Development of a Pichia pastoris-Based Expression System for High-Activity Anti-SARS-CoV-2 Neutralizing Antibodies

Abstract The growing need for rapid and cost-effective antibody production highlights the potential of Pichia pastoris as an alternative to mammalian systems. This study aimed to develop and optimize engineered single-chain antibody fragments (scFvs) against SARS-CoV-2 using Pichia pastoris. Four scFv formats, scFv-His, scFv-CH3-His, scFv-ZIP-His, and scFv-Fc, were evaluated for binding activity, neutralization potency, expression yield, and in vivo stability. Among them, scFv-Fc demonstrated the best overall performance, with strong antigen binding (EC₅₀ = 0.003 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 mg/L 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.