Fast and Automated Characterization of Antibody Variants with 4D HPLC/MS.

Characterization of unknown monoclonal antibody (mAb) variants is important in order to identify their potential impact on safety, potency, and stability. Ion exchange chromatography (IEC) coupled with UV detection is frequently used to separate and quantify mAb variants in routine quality control (QC). However, characterization of the chromatographic peaks resulting from an IEC separation is an extremely time-consuming process, involving many cumbersome steps. Presented here is an online four-dimensional high performance liquid chromatography-mass spectrometry (4D HPLC/MS) approach, developed to circumvent these limitations. To achieve this, a 2D HPLC system was extended through the introduction of additional modules, hence enabling fully automated bioseparation of mAbs, fractionation of peaks, reduction, tryptic digestion, and reversed-phase (RP) separation of resulting peptides followed by MS detection. The entire separation and analytical process for an unknown peak is performed in less than 1.5 h, leading to a significant time savings, with comparable sequence coverage. To show the comparability with the traditional offline process, a proof of concept study with a previously characterized mAb1 is presented in this paper.