Analysis & purification of proteins: HIC, the separation of molecules according to their hydrophobicity

HIC is also known as “Salting out”. The separation is done following the hydrophobicity of the protein. In this method, proteins containing both hydrophilic and hydrophobic regions are applied to an HIC column under high salt buffer conditions Thanks to the salt the hydrophobic regions of the proteins are exposed.
The HIC depends on salt concentration of the mobile phase (eluent), pH, temperature & organic solvents use (if ever some are used).
The hydrophobic ligand on the resin are more often Ether, Butyl or Phenyl groups.

 

A) In contact with hydrophobic groups water is perfectly structured: Hydrophobic group can’t linked together.
B) Salt destroy the structure of the water: hydrophobic group linked together

 

Very useful for analysis & purification of: ADC: Antibody Drug Conjugate: Hydrophobic drug linked on an Antibody DAR: Drug Antibody Ratio Folding & unfolding proteins studies

Advantages Mild conditions, protein friendly Good protein recoveries Biological activity is maintained Sample eluted with low salt concentration Well suited to use before gel filtration, ion exchange and affinity chromatography Disadvantage The sample must be loaded at a high ionic strenght

 

Separation principle

• Proteins get hydrophobic & hydrophilic region.
• In aqueous solution (low salt concentration), these regions are surounded by a thin layer of arranged water molecules.
• At high salt concentration the protein solvatation ratio is decreased (number of water molecules links to salt molecules) so the hydrophobic regions are exposed and they can be adsorbed on an hydrophobic resin.

 

Separation feature

• The model proteins are eluted in order of increasing hydrophobicity, but resolving power of HIC is extremely limited
• The amount of salt required to perform HIC separation is incredibly large (~ 2 M ammonium sulfate)
• The stationary phase should not be too much hydrophobic (C4 bonding with very low ligand density)

 

Factors of influence on Hydrophobic Interaction Chromatography

1. Ligand type & substitution degrees

The binding capacity increase with the alkyl chain length & the substitution degree. A binding capacity plate is reach when the substitution degree is high. This create a multi-point link of the protein & difficulties for its elution. The substitution degree is less than in reverse phase chromatography (about 30 vs. >100 μmol ligand/ml gel)

 

2. Concentration & salts type

 

Proteins are loaded in high concentration salt buffer (under the ppt point). The column is equilibrated with exactly the same buffer. Proteins are eluted with a decreasing salt concentration gradient.

Binding capacity on a phenyl column vs initial salt concentration

Hofmeister serie & effect on the precipitation <——————————————-Precipitation increase (“salting out”) Anions : PO43- > SO42- > CH3COO– > Cl– > Br–, NO3– > ClO4– Cations : NH4+ > Rb+ > K+ > Na+ > Cs+ > Li+ > Mg2+ > Ca2+ > Ba2+ Chaotropic effect increase (“salting in”)————————>   cf: Arch. Biochem. Biophys. 183 (1977) 200–215, Melander, W.,Horvath, C.Separation & Purification Methods 9 (1980) 267–370, Srinivasan, R., Ruckenstein, E.

 

3.pH

pH increase: Hydrophobic interactions decrease. Probably because more negative sites are created. The protein is more hydrophilic.
pH decrease: Hyydrophobic interactions increase. Probably due to a protein conformation changing. The protein is more hydrophobic.

 

4. Temperature

Generally hydrophobic interactions increase when temperature increase due to the entropic effect & Van der Waals strength.
However protein can be denaturated when the temperature increases. Its solubility in solution is modified & lead to its precipitation.

 

 

HIC for ADCs

HIC is particularly well suited for ADCs purifications.

ADCs are complexed molecules composed of a mAb linked via a stable linker with labile bonds, to a biologically active cytotoxic drug. The conjugation can be done on:

• Cystein (e.g. Brentuximab Vedotin)
• Lysine (e.g. Trastuzumab Emtansin)

More than 50 ADCs are currently in clinical trials.

The separation is based on the number of conjugated drugs.

A. Beck et al., Disc. Medicine, 2010, 10, 329-339