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Current Pharmaceutical Biotechnology

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ISSN (Print): 1389-2010
ISSN (Online): 1873-4316

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Research Article

Multivariate Optimization of the Refolding Process of an Incorrectly Folded Fc-Fusion Protein in a Cell Culture Broth

Author(s): Hossein Behrouz, Behnaz Molavi, Ata Tavakoli, Mansoureh Askari, Shayan Maleknia, Fereidoun Mahboudi and Mehdi Khodadadian*

Volume 21, Issue 3, 2020

Page: [226 - 235] Pages: 10

DOI: 10.2174/1389201020666191002144424

Price: $65

Abstract

Background: Protein misfolding is a common problem in large-scale production of recombinant proteins, which can significantly reduce the yield of the process.

Objective: In this work, we aimed at treating a cell culture broth containing high levels (>45%) of incorrectly folded Fc-fusion proteins by a simple redox buffer system in order to increase the proportion of the protein with correct conformation.

Methods: Multi-variable process optimization was firstly conducted at a small scale (25 mL), employing an experimental design methodology. After identifying the key variables using a resolution IV Fractional Factorial Design (FFD), the process was then optimized by the Central Composite Design (CCD).

Results: The optimal conditions for the refolding reaction were 340 mM Tris-base, 6.0 mM L-cysteine, 0.5 mM L-cystine, a buffer pH of 9.0, a reaction temperature of 8.5ºC and a reaction time of 24 h. Based on the treatment conditions obtained at a small scale, the process was further scaled up to 4500- L. The misfolded content was always less than 20%. The reaction can proceed well in the absence of chemical additives, such as chaotropic agents, aggregation suppressors, stabilizers and chelators.

Conclusion: The refolding process increases the fraction of active protein in the original broth reducing the burden on downstream purification steps markedly.

Keywords: Protein misfolding, protein refolding, fusion protein, recombinant DNA technology, cell culture broth composite design.

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