Multivariate optimization of the refolding process of an incorrectly folded Fc-fusion protein in cell culture broth

BACKGROUND: Protein misfolding is a common problem in large-scale production of recombinant proteins, which can significantly reduce yield of the process. OBJECTIVE: In this work, we aimed at treating a cell culture broth containing high levels (>45%) of an incorrectly folded Fc-fusion protein 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 small scale (25 ml) employing an experimental design methodology. Having identified the key variables using a resolution (IV) fractional factorial design (FFD), the process was then optimized by 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 8.5 oC and a reaction time of 24 h. Based upon the treatment conditions obtained at 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 like chaotropic agents, aggregation suppressors, stabilizers and chelators. CONCLUSION: The refolding process increases the fraction of active protein in original broth reducing the burden of downstream purification steps markedly.