Accurate quantification of polyRhamnose (polyRha) is essential for the development of effective Group A Streptococcus vaccines utilizing polyRha conjugation. This study presents the optimization of a High-Performance Anion Exchange Chromatography with Pulsed Amperometric Detection (HPAEC-PAD) assay for polyRha quantification, addressing challenges related to production variability and analytical accuracy.
Temperature and hydrolysis time sensitivity were observed, with rhamnose degradation occurring at high temperatures and extended hydrolysis times, highlighting the importance of controlled hydrolysis conditions. Through systematic optimization, we identified 2N trifluoroacetic acid (TFA) at 100°C for 2 hours as the optimal protocol to maximize rhamnose recovery while minimizing degradation.
Key findings include the establishment of the limit of quantification, and validation of the assay using polyRha batch samples. While the assay demonstrated reliable performance, limitations were noted in measuring undiluted samples at high concentrations, providing direction for future improvements.
This optimized HPAEC-PAD assay offers a robust and reproducible method for polyRha quantification, enabling precise monitoring of vaccine production and supporting the development of effective Group A Streptococcus vaccine.