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A Generic Platform to couple Affinity Chromatography with Native Mass Spectrometry for the analysis of Therapeutic Monoclonal Antibodies

A Generic Platform to couple Affinity Chromatography with Native Mass Spectrometry for the analysis of Therapeutic Monoclonal Antibodies

Recently, the Journal of Pharmaceutical and Biomedical Analysis published research from the team at Regeneron that included Newomics innovative multinozzle M3 emitter and their MnESI source, to achieve nanospray native MS for studying antibody affinity binding.

Newomics MnESI source empowers the M3 emitter to significantly enhance ionization efficiency and achieve high sensitivity, throughput, and robustness for LC/MS analysis. The M3 emitter, unlike the conventional single-tip emitters, has multiple nozzles working together to split a single large flow stream evenly into multiple tiny flows, the way a showerhead works. The MnESI source is designed to simplify the user experience by enabling the plug-and-play operation of the M3 emitter.

In this study, the researchers coupled affinity chromatography with native mass spectrometry for the analysis of therapeutic monoclonal antibodies (mAbs). Affinity chromatography is a separation method based on a specific binding interaction between an immobilized ligand and its binding partner, in this case the mAbs. With affinity chromatography highly complex mAb structures can be studied and it offers insight into their biological relevance. Native mass spectrometry is an emerging analytical technique for studying large biomolecules, while maintaining the native structural features of the analytes and their interactions in the gas phase during the electrospray processes as much as possible. Native mass spectrometry can disclose the composition, stoichiometry, kinetics, stability, and structure of subunits of proteins and protein complexes in a high throughput manner.

Despite the great promise, application of affinity chromatography with native mass spectrometry in routine mAb characterization has been limited, largely due to the complicated experimental set-up. This research introduces a generic platform to facilitate the online coupling of different affinity  separation modes with native mass spectrometry. This platform allows a greatly simplified experimental set-up and facile swapping of affinity separation modes. The utility of this platform was demonstrated by successfully coupling three affinity chromatography methods (protein A, FcγRIIIa, and FcRn) with native mass spectrometry, as shown in the figure below (Figure 1).

Figure 1. Schematic representation of the native affinity LC-MS platform (Source: ScienceDirect)

This study shows that it is possible to use a generic platform to achieve native affinity LC-MS applications. The protein A method can be used to achieve rapid screening of mAb molecules in a “bind-and-elute” mode. It can also be used in a high-resolution resolving mode to study mAb species with altered protein A affinity, such as oxidized mAbs and mAb species with specific Fc mutations. The FcγRIIIa method, glycoform-resolved MS analyses were achieved for both IgG1 and IgG4 subclass molecules. The retention order of various glycoforms, as revealed by extracted ion chromatograms, correlated well with the known relationship between Fc N-glycan features and the FcγRIIIa binding affinity. Finally, the utility of the FcRn method was demonstrated in two examples, where the oxidized mAb showed decreased FcRn binding while the engineered mAb with “YTE” substitution in the Fc region exhibited enhanced FcRn binding.

Due to the generic applicability and simplified experimental set-up, this developed platform should facilitate wider adoption of native affinity LC-MS methods into routine characterization workflows of therapeutic mAbs. Even though only three affinity chromatography methods were investigated during this study, the applicability of this platform can be potentially expanded to other affinity separation modes and also to other protein drug modalities.

Part Numbers & Product Specifications:

SupplierProduct Nr.Specifications
NewomicsIS-T01MnESI Platform for Thermo Scientific New Generation Mass Spectrometers (e.g., Orbitrap Fusion Lumos, Eclipse, Exploris, and TSQ Altis Triple quadrupole mass spectrometers)
 IS-T02MnESI Platform for Thermo Scientific Legacy Mass Spectrometers (e.g., Orbitrap Q Exactive mass spectrometers)
 IS-B01MnESI Platform for Bruker Mass Spectrometers (e.g., maxis II QTOF, timsTOF Pro mass spectrometers)
 IS-A01MnESI Platform for Agilent Mass Spectrometers
 E5N20MU01M3 emitter, 5-nozzle, 20µm ID, 5-20µL
 E8N10MU01M3 emitter, 8-nozzle, 10µm ID, 1-10µL
 E8N20MU01M3 emitter, 8-nozzle, 20µm ID, 10-40µL

Authors & Other Sources:

Victoria C. Cotham, Anita P. Liu, Shunhai Wang & Ning Li
Analytical Chemistry Group, Regeneron Pharmaceuticals Inc., Tarrytown, NY, USA

Full research article: https://www.sciencedirect.com/science/article/pii/S0731708523001061

More information on these products:

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