CN105396128A - Method for purifying poliomyelitis virus liquid - Google Patents
Method for purifying poliomyelitis virus liquid Download PDFInfo
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- CN105396128A CN105396128A CN201510765993.6A CN201510765993A CN105396128A CN 105396128 A CN105396128 A CN 105396128A CN 201510765993 A CN201510765993 A CN 201510765993A CN 105396128 A CN105396128 A CN 105396128A
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Abstract
The invention provides a method for purifying poliomyelitis virus liquid and belongs to the technical field of preparation of biological products. According to the method for purifying poliomyelitis virus liquid, poliomyelitis virus liquid is ultra-filtered, concentrated and subjected to density gradient centrifugation or continuous flow density gradient centrifugation, ultra-filtration sucrose removing, sterilization and filtration, and therefore the purified poliomyelitis virus liquid is obtained. By means of the method, solid virus particles and hollow virus particles in the poliomyelitis virus liquid can be effectively separated, impurity protein can be effectively removed, the content of the solid virus particles in centrifugal products is greatly increased, and the purity degree, safety and effectiveness of sIPV vaccine can be improved easily.
Description
Technical field
The present invention relates to biological product preparation field, particularly, relate to the method for the purification poliovirus liquid in a kind of poliovirus inactivated vaccine preparation process.
Background technology
Poliovirus belongs to Picornaviridae (Picornaviridae) enterovirus genus (Enterovirus), includes single-stranded positive rna gene, and protein coat is icosahedron cubic symmetry structure, without peplos.Poliovirus has 3 kinds of serotypes, is respectively I, II and III type.Poliovirus granule comprises solid construction virion and hollow-core construction virion, and wherein solid construction virion can react with body cell and produce neutralizing antibody.
Poliomyelitis is the disease with very strong infectivity caused by poliovirus, the child of main infection less than five years old.The patient of 1/200 can produce the paralysis of irreversible leg, and more serious meeting causes the difficulty of swallowing and speaking.There is the patient of 5-10% because paralysis of respiratory muscle causes death.There is no the effective means for the treatment of ridge ash at present, the inoculation of poliomyelitis vaccine is the method for the most effectively preventing this disease to occur and infect.
The poliovirus vaccine of current listing has attenuated vaccine (OPV) and inactivated vaccine (IPV).OPV is one of main vaccine kind in majority state planned immunization.Within 1985 to 2003, have and be vaccinated with OPV vaccine more than 5.5 hundred million children in 94 countries.Although OPV is polioeradication played important function, but the OPV relevant case (VAPP) that the inoculation of OPV causes and vaccine-derived polioviruses (VDPV) threaten to be existed all the time, even becomes the problem affecting public health, social stability.In order to thorough polioeradication, WHO has formulated global polioeradication action plan, expects the wild virus of polioeradication in 2015 and at certification in 2018 whole world Poliomyelitis free.In the process, IPV and sIPV will be progressively used to replace OPV.
IPV uses street strain salk strain as raw materials for production, and strict to bio-safety class requirement, therefore the quantity of manufacturer is very limited; In addition, because the price of IPV is relatively high, the planned immunization of developed country and the private market of developing country is mainly used in.
WHO recommends developing country eliminating a kind of novel deactivation OPV produced by attenuated strain (Sabin strain) of ridge ash final stage use to replace the use of OPV vaccine, thus thoroughly eliminates the generation because inoculating VDPV and VAPP that this vaccine causes.This is eliminating final stage of ridge ash and under using the background of inactivated vaccine comprehensively, there is important supplementary meaning: use the Sabin-IPV that attenuated strain produces, comparatively use the IPV inactivated vaccine that street strain's (Salk strain) produces, have and produce the feature safer, cost is cheaper, be more suitable for promoting the use of in developing country.Do not allow within Chinese territory to use street strain to produce IPV, so produce IPV with Sabin strain to become inevitable.
