Mickie Takagi

Purification of capsular polysaccharide from Streptococcus pneumoniae serotype 23F by a procedure suitable for scale-up

Streptococcus pneumoniae is a pathogenic encapsulated bacterium, which causes pneumonia, bacteraemia and meningitis. Capsular polysaccharide conjugated to a carrier protein has been widely used as a vaccine antigen. Serotype 23F is one of the prevalent worldwide pneumococci. A simple and efficient method for capsular polysaccharide serotype 23F purification that can easily be scaled‐up was developed. This method consisted of using culture broth obtained by tangential microfiltration through a 0.22 μm membrane, broth microfiltrate concentration by tangential ultrafiltration in a 30 kDa spiral membrane, fractional ethanol precipitation (28–60%), nuclease and proteinase treatment, and concentration/diafiltration in a 30 kDa cassette membrane. The final polysaccharide recovery was 89%. The final protein and nucleotide contamination was 1.5% (w/w) and 0.3% (w/w) respectively. The final pure polysaccharide meets the requirements of the World Health Organization and residual proteinase was not found in the final product.

Protection induced by pneumococcal surface protein A (PspA) is enhanced by conjugation to a Streptococcus pneumoniae capsular polysaccharide

The currently available anti-pneumococcal vaccines are based on capsular polysaccharide (PS), plain or conjugated to a carrier protein. Conjugated vaccines are expensive products, especially in the case of pneumococcus, in which reasonable coverage requires from 7 to 13 serotypes. To obtain increased coverage with fewer components, we evaluated the immunogenicity of the pneumococcal surface protein A (PspA), conjugated to capsular polysaccharide serotype 23F, aiming at induction of an immune response against both components. Mice immunized with PS23F-rPspA1 conjugate produced antibodies against both PS and rPspA1, comparable or slightly higher than that obtained by free PS. The immunized animals challenged with a lethal dose of a virulent strain bearing a homologous PspA, showed that the PS23F-rPspA1 conjugate induced higher survival than rPspA1 alone or in combination with PS. This increased protection was shown to correlate with the enhanced capacity of the antibodies to bind to the pneumococcal surface and to induce complement deposition. Our results indicate that the use of PS-PspA conjugates may be a way to increase coverage against pneumococci with fewer components.

Purification of capsular polysaccharide produced by Haemophilus influenzae type b through a simple, efficient and suitable method for scale-up

Haemophilus influenzae type b, an encapsulated bacterium, causes meningitis in infants worldwide. The capsular polysaccharide conjugated to a carrier protein is effective in the prevention of such infections. The traditional purification process of polysaccharide from bacterial cultures for vaccine production is based on several selective precipitations with solvents such as: ethanol, phenol, and cationic detergents. The separations of solid and liquid phases are based on continuous centrifugation in explosion proof installations. The lipopolysaccharides are separated by ultracentrifugation. A simple and efficient method that can easily be scaled-up was developed for purification of polysaccharides. The ethanol precipitation was reduced to only two steps. The phenol treatment was substituted by ultrafiltration and enzymatic digestion. Lipopolysaccharide was removed by ultrafiltration together with addition of detergent and chelating agent.

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 kanR, 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.

Characterization of Polysaccharide Production of Haemophilus influenzae Type b and Its Relationship to Bacterial Cell Growth

Haemophilus influenzae type b (Hib) causes invasive infections in infants and young children. Vaccines consisting of Hib capsular polysaccharide (polymer of ribosylribitol phosphate [PRP]) conjugated to a protein are effective in the prevention of such infections. The production of capsular polysaccharide type b was studied in three cultivation conditions: single, glucose pulse, and repeated batch. Specific polysaccharide production (Yp/x) was calculated for all experiments, showing the following values: 67 (single-batch cultivation), 71 (glucose pulse), 75 (repeated-batch cultivation, first batch), and 87 mg of PRP/g of dry cell weight (DCW) (repeated-batch cultivation, second batch). Biomass concentration reached ∼1.8 g of DCW/L, while polysaccharide concentration was about ∼132 mg/L in the three fermentation runs. Polysaccharide synthesis is associated with cell growth in all studied conditions as established by Konos analysis and Luedeking-Pirets model.

