Abstract
Accelerated development of new therapeutics in an increasingly competitive landscape requires the use of high throughput analytical platforms. In addition, the complexity of novel biotherapeutic formats (e.g. fusion proteins, protein-polymer conjugates, co-formulations, etc.) reinforces the need to improve the selectivity and resolution of conventional one-dimensional (1D) liquid chromatography (LC). Liquid chromatography-mass spectrometry (LC-MS)-based technologies such as native LC-MS for intact mass analysis or peptide mapping (also called bottom-up approach)-based multi-attribute methods (MAM) have already demonstrated their potential to complement the conventional analytical toolbox for monoclonal antibody (mAb) characterization. Two-dimensional liquid-chromatography (2D-LC-MS) methods have emerged in the last ten years as promising approaches to address the increasing analytical challenges faced with novel antibody formats. However, off-line sample preparation procedures are still required for conventional 1D and 2D-LC-MS methods for the in-depth variant characterization at the peptide level. Multi-dimensional LC-MS (mD-LC-MS) combine sample preparation and multi-level (i.e. intact, reduced, middle-up and peptide) analysis within the same chromatographic set-up. This review presents an overview of the benefits and limitations of mD-LC-MS approaches in comparison to conventional chromatographic methods (i.e. 1D-LC-UV methods at intact protein level and 1D-LC-MS methods at peptide level). The current analytical trends in antibody characterization by mD-LC-MS approaches, beyond the 2D-LC-MS workhorse, are also reviewed, and our vision on a more integrated multi-level mD-LC-MS characterization platform is shared.
Highlights
Therapeutic antibodies have been effectively used for treatment of a wide range of diseases, including oncology and auto-immune conditions.[1]
With the data generated by the mD-liquid chromatography (LC)-mass spectrometry (MS) platforms, a direct correlation between peptide level and protein level information can be obtained
The capability of mD-Liquid chromatography-mass spectrometry (LC-MS) methods has been demonstrated with the online characterization of charge and size variants by in-line fraction collection and peptide mapping
Summary
Therapeutic antibodies have been effectively used for treatment of a wide range of diseases, including oncology and auto-immune conditions.[1]. Conventional peptide mapping-based MAM can be time consuming and the multiple sample preparation steps can affect the reproducibility of the method.[16] In order to characterize the peaks separated at the intact level (by IEC or SEC for example) and identify corresponding PTMs, an additional off-line fractionation step is needed. The characterization of five charge variants separated by IEC using off-line procedures can require up to 52 hours.[17] In order to streamline this process, multi-dimensional LC-MS approaches have been developed and implemented to perform online fractionation followed by an in-line peptide mapping to characterize antibody variants separated by IEC or SEC.[17,18,19,20] The automated characterization of antibody variants by mD-LC-MS can be performed with a much faster turnaround (typically 9 vs 52 hours17) compared to conventional procedures (manual and/or off-line sample preparation and fraction collection), and has the potential to be used as a more integrated analytical platform for both upstream and downstream applications. In the last part, automated mD-LC-MS approaches allowing the online characterization of mAb variants at multiple levels, i.e. combination of intact and peptide levels, are reviewed
Sign-in/Register to view highlights & summary Sign-in/Register to access full text options 
Free) 
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