Traditional sIPV vaccine purifying process generally includes the steps such as ultrafiltration and concentration, ion-exchange chromatography, aseptic filtration.Separation method cost compare in sIPV vaccine purifying process of ion-exchange chromatography is high, and cannot effectively by solid virion and hollow viral particle separation.In addition, because solid virus can be reacted with body cell, induction body produces neutralizing antibody, and Study On Immunogenicity confirms in rat body: the neutralizing antibody that solid virion produces is at least 10 times of hollow virion.Therefore, urgently need a kind of new purification process, morphology of virus different for sIPV two kinds can be separated, to improve effective titre of poliovirus liquid.
Summary of the invention
The object of the present invention is to provide a kind of method of purification poliovirus liquid.
The traditional purification process of the present invention to sIPV is improved, and original ion-exchange chromatography processing step is replaced with density gradient centrifugation (or Continuous Flow density gradient centrifugation) technique.The density medium that purification poliovirus adopts is sucrose solution, and sucrose is divided into high concentration sucrose solution and low concentration sucrose solution.High concentration sucrose solution is 55% ~ 60%, and low concentration sucrose solution is: 25% ~ 45%.Effectively can be separated according to sucrose density and there is immunogenic solid virion (sedimentation coefficient is about 150S) and immunogenicity is poor or the hollow virion of non-immunogenicity (sedimentation coefficient is about 80S), thus reach the object of selective collection target area.
Present invention efficiently solves chromatography method can not by a difficult problem for solid virion and hollow viral particle separation, not only increase the immunogenicity of unit dose sIPV virus liquid, and greatly reduce the vaccine prepared with this virus liquid use after side reaction, reduce production cost simultaneously.
The invention provides a kind of method of purified virus liquid, comprising:
(1) viral ultrafiltration and concentration liquid to be purified carries out sucrose density gradient centrifugation;
(2) monitor pol, collect the centrifugal liquid merging and be in same position.
The sucrose density gradient centrifugation method of step (1) is:
Surpass from buffer from lateral opening to pump into successively with 50 ~ 100rpm of centrifuge successively, viral ultrafiltration and concentration liquid, low concentration sucrose solution, finally pumps into high concentration sucrose solution until mesopore trickle with 50 ~ 150rpm; Centrifugal rotational speed is 28000 ~ 35000rpm, and centrifugation time is: 6 ~ 20 hours, and centrifuging temperature is 2 ~ 8 DEG C.
In the method for purified virus liquid of the present invention, the volume ratio surpassed from buffer and viral ultrafiltration and concentration liquid is 1:5 ~ 1:10; The volume ratio of virus ultrafiltration and concentration liquid and low concentration sucrose solution is 1:1 ~ 2:1; Can stop adding high concentration sucrose for centrifuge hollow trickle as long as high concentration sucrose adds standard.
Described low concentration sucrose mass percent is 25% ~ 45%, and high concentration sucrose mass percent is 55% ~ 60%.
Surpass the one be selected from from buffer in PB, PBS and PBST of 0.1M ~ 0.5M.
Different according to virion sedimentation coefficient of different nature, it is caused to be in different pol position, and then be separated and different surpass from liquid (centrifugal liquid), in the inventive method, it is collect and merge the centrifugal liquid being in same position that the collection of step (2) merges the centrifugal liquid being in same position, wherein be positioned at the pol position of pol 54% containing the centrifugal liquid of solid virus, the centrifugal liquid containing hollow virus and a small amount of solid virus is positioned at the pol position of 50%-54%.The centrifugal liquid of all the other pols is as liquid waste processing.
After collecting centrifugal liquid, also comprise and adopt ion exchange layer analysis method to carry out purification to centrifugal liquid, be specially: use DEAESepharoseFastFlow, detect 260/280 or 254/280 value of I, II, type III virion respectively, collect stream and wear peak.The pH value of ion exchange is according to the difference of Virus type stability, and the range of choice is pH6.5-7.5.The electrical conductivity of level pad and sample controls within the scope of 15-45ms/cm.
