Optimization of Enzymatic Antibody Fragmentation for Yield, Efficiency, and Binding Affinity.

ABSTRACT: Enzymatic antibody fragmentation has been well studied for various hosts and isotypes, but fragmentation patterns also vary unpredictably by clone, and optimizing Fab or F(ab')₂ production by trial and error consumes large quantities of antibodies. Here, we report a systematic strategy for optimizing functional F(ab')₂ production via pepsin digestion from small quantities of IgG. We tested three key parameters that affect fragmentation, pH, enzyme concentration (% pepsin w/w), and reaction time, and found that pH had the greatest impact on fragmentation yield and efficiency. We then developed a systematic approach to obtaining acceptable yields, digestion efficiency, and binding affinity. Three case studies are described to illustrate the approach. We anticipate that

SUBMITTER: Kinman AWL 

PROVIDER: S-EPMC6718091 | biostudies-literature | 2019 Mar

REPOSITORIES: biostudies-literature