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Reverse Phase HPLC

Reverse Phase HPLC. By Julian Chesterman Natalie McKenzie Harry Zhou. Where Is It?. Why Is It There?. Final Purification Intended to Reduce the Level of Insulin-like Components These are Otherwise Difficult to Remove by Conventional Chromatographic Procedures . Why Does It Work?.

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Reverse Phase HPLC

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  1. Reverse Phase HPLC By Julian Chesterman Natalie McKenzie Harry Zhou

  2. Where Is It?

  3. Why Is It There? • Final Purification • Intended to Reduce the Level of Insulin-like Components • These are Otherwise Difficult to Remove by Conventional Chromatographic Procedures.

  4. Why Does It Work? • Non-Polar Stationary Phase, More Polar Mobile Phase • Highly Polar Particles Elute Rapidly • Polarity of Mobile Phase Reduced Gradually Through Addition of Acetonitrile • Insulin Elutes Before Insulin Derivatives at pH of 3-4

  5. How Does It Work? • 4 Steps in Operation • Loading • Insulin in Water Solution • Elution • Multiple Column Volumes of Solvent for Separation • Gradient vs. Step elution • Washing • One Column Volume of Low Polarity Solvent • Equilibration (aka Reset)

  6. What Are the Design Requirements? • Superpro Specifies 4 Cycles Per Batch • Budgeted to Take 11.1 Hours for Entire Batch • Need to Meet Input Requirements of the Next Unit • 16 kg/batch of Insulin

  7. How Do You Determine a Size? • Used a Paper by Eli Lilly Published in Journal of Chromatography (1989) on Scaling Up RP-HPLC for Insulin Production • Used Scale Up Data in Paper and Linear Regression to Extrapolate to Size Needed • 15 g insulin/L of CV • Mass of packing scaled from required CV • Elution flow rate changed in proportion to CV • Bed height determined from required CV and diameter of available columns

  8. What Is the Final Design? • Recommend a Prochrom LC 1600 from Novasep with a 27 cm Bed Height • Using Agilent Zorbax C8 Stationary Phase (330 kg)

  9. What are the Operating Conditions? • Flow Rate: 720 L/Hr Solvent • Gradient Elution: 18 to 28 weight% Acetonitrile in Water over 6 Column Volumes (4.5 Hrs) • 0.25 Molar Acetic Acid Buffer • Pressure: 50 Bar • Total Solvent Required for One Cycle • 1450 L Acetonitrile • 62 L Acetic Acid • 2850 L

  10. Does It Do Anything Else? • Yes! • Dynamic Axial Compression

  11. Alternatives • None, It’s Integral • Gel Filtration and Ion Exchange Are Complementary, But Can’t Replicate It’s Effectiveness at Separating Highly Similar Proteins

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