CN1966076A - Mass-production method of hydrophobic vaccine - Google Patents
Mass-production method of hydrophobic vaccine Download PDFInfo
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Abstract
The invention relates to a method for producing batch hydrophobia vaccine, wherein it comprises that: (1), in the biological reactor with fixed bed mixing system and Fibra-Cel disks carrier, using cell increment cultivate liquid to cultivate Vero cell; (2), when the Vero cell grows to some density, using cell hold cultivate liquid, seeding hydrophobia vaccine, and affecting cell; (3), increasing virus; (4), obtaining virus continuously; (5), ultra-filter concentrating and inactivating via beta-propanolide; (6), using protamine sulfate or DNA enzyme treatment to remove Vero cell DNA; (7), using Sepharose 4 Fast Flow as stuff to process chromatography purification; (8), adding human blood albumin and sugar as protector to be made into liquid agent; or adding human blood albumin, sugar, and gelatin, as protector and shaping agent to be made into dried agent. The inventive method can produce virus continuously in small biological reactor, to realize batch production.
Description
Technical field
The invention belongs to biological pharmacy technical field, relate to the method for a kind of large-scale production rabies vaccine specifically.
Background technology
Rabies are that a kind of in a single day the people is carried this type of animal bite of rabies virus by zoogenous natural epidemic disease source sexually transmitted diseases such as Canis familiaris L., cat, pig, mouse and Vespertilios, will infection morbidities, and its case fatality rate is 100%.About annual nearly 30,000 people of Asia rabies example, China is the rabies district occurred frequently according to incompletely statistics, and the eighties is annual dead more than 5000 people, enter the nineties and slightly descend, but China's rabies sickness rate is on the rise in recent years.Rabies do not have special effective treatment means, and injecting pstudorabies vaccine is unique effective means of control rubies epidemiology.
Prove that since Louis Pasteur in 1885 the rabbit spinal cord that has infected fixed virus has remarkable effect to exposing the back immunity, Antirabic Vaccine's development history has experienced in 1 hundred 20.Distinguish with training method, the Antirabic Vaccine can be divided into tissue culture's vaccine and cell culture vaccine two big classes.The cell culture vaccine can be divided into HDCV (HDCV), primitive cell culture vaccine and continuous cell line vaccine three major types again.
The Vero cell is the Verorab rabies vaccine, a kind of in the continuous cell line vaccine, and it is a kind of high-purity rabies vaccine, is Antirabic Vaccine comparatively commonly used in the world at present.
The key of producing rabies vaccine is to turn out the high titre rabies virus of q.s.The mass cell cultivation is the important technical links in the vaccine product production technology.In the past during the last ten years, this technology has experienced secondary development, promptly uses attached cells such as roller bottle, CellCube to cultivate from the first generation, develops into second filial generation bioreactor (Bioreactor) and carries out the mass cell cultivation.The key problem of first generation cell culture technology is to be difficult to large-scale production: the one, and can not the mass preparation product when explained hereafter; The 2nd, produce the inhomogeneity that causes product quality easily in batches; The 3rd, be difficult to producing and quality control with criticizing.At present, abroad some vaccine manufacturing companies are developing the extensive animal cell culture technology of the second filial generation, and it is the direction of vaccine product suitability for industrialized production.Zooblast High Density Cultivation The Application of Technology adopts Cytodex 1 suspending carrier more in the world, and cell density can reach 10
7Individual/milliliter.But since in bioreactor loading amount less (3～5 grams per liter), can only do a batch cultivation, and industrialized scale needs the bioreactor of larger volume, generally need reach 1000 liters, equipment and auxiliary facility cost are higher.
Summary of the invention
Purpose of the present invention just is to overcome the defective of above-mentioned prior art, and a kind of method of the large-scale production rabies vaccine of results virus continuously that realizes in small biological reactor is provided.
