Aptamer-based immunoaffinity LC-MS using an ultra-short column for rapid attomole level quantitation of intact mAbs

Jennifer M Nguyen; Xiaoxiao Liu; Maureen DeLoffi; Amarande Murisier; Szabolcs Fekete; Davy Guillarme; Matthew A Lauber

doi:10.1016/j.jchromb.2021.122694

Aptamer-based immunoaffinity LC-MS using an ultra-short column for rapid attomole level quantitation of intact mAbs

J Chromatogr B Analyt Technol Biomed Life Sci. 2021 Apr 5:1173:122694. doi: 10.1016/j.jchromb.2021.122694. Online ahead of print.

Authors

Jennifer M Nguyen¹, Xiaoxiao Liu², Maureen DeLoffi², Amarande Murisier³, Szabolcs Fekete³, Davy Guillarme³, Matthew A Lauber²

Affiliations

¹ School of Science, University of Copenhagen, Rolighedsvej 30, 1958 Frederiksberg, Denmark; Waters Corporation, 34 Maple Street, Milford, MA 01757-3696, United States. Electronic address: Jennifer_Nguyen@waters.com.
² Waters Corporation, 34 Maple Street, Milford, MA 01757-3696, United States.
³ School of Pharmaceutical Sciences, University of Geneva, CMU-Rue Michel Servet 1, 1211 Geneva 4, Switzerland; Institute of Pharmaceutical Sciences of Western Switzerland, University of Geneva, CMU - Rue Michel Servet 1, 1211 Geneva 4, Switzerland.

PMID: 33866109
DOI: 10.1016/j.jchromb.2021.122694

Abstract

Quantification of proteins in biofluids has largely involved either traditional ligand binding assays or "bottom-up" mass spectrometry. Recently, top-down mass spectrometry using reversed-phase liquid chromatography (RPLC) paired with high-resolution mass spectrometry (HRMS) has emerged as a promising technique, due to the potential of better identification of post-translational modifications (PTMs), lack of downstream interferences, and less time-consuming sample preparation and analysis times. However, it can be difficult with this approach to robustly obtain high-fidelity MS data, especially when pushing for low limits of detection. To address these issues, we developed a chromatographic device with an optimized form factor and stationary phase to improve protein recovery, while reducing run times. We have observed that by using this device, it is possible to achieve attomole quantitation of mAbs without the addition of carrier proteins and with over three-fold higher throughput than columns employed in previous studies. Moreover, we have devised a novel affinity capture method, based on repurposing a unique aptamer ligand that can give 93% recovery of mAb using only a 2 h incubation. When hyphenated together, these two technologies greatly improve the ability to analyze proteins in complex matrices.

Keywords: Aptamer immunoaffinity capture; Fast RPLC-MS; Intact protein quantitation; LC-HRMS-based mAb bioanalysis; Mass analysis; Sample preparation.