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Method for removing residual hose cell DNA in recombinant Hansenula polymorpha hepatitis B surface antigen

A technology of HBsAg and Hansenula, applied in peptide preparation methods, retroviral DNA viruses, chemical instruments and methods, etc., can solve difficult problems

Inactive Publication Date: 2016-08-10
北京生物制品研究所有限责任公司
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  • Abstract
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AI Technical Summary

Problems solved by technology

Currently recombinant Hansenula expresses HBsAg mostly through Butyl-Sepharose (Butyl-Sepharose 4B) hydrophobic chromatography purification and purification, but the feed liquid sample after purification and purification by Butyl-Sepharose hydrophobic chromatography still contains More or less free host cell DNA will remain, and the concentration of residual DNA can even be as high as 1000ng / ml, while the concentration of HBsAg is generally 200-600μg / ml. Such a purified feed solution is used to prepare vaccines, and it is often difficult to meet the requirements of the National Pharmacopoeia III. Recombinant Hepatitis B Vaccine (Hansenula) stipulated by the Ministry of Health (2010 version) that the DNA content of each dose should not exceed 10ng

Method used

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  • Method for removing residual hose cell DNA in recombinant Hansenula polymorpha hepatitis B surface antigen
  • Method for removing residual hose cell DNA in recombinant Hansenula polymorpha hepatitis B surface antigen
  • Method for removing residual hose cell DNA in recombinant Hansenula polymorpha hepatitis B surface antigen

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preparation example Construction

[0035] 1.3 Preparation of upper column material liquid

[0036] Through the ultrafiltration device 100KD Vivaflow 50 purchased from Sartorius, Germany, the buffer solution in the sample containing HBsAg feed solution was replaced with a PB solution containing a specific concentration of NaCl, and the replacement flow rate was 50ml / min, thereby preparing the upper column feed solution (sample solution).

[0037] 1.4 Chromatography process

[0038] 1.4.1 Monolith-QA medium balance:

[0039] Equilibrate the CIM-MonolithsQA medium with the same PB solution containing NaCl at a specific concentration as the buffer in the sample solution, and set the flow rate according to the Monolith-QA column specifications (1ml Monolith-QA is 4ml / min; 8ml Monolith-QA is 10ml / min; 80ml Monolith-QA, 80ml / min), the total buffer volume is greater than 10 column bed volumes, and the baseline is a straight line, then the material (sample) can be loaded.

[0040] 1.4.2 Loading (sample):

[0041] Pu...

Embodiment 1

[0066] Using a 100KD Vivaflow 50 ultrafiltration device, 20121010 batches of HBsAg feed liquid samples purified by Butyl-4B hydrophobic chromatography were dissolved in 20mM PB (pH7.2) containing 300, 400, 500 and 600mM NaCl respectively by liquid replacement In, as the sample solution;

[0067] For 1ml monolith-QA columns, use 20mMPB containing 300, 400, 500 and 600mM NaCl as the loading buffer, and 20mM PB (pH7.2) containing 2M NaCl as the elution buffer. for 5ml. The breakthrough peak and elution peak samples were collected respectively, and the DNA level and HBsAg level in the breakthrough peak and elution peak were detected.

[0068] Chromatograms of HBsAg samples loaded under different NaCl concentrations (1ml Monolith-QA column) see figure 1 .

[0069] See Table 1 for the experimental results of chromatography (1ml Monolith-QA column) of HBsAg samples loaded under different NaCl concentration conditions.

[0070] Table 1. Chromatographic experimental results of load...

Embodiment 2

[0076] Take 20100908, 20101009, 20101010 and 20120605 batches of HBsAg feed liquid samples refined and purified by Butyl-4B hydrophobic chromatography, and use a 100KD Vivaflow 50 ultrafiltration device to dissolve the samples in 20mM PB containing 500mM NaCl (pH7.2 ) as the loading solution, and used 8ml Monolith-QA to carry out different loading tests, the loading volumes were 16ml, 24ml and 120ml respectively, the elution buffer was 20mM PB (pH7.2) containing 2MNaCl, and the breakthrough Peak solution and measure its volume, detect breakthrough peak HBsAg concentration and DNA concentration. The DNA level in the breakthrough peak and the HBsAg yield and DNA removal rate were evaluated.

[0077] (1) 16ml sample volume

[0078] For the experimental results of 16ml sample volume, see figure 2 and Table 2.

[0079] Table 2, 8ml Monolith-QA column 16ml HBsAg sample loading chromatography results

[0080]

[0081] from figure 2 And table 2 result can find out, when usin...

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Abstract

The invention provides a method for removing residual hose cell DNA (Deoxyribonucleic Acid) in a recombinant Hansenula polymorpha hepatitis B surface antigen. The method comprises the following steps: step a, dissolving a recombinant Hansenula polymorpha hepatitis B surface antigen sample for removing the residual hose cell DNA into a 10-30 mN of PB solution containing 400-500 mM of NaC1; step b, taking the PB solution containing the NaC1 in which the recombinant Hansenula polymorpha hepatitis B surface antigen sample is dissolved as a loading solution and performing chromatographic purification by utilizing an Monoliths anion exchange column to obtain the recombinant Hansenula polymorpha hepatitis B surface antigen sample for removing the residual hose cell DNA. The method provided by the invention can effectively remove the residual DNA in the recombinant Hansenula polymorpha hepatitis B surface antigen sample, so that prepared vaccine reaches the regulations on the content of the residual DNA in the Part III of the National Formulary (Edition 2010).

Description

technical field [0001] The present invention relates to a method for removing residual DNA of host cells in recombinant Hansenula yeast hepatitis B surface antigen, specifically, a method for removing residual host cell DNA in recombinant Hansenula yeast expressing hepatitis B surface antigen samples by using a chromatographic method DNA method. Background technique [0002] Recombinant Hansenula hepatitis B vaccine is a subunit vaccine of hepatitis B surface antigen (HbsAg). Prepared by recombinant yeast to express HBsAg subunit. [0003] In the preparation technology of recombinant Hansenula hepatitis B vaccine, the purification process is one of the key technologies. According to the National Pharmacopoeia Part III (2010 Edition), the DNA content of each dose (10 μg or 20 μg HBsAg) of the recombinant hepatitis B vaccine (Hansenula) should not exceed 10 ng. Currently recombinant Hansenula expresses HBsAg mostly through Butyl-Sepharose (Butyl-Sepharose 4B) hydrophobic ch...

Claims

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Application Information

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IPC IPC(8): C07K14/02C07K1/18
CPCC07K14/005C12N2730/10151
Inventor 杨旭琴李彩梅张德有李津杨云凯赵铠马锐许宁刘英微王曦
Owner 北京生物制品研究所有限责任公司
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