Development of enhanced capacity affinity microcolumns by using a hybrid of protein cross-linking/modification and immobilization

• A hybrid modification and immobilization method was developed for proteins.
• This method was used to make enhanced capacity affinity supports and microcolumns.
• This approach was optimized by using human serum albumin as a model protein.
• The final supports were used in drug-binding studies and ultrafast affinity extraction.

A hybrid method was examined for increasing the binding capacity and activity of protein-based affinity columns by using a combination of protein cross-linking/modification and covalent immobilization. Various applications of this approach in the study of drug–protein interactions and in use with affinity microcolumns were considered. Human serum albumin (HSA) was utilized as a model protein for this work. Bismaleimidohexane (BMH, a homobifunctional maleimide) was used to modify and/or cross-link HSA through the single free sulfhydryl group that is present on this protein. Up to a 75–113% increase in protein content was obtained when comparing affinity supports that were prepared with BMH versus reference supports that were made by using only covalent immobilization. Several drugs that are known to bind HSA (e.g., warfarin, verapamil and carbamazepine) were further found to have a significant increase in retention on HSA microcolumns that were treated with BMH (i.e., a 70–100% increase in protein-based retention). These BMH-treated HSA microcolumns were used in chiral separations and in ultrafast affinity extraction to measure free drug fractions in drug/protein mixtures, with the latter method giving association equilibrium constants that had good agreement with literature values. In addition, it was found that the reversible binding of HSA with ethacrynic acid, an agent that can combine irreversibly with the free sulfhydryl group on this protein, could be examined by using the BMH-treated HSA microcolumns. The same hybrid immobilization method could be extended to other proteins or alternative applications that may require protein-based affinity columns with enhanced binding capacities and activities.