Simple Fed-Batch Strategy for Production of Capsular Polysaccharide by Haemophilus influenzae b at Pilot Scale

Haemophilus influenzae b (Hib) is a pathogenic bacterium that causes meningitis worldwide, mainly in children less than two years old. The capsular polysaccharide b (PRP) is an essential antigen for vaccine formulation. This study aimed to develop a high-yield, technically accessible production strategy for PRP production to facilitate vaccine manufacturing in non-profit laboratories. Various fed-batch cultivation strategies were evaluated to address metabolic limitations and identify a robust, simplified process suitable for seamless scale-up to pilot scale. Glucose limitation strategies did not reduce inhibitory acetic acid accumulation due to deficiencies in Hib's respiratory chain, whereas oxygen availability was identified as critical parameter. Increasing the specific air flow from 0.5 to 1.0 vvm in constant fed-batch (Cfb) resulted in a 33% yield increase, reaching 1706.40 mg PRP.L^-1. However, the highest PRP concentration was achieved using exponential fed-batch with cell recycling (EfbCR), resulting in 1879.28 mg PRP.L^-1. Although EfbCR offered high productivity, the Cfb strategy emerged to be the most technically feasible and robust solution and was successfully scaled up to an 80 L bioreactor, achieving 1885 mg PRP.L^-1. These results advance understanding of PRP production by Hib and provides valuable insight into an efficient and simplified strategy for producing this key/vital vaccine antigen. The findings support the potential for cost-effective local production in public health initiatives.