LAUSR

Monitoring of post-translational modifications (PTMs) in therapeutic monoclonal antibodies (mAbs) is essential during their production in both upstream and downstream processes. However, characterization of PTMs using conventional peptide mapping procedure requires time-consuming and labor-intensive offline sample preparation steps. This work describes for the first time, the implementation of a Protein-A affinity chromatography column as the first dimension (1D) in a multi-dimensional LC (3D and 4D) setup for the automated characterization of mAb variants from harvest cell culture fluid (HCCF) materials at different purification/production steps. A 4D-LC/MS method (Protein-A - Reduction-RPLC - Digestion - RPLC/MS) was developed to determine PTM levels including oxidation, deamidation, and succinimide formation by on-line peptide mapping analysis. To obtain an accurate and comprehensive profiling of mAb glycosylation patterns at the reduced level, a 3D-LC/MS method (Protein-A - Reduction-RPLC - HILIC/MS) was also developed on the same chroma-tographic system. Overall, the full workflow (data acquisition and analysis) for both 3D and 4D-LC/MS setups can be com-pleted within less than 1-2 days, compared to weeks with the conventional manual approach. This proof of concept study demonstrates that mD-LC/MS has the potential to be used as a powerful tool to perform a fast and reliable monitoring of PTMs during the manufacturing process for both bioreactor control or as a monitoring assay.