Production of Insulin

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

Presented by: Justin McCombRachelle BoltonYoung 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 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

75.1Re

150

ppf

3

2

1

ppDfAQ

LP

1

ReA

QDp

Ergun Equation

Laminar Flow Validation

Pressure Drop Calculation

Summary Table of Organic Modifiers

1 poise = dyne s/cm2 = g/cm s = 1/10 Pa s 1 p = 100 centiPoise

  Density(g/cm3)Viscosity(cP) @ Room

Temperature

Isopropanol 0.78 2.5

Acetonitrile 0.786 0.38

Ethanol 0.789 1.2

O

H HH

HH

H

Resin Design Option #1

Resin: Octadecyl-substituted silica, 5 μm

Column Size: 10 mm x 250 mm

Eluent:

Ethanol, potassium chloride, buffered with Tris, pH 3

Buffer A: 10 mM ammonium acetate, pH 5

Buffer B: 10 mM ammonium acetate, pH 5, 90% Acetonitrile

Gradient: Buffer B 10-60% (50 minutes)

Flow Rate: 100 cm/h, ~2 mL/min

Temperature: 40 oC

Purity: 99%

Resin Design Option #2Resin: Polystyrene-divinyl-benzene

Amberchrome CG300XT, 20 µm

Column Size: 10mm x 250mm

Eluent:

Water

Buffer A: 10% v/v 2-propanol in 50 mM sodium sulfate, 2% acetic acid

Buffer B: 50% v/v 2-propanol in 50 mM sodium sulfate, 2% acetic acid

Gradient: Linear from 40% B to 50% B in 30 column volumes

Flow Rate: 100 cm/h, 1.3 mL/min

Temperature: 20 to 25 oC (ambient)

Purity: 98%

Scale Up

Constant Length

Constant Linear Flowrate

Process Design Considerations

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 DesignResin: Polystyrene-divinyl-benzene

Amberchrome CG300XT, 40 µm

Column Size (D x H): 100 mm x 250mm

Volume: 1.96 L/unit, 1963 mL/unit

Void Fraction: 0.26

Eluent:

Buffer A: 10% v/v 2-propanol in 50 mM sodium sulfate, 2% acetic acid

Buffer B: 50% v/v 2-propanol in 50 mM sodium sulfate, 2% acetic acid

Gradient: Linear from 40% B to 50% B in 30 column volumes

Flow Rate: 100 cm/h

7800 mL/h, 130 mL/min Pressure 750 psi

Temperature: 20 to 25 oC (ambient)

Purity: 98%

Questions

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