CN101695571B - Method for producing swine fever vaccine by using bioreactor and swine fever vaccine product - Google Patents
Method for producing swine fever vaccine by using bioreactor and swine fever vaccine product Download PDFInfo
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- CN101695571B CN101695571B CN 200910193314 CN200910193314A CN101695571B CN 101695571 B CN101695571 B CN 101695571B CN 200910193314 CN200910193314 CN 200910193314 CN 200910193314 A CN200910193314 A CN 200910193314A CN 101695571 B CN101695571 B CN 101695571B
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Abstract
The invention provides a method for producing a swine fever vaccine by using a bioreactor and a swine fever vaccine product. The method comprises the following steps: inoculating cells for manufacturing the vaccine into the bioreactor and a micro carrier after the bioreactor and the micro carrier are sterilized, and adding a cell growth medium for culture; inoculating a maintenance medium containing a swine fever virus lapinized Chinese strain into the bioreactor; 1 to 3days after the inoculation of the viruses, obtaining a cell culture virus liquid in a feeding mode, adding a stabilizer and antibiotics, and obtaining the swine fever vaccine by refrigeration and drying under vacuum. In the method, the cell density and virus concentration are improved greatly, the titer of the vaccine is improved, the side reactions, labor intensity and product cost are reduced, the monitoring performance of vaccine production is improved and uniform and stable product quality is guaranteed. The swine fever vaccine produced by the method has high safety, immune efficacy and an excellent immune and protective effect against the attack by virulent swine fever viruses.
Description
Technical field
The invention belongs to the veterinary biologics technical field, more specifically relate to a kind of method and goods thereof that utilize bioreactor to produce swine fever cell live vaccine.
Background technology
The production of current swine fever cell live vaccine mainly is traditional training method of rolling bottle cell culture, and this training method is that the cultured cell adherent growth is on the inwall of glass culture bottle, though its operating technology requires low and technology maturation is stable; But the rolling bottle culture technique has been used many decades, produce incompatible with current extensive animal vaccine, mainly show as: (1) rolling bottle cell culture, can not accomplish that condition of culture adjusts in good time, can't guarantee that cell culture is in optimum condition, cultured cell density is little, and it is low to cultivate virus titer, and the immune effect of animal is poor; (2) rolling bottle cell culture, cell debris and foreign protein are many, and the impurity of vaccine is many, causes the side reaction of immune animal big; (3) rolling bottle cell culture, the cell differences between batches are bigger, product quality heterogeneity, instability; (4) rolling bottle cell culture, production efficiency is low, the production cost height.
Summary of the invention
For overcoming above-mentioned technological deficiency, the purpose of this invention is to provide a kind of method and swine fever cell live vaccine product that utilizes bioreactor to produce swine fever cell live vaccine.
For achieving the above object, the present invention at first provides a kind of method of utilizing bioreactor to produce swine fever cell live vaccine, and this method comprises the steps:
(1) cultivates the seedling cell with cell growth medium;
(2) the cell inoculation that described step (1) is obtained contains the liquid of keeping of hog cholera lapinised virus seed culture of viruses, and breeds cultivation;
(3) connect poison back 1～3 day or fed-batch mode and gather in the crops the cell culture venom that described step (2) obtains;
(4) in the cell culture venom that described step (3) obtains, add stabilizing agent and antibiotic, obtain swine fever cell live vaccine through lyophilisation;
Wherein, used the bioreactor that contains microcarrier to cultivate in the said method.
Preferably, described step (1) comprises the steps: described bioreactor and microcarrier are inoculated described seedling cell after sterilizing, and the adding cell growth medium is cultivated.
Preferably, described step (2) comprises the steps: the described liquid of keeping that contains the hog cholera lapinised virus seed culture of viruses of described bioreactor inoculation is continued to cultivate.
Preferably, the cultivation temperature in described step (1) and (2) is 36 ℃～37 ℃.
Preferably, the cell growth medium in the described step (1) contains the calf serum of MEM liquid, antibiotics and 8%～10% percent by volume of 90%～92% percent by volume, and the pH value of described cell growth medium is 7.0～7.2.
Preferably, the inoculum concentration the during inoculation in the described step (2) is the production of 1%～2% percent by volume with the liquid of keeping of cell seed culture of viruses.
Preferably, the cell maintenance medium in the described step (2) contains the calf serum of MEM liquid, antibiotics and 2%～5% percent by volume of 95%～98% percent by volume, and the pH value of described cell maintenance medium is 7.2～7.4.
Preferably, described bioreactor is TideCell fixed bed microcarrier bioreactor.The TideCell bioreactor can reach general syringe and the serum-free culture cell mode of only needing that be reduced to, the growth that evenly distributes of the carrier cell of cultivation, its effective dose industry experiment confirm, a TideCell bioreactor, 4 times of the general approximately traditional biological reactor of production capacity.
Preferably, described seedling cell is pig testis cell line ST, porcine kidney cell line PK15 or porcine kidney cell line IBRS-2.
Two of purpose of the present invention provides a kind of swine fever cell live vaccine goods, and this swine fever cell live vaccine obtains by said method.
