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Production of Insulin

Production of Insulin. Reverse Phase – High Pressure Liquid Chromatography Unit (RP-HPLC). Presented by: Justin McComb Rachelle Bolton Young Chang. Overview. Purpose of the Unit Principles of RP-HPLC Design Validation Equations Organic Modifiers Resin Design Options

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Production of Insulin

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  1. Production of Insulin Reverse Phase – High Pressure Liquid Chromatography Unit (RP-HPLC) Presented by: Justin McComb Rachelle Bolton Young Chang

  2. Overview • Purpose of the Unit • Principles of RP-HPLC • Design Validation Equations • Organic Modifiers • Resin Design Options • Process Design Considerations • Cost Analysis • Final Process • Final Design

  3. Purpose of Unit • The unit purifies native insulin by removing impurities such as: • insulin ester • denatured insulin • partially cleaved precursor components • The second RP-HPLC used in the production of insulin is used to purify the human insulin that has been produced.

  4. Principles of RP-HPLC • RP-HPLC is a technique by which differences in polarity of compounds can be used to separate them from a mixture into their components • Chromatography functions through mass transfer between a mobile and stationary phase • Stationary phase (packing): non-polar resin • Mobile phase (solvent): polar liquid • As the mobile phase passes through the column, the components within that phase will have different affinities for the stationary phase.

  5. Principles of RP-HPLC • This will affect the elution time of each compound, and will cause the mixture to separate into its components.

  6. Principles of RP-HPLC

  7. Design Validation Equations Ergun Equation • Rep = Reynolds • fp = friction factor • Q = volumetric flowrate • A = x-sectional area • ρ = density • μ = viscosity • L = column length • ΔP = pressure drop • ε = void fraction • Dp = resin diameter Laminar Flow Validation Pressure Drop Calculation

  8. Summary Table of Organic Modifiers

  9. Resin Design Option #1

  10. Resin Design Option #2

  11. Process Design Considerations Scale Up Constant Length Constant Linear Flowrate

  12. Process Design Considerations

  13. Cost Analysis • Capital Cost (Hamilton estimates): • 20 units x $20000/unit = $400,000 • Operating Costs • Resin Cost: $10,000/unit • Solvent Cost: Encompasses 80% of total operating cost • Energy Cost: • Cold water • Pump (vs. pressure drop)

  14. Final Process • 20,000 mg of the insulin solution is dissolved in 1.5 L water, 10% 2-propanol • Column is regenerated with 0.5N NaOH, washed with water, then washed with 80% isopropyl alcohol containing 0.1% trifluoroacetic acid • Column is equilibrated with 5 column volumes of Buffer A • Insulin solution is applied at 100 cm/h flow rate • Column washed with 3 column volumes of 20% Buffer B and 80% Buffer B • Buffer B increased from 20% to 40% in 1 column volume • Native insulin eluted in a linear gradient of 40-50% buffer B in 30 column volumes • 16,000 mg insulin (98% purity) generated

  15. Final Design

  16. Questions • ????

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