Figure 1. Cleaning in place. A comparison of the capacity of a an
affinity chromatography column with ABD, wild type (ABD) and a
stabilized variant (ABD*) as ligand and HSA as target protein.
The cleaning in place agent used was 0.5M NaOH.

Most protein-based affinity chromatography media show significant fragility towards alkaline conditions. This is a major problem in many industrial applications where it is of great importance to be able to remove contaminants from the chromatography media, usually accomplished by integration of a cleaning-in-place (CIP) protocol. In such a protocol, sodium hydroxide (NaOH) in concentrations ranging from 0.1 to 1 M is the most commonly used agent, resulting in an extremely harsh environment with high pH. The sensitivity of proteins towards such conditions is a challenge when using them as binding ligands, and therefore significant efforts have been put into the development of different synthetic ligands, such as various organic mimics. This approach often results in binders with increased alkaline resistance. However, an encountered drawback is often the significantly decreased affinity of these ligands compared to ligands consisting of protein domains. When considering proteins as affinity ligands, asparagine residues have been recognized as the major contributors to the alkaline sensitivity. The modifications that appear in alkaline conditions may change the function or the potency of a protein or a peptide. We have developed a simple and straightforward strategy consisting in replacing asparagine residues with other amino acids. This strategy dramatically improves the chemical stability of a protein towards alkaline conditions. Such improvements of affinity ligands will be crucial to obtain industrial implementation of protein-based affinity chromatography.