For realizing that the technical scheme that goal of the invention of the present invention is taked is:
The method of a kind of large-scale production rabies vaccine is characterized in that its processing step is:
(1) be in the bioreactor with the basket stirring system of fixed bed of carrier with Fibra-Cel disks (polyester sheet), with cell proliferation culture fluid Cultivation of Vero;
(2) when the Vero cell grows to certain density, use cell maintenance culture solution instead, inoculation rabies virus (CTN-1 strain, aGV strain), and make it infection cell;
(3) make virus multiplication;
(4) results are viral continuously;
(5) ultrafiltration and concentration is also used the beta-propiolactone deactivation;
(6) handle to remove the Vero cell DNA with protamine sulfate or DNA enzyme;
(7) with Sepharose 4 Fast Flow (Chinese name: be that filler carries out chromatography purification agarose gel 4FF);
(8) add the human albumin, sucrose is that protective agent is made liquid preparation, or add human albumin, sucrose, gelatin and make lyophilized formulations as protective agent and excipient.
The Vero cell is the monkey-kidney cells system (being African green monkey kidney cell) that Japanese scholar set up from African green monkey kidney in 1962, in its 93rd generation, be brought to the allergy and the infectious disease institute tropic virus research department of NIH (NIH), in the 113rd generation, be submitted to American type culture collection (ATCC), is numbered ATCC NO.CCL-81.Through comprehensively research evaluation, the Vero cell has nuclear and learns stable, there is not exogenous factor to pollute and the advantage of no oncogenicity in certain generation, meet the rules requirement of The World Health Organization (WHO) fully, and be can be used as the substrate of production of vaccine by the WHO approval about the passage cell that is used for biological product.The Vero cell kind that the present invention uses derives from Chinese typical culture collection center (CCTCC), and can trace back to ATCC, at first sets up Vero cell master seed bank and work seed bank, and it is carried out the calibrating of system.Before the preparation vaccine, need carry out recovery, the rolling bottle amplification cultivation of cell earlier, again with 2～5 * 10
5The density of/ml is seeded in the bioreactor, carry out amplification cultivation with the cell proliferation culture fluid, it is formulated that used cell proliferation culture fluid uses 199 culture medium to add 2～10% Ox blood serums, for preventing germ contamination, in preparation cell proliferation culture fluid, can add an amount of gentamycin sulfate.The cell proliferation culture condition is pH value 6.9～7.4 (preferable is 7.2), 36～38 ℃ of temperature (preferable is 37 ℃), dissolved oxygen (DO) 30～70% (preferable is 50%), mixing speed 30～150rpm (that preferable is 120rpm).
In the method for the invention, used Celligen Plus, BioFlo 4500 and BioFlo Pro bioreactor are available from New Brunswick Scientific Co., Inc. company, the tank volume that Celligen Plus is contained is 2.2 liters, 5 liters, 7.5 liters, 14 liters, the tank volume that BioFlo4500 is contained is 20 liters, 30 liters, the tank volume that BioFlo Pro is contained is 75 liters, 150 liters, 300 liters, all can carry out the automatic control of temperature, pH value, dissolved oxygen, mixing speed.
The Vero cell is an anchorage-dependent cell, and cultivating in bioreactor needs attached to growing on certain carrier, and the used carrier of the present invention is Fibra-Cel disks (polyester sheet), and use amount is every liter of tank volume 30～40 grams.
Vero cell amplification cultivation in bioreactor can be inoculated rabies virus after 6～12 days, and the rabies virus that the present invention uses is CTN-1 strain and aGV strain, and the CTN-1 strain derives from Nat'l Pharmaceutical ﹠ Biological Products Control Institute, and the aGV strain derives from Wuhan Biological Products Inst..Wash with the residual Ox blood serum of flush away with pH7.6 PBS solution or 199 culture medium before the inoculation rabies virus, change cell maintenance culture solution then, it is formulated that used cell maintenance culture solution uses 199 culture medium to add 0.05%～0.5% human albumin, and the rabies virus inoculum concentration is 0.025～0.125MOI infection multiplicity (MOI: the virus numbers that is used to infect and the ratio of cell number) or final concentration 4.5～5.5lgLD
50/ ml (LD
50: half lethal dose).It is pH value 7.2～8.0 (preferable is 7.6) that cell is kept culture condition, and what 32～36 ℃ of temperature were preferable is 35 ℃), dissolved oxygen (DO) 30～70% (preferable is 50%), mixing speed 30～150rpm (that preferable is 120rpm).The growth and breeding of rabies virus on the Vero cell can be kept the long period, but uses bioreactor continous pouring results, can gather in the crops continuously more than 20 days, and virus titer remains on more than the 7lg LD50/ml.