Preferably, the virus described in the inventive method is poliovirus.
Further, described poliovirus is poliovirus attenuation strain Sabin strain I type, II type, type III.
Further, in the inventive method, when poliovirus is Sabin strain poliovirus I, II, the pH value surpassed from buffer is: 6.0 ~ 7.0; When poliovirus is Sabin strain poliovirus III type, the pH value of ultracentrifugation buffer is: 6.5 ~ 7.5.
The vaccine adopting the method for purified virus liquid of the present invention to prepare also belongs to protection scope of the present invention.
The invention provides a kind of poliovirus Sabin strain inactivated vaccine utilizing said method to prepare.
Further, inactivated vaccine of the present invention mixes after the monovalent virus refined solution deactivation of poliomyelitis attenuated strain Sabin strain I type, II type, type III, adds protective agent and prepare.
In poliovirus inactivated vaccine of the present invention, the antigenic content of attenuated strain Sabin strain I type, II type, type III is respectively 7-25DU, 25-68DU, 25-68DU.
Preferably, in poliovirus inactivated vaccine of the present invention, the antigenic content of attenuated strain Sabin strain I type, II type, type III is respectively 12-18DU, 36-54DU, 36-54DU.
More preferably, in poliovirus inactivated vaccine of the present invention, the antigenic content of attenuated strain Sabin strain I type, II type, type III is respectively 15DU, 45DU, 45DU.
The invention provides the application of method in biological product preparation of above-mentioned purified virus liquid.
Further, the invention provides the application of method in preparation poliovirus inactivated vaccine of above-mentioned purified virus liquid.
Particularly; above-mentioned application refers to carries out purification according to the method for purified virus liquid of the present invention respectively by attenuated strain Sabin strain I type, II type, type III antigen in poliovirus inactivated vaccine; mix after deactivation respectively, the poliovirus inactivated vaccine that it is raw material that interpolation protective agent prepares with attenuated strain Sabin strain.
The method of purification poliovirus provided by the invention, its density medium is sucrose solution, and sucrose is divided into high concentration sucrose solution and low concentration sucrose solution.High concentration sucrose solution is 55% ~ 60%, and low concentration sucrose solution is: 25% ~ 45%.Effectively can be separated according to sucrose density and there is immunogenic solid virion (sedimentation coefficient is about 150S) and immunogenicity is poor or the hollow virion of non-immunogenicity (sedimentation coefficient is about 80S), thus reach the object of selective collection target area.The method of density gradient centrifugation provided by the invention, not only effectively can be separated the solid virion of poliomyelitis and hollow virion.And effectively removes impurity protein, substantially increase the content of solid virion in centrifugal product, improve the purity of sIPV vaccine, the safety of vaccine and effectiveness.
By technique scheme, the present invention at least has the following advantages and beneficial effect:
(1) by method provided by the invention can be more simple and effective be separated the solid virion of poliomyelitis I, II and III type virus and hollow virion, viral purity is higher, and effective titre is high, heteroproteins remove more thorough.
(2) the poliovirus granule of method purification provided by the invention is utilized to have stronger immunogenicity.
(3) the Polio virus antigens response rate utilizing method provided by the invention to be separated is high and the safety of vaccine is higher.
(4) utilize the production cost of method provided by the invention separation poliovirus lower, be applicable to large-scale production, attenuated strain Sabin strain can be utilized to produce poliomyelitis vaccine,Salk.Very big reduction enterprise produces the production cost of poliomyelitis vaccine,Salk, overcome and use street strain salk strain as raw materials for production, and problem that limited amount, price higher strict to bio-safety class requirement, produces poliomyelitis vaccine,Salk for adopting attenuated strain Sabin strain and provides a kind of new method.
Detailed description of the invention
Following examples further illustrate content of the present invention, but should not be construed as limitation of the present invention.Without departing from the spirit and substance of the case in the present invention, the amendment do the inventive method, step or condition or replacement, all belong to scope of the present invention.
