Generic approach to the method development of intact protein separations using hydrophobic interaction chromatography

We describe a liquid chromatography method development approach for the separation of intact proteins using hydrophobic interaction chromatography. First, protein retention was determined as function of the salt concentration by isocratic measurements and modeled using linear regression. The error between measured and predicted retention factors was studied while varying gradient time (between 15 and 120 min) and gradient starting conditions, and ranged between 2 and 15%. To reduce the time needed to develop optimized gradient methods for hydrophobic interaction chromatography separations, retention-time estimations were also assessed based on two gradient scouting runs, resulting in significantly improved retention-time predictions (average error < 2.5%) when varying gradient time. When starting the scouting gradient at lower salt concentrations (stronger eluent), retention time prediction became inaccurate in contrast to predictions based on isocratic runs. Application of three scouting runs and a nonlinear model, incorporating the effects of gradient duration and mobile-phase composition at the start of the gradient, provides accurate results (improved fitting compared to the linear solvent-strength model) with an average error of 1.0% and maximum deviation of -8.3%. Finally, gradient scouting runs and retention-time modeling have been applied for the optimization of a critical-pair protein isoform separation encountered in a biotechnological sample.