Decreased solubility and increased adsorptivity of a biotinylated humanized anti-cocaine mAb

Abstract

Biotinylation of proteins, including antibodies, is a very useful and important modification for a variety of biochemical characterizations, including anti-drug antibody (ADA) assays used to detect antibodies raised against therapeutic antibodies. We assessed different degrees of biotin labeling of an anti-cocaine mAb currently under development for treating cocaine use disorders. We noted that higher levels of biotin labeling dramatically decreased mAb solubility, and increased the tendency to bind to surfaces, complicating characterization of the biotinylated antibody. Specifically, in phosphate buffered saline, labeling stoichiometries of more than about 3 biotin/mAb resulted in decreased recoveries due to increased binding to surfaces and decreased mAb solubility. Native gel agarose electrophoresis, differential scanning fluorimetry, and isothermal titration calorimetry all demonstrated changes in the mAb which became more pronounced above a labeling ratio of 3 biotin/mAb. At 3.0 biotin/mAb, there were minimal changes in solubility, adsorptivity, exposure of hydrophobic dye-binding sites, heat stability, and cocaine binding, in stark contrast to labeling with 5.6 biotin/mAb. Thus, the degree of biotinylation should be kept at about 3 biotin/mAb to maintain antigen binding and general structure, solubility, and stability of this mAb, a finding which may be important for other similar mAbs currently in use or under development.