The viral liquid of results concentrates 30～100 times with the ultrafilter membrane that 100K～the 300K molecular weight is held back, and uses beta-propiolactone deactivation in 1: 4000 then.
The key for preparing the biological product safety with passage cell is the content of cell residue DNA, and by the requirement of WHO rules, the substrate DNA of vaccine must be less than the 100pg/ agent.It is to utilize protamine sulfate electrically charged opposite with DNA institute that the present invention removes one of method of Vero cell DNA, and mutually combining forms precipitation, removes by high speed centrifugation.The concrete practice be while stirring in the inactivation of viruses concentrated solution protamine sulfate to final concentration 1～2mg/ml, in 2～8 ℃ of effects 2～4 hours, then in 2～8 ℃ of 7000～8000rpm, centrifugal 15 minutes.Another kind method is with DNase (DNA enzyme) the Vero cell DNA in the concentrated solution to be cut, and at the DNase I that adds final concentration 0.5～1.5U/ml in the viral concentrated solution of deactivation, acts on 6～10 hours under the room temperature in the concrete operations.
Remove behind the Vero cell DNA with Sepharose 4 FastFlow (Chinese name: be that filler carries out chromatography purification agarose gel 4FF) with one of above-mentioned two kinds of methods, testing result shows, the virus stock solution used total protein content reduces approximately more than 99% behind the purification of above-mentioned processing, and Ox blood serum all meets the relevant rules requirement of WHO with Vero cell DNA residual volume.
Virus stock solution used adds the human albumin behind the purification, sucrose is that protective agent is made liquid preparation, or adds human albumin, sucrose, gelatin and make lyophilized formulations as protective agent and excipient.
Above-mentioned Fibra-Cel disks carrier be by Britain BIBBY STERILIN COMPANY according to the cGMP standard production, form by polyester non-woven fibre and polypropylene.Fibra-Cel disks carrier also makes suspension cell attach to carrier easily through Electrostatic Treatment, and is fixed in the fiber system in incubation always.The about 1200cm of every gram Fibra-Cel disks carrier surface area
2, disks diameter 6mm, 10 gram disks carrier bed volumes are 100ml, required cell inoculation density 2～5 * 10
5/ ml can autoclaving.
The present invention is a cell carrier with above-mentioned Fibra-Cel disks, and to select the bioreactor with the basket stirring system of fixed bed for use be culture systems, has the following advantages:
1. the pressure drop that runs through the whole bed body is little.Each position of whole bed body keeps the cell growth microenvironment of homogeneous, and helps the amplification of bioreactor.
2. have higher specific surface area.Increase bioreactor inner cell total amount, thereby significantly increase the output of emiocytosis thing.
3. the suffered shearing force of cell is less.Use bed technology, cell over head and ears in the Fibra-Cel disks carrier, has shielded the turbulent flow that stirring arm rotation and bubble jet are produced.
4. nutrition efficiently and oxygen transmission.Because the shearing force in the fixed bed is very little, can use higher mixing speed, thereby improve the transmission of nutrition and oxygen, make the cell that is positioned at fixed bed bottom and top have same vigor.
5. isolated cell secretory product simply efficiently.Owing to adopt fixed bed, cell fixation is in cultivating on the carrier, and its secretory product discharges to culture fluid, and therefore the clogging that does not have the microactuator suspension carrier to take place does not usually also need by filtering cell to be separated with its secretory product.
6. the cell attachment time lacks, and required cell inoculation density is low.Cell attachment approximately needs 6 hours (inoculum densities 1 * 10 in traditional microcarrier (Cytodex 1 suspending carrier)
6/ ml), and adherent in the Fibra-Cel disks carrier (inoculum density 3 * 10 that only needs 15-60 minute
5/ ml).
