1. Anion Exchanger Kristen Galea

2. Where Are We With in the Process? Entering stream is concentrated with, insulin ester, and denatured proteins This separation technique separates anionic insulin ester from denatured ester and proteins Two streams leave process Impurities Insulin ester for hydrolysis reaction

3. Principles of Ion Exchange Charged resin beads packed in a column Proteins stick to resins by electrostatic interactions If solution is below the isoeletric point of the protein, cation exchange will occur (protein will have net + charge) If solution is above the isoeletric point of the protein, anion exchange will occur (protein will have net – charge)

4. Principles of Ion Exchange Proteins will be exchanged with the free ion around the resin beads – binding will occur Elution of protein involves a change in pH (or addition of a salt or solvent) Changes the charge on the protein – release of the protein

5. Anion Exchange Resins Insoluble polymeric beads Manufactured by polymerization of styrene and divinylbenzene (DVB) DVB acts as a crosslinking agent and without styrene would be water soluble Active groups (+) are covalently attached to bead balanced by equivalent number of oppositely charged (-) free ions

6. Anion Exchanger 3 steps to Anion Exchange Binding Wash Elution

7. Anion Exchanger Binding – inlet to column runs through 100% of insulin esters bind to resin beads Wash with NaOH/Water (high pH) several times Removes everything except insulin ester Elute column with NaOH/Water (pH close to pI of protein – 5.4) Insulin ester changes from a negative charged to neutral Released from beads – 90% yield

8. Anion Exchange Elution process simultaneously replenishes the negatively charged free ions around the resin (OH - ) Purified insulin ester stream then undergoes hydrolysis Multiple columns to make a continuous process

9. Proposed Design Assumptions Batch = 24 hours Flow entering = 67.9 kg/h Bed Capacity = 0.01 kg/L Estimated Dimensions Bed Volume = 300 L (selected) Bed Diameter = 1.5 m Bed Height = 0.17 m Diameter larger than height of bed, to reduce pressure drop

10. Suppliers Column Difficult to find – must be custom made Amersham Biosciences Resin Based on what is being removed Must have high capacity to retain proteins

11. Suppliers Resin Amersham Biosciences – Source 15Q Strong Anion Exchange resin Good for protein separation Problem Binding Capacity: 4.5X10 -3 kg/L Low flow rate

12. Alternatives Different types of elution can be used, however, more sophisticated control systems must be used to ensure appropriate amount of protein is removed More testing required for this process.

13. Questions?