Molecular size distribution assessment of Haemophilus influenzae vaccine containing lactose by HPAEC-PAD and colorimetric assays

Molecular size distribution of Haemophilus influenzae type b (Hib) conjugate vaccine is an important indicator for its immunogenicity and stability. Molecular size distribution was evaluated by High-Performance Protein Chromatography on Sepharose CL-4B column, and fractions were pooled. The use of high flow rate, incorporation of a calibration standard with the injected buffer and pooling method yielded a superior assay compared to conventional pharmacopeial method. The pools were analyzed for determination of distribution coefficient (KD) of 0.2 and 0.5 using two validated techniques: High Performance Anion Exchange Chromatography with pulsed amperometric detection (HPAEC-PAD) for ribitol determination and an optimized colorimetric assay for phosphorus determination. Linearity was achieved over range of 0.10-10.0 mu g/mL and 1.0-8.0 mu g/mL with LOD of 0.03 and 0.28 mu g/mL for HPAEC and colorimetric assays, respectively. The developed assays were successfully applied in quality control monitoring of Hib conjugate vaccine. The optimized colorimetric method had shortened the analysis time to 25 min compared to 3.5 h for the European pharmacopeial assay by modifying the burning process. HPAEC stability results revealed 40% decrease in MSD after stressed storage conditions. The proposed assays offer a reliable and economic platform for monitoring the quality attributes of Hib for biopharma industry.

Automated summary

The molecular size distribution of Hib conjugate vaccine plays a crucial role in its immunogenicity and stability. A superior assay for determining the distribution coefficient (KD) was developed using high flow rate, calibration standards, and pooling method. The assays, including High Performance Anion Exchange Chromatography with pulsed amperometric detection and an optimized colorimetric assay, were successfully applied in quality control monitoring of Hib conjugate vaccine.