Adopt method of the present invention can be implemented in the small biological reactor, results virus realizes large-scale production continuously.
Below for using 5L, 14L, 30L bioreactor pilot production:
Adopt 5L Celligen Plus bioreactor respectively, every canned year Fibra-Cel Disks carrier 192 grams; 14L Celligen Plus bioreactor, every canned year Fibra-Cel Disks carrier 500 grams; 30L BioFlo 4500 bioreactors, every canned year Fibra-Cel Disks carrier 1000 grams; The Vero cell, our company is own, conforms with production of vaccine cellular matrix requirement through evaluation; Work strain aGV
15, our company is own; Cell proliferation culture medium: 199 (Gibco) add 10% new-born calf serum, transfer pH 7.0; Cell is kept culture medium: 199 (Gibco), add 0.4% human albumin, and transfer pH 7.5; New-born calf serum: Hangzhou Sijiqing Biological Engineering Material Co., Ltd.; The human albumin: Shenzhen light is defended actor playing a martial role in Chinese operas's Tetramune company limited.
Adopt the cell proliferation culture medium, setting the bioreactor culture condition is pH 7.0,37 ℃ of temperature, and DO 50%, and mixing speed 90rpm is with density 5 * 10
5/ ml inoculating cell, cultivate 9 days with cell proliferation after, with the 0.025M.O.I virus inoculation, use cell instead and keep culture medium, setting the bioreactor culture condition is pH value 7.5,35 ℃ of temperature, DO 50%, mixing speed 90rpm, continous pouring cultivate, and continue to add cell with the speed of 10L/ days, 30L/ days and 60L/ days respectively and keep culture medium, simultaneously with identical speed sustained yield virus, received poison 24 days continuously, regularly virus titer is surveyed in sampling in the jar, the results are shown in Table 1.
Table 1
In the different tank volume bioreactors, the variation of aGV strain virus titer behind the Vero cell inoculation
| Virus titer (lgLD 50/ml) | |||||||||
| Receive malicious natural law | 1 | 3 | 6 | 9 | 12 | 15 | 18 | 21 | 24 |
| The 5L bioreactor | 7.2 | 7.5 | 8.0 | 8.5 | 8.8 | 8.2 | 7.8 | 7.2 | 6.8 |
| The 14L bioreactor | 7.0 | 7.6 | 8.1 | 8.6 | 8.7 | 8.1 | 7.5 | 7.1 | 6.5 |
| The 30L bioreactor | 7.0 | 7.3 | 7.8 | 8.3 | 8.9 | 8.0 | 7.6 | 7.0 | 6.8 |
As can be seen from Table 1 with 0.025M.O.I infective dose infective dose virus inoculation, the bioreactor of receiving 21 days different tank volume of poison continuously all can be kept virus titer at 〉=7.0lgLD50/ml, can predict to adopt this technology to cultivate scale can to put to bigger.
Description of drawings
The block flow diagram of Fig. 1 preparation method of the present invention.
The specific embodiment
Embodiment 1
(1) earlier the Vero cell is carried out recovery, the rolling bottle amplification cultivation of cell, again with 3.5 * 10
5It is in the Celligen Plus bioreactor jar with the basket stirring system of fixed bed of carrier (capacity can be 2.2 liters, 5 liters, 7.5 liters or 14 liters) that the density of/ml is seeded to Fibra-Cel disks, carry out amplification cultivation with the cell proliferation culture fluid, wherein the use amount of Fibra-Celdisks carrier is every liter of tank volume 30 grams, and used cell proliferation culture fluid uses 199 culture medium to add 6% Ox blood serum and an amount of gentamycin sulfate.The cell proliferation culture condition is a pH value 7.2,37 ℃ of temperature, dissolved oxygen (DO) 50%, mixing speed 120rpm.
