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Hydrophobic Interaction Chromatography

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Improve purity with hydrophobic interaction chromatography

Reducing residual aggregates, product variants, and process-related impurities remains a challenge after capture and intermediate purification. Even following Protein A (ProA) and anion exchange (AEX) chromatography, aggregate levels may remain significant, requiring an orthogonal polishing step to achieve target product quality. In platform monoclonal antibody (mAb) processes, hydrophobic interaction chromatography (HIC) can replace legacy cation exchange (CEX) or even mixed-mode (MCC) polishing steps downstream of AEX. HIC can be run in bind/elute or flow-through modes, giving process development scientists flexibility to tune selectivity and improve productivity within a downstream purification workflow.

Benefits of hydrophobic interaction chromatography

Hydrophobic interaction chromatography complements other steps by offering a separation mechanism orthogonal to ionic or affinity-based interactions. This orthogonality makes HIC particularly effective where charge-based methods reach their limitations, supporting impurity reduction, aggregate clearance, viral clearance contribution, and process efficiency across biologic modalities, including mAbs, antibody-drug conjugates (ADCs), and vaccines.
 

HIC can help with:

  • Separation of closely related species, such as aggregates, product variants, based on innate hydrophobicity differences
  • Recovery across different biomolecules leveraging tunable selectivity
  • Flexible operation in bind/elute or flow-through modes
  • Efficiency gains in buffer consumption and process productivity
  • Potential contribution of viral clearance of endogenous and adventitious viral models

How hydrophobic interaction chromatography works

Hydrophobic interaction chromatography is driven by reversible hydrophobic interactions between proteins and the stationary phase. Understanding the mechanism, operating modes, and process variables helps scientists design effective purification steps.

 

The mechanism

Traditionally, under high-salt loading conditions, kosmotropic salts reduce the solvation of hydrophobic protein surfaces, promoting interaction with hydrophobic ligands on the resin. As salt concentration is decreased during elution, the solvation layer is restored, and proteins desorb in order of increasing hydrophobicity with the least hydrophobic species eluting first. This reversible interaction preserves protein structure and supports the separation of closely related species, such as aggregates and variants.


Operating modes

HIC can be operated in two primary modes depending on process goals. In bind/elute mode, the target molecule is captured under elevated salt conditions during loading, and selectively eluted using a decreasing salt gradient, enabling targeted reduction of co-eluting impurities. In flow-through mode, the target molecule passes through the column while hydrophobic impurities, such as aggregates, are retained on the resin. Flow-through operation supports higher load densities, shorter residence times, and, with modern HIC resins, can be operated at low salt conditions. It can be performed immediately following an AEX step without buffer adjustment, contributing to process intensification. Studies have demonstrated that POROS Benzyl Ultra resin in flow-through mode can achieve greater than 99% monomer purity at load densities up to 125 g/L resin at low salt concentrations (5 mM sodium citrate) and high flow rates (800 cm/hr)1.

 

Process variables

Salt type and concentration drive HIC selectivity and performance. Kosmotropic salts, such as ammonium sulfate, sodium citrate, and sodium acetate, promote hydrophobic interactions to varying degrees. Buffer pH and temperature also affect selectivity and can be adjusted to fine-tune separation. In high-throughput screening studies, sodium citrate at pH 6.8 and conductivity of approximately 2 mS/cm demonstrated high aggregate clearance on POROS Benzyl Ultra resin while enabling direct loading from an AEX flow-through step1. These variables reflect core hydrophobic interaction chromatography principles: increasing salt concentration promotes hydrophobic interactions, while decreasing salt concentration weakens these interactions and enables controlled elution.

Achieve reliable purification with POROS HIC resins


Traditional HIC resins have historically required high-molarity kosmotropic salts to drive hydrophobic interactions, which can challenge product stability and limit operating flexibility. POROS HIC resins operate at lower salt concentrations and with weaker kosmotropic salts, reducing concerns about molecular stability while maintaining high selectivity for aggregate and impurity reduction. Built on an average 50 µm cross-linked polystyrene-divinylbenzene bead, POROS HIC resins enhance flow-rate-independent performance and high dynamic binding capacity (DBC). The portfolio includes POROS Ethyl, POROS Benzyl, and POROS Benzyl Ultra resins.

Supporting diverse modalities and applications

mAbs

Monoclonal antibody purification typically starts with Protein A chromatography to capture the antibody from cell culture fluid. Additional steps, such as ion exchange, are used to help reduce aggregates, variants, or process-related impurities.

Gene Therapy

Gene therapy often relies on viral vectors, such as adeno-associated virus (AAV) or lentivirus, to facilitate the transfer of genetic material. Chromatography separates drug product from product- and process-related impurities in both capture and polishing steps.

mRNA

Chromatography in mRNA production separates full-length transcripts from double-stranded RNA and truncated products. The purified RNA can then be used in formulation or downstream steps, such as lipid nanoparticle assembly.

ADCs

Antibody-Drug Conjugates (ADCs) workflows rely on chromatography steps to separate complex conjugates and reduce impurities. These approaches support consistent production of ADCs for therapeutic research and biomanufacturing.

Vaccines

Chromatography is often used in vaccine manufacturing for both the capture and polishing of the vaccine drug product. The choice of method depends on the platform, for example, protein subunits, viral vectors, or nucleic acid vaccines.

