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Cintra FO. Mathematical modelling and optimization of the production of exopolysaccharide from Haemophilus influenzae type b. [dissertation (Master in Biotechnology)]. São Paulo: Instituto de Ciências Biomédicas, Universidade de São Paulo; 2014. Haemophilus influenzae type b (Hib) is a bacterium that is pathogenic to the human being, causing pneumonia, sepsis and meningitis. The capsular polysaccharide conjugated to a protein is used as antigen in polyvalent vaccines, where it represents more than half of the total price of the formulation. This high cost for the Hib vaccine arises among other reasons from the low recovery ratios throughout the culture, purification and conjugation processes, due to the chemical instability of the polysaccharide. This work considers the interface between culture and purification as the subject for the optimization of the polysaccharide production. First, a kinetic model was selected, that describes the cellular growth as being inhibited by acetate; acetate formation as being of the mixed type; and polyssacharide formation as being non-growth associated and also undergoing acetate inhibition. Then, a design of experiments was carried out in order to evaluate the effects of pH and temperature on the kinetic parameters. It was demonstrated that the rate of growth is maximum at pH 6.73 (0.644 h); the inhibition constant is higher at alkaline pH (9.907 gA.L -1 at pH 7.5 against 5.819 gA.L -1 at pH 6.3); the growth associated acetate formation is also higher at alkaline pH (0.796 gA.gX -1 at pH 7.5 against 0.477 gA.gX -1 at pH 6.3) while the non-associated part is more influenced by temperature, achieving 0.141 gA.gX .h at 37 °C and pH 6.3; the polysaccharide formation presented an optimum at physiological conditions (45.271 mgP.gX .h at pH 7.2 and 37.1 °C), with the inhibition constant also higher at elevated pH (12.083 gA.L -1 at pH 7.5 against 6.541 gA.L -1 at pH 6.3); the rate of polysaccharide release from the cells was also increased at higher pH (88 % at pH 7.5 against 55 % at pH 6.3); both polysaccharide productivity and molecular mass were optimal at pH 7.1, though productivity was increased by temperature (117.89 mgP.L .h at 37 °C against 67,24 mgP.L .h at 29 °C) while the opposite was true for the molecular mass (642 kDa at 29 °C against 472 kDa at 37 °C). From these results, the optimal conditions were defined as pH 7.1 and 30 °C. In these conditions, the molecular mass was 78 % higher (643 kDa) than the described in the literature, with pH 7.5 and 37 °C (361 kDa), achieving the same amount of polysaccharide (1400 mgP.L ). This new scenery resulted in higher recovery at the first step of purification (from 44 to 75 %), thus confirming that the new conditions may also improve the yields in the following steps of purification and conjugation, ensuring better efficiency, enabling reduction of scale and minimizing the final cost of the vaccine. Supported by Fundação Butantan and BNDES (N° 11.2.0322.1/2012).

Modelagem Matemática e otimização da produção de exopolissacarídeo de Haemophilus influenzae tipo b.