If do not specialize, the conventional means that technological means used in embodiment is well known to those skilled in the art; Reagent used in embodiment is commercial goods.
The preparation of embodiment 1 cell work seed and the preparation of virus liquid
1, high-density cells work Spawn preparation
Use 199 culture fluid dilution Vero cell seeds, dilution ratio is 60:1, is inoculated into 175cm
2cultivate in cell square vase, cultivation temperature is 36.0 DEG C ± 1 DEG C.Treat that cell is paved with cell bottle, continue by 1:4 ~ 1:6 4 generations of going down to posterity.In last generation, reaches 5 40 layer cell factory, carries out trypsinization, centrifugally removes supernatant.Use the cryopreserving liquid containing 10%DMSO to carry out resuspended, after mixing, cell concentration is 2.5 × 10
7cells/ml.Cell seed is carried out gradient cooling at programmed cooling instrument, keeps decline per minute 1 DEG C.Move in liquid nitrogen after temperature is down to-196 DEG C and preserve, the effect duration of working cell seed is 10 years.
2, the preparation of poliomyelitis I, II, type III stock solution
(1) one-level cell culture takes microcarrier, spends the night with 0.01MPBS is swelling, with appropriate PBS rinsing 2 times, is placed in the 121 DEG C of sterilizings of high pressure steam sterilization cabinet.Taken out from liquid nitrogen by cell seed, in the water-bath of 39 ± 1 DEG C, shake thawing rapidly, after counting, add in growth-promoting media, carry out one-level cell culture, volume of culture is about 5L.Adjust each culture parameters, temperature is 36.5 DEG C, pH value is 7.20, dissolved oxygen is 50%, rotating speed is 30rpm.Cell culture is to during plateau 2.5 × 10
6cells/ml, carries out had digestive transfer culture.
(2) secondary cell is cultivated cell step (1) cultivated and is prepared into cell suspension through digestion, be seeded to 20L cell reactor, adjust each culture parameters, temperature is 36.5 DEG C, pH value is 7.20, dissolved oxygen is 50%, rotating speed is 30rpm, start to cultivate.Cell culture is to during plateau 5 × 10
6cells/ml, prepares to digest.
(3) three grades of cell culture
Step (2) cultivated after cell be prepared into cell suspension, be seeded in working volume 120L cell reactor, set temperature be 36.5 DEG C, pH value is 7.20, rotating speed is 30rpm, dissolved oxygen is 50%.Cell density is cultured to 1.5 × 10
6cells/ml prepares virus inoculation.
3, virus liquid is cultivated and is inoculated poliovirus Sabin strain I, II, type III seed respectively by MOI0.001, culture parameters is adjusted to rotating speed: 30rpm, temperature: 32.5 DEG C, pH value: 7.40, dissolved oxygen: 25%.Treat that cells on microcarriers pathological changes is more than 95%, stop cultivating, results virus liquid, incubation time is 49 hours.
4, the ultrafiltration and concentration of virus liquid
1) virus liquid is 3-0.8 μm through continuous 3 grades of apertures by clarification, the filter element filtering of 0.8-0.65 μm, 0.65-0.22 μm or centrifugal (comprising continuous flow centrifugation) rotating speed be 2000-5000rpm, centrifugation time is 0.5-4 hour, obtains viral clear liquor;
2) concentrating virus clear liquor concentrates through the Pall slipstream membrane filtration system of 3,000,000 molecular cut offs, and cycles of concentration is 100 times.
