Identifying and optimization of critical process parameters for the modulation of polysaccharide molecular size in Streptococcus pneumoniae serotype-1

Abstract

AbstractMaintaining the molecular size (MS) of Streptococcus pneumoniae capsular polysaccharide within specified range is essential for manufacture of conjugate vaccines, either through physical or acid hydrolysis before use in the conjugation process. Polysaccharide MS typically reduced, with high-pressure homogenization as an approach, for preserving their chemical structure. When the average MS of Pneumococcal capsular polysaccharide serotype-1(CPS1) exceeds 1200 kDa during fermentation, using a high-pressure homogenizer to reduce its MS to 150–250 kDa can become extremely difficult. Even after multiple homogenization cycles, obtaining polysaccharide of the required size can be challenging. Moreover, exceeding a certain number of homogenization cycles can negatively impact the stability, yield, and conjugation efficiency. To control polysaccharide MS, we conducted a design of experiments (DOE) study focused on the optimization of the fermentation process, employing serotype-1 as a represntative case. The successful optimization of these CPPs was achieved in a consistent and reproducible manner. Systematic evaluation by DOE based process optimization has provided valuable insights into precise polysaccharide manufacturing control of polysaccharide MS. Our findings confirm that maintaining Hy-Soy™ at 20-30 g/L and yeast extract at 1–3 g/L in the fermentation media, with a feed concentration of 2–3.5 g/L/H and 0.1–0.5 VVM of air, consistently yields polysaccharide with MS of < 1200 kDa. This strategy that can be extended to other S. pneumoniae serotype polysaccharide production. Graphical abstract