Production of Recombinant
Human Insulin
Design of an Enzyme Reactor
Review
Pro-Insulin (45% w/v)
~825 L
(Trypsin, 0.2 kg)
Product
•Trypsin is added to the aqueous stream of pro-insulin
(@ 0.015 mol%)
•Target 100% conversion to the insulin ester
Enzymatic Digestion of Pro-Insulin
• Trypsin is a serine protease whose catalytic
triad is; Asp, His, Ser.1
+
N
Catalytic pocket
H
N
H
H is
Ser
HO
O
A sp
HO
•Trypsin cleaves the cationic amino acids Arginine
and Lysine (carboxyl side)2
Enzyme Kinetics
•Km values range from; 0.5 - 0.0007 M
•K2 from; 0.2 – 0.05 s-1
•Enzyme functions from pH 4-9 and T 75-160°F
•Iso-electric point, pH 5.4
•Operate above pI; pH~7
•Kinetic data valid for T=101°F, 38.3 ° C
•Bovine trypsin; 23,500 g/mol
Refs 1,3
Stream Composition
Unit Operation
Final Product
Freeze Drying
Basket Centrifuge
Crystallization
Ultrafiltration
RP-HPLC #2
Acid Hydrolysis
Anion Exchanger
Diafilter #2
RP-HPLC #1
Enzyme Reaction
Diafilter #1
Cation Exchanger
Centrifugation
Fermentation Product
Product
Passthrough
Influent Insulin
Analog (kg)
100.00%
100.00%
98.00%
100.00%
100.00%
70.29%
90.00%
98.00%
95.00%
90.00%
100.00%
100.00%
98.00%
189.40
189.40
189.40
193.27
193.27
193.27
274.97
305.52
311.75
328.16
364.62
364.62
364.62
372.06
372.06
Overall Yield (post ferm entation)=
50.91%
*Assumes constant product mass during processing, 5% error
*Process presented in reverse order
Feed
364.62 kg of pro-insulin
(5958 g/mol)
~825 L aq. solution
Product
328.16 kg of insulin ester
(5706 g/mol)
36.45 kg of denatured insulin
(288 g/mol)
~825 L aq. solution
Reactor Choice
•Trypsin is cheap => use the soluble enzyme
PFR
?
Batch
?
CSTR
Inefficient at high X
•Use of the free enzyme renders batch, PFR
kinetics equivalent when Q is variable
V max εV T
Q
VT
Q

VT
Q
 R o X  K m ln(1  X)
 t
Ref 1, 4
Reactor Specification I
Upper Performance Limit
Low er Performance Limit
100.00%
90.00%
80.00%
Conversion (%)
70.00%
60.00%
50.00%
40.00%
30.00%
20.00%
10.00%
0.00%
0.0
2.0
4.0
6.0
8.0
Tim e(hours)
• Best case (X~100%), t=11 h
• Worst case (X~100%), t=40h
• Design for t=25h
10.0
Reactor Specification II
•Long residence time
•High value/conversion product
Batch
Liquid volume= 825L
Add 20% Headspace~ 1000L
Rules of thumb;
HL=DT
DT, HL=1.0m
DT=3Di
HR=1.25m
Wb=0.1DT
Include coils or a jacket
Ref 5
Reactor Cost/Source
•“No-frills,” 1000L s/s reactor ~$95,000 CDN
•Manufacturers;
Pfaudler, De Dietrich, Apache, Northland
•Or buy used from Loeb;
References
1.
Voet et al. Fundamentals of Biochemistry. Toronto: Wiley, 1999.
2.
Swiss Institute of Bioinformatics. Peptide Cutter (simulation software).
http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html#
3.
Sigma Life Sciences. Trypsin from Bovine Pancreas, Prod. T8802.
www.sigmaaldrich.com
4.
Fogler, H.S. Elements of Chemical Reaction Engineering. Upper Saddle
River: Prentice Hall. 1999.
5.
Hasbrouck Engineering. Sample Batch Reactor Drawing.
www.hasbrouckengineering.com