The purification of embodiment 2Sabin strain poliomyelitis I type, II type, III virus liquid and compliance test result
1, low, the high concentration of sucrose solution are respectively 25%, 55%.Density gradient centrifugation buffer is 0.5mol/LPBS buffer, and its pH value is: 6.5.Use Hitachi CP70MX/ME supercentrifuge, 100ml ultracentrifugation buffer is pumped into 100rpm successively from lateral opening, sIPVI type that embodiment 1 ultrafiltration and concentration of about 700ml obtains (or sIPVII type or sIPVIII type) viral ultrafiltrate, low concentration sucrose solution 500ml, finally pump into high concentration sucrose solution until mesopore trickle with 100rpm, at 4 DEG C through the centrifugal 10h of 30000rpm ultrafiltration.Centrifugal complete after, 500ml collected by the first pipe, and collect with 50ml centrifuge tube afterwards, 50ml/ manages, and can collect 25-27 pipe.Detect antigenic content and the protein content of often pipe, the part (pol be 54% surpass from liquid 1) only containing Effective Antigens composition is merged respectively according to the testing result equal-volume of pol, pol, antigenic content, protein content, the HCP content from liquid is surpassed to two kinds with the part (pol is that 50%-54% surpasses from liquid 2) of the non-effective antigenic component containing mixing and Effective Antigens composition, BSA content and DNA content detect, and testing result is in table 1, table 2, table 3.
2, establish controlled trial: adopt traditional handicraft sieve chromatography to carry out viral purification, pillar model is xk16/100, and filler is SepharoseCL6B simultaneously simultaneously.Applied sample amount is 3.5% of column volume.UV detector determined wavelength 260,280 and 254nm.Arranging system flow rate is 15cm/h (0.5ml/min).Collect the 2nd UV absworption peak, i.e. molecular sieve purification liquid.Detectable antigens content, protein content, HCP content, BSA content and DNA content, the results are shown in Table 1, table 2, table 3.
3, ion exchange is in XK50/30 chromatographic column, loads SephasoreDEAEFastFlow chromatographic stuffing, connects AKTAavant150 tomographic system.Balance with the phosphate buffer (pH6.5) of 0.15M, respectively to various surpassing from liquid 1, surpassing and test from liquid 2 and molecular sieve purification liquid, detect uv absorption wavelength 280/254nm absorption value, collection stream is worn peak and is ridge ash I, II and type III viral purification liquid.
Table 1sIPV I type surpasses from liquid 1, surpasses from liquid 2 and molecular sieve purification fluidity matter
Table 2sIPV II type surpasses from liquid 1, surpasses from liquid 2 and molecular sieve purification fluidity matter
Table 3sIPV III type surpasses from liquid 1, surpasses from liquid 2 and molecular sieve purification fluidity matter
By the display of table 1-table 3 result of study, the Effective Antigens component content surpassed from liquid 1 (containing solid virus) is not less than and surpasses from liquid 2 (containing hollow virus and a small amount of solid virus) refined solution, far above molecular sieve purification liquid; And major impurity DNA, BSA, HCP content aspect is significantly lower than surpassing from liquid 2 and molecular sieve purification liquid.
Virus liquid after embodiment 3Sabin strain poliomyelitis I type, II type, III purification is prepared into the indices evaluation of vaccine
1, impurity content
The surpassing from liquid 1 and surpass from liquid 2 of the Sabin strain poliomyelitis I type that embodiment 2 is obtained, II type, type III, and the molecular sieve purification liquid using traditional handicraft to obtain in controlled trial, carry out the purification steps such as ion exchange, ultrafiltration and concentration and deactivation again, obtain monovalent virus bulk.Other unit price stock solution various obtained is carried out dilution and equal proportion mixing according to antigenic content in finished product, and the antigenic content of final finished is I type 36DU, II type 48DU, type III 36DU.Then according to the antigen amount of finished product definition respectively proportioning obtain finished product 1 (only containing Effective Antigens composition), finished product 2 (containing Effective Antigens composition and non-effective antigen composition) and finished product 3 (preparing by traditional handicraft).The impurity content of three finished products the results are shown in Table 4.According to statistical analysis, all there were significant differences and be less than the impurity content (P value is all less than 0.05) of finished product 2 for the impurity of the finished product 1 of I type, II type and type III and finished product 2 (bovine serum albumin is residual, Vero cell protein is residual, protein content, DNA remain); All there were significant differences and be less than the impurity content (P value is all less than 0.05) of finished product 3 for the finished product 1 of I type, II type and type III and the corresponding impurity content of finished product 3.The results are shown in Table 4.
