Method for reducing cell protein and DNA (Deoxyribose Nucleic Acid) residues in rabies virus product

A rabies virus and cell protein technology, applied in the field of reducing cell protein and DNA residues in rabies virus products, can solve problems such as nuclease residues

Active Publication Date: 2022-06-21
SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the use of nuclease treatment will lead to nuclease residues in the product, which needs to...

Method used

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  • Method for reducing cell protein and DNA (Deoxyribose Nucleic Acid) residues in rabies virus product
  • Method for reducing cell protein and DNA (Deoxyribose Nucleic Acid) residues in rabies virus product
  • Method for reducing cell protein and DNA (Deoxyribose Nucleic Acid) residues in rabies virus product

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0063] Embodiment 1. Preparation of human rabies vaccine with Vero cells and the removal method of Vero cell protein and DNA

[0064] 1. Experimental materials and equipment

[0065] After culturing Vero cells using basket bioreactor and sheet carrier culture technology, inoculate virus culture to prepare virus liquid, and the materials used are shown in Table 1.

[0066] Table 1

[0067] name Material Vero cells Seeding density: 5×10E+6 / ml strain rabies virus PM strain cell culture medium Gibco Serum Free Medium (VP SFM AGT) virus maintenance medium Ordinary 199 Medium filter element 0.6μm micropore diameter

[0068] 2. Experimental instruments and equipment

[0069] The virus liquid is concentrated by ultrafiltration through a 300KD membrane to obtain a virus concentrate, inactivated and hydrolyzed, subjected to molecular sieve chromatography, and then added with a stabilizer to obtain a stock solution. The equipment used...

Embodiment 2

[0094] Example 2. Protein and DNA removal under various centrifugal flow rates (sample before ultrafiltration)

[0095] Experimental group: According to the technique of Example 1, after culturing for 48 hours in "(3) Circulating and centrifuging the viral supernatant in the tank", filter through a 0.6 μm microfiltration membrane, and then harvest the virus liquid for measurement ( figure 2 , before ultrafiltration concentration).

[0096] Control group: Cell culture and virus culture were carried out according to the process of Example 1, wherein "(3) Circulating centrifugation of virus supernatant in the tank" was not carried out, and the virus liquid was directly harvested and then subjected to continuous flow centrifugation and 0.6 μm microfiltration membrane filtration .

[0097] In the process of culturing the virus in the bioreactor, the centrifugal force was 10000g, the centrifugal flow rate was 100ml / min (No. 1), 250ml / min (No. 2), 500ml / min (No. 3), and then filter...

Embodiment 3

[0103] Embodiment 3, the mass analysis of sample after ultrafiltration concentration

[0104] Experimental group: The same operation was performed as the experimental group in Example 2 (the supernatant was centrifuged at a centrifugal flow rate of 500 ml / min), and ultrafiltration concentration and gel filtration were also performed after the virus was harvested.

[0105] Control group: The same operation was performed as the control group in Example 2 (the supernatant was centrifuged at a centrifugal flow rate of 500 ml / min), and ultrafiltration concentration and gel filtration were also performed after the virus was harvested.

[0106] The virus solutions of the experimental group and the control group were concentrated by 40 times of concentration and ultrafiltration with a 300KD membrane package, and then purified by a Sepharose 4FF gel chromatography column, and then measured.

[0107] Table 5 shows the contents of Vero cell protein, Vero cell DNA and antigen in the origi...

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Abstract

The invention provides a method for reducing cell protein and DNA residues in a rabies virus product. The method comprises the following steps: continuously performing circulating centrifugation on virus supernate in a tank by using a continuous flow centrifuge in a bioreactor virus culture stage, then perfusing the virus supernate into a bioreactor culture system, and simultaneously harvesting supernate in the tank and filtering the supernate through a proper filtering device. After the method is adopted for treatment, protein residues and DNA residues of virus production cells in the virus harvesting liquid are remarkably reduced, and the protein residues of Vero cells are detected to be far lower than the existing technical standard through ultrafiltration concentration and column chromatography purification of the virus liquid and preparation of the freeze-dried vaccine. Meanwhile, the method effectively reduces the downstream purification cost, effectively ensures that no residues of other substances such as nuclease exist in the virus product, and is suitable for large-scale production.

Description

technical field [0001] The present invention belongs to the field of virology and vaccines, and more particularly, the present invention relates to a method for reducing the residues of cellular proteins and DNA in rabies virus products. Background technique [0002] Rabies is a zoonotic disease caused by the rabies virus with widespread global distribution. More than 55,000 people die from rabies each year, about 95% of which occur in Asia and Africa. Most of the deaths are caused by the bites of dogs infected with rabies virus, and 30-60% of the victims are children under the age of 15. The prevention and treatment of rabies usually involves vaccination against rabies. [0003] In the production of rabies vaccine, the virus is inoculated after the cells are adhered to the fixed bed of the bioreactor, and the supernatant after the virus infects the cells contains the target rabies virus. Impurities mainly include host cell debris, proteins, Vero cell proteins, and Vero ce...

Claims

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Application Information

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IPC IPC(8): C12N7/00C12N7/02A61K39/205A61P31/14C12R1/93
CPCC12N7/00A61K39/12A61P31/14C12N2760/20151C12N2760/20134
Inventor 喻志远朱绍荣刘海东
Owner SHANGHAI RONGSHENG BIOLOGICAL PHARM CO LTD
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