title: Discontinuous and continuous purification of single-chain antibody fragments using immobilized metal ion affinity chromatography.
abstract: This work describes the adsorption-desorption behavior of a histidine-tagged single-chain Fragment variable antibody (D1.3 scFv) on a commercial immobilized metal ion affinity chromatography (IMAC) column. A clarified cell culture supernatant originating from Bacillus megaterium was characterized using single column experiments in a pH-gradient elution mode. The cell culture supernatant containing the antibody fragment D1.3 scFv could be treated in the chromatographic separation process as a pseudo-binary mixture. Adsorption equilibrium constants of the antibody fragment fraction (ABF) and the non-specifically retained protein impurity fraction (IMP) were determined experimentally at constant pH by reinjecting pulses of pooled fractions collected in preliminary batch gradient elution runs. Based on the estimated adsorption equilibrium constants a possible multicolumn open-loop three-zone two-step pH-gradient simulated moving bed (SMB) process is suggested and designed, which possesses the potential to isolate continuously the antibody fragment fraction (ABF) containing the single-chain antibody fragment D1.3 scFv.
authors.affiliation: Max Planck Institute for Dynamics of Complex Technical Systems, Sandtorstr. 1, 39106 Magdeburg, Germany.
authors.forename: Carlos Andrés
authors.initials: CA
authors.lastname: Martínez Cristancho
authors.forename: Florian
authors.initials: F
authors.lastname: David
authors.forename: Ezequiel
authors.initials: E
authors.lastname: Franco-Lara
authors.forename: Andreas
authors.initials: A
authors.lastname: Seidel-Morgenstern
mesh_terms: D001410:Bacillus megaterium / Q000737:chemistry / Q000378:metabolism; D002846:Chromatography, Affinity / Q000379:methods*; D003198:Computer Simulation; D004591:Electrophoresis, Polyacrylamide Gel; D006639:Histidine / Q000737:chemistry; D006863:Hydrogen-Ion Concentration; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D057127:Single-Chain Antibodies / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism
chemical_list: D011993:Recombinant Fusion Proteins; D057127:Single-Chain Antibodies; D006639:Histidine