(2) when the Vero cell grows to certain density, general 6～12 days, use cell maintenance culture solution then instead, with 0.025～0.125MOI infection multiplicity (MOI: the virus numbers that is used to infect and the ratio of cell number) or final concentration 4.5～5.5lgLD
50/ ml (LD
50: inoculation rabies viruses CTN-1 strain half lethal dose), and make it to infect, it is formulated that the used cell maintenance culture solution of cell uses 199 culture medium to add 0.1% human albumin, it is pH value 7.6 that cell is kept culture condition, 35 ℃ of temperature, dissolved oxygen (DO) 50%, mixing speed 120rpm.
Continous pouring results 20 days, virus titer remains on more than the 7lgLD50/ml.
(3) ultrafilter membrane held back with the 200K molecular weight of Shou Huo viral liquid concentrates 30 times, uses beta-propiolactone deactivation in 1: 4000 then.
(4) in the inactivation of viruses concentrated solution, add protamine sulfate while stirring to final concentration 1mg/ml, in 4 ℃ of effects 2 hours, then under 4 ℃ of 7000rpm conditions, centrifugal 15 minutes.
(5) be that filler carries out chromatography purification with Sepharose 4 Fast Flow subsequently.
(6) virus stock solution used adds the human albumin behind the purification, sucrose is that protective agent is made liquid preparation.
Embodiment 2
(1) earlier the Vero cell is carried out recovery, the rolling bottle amplification cultivation of cell, again with 5 * 10
5It is in the 4500 bioreactor jars of the BioFlo with the basket stirring system of fixed bed of carrier (capacity can be 20 liters, 30 liters) that the density of/ml is seeded to Fibra-Cel disks, carry out amplification cultivation with the cell proliferation culture fluid, wherein the use amount of Fibra-Cel disks carrier is every liter of tank volume 40 grams, and used cell proliferation culture fluid uses 199 culture medium to add 2% Ox blood serum and an amount of gentamycin sulfate.The cell proliferation culture condition is a pH value 6.9,36 ℃ of temperature, dissolved oxygen (DO) 30%, mixing speed 30rpm.
(2) when the Vero cell grows to certain density, general 6～12 days, use cell maintenance culture solution then instead, with 0.025～0.125MOI infection multiplicity (MOI: the virus numbers that is used to infect and the ratio of cell number) or final concentration 4.5～5.5lgLD
50/ ml (LD
50: it is formulated that inoculation rabies virus aGV strain half lethal dose), and make it to infect, the used cell maintenance culture solution of cell use 199 culture medium to add 0.05% human albumin, it is pH value 7.2 that cell is kept culture condition, 36 ℃ of temperature, dissolved oxygen (DO) 70%, mixing speed 30rpm.
Continous pouring results 20 days, virus titer remains on more than the 7lgLD50/ml.
(3) ultrafilter membrane held back with the 100K molecular weight of Shou Huo viral liquid concentrates 60 times, uses beta-propiolactone deactivation in 1: 4000 then.
(4) in the inactivation of viruses concentrated solution, add DNase (DNA enzyme) while stirring to final concentration 0.5～1.5U/ml, act on 6～10 hours under the room temperature.
(5) be that filler carries out chromatography purification with Sepharose 4 Fast Flow subsequently.
(6) virus stock solution used adds the human albumin behind the purification, sucrose is that protective agent is made liquid preparation.
Embodiment 3
(1) earlier the Vero cell is carried out recovery, the rolling bottle amplification cultivation of cell, again with 25 * 10
5It is in the BioFlo Pro bioreactor jar with the basket stirring system of fixed bed of carrier (capacity can be 75 liters, 150 liters, 300 liters) that the density of/ml is seeded to Fibra-Cel disks, carry out amplification cultivation with the cell proliferation culture fluid, wherein the use amount of Fibra-Cel disks carrier is every liter of tank volume 30 grams, and used cell proliferation culture fluid uses 199 culture medium to add 10% Ox blood serum and an amount of gentamycin sulfate.The cell proliferation culture condition is a pH value 7.4,38 ℃ of temperature, dissolved oxygen (DO) 70%, mixing speed 150rpm.
