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.
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.
Haemophilus influenzae type b (Hib) is a pathogenic bacterium responsible for meningitis, pneumonia in children aged less than 5 years. The capsular polysaccharide (PSb) is the most important virulence factor and currently it is purified, conjugated to a protein and used as vaccine against Hib. The classical PSb purification process is very complex, which include many ethanol and cationic detergent precipitations (explosion prone and pollutant), phenol extraction (toxic and corrosive) and centrifugation/ultracentrifugation steps. The aim of this work was to improve the process of purification of PSb by total or partial replacement of ethanol precipitations and/or elimination of centrifugation by chromatographies, enzymatic digestion and tangential ultrafiltration (TUF100 kDa) or microfiltration (0.22 µm) membranes. After cell separation, the clarified supernatant was concentrated and diafiltrated by TUF100kDa membrane. The concentrated fraction was submitted to different steps of purification. The ion exchange chromatographies did not give good separation between PSb and contaminants. The resolution in gel filtration was better, but sample volume must be small and flow rate low. Hydrophobic chromatography at neutral pH can be used for elimination of proteins but not for nucleic acids. Ethanol precipitations at 30 and 80% are necessary to achieve the required purity of polysaccharide in respect to proteins and nucleic acid contaminants. In some way, ethanol facilitated the action of hydrolytic enzymes by denaturation of proteins/nucleic acids or make instable some aggregates and improve further ultrafiltration step. The separation of 30% ethanol fraction using new microfiltration membrane hollow fiber 0.22 µm exhibited a better purification than centrifugation, however test of accelerate aging, with the use of whole cell has reduced its efficiency.
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