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.