Purification of O-specific polysaccharide from lipopolysaccharide produced by Salmonella enterica serovar Paratyphi A

Abstract

The O specific polysaccharide (OSP) of the lipopolysaccharide (LPS) of Salmonella enterica serovar Paratyphi A is a protective antigen and the target for vaccine development. LPS is the major constituent of the outer membrane of S. Paratyphi A with the OSP exposed on the surface, in addition to the cell associated LPS a large amount of free LPS was present in the fermentation broth. A purification method was developed to take advantage of both sources of LPS and to maximize recovery of OSP. After fermentation the bacterial cells were concentrated and washed, the permeate containing the free LPS was processed separately from the cells. The free LPS was concentrated and washed on a 100kD ultrafiltration membrane to remove low molecular weight impurities. The LPS was then detoxified by separation of the lipid A from the OSP using acid hydrolysis at 100°C, the precipitated lipid A was removed by 0.2μm membrane filtration. Contaminants were then removed by acid precipitation in the presence of sodium deoxycholate. The OSP was concentrated and washed with 1M NaCl then water using a 10kD ultrafiltration membrane then sterile filtered through a 0.2μm membrane filter. The cells were treated by acid hydrolysis at 100°C, the remaining cells, cell debris and precipitate was removed by centrifugation. The filtrate was then treated in the same way as described above for the free LPS. The combined yield of purified OSP from free LPS plus the cells was greater than 880mg/L of culture broth. The method developed yields large amounts of OSP, is scalable and compatible with cGMP so would be readily transferrable to developing country vaccine manufacturers for low cost production of vaccine against S. Paratyphi A.