The first step in the optimization process was to select a resin that was more hydrophobic than the Phenyl Sepharose FF HS resin used in the existing process. 20 For our examination of the effects of pH adjustment, we selected a very hydrophobic resin to promote maximum interaction with the stationary phase under no-salt conditions. In practice, however, it can influence protein retention by titrating charged patches close to the hydrophobic patches on the protein surface. The effect of pH on retention in HIC is usually unpredictable and thus pH is not frequently studied as a parameter during HIC optimization. Instead of the addition of salt, the pH of the mobile phase was modulated to alter the surface charge of the protein, and thereby influence selectivity. Here, we report a novel use of HIC in the flowthrough mode with no kosmotropic salt in the mobile phase. 19 used HIC at low salt concentration for capture of mAbs using a critical hydrophobicity approach, but with limited success. Recently, Gagnon 18 reported the use of glycine in HIC systems to keep conductivities low. Arakawa and researchers 16, 17 tried to use arginine to promote binding and facilitate elution in HIC systems. 13Д®fforts to operate HIC under reduced or no-salt conditions have been reported. 15 In addition, the presence of salt in the load material, elution pool or the FT pool from the HIC step also complicates sample manipulation and requires significant dilution, or an ultrafiltration/ diafiltration unit operation, between processing steps. Due to municipal waste water concerns, it is very expensive to dispose of ammonium sulfate, the most commonly used kosmotropic salt. 14 Second, the use of high concentrations of salts is highly undesirable in any manufacturing process because it can cause corrosion of stainless steel tanks. This strategy has been particularly popular as a polishing step in antibody processes since aggregates are usually more highly retained on HIC. 13 To circumvent this issue, HIC is sometimes used in the flowthrough mode in which the product of interest flows while the more hydrophobic impurities remain bound to the column. 10, 11 Resin vendors have lately tried to optimize the pore size and ligand density in an effort to maximize capacity 12 however, 10% breakthrough capacities of > 40 mg/mL of resin have not yet been reported. In general, binding capacity has been traditionally limited on HIC, especially in comparison to ion exchange chromatography (IEX). 9 Elution is usually facilitated by decreasing salt concentration or by use of organic mobile phase modifiers.Дespite its orthogonal selectivity, the use of HIC in any purification process presents two primary challenges. 8 Kosmotropic salts interact with water molecules to reduce solvation of protein molecules in solution and expose their hydrophobic patches to promote binding. 7 Interactions of proteins on HIC are usually promoted by kosmotropic salts, e.g., ammonium sulfate, sodium citrate, potassium phosphate. 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.

Capto butyl impres