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Design of a Well-Mix Reactor to Convert Insulin-Precursor to Crude Insulin via Transpeptidation

Design of a Well-Mix Reactor to Convert Insulin-Precursor to Crude Insulin via Transpeptidation. Monday, April 3, 2006 8:30-9:30am. Presented by: Andrea Evans Rob Galloway Imran Ratanshi Department of Chemical Engineering Queen’s University, Kingston, ON.

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Design of a Well-Mix Reactor to Convert Insulin-Precursor to Crude Insulin via Transpeptidation

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  1. Design of a Well-Mix Reactor to Convert Insulin-Precursor to Crude Insulin via Transpeptidation Monday, April 3, 2006 8:30-9:30am Presented by: Andrea Evans Rob Galloway Imran Ratanshi Department of Chemical Engineering Queen’s University, Kingston, ON Well-Mix Reactor Design

  2. Overview • Background • Reactor Options • Reaction Kinetics • Design Methodology • Recommended Size • Costs • Process Alternatives • Conclusions • References • Questions

  3. Insulin Production Process Figure 1. Proposed recombinant human insulin production process.

  4. Background • Our Reaction Insulin Precursor Crude Insulin + “Denatured” Insulin (90%) (10%) • Aqueous reaction • Other info: • 100% Yield • Reaction is catalyzed by Trypsin transpeptidation

  5. The Reaction • Enzyme Trypsin cleaves precursor after: • 29th Amino acid (Lysine) of B-chain • 22nd Amino acid (Glycine) of A chain • Cleavage at alternate site (Gly-Arg) results in ‘denatured form) • 10% of output product

  6. Reactor Options • Batch vs. Continuous

  7. Reactor Options • Want to avoid cleavage of insulin at secondary site by avoiding product inhibition • Use Batch reactor

  8. Process Specifications • Reactor Yield ~ 100% • Overall process output = 44 kg/batch • Neglect insulin ‘defolding’ and ‘refolding’ unit operations • Assume fermentor operation was process limiting • Reactor operating time should be < 60hrs • Reactor Input = 45% (w/v) • Assume complete dissolution of insulin precursor • Fluid density ~ 1kg/L

  9. Reaction Kinetics • Enzymatic reaction [E] + [S] [ES] [E] + [P] - Michaelis-Mention Kinetics • Vmax = Maximum Conversion Rate (Enzyme saturation) • Vmax = k2[ET] • Km = Michaelis-Menton Constant (Measure of binding) • [S] = Substrate Concentration (Insulin precursor) • Assumptions: • No Competitive/Non-competitive Inhibition k1 k2

  10. Design Methodology • Batch process (Fogler, 1999): • General Mole Balance Equation • Combine with Rate Expression

  11. Design Methodology 3. Separate variables and solve for time t • Process Parameters (Schilling and Mitra, 1991) • Vmax = f ([ET]) = 1.41 x 10-5M min-1 • Km = 9.9 x 10-5M

  12. Design Methodology • To produce 11.31kg, Petrides (2000) required a fluid volume of 4,300L to convert insulin precursor to crude human insulin • E.coli (intracellular) production process • To produce ~44kg, we scaled up this volume by a factor of 4 • Approximate fluid volume is 17,200L

  13. Design Summary Table 1. Summary of reactor design parameters.

  14. Reaction Time Figure 2. Plot of insulin precursor concentration as a function of time.

  15. Cost Analysis • Perry’s Chemical Engineering Handbook • q1= 100 US Gal q2= 5679.7 US Gal C1= $9300 (US) n= 0.53 Therefore C2= $79118

  16. Cost Analysis • Problems with this model: • Oversimplification by correlating in terms of a single variable • Simple exponential relationship • Fails to include technological improvement • Reactor modeled as a carbon steel reactor instead of stainless steel

  17. Cost Analysis • ChemEcon • Modeled as a stainless steel vessel • Height (m) = 4.78 • Diameter (m) = 2.39 • Pressure (bar) = 1.013 • Bare Module Cost = $152 067

  18. Cost Analysis • SuperPro Designer • Purchase Cost = $100 578 • Unit Cost = $101 000 • Operating Hours = 120 hours/year • Power = 4.9 kW • Annual = 11703 kWh • Cost of Power = $1000/year at 10 cents/kWh

  19. Process Alternatives • Enzymatic decomposition of proinsulin by trypsin. In large-scale production it is more economical to use immobilized enzymes.

  20. References • Fogler, S.H. Elements of Chemical Reaction Engineering, 3rd Ed. Prentice Hall. New Jersey. (1999). 1-393. • Kudryavtseva, N.E. and L. S. Zhigis, V. P. Zubov, A. N. Vul'fson, K. V. Mal'tsev and L. D. Rumsh. Immobilization of trypsin and carboxypeptidase B on modified silicas and their use in converting human recombinant proinsulin into insulin. Pharmaceutical Chemistry Journal. 29:1 (1995) 70-73 • Petrides, D. Bioprocess Design. Intelligen, Inc. • Schilling, R.J. and A.K. Mitra. Degradation of insulin by trypsin and alpha-chymotrypsin. Pharmaceutical Research. 8:6 (1991) 721-727

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