Isolation and purification of capsular polysaccharide of Haemophilus influenzae type b (Hib) by hexadecyltrimethylammonium bromide (CTAB) precipitation and chromatography

Polyribosylribitol phosphate (PRP) is a capsular component from Haemophilus influenzae type b (Hib) which is used as an active ingredient in Hib vaccine. PRP was isolated and purified from harvested bulk. This experiment was done to observe the profile of purified PRP. Purification process consists of several steps, which were CTAB (cetyltrimethyl-ammonium bromide) precipitation, chromatography using XK-50 column (Amersham, GE) with CTAB-celite slurry as a stationary phase. CTAB solution and gradient concentration of ethanol in sodium acetate 0.05M was used as mobile phase. Ethanol 60% fraction then precipitated with sodium chloride and ethanol absolute, concentrated, diafiltrated and sterilized with filtration. From three batches of 10 liters purification, about 4.84-7.44 g of PRP could be isolated. PRP was analyzed by orcinol assay. The impurities, nucleic acid and protein, were removed to the minimum level as recommended by WHO guideline for Hib vaccine : less than 1% for both parameter determined by UV scan at 260 nm for DNA and Lowry method respectively. Molecular size of PRP was determined by High Performance Gel Permeation Chromatography (HPGPC) with the result around 165-370 kDa. In conclusion this method could be used to isolate and purify the PRP.