Bioprocess Optimization for Scalable O-Antigen Production in Vaccine Development

O-polysaccharides are the main components in bacterial pathogenesis and vaccine formulations, making their large-scale production essential for polysaccharide and conjugate vaccine development. However, exact optimization of bacterial growth conditions, fermentation techniques and hydrolysis parameters is necessary to achieve high-yield and high-purity polysaccharide extraction. This study focuses on systematically enhancing O-polysaccharide production from Salmonella enterica serovar Paratyphi by refining key growth factor variables. The effects of temperature, pH, aeration, nutrient supplementation and glucose feeding on bacterial growth and polysaccharide biosynthesis were assessed using a thorough experimental design. Large-scale fed-batch fermentation yielded superior results over batch fermentation, ensuring sustained bacterial growth and continuous glucose availability for enhanced O-polysaccharide. The acid hydrolysis process was optimized to prevent polysaccharide degradation while maintaining structural integrity, with a 2-hour hydrolysis duration at pH 5.0 and 95°C identified as the optimal condition. Size-exclusion chromatography (SEC) analysis confirmed that prolonged hydrolysis led to molecular fragmentation, reinforcing the need for controlled extraction conditions. This study presents optimized factors for bioprocess development combined with scalable and reproducible approach for maximizing O-polysaccharide production, aligning with industrial demands for efficient bioprocess development.