High resolution reversed phase analysis of recombinant monoclonal antibodies by ultra-high pressure liquid chromatography column coupling.

Monoclonal antibodies (mAbs) represent one of the fastest growing areas of new drug development. However, their analytical characterization is complex and generally requires an array of orthogonal analytical techniques. Reversed phase liquid chromatography is a valuable strategy due to its high resolving power and straightforward compatibility to mass spectrometry. The present study demonstrates that high peak capacity can be attained with intact mAb of ~150 kDa, reduced mAb fragments of ~25-50 kDa and also digested mAb generating numerous peptides of ~0.5-3 kDa. Several long columns packed with fully porous wide-pore sub-2 μm particles were coupled in series to evaluate the effect of column length on peak capacity. By using three columns of 150 mm length, a mobile phase temperature of 80 °C and a gradient time of around 20 min, peak capacities of 117 and 128 were obtained for a commercial intact mAb and its reduced mAb fragments, respectively. On the other hand, when peptide mapping was performed at 50 °C, with a gradient time of 270 min and a column length of 450 mm, a peak capacity of more than 700 was achieved.