Figure 1. Flow rate independent performance. Separation comparison of POROS benzyl resin at different flow rates showing good resolution and flow rate independent performance. Experimental details: load buffer: 1.7 M ammonium sulfate in 50 mM sodium phosphate, pH 7; buffer gradient: load buffer to 50 mM sodium phosphate, pH 7, in 10 CVs; format: 0.46 cm D x 20 cm L; flow rate: 200, 400, 600 cm/hr. Protein mixture: ribonuclease A, lysozyme, and chymotrypsinogen A.
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 the capture step, intermediate steps, and final polish purification.
POROS hydrophobic interaction resins
Thermo Scientific POROS hydrophobic interaction chromatography (HIC) resins are based on the 50 μm POROS base bead, utilizing a novel coating procedure to enable functionalization with unique hydrophobic ligands. These resins are suitable for bind/elute and flow-through applications at lower salt concentrations and have higher binding capacity and resolution than classical HIC resins, thereby enabling more flexibility around process operating conditions.
Key features
- Three unique resins covering a wide range of hydrophobicity
- High resolution, even with lower conductivity conditions
- High dynamic binding capacity and superior pressure-flow characteristics
Key applications
- Monoclonal antibodies, bispecific antibodies, and antibody–drug conjugates (ADCs)
- Product-related impurities and aggregate removal
- Plasmids, RNAi, and oligonucleotides
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 used for the purification of a wide variety of biomolecules including therapeutic proteins, antibody fragments, antibody drug conjugates (ADCs), and other large molecules.
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 are being developed.
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Unique resin design
The 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. POROS HIC resins enable outstanding 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 and may help reduce cost of goods
- 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 exceptional resolution
- Flow rate independent performance
- Robust stability
Figure 2. Dynamic binding capacity. Lysozyme dynamic binding capacity at 5% breakthrough of all three POROS HIC resins, compared to their leading comparable alternative resins. Experimental details: Protein sample 1.5 mg/mL lysozyme; Buffer: 1.5 M ammonium sulfate, 50 mM sodium phosphate pH 7.0; Linear velocity: 300 cm/hr; Column format: 0.66 cm D x 20 cm L; Protein loaded until breakthrough, measurement taken at 5% breakthrough.
Ordering information
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.
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Related resources
Article: Monoclonal Antibody Aggregate Polish and Viral Clearance Using Hydrophobic-Interaction Chromatography
Article: Novel Hydrophobic Interaction Chromatography Resins for next generation biotherapeutic challenges
Application note: Efficient removal of aggregates from monoclonal antibodies by hydrophobic interaction chromatography in flow-through mode Purification
Poster: Viral Clearance Capability of POROS HIC resins
Poster: Efficient Monoclonal Antibody Aggregate Removal by Hydrophobic Interaction Chromatography
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