The TAGZyme System

September 2004

COOHTarget ProteinPADAPaseDAPase

The TAGZyme system (I)

QQHH HH QQ HH QQMM KK HH QQ HH QQ HH QQDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPase DAPaseDAPaseDAPaseDAPase

COOHTarget ProteinIK

COOHTarget ProteinLR

Natural DAPase

stop

Natural DAPase

stop

QQQQpGAPasepGAPaseQCyclaseQCyclaseDAPaseDAPase

The TAGZyme system (II)

QQHH HH QQ HH QQMM KK HH QQ HH QQ HH QQDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPaseDAPase DAPaseDAPaseDAPaseDAPase COOHNH2 Target Protein

QCyclaseQCyclaseQCyclaseQCyclase

QCyclaseQCyclaseQCyclaseQCyclase

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

CrudeHis-tag protein

PurifiedHis-tag protein

COOHNH2Target Protein

Purified

Production of human growth hormone (hGH)

Skip DSP

Histag-hGH

hGH

1 2 3 54 6 7

Fast buffer-exchange

TAGZyme cleavage/refolding

Subtractive IMAC

Crude urea/GuClSolubilized Histag-hGH

PurifiedHistag-hGH

IMAC purification

Fast buffer-exchange

COOHNH2hGH

Lane 2

Lane 3: 5 min

Lane 5: 20 minLane 4: 10 min

Lane 6: 30 min

Lane 7

Purification scales•Up to 200 mg

Typical recoveries•IMAC purification : 95-98 %•Cleavage/subtractive IMAC : 90-95 %

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

QQQQHH HH QQ HH QQMM KK HH QQ HH QQ HH QQ QQCOOHNH2

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, PhDUnizyme Laboratories A/SDr. Neergaardsvej 17DK-2970 HørsholmDENMARK

Email: ja@unizyme.dkWeb: http://www.unizyme.com

Tel: +45 45760154Mobile: +45 40176233Fax: +45 45761407