Development of a multidimensional online method for the characterization and quantification of monoclonal antibodies using immobilized flow-through enzyme reactors.

Abstract

Complete characterization and quantification of monoclonal antibodies often rely on enzymatic digestion with trypsin. In order to accelerate and automate this frequently performed sample preparation step, immobilized enzyme reactors (IMER) compatible with standard HPLC systems were used. This allows an automated online approach in all analytical laboratories. We were able to demonstrate that the required digestion time for the model monoclonal antibody rituximab could be reduced to 20min. Nevertheless, a previous denaturation of the protein is required, which also needs 20min. Recoveries were determined at various concentrations and were 100% ± 1% at 100ng on column, 96% ± 7% at 250ng on column and 98% ± 2% at 450ng on column. Despite these good recoveries, complete digestion was not achieved, resulting in a poorer limit of quantification. This is 50ng on column under optimized IMER conditions, whereas an offline digest on the same system achieved 0.3ng on column. Furthermore, our work revealed that TRIS buffers, when used with an IMER system, led to alteration of the peptides and induced modifications in the peptides. Therefore, the addition of TRIS should be avoided when working at elevated temperatures of about 60°C. Nevertheless, our results have shown that the recovery is not significantly influenced whether TRIS is used or not (recovery: 96 ± 7% with TRIS vs. 100 ± 9% without TRIS).