Table 46 crowd vaccine finished product major impurity residual quantity result *
If numerical value is for being less than (<) value when * calculating P value, calculate according to this value.If numerical value is value range, calculate according to the meansigma methods of scope bound.
2, immunogenicity
According to the antigenic content that finished product 1, finished product 2 and finished product 3 detect, prepare immunogenicity sample respectively, above-mentioned sample is carried out 3 times of serial dilutions, totally 4 dilution factors; International standard substance is diluted to simultaneously I, II, type III antigenic content to 15DU, 45DU, 45DU, and carry out 3 times of serial dilutions on this basis, totally 4 dilution factor samples, each dilution factor sample carries out single two hindlimb muscle immune rat respectively, and only, 10 (male and female half and half) are dilution factor often for 0.5ml/, immunity blood sampling in latter 21 days, separation of serum, detects the NAT of anti-I, II, type III in serum respectively, calculates serological conversion rate.The serological conversion rate of finished product 1, finished product 2 and finished product 3 and neutralizing antibody geometrical mean GMT level are in table 5.According to statistical analysis, the finished product 1 of I type, II type and type III and the neutralizing antibody GMT value of finished product 2 are all without significant difference (P value is all greater than 0.05); The finished product 1 of I type, II type and type III and the neutralizing antibody GMT value of finished product 3 are all without significant difference (P value is all greater than 0.05).The immunogenicity of three kinds of finished products the results are shown in Table 5.
Table 5 finished product 1, finished product 2 and finished product 3 immunogenicity result
3, stability
Under finished product 1, finished product 2 and finished product 3 are stored in 2-8 DEG C of environment, temporally sample detectable antigens content, outward appearance, pH value, osmotic pressure molar density, bacterial endotoxin, content of formaldehyde, BSA content, HCP content, DNA content, protein content etc.Record data to 18 month, the results are shown in Table 6-table 12.Finished product 1 (only containing solid virus), finished product 2 and finished product 3 are stored 18 months under 2-8 DEG C of environment, and the stability of finished product 1 and finished product 2, finished product 3 are quite showed no obvious decline; Meanwhile, major impurity content finished product 1 is far below finished product 2, finished product 3.Stability result is in Table 6-table 12.
The D-antigenic content testing result (percentage ratio of labelled amount) of table 62 ~ 8 DEG C finished product 1
* the percent value of labelled amount is the percent value compared with 0 month value.
The D-antigenic content testing result (percentage ratio of labelled amount) of table 72 ~ 8 DEG C finished product 2
* the percent value of labelled amount is the percent value compared with 0 month value.
The D-antigenic content testing result (percentage ratio of labelled amount) of table 82 ~ 8 DEG C finished product 3
* the percent value of labelled amount is the percent value compared with 0 month value.
Table 92 ~ 8 DEG C protein content testing result
Table 102 ~ 8 DEG C DNA content testing result
Table 112 ~ 8 DEG C BSA content detection result
Table 122 ~ 8 DEG C HCP content detection result
Finished product 1 (only containing solid virus), finished product 2 and finished product 3 are stored 18 months under 2-8 DEG C of environment, and the stability of finished product 1 and finished product 2, finished product 3 are quite showed no obvious decline; Meanwhile, major impurity content method, finished product 1 is far below finished product 2, finished product 3.
Although above the present invention is described in detail with a general description of the specific embodiments, on basis of the present invention, can make some modifications or improvements it, this will be apparent to those skilled in the art.Therefore, these modifications or improvements without departing from theon the basis of the spirit of the present invention, all belong to the scope of protection of present invention.
