Capillary Isoelectric Focusing-Mass Spectrometry Method for the Separation and Online Characterization of Intact Monoclonal Antibody Charge Variants

Abstract

We report a new online capillary isoelectric focusing-mass spectrometry (CIEF-MS) method for monoclonal antibody (mAb) charge variant analysis using an electrokinetically pumped sheath-flow nanospray ion source and a time-of-flight MS with pressure-assisted chemical mobilization. To develop a successful, reliable CIEF-MS method for mAb, we have selected and optimized many critical, interrelating reagents and parameters that include (1) MS-friendly anolyte and catholyte; (2) a glycerol enhanced sample mixture that reduced non-CIEF electrophoretic mobility and band broadening; (3) ampholyte selected for balancing resolution and MS sensitivity; (4) sheath liquid composition optimized for efficient focusing, mobilization, and electrospray ionization; (5) judiciously selected CIEF running parameters including injection amount, field strength, and applied pressure. The fundamental premise of CIEF was well maintained as verified by the linear correlation (R = 0.99) between pI values and migration time using a mixture of pI markers. In addition, the charge variant profiles of trastuzumab, bevacizumab, infliximab, and cetuximab, obtained using this CIEF-MS method, were corroborated by imaged CIEF-UV (iCIEF-UV) analyses. The relative standard deviations (RSD) of absolute migration time of pI markers were all less than 5% (n = 4). Triplicate analyses of bevacizumab showed RSD less than 1% for relative migration time to an internal standard and RSD of 7% for absolute MS peak area. Moreover, the antibody charge variants were characterized using the online intact MS data. To the best of our knowledge, this is the first time that direct online MS detection and characterization were achieved for mAb charge variants resolved by CIEF as indicated by a well-established linear pH gradient and correlated CIEF-UV charge variant profiles.

Recent advances in capillary electrophoresis‐mass spectrometry: Instrumentation, methodology and applications

“…Dai et al. reported a CIEF‐MS application for the separation and characterization of mAb charge variants as can be seen in Fig. .…”

“…Due to a larger injection volume applicable to the iCIEF system, low-abundance basic variants of stressed trastuzumab could be characterized. Dai et al reported a CIEF-MS application for the separation and characterization of mAb charge variants [73] as can be seen in Fig. 4.…”

Analysis of four therapeutic monoclonal antibodies by online capillary isoelectric focusing directly coupled to quadrupole time‐of‐flight mass spectrometry

“…A sandwich configuration containing the catholyte segment, the sample segment containing analyte and carrier ampholytes, and the anolyte segment in the IEF capillary was used to avoid the exchange step and achieve full automation . Direct online cIEF–MS analysis of intact mAbs have been reported using either a stainless‐steel flow‐through microvial interface , or a pulled‐glass electrokinetically pumped sheath‐flow interface . The reported total ion chromatograms (TIC) containing structural information demonstrated high similarity to imaging IEF traces of identical mAbs .…”

Preparation of a capillary isoelectric focusing column with monolithic immobilized pH gradient and its application on protein separation based on an online capillary isoelectric focusing platform

“…In this case, the cIEF column with immobilized CAs could be carried out with high detection sensitivity even at low wavelengths. At the same time, coupling cIEF with MS could be also easier to implement, which would provide an important information on both isoelectric points (pIs) and molecular weights of target proteins, and then would greatly facilitates the profiling of proteome and the discovery of biomarker .…”