The present invention utilizes bioreactor large scale and high density culture technique, can improve unit volume cell stand density in the bioreactor to greatest extent, have very big potentiality and advantage in viral vaccine production, be embodied in: (1) cell density increases greatly, and virus concentration greatly improves; (2) vaccine valence improves, and side reaction reduces; (3) reduce labor intensity, reduce production cost; (4) the production of vaccine property monitored raising; (5) guarantee the product quality stable homogeneous.The swine fever cell live vaccine safety that utilizes the present invention to produce is good, immune efficacy is high, and the swine fever strong virus attack is had immanoprotection action preferably.
The specific embodiment
For making the present invention easier to understand, below in conjunction with specific embodiment, further set forth the present invention.Should be understood that these embodiment only to be used for explanation the present invention and be not used in and limit the scope of the invention that NM concrete experimental technique in the following example carries out according to the normal experiment method usually.
Utilize bioreactor mass cell culture technique, produce production Technology and the method for swine fever cell live vaccine, comprise the steps:
(1) selects bioreactor: TideCell fixed bed microcarrier bioreactor.
(2) select cell line as the seedling cell: to select pig testis (ST) cell line as the seedling cell in the present embodiment.In other two embodiment, select Ren sus domestica (PK15) cell line and Ren sus domestica (IBRS-2) cell line respectively for use.
(3) utilize bioreactor culture seedling cell: select suitable cell line, bioreactor and microcarrier are inoculated suitable cell concentration (10 after sterilizing
6～8 * 10
6Individual cell/ml) also adds an amount of culture medium, and by adjusting each condition of culture in the cell culture system: oxyty is 50%～100%, CO
2Concentration is 5%, glucose content is that 500～3000mg/L and pH value are 7.0～7.4, cultivates highdensity cell line as the seedling cell.
(4) select the seedling seed culture of viruses: the hog cholera lapinised virus strain.
(5) bioreactor breeding seedling venom: the cell growth medium that well-grown above-mentioned passage cell microcarrier in the bioreactor is cultivated in irritating discards, and inoculation contains the liquid of keeping of an amount of seed culture of viruses, continues to cultivate; Connect poison back 1～3 day or fed-batch mode harvesting and cultivate venom, the venom of results is put below-15 ℃ and is preserved;
The check of seedling venom: test by " People's Republic of China's veterinary drug allusion quotation " (version in 2005) appendix 15,19 pages, no antibacterial, mycete, mycoplasma are grown.The cell venom has no side effect safely to pig, and each is received time virus-culturing fluid and tires with rabbit mensuration respectively, and every 1ml contains virus 〉=2 * 10
6Rabbit body infective dose (RID).
(6) join Seedling, packing and lyophilizing: with the virus-culturing fluid that is up to the standards, be mixed in the same container, add stabilizing agent, add antibiotic simultaneously, fully shake up, quantitatively packing; Carry out rapidly after the packing getting product after the lyophilisation.
Product inspection: test by " People's Republic of China's veterinary drug allusion quotation " (version in 2005), meet the regulation of " swine fever cell live vaccine ", pig is had no side effect safely, every part vaccine contains virus 〉=1.5 * 10
4RID.
Wherein, the concrete condition that relates in the said method is as follows:
Passage cell cultivation temperature described in step (3), (5) is 36～37 ℃.
Inoculum concentration described in the step (5) is to contain 1%～2% liquid of producing with the cell seed culture of viruses of keeping.
The prescription of used growth-promoting media is in the step (3): 90%～92%MEM liquid, 8%～10% calf serum, add an amount of antibiotics, pH value is adjusted into 7.0～7.2.
The used prescription of keeping liquid is in the step (5): 95%～98%MEM liquid, 2%～5% calf serum, add an amount of antibiotics, pH value is adjusted into 7.2～7.4.
Last institute should be noted that; above embodiment is only in order to illustrate technical scheme of the present invention but not limiting the scope of the invention; although with reference to preferred embodiment the present invention has been done detailed description; those of ordinary skill in the art is to be understood that; can make amendment or be equal to replacement technical scheme of the present invention, and not break away from essence and the scope of technical solution of the present invention.
Claims (7)
1. a method of utilizing bioreactor to produce swine fever cell live vaccine is characterized in that, comprises the steps:
(1) cultivates the seedling cell with cell growth medium;
(2) the cell inoculation that described step (1) is obtained contains the liquid of keeping of hog cholera lapinised virus seed culture of viruses, and breeds cultivation;
(3) connect poison back 1～3 day or fed-batch mode and gather in the crops the cell culture venom that described step (2) obtains;
(4) in the cell culture venom that described step (3) obtains, add stabilizing agent and antibiotic, obtain swine fever cell live vaccine through lyophilisation;
Wherein, used TideCell fixed bed microcarrier bioreactor to cultivate in the said method.
2. a kind of method of utilizing bioreactor to produce swine fever cell live vaccine according to claim 1, it is characterized in that, described step (1) comprises the steps: described bioreactor and microcarrier are inoculated described seedling cell after sterilizing, and the adding cell growth medium is cultivated.