(2) when the Vero cell grows to certain density, general 6～12 days, use cell maintenance culture solution then instead, with 0.025～0.125MOI infection multiplicity (MOI: the virus numbers that is used to infect and the ratio of cell number) or final concentration 4.5～5.5lgLD
50/ ml (LD
50: inoculation rabies viruses CTN-1 strain half lethal dose), and make it to infect, it is formulated that the used cell maintenance culture solution of cell uses 199 culture medium to add 0.5% human albumin, it is pH value 8.0 that cell is kept culture condition, 32 ℃ of temperature, dissolved oxygen (DO) 30%, mixing speed 150rpm.
Continous pouring results 20 days, virus titer remains on more than the 7lgLD50/ml.
(3) ultrafilter membrane held back with the 300K molecular weight of Shou Huo viral liquid concentrates 100 times, uses beta-propiolactone deactivation in 1: 4000 then.
(4) in the inactivation of viruses concentrated solution, add protamine sulfate while stirring to final concentration 2mg/ml, in 8 ℃ of effects 2 hours, then under 2 ℃ of 8000rpm conditions, centrifugal 15 minutes.
(5) be that filler carries out chromatography purification with Sepharose 4 Fast Flow subsequently.
(6) virus stock solution used adding human albumin, sucrose, gelatin are made lyophilized formulations as protective agent and excipient behind the purification.
Above-mentioned 199 used culture medium are selected from the Beijing Qingdatianyi Bioisystech Co., Ltd, and its concrete preparation consists of:
| Composition | Content (mg/L) | Composition | Content (mg/L) |
| Calcium chloride | 200.00 | The D-glucose | 1000.00 |
| Nine water ferric nitrates | 0.70 | Glutathion (reduced form) | 0.05 |
| Potassium chloride | 400.00 | Guanine hydrochloride | 0.30 |
| Anhydrous magnesium sulfate | 97.67 | Hypoxanthine | 0.30 |
| Potassium dihydrogen phosphate | - | Phenol red | 20.00 |
| Sodium chloride | 6800.00 | D-ribose | 0.50 |
| AMSP | 121.74 | Sodium acetate | 50.00 |
| Sodium hydrogen phosphate | - | Thymus pyrimidine | 0.30 |
| The L-alanine | 25.00 | Tween 80 | 20.00 |
| The L-arginine monohydrochloride | 70.00 | Uracil | 0.30 |
| The L-aspartic acid | 30.00 | Xanthine | 0.30 |
| The L-cysteine hydrochloride | 0.11 | Vitamin C | 0.05 |
| The L-cystine hydrochloride | 26.00 | Vitamin E | 0.01 |
| L-glutamic acid | 75.00 | Biotin | 0.01 |
| L-glutaminate | 100.00 | Vitamin D2 | 0.10 |
| Glycine | 50.00 | The D-calcium pantothenate | 0.01 |
| The L-histidine hydrochloride | 21.88 | Choline chloride | 0.50 |
| The L-hydroxyproline | 10.00 | Folic acid | 0.01 |
| The L-isoleucine | 40.00 | The i-inositol | 0.05 |
| The L-leucine | 60.00 | Vitamin K3 | 0.01 |
| L-lysine hydrochloride | 70.00 | Nicotinic acid | 0.025 |
| The L-methionine | 15.00 | Nicotiamide | 0.025 |
| The L-phenylalanine | 25.00 | Para-amino benzoic acid | 0.05 |
| The L-proline | 40.00 | Pyridoxal hydrochloride | 0.025 |
| The L-serine | 25.00 | Pyridoxine hydrochloride | 0.025 |
| The L-threonine | 30.00 | Riboflavin | 0.01 |
| The L-tryptophan | 10.00 | Thiamine hydrochloride | 0.01 |
| L-tyrosine | 40.00 | Vitamin A acetate | 0.14 |
| The L-valine | 25.00 | ||
| Adenine sulfate | 10.00 | PH (no sodium bicarbonate) | 4.2±0.3 |
| Adenylic acid | 0.20 | PH (adding sodium bicarbonate) | 7.3±0.3 |
| Adenosine triphosphate disodium salt | 1.00 | Osmotic pressure (no sodium bicarbonate) | 250±5％ |
| Cholesterol | 0.20 | Osmotic pressure (adding sodium bicarbonate) | 288±5％ |
| Deoxyribose | 0.50 |
Claims (11)
1, the method for a kind of large-scale production rabies vaccine is characterized in that its processing step is:
(1) be in the bioreactor with the basket stirring system of fixed bed of carrier with Fibra-Cel disks, with cell proliferation culture fluid Cultivation of Vero;
