S-Sepharose C-101 Andy Hunter Alec Mackenzie Trevor Seelert

Purpose of Unit Separate Proinsulin and other proteins from basic impurities Prepare the stream for further downstream purification

Cation exchange Chromatography Equilibrate Load Rinse Elute Wash Regenerate

Process Flow Sheet

New Design

Process Flow Sheet New Design

S-Sepharose Design Scale-up keeping constant values Constant ParameterVairable Parameters Column Height Column diameter Residence time Volumetric flow rates Linear velocities Amount of eluent Rinse bed volume

S-Sepharose Design ParameterLab ScaleProcess Scale Column Height 0.8 m Residence time h Load linear velocity cm/h Eluent linear velocity cm/h Column Diameter 0.1 m1.574 m Load Volumetric Flowrate N/A m3/h Scaled-up from US Patent

S-Sepharose Design GE Chromaflow GE AxiChrom

S-Sepharose Design GE Chromaflow Column Currently max D = 1200mm, H = 30cm Custom Design available for D > 1200mm

Economics and Suppliers Unit ComponentUnit Cost S-Sepharose$1000/L Annual Report L/yr Total$ /yr Chromatography column$ Other plant direct costs$ First Year TOTAL$ S-Sepharose available from GE Healthcare Chromaflow supplied by GE

IEC Alternatives SP-Sepharose: Sulphopropyl  –O–CH 2 CHOHCH 2 OCH 2 CH 2 CH 2 SO 3 –  S-Sepharose: Sulphoethyl  no longer produced  –O–CH 2 –CH 2 –SO 3 –  Replaced by SP-Sepharose Anion exchange resin  Q-sepharose  DEAE HIC Columns SEC/Gel Filtration

IEC Alternatives DEAE: in accordance with German Patent S-sepharose: in accordance with US Patent ExchangerDEAE anion exchanger S-Sepharose cation exchanger MatrixVinyl polymerCrosslinked agarose gel Buffer, gradientpH 9, M NaClpH 3.5, M NaCl Loading1 g/l3 g/l Yield60%80% Purity80-85%90-95% SeparationIncomplete separationBase line separation Comparison of IEC

No Questions Allowed Disclaimer: The views presented in this slide are not necessarily those of any or all team members.

Just Kidding!