A discussion of hydrophobic interaction chromatography (HIC) theory and its application to the analysis of proteins and biomolecules is presented.
A discussion of hydrophobic interaction chromatography (HIC) theory and its application to the analysis of proteins and biomolecules is presented.
Hydrophobic interaction chromatography (HIC) is a very versatile technique capable of separating protein molecules according to differences in hydrophobicity (in a similar way to reversed-phase separations). But, unlike reversedâphase separations, HIC utilizes nondenaturing mobile phases and is often employed for protein purification, within a capture, intermediate purification, polishing regime. Furthermore, because of its ability to discriminate between analytes with only minor differences in hydrophobicity, HIC has advantages in separating variants with subtle changes that can expose more, or less, of a hydrophobic surface. These variants are difficult to resolve using other chromatographic approaches, including oxidized species (such as methionine), deamidation (asparagineâaspartic acid), and isoaspartate formation; as well as the separation of antibody–drug conjugate (ADC) variants, and for drugâtoâantibody ratio (DAR) analysis.
HIC uses a somewhat hydrophobic stationary phase with typically shorter ligands than in reversed-phase chromatography (C4 ligands are popular), and typically the ligand binding density is lower (bonded phases ligands are spaced further apart on the stationary phase surface). A salt solution is used as the strong eluent to promote adsorption between the protein hydrophobic regions and the bonded phase. The salt concentration is gradually reduced throughout the run (typically to 0%) to allow the protein to elute, and elution order in HIC is usually least hydrophobic to the most hydrophobic. Crucially, unlike reversedâphase HPLC, the eluent does not contain a denaturing organic solvent, and the native protein structure will be retained.
With a sufficiently high eluent salt concentration, the hydrophobic regions of the protein will be encouraged to interact with the hydrophobic stationary phase, and will concentrate to ensure sharp chromatographic peaks. As the salt concentration decreases during the gradient elution, adsorption to the stationary phase becomes less favourable, and the proteins will elute in hydrophobicity order (least hydrophobic first). The second law of thermodynamics also plays a part in protein retention in HIC, and the highly ordered water cages around the protein and the stationary phase ligands makes interaction, and therefore retention, much less favourable. However, at higher salt concentrations, these water cages are disrupted (less ordered), and therefore retention is more favourable.
Ammonium sulfate is typically used as the salt in HIC, as it is very effective at “salting out” the protein (Hofmeister series for protein retention is LiCl < NaCl < Na2HPO3 < (NH4)2SO4 < Na2SO4), which promotes the protein stationary phase interaction without causing protein denaturation (2 M is a typical starting concentration), while being highly soluble in aqueous solutions. Care should be taken to avoid elevated salt concentrations, which may lead to precipitation due to protein agglomeration.
Proteins possess complex threeâdimensional structures that result in areas rich in amino acids possessing hydrophobic side chains, or areas rich in ionic side chains. Consequently, proteins may well be poorly soluble in water alone, with hydrophobic interactions as well as ionic interactions able to occur. Small amounts of buffer help to disrupt the weak interactions that can cause insolubility. This process is often referred to as “salting in” and improves the protein solubility. It is also important to maintain a constant pH and so the use of a buffer is also typical in HIC chromatography, with 50 mM sodium phosphate at pH 7 added to both eluent A and eluent B a typical example.
The pH of the eluent will influence the degree of ionization of ionogenic amino acid residues, and therefore retention will alter as the protein hydrophobicity changes. Eluent pH values below 5 or above 8.5 tend to dramatically alter retention and therefore the eluent pH is typically maintained between these values and should be optimized on a case-by-case basis.
Due to the thermodynamic dependency of the retention mechanism in HIC, variations in temperature will also alter retention behaviour. The relationship between retention and temperature is complex, and therefore should be optimized on a caseâbyâcase basis, using a controlled room temperature (20–25 oC) as a reasonable starting point. It is important that temperature is carefully controlled in order to ensure run-to-run or batchâtoâbatch retention time stability.
Water-miscible additives, such as detergents, can reduce the binding of proteins even at low concentrations, as they compete with the protein for binding sites on the stationary phase surface. Therefore, addition of such species can substantially improve the elution efficiency of the protein, sharpening peaks and often improving resolution.
Lately, HIC has been extensively used for DAR determination in ADCs. Typically, the protein, usually a monoclonal antibody (mAb), is fully reduced prior to analysis of light and heavy chains to determine the number, and position, of conjugated small molecules, which can affect the potency of the biotherapeutic.
AI and GenAI Applications to Help Optimize Purification and Yield of Antibodies From Plasma
October 31st 2024Deriving antibodies from plasma products involves several steps, typically starting from the collection of plasma and ending with the purification of the desired antibodies. These are: plasma collection; plasma pooling; fractionation; antibody purification; concentration and formulation; quality control; and packaging and storage. This process results in a purified antibody product that can be used for therapeutic purposes, diagnostic tests, or research. Each step is critical to ensure the safety, efficacy, and quality of the final product. Applications of AI/GenAI in many of these steps can significantly help in the optimization of purification and yield of the desired antibodies. Some specific use-cases are: selecting and optimizing plasma units for optimized plasma pooling; GenAI solution for enterprise search on internal knowledge portal; analysing and optimizing production batch profitability, inventory, yields; monitoring production batch key performance indicators for outlier identification; monitoring production equipment to predict maintenance events; and reducing quality control laboratory testing turnaround time.