(2) when the Vero cell grows to certain density, use cell maintenance culture solution instead, the inoculation rabies virus, and make it infection cell;
(3) make virus multiplication;
(4) results are viral continuously;
(5) ultrafiltration and concentration is also used the beta-propiolactone deactivation;
(6) handle to remove the Vero cell DNA with protamine sulfate or DNA enzyme;
(7) be that filler carries out chromatography purification with Sepharose 4 Fast Flow;
(8) add the human albumin, sucrose is that protective agent is made liquid preparation, or add human albumin, sucrose, gelatin and make lyophilized formulations as protective agent and excipient.
2, the method for large-scale production rabies vaccine in accordance with claim, it is characterized in that: it is 2.2 liters, 5 liters, 7.5 liters, 14 liters Celligen Plus bioreactors that described bioreactor with the basket stirring system of fixed bed can be selected tank volume for use, and tank volume is any one in the BioFlo Pro bioreactor of 75 liters, 150 liters, 300 liters of 20 liters, 30 liters BioFlo 4500 bioreactors and tank volume.
3, according to the method for the described large-scale production rabies vaccine of claim 1, it is formulated to it is characterized in that above-mentioned cell proliferation culture fluid uses No. 199 culture medium to add 2～10% Ox blood serum, its condition of culture is a pH value 6.9～7.4,36～38 ℃ of temperature, dissolved oxygen 30～70%, mixing speed 30～150rpm.
4,, it is characterized in that also adding an amount of gentamycin in the above-mentioned cell proliferation culture fluid according to the method for claim 1 or 3 described large-scale production rabies vaccine.
5, according to the method for the described large-scale production rabies vaccine of claim 1, the use amount that it is characterized in that above-mentioned Fibra-Cel disks carrier is every liter of tank volume 30～40 grams.
6,, it is characterized in that above-mentioned Vero cell is with 2～5 * 10 according to the method for the described large-scale production rabies vaccine of claim 1
5The density of/ml is seeded in bioreactor, and back amplification cultivation can be inoculated rabies virus after 6～12 days.
7, according to the method for the described large-scale production rabies vaccine of claim 1, it is formulated to it is characterized in that cell maintenance culture solution uses 199 culture medium to add 0.05%～0.5% human albumin, its condition of culture is a pH value 7.2～8.0,32～36 ℃ of temperature, dissolved oxygen 30～70%, mixing speed 30～150rpm.
8, according to the method for the described large-scale production rabies vaccine of claim 1, it is characterized in that the rabies virus of selecting for use is CTN-1 strain or aGV strain, this rabies virus is with infection multiplicity or the final concentration 4.5～5.5lgLD of 0.025～0.125MOI
50/ ml infects described cell.
9,, it is characterized in that described ultrafiltration and concentration is meant that the employing ultrafilter membrane that 100K～the 300K molecular weight is held back concentrates 30～100 times according to the method for the described large-scale production rabies vaccine of claim 1.
10, according to the method for the described large-scale production rabies vaccine of claim 1, it is characterized in that the described detailed process of handling to remove the Vero cell DNA with protamine sulfate is: in the inactivation of viruses concentrated solution, add protamine sulfate while stirring to final concentration 1～2mg/ml, act on 2～4 hours down in 2～8 ℃, then in 2～8 ℃, under 7000～8000rpm condition, centrifugal 15 minutes.
