Biomolecules such as proteins, peptides, and oligonucleotides are the physiological building blocks at the heart of the biopharmaceutical revolution. Fine control over these complex structures is driving the development of our latest therapeutic applications. The differences in their physical, chemical, and functional properties serve as the basis for their separation. Various liquid chromatography techniques, including HIC, are crucial in separating and purifying these fundamental structures. As therapeutic applications for these classes of biomolecules are being developed, chromatographic techniques are at the forefront of this discovery. Size exclusion, affinity, and ion exchange chromatography are employed to separate biomolecules according to their size (molecular weight), shape (selectivity to ligands), and overall charge (positive or negative).
Reversed-phase (RPC) and hydrophobic interaction chromatography (HIC) rely on hydrophobic interactions between the analyte and stationary phase media to control separation. This discussion will focus on hydrophobic interaction chromatography (HIC) as it applies to separating biomolecules, sketch out some differences between HIC and RPC, and highlight some areas of research where HIC innovatively solves issues related to other chromatographic modes.
Mechanisms of Action: Hydrophobic Interaction Chromatography vs Reversed-Phase Chromatography
HIC relies on weak interactions between the target analytes' hydrophobic (non-polar) regions and hydrophobic groups or surfaces of the stationary phase media. This technique employs water-based mobile phases at pH 6 to 8, operated with a salt gradient.
In HIC separations (Figure 1), biomolecules generally stick to columns in water solutions with lots of salt. The salt, a key factor in this process, boosts the number of ions in the solution and creates an environment less capable of keeping analytes in a dissolved state. In these conditions, biomolecules favorably bind to the media. The hydrophobic character of the molecule determines the strength of this binding. More hydrophobic biomolecules bind stronger than less hydrophobic biomolecules.
As the gradient reduces the salt concentration in the mobile phase, conditions change, and the biomolecules lose their grip on the column and start eluting off in a signature order.
Using proteins to exemplify HIC retention and elution, we explain the interplay between the surface of proteins, the surface of the stationary phase media (packed in a column), and the salt concentration of the mobile phase (Figure 2).
Proteins with the weakest grip on the media (least hydrophobic) come off first. Those with the most robust grip on the media (most hydrophobic) come off last. Method development can be used to define signature elution times for particular proteins under set mobile phase conditions.
HIC is performed in aqueous solutions containing salt and buffer. This “mild” solution keeps proteins in their native state. The mobile phase does not unfold or denature proteins, which is a crucial attribute in HIC, although some denaturing may occur at the binding sites.
In contrast, RPC separations control analyte retention using organic solvents instead of salt. The organic solvent defines how “favorable” it is for a particular analyte to travel with the mobile phase versus staying on the column. The stationary phase media of the column is essentially set in its surface character. Extremely hydrophobic materials are used in the column for RPC separations. The amount of organic solvent added makes the mobile phase more or less attractive to the analytes than the column. This competition between the mobile and stationary phases defines the separation of the various analytes in the mix. Like HIC, the least hydrophobic analytes have the weakest hold on the column, so they come off first. The most hydrophobic analytes have the strongest hold on the column and come off last. However, unlike HIC, the organic solvents in RPC can unfavorably interact with biomolecules. The presence of organic solvents can initiate conformational changes depending on the nature of the protein, peptide, or oligonucleotide. Hydrophobic regions of the biomolecule’s structure will twist “outward” to interact with non-polar organic solvents. This may or may not lead to irreversible structural changes.
Media Used in HIC
HIC separations usually use columns with silica or polymeric supports modified with functional groups such as short-chain alkyl or phenyl groups. These bonded attachments are less hydrophobic (containing 2 – 6 carbon atoms) than groups used in RPC, which contain 4 – 18 carbon atoms. As stated earlier, the goals in HIC usually involve maintaining the shape of the biologic product, so a gentler touch is applied. One must consider that the overall hydrophobicity of the chosen column may impact the rate of protein unfolding in the adsorbed state. The strength of hydrophobic interactions between the analyte and stationary phase functional groups (alkyl, phenyl, etc.) can be controlled by the functional group's density or the nature of the support material itself.
With silica-based support, a wide variety of functional groups are available to optimize selectivity in HIC applications. Additionally, silica offers superior mechanical stability for operations in the UPLC or UHPLC pressure ranges (> 600 bar). Polymeric-based supports are available in similar functional group options, are typically composed of naturally hydrophobic surfaces, and offer superior stability over pH extremes.