Claims (10)
1. a method for purified virus liquid, is characterized in that, comprises the following steps:
(1) viral ultrafiltration and concentration liquid to be purified carries out sucrose density gradient centrifugation;
(2) monitor pol, collect the centrifugal liquid merging and be in same position.
2. method according to claim 1, is characterized in that, the sucrose density gradient centrifugation method of step (1) is:
Surpass from buffer from lateral opening to pump into successively with 50 ~ 100rpm of centrifuge successively, viral ultrafiltration and concentration liquid, low concentration sucrose solution, finally pumps into high concentration sucrose solution until mesopore trickle with 50 ~ 150rpm; Centrifugal rotational speed is 28000 ~ 35000rpm, and centrifugation time is: 6 ~ 20 hours, and centrifuging temperature is 2 ~ 8 DEG C.
3. method according to claim 2, is characterized in that, the volume ratio surpassed from buffer and viral ultrafiltration and concentration liquid is 1:5 ~ 1:10; The volume ratio of virus ultrafiltration and concentration liquid and low concentration sucrose solution is 1:1 ~ 2:1; Can stop adding high concentration sucrose for centrifuge hollow trickle as long as high concentration sucrose adds standard.
4. method according to claim 2, is characterized in that, described low concentration sucrose mass percent is 25% ~ 45%, and high concentration sucrose mass percent is 55% ~ 60%.
5. method according to claim 2, is characterized in that, surpasses the one be selected from from buffer in PB, PBS and PBST of 0.1 ~ 0.5M.
6. according to the arbitrary described method of claim 1-5, it is characterized in that, it is collect and merge the centrifugal liquid being in same position that the collection of step (2) merges the centrifugal liquid being in same position, wherein be positioned at the pol position of pol 54% containing the centrifugal liquid of solid virus, the centrifugal liquid containing hollow virus and a small amount of solid virus is positioned at the pol position of 50%-54%.
7., according to the arbitrary described method of claim 1-5, it is characterized in that, described virus is poliovirus.
8. method according to claim 7, is characterized in that, described poliovirus is poliovirus attenuation strain Sabin strain I type, II type, type III.
9. method according to claim 8, is characterized in that, when poliovirus is Sabin strain poliovirus I, II, the pH value surpassed from buffer is: 6.0 ~ 7.0; When poliovirus is Sabin strain poliovirus III type, the pH value of ultracentrifugation buffer is: 6.5 ~ 7.5.
10. the application of the arbitrary described method of claim 1-9 in preparation poliovirus inactivated vaccine.
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
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| CN108607697A (en) * | 2018-05-09 | 2018-10-02 | 宜兴市华鼎机械有限公司 | A kind of centrifugal immunogenic production process |
| CN110168081A (en) * | 2016-11-04 | 2019-08-23 | 百深公司 | Adeno-associated virus purification process |
| CN113227349A (en) * | 2018-10-19 | 2021-08-06 | 尤尼沃尔塞尔斯技术股份公司 | Method for cleaning a biomolecule production system and system suitable for cleaning |
-
2015
- 2015-11-11 CN CN201510765993.6A patent/CN105396128A/en active Pending
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| 石慧颖等: "大规模区带离心纯化Vero细胞乙脑疫苗", 《中国病毒学》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110168081A (en) * | 2016-11-04 | 2019-08-23 | 百深公司 | Adeno-associated virus purification process |
| CN108607697A (en) * | 2018-05-09 | 2018-10-02 | 宜兴市华鼎机械有限公司 | A kind of centrifugal immunogenic production process |
| CN108607697B (en) * | 2018-05-09 | 2019-11-01 | 宜兴市华鼎机械有限公司 | A kind of centrifugal immunogenic production process |
| CN113227349A (en) * | 2018-10-19 | 2021-08-06 | 尤尼沃尔塞尔斯技术股份公司 | Method for cleaning a biomolecule production system and system suitable for cleaning |
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