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A Novel Purification Process for Pneumococcal Polysaccharide Antigen

IP.com Disclosure Number: IPCOM000237738D
Publication Date: 2014-Jul-08
Document File: 2 page(s) / 41K

Publishing Venue

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Abstract

For purification of Pneumococcal polysaccharide antigens, a chromatographic step using CaptoTM adhere, a multimodal anion exchanger, has been developed to replace the traditional hazardous step of phenol extraction.

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A novel purification process for Pneumococcal polysaccharide antigen

The current process is an improved process for purification of 23-valent (1,2,3,4,5,6b,7F,8,9N,9V,10A,12F,14,15B,17F,18C,19A,19F,20,22F,23F,33F) Pneumococcal polysaccharide to be used as an antigen in Pneumococcal vaccines. After fermentation and harvest, the traditional purification process involves precipitation with ethanol or CTAB, which removes DNA and part of the protein content. Then phenol is added to the crude polysaccharide to remove residual proteins and recovering a refined polysaccharide. The handling of phenol involves both occupational health and environmental hazards and the traditional process also has a low efficiency and recovery. Removal of phenol from the antigen is accomplished by dialysis, which requires 10-15 days. By replacing the last step with modern chromatography methods, a high purity polysaccharide can be produced with high efficiency and without the hazards associated with phenol use.

Starting from the crude Pneumococcal polysaccharide, the new process involves: 1) dissolution of the sample, 2) pretreatment of the sample, 3) column purification on CaptoTM adhere, a multimodal anion exchanger, and 4) desalting to arrive at the purified polysaccharide.


1) Dissolution of crude sample

Dissolve 10 mg/ml crude polysaccharide in saturated sodium acetate solution diluted 1:10 and then adjust the pH to 7.0. Depending on the type of polysaccharide, dissolution may require 4-10 hours.


2) Sample pretreatment

Add 10% sodium deoxycholate (SDC) stock solution to provide a final SDC concentration of 0.5%. Mix and then incubate on ice for 5-10 min. Adjust the pH to 6.1-6.2 with 1M phosphoric acid. Mix thoroughly and then incubate on ice for 3-6 hours. The pretreatment step is a key step to remove proteins and nucleic acids. Centrifuge at 12.000 g for 30 min. If the viscosity of the solution is very high, dilute with water. Repeat the step if necessary. If it is difficult to get a clear solution after centrifugation, filter on a 1 micrometer glass fiber membrane. Then pass the solution through an activated carbon filter to clarify the sample and remove part of the DNA.


3) Chromatography

For the components to be used in a 23-valent va...