title: Continuous processing of recombinant proteins: Integration of inclusion body solubilization and refolding using simulated moving bed size exclusion chromatography with buffer recycling.
abstract: An integrated process which combines continuous inclusion body dissolution with NaOH and continuous matrix-assisted refolding based on closed-loop simulated moving bed size exclusion chromatography was designed and experimentally evaluated at laboratory scale. Inclusion bodies from N(pro) fusion pep6His and N(pro) fusion MCP1 from high cell density fermentation were continuously dissolved with NaOH, filtered and mixed with concentrated refolding buffer prior to refolding by size exclusion chromatography (SEC). This process enabled an isocratic operation of the simulated moving bed (SMB) system with a closed-loop set-up with refolding buffer as the desorbent buffer and buffer recycling by concentrating the raffinate using tangential flow filtration. With this continuous refolding process, we increased the refolding and cleavage yield of both model proteins by 10% compared to batch dilution refolding. Furthermore, more than 99% of the refolding buffer of the raffinate could be recycled which reduced the buffer consumption significantly. Based on the actual refolding data, we compared throughput, productivity, and buffer consumption between two batch dilution refolding processes - one using urea for IB dissolution, the other one using NaOH for IB dissolution - and our continuous refolding process. The higher complexity of the continuous refolding process was rewarded with higher throughput and productivity as well as significantly lower buffer consumption compared to the batch dilution refolding processes.
authors.affiliation: Austrian Centre of Industrial Biotechnology (ACIB), Muthgasse 11, 1190 Vienna, Austria.
authors.forename: Martin
authors.initials: M
authors.lastname: Wellhoefer
authors.forename: Wolfgang
authors.initials: W
authors.lastname: Sprinzl
authors.forename: Rainer
authors.initials: R
authors.lastname: Hahn
authors.forename: Alois
authors.initials: A
authors.lastname: Jungbauer
mesh_terms: D002021:Buffers; D002850:Chromatography, Gel / Q000379:methods*; D017510:Protein Folding; D011994:Recombinant Proteins / Q000737:chemistry / Q000302:isolation & purification*; D012995:Solubility
chemical_list: D002021:Buffers; D011994:Recombinant Proteins
keywords: Autoprotease fusion protein; Buffer recycling; Continuous refolding; Inclusion body; Simulated moving bed