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
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.