2.5.1. Hydrophobic Interaction Chromatography—HIC

The CIMmultus OH monolith column (1 mL, 2-µm pores; BIA separations, Ajdovščina, Slovenia) was used for HIC. The sample was diluted 2-fold with 3-M K₂HPO₄ (Merck, Darmstadt, Germany), 2% glucose (Sigma-Aldrich Chemie GmbH, Steinheim, Germany), pH 7.0, and then filtrated over a 0.45-µm mPES membrane filter. After equilibration with 1.5-M K₂HPO₄ and 1% glucose, pH 7.0, the sample was loaded onto the column with a flow rate of 2 mL/min. For elution, the flow rate was set to 3 mL/min, and the gradient was run in 20 column volumes (CV) from 0% to 100% 50-mM K₂HPO₄ and 1% glucose, pH 7.0. The columns were washed in the cleaning in place (CIP) mode with 1-M NaOH (VWR, Geldenaaksenbaan, Belgium) for reuse [23].