Effect of pH on the yield of capsular polysaccharide precipitated with CTAB from Haemophilus influenzae type b culture supernatant

doi:10.5246/jcps.2026.02.010

Abstract

Abstract:

Haemophilus influenzae type b (Hib) conjugate vaccines can protect children from Hib infection, which was once the leading cause of severe invasive diseases in children under 5 years of age. The pH of the culture supernatant, in combination with the cationic surfactant cetyltrimethylammonium bromide (CTAB), plays a critical role in determining the yield of capsular polysaccharide (CP). Twelve samples collected from three batches of culture supernatant were adjusted to various pH values (ranging from 5.25 to 8.0). CP purification was carried out according to Gotschlich’s procedure. To confirm the effects of pH, ¹H NMR analyses were performed on CP samples obtained at pH 5.25, 7.0, and 8.0. The P-values for the three parallel experiments were all < 0.01 (0.001, 0.001, and 0.001 for crude samples; 0.002, 0.01, and 0.001 for purified samples), indicating that pH had a significant effect on CP precipitation. When the pH of the supernatant was adjusted to 6.5, CP recovery was maximized, and its structural integrity was maintained. In contrast, ¹H NMR spectra revealed that the structure of CP was altered at pH 8.0. These findings demonstrated that both the yield and quality of CP were strongly influenced by the pH of the culture supernatant.

Key words: pH, Capsular polysaccharide, Haemophilus influenzae type b, CTAB

Supporting:

Dong Feng. Effect of pH on the yield of capsular polysaccharide precipitated with CTAB from Haemophilus influenzae type b culture supernatant[J]. Journal of Chinese Pharmaceutical Sciences, 2026, 35(2): 144-150.

Figures/Tables 6

Table 1. Pentose assay results for final bulk Hib polysaccharide from three consistency runs. Component percentages are expressed relative to the dry weight of the polysaccharide.

Table 2. Results of protein assays performed on final bulk Hib produced for three consistency runs. Percentage of each component was calculated on the dry weight of Hib polysaccharide.

Table 3. Nucleic acid assay results of final bulk Hib polysaccharide from three consistency runs. Percentages are expressed relative to the dry weight of the polysaccharide.

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