Hydrophobic Interaction Chromatographic Media for Bioseparation with High Resolution

Ke Yang, Sepax Technologies, Inc., USA
Sixi Wang, Sepax Technologies, Inc., USA
Zhong Ding, Sepax Technologies, Inc., USA
Xueying Huang Sepax Technologies, Inc., USA
Yair Shachar, Yair Technologies, Israel

Hydrophobic interaction chromatographic (HIC) separation applies to all stages of biological sample purification process including capturing, polishing protein purifications. Aggregates, fragments and different forms of proteins can be isolated and analyzed based on the hydrophobic interaction between the samples and the resin solid support. The hydrophobic interaction can be controlled by a number of factors such as protein structure, salt concentration, pH, temperature and organic solvent additives. HIC separation is a great addition to the native protein purification process including ion exchange, size exclusion, and affinity chromatography. The current commercially available HIC medias for pilot and process purification are based on soft agarose and methacrylate. These resins can not tolerate high flow rates and the separation efficiency is low. As such, Sepax has developed an alternative methacrylate based HIC media, capable to withstand higher flow rate application.

Recently we have developed HIC resins of 10 µm particle size and 800 Å pore size with very narrow dispersity (D90/D10<1.3). These HIC resins are composed of hydrophilic methacrylate polymer beads. On the resin surface, alkyl groups or aryl groups can be chemically attached to enable hydrophobic interactions with biomolecules. These unique HIC medias show a linear relationship between backpressure and flow rate up to 50 bars, which offers an advantage for achieving faster pilot and process purification. This new narrow dispersed 10 µm particle size resin provides better resolution compared to 30 µm HIC series.

In this poster, we present the characteristics of three selected 10 µm particle size HIC resin series, Generik MC10-HIC Butyl, Polar MC10-HIC Butyl and Polar MC10-HIC Ether. These three HIC phases are of decreasing hydrophobicity, providing a wide range for HIC interactions for different bio-molecules. Compared to their larger particle counterparts, MC10-HIC resins provide higher efficiency with excellent recovery of bio-molecules such as mAb (monoclonal antibody), ADC (antibody drug conjugate) and their related protein fragments, and oligonucleotides. The MC10-HIC media also can tolerate high-pressure operation up to 50 bars. All three phases are applicable at different stages from laboratory discovery, to pilot scale purification and industrial process chromatography