Cell Therapy

Cell therapies utilize living cells as treatments, necessitating strict control over inputs and processing steps. Chromatography is applied to prepare ancillary materials, such as cytokines or viral vectors, that are used during cell manufacturing.

Explore other chromatography techniques

Affinity chromatography

Affinity chromatography is a highly selective capture technique based on specific biological interactions between the target molecule and a ligand. Widely used as the initial purification step for mAbs and other biologics, it can achieve high purity and yield in a single step.

Ion exchange chromatography

Ion exchange chromatography separates biomolecules based on charge interactions with functional groups on the resin matrix. Available in both strong and weak acid or base chemistries, it is often applied as a polishing step following affinity capture.

Mixed-mode chromatography

Mixed-mode chromatography combines ionic and hydrophobic interactions within one chemistry to achieve greater selectivity for difficult feedstreams. It is designed for polishing and can reduce aggregates, host cell proteins (HCPs), and impurities while supporting high product recovery.

Explore more chromatography resins

Thermo Fisher Scientific offers POROS ion-exchange, hydrophobic interaction, and mixed-mode chromatography resins for large-scale bioseparations, along with MabCaptureC and CaptureSelect affinity resins designed for efficient purification of mAbs, advanced antibody variants, recombinant proteins, mRNA, viruses, and other biologics.
 

Find the right chromatographic method for your process

Selecting the optimal chromatography approach is important for achieving consistent yield, purity, and scalability in downstream purification. Whether optimizing a polishing step or implementing continuous operation, the right combination of resins and formats can support process intensification, reducing cycle times, improving throughput, and minimizing buffer use. Thermo Fisher Scientific offers chromatography solutions that align with your productivity goals and accelerate progress from development through cGMP manufacturing.

Explore supporting chromatography resins

Affinity chromatography resins

Affinity resins can achieve high purity and yield in a single capture step. CaptureSelect technology supports purification of antibodies, bispecific antibodies, fragments, Fc-fusion proteins, recombinant proteins, and viral vectors using mild elution conditions.
 

Ion exchange chromatography resins

Ion exchange chromatography separates biomolecules through charge interactions with functional groups. These resins are available in both strong and weak base or acid chemistries, suitable for bind/elute or flow-through purification of antibodies, vaccines, viral vectors, and other biologics.

Mixed-mode chromatography resin

Mixed-mode chromatography uses ion exchange and hydrophobic interactions within a single chemistry to improve selectivity in complex samples. POROS mixed-mode cation-exchange resin is designed for polishing and can reduce aggregates, HCPs, and other impurities.

Frequently asked questions

Hydrophobic interaction chromatography separates biomolecules based on differences in surface hydrophobicity. Traditionally, under high-salt loading conditions, hydrophobic regions of proteins interact with hydrophobic ligands on the resin. As salt concentration decreases, the strength of these interactions is disrupted, allowing molecules to elute in order of increasing hydrophobicity, with the least hydrophobic species eluting first. This controlled, reversible separation supports high-resolution purification while maintaining protein stability throughout the process.

HIC is commonly applied as a polishing step following initial capture and intermediate purification, for example, after ProA and AEX steps. It is particularly valuable for reducing aggregates, product variants, and other impurities that remain after earlier steps, including challenging species that charge-based methods may not address. HIC can be run in bind/elute mode to target specific impurities or in flow-through mode for higher load capacity and process productivity. Flow-through HIC can be coupled to an AEX step without buffer adjustment, supporting process intensification in mAb workflows.

Traditional HIC resins have historically required high-molarity kosmotropic salts that can challenge product stability for certain molecules. POROS HIC resins are designed to operate effectively at lower salt concentrations and with weaker kosmotropic salts, reducing concerns about molecular stability during the polishing step. POROS Benzyl Ultra resin in particular is designed for operation under low-salt, low-conductivity conditions, enabling direct loading from an AEX flow-through step at approximately 2 mS/cm. Flow-through mode operation at low conductivity conditions can further support process intensification.

HIC separates monomers from aggregates by exploiting subtle differences in surface hydrophobicity. Aggregated proteins typically expose more hydrophobic surface area than monomers, leading to stronger binding under controlled salt conditions while monomers elute earlier in the separation. Studies using POROS Benzyl Ultra resin in flow-through mode demonstrated that, from an AEX eluate feedstock with 12% aggregate content, greater than 99% monomer purity was achieved up to a load density of 125 g/L resin at a low salt concentration of 5 mM sodium citrate run at high flow rates of 800 cm/hr.1 These characteristics support reproducible impurity reduction and consistent process outcomes across development and manufacturing scales.

Hydrophobic residues exist on viruses, particularly enveloped viruses, making HIC a viable contributing clearance step in a downstream purification process. Studies using POROS HIC resins have demonstrated XMuLV, an enveloped virus with higher surface hydrophobicity, showed clearance greater than 5 log reduction value (LRV) in both bind/elute and flow-through modes2. MVM, a non-enveloped parvovirus with lower hydrophobicity, showed minimal binding under the same conditions. In conjunction with process optimization, including salt type, concentration, and operating mode, HIC can be optimized to achieve viral clearance of both endogenous and adventitious viruses.

Chromatography resources


Access resources that cover chromatography performance, purification strategies, and method development. These resources empower scientists to select and apply chromatography resins from research to cGMP manufacturing.

Optimize your hydrophobic interaction chromatography process

For research and development use only in support of FDA-regulated end uses. Not for diagnostic use or direct administration to humans or animals.