Presentation on theme: "Production of Insulin Reverse Phase – High Pressure Liquid Chromatography Unit (RP-HPLC) Presented by:Justin McComb Rachelle Bolton Young Chang."— Presentation transcript:
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 Process Design Considerations Cost Analysis Final Process Final Design
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.
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.
Principles of RP-HPLC This will affect the elution time of each compound, and will cause the mixture to separate into its components.
Principles of RP-HPLC
Design Validation Equations Re p = Reynolds f p = friction factor Q = volumetric flowrate A = x-sectional area ρ = density μ = viscosity L = column length ΔP = pressure drop ε = void fraction D p = resin diameter Ergun Equation Laminar Flow Validation Pressure Drop Calculation
Summary Table of Organic Modifiers 1 poise = dyne s/cm 2 = g/cm s = 1/10 Pa s 1 p = 100 centiPoise Density(g/cm 3 Room Temperature Isopropanol Acetonitrile Ethanol
Resin Design Option #1
Resin Design Option #2
Scale Up Constant Length Constant Linear Flowrate Process Design Considerations
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)
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