title: Purification of single-chain antibody fragments exploiting pH-gradients in simulated moving bed chromatography.
abstract: This paper deals with the theoretical design and experimental validation of an affinity-based continuous multi-column chromatography process for the purification of single-chain Fragment variable (scFv) antibodies. An open-loop 3-zone pH-gradient simulated moving bed (SMB) process was investigated exploiting the highly specific affinity of metal ions toward histidine-tagged recombinant proteins. The separation problem was simplified by considering the cell culture supernatant as a pseudo-binary mixture. The influence of mobile phase pH on the adsorption isotherm parameters was estimated by the inverse method using recorded pH-gradient batch elution profiles. Suitable operating parameters for the SMB process were identified using an equilibrium stage model and subsequently validated in a lab-scale SMB unit. Finally, the performance of the pH-gradient SMB process was compared against a non-optimized batch process. Biologically active single-chain Fragment variable antibody formats were purified continuously with 9% more recovery, 11 times more productivity (576 mg of purified scFv per day and liter stationary phase in SMB) and enriched by a factor of 2.5 compared to those obtained in the non-optimized batch process.
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany; Evonik Technology & Infrastructure GmbH, Rodenbacher Chaussee 4, 63457 Hanau, Germany.
authors.forename: Carlos Andrés
authors.initials: CA
authors.lastname: Martínez Cristancho
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany; Institute of Process Engineering, Otto-von-Guericke University, P.O. Box 4120, 39106 Magdeburg, Germany. Electronic address: seidel-morgenstern@mpi-magdeburg.mpg.de.
authors.forename: Andreas
authors.initials: A
authors.lastname: Seidel-Morgenstern
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D002850:Chromatography, Gel / Q000379:methods*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D057127:Single-Chain Antibodies / Q000302:isolation & purification*
chemical_list: D057127:Single-Chain Antibodies
keywords: Histidine-tagged recombinant protein purification; Immobilized metal ion affinity chromatography; Simulated moving bed chromatography; Single-chain antibody fragments