Charge heterogeneity profiling of monoclonal antibodies using low ionic strength ion-exchange chromatography and well-controlled pH gradients on monolithic columns.

In this work, the suitability of employing shallow pH gradients generated using single component buffer systems as eluents through cation-exchange (CEX) monolithic columns is demonstrated for the high-resolution separation of monoclonal antibody (mAb) charge variants in three different biopharmaceuticals. A useful selection of small molecule buffer species is described that can be used within very narrow pH ranges (typically 1 pH unit) defined by their buffer capacity for producing controlled and smooth pH profiles when used together with porous polymer monoliths. Using very low ionic strength eluents also enabled direct coupling with electrospray ionisation mass spectrometry. The results obtained by the developed pH gradient approach for the separation of closely related antibody species appear to be consistent with those obtained by imaged capillary isoelectric focusing (iCE) in terms of both resolution and separation profile. Both determinants of resolution, i.e., peak compression and peak separation contribute to the gains in resolution, evidently through the Donnan potential effect, which is increased by decreasing the eluent concentration, and also through the way electrostatic charges are distributed on the protein surface. Retention mechanisms based on the trends observed in retention of proteins at pH values higher than the electrophoretic pI are also discussed using applicable theories. Employing monolithic ion-exchangers is shown to enable fast method development, short analysis time, and high sample throughput owing to the accelerated mass transport of the monolithic media. The possibility of short analysis time, typically less than 15 min, and high sample throughput is extremely useful in the assessment of charge-based changes to the mAb products, such as during manufacturing or storage.