Optimization of affinity chromatography using HABA ligand for avidin purification
Pamela A Kikot, Ezequiel M Rios, Diego S Vazquez, Mariano Grasselli Abstract
BACKGROUND
Avidin is a commercially available protein used as a specific linker to immobilise molecules. It is currently used in diagnostic kits. The high strength of the avidin‐biotin interaction is essential for assay performance; however, it is a drawback for its purification by affinity techniques.
RESULTS
This work prepared an affinity matrix based on a low‐binding affinity ligand, 4‐hydroxyazobenzene‐2‐carboxylic acid (HABA) bound to agarose. The effect of the space arm on the purity and recovery of avidin from a starting material was studied by preparing HABA‐agarose matrices bound with three different spacers based on ethylene diamines: ethylenediamine, triethylenetetramine, and pentaethylenehexamine. The intermediate length of the spacer reached the best results in agreement with in silico analysis. The optimal adsorption conditions of avidin to the affinity matrix were found using response surface methodology, corresponding to citrate buffer 50 mM pH 5.6 + 425 mM NaCl, allowing the best adsorption performance and simultaneously reducing nonspecific adsorption. The role of pH in the elution buffer was demonstrated by a pH gradient elution, showing AV desorption at pH 10. A final AV purity of 82% was achieved.
CONCLUSION
The HABA‐based affinity matrix attached to agarose via a triethylenetetramine space arm proved to be the most suitable material for the recovery of avidin from an egg‐based starting material solution. The optimal affinity chromatography conditions described in this report are potentially applicable in an integrated purification process for obtaining avidin from egg white, considering its industrial production. © 2025 Society of Chemical Industry (SCI).