title: 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.
abstract: 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.
authors.affiliation: TECNOGEN S.p.A., Località La Fagianeria, 81013 Piana di Monte Verna (CE), Italy.
authors.forename: Biagio
authors.initials: B
authors.lastname: D'Agostino
authors.forename: Piero
authors.initials: P
authors.lastname: Bellofiore
authors.forename: Tiziana
authors.initials: T
authors.lastname: De Martino
authors.forename: Carmine
authors.initials: C
authors.lastname: Punzo
authors.forename: Vincenzo
authors.initials: V
authors.lastname: Rivieccio
authors.forename: Antonio
authors.initials: A
authors.lastname: Verdoliva
mesh_terms: D000911:Antibodies, Monoclonal / Q000235:genetics / Q000302:isolation & purification*; D040761:Biomimetic Materials; D002153:Calorimetry, Indirect; D002846:Chromatography, Affinity / Q000379:methods*; D004247:DNA / Q000032:analysis; D004591:Electrophoresis, Polyacrylamide Gel; D004797:Enzyme-Linked Immunosorbent Assay; D007074:Immunoglobulin G / Q000302:isolation & purification*; D008024:Ligands; D010455:Peptides / Q000737:chemistry*; D013205:Staphylococcal Protein A / Q000737:chemistry*; D013816:Thermodynamics
chemical_list: D000911:Antibodies, Monoclonal; D007074:Immunoglobulin G; D008024:Ligands; D010455:Peptides; D013205:Staphylococcal Protein A; D004247:DNA