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Purification with hydrophobic interaction chromatography
Downstream purification requires methods that maintain yield and molecular stability across a diverse range of biologics. Hydrophobic interaction chromatography (HIC) is used in bioprocessing as a polishing step to improve purity and resolution by exploiting differences in hydrophobicity between the target molecule and impurities. A variety of HIC resins are available that use chemistries with differing levels of hydrophobicity. HIC can be run in bind/elute or flow-through modes, giving scientists flexibility for enhancing selectivity and productivity of a particular process.
Benefits of hydrophobic interaction chromatography
Hydrophobic interaction chromatography complements other purification methods by enabling selective reduction of aggregates, product variants, and process-related impurities through differences in hydrophobicity rather than ionic or affinity-based interactions.
HIC can help with:
- Separation of closely related species, such as aggregates or product variants, based on their innate hydrophobicity differences
- Tunable selectivity to balance recovery and resolution across biomolecules
- Flexible operation in bind/elute or flow-through modes
- Process consistency across development and manufacturing scales using POROS HIC resins
- Efficiency gains in buffer consumption and process productivity
- Viral clearance of a process
Resins designed for selectivity and process efficiency
POROS HIC resins are built on a 50 µm cross-linked polystyrene-divinylbenzene bead that enables scalable operation at high linear velocities. These beads are functionalized to varying levels of hydrophobicity, demonstrating high binding capacities while supporting efficient impurity reduction and reproducible resolution across a broad pH range. When optimized to use with lower salt concentrations and weaker lyotropic salts, the design can help to improve recovery and reduce cycle times in downstream processes.
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.
Figure 2: Dynamic binding capacity comparison.
Lysozyme dynamic binding capacity at 5% of 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%.
Wide hydrophobicity range of POROS HIC resins
Each of the three POROS hydrophobic interaction chromatography resins contain distinct chemistries, offering process developers flexibility to fine-tune selectivity, resolution, and yield. POROS Ethyl, POROS Benzyl, and POROS Benzyl Ultra resins range from very low to high hydrophobic interaction strength.
Figure 3: Range of commercially available HIC resins from least to most hydrophobic
Ordering information
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Frequently asked questions
Hydrophobic interaction chromatography separates biomolecules based on differences in surface hydrophobicity. Under your selected salt conditions, hydrophobic regions of proteins interact with the varying hydrophobicity of the different ligands on POROS HIC resins. As the salt concentration decreases, the strength of these interactions is disrupted, allowing molecules to elute according to their relative hydrophobicity. This controlled separation enables high-resolution purification while maintaining protein stability.
HIC is commonly applied as a polishing step following initial capture and intermediate purification. It helps reduce aggregates, variants, and other process-related impurities that remain after affinity or ion exchange chromatography. It can be run in bind/elute mode with salts selected to target specific impurities or in flow-through mode for gains in capacity and productivity. This makes it particularly valuable for refining final bioprocessing workflows for manufacturing.
POROS Benzyl Ultra HIC resin is distinct in its very high hydrophobicity, designed to operate in low salt conditions. Additionally, the flow-through operational mode of this resin can contribute to process intensification by reducing time for load adjustment and buffer use.
HIC separates monomers from aggregates by exploiting subtle differences in surface hydrophobicity. Aggregated proteins generally exhibit more exposed hydrophobic regions than monomers, leading to stronger binding and minimizing the formation of aggregates under controlled salt gradients. Under these conditions, monomers elute earlier, improving purity and consistency. HIC resins offer high binding capacity and dynamic performance across a range of hydrophobicity, supporting reproducible impurity reduction and consistent process outcomes.
Hydrophobic residues do exist on viruses. Therefore, applicable model viruses should be selected to account for this property. In conjunction with process optimization, HIC too can be optimized to achieve contributing virus clearance for endogenous and adventitious virus models. This is true whether operating in bind/elute or flow-through mode.
Hydrophobic interaction resin samples are available. Request a sample through the Biotherapeutics Sample Request Form.
Additional bioprocessing resources
Bioprocessing resources
For Research Use or Further Manufacturing. Not for use in diagnostic procedures.