(545g) In Vitro Folding of Methionine-Arginine Human Lyspro Proinsulin S-Sulfonate

The in vitro disulfide formation pathways of Methionine-Arginine Human Lyspro Proinsulin S-Sulfonate (MR-KPB-hPSS) were elucidated. Folding intermediates were detected and characterized using Matrix Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS), Reversed Phase Chromatography (RPC), Size Exclusion Chromatography (SEC), and SDS-PAGE. The disulfide forming pathways and the yield of correctly folded proinsulin (MR-KPB-hPI) depended on the redox concentration (cysteine to proinsulin-SH molar ratio). At a cysteine to proinsulin-SH ratio of 3.5:1, MALDI-MS analysis of intact intermediates indicated a sequential formation of intermediates with one, two, and three disulfide bonds. MALDI-MS analysis of digested intermediates indicated that an intra-A-chain disulfide bond between A6, A7, and A11 was likely to form first. Various mismatched intra-A, intra-B, and inter-AB disulfide bonds were observed in intermediates with two disulfide bonds. Intermediates with three disulfide bonds were found to contain non-native intra-A-chain (A20 with A6, A7, or A11) and intra-B-chain (B7 with B19) disulfide bonds. The intermediates with two or more non-native disulfide bonds were converted to correctly folded proinsulin via disulfide bond reshuffling, which was the rate limiting step. At a high cysteine to proinsulin-SH ratio (17.5:1), species with two or more free thiols were formed. Aggregation due to formation of inter-molecular disulfide bonds of the early intermediates was the major cause of yield loss.