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Polishing with hydrophobic interaction chromatography resins
Downstream purification requires methods that maintain yield and molecular stability across a diverse range of biologics. Hydrophobic interaction chromatography (HIC) is used as an orthogonal polishing step to improve purity by exploiting differences in surface hydrophobicity between the target molecule and impurities and can be operated in bind/elute or flow-through modes. Traditional HIC resins have historically required high-molarity kosmotropic salts, which can challenge molecular stability and limit operating flexibility. POROS HIC resins are designed to operate at lower salt concentrations with weaker lyotropic salts while supporting high selectivity for aggregate and impurity reduction.
Wide hydrophobicity range of POROS HIC resins
The three POROS HIC resins use distinct chemistries, allowing process developers 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 1: Range of commercially available HIC resins from least to most hydrophobic
Benefits of POROS HIC resins
By operating at lower salt concentrations than traditional HIC resins and supporting both bind/elute and flow-through modes, these resins can facilitate process intensification. The portfolio covers a broader hydrophobicity range than other commercially available HIC resins.
POROS HIC resins can help with:
- Separation of closely related species, such as aggregates or product variants, based on their innate hydrophobicity differences
- Recovery across different biomolecules leveraging tunable selectivity
- Flexible operation in bind/elute or flow-through modes
- Process consistency across development and manufacturing scales
- Efficiency gains in buffer consumption and process productivity
- Potential contribution of viral clearance of endogenous and adventitious viral models
Resins designed for performance
POROS HIC resins are built on a 50 µm (nominal) cross-linked polystyrene-divinylbenzene bead that supports scalable operation across a broad range of operating pressures and flow rates for large-scale manufacturing. A proprietary coating procedure enables distinct hydrophobic ligand functionalization across the three resin chemistries. These beads are functionalized to different hydrophobicity levels, supporting high dynamic binding capacity (DBC), efficient mass transfer, exceptional peak resolution, and reproducibility across pH ranges. Predictable linear pressure-flow characteristics allow columns to be operated across a range of conditions without compromising purity or recovery and make production scale-up activities easier. When used with lower salt concentrations and weaker lyotropic salts, the design can improve recovery and reduce cycle times.
Figure 2: 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 3: 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%.
Ordering information
Related resource
Applications across bioprocessing workflows
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Frequently asked questions
POROS HIC resins share the same average 50 µm cross-linked polystyrene-divinylbenzene base bead but differ in their hydrophobic ligand chemistry and interaction strength. POROS Ethyl is the least hydrophobic of the three. POROS Benzyl is intermediate, and POROS Benzyl Ultra is the most hydrophobic. This range gives process development scientists flexibility to screen selectivity and optimize resolution, recovery, and purity for a specific molecule and impurity profile. Resin selection typically begins with a screening study across the three chemistries to identify an appropriate starting point.
Traditional HIC resins typically require high-molarity kosmotropic salts to drive hydrophobic interactions, raising concerns about molecular stability, hardware corrosion, and waste disposal at the manufacturing scale. POROS HIC resins are designed to operate at lower salt concentrations with weaker lyotropic salts, while offering high dynamic binding capacity, strong resolution, and predictable linear pressure-flow characteristics with a broader range of operating pressures and flow rates for large-scale manufacturing operations compared to alternatives. POROS HIC resins support both bind/elute and flow-through modes with consistent lot-to-lot performance from process development through cGMP manufacturing.
POROS HIC resins are designed for consistent, reproducible performance across scales. Predictable dynamic binding capacity and resolution across a broad range of operating pressures and flow rates support scalable operation without compromising purity or recovery, supporting process intensification objectives. The same resin chemistry is available from screening through manufacturing-scale bulk resin.
Performance data involves various molecules purified by POROS HIC resins in both bind-and-elute and flow-through modes, including: a study demonstrating greater than 99% monomer purity with loading densities higher than 100 g/L at low salt concentrations (5 mM) 1 and high flow rates (800 cm/hr) from an AEX eluate containing 12% aggregates; an additional study showing effective reduction of a difficult-to-remove product-related HMW impurity, while simultaneously contributing significantly to viral clearance, reducing XMuLV by greater than 5 logs (LRV)1.
The three POROS HIC resin chemistries, POROS Ethyl, POROS Benzyl, and POROS Benzyl Ultra, are available in three formats. Bulk resin is available in sizes from 25 mL to 10 L to support process development through manufacturing-scale operations. POROS GoPure pre-packed columns are designed for rapid method development, reducing preparation time. RoboColumns are designed for high-throughput manual and automated screening. These formats use the same resin chemistry to support continuity from screening through production scale.
POROS HIC resin samples are available for evaluation through the Biotherapeutics Sample Request Form. Process development scientists are encouraged to evaluate resin performance within their specific workflow and with their target molecule before purchasing.
Additional bioprocessing resources
Bioprocessing resources
For Research Use Only. Not for use in diagnostic procedures.
References
- Thermo Fisher Scientific. Efficient removal of aggregates from monoclonal antibodies by hydrophobic interaction chromatography in flow-through mode. Thermo Fisher Scientific Application Note. 2018.