PEGylation Prolongs the Half-Life of Equine Anti-SARS-CoV-2 Specific F(ab')₂

Therapeutic antibodies-F(ab')₂ obtained from hyperimmune equine plasma could treat emerging infectious diseases rapidly because of their high neutralization activity and high output. However, the small-sized F(ab')₂ is rapidly eliminated by blood circulation. This study explored PEGylation strategies to maximize the half-life of equine anti-SARS-CoV-2 specific F(ab')₂. Equine anti-SARS-CoV-2 specific F(ab')₂ were combined with 10 KDa MAL-PEG-MAL in optimum conditions. Specifically, there were two strategies: Fab-PEG and Fab-PEG-Fab, F(ab')₂ bind to a PEG or two PEG, respectively. A single ion exchange chromatography step accomplished the purification of the products. Finally, the affinity and neutralizing activity was evaluated by ELISA and pseudovirus neutralization assay, and ELISA detected the pharmacokinetic parameters. The results displayed that equine anti-SARS-CoV-2 specific F(ab')₂ has high specificity. Furthermore, PEGylation F(ab')₂-Fab-PEG-Fab had a longer half-life than specific F(ab')₂. The serum half-life of Fab-PEG-Fab, Fab-PEG, and specific F(ab')₂ were 71.41 h, 26.73 h, and 38.32 h, respectively. The half-life of Fab-PEG-Fab was approximately two times as long as the specific F(ab')₂. Thus far, PEGylated F(ab')₂ has been prepared with high safety, high specificity, and a longer half-life, which could be used as a potential treatment for COVID-19.