Haroldo Hiss

Purification of meningococcal group C polysaccharide by a procedure suitable for scale-up

Abstract Neisseria meningitidis group C capsular polysaccharide is the antigen for the vaccine. An easier method has been developed for the purification of N. meningitidis group C capsular polysaccharide. In this method, two steps of the traditional procedure have been modified: the removal of protein and lipopolysaccharide. The phenol extraction for removal of contaminant protein was substituted by proteinases digestion using three different proteinases: proteinase K, nagarse and trypsin. Tangential ultrafiltration in hollowfiber 100 kDa cutoff was used instead of ultracentrifugation. Extensive diafiltration on a 100 kDa cutoff in Tris-HCl buffer containing 0.5% of deoxycholate was able to eliminate lipopolysaccharide as well as low molecular weight protein. The resultant purified polysaccharide contained around 2% of protein, 1.5% of nucleic acid, and it passed the pyrogen test for lipopolysaccharide in rabbit.

Polysaccharide production in batch process of Neisseria meningitidis serogroup C comparing Frantz, modified Frantz and Cartlin 6 cultivation media

Polysaccharide of N. meningitidis serogroup C constitutes the antigen for the vaccine against meningitis. The goal of this work was to compare three cultivation media for production of this polysaccharide: Frantz, modified Frantz medium (with replacement of glucose by glycerol), and Catlin 6 (a synthetic medium with glucose). The comparative criteria were based on the final polysaccharide concentrations and the yield coefficient cell/polysaccharide (YP/X). The kinetic parameters: pH, substrate consumption and cell growth were also determined. For this purpose, 9 cultivation runs were carried out in a 80 L New Brunswick bioreactor, under the following conditions: 42 L of culture medium, temperature 35oC, air flow 5 L/min, agitation frequency 120 rpm and vessel pressure 6 psi, without dissolved oxygen or pH controls. The cultivation runs were divided in three groups, with 3 repetitions each. The cultivation using the Frantz medium presented the best results: average of final polysaccharide concentration = 0.134 g/L and YP/X=0.121, followed by Catlin 6 medium, with results of 0.095 g/L and 0.067 respectively. Considering the principal advantages in the use of the synthetic medium, i.e. facilitation of a cultivation and purification steps of the polysaccharide production process, there is a possibility that in the near future, Catlin 6 will replace the traditional Frantz medium.

Nitrogen consumption during batch cultivation of Neisseria meningitidis (serogroup C) in Frantz medium

Polissacarideo capsular, extraido de cultivos microbianos, e o principal antigeno para o preparo da vacina contra a doenca causada por Neisseria meningitidis sorogrupo C. O conteudo final de proteina permitido nessa vacina e de 1%. De modo a encontrar uma relacao entre o consumo de nitrogenio, o crescimento microbiano (incluindo a producao de polissacarideo) e o conteudo de nitrogenio celular, cultivos foram realizados em um fermentador de 80 L (capacidade total), sob as seguintes condicoes: meio de Frantz; temperatura de 35oC; vazao de ar de 5 L/min (0,125 vvm); frequencia de agitacao de 120 rpm e pressao da dorna de 6 psi (kLa = 0.07 min-1). Durante os cultivos, foram determinadas as concentracoes de biomassa, polissacarideo, nitrogenio celular, nitrogenio residual orgânico e inorgânico no meio. De cinco fermentacoes realizadas sob as mesmas condicoes, encontrou-se uma porcentagem media de nitrogenio celular de 12,6% (p/p) em relacao a biomassa seca. O nitrogenio inorgânico do meio nao apresentou variacao significativa ao longo do tempo de cultivo, enquanto que o consumo de nitrogenio orgânico foi linearmente relacionado com o crescimento da populacao celular, com fatores de conversao constantes (media de 8,44). A cinetica de producao de polissacarideo acompanhou a de crescimento da cultura celular ate o comeco da fase estacionaria de crescimento e, a partir desse momento ate o fim do cultivo, foi observada uma producao suplementar de polissacarideo, mas sem absorcao de nitrogenio pelas celulas. Assim, os resultados indicaram uma estrategia de producao de polissacarideo em duas etapas, sendo a primeira crescimento celular seguida da producao de polissacarideo nao-associado ao crescimento microbiano.

INFLUENCE OF INITIAL L-ASPARAGINE AND GLYCEROL CONCENTRATIONS ON THE BATCH GROWTH KINETICS OF MYCOBACTERIUM BOVIS BCG

The influences of the L-asparagine and glycerol initial concentrations in Sauton medium on the productivities of biomass and colony forming units were studied. The submerged batch cultivations of Mycobacterium bovis were carried out in a 20 L bioreactor. The L-asparagine and glycerol initial concentrations of 4.54 g/L and 25 mL/L, respectively, corresponded to the best biomass productivity, namely 2.5 g/L.day. On the other hand, the concentrations of 2.27 g/L and 25 mL/L, respectively, led to the highest productivity in terms of colony forming units, namely 2.7·106 colonies/mg.day. In addition, by means of the relative consumption analysis of L-asparagine and glycerol (50 and 26% respectively), it was concluded that the concentrations of such components could be reduced, with respect to the original Sauton medium composition, aiming the obtainment of an optimal BCG vaccine production in the bioreactor.

Polysaccharide Production in Pilot Scale Bioreactor Cultivations of Neisseria meningitidis Serogroup C

Serogroup C polysaccharide from Neisseria meningitidis (PS) constitutes the antigen for the respective vaccine production. In order to investigate the enhancement of the final PS concentration (Pf), as well as the overall yield factor (PS/biomass) (YP/X), 13 total cultivations distributed in 6 series (from A to F) were carried out in Frantz medium (40 L plus inoculum) in a 80L bioreactor at 35oC, 0.4 atm, 120 rpm, airflow rate of 5 L/min and KLa = 4.2 h-1. The series (A-F) correspond to different experimental conditions as follows: A) without pH and dissolved O2 controls; B) pH control at 6.5; C) pH control at 6.5 and glucose pulse at the 10th hour; D) dissolved O2 control at 10% saturation value; E) pH control at 7.4; F) dissolved O2 limitation (set rotation at 55 rpm). Concentrations of dry biomass, PS, cellular nitrogen, residual glucose, organic and inorganic nitrogen in the medium were measured. The best results were represented by series A (averages of Pf = 0.15 g/L and YP/X = 107 mg/g). The presented findings could be useful for a proper Frantz medium reformulation in order to obtain a greater amount of PS and improve the vaccine development in industrial scale-up production.

Semicontinuous alcoholic fermentation of sugar cane blackstrap molasses by pressed yeast

SummaryA third degree polynomial equation correlates the fermentation time and the volume fraction of inoculum in semicontinuous ethanol fermentation of sugar cane blackstrap molasses by pressed yeast. The volume of the inoculum varied from 40% to 92% of the reactor working volume. The highest ethanol productivity was obtained when the volume of the inoculum was 58% of the working volume.

Semicontinuous ethanol fermentation of sugar cane blackstrap molasses by pressed yeast. II: Mathematical model

SummaryA mathematical model is proposed to explain the influence of the volume fraction of inoculum on the fermentation time and ethanol productivity in semicontinuous ethanol fermentation of sugar cane blackstrap molasses by pressed yeast.