Affinity purification of IgG monoclonal antibodies using the D-PAM synthetic ligand: chromatographic comparison with protein A and thermodynamic investigation of the D-PAM/IgG interaction.

This study investigates the applicability of D-PAM, the inverso form of the Protein A Mimetic synthetic peptide affinity ligand (PAM) obtained from the screening of a multimeric combinatorial peptide library, in monoclonal IgG isolation from ascitic fluids and cellular supernatants. D-PAM affinity columns, prepared by immobilizing the all-D peptide on the commercially available support Emphaze, were able to capture monoclonal antibodies in a single chromatographic step, with a recovery yield and purity degree above 90% and full recovery of antibody activity. D-PAM/Emphaze resin showed a host cell protein (HCP) and DNA reduction similar to protein A sorbent. Indeed, column capacity, determined by applying a large excess of purified antibodies to 1 mL of column bed volume, was always higher than 50 mg/mL. D-PAM/IgG interaction was characterized by isothermal titration calorimetry (ITC) and an analysis of binding isotherms, obtained for titration of ST2146, ST1485 and 7H3 IgG monoclonal antibodies, suggested that two peptides bind simultaneously to the IgG molecule, with a K(A) (equilibrium association constant) of 3.4, 6.2 and 3.4 x 10(4) M(-1), and a DeltaH (change in enthalpy) of -1.3, -4.2 and -4.1 kcal mol(-1), respectively.

Identification and refinement of a peptide affinity ligand with unique specificity for a monoclonal anti-tenascin-C antibody by screening of a phage display library.

Using phage display technology, a 22-mer peptide was selected as a ligand with unique specificity for the murine monoclonal ST2146 antibody that recognizes the EGF repeats region of the human tumor-associated antigen tenascin-C. This peptide, synthesized in an 8-branched form to enhance its binding properties, is useful in replacing the native antigen in the affinity and immunoreactivity characterization of the ST2146 antibody and its biotinylated derivatives. Affinity resins, prepared by immobilizing the mimotope or its shorter 10-mer binding unit on a chromatographic support, were able to capture ST2146 directly from the hybridoma supernatant, with antibody recovery and host cell protein (HCP) reduction similar to or better than protein A sorbent, a purity degree exceeding 95%, and full recovery of antibody activity. The affinity constants of both peptides, as determined by frontal analysis of broad-zone elution affinity chromatography and BiaCore measurements, were very similar and included in a range suitable for affinity ligands. Column capacity, determined by applying a large excess of purified ST2146 to 1 mL of column bed volume, was close to 50 mg/mL for both resins. These matrices retain their ST2146 binding properties after various treatments, including sanitization, thus indicating very high stability in terms of ligand leakage and degradation. Moreover, the short form shows higher enzymatic stability, thus proving more suitable as ligand for ST2146 affinity purification.