High efficiency reduction capability for the formation of Fab׳ antibody fragments from F(ab)₂ units

Antibodies have widespread applications in areas ranging from therapeutics to chromatography and protein microarrays. Certain applications require only the fragment antigen-binding (Fab) units of the protein. This study compares the cleavage efficacy of dithiothreitol (DTT), mercaptoethylamine (MEA), and dithiobutylamine (DTBA) - a relatively new reducing agent synthesized in 2012. Pseudo-first order kinetic analyses show DTBA to be ~213 times faster than DTT and ~71 times faster than MEA in the formation of Fab׳ antibody fragments from polyclonal rabbit antibodies. Monoclonal mouse antibodies were also used to show the feasibility of the reduction process on antibodies from a different species and with a different clonality. DTBA cleaved the monoclonal mouse F(ab)₂ units most efficiently, ~2 times faster than DTT ~10 times faster than MEA. Due to the extremely quick reactivity of all the reducing agents in the first five minutes of monoclonal antibody reductions as well as for the DTBA reductions of the polyclonal rabbit antibodies, the pseudo-first order kinetic analyses should be interpreted qualitatively for these results. Nucleophilic sulfides on Fab׳ fragments are preserved in the DTBA reduction process, demonstrated by their reactivity with Ellman׳s reagent. Degradation of the Fab׳ fragments was observed with the monoclonal mouse antibodies after reduction with DTBA or DTT. In conclusion, DTBA is the more efficient reducing agent compared to DTT and MEA, however, the reduction process should be optimized as degradation of the Fab׳ fragments is possible.