column with the equilibrium liquid of 2-5 times of column volume.
(3) pre- elution：The chromatographic column for having adsorbed virus is eluted in advance with pre- eluent, pre- eluent is：PH8.0's
20mMol/L phosphate buffers (being 50mMol/L containing sodium chloride).
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.2's
20mMol/L phosphate buffers (being 300mMol/L containing sodium chloride), are received according to the optical absorption peak of tomographic system detector instruction
Collect eluent, obtains product A.
3, hydroxyapatite chromatography
(1) column equilibration：CHT chromatographic columns are balanced, equilibrium liquid is：The 5mMol/L phosphate buffers of pH7.2.
(2) viruses adsorption：Product A will be crossed and flow through the CHT chromatographic columns after balance, after sample-adding, with 2-5 times of column volume
Equilibrium liquid continues to balance chromatographic column.
(3) pre- elution：The chromatographic column for having adsorbed virus is eluted in advance with pre- eluent, pre- eluent is：PH7.2's
5mMol/L phosphate buffers.
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.2's
150mMol/L phosphate buffers collect eluent according to the optical absorption peak of tomographic system detector instruction, obtain target viral
Grain component.
Fig. 4 be 2 Vero cell flask cultures of embodiment harvest japanese encephalitis virus through different purification process after purification
SDS-PAGE electrophoresis silver staining analysis results (Marker：Protein molecular weight standard (Mr：Molecular weight, unit KD)；1 this method
Purification of samples；2 gel filtration method purification of samples；3 Ultracentrifugation Method purification of samples, 4 virus harvest liquids).It can from detection knot
To find out, compared with other purifying (separation) methods, the method for the present invention obtains in elution of virus liquid, and only there are one band, explanations
It is practically free of impurity in the sample, effect is isolated and purified with fabulous.
Fig. 5 is that the purified restrovirus particle Electronic Speculum of japanese encephalitis virus of 2 Vero cell flask cultures of embodiment harvest is shone
Piece.It can be seen from the figure that the virion obtained is complete, the form of different virus particle has typical without marked difference
Japanese encephalitis virus particle shape.The method of further illustrating the present invention can isolate and purify to obtain complete virion.
Embodiment 3
The present embodiment is by by taking the purifying of influenza virus particles as an example, and the present invention is further illustrated.
This experiment makees further the purification process of the present invention by taking the H1N1 influenza virus liquid of chick embryo culture as an example
It is bright.
1, virus-culturing fluid pre-processes
The virus-culturing fluid of harvest is filtered clarification with 1.2 μm of miillpore filter, obtains viral pretreatment fluid.
2, anion-exchange chromatography
(1) column equilibration：Capto-DEAE chromatographic columns are balanced, equilibrium liquid is：The 20mMol/L phosphoric acid buffers of pH7.6
Liquid.
(2) viruses adsorption：Virus liquid after filtering is clarified flows through the Capto-DEAE chromatographic columns after balance, and sample-adding terminates
Afterwards, continue to balance chromatographic column with the equilibrium liquid of 2-5 times of column volume.
(3) pre- elution：The chromatographic column for having adsorbed virus is eluted in advance with pre- eluent, pre- eluent is：PH7.6's
20mMol/L phosphate buffers (being 120mMol/L containing sodium chloride).
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.6's
20mMol/L phosphate buffers (being 500mMol/l containing sodium chloride), are received according to the optical absorption peak of tomographic system detector instruction
Collect eluent, obtains product A.
3, hydroxyapatite chromatography
(1) column equilibration：CHT chromatographic columns are balanced, equilibrium liquid is：The 20mMol/L phosphate buffers of pH7.6.
(2) viruses adsorption：Product A will be crossed and flow through the CHT chromatographic columns after balance, after sample-adding, with 2-5 times of column volume
Equilibrium liquid continues to balance chromatographic column.
(3) pre- elution：The chromatographic column for having adsorbed virus is eluted in advance with pre- eluent, pre- eluent is：PH7.6's
20mMol/L phosphate buffers.
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.6's
200mMol/L phosphate buffers collect eluent according to the optical absorption peak of tomographic system detector instruction, obtain target viral
Grain component.
Fig. 6 is the influenza virus of 3 chick embryo culture of embodiment harvest through different purification process SDS-PAGE Gel Electrophoresis Silvers after purification
Contaminate analysis result (Marker：Protein molecular weight standard (Mr：Molecular weight, unit KD)；1 virus harvest liquid；2 ultracentrifugations
Method purification of samples；3 gel filtration method purification of samples, 4 this method purification of samples).It can be seen from the figure that this method purifies
The amount of obtained viral contaminations substantially reduces, and viral associated proteins concentration significantly improves.
Fig. 7 is the influenza virus virion electromicroscopic photograph after purification of 3 chick embryo culture of embodiment harvest.As can be seen that obtaining
The virion obtained is complete, and the form of different virus particle has typical influenza virus form without marked difference.
Other enveloped virus, as measles virus, rubella virus, varicella virus, mumps virus, dengue fever virus or
Separation, the purifying of AIDS virus etc., can also be realized with reference to above-described embodiment under the premise of not having material alterations.
Claims (20)
1. a kind of method for separate coating virion, including virus liquid is subjected to ion-exchange chromatography and hydroxyapatite
Chromatographic runs, wherein ion-exchange chromatography carries out before hydroxyapatite chromatography；Or ion-exchange chromatography is in hydroxyapatite
It is carried out after chromatography.
