A generic method for intact and subunit level characterization of mAb charge variants by native mass spectrometry.

Monoclonal antibodies (mAbs) are heterogeneous macromolecules that display a complex isoform profile as a result of the large series of modifications they can undergo. Product-related charge variants that are associated with a loss of biological activity or affected half-life and immunogenicity are especially important. Consequently, they are often considered critical quality attributes such that acceptance criteria and controls should be established. The characterization of mAbs charge variants has long been a time and resource consuming task. Recent successes in the use of salt mediated pH gradient ion exchange chromatography with volatile mobile phases have shown there to be significant promise in using online mass spectrometric (MS) detection to facilitate peak detection. In this study, a newly developed 3 µm non-porous cation exchange column technology was investigated for its capability to be hyphenated to MS for the purpose of characterizing mAb charge variants. A 2 mm ID format was selected for the ease of configuring it to classical MS ESI ion sources. A monoclonal antibody reference material from NIST (RM 8671; NISTmAb) was used in its intact and IdeS/IgdE-digested forms to test for column performance and MS sensitivity. Furthermore, three different mAbs with highly basic isoelectric points (pI) were analyzed in their native and proteolyzed forms to demonstrate the straightforward application of the developed technique even with mAbs having strong retention on cation exchange media. The MS detection of low-abundance charge variant species (<0.1%) demonstrated there to be acceptable sensitivity and dynamic range even from routine analyses. The capability of the column to separate different mAbs having high basic pI was demonstrated, and it was found that slight adjustment of ammonium acetate concentration in the eluent can be a convenient way to rapidly optimize a separation if necessary. Linearity was shown to exist between protein mass loads of 2.5 and 50 µg while an optimal balance between chromatographic resolution and MS sensitivity was observed between 5 and 10 µg. Excellent run-to-run and column-to-column repeatability was achieved in terms of retention times, resolution and recovery.