Purification of a phosphated biopolymer by selective ethanol precipitation in presence of surfactant and sodium acetate

Abstract

Bacteria have the ability to produce biopolymers with different chemical properties, for different purposes and vary according to the bacterial strains and their physiological status, and these can be used as vaccine antigens. Haemophilus influenzae type b is a microorganism pathogenic to humans, which causes several types of infections. It is classified into six serotypes, the biopolymer of serotype b (Hib) being the most virulent, known as Poly Ribosylribitolphosphate (PRP). The aim of this work was to evaluate different candidate surfactants to be used in the PRP purification step, as well as the effects of ethanol in combination with sodium acetate. From all the surfactants used, 0.5% SDS proved to be potent in eliminating protein impurities and nucleic acids and in accordance with criteria of regulatory agencies. Regarding the combination of ethanol and sodium acetate to precipitate impurities, in the first fractionation step and polysaccharide, in the second fractionation step; the best conditions were: 40% ethanol without sodium acetate in the first stage and 60% ethanol containing 7% sodium acetate in the second stage. This improved condition resulted in nearly 100% polysaccharide recovery with relative purities higher than 100 for both protein and nucleic acid. In the traditional PRP purification process the final polysaccharide recovery was around 20% at the end of the process, while the new condition will result in at least 80% and within the purity criteria established by WHO for this polysaccharide.