Development of an efficient and scalable method for processing and purification of Vi capsular polysaccharide

A method for the purification of Vi capsular polysaccharide produced during fermentation of Salmonella typhi was developed. A number of wild type isolates of S. typhi were obtained from National Institute of Cholera and Enteric Diseases (NICED) India and screened for their capacity to produce Vi. One isolate designated C6524 produced more Vi than any of the other isolates so was selected as a potential seed lot for vaccine production. The first step in the Vi purification process was to separate the S typhi cells from the crude Vi and this was achieved by crossflow microfiltration followed by diafiltration against high salt to assist passage of the Vi through the membrane into the permeate. The next step was to precipitate the Vi which has a negative charge at neutral pH with the positively charged cetavlon. During this procedure most of the protein, nucleic acid and LPS impurities remained in solution and were easily separated from the precipitated Vi after washing of the precipitate with 20% ethanol. The precipitated Vi was then dissolved in 60% ethanol. The Vi was then precipitated a second time by increasing the ethanol concentration to 75%, the precipitate was allowed to settle and the ethanol decanted, the Vi was then dissolved in water. To achieve satisfactory nucleic acid levels ammonium sulfate was added and the precipitate containing nucleic acid was removed by filtration. The purified Vi produced by this method met the WHO requirements for Vi polysaccharide vaccine with a high yield of Vi equating to low production costs and an affordable vaccine for communities living in typhoid endemic areas.