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Method for removing residual DNA from hydrophobia vaccine

A rabies vaccine and residual technology, applied in the field of bioengineering, can solve the problems of low antigen recovery rate, difficult to meet quality standards, high DNA residue, etc., achieve good DNA removal effect, reduce sample viscosity, and high antigen recovery rate Effect

Inactive Publication Date: 2010-01-13
CHANGCHUN ZHUOYI BIOLOGICAL CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] Using a single molecular sieve gel chromatography method, since the molecular weight of rabies virus is greater than that of other miscellaneous proteins, during the elution process, when the protein concentration is low, rabies virus and miscellaneous proteins can be separated, but in the actual production process The protein content is often high, and the sample viscosity is high, which makes the separation effect of the virus protein by single molecular sieve gel chromatography not good, and it is difficult to meet the existing national quality standards. At the same time, there is a problem of DNA exceeding the standard. , restricting the scale of production
[0004] At present, there is also a method that combines anion and molecular sieve gel chromatography to remove residual DNA. Protamine sulfate is used to bind residual DNA in the sample, and then gradient elution is performed with buffers of different salt concentrations to harvest virus penetration. However, it was found that protamine sulfate combined with DNA and part of the viral glycoprotein, which greatly reduced the recovery rate of the antigen. At the same time, different salt concentrations and pH also had a certain impact on the stability of the antigen.
[0005] The invention solves the problems of low antigen recovery rate and high DNA residue in the purification process

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] 1. Use the pellicon2 system to concentrate the virus harvest solution and remove some impurities. The virus harvest liquid is selected from the virus harvest liquid continuously harvested by Celligen Plus bioreactor. Firstly, the ultrafiltration system is cleaned and disinfected for later use. This experiment uses a pellicon2 ultrafiltration system with a membrane area of ​​0.5m 2 , cut-off quality 300KD, select 30L virus harvest liquid for 30-fold concentration, concentrate to 1L, and use β-propiolactone for virus inactivation.

[0018] 2. Divide the concentrated solution obtained in step 1 into two parts for experimentation, numbered as sample ① and sample ②, and sample ① is subjected to a single ultrafiltration purification using the original process, as the control group of the experiment, and the obtained concentrated solution is numbered as N-1, the purified solution number is C-1.

[0019] 3. Add non-restrictive endonuclease at 50U / ml to sample ②, and add MgCl ...

Embodiment 2

[0032] 1. Prepare 10L of rabies virus stock solution, divide it into two groups of Y-1 and Y-2 for experiment, and Y-1 still uses the original process for single ultrafiltration purification.

[0033] 2. Add non-restrictive endonuclease to the Y-2 sample at 5U / ml, and add Mg with a final concentration of 2mmol / L to the sample at the same time 3 (PO 4 ) 2 (provides the activator Mg for the enzyme 2+ ), put the sample at 20°C for DNA degradation.

[0034] After 3.4 hours, the enzymolysis sample was concentrated with the pellicon2 system and washed with 8 volumes of PBS.

[0035] 4. The washed sample is eluted by column chromatography to remove impurity proteins, residual enzymes and degraded DNA in the sample. Sampling was carried out according to the detection method of Example 1 for comparison.

[0036] sample name

Embodiment 3

[0038] 1. Use the pellicon2 system to concentrate the virus harvest solution and remove some impurities. The virus harvest liquid is selected from the virus harvest liquid continuously harvested by Celligen Plus bioreactor. Firstly, the ultrafiltration system is cleaned and disinfected for later use. This experiment uses a pellicon2 ultrafiltration system with a membrane area of ​​0.5m 2 , cut-off quality 300KD, select 30L virus harvest liquid for 30-fold concentration, concentrate to 1L, and use β-propiolactone for virus inactivation.

[0039] 2. Purify the sample obtained in step 1 by column chromatography, and divide it into two parts for experimentation. The sample numbers are C-1 and C-2.

[0040] 3. In the C-2 sample, add non-restrictive endonuclease by 1500U / ml, add the (CH 3 COO) 2 Mg·4H 2 O (provides the activator Mg for the enzyme 2+ ), put the sample at 4°C for DNA degradation. After 30 hours, samples were taken for detection and comparison (the method is the...

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PUM

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Abstract

The invention belongs to the technical field of biological engineering, in particular to a purification method during vaccine production, solving the problems that DNA is difficult to be removed and protein is easy to be polymerized and precipitated because column chromatography method is singly adopted during the previous production process of the hydrophobia vaccine. The method comprises the following steps: adopting a 750KD hollow fibre ultrafiltration column or a 300KD ultrafiltration membrane for concentrating virus harvested liquid or removing part of impurities, adopting non-restriction endonuclease for degrading DNA, and then removing nuclease by the method of ultrafiltration or column chromatography. The method is characterized by easy magnification, high product purity and obvious DNA removing effect, and greatly improves the safety of the vaccine.

Description

technical field [0001] The invention relates to the field of bioengineering, in particular, the invention is a method for removing residual DNA in the vaccine production process. technical background [0002] At present, the purification of rabies vaccine generally adopts a single molecular sieve gel chromatography or a method of combining anion and molecular sieve, both of which have certain defects: [0003] Using a single molecular sieve gel chromatography method, since the molecular weight of rabies virus is greater than that of other miscellaneous proteins, during the elution process, when the protein concentration is low, rabies virus and miscellaneous proteins can be separated, but in the actual production process The protein content is often very high, and the sample viscosity is high, which makes the separation effect of the virus protein by the single molecular sieve gel chromatography method not good, and it is difficult to meet the existing national quality stand...

Claims

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Application Information

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IPC IPC(8): A61K39/205A61P31/14
Inventor 陈明徐秀芝刘华张巍张景芝卢志平杨文杰
Owner CHANGCHUN ZHUOYI BIOLOGICAL CO LTD
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