Submit Manuscript  

Article Details


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

[ Vol. 21 , Issue. 3 ]

Author(s):

Hossein Behrouz, Behnaz Molavi, Ata Tavakoli, Mansoureh Askari, Shayan Maleknia, Fereidoun Mahboudi and Mehdi Khodadadian*   Pages 226 - 235 ( 10 )

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 8.5ÂșC 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.

Affiliation:

Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj, Biopharmaceutical Research Center, AryoGen Pharmed Inc., Alborz University of Medical Sciences, Karaj

Graphical Abstract:



Read Full-Text article