The Evaluation of Pressure-Assisted Enzymatic Digestion for Optimal Digestion of Monoclonal Antibodies

摘要

These results indicate the superiority of the PAED approach for the rapid enzymatic digestion of mAb. When the reduction and alkylation of the mAb was performed in PBS, results obtained using the 60 min PAED approach were comparable to those obtained from the overnight-bench top digestion. Also, the PAED approach is very suitable for native digestions which is important for disulfide mapping. When the sample was R&A in a denaturing buffer containing GuHCl, 80percent sequence coverage was obtained within 20 min of pressure cycling. Performing the reduction and alkylation in a denaturing buffer also decrease the number of under-cleaved peptides. The PAED approach is very effective in reducing the total time required to digest proteins and will expedite the complete characterization of mAbs.