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An Efficient and Rapid Purification and Analysis Method for Polysaccharide-Protein Conjugated Vaccines

A polysaccharide protein and analysis method technology, which is applied in the field of purification and analysis of polysaccharide protein conjugates, can solve the problems of lack of universal prevention, high cost of pneumonia vaccine, high cost of imported vaccination, etc., to achieve simple operation and save manpower and material resources The effect of large consumption and processing volume

Active Publication Date: 2020-01-07
李红臣
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] Currently commercially available pneumonia polysaccharide-protein conjugated vaccines mainly rely on foreign imports. Imported vaccines are expensive to inoculate and cannot achieve the effect of universal prevention. Therefore, the development of serotype conjugate vaccines with a high incidence of pneumonia in my country has formed a product that can compete with foreign countries to solve the problem of vaccination. The problem of high cost of pneumonia vaccine is imminent

Method used

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  • An Efficient and Rapid Purification and Analysis Method for Polysaccharide-Protein Conjugated Vaccines
  • An Efficient and Rapid Purification and Analysis Method for Polysaccharide-Protein Conjugated Vaccines
  • An Efficient and Rapid Purification and Analysis Method for Polysaccharide-Protein Conjugated Vaccines

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Embodiment 1

[0060] Example 1. Preparation of CPS9V-CRM197 polysaccharide-protein conjugate by amine reduction method

[0061] Weigh 40 mg of CPS9V dry powder and dissolve it in 8 mL of phosphate buffer (pH 7.0) to form a polysaccharide solution with a concentration of 5 mg / mL. Add sodium periodate to the polysaccharide solution for oxidation, and then add vinyl glycol to terminate the reaction. The above reaction solution was transferred to a dialysis bag for dialysis at room temperature, and each dialysis was carried out for 1 hour for a total of 3 times.

[0062] Add carrier protein CRM to the above dialysate 197 After mixing, 100 mM sodium cyanoborohydride was added to react at room temperature for 5 days. After the reaction, 100 mM sodium borohydride was added to remove the remaining aldehyde groups in the reaction solution, and then the reaction solution was freeze-dried and stored for further purification.

Embodiment 2

[0063] Embodiment 2, CPS9V-CRM 197 Purification of polysaccharide-protein conjugates

[0064] Our company has developed a two-step combined purification process. The first step is to use the Minimate TFF ultrafiltration system (molecular cut-off of 1000kDa) to remove unreacted carrier proteins and small molecules. The second step is to use DEAE ion exchange Column chromatography removes free unbound CPS9V polysaccharide, and the column is eluted to collect CPS9V-CRM 197 conjugates.

Embodiment 3

[0065] Embodiment 3, CPS9V-CRM 197 Analysis of polysaccharide-protein conjugates

[0066] 3.1 CPS9V-CRM 197 Quantitative analysis of polysaccharide-protein conjugates: the content of capsular polysaccharide was determined by phenol-sulfuric acid method, and the content of protein was determined by BCA method. The purity of the conjugate was separated by CL-4B chromatography, and the absorption peaks were detected at dual wavelengths of ultraviolet light at 280nm and 206nm.

[0067] The results of quantitative analysis showed that: CPS9V pneumonia capsular polysaccharide and carrier protein CRM 197 Combined, separated by CL-4B chromatographic column and detected at dual wavelengths of 280nm and 206nm. Free CPS9V and CPS9V-CRM 197 The elution volumes of the conjugates were 50.34 mL and 47.82 mL, respectively.

[0068] For specific results, see the attached Figure 1a and 1b , Figure 1a It is the detection result of 280nm and 206nm dual wavelength after separation of fr...

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Abstract

The invention discloses a highly efficient and fast purification analysis method for polysaccharide conjugate vaccine. The method includes the following steps: first using an ultrafiltration system to remove carrier proteins and other small molecular substances not combined with the polysaccharide protein to acquire the polysaccharide protein and free polysaccharide not participating the conjugation, then enabling the ultrafiltrate flowing through the anion exchange resin to allow the free polysaccharide to pass through gel column to make the polysaccharide protein adsorbed to gel base, eluting and concentrating the eluent acquire the purified polysaccharide protein conjugate. The method has the advantages of fast speed, high efficiency, large processing quantity, low requirement of equipment, simple operation and the like. The method solves the problems of long purification time, low efficiency and high costs prevalent in the research, medium test and development and industrialized production of the polysaccharide conjugate vaccine.

Description

technical field [0001] The invention relates to the technical field of molecular bioengineering, in particular to a method for purifying and analyzing polysaccharide-protein conjugates. Background technique [0002] Pneumococcal infection is one of the important causes of death worldwide, and one of the main causes of pneumonia, meningitis, and otitis media. It is a global infectious disease and the population is generally susceptible. The elderly over the age of 10 are the main susceptible population. According to observations in the United States, it is estimated that 400,000 to 500,000 people suffer from pneumococcal pneumonia every year, with a case fatality rate of 5% to 10%. Diseases caused by pneumococcus rank first among vaccine-preventable childhood deaths. At present, with the dependence and abuse of antibiotics, drug-resistant strains are increasing day by day. The medical community is actively paying attention to and starting the development of pneumococcal vac...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/34C07K1/18C07K14/34A61K39/09A61P31/04G01N33/68G01N33/535
CPCA61K39/092C07K1/18C07K1/34C07K14/34G01N33/535G01N33/68
Inventor 李红臣寇佳琳黄前荣张永强任新刚葛俊男
Owner 李红臣