Hydrophobic interaction chromatography (HIC) is a versatile method for the purification and separation of biomolecules by using the function of hydrophobicity. The proteins containing both hydrophilic and hydrophobic regions are applied to a HIC column under specified salt buffer conditions, which promotes the binding of the biomolecule to the HIC resin but also has a stabilizing influence on the molecule’s structure. HIC steps are commonly used at all steps in the process including capture step, intermediate and final polish purification.
Webinar: Mastering monoclonal antibody aggregate removal using hydrophobic interaction chromatography
Register to watch this webinar about a process development case study using POROS HIC in bind-elute mode for high-aggregate mAb polishing. We will highlight the differences and specifics of the POROS HIC resins that address current purification challenges as more diversified classes of biomolecules being developed.
Thermo Fisher Scientific provides a suite of POROS HIC resins offering a differentiating range in hydrophobicity suitable for bind/elute and flow-through applications. The resins can be utilized at lower salt concentrations for the purification of a wide variety of biomolecules including therapeutic proteins, antibody fragments, antibody drug conjugates (ADCs), and other large molecules.
POROS HIC Resin
Bind/elute mode of moderately to considerably hydrophobic molecules
Low density benzyl/aromatic
Bind/elute or flow-through mode depending on molecule
POROS Benzyl Ultra
High density benzyl/aromatic
Flow-through mode in lower salt to bind impurities such as aggregates
Figure 1: Order of hydrophobicity of POROS HIC resins. Hydrophobicity based on lysozyme gradient elution.
The new 50 µm resin backbone consists of cross‐linked poly(styrene-divinylbenzene) with a unique pore structure that provides rapid mass transport and enables enhanced productivity. The particle surface is coated with a novel polymer coating, which is then further derivatized with a range of hydrophobic ligands for flexible purification process design (Figure 2). POROS HIC resins deliver superior performance independent of flow rate, a major improvement in the purification tool box for impurity removal.
- Differentiating selectivity and superior resolution capability which helps improve yield and purity, reduce cost of goods (COG)
- High capacity for a range of molecules leading to reduced column size and smaller footprint
- Use of lower salt concentrations and weaker lyotropic salts allowing for increased process optimization flexibility with lower conductivity while maintaining superior resolution
- Flow rate independent performance
- Robust stability
Figure 2. Separation of a mixture of standard proteins using POROS Ethyl, POROS Benzyl and POROS Benzyl Ultra resins. Column size: 0.46cmD X 20cmL; Protein Mixture: Ribonuclease A, Lysozyme, Chymotrypsin and Chymotrypsinogen in 1.7M ammonium sulfate, 50mM sodium phosphate pH 7.0; Gradient Elution: 1.7M ammonium sulfate/50 mM sodium phosphate pH 7.0 to 50 mM sodium phosphate pH 7.0 over 10 column volumes (CV); Flow rate: 100 cm/hr.
Events & Webinars
Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only.
For research use only. Not for use in diagnostic procedures.