1 / 13

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

gersemi
Download Presentation

Reversed Phase HPLC 2

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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?

More Related