No abstract

In therapeutic monoclonal antibody (mAb) development, charge heterogeneity of a mAb molecule is often associated with critical quality attributes and is therefore monitored throughout development and during QC release to ensure product and process consistency. Elucidating the cause of each charge variant species is an involved process that often requires offline fractionation by ion exchange chromatography (IEX) followed by mass spectrometry (MS) analysis, largely due to the incompatibility of conventional IEX buffers for direct MS detection. In this study, we have developed a method that combines a generic strong cation exchange (SCX) chromatography step with ultrasensitive online native MS analysis (SCX-MS) optimized for mAb separation and detection. As demonstrated by analyzing mAb molecules with a wide range of pI (isoelectric point) values, the developed method can consistently achieve both high-resolution IEX separation and ultrasensitive MS detection of low-abundance charge variant species. Using this method, we analyzed the charge heterogeneity of NISTmAb reference material 8671 (NISTmAb) at both whole antibody and subdomain levels. In particular, due to the high sensitivity, a nonconsensus Fab glycosylation site, present at a very low level (<0.1%), was directly detected in the NISTmAb sample without any enrichment. The structure and location of this Fab glycosylation was further characterized by peptide mapping analysis. Despite the extensive characterization of NISTmAb material in previous studies, this is the first time that this Fab-glycosylated variant has been identified in the NISTmAb, demonstrating the value of this new method in achieving a more comprehensive characterization of charge heterogeneity for therapeutic mAbs.

LC−MS based analysis of protein biopharmaceuticals could benefit from improved data quality, which can subsequently lead to improved drug characterization with higher confidence and less ambiguity. In this study, we created a simple device to modify the desolvation gas on a Q-Exactive mass spectrometer and to demonstrate the utility in improving both peptide mapping analysis and intact mass analysis, the two most routinely and widely applied LC−MS techniques in protein biopharmaceutical characterization. By modifying the desolvation gas with acid vapor from propionic acid (PA) and isopropanol (IPA), the ion suppression effects from trifluoroacetic acid (TFA) in a typical peptide mapping method can be effectively mitigated, thus leading to improved MS sensitivity. By modifying the desolvation gas with base vapor from triethylamine (TEA), the charge reduction effect can be achieved and utilized to improve the spectral quality from intact mass analysis of protein biopharmaceuticals. The approach and device described in this work suggests a lowcost and practical solution to improve the LC−MS characterization of protein biopharmaceuticals, which has the potential to be widely implemented in biopharmaceutical analytical laboratories.

Traditional SDS-PAGE method and its modern equivalent CE-SDS method are both widely applied to assess the purity of therapeutic monoclonal antibody (mAb) drug products. However, structural identification of low molecular weight (LMW) impurities using those methods has been challenging and largely based on empirical knowledges. In this paper, we present that hydrophilic interaction chromatography (HILIC) coupled with mass spectrometry analysis is a novel and orthogonal method to characterize such LMW impurities present within a purified mAb drug product sample. We show here that after removal of N-linked glycans, the HILIC method separates mAb-related LMW impurities with a size-based elution order. The subsequent mass measurement from a high-resolution accurate mass spectrometer provides direct and unambiguous identification of a variety of low-abundance LMW impurities within a single LC-MS analysis. Free light chain, half antibody, H2L species (antibody possessing a single light chain) and protein backbone-truncated species can all be confidently identified and elucidated in great detail, including the truncation sites and associated post-translational modifications. It is worth noting that this study provides the first example where the H2L species can be directly detected in a mAb drug product sample by intact mass analysis without prior enrichment.

Despite the recent success of coupling anion exchange chromatography with native mass spectrometry (AEX-MS) to study anionic proteins, the utility of AEX-MS methods in therapeutic monoclonal antibody (mAb) characterization has been limited. In this work, we developed and optimized a salt gradient-based AEX-MS method and explored its utility in charge variant analysis of therapeutic mAbs. We demonstrated that, although the developed AEX-MS method is less useful for IgG1 molecules that have higher isoelectric points (pIs), it is an attractive alternative for charge variant analysis of IgG4 molecules. By elevating the column temperature and lowering the mAb pI through PNGase F-mediated deglycosylation, the chromatographical resolution from AEX separation can be significantly improved. We also demonstrated that, after PNGase F and IdeS digestion, the AEX-MS method exhibited excellent resolving power for multiple attributes in the IgG4 Fc region, including unprocessed C-terminal Lys, N-glycosylation occupancy, and several conserved Fc deamidations, making it ideally suited for multiple attribute monitoring (MAM). Through fractionation and peptide mapping analysis, we also demonstrated that the developed AEX-MS method can provide site-specific and isoform-resolved separation of Fc deamidation products, allowing rapid and artifact-free quantitation of these modifications without performing bottom-up analysis.