DEVELOPMENT OF A METHOD FOR PRODUCING FAB/F(AB’)2 FRAGMENTS FROM A FULL-LENGTH MONOCLONAL ANTIBODY FOR BIOANALYTICAL ASSAYS

Abstract Background Monoclonal antibodies (mAb) comprise of two Fab fragments and one Fc fragment or one F(ab)’2 fragment and one Fc fragment. While a full-length mAb is frequently used as an assay reagent for bioanalysis, mAb fragments are required in certain cases. For example, to build a sandwich assay for detection of anti-drug antibodies (ADA) for therapeutic antibodies, Fab or F(ab)’2 fragment is used instead of a full-length mAb as capture reagent. This is because therapeutic antibodies, either humanized or fully human, are in many ways indistinguishable from the ADA generated in patients, especially in Fc fragment. When ADA detection methods utilizes anti-human Fc antibodies as the detection reagent, the full-length mAb drug will be directly bound by the detection reagent, causing interference. Preparation of a Fab or F(ab)’2 fragment is therefore needed. Methods and results We are developing a method for enzymatic digestion of therapeutic antibodies to generate monovalent Fab or bivalent F(ab’)2 fragments in this study. With such reagents becoming available, a sandwich ADA assay formats can be expanded to allow anti-human Fc antibodies as detection regents. To standardize the method, we explored various enzymatic conditions, including type of enzymes (i.e., pepsin, papain, and IdeS Protease), digestion-time (1, 2, 4, and 6 h), enzyme to antibody ratio (1:10, 1:20, and 1:40 w/w), IgG species and isotypes (human IgG1-κ, IgG1-λ, and IgG4-κ). The enzymatic hydrolysates were quantified by NanoDrop and purified by dialysis (10K MWCO) and Protein A/G/L magnetic bead methods. The effective recovery of truncated antibodies was > 90%, as assessed by reduced/non-reduced SDS-PAGE and ELISA analysis. Digestion of human IgG1 and IgG4 with pepsin resulted into a complete cleavage into F(ab')2 fragments and degradation of Fc fragments. While IdeS Protease produced an equivalent quantity of F(ab’)2 and Fc fragments with a similar efficiency, removal of the intact Fc fragment was required as an additional step. If the Fab fragments were desired, papain could be used with yield being over 90%. We have subsequently utilized either Fab or F(ab’)2 as a capture reagent for ADA detection. Conclusion We have successfully developed the enzymatic digestion method to prepare Fab or F(ab’)2 fragments. The optimized conditions described here are broadly applicable to different IgG isotypes across many therapeutic antibodies.