ABSTRACT
Haemophilus influenzae type b (Hib) is a human pathogen that causes meningitis in infants worldwide. Capsular polysaccharide linked to a protein has been used as an efficient vaccine, and this approach has reduced the incidence of Hib disease since its inclusion in national immunisation campaigns. The traditional polysaccharide downstream process is based on several ethanol precipitations, treatment with detergents and centrifugation. The aim of this study was to introduce tangential microfiltration (TMF) in the place of centrifugation to simplify handling and to scale up the process. The purity of the polysaccharide was RPNA=1747.2 and RPPrt=196.1 for nucleic acid and protein, respectively, meeting the quality requirements for this polysaccharide. Moreover, the polysaccharide was recognised by at specific antibody, and the ribose and phosphate contents were within the expected limits. Thus, we established a process for the purification of capsular polysaccharide produced by H. influenzae type b that is effective, robust and feasible to be scaling up.
Subject(s)
Haemophilus influenzae type b , Polysaccharides, Bacterial/isolation & purification , Bacterial Proteins/analysis , Bioreactors , Chemical Precipitation , Filtration , Haemophilus influenzae type b/metabolism , Nucleic Acids/analysis , Phosphorus/analysis , Polysaccharides, Bacterial/metabolismABSTRACT
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.
Subject(s)
Bacterial Capsules/isolation & purification , Haemophilus influenzae type b/metabolism , Ultrafiltration/methods , Bacterial Capsules/biosynthesis , Glucose/pharmacology , Haemophilus influenzae type b/drug effects , Haemophilus influenzae type b/growth & developmentABSTRACT
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.
Subject(s)
Centrifugation/methods , Haemophilus influenzae type b/chemistry , Polysaccharides, Bacterial/isolation & purification , Ultrafiltration/methods , Bioreactors/microbiology , Chemical Precipitation , Ethanol/chemistry , Haemophilus influenzae type b/metabolism , Lipopolysaccharides/chemistry , Lipopolysaccharides/isolation & purification , Polysaccharides, Bacterial/chemistryABSTRACT
Haemophilus inXuenzae type b, an encapsulatedbacterium, causes meningitis in infants worldwide. The capsularpolysaccharide conjugated to a carrier protein is eVectivein the prevention of such infections. The traditional puriWcationprocess of polysaccharide from bacterial cultures for vaccineproduction is based on several selective precipitationswith solvents such as: ethanol, phenol, and cationic detergents.The separations of solid and liquid phases are based oncontinuous centrifugation in explosion proof installations. Thelipopolysaccharides are separated by ultracentrifugation. Asimple and eYcient method that can easily be scaled-up wasdeveloped for puriWcation of polysaccharides. The ethanolprecipitation was reduced to only two steps. The phenol treatmentwas substituted by ultraWltration and enzymatic digestion.Lipopolysaccharide was removed by ultraWltrationtogether with addition of detergent and chelating agent.
