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Method for preparing polyvalent vaccine of primary hamster kidney cells of flu

A technology of influenza and hamster kidney cells, applied in the biological field, can solve the problems of vaccine retrovirus contamination, slow adaptation of influenza virus, insufficient source of chicken embryos, etc., to achieve large-scale production, sufficient kidney sources, and reduce The effect of production costs

Inactive Publication Date: 2011-07-06
深圳市孚沃德生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

This method uses primary hamster kidney cells as the proliferation matrix of influenza virus, which solves the problems of insufficient source of chicken embryos and the vulnerability of vaccine to retrovirus contamination, and also solves the problems of slow adaptation of influenza virus in passaged cells and weak virus titer The problem

Method used

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  • Method for preparing polyvalent vaccine of primary hamster kidney cells of flu
  • Method for preparing polyvalent vaccine of primary hamster kidney cells of flu
  • Method for preparing polyvalent vaccine of primary hamster kidney cells of flu

Examples

Experimental program
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preparation example Construction

[0056] 1. Preparation of Hamster Kidney Cells

[0057] Choose healthy golden hamsters aged 10-14 days, kill them with drinking water, wash them 1-2 times, and disinfect them with 1‰ bromogeramine for 1-3 times, each time for 3-8 minutes. In a sterile environment, use sterile scissors to dissect the hamster and take out the kidney, cut it into pieces, add a digestive solution composed of 0.1% to 0.5% trypsin and 0.01% to 0.05% EDTA, and place it in a cold place at 2 to 8°C. Digest for 15-20 hours, discard the digestion solution, add growth solution to disperse the cells, and prepare 1.0×10 7 ~1.0×10 8 cells / ml of cell suspension. Take the primary cell suspension and inoculate it in a 3L or 10L spinner bottle or in a cell bioreactor according to the ratio of cell suspension: growth liquid of 1:20 to 1:100, and then add growth liquid to make the cell The initial concentration is 1.0×10 5 ~5.0×10 6 pieces / ml. Spinner bottle at 37°C with CO 2 The cell culture is carried out ...

Embodiment 1

[0062] Example 1: Preparation of virus seeds adapted to primary hamster kidney cells of influenza virus

[0063] Primal Poison Seed Armor 1 (H 1 N 1 ) type is IVR-116, which is the 8th generation chicken embryo allantoic fluid freeze-dried and preserved virus species; 3 (H 3 Type N2) is NYMC X-15F, which is the 8th generation chicken embryo allantoic fluid freeze-dried preservation virus species; B type is B / Jiangsu / 10 / 2003, which is the 6th generation chicken embryo allantoic fluid freeze-dried preservation virus species.

[0064] The above-mentioned three virus species were unsealed in a special aseptic room, and adaptive passages were carried out on SPF chicken embryos for 2 times. The finally harvested viral allantoic fluid was subjected to sterility test and hemagglutination titer (HA titer) measurement respectively. The sterility test is qualified, the hemagglutination titer of A1 and A3 are both 1:640, and the hemagglutination titer of type B is 1:320, thus establi...

Embodiment 2

[0070] Example 2: Preparation of virus seeds adapted to primary hamster kidney cells of influenza virus

[0071] Primal Poison Seed Armor 1 (H 1 N 1 ) type is IVR-116, which is the 8th generation chicken embryo allantoic fluid freeze-dried preservation virus species: A 3 (H 3 N 2 ) type is NYMC X-15F, which is lyophilized and preserved in the 8th generation of chicken embryo allantoic fluid; type B is B / Jiangsu / 10 / 2003 (recommended by WHO in 2004), which is lyophilized and preserved in the 6th generation of chicken embryo allantoic fluid Poison species.

[0072] The main generation seeds are the three types of influenza main generation seeds prepared in Example 1.

[0073] The anatomy of the hamster kidney, the preparation of the cell suspension, and the inoculation of the glass bottle for culture are the same as in Example 1.

[0074] When the cells in the culture flask occupy 60% to 80% of the culture surface, replace with maintenance solution I after rinsing, and use...

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Abstract

The invention discloses a method for preparing a flu vaccine. The method comprises the following steps of: a) performing adaptability passage on the original viruses of flu viruses through unspecific pathogenic chick embryos to serve as main-generation seeds; b) preparing primary hamster kidney cells, and culturing by using a culture flask or a cell biological reactor; c) infecting the by using the main-generation seeds, and performing adaptability passage until viruses with the virus clotting titer of not lower than 1:640 are obtained and used as working seeds of the vaccine; d) infecting the primary hamster kidney cells by using the working seeds, performing virus amplification until vaccine monovalent stock solution with the virus clotting titer of not lower than 1:320 is obtained; ande) concentrating, inactivating and purifying the vaccine monovalent stock solution, mixing different types of monovalent virus solution, and sub-packing into finished products. The primary hamster kidney cells have adequate sources and are easy to produce on a large scale. The prepared vaccine does not contain reproducible deoxyribonucleic acid (DNA) which has tumorigenicity on human bodies or animal bodies and has high safety.

Description

[0001] This application is a divisional application of Chinese patent application CN200610112125.9. technical field [0002] The invention belongs to the field of biotechnology, and in particular relates to a method for preparing influenza primary hamster kidney cell vaccine and the prepared vaccine. Background technique [0003] Influenza is called flu for short, is a kind of serious respiratory infectious disease. In the influenza pandemic of 1918-1919, more people died due to infection with the influenza virus than in World War II, which lasted 4 years. Influenza is often characterized by repeated epidemics, sudden onset, and rapid spread, ranging from small local epidemics to large ones all over the world. Infants and the elderly have a high case fatality rate of tens of thousands every year. At present, influenza is still an important issue affecting public health in the world, and the recent highly pathogenic avian influenza has a great danger of invading humans, so ...

Claims

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Application Information

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IPC IPC(8): A61K39/145C12N7/08A61P31/16
Inventor 李云英王玉清吴歧
Owner 深圳市孚沃德生物技术有限公司
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