production of insulin reverse phase – high pressure liquid chromatography unit (rp-hplc) presented...
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Production of Insulin
Reverse Phase – High Pressure Liquid Chromatography Unit (RP-HPLC)
Presented by: Justin McCombRachelle BoltonYoung 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 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.
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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 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
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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
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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%
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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%
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Scale Up
Constant Length
Constant Linear Flowrate
Process Design Considerations
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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 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%
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Questions
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