Cation-exchange chromatography of monoclonal antibodies: characterisation of a novel stationary phase designed for production-scale purification.
A novel cation-exchange resin, Eshmuno™ S, was compared to Fractogel® SO3(-) (M) and Toyopearl GigaCap S-650M. The stationary phases have different base matrices, and carry specific types of polymeric surface modifications. Three monoclonal antibodies (mAbs) were used as model proteins to characterize these chromatographic resins. Results from gradient elutions, stirred batch adsorptions and confocal laser scanning microscopic investigations were used to elucidate binding behaviour of mAbs onto Eshmuno™ S and Fractogel® SO3(-) and the corresponding transport mechanisms on these two resins. The number of charges involved in mAb binding for Eshmuno™ S is lower than for Fractogel® SO3(-), indicating a slightly weaker electrostatic interaction. Kinetics from batch uptake experiments are compared to kinetic data obtained from confocal laser scanning microscopy images. Both experimental approaches show an accelerated protein adsorption for the novel stationary phase. The influence of pH, salt concentrations and residence times on dynamic binding capacities was determined. A higher dynamic binding capacity for Eshmuno™ S over a wider range of pH values and residence times was found compared to Fractogel® SO3(-) and Toyopearl GigaCap S-650M. The capture of antibodies from cell culture supernatant, as well as post-protein A eluates, were analyzed with respect to their host cell protein (hcp) removal capabilities. Comparable or even better hcp clearance was observed at much higher protein loading for Eshmuno™ S than Fractogel® SO3(-) or Toyopearl GigaCap S-650M.
Urmann
Marina
M
Institute for Biochemistry, University of Applied Sciences Mannheim, Germany.
Graalfs
Heiner
H
Performance & Life Science Chemicals, Merck KGaA, Germany.
Joehnck
Matthias
M
Performance & Life Science Chemicals, Merck KGaA, Germany.
Jacob
Lothar R
LR
Performance & Life Science Chemicals, Merck KGaA, Germany.
Frech
Christian
C
Institute for Biochemistry, University of Applied Sciences Mannheim, Germany.
D000327:Adsorption; D000818:Animals; D000911:Antibodies, Monoclonal / Q000737:chemistry* / Q000302:isolation & purification; D002411:Cation Exchange Resins / Q000737:chemistry*; D002412:Cations / Q000737:chemistry; D002852:Chromatography, Ion Exchange / Q000379:methods*; D017077:Culture Media, Conditioned / Q000032:analysis; D006801:Humans; D006863:Hydrogen-Ion Concentration; D007163:Immunosorbent Techniques; D007700:Kinetics; D018613:Microscopy, Confocal / Q000379:methods*; D011108:Polymers / Q000737:chemistry*; D011485:Protein Binding; D055672:Static Electricity
D000911:Antibodies, Monoclonal; D002411:Cation Exchange Resins; D002412:Cations; D017077:Culture Media, Conditioned; D011108:Polymers