1. Refolding Production of recombinant human insulin from E.coli Matt Perry, James Ragan and Mike Trowell

2. Purpose • Remove SO3 units from proinsulin-SSO3 • Refold proinsulin into correct form • Form disulfide bridges

3. Why is proinsulin not initially folded correctly? • Proinsulin is not folded correctly when produced by E.coli • Sulfitolysis step denatures proinsulin with guanidine•HCl • Sulfite bridges broken and SO3 added • Urea added to prevent refolding

4. How does it work? • ProinsulinSSO3 diluted with WFI to prevent protein interactions • Mercaptoethanol used to remove SO3 groups • Denatureants (MrEtOH and urea) filtered out • Proinsulin refolds into correct configuration

5. Mercaptoethanol • 20 – 100 fold excess MrEtOH to SO3 required 218 kg MrEtOH per Batch

6. Reactor Sizing • Low protein concentration required to prevent cross-folding • Yield is based on protein concentration • Reactor size determined by desired yield (Kim et al., 1999)

7. Reactor Sizing • Add 20% head space for reactor • Volume = 100 m3 • Use two 50m3 vessels • Stainless steel construction • Cooling system required

8. Reaction Conditions • Temperature: 4°C • Low temperature prevents aggregation of proteins • Time: 16 hrs (Cowley & Mackin, 1997) • pH: 10.5 • Glycine buffer used • Yield: 83% • Conversion of proinsulin-SS03 to proinsulin • Nitrogen Sparge • Oxygen inhibits correct reduction reaction • Slow Mixing Required

9. Economics Reactor Cost (1994 $US) = 10α × capacity (m3)β Where α=4.41 and β=0.59 for refolding reactor (Kotlarski et al, 1997) • Cooling Cost • Use glycol at -10°C • 7885kg/hr during reactor loading • Reactant Cost • 218 MrEtOH kg/batch • $654 per batch at $3/kg $260 000 = 104.41 × 500.59 $370 000 per reactor

10. Filter Specifications • 8 hour filtering process • Flow through filter 10.4 m3/h • Diafilter removes: • 98% of denaturants (Urea and MrEtOH) • 87% of water • 98% of salt

11. Questions?