Cation Exchange Column
CHEE450
Leslie Davis
Cation Exchange
• Following removal of biomass – processes supernatant
• Separate insulin precursor from glucose, salts & proteins
• Sends precursor to the diafilter
What is Cation Exchange
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Type of ion exchange chromatography
Separates biomolecules
Based on electrostatic interactions
Charged stationary phase
Most widely employed chromatographic
separation technique
Cation Exchange Chromatography
• Employs a negatively charged
surface
• Separates positively charged
solutes
• Positively charged proteins
(mobile phase) bond to
negatively charged stationary
phase
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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
Process Considerations
• Stationary Phase
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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
Process Considerations
• SP SepharoseTM Fast Flow Resin
• Preparative purification with high flow rates
• For purification of crude samples
• Easy to scale up
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6% highly cross-linked agarose
45-165 μm bead size
Strong cationic gel
Charged group: -CH2CH2SO3- (sulfopropyl)
70 mg ribonuclease A/mL gel
Process Considerations
• Selection Kits
• Screen different ion
exchange media
• 1 - 5mL columns
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
Column Sizing
Required protein throughput
Number of columns in parallel
Cycle time
Operating Gel capacity
442000 g per 24 hours
3
2.5 h
70 g/L
Bed Volume
Bed Diameter
Bed Height
274.06 L
100 cm
40.13 cm
D/H ratio
Flow Rate
Actual volume
2.87
320.31 L/column/h
315.17 L
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
Column Selection
• Amersham Pharmacia biotech
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)
The End
Questions?