1. Cation Exchange Column CHEE450 Leslie Davis

2. Cation Exchange Following removal of biomass – processes supernatant Separate insulin precursor from glucose, salts & proteins Sends precursor to the diafilter

3. What is Cation Exchange Type of ion exchange chromatography Separates biomolecules Based on electrostatic interactions Charged stationary phase Most widely employed chromatographic separation technique

4. Cation Exchange Chromatography Employs a negatively charged surface Separates positively charged solutes Positively charged proteins (mobile phase) bond to negatively charged stationary phase - - - - - - - - - - + + + + + + + + + + + + + + + +

5. Cation Exchange Chromatography Protein: ampholytic (+ & -) pH & isolectric point determine protein surface charge pH kept below isoelectric point (5.3-5.35) Cause protein to become

6. Process Considerations Stationary Phase Rigid to support high flow rates Provide sufficient selectivity and capacity Not denature protein Stable over a wide range of operating conditions Amenable to rigorous cleaning Should not leach materials

7. Process Considerations SP Sepharose TM Fast Flow Resin Preparative purification with high flow rates For purification of crude samples Easy to scale up 6% highly cross-linked agarose 45-165 μm bead size Strong cationic gel Charged group: -CH 2 CH 2 SO 3 - (sulfopropyl) 70 mg ribonuclease A/mL gel

8. Process Considerations Selection Kits Screen different ion exchange media 1 - 5mL columns

9. Process Considerations Sizing: D/H range between 2 – 4 Short columns recommended to maximize throughput Taller columns: cause increase in cycle time and higher pressure drop Shorter columns: uneven flow pattern and breakthrough of product

10. Column Sizing Required protein throughput442000g per 24 hours Number of columns in parallel3 Cycle time2.5h Operating Gel capacity70g/L Bed Volume274.06L Bed Diameter100cm Bed Height40.13cm D/H ratio2.87 Flow Rate320.31L/column/h Actual volume315.17L

11. Process Wasted material: salts, glucose, biomass, 100% protein contaminants Buffer 0.1M acetate (pH 5.0) Elution 0.1 M NaOH, 2 M NaCl → 5.3 22 kg/batch → 3.2 kg/column/cycle Cleaning or Column Regeneration Washing with 0.1M NaOH, 1M NaCl and 50% EtOH

12. Column Selection Amersham Pharmacia biotech

13. Process Alternatives Anion exchange high negative charge and good solubility of protein Affinity Chromatography Ultra high resolution, removes bioactive contaminants Expensive, not readily amenable to scale up and harsh manufacturing environment (CIP)

14. The End Questions?