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 ana-lytical techniques. Reversed phase liquid chromatography is a valuable strategy due to its high resolvingpower and straightforward compatibility to mass spectrometry. The present study demonstrates thathigh peak capacity can be attained with intact mAb of ∼150 kDa, reduced mAb fragments of ∼25–50 kDaand also digested mAb generating numerous peptides of ∼0.5–3 kDa. Several long columns packed withfully porous wide-pore sub-2 µm particles were coupled in series to evaluate the effect of column lengthon peak capacity. By using three columns of 150 mm length, a mobile phase temperature of 80◦C and agradient time of around 20 min, peak capacities of 117 and 128 were obtained for a commercial intactmAb and its reduced mAb fragments, respectively. On the other hand, when peptide mapping was per-formed at 50◦C, with a gradient time of 270 min and a column length of 450 mm, a peak capacity of morethan 700 was achieved