Abstract

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 a 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 multidimensional 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 online 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 chromatographic system. Overall, the full workflow (data acquisition and analysis) for both 3D and 4D-LC/MS setups can be completed 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.

Talk to us

Join us for a 30 min session where you can share your feedback and ask us any queries you have

Schedule a call

Disclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.