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.
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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.
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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 | Surface Chemistry | Key Application |
POROS Ethyl | Novel ethyl | Bind/elute mode of moderately to considerably hydrophobic molecules |
POROS Benzyl | 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.
A32552,A32553,A32554,A32555,A32556,A32557,A32558,A32559,A32560,A32561,A32562,A32563,A32564,A32565,A32566,A32567,A32568,A32569
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Pharmaceutical Grade Reagent. For Manufacturing and Laboratory Use Only.
For research use only. Not for use in diagnostic procedures.