1. S-Sepharose C-101 Andy Hunter Alec Mackenzie Trevor Seelert

2. Purpose of Unit Separate Proinsulin and other proteins from basic impurities Prepare the stream for further downstream purification

3. Cation exchange Chromatography Equilibrate Load Rinse Elute Wash Regenerate

4. Process Flow Sheet

5. New Design

6. Process Flow Sheet New Design

7. S-Sepharose Design Scale-up keeping constant values Constant ParameterVairable Parameters Column Height Column diameter Residence time Volumetric flow rates Linear velocities Amount of eluent Rinse bed volume

8. S-Sepharose Design ParameterLab ScaleProcess Scale Column Height 0.8 m Residence time 0.376991h Load linear velocity 212.207 cm/h Eluent linear velocity 11 936.62 cm/h Column Diameter 0.1 m1.574 m Load Volumetric Flowrate N/A4.1288 m3/h Scaled-up from US 101013 Patent

9. S-Sepharose Design GE Chromaflow GE AxiChrom

10. S-Sepharose Design GE Chromaflow Column Currently max D = 1200mm, H = 30cm Custom Design available for D > 1200mm

11. Economics and Suppliers Unit ComponentUnit Cost S-Sepharose$1000/L Annual Report601.35 L/yr Total$601 350/yr Chromatography column$241 000 Other plant direct costs$488 000 First Year TOTAL$1330350 S-Sepharose available from GE Healthcare Chromaflow supplied by GE

12. IEC Alternatives SP-Sepharose: Sulphopropyl  –O–CH 2 CHOHCH 2 OCH 2 CH 2 CH 2 SO 3 –  S-Sepharose: Sulphoethyl  no longer produced  –O–CH 2 –CH 2 –SO 3 –  Replaced by SP-Sepharose Anion exchange resin  Q-sepharose  DEAE HIC Columns SEC/Gel Filtration

13. IEC Alternatives DEAE: in accordance with German Patent 26 29 568 S-sepharose: in accordance with US Patent 5 101 013 ExchangerDEAE anion exchanger S-Sepharose cation exchanger MatrixVinyl polymerCrosslinked agarose gel Buffer, gradientpH 9, 0.1-0.25 M NaClpH 3.5, 0.15-0.35M NaCl Loading1 g/l3 g/l Yield60%80% Purity80-85%90-95% SeparationIncomplete separationBase line separation Comparison of IEC

14. No Questions Allowed Disclaimer: The views presented in this slide are not necessarily those of any or all team members.

15. Just Kidding!