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Method for producing porcine parvovirus inactivated vaccine by using torrent bioreactor

A bioreactor, parvovirus technology, applied in biochemical equipment and methods, microorganisms, antiviral agents, etc., can solve the problems of unstable product quality, low vaccine production efficiency, and easily contaminated products, and achieve rapid expansion of production. Scale, beneficial to continuous reproduction, easy cell growth and effect of nutrient supply

Active Publication Date: 2014-05-28
山东滨州沃华生物工程有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] The purpose of the present invention is to address the deficiencies in the prior art, and propose a method for producing porcine parvovirus inactivated vaccines using a torrent bioreactor, which can solve the problems of low vaccine production efficiency, unstable product quality, and low virus titer. Low cost, cumbersome operation, many passage links, and products are easily contaminated, etc.

Method used

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  • Method for producing porcine parvovirus inactivated vaccine by using torrent bioreactor
  • Method for producing porcine parvovirus inactivated vaccine by using torrent bioreactor
  • Method for producing porcine parvovirus inactivated vaccine by using torrent bioreactor

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0025] Embodiment 1: Cultivate IBRS-2 cells in a torrent bioreactor

[0026] 1. Cell Recovery

[0027] Take out the IBRS-2 cell cryopreservation tube from the liquid nitrogen tank, quickly put it into 37°C water, shake it constantly to thaw it quickly, use a pipette to separate the cell suspension, put it into a sterile centrifuge tube, and add 9ml of cell growth centrifuge at 1000rpm for 10 minutes, discard the supernatant, add cell culture medium (containing 10% newborn bovine serum) to suspend the cells, transfer them to a disposable cell culture bottle, and store at 37°C 5% CO 2 Cultivate in a constant temperature incubator, replace the cell growth medium once after 6 hours, and continue to cultivate.

[0028] 2. Cell passage

[0029] Take a well-grown IBRS-2 cell monolayer, discard the medium, wash twice with PBS, add an appropriate amount of trypsin digestion solution, and place it flat for about 2 minutes to observe the cell digestion. If the cell layer is blurred, d...

Embodiment 2

[0035] Example 2: Cultivation of Porcine Parvovirus Cells in a Rapid Flow Bioreactor

[0036] 1. Preparation of porcine parvovirus seed virus

[0037] First transfer the IBRS-2 cells to the bottle for passage, and when the cells grow into a single layer, discard the culture medium, inoculate the cells with the porcine parvovirus CP-99 strain, the inoculation dose is 0.01MOI, and replace the maintenance solution with 2% serum after inoculation Continue to cultivate. Harvest the virus liquid when the cell lesion reaches more than 80%, and carry out virus titer and sterility test on the harvested virus liquid, and the TCID of the virus liquid 50 ≥ 10 6.5 TCID 50 / ml, if the sterility test is qualified, it can be used as a seed virus for production.

[0038] 2. Bioreactor cells were inoculated with porcine parvovirus

[0039] When the cells in the torrent bioreactor were continuously cultured for 5 days and the measured glucose consumption reached a stable level, the cell den...

Embodiment 3

[0043] Example 3: Optimization test of PPV proliferation conditions in AMP AP20C torrent bioreactor

[0044] 1. The effect of the number of cells inoculated on the proliferation of PPV

[0045] Three groups (groups A, B and C) were selected for the experiment with different inoculum volumes of cells, which were 1×10 6 cells / ml, 1×10 7 cells / ml, 2×10 7 cells / ml, the virus fluid was harvested at 54h, 60h, 66h, and 72h after virus inoculation, and the virus titer was determined to study the effect of the number of cells inoculated on the proliferation of PPV.

[0046] Experimental data:

[0047] Table 1 Effect of different cell densities on PPV proliferation

[0048]

[0049] The experimental results showed that the virus liquid harvested after 60 hours of virus inoculation had higher toxicity, and the cell density was 1×10 7 When cells / ml is lower, the virus liquid has a higher toxicity.

[0050] 2. Influence of poisoning dose on PPV proliferation conditions

[0051] T...

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Abstract

The invention relates to a method for producing a porcine parvovirus inactivated vaccine by using a torrent bioreactor, comprising the following steps: inoculating the porcine parvovirus into the IBRS-2 cell, and culturing to obtain a production virus strain; inoculating the IBRS-2 cell into the torrent bioreactor; inoculating the prepared porcine parvovirus strain into the IBRS-2 cell cultured in the torrent bioreactor, and proliferating and culturing the virus; harvesting the proliferated virus solution; inactivating; and matching to obtain the porcine parvovirus inactivated vaccine. According to the method, because the torrent bioreactor and a paper vector are used, the shearing force generated by a microcarrier bioreactor used for culturing the cell is avoided, the density and the viability of the cell are further improved, and the capacity of proliferating the viruses by the cell is enhanced. The reactor can be used for monitoring and automatically regulating the optimal condition for growing the cells, thus the viruses harvested in all bathes have small difference, the quality stability of the vaccine is improved, the vaccine production efficiency is improved, and the vaccine production cost is reduced.

Description

technical field [0001] The invention relates to the technical field of animal biological product cultivation, in particular to a method for producing porcine parvovirus inactivated vaccine by using a torrent bioreactor. Background technique [0002] Porcine parvovirus (PPV) is one of the only diseases that cause reproductive disorders in sows. The main manifestations are infected sows, especially multiparous sows and seronegative multiparous, abortion, stillbirth, deformed fetus, mummified fetus, weak fetus and repeated infertility, etc. PPV infection is also harmful to the fetus. Pigs of other ages generally do not show obvious clinical symptoms after infection. The disease is widely distributed in the world, and most of the infected pig farms are endemic, and it is difficult to purify the infected pigs, which causes continuous economic losses and seriously hinders the healthy development of the global pig industry. PPV has a single serotype and rarely mutates, but there ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/23A61K9/107A61P31/20C12N7/00C12N7/06
Inventor 徐明明陈申秒董新荣
Owner 山东滨州沃华生物工程有限公司
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