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Production of Recombinant Human Insulin

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.

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Production of Recombinant Human Insulin

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  1. Production of Recombinant Human Insulin Design of an Enzyme Reactor

  2. 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

  3. Enzymatic Digestion of Pro-Insulin • Trypsin is a serine protease whose catalytic triad is; Asp, His, Ser.1 Catalytic pocket • Trypsin cleaves the cationic amino acids Arginine and Lysine (carboxyl side)2

  4. 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

  5. Stream Composition 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

  6. 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 Ref 1, 4

  7. Reactor Specification I • Best case (X~100%), t=11 h • Worst case (X~100%), t=40h • Design for t=25h

  8. 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

  9. Reactor Cost/Source • “No-frills,” 1000L s/s reactor ~$95,000 CDN • Manufacturers; • Pfaudler, De Dietrich, Apache, Northland • Or buy used from Loeb;

  10. References • Voet et al. Fundamentals of Biochemistry. Toronto: Wiley, 1999. • Swiss Institute of Bioinformatics. Peptide Cutter (simulation software). http://ca.expasy.org/tools/peptidecutter/peptidecutter_enzymes.html# • Sigma Life Sciences. Trypsin from Bovine Pancreas, Prod. T8802. www.sigmaaldrich.com • Fogler, H.S. Elements of Chemical Reaction Engineering. Upper Saddle River: Prentice Hall. 1999. • Hasbrouck Engineering. Sample Batch Reactor Drawing. www.hasbrouckengineering.com

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