Introduction of air in the anaerobic culture of Streptococcus pneumoniae serotype 23F induces the release of capsular polysaccharide from bacterial surface into the cultivation medium.

Aim:  An approach to increase Streptococcus pneumoniae capsular polysaccharide (CPS) in the culture medium during fed‐batch cultivation in bioreactor.

Production of native flagellin from Salmonella Typhimurium in a bioreactor and purification by tangential ultrafiltration

Flagellin is the structural protein and most abundant component of bacterial flagella. The flagellum filament contains around 20,000 - 100,000 subunits of 50 kDa flagellin that can have diverse biotechnological applications such as vaccine adjuvant and cellular protector during chemo- and radiotherapy. The main aim of this work was to study a production process of purified native FliC flagellin of Salmonella Typhimurium. The culture conditions in shakers were established with medium devoid of animal-derived components. In bioreactors, culture conditions were established in order to obtain flagellin from the culture supernatant by tangential ultrafiltration (TUF). The concentrated 750 kDa cut-off TUF fraction had a purification factor of 1.5 and a recovery yield of 52.2% for flagellin. The volumetric production of flagellin using the described procedure achieved around 307 mg/L of culture, which represented a significant improvement over previously reported methods. These results permit the development of production and purification processes that can be easily scaled up.

Study of the chemical stability of the capsular polysaccharide produced by Haemophilus influenzae type b.

Haemophilus influenzae type b (Hib) is a human pathogen that causes severe infections such as pneumonia, sepsis and meningitis. Vaccines for Hib infections are based on its capsular polysaccharide conjugated to a protein. This conjugated Hib antigen is included as one of the components of polyvalent vaccines and accounts for more than 50% of the total cost of the formulations. The instability of the polysaccharide is responsible for the high cost of the vaccine. In this study, the factors affecting the spontaneous degradation of the polysaccharide from Hib were evaluated based on the decrease in its molecular mass, as measured by size-exclusion chromatography. Temperature and pH were found to be the most significant variables, and the results showed that the conditions of bacterial cell growth (37 °C and pH 7.5) are favourable for depolymerization. An increase in the concentration of sodium ions up to 200 mM intensified the effect of pH, allowing higher rates of depolymerization at lower pH values, whereas the presence of magnesium ions showed no effects.

Improvement in the Purification Process of the Capsular Polysaccharide from Haemophilus influenzae Type b by Using Tangential Ultrafiltration and Diafiltration

Capsular polysaccharide produced by Haemophilus influenzae b (Hib) is the main virulent agent and used as the antigen in the vaccine formulation. In this study, an improved process of polysaccharide purification was established based on tangential flow ultrafiltration using detergents (cocamidopropyl betaine and sodium deoxycholate), two selective ethanol precipitations steps, and extensive enzymatic hydrolysis as strategy. The relative purity (RP) related to protein and nucleic acids were 122∼263 and 294∼480, respectively, and compatible with the specifications established by the World Health Organization for Hib vaccine, RP ≥ 100. These results make this process simple, cheaper, efficient, environmentally friendly, and prone to be scaled up.

Purification of porcine plasma factor VIII using chromatographic methods

Factor VIII was purified from porcine plasma using adsorption on aluminium hydroxide with CM-cellulose as a filtration aid, cold ethanol precipitation, and two anion-exchange (Q-Sepharose fast flow) chromatographies. The final product was purified 264-fold and had a specific activity of 10 U mg−1. The method is suitable to produce purified porcine FVIII by an easy process where all steps can be scaled up. The final product is free of von Willebrand factor that is responsible for the main side effects in patients. Finally, this method can be used to obtain purified porcine plasma FVIII for use in haemophilic patients with inhibitors.