3 - 6 HIC is based on interactions between hydrophobic (aliphatic or aromatic) ligands on the stationary phase with hydrophobic patches on the surface of proteins. 1, 2 This mode of chromatography is particularly useful for aggregate removal, and it provides good clearance of other process-related impurities such as host cell protein (HCP), leached Protein A and endogenous viruses. Hydrophobic interaction chromatography (HIC) occupies a unique niche as a polishing step in many monoclonal antibody (mAb) purification processes. Performance was comparable to that observed using conventional HIC conditions with high salt. This strategy was tested with a panel of antibodies with varying pI and surface hydrophobicity. Optimum pH conditions were chosen under which the antibody product of interest flowed through while impurities such as aggregates and host cell proteins bound to the column. Under the pH conditions tested (pH 6.0 and below), antibodies typically become positively charged, which has an effect on its polarity and overall surface hydrophobicity. A very hydrophobic resin is selected as the stationary phase and the pH of the mobile phase is modulated to achieve the required selectivity. Here, we report an unconventional way of operating HIC in the flowthrough (FT) mode with no kosmotropic salt in the mobile phase. These salts often pose a disposal concern in manufacturing facilities and at times can cause precipitation of the product. HIC, however, suffers from the limitation of use of high concentrations of kosmotropic salts to achieve the desired separation. HIC offers an orthogonal selectivity to ion exchange chromatography and can be an effective step for aggregate clearance and host cell protein reduction. Hydrophobic interaction chromatography (HIC) is commonly used as a polishing step in monoclonal antibody purification processes.

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