Hydrophobic interaction chromatography (HIC) is frequently used for the purificaiton and analytical characterization of proteins. This chromatographic mode relies on interactions between hydrophobic patches on the protein’s surface with immobilized hydrophobic ligands. The specific interaction is influenced by the addition of highly concentrated electrolytes and additives. In contrast to other chromatographic modes, the salt type and potential additves can be varied in a wide range. This offers additional modulation opportunities for the separation of proteins. (Muller, J. Sep Sci, 36: 1327-1334 (2013).

Factors which affect Dynamic Binding Capacity:

The term “dynamic capacity” of a HIC column refers to the maximum amount of protein in solution which can be loaded without significant breaktrhough or leakage of the protein into the solution phase of the column before elution. 

The dynamic binding capacity (DBC) of HIC resins is affected by many factors such as mobile phase composition and resin characteristics. Both binding affinity and DBC are higher with stronger salting-out salts and increase with salt concentration (Senezuk, Biotechnology and Bioengineering, 103(5), 2009)

For antibodies: Traditional HIC media typically exhibit dynamic IgG binding capacities ranging from about 15 to 40 g/L at 10% breakthrough, with most monoclonals exhibiting capacities of 20-30 g/L typically at 5% break-through. Improved HIC resins such as Toyopearl® has been shown to have DBC of about 40 g/L at 10% break-through for mAbs and 58 g/L for lysocyme. Monoliths are denoted as the fourth generation chromatography material. (Lu, Current Pharmaceutical Biotechnology, 2009, 10(4)). 

Effect of pore size:  HIC resins with optimized pore size have significantly imporved binding capacity which can increase HIC purificaiton unit operation efficiency (Chn, J Chromatogr A 2008, 1177(2), 272-81, 2007).

Effect of temperature: Temperature is known to affect HIC binding capacity and increased temperature generally leads to higher binding capacity due to the entropy driven characteristics of the hydrophobic interactions. However, the response (ie., change in retentin time, of any given protein to temperature is highly individual (Lu, Current Pharmaceutical Biotechnology, 2009, 10(4)).

Use of Dual Salts: during HIC have been reported to increase dynamic binding capacity to up to 3 times over that of single salt. (Lu, Current Pharmaceutical Biotechnology, 2009, 10(4)). For example, Senzuk (Biotechnology and Bioengineering, 103(5), 2009; see also US 2010/01311953) report that combining two salting out salts had a remarkable benefit on protein solubility and binindg of a given protein to HIC resin. 

 Operating Conditions:

HIC seperates molecules based on hydrophobicity of molecules. Hydrophobic regions in the molecuel of interest bind to the HIC resin through hydrophobic interaction. Strenght of the itneraction depends on operating conditions such as pH, ionic strengh, and salt. (Welsh, US 20230077205)

Binding Conditions:

Salts:

Some additive like alcohols and polymers greatly impact the protein soluility as well as adsorption to HIC media. They can either increase or decrease the binding apacity and their use may lead to an increase in recvoery. Ammonium sulfate is freequenly employed for protein preciptiation and HIC because it is readily soluble at high concentraitons. Further, it precipitates proteins at high pH and it is availabe at relatively hihg purity and in large quantites. Several other salts like sodium citrate, sodium phosphate, or sodium chloride provide an alternative. the most prominanet example for salt classificaiton is the Hofmeister series. Sales are classified as “kosmotripc” or chaotropic”. Kosmotropic salts are usually emmployed for precipitation of proteins. In constrast, chaotropic salts support protein solubilziation, In HIC, mianly kosmotropic salts are used because they convey the interaction between protein and resin thereby increasing the binding capacity. The Hofmister series may vary from different prtoeins. Further, the relative salting out effectiveness also depends on the isoelectric point of a certain protein and the experimentally applied pH value. (Muller, J. Sep Sci, 36: 1327-1334 (2013).

Compared to the single salt solution, simialr or higher DBCs can be achieved for salt mixtures of chatropic and kosmotropic. For lysozyme and mAb appropriate concetnraitons of the salt with the low surface tension to be added is around 1M whereas the concetnraiton of salt with the high surface tension depends on the solubility of the protein. In addition to sodium sulfate-sodium acetate, the salt combiantions of sodium citrate-sodium chloride, ammonium sulfate-sodium chhloride and ammonium sulfate-glycine elasd to the most promising resutls regarding increase in resin capacity. (Muller, J. Sep Sci, 36: 1327-1334 (2013).