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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 this work will provide a quick and cost-effective method for researchers to produce antibody fragments from whole IgG, avoiding haphazard trial and error.

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