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Reversed Phase HPLC 2

Reversed Phase HPLC 2. CHEE 450 Engineering Biology Thomas Cooper Pedro Isaza. Purpose & Alternatives . Final insulin product must be purified Mixture contains many impurities Unreacted insulin esters Precursors Deamidated insulin

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Reversed Phase HPLC 2

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  1. Reversed Phase HPLC 2 CHEE 450 Engineering Biology Thomas Cooper Pedro Isaza

  2. Purpose & Alternatives • Final insulin product must be purified • Mixture contains many impurities • Unreacted insulin esters • Precursors • Deamidated insulin • RP-HPLC demonstrated to separate insulin and insulin-like compounds differing by one amino acid • Also possible with ion exchange chromatography • Loss of product • Lower yields

  3. How it Works • HPLC able to separate based on charge, size, and hydrophobic character • Hydrophobic analytes interact strongly with resin • Hydrophilic pass freely through the column • Organic solvents are used to elute hydrophobic molecules

  4. Design Requirements • Annual insulin production of 4000 kg at 99% purity • Insulin enters RP-HPLC 2 at 530 g/h • Minimum yield of 88% required • Assume 100% binding Fig. 1: Overall annual insulin production as a function of the RP-HPLC 2 unit recovery

  5. Design Considerations • Column Type • Fixed-bed vs. Axial Compression • Large size creates difficulties for efficient and reproducible packing • Axial compression eliminates this problem • General Operating Parameters • Temperature range of 15 to 20 oC • Pressure range of 20 to 100 bar Fig. 2: Illustration of “self-packing” axial compression column (TechniKrom, 2007).

  6. Design Considerations • Packing (Stationary Phase) • Lipophilically modified silica gels commonly used • For insulin purification, C8 to C18 yield best results • Ideal particle size ≤ 12 µm and pore sizes of 120 to 150 Å Fig. 3: Structure of C18 resin • Alternative option is Amberchrom HPR10 • Recommended for polishing stages of insulin purification • Both display similar performance and cost • More data available for silica-based materials • C8 packing selected

  7. Design Considerations • Column Size • C8 suggested loading is 17 mg insulin/mL • For 530 g/batch, 32 L of packing is required • Mobile-phase pH • Acidic conditions elute insulin before impurities • Early elution improves yields • Ideal pH range of 3 to 4 • Well below isoelectric point of insulin (pH 5.4) • Compatible with chosen silica resin (pH 2 to 8) • high pH: dissolution of the silica • very low pH: hydrolysis of attached C8 chains • May lead to insulin deamidation • Not significant problem due to short exposure times

  8. Design Considerations • Organic Modifier for Elution • Requirements: good insulin selectivity and low viscosity • Acetone or acetonitrile are recommended • Acetonitrile • Well-documented analytical insulin separation • High yields obtained at production scale • Acetone • Lower yields due to poor insulin solubility • Loading, Elution, and Regeneration Scheme • Load insulin onto column in aqueous-alcoholic, or purely aqueous, buffer solution • Linear gradient ranging from 15 to 30% acetonitrile effectively separates insulin in 1 CV or less • Regeneration with 60% acetonitrile and pH 7.4

  9. Design Considerations • Confirmation • Product stream monitored via spectroscopy • At 280 nm for insulin • Final Yields • Minimum 88% yield required • Yields from 83 to 98% documented for C8 Insulin Peak Fig. 4: Elution of insulin and insulin derivatives under acidic conditions (Kroeff et al., 1989)

  10. Eluting Regenerating Loading Final Design – Batch Time • 3 columns are required • 4 recommended for unexpected failures • To find number of columns required for continuous operation • At inlet flowrate: loading time is 1 h • Elution in 1 CV at 1.5 CV/h: elution time is 40 min • Similarly, column regeneration time is 40 min • Total HPLC cycle is 140 min

  11. Final Design – Cost Analysis • Axial compression column • 32 L required • TechniKrom 35 L (30 cm ID x 50 cm): $80,000 • Packing • Kromasil C8 (10 µm, 100 Å): $150,000/column • 18 kg per column • Quoted price of $8.02/g • Average lifetime: 300 cycles • 7 re-packings required annually • Chemicals • High purity water (WFI): $50,000/year • 120 kg WFI/h • $0.05/kg • Note: Neglected acetonitrile, acetone, and acetic acid

  12. Final Design – Cost Summary Table 1: Summary of costs associated with RP-HPLC 2

  13. Questions?

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