Automated tryptic digestion procedure for HPLC/MS/MS peptide mapping of immunoglobulin gamma antibodies in pharmaceutics

The rapid growth of antibody drugs and drug candidates in the biopharmaceutical industry has created a demand for automated proteolytic digestion to assist in pharmaceutical stability studies, identity assays and quality control of these therapeutic proteins. Here, we describe the development of a fully automated proteolytic digestion procedure for monoclonal antibodies in solution, which requires a high concentration of denaturants for unfolding. The antibody samples were placed in a 96-well plate or in 0.5-mL Eppendorf tubes. The proteins were then reduced and alkylated in a denaturing solution of 6 M guanidine HCl. The denaturing solution was replaced with a digestion buffer using a custom-designed 96-well size-exclusion plate for desalting. The sample was digested for 5 h with two additions of trypsin. The completeness and reproducibility of digestion were verified by reversed-phase high-performance liquid chromatography tandem mass spectrometry (HPLC/MS) analysis of the digestion products. The performance of the automatic digestion was comparable to the currently used manual digestion procedure, but saved time, reduced manual labor, and increased the reproducibility of the tryptic digests. Our method should be useful not only for high-throughput analysis of antibodies, but for other therapeutic protein samples as well. Other applications like gel-free proteomics, where the analysis of a large number of samples is often needed and the completeness of the liquid digestion is critical for the identification of a large number of different proteins, should also benefit from this fully automated liquid proteolytic digestion procedure.