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Method for measuring residual amount of Triton X-100 in HBsAg stock solution expressed by recombinant Saccharomyces cerevisiae

A technology of recombinant Saccharomyces cerevisiae and an assay method, applied in the field of preparation of recombinant genetic engineering hepatitis B vaccine, can solve the problems of accuracy interference, inability to effectively remove impurity interference, system inconsistency, etc., and achieve the effect of accurate residual amount

Active Publication Date: 2015-03-25
深圳鑫泰康生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] However, the inventors found in the process of researching the present invention that the hepatitis B surface antigen expressed by recombinant Saccharomyces cerevisiae was purified to obtain the HBsAg stock solution for preparing vaccines, the protein content of which was between 20 μg / ml and 250 μg / ml, as Macromolecular substances and high-concentration protein content will not only seriously interfere with the accuracy of the determination of Triton X-100 residues by high performance liquid chromatography, but also the impurity washing method of the prior art solid phase extraction method cannot effectively remove impurities At the same time, in the determination step of high performance liquid phase method, the solvent for preparing the reference substance is water, and the solution of the test product is methanol solution, the system is inconsistent, which affects the accuracy

Method used

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Examples

Experimental program
Comparison scheme
Effect test

example 1

[0064] Instrument:

[0065] Waters Alliance 2695 high performance liquid chromatograph, Waters PDA2996 detector, Waters Empower software, Mettler-Toledo XP205 electronic analytical balance.

[0066] Reagents and reagents:

[0067] Triton X-100 reference substance: purchased from Sigma Company, batch number 100M0128V; methanol: HPLC grade, purchased from Merck Company; water: ultrapure water.

[0068] process:

[0069] In the preparation step of the reference substance solution, the Triton X-100 reference substance was dissolved in 90% methanol solution to make a series of concentrations of 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml Reference substance solution.

[0070] In the activation step, select Waters HLB 1cc solid phase extraction column, first pass through 1ml of methanol, then pass through with 1ml of water.

[0071] In the protein content detection step, it is detected that the protein content in the stock solution A of the test product is 20 μg / ml; t...

example 2

[0080] Instrument:

[0081] Waters Alliance 2695 high performance liquid chromatograph, Waters PDA2996 detector, Waters Empower software, Mettler-Toledo XP205 electronic analytical balance.

[0082] Reagents and reagents:

[0083] Triton X-100 reference substance, purchased from Sigma Company, batch number 100M0128V; methanol: HPLC grade, purchased from Merck Company; water: ultrapure water.

[0084] process:

[0085] In the preparation step of the reference substance solution, the Triton X-100 reference substance was dissolved in 90% methanol solution to make a series of concentrations of 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml Reference substance solution.

[0086] In the activation step, select Waters HLB 1cc solid phase extraction column, first pass through 1ml of methanol, then pass through with 1ml of water.

[0087] In the protein content detection step, it is detected that the protein content in the stock solution C of the test product is 20 μg / ml; t...

example 3

[0096] Instrument:

[0097] Waters Alliance 2695 high performance liquid chromatograph, Waters PDA2996 detector, Waters Empower software, Mettler-Toledo XP205 electronic analytical balance.

[0098] Reagents and reagents:

[0099]Triton X-100 reference substance: purchased from Sigma Company, batch number 100M0128V; methanol: HPLC grade, purchased from Merck Company; water: ultrapure water.

[0100] process:

[0101] In the preparation step of the reference substance solution, the Triton X-100 reference substance was dissolved in 90% methanol solution to make a series of concentrations of 1 μg / ml, 2.5 μg / ml, 5 μg / ml, 10 μg / ml, and 20 μg / ml Reference substance solution;

[0102] In the activation step, select Waters HLB 1cc solid phase extraction column, first pass through 1ml of methanol, then pass through with 1ml of water.

[0103] In the protein content detection step, it is detected that the protein content in the stock solution E of the test product is 20 μg / ml; th...

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PUM

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Abstract

The invention discloses a method for measuring the residual amount of a Triton X-100 in a HBsAg stock solution expressed by recombinant Saccharomyces cerevisiae. The method includes the step of preparing reference substance solution preparation, the step of preparing the solution of substances to be measured and the step of measuring Triton X-100. The step of preparing the solution of the substances to be measured includes the steps that the protein content in each set of substances to be measured is detected, and the substances, with the protein content larger than a threshold value, to be measured and the substances, with the protein content larger than the threshold value, to be measured are discriminated; one part of water is used for conducting first time of washing and impurity removing on each set of substances to be measured through a solid phase extraction column; then one part of water is used for conducting second time of washing and impurity removing on each set of substances to be measured for the substances, with the protein content larger than the threshold value, to be measured; one part of methanol solution with the volume concentration being 2-70 percent is used for conducting third time of washing and impurity removing on each set of substances to be measured. The method can effectively remove the influences of protein macromolecule matter and other interference matter in the stock solution on measuring of the residual amount of Triton X-100, the recovery rate is high, the residual amount of Triton X-100 in the stock solution can be accurately measured, and the limit of detection of the residual amount of Triton X-100 can reach the 1mg / ml level.

Description

technical field [0001] The invention relates to the preparation technology of recombinant genetic engineering hepatitis B vaccine, in particular to a method for determining the residual amount of Triton X-100 in the stock solution of HBsAg expressed by recombinant Saccharomyces cerevisiae. Background technique [0002] my country is a high-prevalence area of ​​hepatitis B, with an average of more than 500,000 cases reported each year, and hepatitis B vaccination is the most effective and economical way to prevent hepatitis B. Using recombinant Saccharomyces cerevisiae to express hepatitis B surface antigen (hepatitis B surface antigen, HBsAg) in large-scale industrial production of hepatitis B vaccine is a kind of hepatitis B vaccine production process with easy access to medium, low cost and high productivity, which can meet the requirements of my country's hepatitis B vaccine massive demand. [0003] Polyethylene glycol octyl phenyl ether (Triton X-100) is a non-ionic surf...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N30/02G01N30/06
Inventor 饶洪冲朱征宇段琰黄龙
Owner 深圳鑫泰康生物科技有限公司