title: Continuous processing of recombinant proteins: integration of refolding and purification using simulated moving bed size-exclusion chromatography with buffer recycling.
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, N(pro) 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 N(pro) fusion pep6His and N(pro) fusion MCP-1. Refolding solution, which contained residual N(pro) fusion peptide, the cleaved autoprotease N(pro), 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.
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Wellhoefer
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sprinzl
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria.
authors.forename: Rainer
authors.initials: R
authors.lastname: Hahn
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190 Vienna, Austria; Department of Biotechnology, University of Natural Resources and Life Sciences, Vienna, Muthgasse 18, 1190 Vienna, Austria. Electronic address: alois.jungbauer@boku.ac.at.
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D002021:Buffers; D002850:Chromatography, Gel; D005240:Feasibility Studies; D002479:Inclusion Bodies / Q000737:chemistry; D010455:Peptides / Q000032:analysis; D058849:Protein Refolding; D011993:Recombinant Fusion Proteins / Q000032:analysis* / Q000302:isolation & purification
chemical_list: D002021:Buffers; D010455:Peptides; D011993:Recombinant Fusion Proteins
keywords: Autoprotease fusion protein; Buffer recycling; Continuous purification; Process intensification; Simulated moving bed