In large-scale process it is compulsory with a Cleaning-in-place (CIP) step to a definite removal of contaminants, including non-eluted molecules, bacteria and viruses. This includes in most cases a washing step with NaOH solutions, ranging from 0.1 to 1 M in concentration. Since most affinity purification strategies are based on protein ligands which are sensitive to alkaline conditions, this technique is not very often used in large scale purification. Therefore a general method to stabilize protein ligands would be of great technical and economical interest. Previous studies have shown that the most sensitive residues are the asparagines and glutamines.

To determine which amino acids in a ligand candidate that are sensitive to NaOH exposure a stability analysis including mass-spectrometry and amino acid sequencing was carried out. To stabilize the protein of interest a protein engineering strategy was employed. This procedure consists of substituting all or most of the aspargine residues by PCR technique and/or by help of phage-display. By applying this general method on different ligands we have increased the ligands stability considerably without affecting its selectivity.