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Continuous processing of recombinant proteins: Integration of refolding and purification using simulated moving bed size-exclusion chromatography with buffer recycling

Authors
  • Wellhoefer, Martin
  • Sprinzl, Wolfgang
  • Hahn, Rainer
  • Jungbauer, Alois1, 2
  • 1 Austrian Centre of Industrial Biotechnology (ACIB)
  • 2 Department of Biotechnology, University of Natural Resources and Life Sciences Vienna
Type
Published Article
Journal
Journal of Chromatography A
Publisher
Elsevier
Publication Date
Jan 01, 2014
Accepted Date
Feb 05, 2014
Volume
1337
Pages
48–56
Identifiers
DOI: 10.1016/j.chroma.2014.02.016
Source
Elsevier
Keywords
License
Unknown

Abstract

Continuous processing of recombinant proteins was accomplished by combining continuous matrix-assisted refolding and purification by tandem simulated moving bed (SMB) size-exclusion chromatography (SEC). Recombinant proteins, Npro fusion proteins from inclusion bodies were dissolved with NaOH and refolded in the SMB system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling of the refolding buffer of the raffinate by tangential flow filtration. For further purification of the refolded proteins, a second SMB operation also based on SEC was added. The whole system could be operated isocratically with refolding buffer as the desorbent buffer, and buffer recycling could also be applied in the purification step. Thus, a significant reduction in buffer consumption was achieved. The system was evaluated with two proteins, the Npro fusion pep6His and Npro fusion MCP-1. Refolding solution, which contained residual Npro fusion peptide, the cleaved autoprotease Npro, and the cleaved target peptide was used as feed solution. Full separation of the cleaved target peptide from residual proteins was achieved at a purity and recovery in the raffinate and extract, respectively, of approximately 100%. In addition, more than 99% of the refolding buffer of the raffinate was recycled. A comparison of throughput, productivity, and buffer consumption of the integrated continuous process with two batch processes demonstrated that up to 60-fold higher throughput, up to 180-fold higher productivity, and at least 28-fold lower buffer consumption can be obtained by the integrated continuous process, which compensates for the higher complexity.

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