The TAGZyme System September 2004. COOH Target Protein PA DAPase The TAGZyme system (I) Q Q H H H H Q Q H H Q Q M M K K H H Q Q H H Q Q H H Q Q DAPase.

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The TAGZyme System September 2004

COOH Target Protein PA DAPase The TAGZyme system (I) Q Q H H H H Q Q H H Q Q M M K K H H Q Q H H Q Q H H Q Q DAPase COOH Target Protein IK COOH Target Protein LR Natural DAPase stop Natural DAPase stop

Q Q Q Q pGAPase QCyclase DAPase The TAGZyme system (II) Q Q H H H H Q Q H H Q Q M M K K H H Q Q H H Q Q H H Q Q DAPase COOHNH 2 Target Protein QCyclase

The TAGZyme downstream process Skip DSP IMAC purification (immobilized metal affinity chromatography) Cleavage with His-tagged DAPase Removal of enzymes and contaminants by subtractive IMAC Crude His-tag protein Purified His-tag protein COOH NH 2 Target Protein Purified

Production of human growth hormone (hGH) Skip DSP  Histag-hGH  hGH Fast buffer-exchange TAGZyme cleavage/refolding Subtractive IMAC Crude urea/GuCl Solubilized Histag-hGH Purified Histag-hGH IMAC purification Fast buffer-exchange COOH NH 2 hGH Lane 2 Lane 3: 5 min Lane 5: 20 min Lane 4: 10 min Lane 6: 30 min Lane 7 Purification scales Up to 200 mg Typical recoveries IMAC purification: % Cleavage/subtractive IMAC: %

The TAGZyme system Used for both intracellular and secreted proteins Successfully tested in different production hosts (E. coli, insect cells, etc) Scaled up to 2 gram scale Successfully tested on more than 200 different proteins The TAGZymes can be produced in bulk quantities For more than 10 years, DPPI has been used for production of a pharmaceutical protein (~10 kg/year) Currently being tested at other pharmaceutical companies Q Q Q Q H H H H Q Q H H Q Q M M K K H H Q Q H H Q Q H H Q Q Q Q COOHNH 2 Target Protein

The TAGZyme downstream process: strengths IMAC matrices have high protein-binding capacity and selectivity Robust and simple chromatographic procedures with high recovery (> 90 %) IMAC matrices are chemically stable to prolonged CIP procedures Tag sequences can be optimized for efficient expression levels Complete and specific removal of N-terminal His-tags, -i.e. the correct N-terminus is obtained without non-specific internal cleavage Simultaneous removal of both processing enzymes and residual contaminants by subtractive IMAC Process is scalable from R&D to production

Contact information: José Arnau, PhD Unizyme Laboratories A/S Dr. Neergaardsvej 17 DK-2970 Hørsholm DENMARK Web: Tel: Mobile: Fax: