CN108531464A - A kind of purification process of rabies viruses particle - Google Patents
A kind of purification process of rabies viruses particle Download PDFInfo
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Abstract
The invention discloses a kind of purification process of rabies viruses particle, including by virus liquid into the interchangeable anion-exchange chromatography of row order and hydroxyapatite chromatography.The advantageous effect of the method for the present invention is：The pre-treating technology requirement of rabies venom is low, and secondary destruction will not be caused to rabies viruses particle, 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 virion of textural anomaly efficiently separates by the structural homogeneity for improving rabies viruses particle.
Description
Technical field
The invention belongs to viral biology technical fields, and in particular to a kind of purification process of rabies viruses particle.
Background technology
Rabies viruses is a kind of enveloped virus, complicated and change greatly, and therefore, it is high, structure homogeneous to obtain purity
Rabies viruses particle is extremely difficult.
Rabies viruses particle is made of a single strand RNA molecule (about 11930 nucleotide) and 5 kinds of protein, wherein 20
The nucleocapsid that~150 L albumen, 950 P albumen and single stranded RNA composed structure are stablized, nucleocapsid outer wrapping are thin from host
The bilayer lipid membrane of born of the same parents adds between nucleocapsid and lipid film and has filled 1650 M albumen；Lipid film surface inserts different number
G-protein is connected to the sugar chain of different number and different length in G-protein.The quantity and degree of glycosylation of G-protein are to virion
Biological characteristics and immunological characteristic have significant impact.
The used medium matter of rabies viruses culture at present includes mouse brain, chicken embryo, duck embryos, suslik kidney primary cell, chicken embryo
No matter primary fibroblast virus, human diploid cell and Vero cells etc. use which kind of culture substrate, rabies viruses harvest
Liquid is all the mixture system of a complicated component.It includes the rabies viruses particle of various structures and various impurity.
Complete rabies viruses granular size about 75 × 180nm, molecular weight 5~8 × 108, sedimentation coefficient 600S, in sucrose
1.14~1.17g/ml of buoyant density in solution.Cast-off cells directly through about 5 μm, most cells fragment directly through 0.8 μm with
On, equal significantly greater than virions；Most of host cell proteins matter, carbohydrate, lipid and RNA (mainly tRNA and rRNA) point
Hereinafter, being significantly less than virion, (mRNA molecular weight is larger, but extremely unstable, the content in harvest liquid in 1000KD for son amount
Pettiness)；Host cell DNA then contains the series of components for being more than, being less than and being equivalent to virion size；Product related impurities
It is also the series of components of size unevenness, including is more than (viral agglomerate), is less than that (virolysis is crushed, without being made into virion
Subunit's macromolecular) and be equivalent to active ingredient size (virion size be close but G-protein copy number is low, glycosylation is different
Often, immunogenicity is low, therefore is generally classified as impurity) various composition.
Currently, the separation principle of rabies viruses 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.Due to disease
The molecular sieve property of part big molecular impurity is very close with active ingredient in malicious harvest liquid, is difficult by molecular sieve principle merely
It is 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 rabies viruses is derived from.During Virus culture, it will usually it is endless to generate a part of structure
Whole virion, as G-protein copy number is too low, glycosylation lacks or incomplete virus.The incomplete virus of these structures
The virion of particle and structural integrity has marked difference, but its molecular sieve in biological property and immunological properties etc.
Matter is sufficiently close to the normal virion of structure, is not readily separated.
Based on the above reason, existing rabies viruses particle purification process is difficult to have rabies viruses particle and other impurities
Effect separation, it more difficult to accomplish the different or abnormal rabies viruses particle 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 rabies viruses
It is very fragile, it is easily broken during being concentrated by ultrafiltration, causes the heterogeneity of rabies viruses particle even more serious.It is existing
The rabies venom requirement that purifying process obtains culture is relatively high, is only used for the rabies viruses of processing ad hoc approach culture
Liquid, once cultural method changes, then purifying process needs are changed on a large scale.Even if being obtained for Same Way culture
Rabies venom is purified, it is also difficult to be ensured the stability between product batches, can not be obtained the highly stable rabies of quality
Malicious particle.
Therefore, develop that a kind of purification technique route is succinct, separative efficiency is high, separation selectivity is good, to rabies viruses structure
Destructive small rabies viruses purification process has very important significance.
Invention content
It is an object of the invention to overcome the deficiencies of the prior art and provide a kind of purification process of rabies viruses particle.
