Determination of free polysaccharides in the AC-Hib conjugate vaccine using high performance anion-exchange chromatography
YUAN Su-ying
YANG Yao
YANG Li-xia
SHAO Zhong-qi
Abstract：Objective: To develop a method for the determination of free polysaccharides in the AC-Hib conjugate vaccine using high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Methods: Fifteen vials of the vaccine product were combined and concentrated with 3 kDa ultrafiltration centrifuge tubes. The sample was treated with cold phenol to remove the polysaccharides conjugated to the carrier protein. The free polysaccharides in the supernatant were hydrolyzed with 2 mol·L-1HCl, and the monosaccharide was analyzed by HPAEC-PAD. The hydrolyzed free polysaccharide was analyzed by HPAEC-PAD method. The content of free polysaccharide was calculated according to the peak area. The mole ratio of monosaccharide after hydrolysis which was used to calculate the content of free polysaccharide was determined through the chemical structure formula. Results: The standard curves generated had a good linearity, R2>0. 99; all the recovery rates of the three polysaccharides were in the range of 80％～120％. The method showed a high repeatability and intermediate precision, with relative standard deviation (RSD)less than 10％ and 15％, respectively. The quantitative limits of A, C and Hib polysaccharides were 0. 025, 0. 03 and 0. 05 μg·mL-1, respectively. Conclusion: The combination of the cold phenol treatment and HPAEC-PAD can be used to determine the free polysaccharides in the AC-Hib conjugate vaccine.
Keywords：HPAECfree polysaccharidecold phenol treatmentpolysaccharide-protein conjugate vaccine
Publication Date：2018-01-01
Online Publishing Date：2026-08-14(First online date of this platform, not the publication date of the document)
Pages：8( 874-881 )