Proof of Concept To Achieve Infinite Selectivity for the Chromatographic Separation of Therapeutic Proteins

Reversed phase liquid chromatography (RPLC) is a widely used technique for the analytical characterization of proteins biopharmaceuticals, due to its inherent compatibility with mass spectrometry (MS). However, this chromatographic mode suffers from limited selectivity when analyzing large molecules. Due to the on/off mechanism observed with large solutes in RPLC (S values were higher than 100 for intact proteins), we have developed a new analytical strategy based on the use of multi-isocratic elution mode, to achieve arbitrary selectivity for protein variants. In this work, it has been demonstrated that the combination of multi-isocratic steps and very short steep gradient segments at solute elution allows one to set the selectivity as desired, while maintaining sharp peaks due to significant band compression effects. The strategy was successfully applied to the analysis of intact and subunits of monoclonal antibodies (mAbs) as well as antibody-drug conjugates (ADCs), illustrating the possibility to achieve a uniform peak distribution (equidistant band spacing) and much higher resolution than in the case of common linear, multilinear, or nonlinear gradients