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Purification and Visualization of Lipopolysaccharide from Gram-negative Bacteria by Hot Aqueous-phenol Extraction
Published on: May 28, 2012
Purification of capsular polysaccharides isolated from S. pneumoniae serotype 2 by hydrogen peroxide and endonuclease
Walmik Karbhari Gaikwad1, Swapan K Jana2, Rajeev M Dhere2
1Department of Technology, Savitribai Phule Pune University, Pune 411007, India; Research and Development Department, Serum Institute of India Pvt. Ltd., Hadapsar, Pune 411028, Maharashtra, India.
Abstract:
A high-quality and cost-effective purification procedure is one of the most important requirements for manufacturing glycoconjugate vaccines. The goal of the present work was to devise a method for removing impurities such as protein and nucleic acid from Streptococcus pneumoniae serotype 2 capsular polysaccharides (CPS). The use of hydrogen peroxide for the reduction of impurities of crude CPS was investigated. Centrifugation followed by filtration decreased protein contaminant of the hydrogen peroxide-treated CPS to meet the limit specified by WHO. The nucleic acid impurity remaining was removed by a further step of endonuclease treatment to yield the purified CPS. Characterization of purified CPS was evaluated by various analytical techniques including 1H NMR and antigenicity by competitive inhibition assay. Various hydrogen peroxide concentrations have significant impact on the antigenic property of CPS. Whereas, optimum process conditions can preserve the native characteristics of CPS.
