High Pressure-Accelerated Digestion of Unreduced IgG by Lys-C and Trypsin

High Pressure-Accelerated Digestion of Unreduced IgG by Lys-C and Trypsin

Introduction: Efficient and rapid proteolytic digestion of disulfide-intact monoclonal antibodies could significantly improve overall routine workflows in protein characterizationand biopharmaceutical quality control. Conventional approaches use extensive denaturation by guanidine and/or urea, followed by overnight incubation with highconcentrations of enzyme(s). Such protocols frequently produce sporadic miscleavages and other artifacts of long protocols, as deamidation, methionineoxidation, and disulfide scrambling. Pressure-induced protein denaturation leads to better access of enzymes to previously inaccessible, or poorly accessible, sites,resulting in rapid and reproducible digestion. For proteins such as unreduced IgG, higher pressure levels may be necessary for effective unfolding while keepingthe disulfide bridges intact. Since pressure exerts a strong denaturing effect, IgG digestion by Lys-C or trypsin at pressures up to 90,000psi was evaluated. Inaddition, pressure is known to be synergistic with chaotropes, organic solvents, or detergents in denaturing not only substrate proteins, but also affecting theenzyme activity. Thus, it is important to determine which reagents, and concentrations, are compatible with pressure-enhanced digestion of difficult proteins.