production of recombinant human insulin
DESCRIPTION
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. - PowerPoint PPT PresentationTRANSCRIPT
Production of Recombinant Human Insulin
Design of an Enzyme Reactor
Review
Pro-Insulin (45% w/v)~825 L
Product
•Trypsin is added to the aqueous stream of pro-insulin (@ 0.015 mol%)•Target 100% conversion to the insulin ester
(Trypsin, 0.2 kg)
Enzymatic Digestion of Pro-Insulin
• Trypsin is a serine protease whose catalytic triad is; Asp, His, Ser.1
N
N+
H
H
OOHOH
His
Asp
Ser
•Trypsin cleaves the cationic amino acids Arginine and Lysine (carboxyl side)2
Catalytic pocket
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/molRefs 1,3
Stream CompositionUnit Operation
Product Passthrough
Influent Insulin Analog (kg)
Final Product 189.40Freeze Drying 100.00% 189.40Basket Centrifuge 100.00% 189.40Crystallization 98.00% 193.27Ultraf iltration 100.00% 193.27RP-HPLC #2 100.00% 193.27Acid Hydrolysis 70.29% 274.97Anion Exchanger 90.00% 305.52Diafilter #2 98.00% 311.75RP-HPLC #1 95.00% 328.16Enzyme Reaction 90.00% 364.62Diafilter #1 100.00% 364.62Cation Exchanger 100.00% 364.62Centrifugation 98.00% 372.06Fermentation Product 372.06
50.91%
*Process presented in reverse order
Overall Yield (post fermentation)=
*Assumes constant product mass during processing, 5% error
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? ?
•Use of the free enzyme renders batch, PFR kinetics equivalent when Q is variable
Inefficient at high X
tQ
V
Q
V
X)ln(1KXRQ
εVV
TT
moTmax
Ref 1, 4
Reactor Specification I
0.00%
10.00%
20.00%
30.00%
40.00%
50.00%
60.00%
70.00%
80.00%
90.00%
100.00%
0.0 2.0 4.0 6.0 8.0 10.0
Time(hours)
Co
nve
rsio
n (
%)
Upper Performance Limit Low er Performance Limit
• Best case (X~100%), t=11 h• Worst case (X~100%), t=40h• Design for t=25h
Reactor Specification II
•Long residence time•High value/conversion product
Batch
Liquid volume= 825LAdd 20% Headspace~ 1000L
Rules of thumb;HL=DT DT, HL=1.0mDT=3Di HR=1.25mWb=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