Design of ligands for the purification of anti-MUC1 antibodies by peptide epitope affinity chromatography

The fine specificity of epitope recognition of the anti-MUC1 mucin monoclon al antibody, C595 has been studied using solid-phase replacement net (RNET) analysis. Two peptides (RAAP and RPPP) showed increased reactivity with C5 95 antibody compared with the native epitope (RPAP). These were synthesized as integral motifs within MUC1 immunodominant peptides and analyzed by flu orescence quenching (FQ) and circular dichroism (CD). They were also tested as ligands for the purification of C595 antibody using epitope affinity ch romatography. Affinity matrices were compared with respect to capacity, aff inity, and quality of the purified product. In FQ tests the native epitope peptide (APDTRPAPG) and the alanine substitu ted peptide had similar association constants when reacting with C595 antib ody, whereas the proline substituted peptide (APDTRPPPG) had a higher assoc iation constant. This order of affinity for C595 was confirmed in chromatog raphy experiments in which antibody was eluted from the former two peptide matrices at approximately the same point on the NaSCN elution gradient, whe reas antibody was desorbed from APDTRPPPG at a higher NaSCN concentration. Circular dichroism analysis showed that the thermodynamically preferred con formation of these peptides in aqueous solution is the P-II extended helix, the conformation preferred for an extended bound form of the peptide held by interactions with the peptide amides. The stronger binding peptide (APDT RPPPG) has the higher population of the P-II helix in solution. In conclusion, RNET analysis is useful in the rational design of peptide li gands so that the performance of affinity matrices may be regulated.