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