1. Anion Exchanger Mike LaVallee Shane Nagle Matt Rossiter

2. Outline Background Why an anion exchanger? Alternatives Design Resins Cost of chosen design Questions

3. Background Ions are electrostatically bound to an insoluble and chemically inert matrix – Anion exchangers bear positively charged functional groups that bind negatively charged particles. During elution the anion exchanger can be described by the following chemical equation:

4. Why an anion exchanger? Assumptions Denatured insulin had a 0% binding efficiency Insulin-Ester had a 100% binding efficiency and 90% recovery Isoelectric point of Denatured Insulin is higher than Insulin-Ester Component Flowrate (kg/batch) Mass Percent (%) Conc. (g/L) Denatured Insulin0.43590.030.30 Insulin-Ester296.3820.22202.25 Water1168.6579.75797.46

5. Anion Exchanger Advantages Capable of handling large volumes Efficient, precise separation – Accurate separation of Insulin ester and denatured Insulin 1

6. Large pressure drop – Require expensive pumps High resolution requires small beads – Small beads decrease flow rate Each cycle requires several stages – Charging, sample input, washing, elution, cleaning Anion Exchanger Disadvantages

7. Alternatives Membrane Chromatography Exclusion Chromatography Affinity Chromatography 23 4

8. Membrane Chromatography Allows for much larger throughput (about 100 times that of ion exchange) and a higher efficiency Uses microporous membrane Small pressure drop compared to traditional ion exchangers Scaling up separation is simplified

9. Exclusion Chromatography Separates on basis of Molecular Mass Column is filled with semi- solid beads of a polymeric gel – The porosity of the gel can be changed as to exclude molecules of a set size Useful for samples containing many types of proteins 5

10. Affinity Chromatography Matrix anchored ligand – Specifically binds to protein of interest Can exploit protein’s unique biochemical properties instead of charge in ion exchange Requires low flowrates 8

11. Anion Exchanger Design Ergun Equation for pressure drop in a packed bed

12. Design Considerations PropertyDimension Bead Diameter, D P 35μm Column Lenth, L3.47m Column Diameter, D1.3 m Pressure Drop, ΔP1718 kPa Void Fraction, ε0.4 Vessel Volume4604 L All constants were taken from Perry’s Chemical Engineering Handbook 7

13. Vessel Sizing Exchanger is very large – Much larger than needed, however, this will save on pumping costs Total flow rate through the anion exhanger is 1.721 x 10 -5 m 3 /s – Based on 80h batch time and 826kg/batch – Scaled up by a factor of 6

14. Vessel Schematic

15. P&ID 1

16. Resins Serves as the media for stationary phase – Polymeric matrix with immobilized charged functional groups Quaternary Ammonium functional group Diethylaminoethane functional group Can be regenerated in the columns and used for many production cycles – anionic resin: 500 - 1,000 cycles 8

17. Estimated Costs for Anion Exchanger UnitCost/Unit# of UnitsTotal Anion Exchanger$261,0001 Resin~9 $/L110$910 Peripheral Equip.$50,000N/A$50,000 Shipping$2,0001 Installation$100,000N/A$100,000 Controls/Software$25,0001 Total Equip. Cost$438,910

18. Estimated Operation Costs / Year OperationTotal Utilities$10,000 Maintenance$5,000 Supplies$1,000 Total Operations$16,000 *Overhead costs, taxes and insurance costs have not been included in this estimate The total cost associated with the purchase of an Anion Exchanger with one year of operation is approximately $454,910.

19. References [1] PowerMax, 2006 http://www.usfilter.com/ [2] Pall Corporation, 2006 (http://www.pall.com/datasheet_biopharm_36585.asp) [3] Protein Chemistry, 2006 (http://fig.cox.miami.edu/~cmallery/255/255tech/255techniques.htm) [4] Voet & Voet Biochemistry 3 rd Edition, Wiley, 2004 [5] Waters Corporation, 2006 (http://www.waters.com/watersdivision/) [6] Novasep Technologies, 2006 (http://www.novasep.com/technologies/ion-exchange.asp)http://www.novasep.com/technologies/ion-exchange.asp [7] Graver Technologies, 2006 (http://www.gravertech.com/pdfs/literature/ionex/IonExchangeResinSelection.pdf)http://www.gravertech.com/pdfs/literature/ionex/IonExchangeResinSelection.pdf [8] OR-Live, 2003 http://www.biopharminternational.com/

20. Questions ?