The technical solution used in the present invention is：
A kind of purification process for rabies viruses particle, including rabies viruses virus liquid is subjected to anion-exchange chromatography
It is operated with hydroxyapatite chromatography, wherein anion-exchange chromatography carries out before hydroxyapatite chromatography；Or anion exchange
Chromatography carries out after hydroxyapatite chromatography.
As being further improved for the above method, anion-exchange chromatography includes：
It is optional to pre-equilibrate operation：Pre- put down is carried out to anion exchange chromatography including using ion exchange to pre-equilibrate liquid
Weighing apparatus；
Loading operates：By virus liquid or sample liquid loading after hydroxyapatite chromatography to anion exchange chromatography；
Optional balancing run：Including using ion-exchange equilibrium liquid to be balanced anion exchange chromatography；
Optional pre- elution action：Including using the pre- eluent of ion exchange to carry out prewashing to anion exchange chromatography
It is de-；
Elution action：Anion exchange chromatography is eluted using ion-exchanging eluent.
As being further improved for the above method, further comprise using the second ion-exchanging eluent to anion exchange
Chromatographic 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 anion-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.
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, there is no middle layer between anion-exchange chromatography and hydroxyapatite chromatography
Analysis operation.
As being further improved for the above method, without intermediate behaviour between anion-exchange chromatography and hydroxyapatite chromatography
Make.
As being further improved for the above method, ion-exchange chromatography ion exchange pre-equilibrate liquid, ion-exchange equilibrium liquid,
The pre- eluent of ion exchange, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxyapatite equilibrium liquid, hydroxy-apatite
The pre- eluent of stone, hydroxyapatite eluent pH stand 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.
A kind of rabies viruses grain fraction, is prepared by above-mentioned method.
A kind of rabies viruses grain fraction, is prepared by the process described above.
The beneficial effects of the invention are as follows：
(1) in the method for the present invention, the pre-treating technology requirement of rabies venom is low, and treatment conditions are mild, will not be to mad dog
Virion causes secondary destruction, 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 rabies viruses particle, passes through ion-exchange chromatography and hydroxyl phosphorus
Lime stone chromatographs, and target viral particle can be approached with molecular weight but the virion of textural anomaly efficiently separates.
(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 adhesive coomassie brilliant blue staining results that embodiment 1 purifies obtained target viral component
(band 1-3 passes through result after purification for the virus liquid that Vero cell flask cultures obtain；Band 4 is albumen Marker；Band
5-7 passes through result after purification for the virus liquid that Vero cell spinner bottle cultures obtain；Band 8-10 is that protein content is Vero thin
The virus liquid that born of the same parents' bioreactor culture obtains is by result after purification).
Fig. 2 is the virus liquid difference purification phase SDS-PAGE eggs that 1 Vero bioreactor cultures of embodiment obtain
White glue Coomassie brilliant blue analysis result (Marker：Protein molecular weight standard (Mr：Molecular weight, unit KD)；1 receives for virus
Obtain liquid；2 be ion-exchange chromatography elution fraction；3-4 is CHT elution fractions, and 3 be the sample before desalination, and 4 be the sample after desalination
Product).
Fig. 3 is the electricity for the virion that the virus liquid that 1 Vero bioreactor cultures of embodiment obtain purifies
Mirror photo.
Specific implementation mode
A kind of purification process for rabies viruses particle, including rabies viruses virus liquid is subjected to anion-exchange chromatography
It is operated with hydroxyapatite chromatography, wherein anion-exchange chromatography carries out before hydroxyapatite chromatography；Or anion exchange
Chromatography carries out after hydroxyapatite chromatography.
Anion exchange chromatography used in the technology of the present invention can be various existing anion exchange chromatography.Show
Example property anion exchange chromatography include but not limited to：DEAE cellulose, the Poros of Applied Biosystems
PI 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
Hydroxyapatite chromatography column includes but not limited to that (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, anion-exchange chromatography includes：
A) optional to pre-equilibrate operation：Pre- put down is carried out to anion exchange chromatography including using ion exchange to pre-equilibrate liquid
Weighing apparatus；
B) loading operates：By virus liquid or sample liquid loading after hydroxyapatite chromatography to anion-exchange chromatography
Column；
C) optional balancing run：Including using ion-exchange equilibrium liquid to be balanced anion exchange chromatography；
D) optional pre- elution action：Including using the pre- eluent of ion exchange to carry out prewashing to anion exchange chromatography
It is de-；
E) elution action：Anion exchange chromatography is eluted using ion-exchanging eluent.
