Clinical Pharmacology & Biopharmaceutics
Open Access

Abstract

Correct formation of disulfide bonds is crucial in the refolding of multi disulfide bonded inclusion body proteins. Covalent aggregation due to wrong cross linking of disulfide bonds leads to a low refolding yield. On-column refolding can be used to refold complex proteins under controlled conditions. This study reports the development of a multi gradient strategy for on-column refolding of high disulfide bonded proteins using size exclusion chromatography. Recombinant tissue plasminogen activator (r-PA, reteplase) with 9 disulfide bonds was used as the protein model. Applying a four-gradient strategy, including decreasing linear gradient of urea concentration and increasing linear gradients of pH, cysteine, and arginine concentration at the same time, resulted in 49.72% activity recovery and 91% mass recovery, which was 4 and 2.5-fold more than conventional on-column refolding with non-gradient and urea-arginine gradient strategies respectively. Successful application of multi gradient strategy in improving refolding yield of reteplase demonstrated controllability of the refolding process using size exclusion chromatography with a rationally designed gradient strategy which can be used in the refolding of other complex proteins too.

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