Salts Used in HIC
Biomolecule–column interactions are governed by the type and concentration of salt used in the mobile phase. The amount of protein, for example, bound to a column increases with salt concentration. In HIC separations, the mobile phase contains a “salting-out” agent, which increases the hydrophobic interaction between the protein and stationary phase at high concentrations. The ability of salt to promote these hydrophobic interactions depends on the ionic molecules present and their concentration. Mobile phases are generally run at 1M – 3M salt content to optimize selectivity. Increasing salt concentration can promote longer retention times if the target analytes eluting too early and resolution is compromised.
The type of salt also plays a role in its ability to influence the hydrophobic interactions. It has been proposed that salts that promote higher surface tension induce a higher dynamic loading capacity for HIC columns. Salts that cause a more significant change to the structure of the water molecules themselves play a more greater role in increasing hydrophobic interactions. The Hofmeister series describes the order of these effects. Method development usually involves testing different salts or mixtures to improve the HIC separation. Commonly used salts with relative surface tensions include KCL < NaCL < Na2HPO4 < (NH4)2SO4 < Na3PO4 [2,3].
Applications of HIC
Hydrophobic interaction chromatography offers the possibility of working under non-denaturing conditions, and it is a widely used chromatographic technique that finds application at different stages of both upstream and downstream processing. HIC has been successfully employed in polishing steps to obtain ultra-pure recombinant antibodies, resolving proteins at different oxidized forms, discriminating between antibody drug conjugates at differing drug-to-antibody ratios, purifying peptides (e.g., GLP-1 analogues), and isolating oligonucleotides among many others [3,4-5].
HIC is particularly useful for separating analytes with similar charge or size, as it provides an additional dimension of separation based on hydrophobic characteristics. Ion-exchange chromatography (IEC) and Ion-pair reversed-phase chromatography (IP-RPC) have been the standard methods for performing analytical separations of oligonucleotides. However, these charge-selection techniques quickly confront limitations as the length of oligonucleotides increases. Resolution between closely related oligonucleotide species suffers with IEC and IP-RPC as the differences in charge drop off quickly with the additional length of each nucleotide. However, the unique hydrophobic regions associated with nucleotide residuals can be exploited to discriminate between analytes using HIC [6].
The HIC mode is applied over all stages of purification processes, performing both analytical and preparative separations. Most of its applications are seen in intermediate purification and polishing rather than capture, where handling crude feedstocks would be complex and expensive. Because of the high salt concentrations used, HIC is generally incompatible with mass spectrometry (MS) detection. However, studies implementing MS-compatible salts such as ammonium tartrate have found success in HIC–MS analysis of intact proteins [7].
At Millennial Scientific, we develop next-generation products for separation and purification of biomolecules. Get in touch with us to discuss how we can support your HIC and reverse phase chromatography separation needs. For more information or to request samples, please email us at inquiry@millennialscientific.com, call us at 855 388 2800, or fill in our online form.
Author
Michael Jack Parente
References
[1] Fekete, S., Murisier, A., Verscheure, L., Sandra, K. & Guillarme, D. Hydrophobic Interaction Chromatography (HIC) for the Characterization of Therapeutic Monoclonal Antibodies and Related Products, Part 2: Practical Considerations. (2021).
[2] Kazakevich, Y. V. & Lobrutto, R. HPLC for pharmaceutical scientists. (John Wiley & Sons, 2007).
[3] Ewonde Ewonde, R. et al. Selectivity and Resolving Power of Hydrophobic Interaction Chromatography Targeting the Separation of Monoclonal Antibody Variants. Analytical Chemistry 96, 1121-1128 (2024).
[4] Zhou, J. et al. Purification and bioactivity of exendin-4, a peptide analogue of GLP-1, expressed in Pichia pastoris. Biotechnol Lett 30, 651-656 (2008).
[5] Savard, J. M. & Schneider, J. W. Sequence‐specific purification of DNA oligomers in hydrophobic interaction chromatography using peptide nucleic acid amphiphiles: Extended dynamic range. Biotechnology and bioengineering 97, 367-376 (2007).
[6] Gilar, M., Koshel, B. M. & Birdsall, R. E. Ion-pair reversed-phase and hydrophilic interaction chromatography methods for analysis of phosphorothioate oligonucleotides. Journal of Chromatography A 1712, 464475 (2023).
[7] Watson, D. Hydrophobic interaction chromatography for biopharmaceutical analysis. LCGC North America 35, 278-278 (2017).
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