A Novel Phenyl-Based RPLC Stationary Phase for High Throughput, High Resolution Characterization of Protein Therapeutics

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Jennifer Nguyen, Susan Rzewuski, Dan Walsh, Jim Cook, Maureen DeLoffi, Gary Izzo, Yuehong Xu, and Matthew A. Lauber
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Protein therapeutics can be effectively characterized using the capabilities of liquid chromatography (LC).  Because of its high resolving power and amenability to mass spectrometric (MS) detection, reversed phase liquid chromatography (RPLC) has become one of the most heavily relied upon techniques. However, RPLC stationary phases notoriously suffer from performance limitations, including their strong dependence on ion pairing and elevated separation temperatures that can cause on-column degradation.

To address these limitations, a novel column technology has been designed.  This column technology is based on an optimized 2.7 µm superficially porous particle that by van Deemter analyses has proven to be effective in minimizing intra-particle diffusion, thereby affording a kinetic efficiency advantage.  For the porous layer of this stationary phase, an optimal pore diameter has also been carefully selected.  Comprehensive analysis of intact and IdeS-digested monoclonal antibodies (mAbs) has shown that the average pore diameter needs to be at least 400 Å, particularly when ion pairing is minimized for MS compatibility, where proteins are more likely to adopt extended structures.  Moreover, the capability of this column technology is augmented by a novel surface chemistry that is synthesized using a multistep silanization process to yield a phenyl-based bonded phase which is both high in coverage (up to 6 µmol phenyl moiety/m2) and comprised of rigidly constrained carbons.  This novel bonded phase is believed to limit silanol interactions by extensively masking the silica base particle, to facilitate more discrete desorption at lower temperatures by minimizing the conformational heterogeneity of protein adsorption, and to improve resolving power by being highly retentive.  Using either HPLC or UHPLC instrumentation, it will be shown that this technology has made it possible to better characterize mAb and ADC therapeutics by delivering unprecedented resolution as well as higher fidelity, higher quality data.

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