Application of sample displacement batch chromatography for fractionation of proteoforms

Fractionation of proteoforms is currently the most challenging topic in the field of protein purification. The need for considering the existence of proteoforms into experimental approaches is not only important in Life Science research in general but especially in the manufacturing of therapeutic proteins (TPs) like recombinant therapeutic antibodies (mAbs). Some of the proteoforms of TPs have significantly decreased actions or even cause side effects. The identification and removal of proteoforms differing from the main species, having the desired action, is challenging because the difference in the composition of atoms often is very small and their concentration in comparison to the main proteoform can be small. In this study we demonstrate that sample displacement batch chromatography (SDBC) is an easy to handle, economic and efficient method for fractionating proteoforms. As a model sample a commercial ovalbumin fraction was used, containing many ovalbumin proteoforms. The most promising parameters for the SDBC were determined by a screening approach and applied for a 10-segment fractionation of the ovalbumin with cation exchange chromatography resin. Mass spectrometry of intact proteoforms was used for characterizing the SDBC fractionation process. By SDBC a significant separation of different proteoforms was obtained.