A protocol for setting‐up robust hydrophobic interaction chromatography targeting the analysis of intact proteins and monoclonal antibodies

Hydrophobic interaction chromatography (HIC) is a chromatographic technique that mainly targets the separation of biomolecules (intact proteins, monoclonal antibodies, etc.) based on difference in surface hydrophobicity while applying non-denaturing conditions. This protocol paper provides guidelines for setting-up robust HIC analysis and considers instrument configuration, mobile-phase sample preparation, as well conditions settings. The mixture intact proteins antibodies demonstrated by conventional conditions, is, using mildly hydrophobic (C4) stationary phase combination with an inverse ammonium sulphate gradient dissolved aqueous phosphate buffer. effect sample-preparation breakthroughs presented. Finally, good run-to-run repeatability (relative standard deviation < 2%) five different columns obtained from three column lots, considering retention, peak width, area pressure.