1. IEX Chromatography Presented by: Nikki Apostolakis Helen So Tiffany Yu CHEE450: Engineering Biology

2. Chromatography Introduction The term “chromatography” applies to a wide range of separation techniques that are based on the differential interaction of molecules between a moving phase and a stationary phase. Different column chromatography methods exploit different properties of the protein; Ion exchange chromatography separates molecules based on charge. Column chromatography is a commonly used technique for protein purification. Anion Exchanger Cation Exchanger vs.

3. Ion Exchange Principles There are four main steps to IEX Chromatography: o Equilibration o Load and Wash o Elution o Regeneration Resins are the medium for separation and represent the stationary phase. The surface of these particles provide sites for selective binding. Separation is based on reversible adsorption of ions onto resin

4. Ion Exchange Principles EquilibrationStationaryLoadWashElutionRegeneration

5. Ion Exchange Purpose Intermediate separation and purification of MABs from bulk of impurities Inlet stream Composition: o Acetic Acid o Impurities o MAB o Polysorbate 80 o WFI

6. Design Considerations COLUMN DESIGN Flow rates Aspect ratio (H, d) Column material Space constraints COLUMN INTERNALS Packing material Buffer pH’s Buffer composition

7. Chosen Design Chromaflow Columns validated Easily validated Cost-effective Cost-effective, safe packing/unpacking Appropriate Appropriate for most chromatography techniques Suitable Suitable for large scale GMP production

8. Chosen Design Resin Type: Sepharose High Performance Resin Volume: 207.88 L Column Type: GE Chromaflow Column Dimensions: Inner diameter: 1800 mm Height: 100-300mm Pressure Rating: 3 bar Chromaflow Columns

9. IEX Column Costing BPG BioProcess Columns

10. Alternatives Can make batch process continuous by running columns in parallel Change resin to have impurities bind and allow MAB to pass CONFIGURATION SEPARATION Use a different basis for separation (e.g. molecular size) Downstream Processing

11. THE END THANKS FOR LISTENING. ANY QUESTIONS?