Techniques
The separation techniques chosen have potential for further optimisation to meet the overall goals of the purification process.
Ion exchange chromatography
(Sepharose™ Fast Flow, High Performance, Big Beads, SOURCE 30 or 15).
High capacity. Relatively insensitive to flow rate and bed height. Counter ions and pH important for selectivity. Resolution dependent on conditions during binding, washing and the step or gradient during elution. CIP with NaOH.
Hydrophobic interaction chromatography
(Sepharose™ Fast Flow, High Performance, Big Beads, SOURCE 15)
Can have high capacity. Protein:protein binding can occur at high loads, and even precipitation onto the medium, affecting recovery and selectivity. Binding/release kinetics can be slow. Type of salt effects strength of binding and selectivity. Binding conditions extremely important for capacity and resolution. Washing and elution conditions also important for resolution. Step gradient are frequently used. Dilute organic solvent (ethanol, isopropanol, acetonitrile) can be used during elution. CIP with NaOH.
Affinity chromatography
(Sepharose™ Fast Flow, MabSelect™)
Can have high capacity. Binding conditions affect selectivity, capacity and resolution. Washing steps are important to achieve selectivity. Step elution is the norm. Elution conditions can be tough on the product. Special CIP procedures required.
Reversed phase chromatography
(SOURCE™ 30 or 15).
Typically used for polishing. Need for high resolution limits sample capacity. Bed height might be used to achieve resolution. Acetonitrile, isopropanol or ethanol mobile phase. Ion pairing agents increase complexity, difficult to remove from product. Gradient or isocratic or step elution. Ionic strength, salt-type, pH are important variables. Silica-based media cannot be exposed to NaOH.
Gel filtration/ Desalting
(Superdex™, Sephadex™ or Sephacryl™)
Simplest. Only sample volume, bed height and flow velocity need optimization for resolution, speed, capacity, recovery. CIP with NaOH. |
