Production of Insulin

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Production of Insulin

• Reverse Phase High Pressure Liquid
  Chromatography Unit (RP-HPLC)

Presented by Justin McComb Rachelle
Bolton Young Chang
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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

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Purpose of Unit

• The second RP-HPLC used in the production of
  insulin is used to purify the human insulin that
  has been produced.

• The unit purifies native insulin by removing
  impurities such as
• insulin ester
• denatured insulin
• partially cleaved precursor components

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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.

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Principles of RP-HPLC

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

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Principles of RP-HPLC
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Design Validation Equations
Ergun Equation

• Rep Reynolds
• fp friction factor
• Q volumetric flowrate
• A x-sectional area
• ? density
• µ viscosity
• L column length
• ?P pressure drop
• e void fraction
• Dp resin diameter

Laminar Flow Validation
Pressure Drop Calculation
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Summary Table of Organic Modifiers
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Resin Design Option 1
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Resin Design Option 2
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Process Design Considerations
Scale Up Constant Length Constant
Linear Flowrate
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Process Design Considerations
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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)

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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

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Final Design
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Questions

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