Understanding ligand-protein interactions in affinity membrane chromatography for antibody purification.
Affinity chromatography with Protein A beads has become the conventional unit operation for the primary capture of monoclonal antibodies. However, Protein A activated supports are expensive and ligand leakage is an issue to be considered. In addition, the limited production capabilities of the chromatographic process drive the research towards feasible alternatives. The use of synthetic ligands as Protein A substitutes has been considered in this work. Synthetic ligands, that mimic the interaction between Protein A and the constant fragment (Fc) of immunoglobulins, have been immobilized on cellulosic membrane supports. The resulting affinity membranes have been experimentally characterized with pure immunoglobulin G (IgG). The effects of the membrane support and of the spacer arm on the ligand-ligate interaction have been studied in detail. Experimental data have been compared with molecular dynamic simulations with the aim of better understanding the interaction mechanisms. Molecular dynamic simulations were performed in explicit water, modelling the membrane as a matrix of overlapped glucopyranose units. Electrostatic charges of the ligand and spacer were calculated through ab initio methods to complete the force field used to model the membrane. The simulations enabled to elucidate how the interactions of surface, spacer and ligand with IgG, contribute to the formation of the bond between protein and affinity membrane.
Boi
Cristiana
C
Dipartimento di Ingegneria Chimica, Mineraria e delle Tecnologie Ambientali, Università di Bologna, via Terracini 28, 40131 Bologna, Italy. cristiana.boi@unibo.it
Busini
Valentina
V
Salvalaglio
Matteo
M
Cavallotti
Carlo
C
Sarti
Giulio C
GC
D000327:Adsorption; D000627:Aminophenols / Q000737:chemistry; D001665:Binding Sites; D002482:Cellulose / Q000737:chemistry; D002846:Chromatography, Affinity / Q000379:methods*; D007074:Immunoglobulin G / Q000737:chemistry / Q000302:isolation & purification*; D007700:Kinetics; D008024:Ligands; D008567:Membranes, Artificial; D008968:Molecular Conformation; D056004:Molecular Dynamics Simulation; D017434:Protein Structure, Tertiary; D012996:Solutions; D013205:Staphylococcal Protein A / Q000378:metabolism*; D055672:Static Electricity; D013816:Thermodynamics; D013997:Time Factors
D000627:Aminophenols; D007074:Immunoglobulin G; D008024:Ligands; D008567:Membranes, Artificial; D012996:Solutions; D013205:Staphylococcal Protein A; D002482:Cellulose