From proof of concept to the routine use of an automated and robust multi-dimensional liquid chromatography mass spectrometry workflow applied for the charge variant characterization of therapeutic antibodies

Abstract

The identification and quantification of post-translational modifications (PTMs) is a crucial step required during the development of therapeutic proteins. In particular, the characterization of charge variants separated by cation exchange chromatography (CEX) is a tedious process commonly performed with an off-line manual fraction collection followed by peptide mapping. To improve the efficiency of this time-consuming approach, a generic on-line multi-dimensional LC/MS approach was developed for the characterization of various monoclonal antibody (mAb) isotypes and a bi-specific antibody (BsAb). Fractions collected from ¹D CEX analysis were consecutively reduced on a ²D reversed phase liquid chromatography (RPLC) column (polyphenyl), digested within 1-2 min using a ³D immobilized trypsin cartridge, and finally the obtained peptides were separated on another ⁴D RPLC column (C18), and simultaneously identified with a Q Exactive™ mass spectrometer. ²D RPLC columns and ³D trypsin cartridges from different suppliers were compared, as well as the effects of reducing agents. The effect of ²D and ⁴D RPLC column temperature, and ²D RPLC column mass load were also systematically studied. Under optimal conditions, the multi-dimensional LC/MS system described in this paper is a robust tool for the on-line digestion of proteins and shows high repeatability. Similar levels of oxidation and deamidation were measured using the off-line and on-line approaches for the same stressed samples. The lower amounts of deamidation and isomerization measured at some asparagine and aspartic acid residues by the on-line approach compared to the manual off-line procedure suggest reduced artifacts using the on-line methodology. The multi-dimensional LC/MS described here enables fast, on-line, automated characterization of therapeutic antibodies without the need for off-line fraction collection and sample pre-treatment (manual approach). The entire workflow can be completed within less than one day, compared to weeks with the manual off-line procedure.

Keywords: Automation; Charge variants; Monoclonal antibodies; Multi-dimensional chromatography; PTM.