3. a kind of method of utilizing bioreactor to produce swine fever cell live vaccine according to claim 1 is characterized in that the cultivation temperature in described step (1) and (2) is 36 ℃～37 ℃.
4. a kind of method of utilizing bioreactor to produce swine fever cell live vaccine according to claim 1, it is characterized in that, cell growth medium in the described step (1) contains the calf serum of MEM liquid, antibiotics and 8%～10% percent by volume of 90%～92% percent by volume, and the pH value of described cell growth medium is 7.0～7.2.
5. a kind of method of utilizing bioreactor to produce swine fever cell live vaccine according to claim 1 is characterized in that, the inoculum concentration during inoculation in the described step (2) is the production of 1%～2% percent by volume with the liquid of keeping of cell seed culture of viruses.
6. a kind of method of utilizing bioreactor to produce swine fever cell live vaccine according to claim 1, it is characterized in that, cell maintenance medium in the described step (2) contains the calf serum of MEM liquid, antibiotics and 2%～5% percent by volume of 95%～98% percent by volume, and the pH value of described cell maintenance medium is 7.2～7.4.
7. according to the described a kind of method of utilizing bioreactor to produce swine fever cell live vaccine of one of claim 1～6, it is characterized in that described seedling cell is pig testis cell line ST, porcine kidney cell line PK15 or porcine kidney cell line IBRS-2.
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Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101797380A (en) * | 2010-01-28 | 2010-08-11 | 洛阳普莱柯生物工程有限公司 | Method for preparing hogcholera vaccine |
| CN101905020B (en) * | 2010-08-05 | 2012-09-05 | 中国兽医药品监察所 | Method for producing swine fever vaccines |
| CN102038944B (en) * | 2010-09-15 | 2013-07-03 | 武汉中博生物股份有限公司 | Method for industrially producing swine fever live vaccine by using bioreactor |
| CN103083653B (en) * | 2011-10-28 | 2015-07-22 | 辽宁成大动物药业有限公司 | Method for producing classical swine fever live vaccine by using microcarrier high-density cell culture technology |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101028514A (en) * | 2006-06-30 | 2007-09-05 | 辽宁成大生物股份有限公司 | Method for producing vaccine for man by biological reactor |
| CN101181637A (en) * | 2007-11-30 | 2008-05-21 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
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| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101028514A (en) * | 2006-06-30 | 2007-09-05 | 辽宁成大生物股份有限公司 | Method for producing vaccine for man by biological reactor |
| CN101181637A (en) * | 2007-11-30 | 2008-05-21 | 中国兽医药品监察所 | Method for producing swine fever live vaccine with cell line |
Non-Patent Citations (1)
| Title |
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| 北京清大天一科技有限公司.120L反应器微载体培养ST细胞生产猪瘟病毒技术.《120L反应器微载体培养ST细胞生产猪瘟病毒技术》.2009,第2页第三节. * |
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Inventor after: Wu Wenfu Inventor after: Situ Jianmo Inventor after: Ning Yibao Inventor after: Zhang Yujin Inventor after: Lin Xuye Inventor after: Cen Xiaoqing Inventor after: Ren Xiangyang Inventor after: You Qiyou Inventor after: Zhang Muhui Inventor after: Liu Qiuyan Inventor before: Wu Wenfu Inventor before: Situ Jianmo Inventor before: Zhang Yujin Inventor before: Lin Xuye Inventor before: Cen Xiaoqing Inventor before: Ren Xiangyang Inventor before: You Qiyou Inventor before: Zhang Muhui Inventor before: Zhou Zebiao Inventor before: Liu Qiuyan |
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Free format text: CORRECT: INVENTOR; FROM: WU WENFU ZHANG YUJIN LIN XUYE CEN XIAOQING REN XIANGYANG YOU QIYOU ZHANG MUHUI ZHOU ZEBIAO LIU QIUYAN SITU JIANMEI TO: WU WENFU NING YIBAO ZHANG YUJIN LIN XUXU CEN XIAOQING REN XIANGYANG YOU QIYOU ZHANG MUHUI LIU QIUYAN SITU JIANMEI |
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Effective date of registration: 20101214 Address after: 511356 Guangzhou, Luogang District Yonghe Economic Zone West Garden Road, No. 35 Applicant after: Guangdong Winsun Bio-Pharmaceutical Co., Ltd. Co-applicant after: China Veterinery Drug Inspection Office Address before: 511356 Guangzhou, Luogang District Yonghe Economic Zone West Garden Road, No. 35 Applicant before: Guangdong Winsun Bio-Pharmaceutical Co., Ltd. |
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Address after: 511356 Guangzhou, Luogang District Yonghe Economic Zone West Garden Road, No. 35 Applicant after: GUANGDONG WINSUN BIOPHARMACEUTICAL CO., LTD. Co-applicant after: China Veterinery Drug Inspection Office Address before: 511356 Guangzhou, Luogang District Yonghe Economic Zone West Garden Road, No. 35 Applicant before: Guangdong Winsun Bio-Pharmaceutical Co., Ltd. Co-applicant before: China Veterinery Drug Inspection Office |
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