S-SepharoseC-101Andy HunterAlec MackenzieTrevor Seelert

Purpose of UnitSeparate Proinsulin and other

proteins from basic impuritiesPrepare the stream for further

downstream purification

Cation exchange Chromatography

EquilibrateLoadRinseEluteWashRegenerate

Process Flow Sheet

Process Flow Sheet

NewDesign

Process Flow Sheet

NewDesign

S-Sepharose DesignScale-up keeping constant values

Constant Parameter

Vairable Parameters

Column Height Column diameter

Residence time Volumetric flow rates

Linear velocities Amount of eluent

Rinse bed volume

S-Sepharose Design

Parameter Lab Scale Process Scale

Column Height 0.8 m 0.8 m

Residence time 0.376991h 0.376991h

Load linear velocity 212.207 cm/h 212.207 cm/h

Eluent linear velocity 11 936.62 cm/h 11 936.62 cm/h

Column Diameter 0.1 m 1.574 m

Load Volumetric Flowrate

N/A 4.1288 m3/h

Scaled-up from US 101013 Patent

S-Sepharose Design

GE Chromaflow GE AxiChrom

S-Sepharose DesignGE Chromaflow ColumnCurrently max D = 1200mm, H =

30cmCustom Design available for D >

1200mm

Economics and Suppliers

Unit Component Unit Cost

S-Sepharose $1000/L

Annual Report 601.35 L/yr

Total $601 350/yr

Chromatography column $241 000

Other plant direct costs $488 000

First Year TOTAL $1330350

S-Sepharose available from GE Healthcare

Chromaflow supplied by GE

IEC AlternativesSP-Sepharose: Sulphopropyl

–O–CH2CHOHCH2OCH2CH2CH2SO3–

S-Sepharose: Sulphoethyl no longer produced –O–CH2–CH2–SO3–

Replaced by SP-SepharoseAnion exchange resin

Q-sepharose DEAE

HIC ColumnsSEC/Gel Filtration

IEC Alternatives

DEAE: in accordance with German Patent 26 29 568

S-sepharose: in accordance with US Patent 5 101 013

Exchanger DEAE anion exchanger S-Sepharose cation exchanger

Matrix Vinyl polymer Crosslinked agarose gel

Buffer, gradient pH 9, 0.1-0.25 M NaCl pH 3.5, 0.15-0.35M NaCl

Loading 1 g/l 3 g/l

Yield 60% 80%

Purity 80-85% 90-95%

Separation Incomplete separation Base 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!