Method for removing residual dna in Japanese encephalitis vaccine products by anion exchange chromatography

An exchange chromatography, anion technology, applied in the direction of resistance to vector-borne diseases, virus antigen components, etc., can solve the problems of large side effects of vaccines, low removal rate of impurity proteins, and high host DNA content, to achieve removal content and improve product quality. and pass rate, the effect of breaking through the quality bottleneck

Active Publication Date: 2017-05-17
LIAONING CHENGDA BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] 1. The residual host DNA content after virus vaccine extraction is too high
[0005] 2. After the virus vaccine is extracted, it still contains too much host protein, and the removal rate of miscellaneous proteins is not high, which leads to relatively large side effects of the vaccine after clinical use
[0006] 3. The above defects have affected the quality of virus vaccine products and restricted the production of vaccine products

Method used

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  • Method for removing residual dna in Japanese encephalitis vaccine products by anion exchange chromatography
  • Method for removing residual dna in Japanese encephalitis vaccine products by anion exchange chromatography
  • Method for removing residual dna in Japanese encephalitis vaccine products by anion exchange chromatography

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Use the AKTApilot automatic chromatography equipment to pass 2000mL JE vaccine concentrate (lot number S1) through the BPG200 chromatography column, Sepharose 4Fast Flow gel filtration chromatography, the ultraviolet detection wavelength is 280nm, the flow rate is 6cm / h, and the mobile phase is pH7.2 10mmol / L PBS (NaCl 0.2mol / L), collect the first absorption peak, that is, the preliminary purification solution of Japanese encephalitis virus protein (batch number S2). Then use 10mmol / L PBS (NaCl0.2mol / L) with pH 7.2 to equilibrate the DEAE Sepharose Fast Flow chromatography column (column bed volume 1000mL) to UV and conductance balance, flow rate 3cm / h. Then the preliminary purified liquid is loaded into the DEAE Sepharose Fast Flow chromatography medium by 2 times column bed volume (2000mL), the flow velocity is 2cm / h, and the host DNA is firmly adsorbed on the DEAE Sepharose Fast Flow chromatography medium, while the Japanese encephalitis virus protein and The binding...

Embodiment 2

[0028] Use AKTApilot automatic chromatography equipment to pass 2000mL JE vaccine concentrate through BPG200 chromatography column, Sepharose 4Fast Flow gel filtration chromatography, UV detection wavelength 280nm, flow rate 6cm / h, mobile phase 10mmol / L PBS with pH 7.4 (NaCl 0.3mol / L), collect the first absorption peak, i.e. the preliminary purified liquid of Japanese encephalitis virus protein. Then equilibrate the QSepharose Fast Flow chromatography column with pH 7.4 10mmol / L PBS (NaCl 0.3mol / L) to reach UV and conductance balance, and flow rate 3cm / h. Then the preliminary purified solution was loaded into the Q Sepharose Fast Flow chromatography medium by 2 times the column bed volume (2000mL), and the flow rate was 2cm / h. The host DNA was firmly adsorbed on the Q Sepharose Fast Flow chromatography medium, while the Japanese encephalitis virus protein The binding site with the ion exchange medium is replaced by the ions in the buffer, directly penetrated, the ultraviolet d...

Embodiment 3

[0030] Use AKTApilot automatic chromatography equipment to pass 2000mL JE vaccine concentrate through BPG200 chromatography column, Sepharose 4Fast Flow gel filtration chromatography, UV detection wavelength 280nm, flow rate 6cm / h, mobile phase 10mmol / L PBS with pH 7.4 (NaCl 0.4mol / L), collect the first absorption peak, i.e. the preliminary purified liquid of Japanese encephalitis virus protein. Then equilibrate the CaptoQ chromatography column with 10 mmol / L PBS (NaCl 0.4 mol / L) of pH 7.4 until the UV and conductance are balanced, and the flow rate is 3 cm / h. Then the preliminary purified liquid is loaded into the Capto Q chromatography medium by 2 times the column bed volume (2000mL), the flow rate is 2cm / h, the host DNA is firmly adsorbed on the Capto Q chromatography medium, and the JE virus protein and the ion exchange medium The binding site is replaced by ions in the buffer, directly penetrated, the UV detection wavelength is 280nm, and the absorption peak is collected....

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Abstract

The invention relates to a method for removing residual DNA in an encephalitis B vaccine product by utilizing anionic exchange chromatography. The method is characterized in that a preliminary purifying liquid obtained through the gel filtration chromatography of an encephalitis B vaccine concentrate undergoes anionic exchange chromatography to make DNA firmly adsorbed on an ion exchange medium and encephalitis B virus proteins directly penetrate the ion exchange medium, so host DNA is removed. The method is provided against the problems that only a certain proportion of free DNA can be removed, host DNA combined with antigen proteins cannot be removed, and clinic untoward effect phenomena common occur of present encephalitis B vaccine extraction methods comprising concentration, gel filtration chromatography and the like, improves the quality of the vaccine product on the premise of guaranteeing the vaccine titer, removes a large protein of other proteins and the host DNA to make the content of the residual DNA in the vaccine product reach below 50pg per dose, and solves the quality problem of the current encephalitis B vaccine industry.

Description

technical field [0001] The invention relates to a method for removing host DNA and miscellaneous proteins in vaccine products, in particular to a method for removing residual DNA in JE vaccine products by using anion exchange chromatography. Background technique [0002] The Japanese encephalitis vaccine is to inoculate the Japanese encephalitis virus on suitable animal cells, and the virus is released into the culture medium after reproduction. At the same time, some residual host cells or their fragments will also enter the culture medium. After clarification, ultrafiltration After concentration and gel filtration chromatography, free host DNA and host DNA combined with antigenic protein will be produced in the extracted JE vaccine stock solution. The stock solution also contains a large amount of host protein, and the process of concentration and gel filtration chromatography will Remove a certain proportion of free DNA, but there will still be residual DNA, especially th...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): A61K39/12
CPCY02A50/30
Inventor 高军白珠穆李旭李庆岸杜鹏于海
Owner LIAONING CHENGDA BIOTECH
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