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

Final Design

Questions  ????