CN104353068A - Large-scale production method of rabies vaccines by using human diploid cells - Google Patents

Abstract

The invention discloses a large-scale production method of rabies vaccines by using human diploid cells. The method comprises the following steps: culturing and amplifying resuscitated human diploid cells in a bioreactor, and then inoculating rabies viruses to the human diploid cells; cultivating the rabies viruses, and after a virus fluid is obtained, adding a formaldehyde solution with a final concentration of 0.1% for inactivating for 24 h, and then carrying out separation and purification; and adding an adjuvant for adsorption and a vaccine protectant, wherein the vaccine protectant comprises 2% of human albumin, 0.5% of cysteine, 3% of fructose, 0.2% of sodium glutamate and 1% of acesulfame potassium. According to the preparation method disclosed by the invention, the stability of vaccines can be enhanced, the retention time is long, and the titer reduction is slow.

Description

A kind of method utilizing human diploid cell large-scale production rabies vaccine

Technical field

The present invention relates to technical field of vaccines, be specifically related to a kind of method utilizing human diploid cell large-scale production rabies vaccine.

Background technology

Rabies have another name called hydrophobia, are the Natur al foca infecting both domestic animals and human acute infectious disease caused by rabies virus.Popular wide, case fatality rate is high, is almost 100%.Rabies cause serious threat to people's life health.Human rabies is usually passed to people's human body by sick beast in the mode of biting and is infected.Clinical manifestation is distinctive hydrophobia, probably sound, aversion to wind, frightened and restless, pharyngismus, Progressive symmetric erythrokeratodermia paralysis etc.

Rabies are once morbidity there is no method treatment at present, and its control depends on vaccination.Anti-rabies immune inoculation is one of successful example in early immune preventive medicine history.Within 1885, first the Pedis Canitis tissue having infected rabies virus is utilized to infect rabbit by intracerebral injection by Pasteur, then by immune Canis familiaris L. after the spinal cord drying of rabbit, and pass through the virulence attenuation of of rabies virus after monkey interior generation repeatedly.The immunity of Here it is the most original rabies vaccine.Within 1908, Dr.Fermi utilizes 1% phenol by the viral aliquots deactivation in Medulla Leporis seu Oryctolagi tissue.Dr.Semple in 1911 by cerebral tissue suspension in 37 DEG C with phenol fix, deactivation, prepared avirulent Semple Seedling.Nineteen twenty-five Dr.Hempt, by adding ether process, further ensures the avirulence of vaccine.China, from 1949, uses the Semple vaccine prepared by Medulla caprae seuovis always, until 1980 stop using, is replaced by primary hamster kidney cell vaccine.In order to reduce the anaphylaxis to marrowbrain tissue, nineteen fifty-nine Dr.Peck adopts duck embryo tissue and beta-propiolactone (β-propiolatone) inactivation of viruses method to prepare duck embryo and organizes mad dog Seedling (DEV, Duck EmbryoVaccine), it is 1.5% be down to 1/25000 that result side reaction reduces many side reactions namely caused from nervous tissue's Seedling (NTV, Nerve Tissue Vaccine).But the effect of DEV and protectiveness bad.In addition, owing to having report when duck egg causes allergic reaction also in vain.Being that the vaccine of substrate has had and develops on a large scale very much with cultured cell after nineteen sixty, is also using with widely used at present.The sixties, Dr.Kissling and Fenje first trial histiocyte cultivates rabies virus.Rabies virus after primary hamster kidney cell and DEC are cultivated, through Formalin inactivation, is prepared vaccine by two scholars.1964, Dr.Wiktor etc. by Pasteur in 1882 from Medulla Leporis seu Oryctolagi the fixing strain of isolated Pitman-Moore (PM) adapt to human embryonic lung diploid fibroblast 2BS (Wistar-38), namely utilize and infected the cell of rabies virus and fresh cell co-cultivation, after 47 generations of going down to posterity, virus has adapted to 2BS cell completely, and virus also can be allowed to go down to posterity under acellular state.Reliable immunne response can be produced after the vaccination prepared thus, produce the neutralizing antibody of high titre.In the 1980's, French Merieux institute takes the lead in studying and uses passage cell---Vero cells produce rabies vaccine, and WHO formulates the passage cell code seen and produce vaccine thereafter, and formulates the code of passage cell production rabies vaccine in 1987.China carried out people with refining Vero cell culture rabies vaccine development from age end in 1980, to be given the ratification production in nineteen ninety for end.

Passage cell at present for the production of people mostly is Vero cell, and it is the cell line of African green monkey kidney cell, is generally regarded as safe through repeatedly identifying.Such as, in patent 200510080058.2 disclosed in, the vaccine protectant of this patent have selected conventional protective agent, and effect is poor.In the preparation process of vaccine, the protective agent of vaccine often has important impact, has very great effect to the thermostability of vaccine, storage life and active protection.

Summary of the invention

The object of this invention is to provide a kind of method utilizing human diploid cell large-scale production rabies vaccine, the stability of vaccine can be made better, be convenient to preserve after lyophilizing, tiring, it is slower to reduce.

For reaching above-mentioned purpose, providing a kind of method utilizing human diploid cell large-scale production rabies vaccine in one embodiment of the present of invention, comprising the following steps:

Human diploid cell after recovery is cultivated amplification in bioreactor, on human diploid cell, then inoculates rabies virus; Rabies virus is cultivated, adds the formalin fire extinguishing 24h that final concentration is 0.1% after results virus liquid, then carry out purifies and separates;

Purge process is: inactivation of viruses makes thick vaccine, and then clarification filtration, ultrafiltration purification carry out anion exchange and column chromatography purification, then adds adjuvant absorption and vaccine protectant; Lyophilizing subpackage;

Wherein, described vaccine protectant is for comprising human albumin 2%, cysteine 0.5%, fructose 3%, sodium glutamate 0.2% and acesulfame potassium 1%.