Optional operating procedure can be according to the type of different anions displacement chromatography column, concrete model, virus liquid or sample
The case where liquid etc., is adjusted correspondingly.
As being further improved for the above method, further comprise using the second ion-exchanging eluent to anion exchange
Chromatographic 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 rabies viruses particle 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 anion-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.
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.
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 rabies viruses particle of different structure, composition or purity.
As being further improved for the above method, there is no middle layer between anion-exchange chromatography and hydroxyapatite chromatography
Analysis operation.
As being further improved for the above method, without intermediate behaviour between anion-exchange chromatography and hydroxyapatite chromatography
Make.
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
Some specific chromatography conditions include but not limited to the concrete model of anion exchange chromatography, hydroxyapatite chromatography column it is specific
Model, rabies viruses particle degree of glycosylation, phosphorylation degree, the composition of each solution and dosage etc..By to each pH value of solution
The adjusting of value obtains different solution properties, meets different 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 it is cloudy from
The concrete model of sub- displacement chromatography column, the concrete model of hydroxyapatite chromatography column, rabies viruses particle degree of glycosylation, phosphoric acid
Change degree, the composition of each solution and dosage etc..By the adjusting to each solution ph, different solution properties are obtained, are met
Different needs.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 (as it is cloudy from
Son, phosphate anion) concentration be about 1-20mM, 1-10mM, 2-8mM, 4-6mM or 5mM.
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.By adding water soluble salt in the solution, can further adjust each molten
The characteristic of liquid meets different needs.
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-
9.0,7.0-8.5,7.0-8.0,72-7.8,7.4-7.7,7.5-7.6 or 7.6.In some instances, the pre- eluent of ion exchange
PH value 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 elutes in advance
In liquid added with concentration be about 1-700mM, 10-600mM, 50-500mM, 100-350mM, 150-300mM, 175-275mM or
The NaCl of 250-300mM.In some instances, it is about 1- added with concentration in the pre- eluent of ion exchange (such as phosphate buffer)
The salt of 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、150-250mM、180-220mM、250-350mM、280-380mM、75-
225mM, 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.
A kind of rabies viruses grain fraction, is prepared by above-mentioned method.
With reference to embodiment, the present invention is further illustrated, and however, it is not limited to this.
In following embodiment, viral purity and virus protein proportion grading method are：By the virus component warp of purifying harvest
Denaturation SDS- polyacrylamide gel electrophoresises (SDS-PAGE) analysis is crossed, after Silver stain or coomassie brilliant blue staining, gel is set
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 viral eggs
The sum of white 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.
Embodiment 1
The present embodiment is with Vero cells (spinner culture, flask culture, bioreactor culture), human diploid cell, chicken
For the CTN-1V strain rabies venom of embryo 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 (sodium chloride containing 150mMol/L).
(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 (sodium chloride containing 200mMol/L).
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.6's
20mMol/L phosphate buffers (sodium chloride containing 500mMol/L) 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. 3.
Table 1, different culture media matter culture the characteristic of target viral grain fraction that obtains after purification of virus liquid
Fig. 1 is the SDS-PAGE protein adhesive coomassie brilliant blue staining results that embodiment 1 purifies obtained target viral component
(band 1-3 passes through result after purification for the virus liquid that Vero cell flask cultures obtain；Band 4 is albumen Marker；Band
5-7 passes through result after purification for the virus liquid that Vero cell spinner bottle cultures obtain；Band 8-10 is that protein content is Vero thin
The virus liquid that born of the same parents' bioreactor culture obtains is by result after purification).It can be seen from the figure that different culture media matter culture
After identical method isolated or purified, protein band position almost overlaps obtained rabies viruses particle.Fully
Illustrate by the rabies viruses particle of the method for the present invention after purification there is high purity, the method for the present invention can be adapted for purifying
Or the rabies viruses that separation different culture media matter culture obtains, substantially increase technique adaptability.
