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