Wherein add gentamycin during bioreactor culture; Human diploid cell is WI-38.

Preparation method of the present invention, can strengthen the stability of vaccine, the holding time is long, and tiring, it is slow to reduce.

Detailed description of the invention

Embodiment 1

The acquisition of rabies vaccine

Human diploid cell after recovery is cultivated amplification in bioreactor, on human diploid cell, then inoculates rabies virus; Rabies virus is cultivated, adds the formalin fire extinguishing 24h that final concentration is 0.1% after results virus liquid, then carry out purifies and separates;

Wherein, purge process is: inactivation of viruses makes thick vaccine, and then clarification filtration, ultrafiltration purification carry out anion exchange and column chromatography purification, then add adjuvant absorption and vaccine protectant carry out lyophilizing;

Wherein, vaccine protectant is for comprising human albumin 2%, cysteine 0.5%, fructose 3%, sodium glutamate 0.2% and acesulfame potassium 1%.

The lyophilizing of vaccine

By the vaccinogen liquid adding adjuvant and vaccine protectant by the following method or other existing lyophilizing modes carry out lyophilizing.

Aforementioned freeze-dry process comprises

Pre-freeze: be distributed into by treated vaccinogen liquid in lyophilizing bottle, lyophilizing bottle is placed in freeze dryer, makes its pre-freeze 4h in subzero 20 DEG C, then pre-freeze 2h in subzero 35 DEG C; Open vacuum machine simultaneously, make the vacuum of freeze dryer maintain 8pa;

Primary drying: regulate baffle temperature, control baffle temperature and maintain subzero 30 DEG C, vacuum maintains 15Pa, insulation 10h;

Redrying: regulate baffle temperature, maintain between subzero 25 DEG C, vacuum maintains 15Pa, insulation 15h;

Parsing-desiccation: baffle temperature is warming up to 15 DEG C, vacuum is 15Pa, maintains 2h; Then baffle temperature is warming up to 30 DEG C, vacuum is 10Pa, insulation 5h.

From rabies vaccine lyophilized formulations product, choose three freeze-dried powders test, its inspection item comprise initially tire, tire after heat stabilization test, outward appearance etc.Its assay is as table 1.Meanwhile, the finished product in embodiment 1 is carried out to the evaluation of drug safety, carry out zoopery and check its safety.Vaccine is carried out respectively the experiment of vaccine intracerebral injection, anaphylaxis experiment and injection site irritation test, result shows, Novel presentation does not appear in mice, and Continuous Observation 2 weeks, experiment mice is all healthy.Wherein, by rabies vaccine intraperitoneal injection of mice, and it is observed 14 days.Mice is all good in 14 days and deposits, and Normal-weight increases.

Matched group

Human diploid cell after recovery is cultivated amplification in bioreactor, on human diploid cell, then inoculates rabies virus; Rabies virus is cultivated, adds the formalin fire extinguishing 24h that final concentration is 0.1% after results virus liquid, then carry out purifies and separates;

Wherein, purge process is: inactivation of viruses makes thick vaccine, and then clarification filtration, ultrafiltration purification carry out anion exchange and column chromatography purification, then add adjuvant absorption and vaccine protectant carry out lyophilizing;

Wherein, vaccine protectant is the human albumin 5% comprising weight ratio.

The lyophilizing of vaccine

By the vaccinogen liquid adding adjuvant and vaccine protectant by the following method or other existing lyophilizing modes carry out lyophilizing.

Aforementioned freeze-dry process comprises

Pre-freeze: be distributed into by treated vaccinogen liquid in lyophilizing bottle, lyophilizing bottle is placed in freeze dryer, makes its pre-freeze 4h in subzero 20 DEG C, then pre-freeze 2h in subzero 35 DEG C; Open vacuum machine simultaneously, make the vacuum of freeze dryer maintain 8pa;

Primary drying: regulate baffle temperature, control baffle temperature and maintain subzero 30 DEG C, vacuum maintains 15Pa, insulation 10h;

Redrying: regulate baffle temperature, maintain between subzero 25 DEG C, vacuum maintains 15Pa, insulation 15h;

Parsing-desiccation: baffle temperature is warming up to 15 DEG C, vacuum is 15Pa, maintains 2h; Then baffle temperature is warming up to 30 DEG C, vacuum is 10Pa, insulation 5h.

Rabies vaccine lyophilized formulations product is tested, its inspection item comprise initially tire, tire after heat stabilization test, outward appearance etc., and to compare with the product of embodiment 1.

Table 1 heat stability experimental result

As can be seen from above-mentioned experiment, the heat stability experimental result that rabies vaccine of the present invention carries out at identical conditions is obviously better than currently available vaccines, existing vaccines preparation method.

Claims (3)

1. utilize a method for human diploid cell large-scale production rabies vaccine, comprise the following steps:

Human diploid cell after recovery is cultivated amplification in bioreactor, on human diploid cell, then inoculates rabies virus; Rabies virus is cultivated, adds the formalin fire extinguishing 24h that final concentration is 0.1% after results virus liquid, then carry out purifies and separates;

Described purge process is: inactivation of viruses makes thick vaccine, and then clarification filtration, ultrafiltration purification carry out anion exchange and column chromatography purification, then adds adjuvant absorption and vaccine protectant; Lyophilizing subpackage;

Wherein, described vaccine protectant is for comprising human albumin 2%, cysteine 0.5%, fructose 3%, sodium glutamate 0.2% and acesulfame potassium 1%.

2. the method for claim 1, is characterized in that: add gentamycin during described bioreactor culture.

3. the method for claim 1, is characterized in that: described human diploid cell is WI-38.