Journal
JOURNAL OF BIOTECHNOLOGY
Volume 163, Issue 2, Pages 233-242Publisher
ELSEVIER SCIENCE BV
DOI: 10.1016/j.jbiotec.2012.08.022
Keywords
Single-chain Fragment variable antibody; Immobilized metal ion affinity chromatography; Simulated moving bed; Histidine-tagged recombinant protein purification; Bacterial expression; Adsorption equilibrium constants
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Funding
- Deutsche Forschungsgemeinschaft (DFG) [Sonderforschungsbereich 578, C2]
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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. (C) 2012 Elsevier B.V. All rights reserved.
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