CHEE450: Engineering Biology IEX Chromatography Presented by: Nikki Apostolakis Helen So Tiffany Yu 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. Column chromatography is a commonly used technique for protein purification. Different column chromatography methods exploit different properties of the protein; Ion exchange chromatography separates molecules based on charge. Anion Exchanger vs. Cation Exchanger 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 Ion Exchange Principles Stationary Equilibration Load Wash Elution Regeneration 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 Design Considerations COLUMN DESIGN • • • • Flow rates Aspect ratio (H, d) Column material Space constraints COLUMN INTERNALS • Packing material • Buffer pH’s • Buffer composition Chosen Design Chromaflow Columns Why the Chromaflow line? • Easily validated • Cost-effective, safe packing/unpacking • Appropriate for most chromatography techniques • Suitable for large scale GMP production Chosen Design Chromaflow Columns Specifications 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 IEX Column Costing BPG BioProcess Columns BPG Family (Model #) ID (mm) Bed Height (cm) Packing Volume (L) Purchase Cost ($) BPG 100/500 100 0-26 0-2 $5,678.40 BPG 100/950 100 45-54 3.5-4.2 $6,171.90 BPG 200/500 200 0-26 0-8.2 $7,677.60 BPG200/950 200 45-54 14.1-16.9 $8,922.90 BPG 300/500 296 0-26 0-17.9 $15,682.80 BPG 300/950 296 45-54 31-37.2 $18,244.80 BPG 450/500 446 3-23 4.7-35.9 $35,631.75 BPG 450/1000 446 47-58 73.4-90.6 $43,548.75 Alternatives CONFIGURATION Can make batch process continuous by running columns in parallel SEPARATION Change resin to have impurities bind and allow MAB to pass Use a different basis for separation (e.g. molecular size) Downstream Processing THE END THANKS FOR LISTENING. ANY QUESTIONS?