Online coupling of analytical hydrophobic interaction chromatography with native mass spectrometry for the characterization of monoclonal antibodies and related products

• Published in: Journal of pharmaceutical and biomedical analysis 2020-07, Vol.186, p.113313-113313, Article 113313
• Main Authors: Yan, Yuetian, Xing, Tao, Wang, Shunhai, Daly, Thomas J., Li, Ning
• Format: Article
• Language: English
• Subjects: Antibodies, Monoclonal - analysis; Antibodies, Monoclonal - chemistry; Chromatography - methods; Cys-linked ADC; DAR; HIC-MS; Hydrophobic and Hydrophilic Interactions; Hydrophobic interaction chromatography; Immunoconjugates - analysis; Immunoconjugates - chemistry; Mass Spectrometry - methods; Monoclonal antibody; Native MS; Post-translational modification; Quality Control
• ISSN: 0731-7085; 1873-264X
• DOI: 10.1016/j.jpba.2020.113313

[Display omitted] •Successful online coupling of analytical HIC to native MS for the characterization of mAbs and mAb-related products.•Up to 3 M of ammonia acetate salt was tolerated by an innovative makeup and splitting flow design.•mAb mixture and mAb variants were successfully characterized.•The first HIC-MS characterization of Cys-linked ADC mimic that generated separation profile highly comparable to a conventional HIC-UV method. Native mass spectrometry (native MS) has seen tremendous development and an increase in application over the past decade for the study of proteins and protein complexes. Although conventionally performed using a static nanospray emitter in an offline fashion, native MS has been increasingly applied in hyphenated methods, where a wide variety of separation techniques are directly coupled to online native MS detection. Those new developments have greatly expanded the utility of native MS in protein biopharmaceutical characterization. Analytical hydrophobic interaction chromatography (HIC) method, although frequently used for the characterization of monoclonal antibodies (mAbs) and antibody-drug-conjugates (ADCs), has rarely been explored for online coupling with native MS. This is largely due to the high salt concentrations used in HIC analysis that are not compatible with direct MS detection. In this study, we overcame this challenge via an innovative makeup and splitting flow design and successfully achieved online coupling of analytical HIC separation with native MS detection. The development and experimental setup of this HIC-MS method is outlined in detail to elucidate how this design could tackle the high salt concentrations used in HIC separation and ultimately achieve both good chromatographic resolution and MS data quality. Subsequently, the utility of this HIC-MS method was demonstrated in three different applications, where a mAb mixture, mAb molecular variants resulting from PTMs, and a Cys-based ADC mimic were all readily characterized in detail. Unlike previously reported HIC-MS methods, this newly developed method utilizes an analytical scale HIC column with conventional ligand so that the achieved separation profile is highly comparable to those obtained by a standard HIC-UV method. As a result, this HIC-MS method not only provides an alternative approach for in-depth characterization of mAbs and related products during their development but could also be readily applied to assist peak assignment and identity e