Journal
JOURNAL OF CHROMATOGRAPHY A
Volume 1434, Issue -, Pages 29-38Publisher
ELSEVIER
DOI: 10.1016/j.chroma.2016.01.001
Keywords
Single-chain antibody fragments; Immobilized metal ion affinity chromatography; Simulated moving bed chromatography; Histidine-tagged recombinant protein purification
Funding
- Deutsche Forschungsgemeinschaft (DFG) in the frame of the project From Gene to Product [SFB578]
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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. (C) 2016 Elsevier B.V. All rights reserved.
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