11, according to the method for the described large-scale production rabies vaccine of claim 1, it is characterized in that the described detailed process of handling to remove the Vero cell DNA with the DNA enzyme is: in the inactivation of viruses concentrated solution, add the DNA enzyme while stirring to final concentration 0.5～1.5U/ml, at room temperature act on 6～10 hours.
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| WO2009109086A1 (en) * | 2008-03-05 | 2009-09-11 | 辽宁依生生物制药有限公司 | Method for extracting rabies viruses |
| CN102038947A (en) * | 2010-09-15 | 2011-05-04 | 武汉中博生物股份有限公司 | Method for industrially producing animal rabies vaccine by utilizing bioreactor |
| CN102154220A (en) * | 2010-12-30 | 2011-08-17 | 浙江易邦生物技术有限公司 | Method and equipment for ultrahigh-density and large-scale production of porcine reproductive and respiratory syndrome virus (PRRSV) |
| CN102205116A (en) * | 2010-03-29 | 2011-10-05 | 杭州安普生物工程有限公司 | Method for producing vaccine by virtue of culturing animal cells |
| CN102228686A (en) * | 2011-06-30 | 2011-11-02 | 金宇保灵生物药品有限公司 | Process for preparing veterinary rabies inactivated and freeze-dried vaccine through suspension culture cell |
| CN102240400A (en) * | 2011-05-25 | 2011-11-16 | 长春生物制品研究所 | Method for producing rabies vaccine by applying bioreactor and sheet carrier |
| WO2012019354A1 (en) * | 2010-08-12 | 2012-02-16 | Yisheng Biopharma Holdings Ltd. | Method for reducing dna impurities in viral compositions |
| CN102949716A (en) * | 2011-08-26 | 2013-03-06 | 唐山怡安生物工程有限公司 | Method for preparing inactivated rabies vaccine for animal by bioreactor |
| CN103083654A (en) * | 2012-08-27 | 2013-05-08 | 万里明 | Process for preparing human diploid cell rabies vaccine through Celligen310 bioreactor |
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| WO2012019354A1 (en) * | 2010-08-12 | 2012-02-16 | Yisheng Biopharma Holdings Ltd. | Method for reducing dna impurities in viral compositions |
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| CN102949716A (en) * | 2011-08-26 | 2013-03-06 | 唐山怡安生物工程有限公司 | Method for preparing inactivated rabies vaccine for animal by bioreactor |
| CN103083654A (en) * | 2012-08-27 | 2013-05-08 | 万里明 | Process for preparing human diploid cell rabies vaccine through Celligen310 bioreactor |
| CN108434106A (en) * | 2017-04-25 | 2018-08-24 | 广州瑞贝斯药业有限公司 | A kind of lyophilized preparation of rabies vacciness |
| CN108434106B (en) * | 2017-04-25 | 2021-07-30 | 广州瑞贝斯药业有限公司 | Freeze-dried preparation of rabies vaccine |
| CN110101864A (en) * | 2019-05-31 | 2019-08-09 | 辽宁茂康源生物科技有限公司 | The protective agent of serum-free Antirabic Vaccine a kind of and its application |
| CN111840535A (en) * | 2020-08-07 | 2020-10-30 | 成都柏奥特克生物科技股份有限公司 | Process for preparing rabies vaccine by using serum-free Vero cells and rabies viruses rPV-2061 |
| CN111840535B (en) * | 2020-08-07 | 2021-08-03 | 成都柏奥特克生物科技股份有限公司 | Process for preparing rabies vaccine by using serum-free Vero cells and rabies viruses rPV-2061 |
| CN114181287A (en) * | 2021-12-10 | 2022-03-15 | 商丘安华生物疫苗有限公司 | Preparation method of DNA virus vaccine |
| CN114181287B (en) * | 2021-12-10 | 2023-09-19 | 商丘美兰生物工程有限公司 | Preparation method of DNA virus vaccine |
| CN115287269A (en) * | 2022-09-16 | 2022-11-04 | 江苏康润生物科技有限公司 | Method for producing rabies virus by high-density culture of Vero cells by microcarrier |
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