Development of a Komagataella phaffii-based expression system for high-activity anti-SARS-CoV-2 neutralizing antibodies.

The growing need for rapid and cost-effective antibody production highlights the potential of Komagataella phaffii (syn. Pichia pastoris) as an alternative to mammalian systems. This study aimed to develop and optimize engineered scFvs (single-chain antibody fragments) against SARS-CoV-2 using K. phaffii. Four scFv formats, scFv-His, scFv-CH3-His, scFv-ZIP-His, and scFv-Fc, were evaluated for binding activity, neutraliation potency, expression yield, and in vivo stability. Among them, scFv-Fc demonstrated the best overall performance, with strong antigen binding (EC50 = 0.0034 µg/mL), effective neutralizing activity (IC50 = 0.570 µg/mL), and a prolonged serum half-life exceeding 5 days. Optimization of fermentation conditions increased its expression from 23 to 110 mg/L. These results demonstrate the feasibility of engineering scFv-based therapeutics using yeast systems and establish a framework for the rapid and economical production of next-generation antibody formats.