Superheated reversed phase chromatography with ultrashort columns for the analysis of therapeutic proteins

Mild or elevated temperatures are routinely used for the analysis of therapeutic proteins by reversed phase liquid chromatography. Generic conditions can be used for the analysis of monoclonal antibodies, and may be adapted for species derived thereof, for instance their immuno-conjugates. Beyond platform monoclonal antibodies, many novel, non-covalent protein complexes are also frequently pursued as protein therapeutics. These complexes, in reverse phased chromatography, may require extremely harsh, superheated conditions to dissociate and elute as interpretable profiles. In order to minimize on-column degradation under superheated conditions, the analysis time has to be reduced as much as possible. Using ultrashort columns and fast gradients is a promising approach in achieving informative separations within a minute, or even faster. Here the applicability of this approach, which supports maintaining levels of degradation products close to the intrinsic sample composition without further on-column degradation is demonstrated. NISTmAb as conventional IgG, a bispecific homodimer and a bispecific homotetramer were used for demonstrating differences in the elution characteristics and the necessity of using the proposed approach. The analysis of the bispecific homodimer was discussed in detail as a case study.