Biochemical and Immunological Characters of Different Fractions During Purification of Haemophilus influenzae Type b(Hib) Conjugate

Objective To analyze the biochemical and immunological characters of different fractions during the purification of Haemophilus influenzae type b(Hib) conjugate.Methods Purify Hib conjugate by Sepharose CL-4B column chromatography and collect five different fractions for the determination of biochemical and immunological characters.Results The ratios of polysaccharide to protein in fractions 1,2 and 3 were about 0.4,and the high molecular conjugate contents were more than 80%.HPLC proved that all the relative molecular weights of the three fractions were more than 1 400 000.Good immune responses were induced in the mice immunized with the three fractions.The fraction 4,with a high molecular conjugate content of about 70% and a relative molecular weight of about 670 000,also induced good immune response in mice.However,the fraction 5,with a high molecular conjugate content of 23.7% and a relative molecular weight of 50 000,was mainly polysaccharide and vector protein in free form and only induced low immune response in mice.Conclusion The fractions 1,2 and 3 were needed to be collected during purification of Hib conjugate.The fraction 4 was conjugate with a low relative molecular weight.However,the fraction 5 was mainly free polysaccharide and protein.