DEVELOPMENT OF A NEW PROCESS FOR PURIFICATION OF CAPSULAR POLYSACCHARIDE FROM Streptococcus pneumoniae SEROTYPE 14

The main virulence factor of Streptococcus pneumoniae is the capsular polysaccharide (PS), which is the antigen of all current vaccines that are prepared with PS purified from serotypes prevalent in the population. In this work, three purification strategies were evaluated and a new process was developed for purification of serotype 14 PS (PS14), responsible for 39.8% of diseases in children of 0-6 years old in Brazil. The developed method consists of cell separation by tangential microfiltration, concentration of the microfiltrate by tangential ultrafiltration (50 kDa), diafiltration in the presence of sodium dodecyl sulfate using a 30 kDa ultrafiltration membrane, precipitation with 5% trichloroacetic acid, precipitation with 20% and 60% ethanol, and anion exchange chromatography. The required purity regarding nucleic acids (<= 2%) and proteins (<= 3%) was achieved, resulting in a relative purity of 439 mg PS14/mg nucleic acids and 146 mg PS14/mg proteins. The final polysaccharide recovery was 65%, which is higher than the recovery of the majority of processes described in the literature

Recombinant protein production by engineered Escherichia coli in a pressurized airlift bioreactor: A techno-economic analysis

Standard cultivation protocols for high cell-density cultures of recombinant E. coli rely on air enrichment with oxygen to prevent oxygen limitation. This way of increasing oxygen supply impacts on process economics due to the high cost of pure oxygen. Reactor pressurization is an alternative approach to improve oxygen mass transfer. In the present study, the performance of pressurized airlift bioreactor in growing rE. coli cells was evaluated. Experiments were performed in a pressurized internal-loop airlift bioreactor (ALB) and in a non-pressurized stirred tank reactor (STR), both with 5 L working volume, equipped with a system for automatic control and monitoring of pressure, temperature, pH, and dissolved oxygen. Pressurization showed to be crucial to improve ALB performance in biomass formation (29 g(DCW) L-1) and protein production (201 mg(protein) g(DCW)(-1)), resulting in protein productivity (240 mg(protein)(-1) h(-1)) and energy efficiency (11 g(protein) kWh(-1)) similar to the achieved in STR (200 Mg-protein L-1 h(-1) and 13 g(protein) kWh(-1), respectively). Due to the high cost of pure oxygen, air enrichment revealed to be economically unfeasible for ALB. By pressurizing the bioreactor up to 0.41 MPa, without pure oxygen supply, an 8.7-fold increase in economic efficiency is estimated, what shows the potential of this innovative strategy for aerobic cultures. (C) 2015 Elsevier B.V. All rights reserved.

Pneumococcal whole-cell vaccine optimization of cell growth of unencapsulated Streptococcus pneumoniae in bioreactor using animal-free medium

The high cost of the available pneumococcal conjugated vaccines has been an obstacle in implementing vaccination programs for children in developing countries. As an alternative, Malley et al. proposed a vaccine consisting of inactivated whole-cells of unencapsulated S. pneumoniae, which provides serotype-independent protection and involves lower production costs. Although the pneumococcus has been extensively studied, little research has focused on its large-scale culture, thus implying a lack of knowledge of process parameters, which in turn are essential for its successful industrial production. The strain Rx1Al- eryR was originally cultured in Todd-Hewitt medium (THY), which is normally used for pneumococcus isolation, but is unsuitable for human vaccine preparations. The purposes of this study were to compare the strains Rx1Al- eryR and kan R, develop a new medium, and generate new data parameters for scaling-up the process. In static flasks, cell densities were higher for eryR than kanR. In contrast, the optical density (OD) of the former decreased immediately after reaching the stationary phase, and the OD of the latter remained stable. The strain Rx1Al- kanR was cultivated in bioreactors with medium based on either acid-hydrolyzed casein (AHC) or enzymatically hydrolyzed soybean meal (EHS). Biomass production in EHS was 2.5 times higher than in AHC, and about ten times higher than in THY. The process developed for growing the strain Rx1Al- kanR in pH-controlled bioreactors was shown to be satisfactory to this fastidious bacterium. The new culture conditions using this animal-free medium may allow the production of the pneumococcal whole-cell vaccine.