Fig. 2 is the virus liquid difference purification phase SDS-PAGE eggs that 1 Vero bioreactor cultures of embodiment obtain
White glue Coomassie brilliant blue analysis result (Marker：Protein molecular weight standard (Mr：Molecular weight, unit KD)；1 receives for virus
Obtain liquid；2 be ion-exchange chromatography elution fraction；3-4 is CHT elution fractions, and 3 be the sample before desalination, and 4 be the sample after desalination
Product).It can be seen from the figure that the method for the present invention can effectively remove the impurity in virus liquid, the good rabies of homogenieity are obtained
Malicious particle.
Fig. 3 is the electricity for the virion that the virus liquid that 1 Vero bioreactor cultures of embodiment obtain purifies
Mirror photo.It can be seen from the figure that virus particle structure is complete, there is typical rabies viruses particle shape.
Embodiment 2
The present embodiment by taking the CTN-1V of Vero cell culture strain rabies venom as an example, to the present invention purification process make into
The explanation of one 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 (sodium chloride containing 150mmol/L).
(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 (sodium chloride containing 200mmol/L).
(4) elution of virus：The chromatographic column after eluting in advance is eluted with eluent, eluent is：PH7.6's
20mmol/L phosphate buffers (sodium chloride containing 500mmol/L) 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 table 2, different elution fractions
Examples 1 and 2 the experimental results showed that, the present invention has the following advantages：
(1) pre-treating technology of the method for the present invention rabies venom requires low, in above-described embodiment, it is only necessary to use filtering
Method so that virus liquid is clarified, secondary destruction will not be caused to rabies viruses particle, while having that treating capacity is big, processing cost is low
The characteristics of.
(2) the method for the present invention improves the structural homogeneity of rabies viruses particle, passes through ion-exchange chromatography and hydroxyl phosphorus
Lime stone chromatographs, and target viral particle can be approached with molecular weight but the virion of textural anomaly efficiently separates.
(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.
(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.
Claims (14)
1. a kind of purification process for rabies viruses particle, including by rabies viruses virus liquid carry out anion-exchange chromatography and
Hydroxyapatite chromatography operates, wherein anion-exchange chromatography carries out before hydroxyapatite chromatography；Or anion exchange layer
Analysis carries out after hydroxyapatite chromatography.
2. according to the method described in claim 1, it is characterized in that, anion-exchange chromatography includes：
It is optional to pre-equilibrate operation：Anion exchange chromatography is pre-equilibrated including using ion exchange to pre-equilibrate liquid；
Loading operates：By virus liquid or sample liquid loading after hydroxyapatite chromatography to anion exchange chromatography；
Optional balancing run：Including using ion-exchange equilibrium liquid to be balanced anion exchange chromatography；
Optional pre- elution action：Including using the pre- eluent of ion exchange to elute anion exchange chromatography in advance；
Elution action：Anion exchange chromatography is eluted using ion-exchanging eluent.
3. according to the method described in claim 2, it is characterized in that, further comprising using the second ion-exchanging eluent to the moon
Ion exchange 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：
It is optional to pre-equilibrate operation：Hydroxyapatite chromatography column is pre-equilibrated including using hydroxyapatite to pre-equilibrate liquid；
Loading operates：By virus liquid or sample liquid loading after anion-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 elute hydroxyapatite chromatography column in advance；
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 anion-exchange chromatography and hydroxyapatite
Without intermediate chromatographic runs between chromatography.
9. according to the method described in claim 8, it is characterized in that, not having between anion-exchange chromatography and hydroxyapatite chromatography
There is intermediary operation.
10. according to claim 1~9 any one of them method, which is characterized in that ion-exchange chromatography ion exchange pre-equilibrates
Liquid, ion-exchange equilibrium liquid, the pre- eluent of ion exchange, ion-exchanging eluent, hydroxyapatite pre-equilibrate liquid, hydroxy-apatite
The pre- eluent of stone equilibrium liquid, hydroxyapatite, hydroxyapatite eluent pH stand alone as 7.0~9.5.
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 stand alone as phosphate buffer, Tris-HCl buffer solutions, Tricine buffer solutions, Hepes
Buffer solution, glycine buffer, TEA buffer solutions, veronal buffer.
12. according to the method for claim 11, 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.
13. according to claim 10~13 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.
14. a kind of rabies viruses grain fraction is prepared by claim 1~13 any one of them method.
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| 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 |
| 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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| CN108524929A (en) | 2018-09-14 |
| CN108531464B (en) | 2019-04-23 |
| CN108524929B (en) | 2019-05-07 |
| CN108531463B (en) | 2019-04-23 |
| CN108531463A (en) | 2018-09-14 |
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