Transcript Refolding

Production of recombinant human insulin from E.coli
Matt Perry, James Ragan and Mike Trowell
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Remove SO3 units from proinsulin-SSO3
Refold proinsulin into correct form
Form disulfide bridges
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Proinsulin is not folded correctly when
produced by E.coli
Sulfitolysis step denatures proinsulin with
guanidine•HCl
◦ Sulfite bridges broken and SO3 added
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Urea added to prevent refolding
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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
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20 – 100 fold excess MrEtOH to SO3 required
218 kg MrEtOH per Batch
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Low protein concentration required to prevent
cross-folding
Yield is based on protein concentration
Reactor size determined by desired yield
100
Yield
(%)
95
90
83
53
20
8
(Kim et al., 1999)
90
80
70
Yield (%)
Protein Concentration
(mg/mL)
0.1
0.2
0.5
1
2
4
60
50
40
30
y = 101.33e-0.666x
20
10
0
0
1
2
3
Protien Concentration (mg/mL)
4
5
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Add 20% head space for reactor
Volume = 100 m3
Use two 50m3 vessels
Stainless steel construction
Cooling system required
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Temperature: 4°C
◦ Low temperature prevents aggregation of proteins
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Time: 16 hrs
pH: 10.5
(Cowley & Mackin, 1997)
◦ Glycine buffer used
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Yield: 83%
◦ Conversion of proinsulin-SS03 to proinsulin
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Nitrogen Sparge
◦ Oxygen inhibits correct reduction reaction
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Slow Mixing Required
Reactor Cost (1994 $US) = 10α × capacity (m3)β
Where α=4.41 and β=0.59 for refolding reactor (Kotlarski et al, 1997)
$260 000 = 104.41 × 500.59
$370 000 per reactor
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Cooling Cost
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Reactant Cost
◦ Use glycol at -10°C
◦ 7885kg/hr during reactor loading
◦ 218 MrEtOH kg/batch
◦ $654 per batch at $3/kg
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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