Design and scale-up of a mixed-substrate fed-batch strategy for enhanced HPV VLP production in Komagataella phaffii.

Production of human papillomavirus (HPV) virus-like particles (VLPs) in Komagataella phaffii is often constrained by methanol-associated toxicity and suboptimal feeding strategies during induction. In this study, a microbioreactor-based approach was employed to systematically design and evaluate mixed-substrate fed-batch strategies for enhanced HPV16 L1 VLP production. Four methanol-based feeding compositions, including methanol-only and combinations with glycerol and sorbitol, were investigated under pulse and linear feeding modes. Among the tested conditions, a linear feeding strategy using 70% methanol and 30% glycerol (70 M + 30G) resulted in the highest biomass (DCW: 90.36 g/L) and VLP titer (42 mg/L), corresponding to a two-fold improvement over methanol-only feeding. Enhanced performance was attributed to reduced methanol-induced metabolic stress and improved substrate utilization, leading to more stable dissolved oxygen (DO) profiles and sustained cellular activity. In contrast, pulse feeding resulted in significant DO fluctuations, indicating transient metabolic imbalances and lower overall productivity. The optimized feeding strategy was successfully translated to a 1 L stirred-tank bioreactor, achieving a DCW of 120 g/L and a VLP titer of 49 mg/L, with a ~ 2.3-fold increase compared to the control. The consistency between microscale and bench-scale results demonstrates the predictive capability of microbioreactor systems for fed-batch process development. Overall, this study presents a rational and scalable feeding strategy that improves recombinant VLP production while reducing methanol usage, providing a practical framework for efficient bioprocess design in K. phaffii.