2. according to the method described in claim 1, it is characterized in that, ion-exchange chromatography includes：
A) optional to pre-equilibrate operation：Ion exchange column is pre-equilibrated including using ion exchange to pre-equilibrate liquid；
B) loading operates：By virus liquid or sample liquid loading after hydroxyapatite chromatography to ion exchange column；
C) optional balancing run：Including using ion-exchange equilibrium liquid to be balanced ion exchange column；
D) optional pre- elution action：Including using the pre- eluent of ion exchange to elute ion exchange column in advance；
E) elution action：Ion exchange column is eluted using ion-exchanging eluent.
3. according to the method described in claim 2, it is characterized in that, further comprise using the second ion-exchanging eluent to from
Sub- displacement chromatography column is eluted.
4. according to the method described in claim 3, it is characterized in that, collecting the first ion-exchanging eluent and of wash-off respectively
Two ion-exchanging eluents obtain the virion of different structure, composition or purity.
5. according to Claims 1 to 4 any one of them method, which is characterized in that hydroxyapatite chromatography includes：
A) optional to pre-equilibrate operation：Pre- put down is carried out to hydroxyapatite chromatography column including using hydroxyapatite to pre-equilibrate liquid
Weighing apparatus；
B) loading operates：By virus liquid or sample liquid loading after ion-exchange chromatography to hydroxyapatite chromatography column；
C) optional balancing run：Including using hydroxyapatite equilibrium liquid to be balanced hydroxyapatite chromatography column；
D) optional pre- elution action：Including using the pre- eluent of hydroxyapatite to carry out prewashing to hydroxyapatite chromatography column
It is de-；
E) elution action：Hydroxyapatite chromatography column is eluted using hydroxyapatite eluent.
6. according to the method described in claim 5, it is characterized in that, further comprising using the second hydroxyapatite eluent pair
Hydroxyapatite chromatography column is eluted.
7. according to the method described in claim 6, it is characterized in that, collect respectively wash-off the first hydroxyapatite eluent and
Second hydroxyapatite eluent obtains the virion of different structure, composition or purity.
8. according to claim 1~7 any one of them method, which is characterized in that ion-exchange chromatography and hydroxyapatite layer
Without intermediate chromatographic runs between analysis.
9. according to the method described in claim 8, it is characterized in that, not having between ion-exchange chromatography and hydroxyapatite chromatography
Intermediary operation.
10. according to claim 1~9 any one of them method, which is characterized in that ion-exchange chromatography is anion exchange layer
Analysis.
11. according to the method described in claim 10, it is characterized in that, ion exchange pre-equilibrate liquid, ion-exchange equilibrium liquid, from
Son exchanges pre- eluent, ion-exchanging eluent, hydroxyapatite and pre-equilibrates liquid, hydroxyapatite equilibrium liquid, hydroxyapatite
Pre- eluent, hydroxyapatite eluent pH value stand alone as 7.0~9.5.
12. the method according to claim 10 or 11, which is characterized in that ion exchange pre-equilibrates liquid, ion-exchange equilibrium
The pre- eluent of liquid, ion exchange, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, hydroxyl phosphorus
The pre- eluent of lime stone, hydroxyapatite eluent stand alone as phosphate buffer, Tris-HCl buffer solutions, Tricine buffer solutions,
Hepes buffer solutions, glycine buffer, TEA buffer solutions, veronal buffer.
13. according to the method for claim 12, which is characterized in that ion exchange pre-equilibrate liquid, ion-exchange equilibrium liquid, from
Son exchanges pre- eluent, ion-exchanging eluent, hydroxyapatite and pre-equilibrates liquid, hydroxyapatite equilibrium liquid, hydroxyapatite
Pre- eluent, hydroxyapatite eluent are phosphate buffer.
14. according to claim 11~13 any one of them method, which is characterized in that ion exchange pre-equilibrates liquid, ion is handed over
Change the pre- eluent of equilibrium liquid, ion exchange, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid,
The pre- eluent of hydroxyapatite, hydroxyapatite eluent are independently added with water soluble salt.
15. according to claim 1~14 any one of them method, which is characterized in that enveloped virus includes rabies viruses, influenza
Virus, japanese encephalitis virus, measles virus, rubella virus, varicella virus, mumps virus, dengue fever virus or AIDS
Poison.
16. a kind of enveloped virus composition, which is characterized in that including being detached by any one of claim 1~15 the method
The virion arrived.
17. enveloped virus composition according to claim 16, which is characterized in that further include stabilizer.
18. enveloped virus composition according to claim 17, which is characterized in that stabilizer includes sucrose and albumin.
19. enveloped virus composition according to claim 18, which is characterized in that the mass percentage of sucrose is 0.5
~10%.
20. the enveloped virus composition according to claim 18 or 19, which is characterized in that the mass percentage of albumin
It is 1~20%.
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| CN112798787A (en) * | 2019-11-14 | 2021-05-14 | 安徽智飞龙科马生物制药有限公司 | Rabies vaccine antigen content detection method and reagent or kit |
| 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 |
| CN113117067A (en) * | 2021-04-16 | 2021-07-16 | 深圳市卫光生物制品股份有限公司 | Method for removing chick embryo host protein in rabies vaccine product |
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