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The TAGZyme  System

The TAGZyme  System. September 2004. Natural DAPase stop. M. K. H. Q. H. Q. H. Q. H. Q. H. Q. H. Q. Target Protein. COOH. A. P. Target Protein. COOH. K. I. Natural DAPase stop. Target Protein. COOH. R. L. The TAGZyme  system (I). DAPase. DAPase. DAPase.

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The TAGZyme  System

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  1. The TAGZyme System September 2004

  2. Natural DAPase stop M K H Q H Q H Q H Q H Q H Q Target Protein COOH A P Target Protein COOH K I Natural DAPase stop Target Protein COOH R L The TAGZyme system (I) DAPase DAPase DAPase DAPase DAPase DAPase DAPase DAPase DAPase

  3. QCyclase QCyclase M K H Q H Q H Q H Q H Q H Q Target Protein NH2 COOH QCyclase QCyclase The TAGZyme system (II) DAPase DAPase DAPase DAPase DAPase DAPase DAPase DAPase DAPase pGAPase QCyclase Q Q

  4. Crude His-tag protein Purified His-tag protein Target Protein COOH NH2 The TAGZyme downstream process IMAC purification (immobilized metal affinity chromatography) Cleavage with His-tagged DAPase Removal of enzymes and contaminants by subtractive IMAC Purified Skip DSP

  5. Crude urea/GuCl Solubilized Histag-hGH 1 2 3 4 5 6 7 Purified Histag-hGH Lane 2 Histag-hGH  hGH Lane 3: 5 min Lane 4: 10 min Lane 5: 20 min Lane 6: 30 min Lane 7 hGH COOH NH2 Production of human growth hormone (hGH) Fast buffer-exchange IMAC purification Fast buffer-exchange TAGZyme cleavage/refolding Subtractive IMAC • Purification scales • Up to 200 mg • Typical recoveries • IMAC purification : 95-98 % • Cleavage/subtractive IMAC : 90-95 % Skip DSP

  6. Target Protein Q Q M K H Q H Q H Q H Q H Q H Q NH2 COOH 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

  7. 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

  8. Contact information: José Arnau, PhD Unizyme Laboratories A/S Dr. Neergaardsvej 17 DK-2970 Hørsholm DENMARK Email: ja@unizyme.dk Web: http://www.unizyme.com Tel: +45 45760154 Mobile: +45 40176233 Fax: +45 45761407

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