|Advancing hydrophobic interaction chromatography methods to characterize biotechnology enzyme mixtures and to profile biotherapeutics
|(R)evolutions in Biopharmaceutical Analysis (KVCV)
|Prof Sebastiaan Eeltink
|Vrije Universiteit Brussel
Abstract Information :
The need for comprehensive characterization of protein-derived macromolecules applied in the biopharmaceutical and biotechnology industries is increasing rapidly. The biological activity and/or enzymatic reactivity of these biomolecules strongly depend on their chemical composition and three-dimensional structure. To obtain information on those characteristics it is important to maintain the original structural composition of bio-macromolecules during the analysis. Hydrophobic interaction chromatography (HIC) capitalizes the interaction between hydrophobic patches of proteins and weakly hydrophobic ligands attached to the stationary phase. HIC method development is a major bottleneck since effects of salt systems, buffers, and stationary-phase chemistry on protein retention are poorly understood.
The effects of kosmotropic/chaoptropic salt systems and the addition of mobile-phase additives (calcium and isopropanol) on retention have been assessed. The hydrophobicity index appeared to be a good indicator to predict the elution order of intact proteins in HIC mode. Furthermore, the effect of adding additives in the mobile phase, such as calcium chloride (stabilizing the 3D conformation of -lactalbumin) and isopropanol, on retention properties has been assessed. Results indicate that HIC retention is also governed by conformational changes in the proteins which affect the number of accessible hydrophobic moieties. Furthermore, method-development (MD) strategies will be discussed and the possibilities and limitations to develop methods for the separation of intact proteins in HIC mode, based on retention-time models in combinations gradient scouting runs. Finally, the potential of HIC has been assessed to profile biotechnology mixtures and biotherapeutics, including monoclonal antibodies and anti-body drug conjugates