RESUMO
Haemophilus influenzae b (Hib), an encapsulated Gram-negative coccobacillus, is one of the most common agents of meningitis worldwide. The Hib vaccine is included in the routine immunization schedule of several countries. The capsular polysaccharide, Poliribosyl-Ribitol-Phosphate (PRP), conjugated to a carrier protein is the antigen of the vaccine against Hib. Currently in industrial processes, Hib is cultivated in a soy peptone and yeast-extract based complex medium (MMP). This work aimed to evaluate whether a chemically defined medium could replace the MMP medium in the PRP production. The use of a defined media could offer several advantages in the whole process and would be of great interest to the industrial PRP production in vaccination programs. The PRP production ranged from 100 to 290 mg L-1 in the studied semi-defined and defined media. The formulation of several chemically defined media based on literature led to a promising composition as is the case of medium G which presented a production of 295 mg PRPL-1 with a lower biomass value of 4.8 UA (O.D.540 nm) when compared to MMP medium with 395 mg PRPL-1 and 6.45 UA respectively.
RESUMO
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.