Unizyme Laboratories A/S and its spin-out Prozymex A/S

Publiceret Oktober 2001

UNIZYME Laboratories A/S (www.unizyme.com) is an R&D-based biotechnology firm, established in 1989. UNIZYME develops peptidases and accompanying enzymatic methods for use in the biopharmaceutical industry as a means of producing recombinant protein drugs. The main part of UNIZYME's enzyme production is carried out in accordance with GMP (Good Manufacturing Practice).

UNIZYME is a small firm with 8 employees in R&D, production, quality control and Business Development. About 65% of the firm's resources are devoted to research and development in gene technology, protein expression and protein chemistry.

For a small company like UNIZYME to be able to develop new innovative products, collaboration with research institutions and other biotech firms is essential. Over the years, UNIZYME has established links with a number of research institutions and companies, including the Department of Molecular Biology at Aarhus University, the Centre for Crystallographic Studies (Sine Larsen) and Dept. of Chemistry (Jens Jørgen Led) at HCØ Institute, the J. Stefan Institute (Slovenia), Pharmarex (Hørsholm, Denmark; previously M&E Biotech), Bioteknologisk Institut (Hørsholm, Denmark), University of Southampton, the University of Southern Mississippi and University of Nottingham.

UNIZYME has an 800 m2 laboratory complex at the Research Centre at Hørsholm, which contains R&D laboratories, and production and administration. The R&D laboratories can be expanded from 225 m2 to about 450 m2 within the existing building.

UNIZYME?s most important enzyme product to date is dipeptidyl peptidase I (DPPI; also called dipeptidyl aminopeptidase I, DAPI or cathepsin C), a cysteine protease which selectively splits off dipeptides from the N-terminal of proteins and peptides. This enzyme has exciting possibilities in connection with methods for fast and convenient purification of proteins, as described below.

Cleavage of poly-histidine tags (HisTags) from recombinant proteins

To facilitate functional and structural studies of proteins derived from the rapidly growing number of genes coming out of genome programmes such as the Human Genome Project, efficient and robust production and purification strategies are necessary. For industrial production of recombinant proteins, simple and fast purification methods introduced as an early unit operation can improve the overall economy of the process. One powerful purification technique, made possible by genetic engineering, is to purify the target protein by means of a genetically fused affinity fusion partner. Such fusion proteins can often be purified to near homogeneity from crude biological mixtures by a single, and fusion-partner-specific, affinity chromatography step.

The addition of a histidine-rich peptide tag (HisTag) to the target protein is a simple and well-established approach for generating a novel affinity for metal ions, making one-step purifications possible by using immobilized metal affinity chromatography (IMAC). IMAC matrices have a number of advantages, including high protein-binding capacity and ligand stability, low cost, and use of mild elution conditions. Furthermore, because of their chemical nature, IMAC matrices can easily be sanitized and regenerated, making them suitable for large-scale applications. For some applications of the purified proteins, the HisTag does not need to be removed. However, when the recombinant protein is intended for structural/physiological studies or pharmaceutical use, the HisTag must be removed to obtain the protein with the correct amino acid sequence, thus avoiding unpredictable properties.

UNIZYME Laboratories A/S have developed a specific method (The TAGZymeTM system) for the removal of N-terminal HisTags by means of exopeptidases. This method is based on the use of DPPI alone or in combination with glutamine cyclotransferase (GCT) and pyroglutamyl aminopeptidase (PGAP). These enzymes all have the ability to bind to IMAC matrices through an engineered HisTag in recombinant forms of DPPI (HT-DPPI, DAPaseTM), PGAP (HT-PGAP, pGAPaseTM) and GCT (HT-GCT, QcyclaseTM). This feature has been utilised in the design of a simple process consisting of aminopeptidase cleavage and subtractive IMAC. The system has the potential of forming the backbone in a combined IMAC/TAGZyme strategy for the efficient production of highly purified and homogeneous recombinant proteins.

Production and distribution

Today, DPPI extracted from natural sources is used in the production of a well-established pharmaceutical protein preparation, of which more than 10 kg a year is produced. In addition to this, a recombinant version of DPPI is used in combination with PGAP and GCT in the above-mentioned TAGZymeTM system, which is used and tested by more than 20 biopharmaceutical firms. UNIZYME market the TAGZyme system in the large-scale market in Denmark itself, while from summer 2001 it has been marketed worldwide in both the R&D and large-scale market by QIAGEN (www.qiagen.com), a major biotech company, which already sells IMAC column matrices and other reagents used in connection with polyhistidine tags.

Prozymex A/S – a spinout of UNIZYME Laboratories A/S

In addition to biotechnological applications, DPPI also plays a central role in several immune/inflammatory conditions. DPPI is expressed in many tissues and has generally been associated with protein degradation in the lysosomes. More recently, DPPI has also been assigned an important role in the activation of many granule-associated serine proteinases, including chymase and tryptase from mast cells, cathepsin G and elastase from neutrophils, and granzyme A, B and K from cytotoxic lymphocytes (CTL, NK and LAK cells). These immune/­inflammatory cell proteinases are translated as inactive zymogens and the final step in the conversion to their active forms is a DPPI-catalysed removal of an activation dipeptide from the N-terminus of the zymogens. DPPI -/- knock-out mice have been shown to accumulate the inactive, dipeptide extended proforms of the mast cell specific proteases tryptase and chymase and the pro-apoptopic proteases granzyme A and B.

Many of the granule-associated proteases, which are activated by DPPI, serve important biological functions and inhibition of DPPI may thus be a general means of controlling the activities of these proteases in certain immune/inflammatory diseases and conditions.

In 1999, UNIZYME therefor set up PROZYMEX A/S with a view to further commercializing the knowledge and know-how related to DPPI. From UNIZYME, PROZYMEX has acquired considerable and unique knowledge on the enzyme and its properties, including manufacture by means of recombinant techniques. PROZYMEX have been able to isolate the pro-form of DPPI and to solve the activation pathway of DPPI in collaboration with the J. Stefan Institute (Slovenia). Furthermore the Centre for Crystallographic Studies at HCØ and the J. Stefan Institute in collaboration with PROZYMEX have solved the three-dimensional crystal structure of DPPI. PROZYMEX has filed patent applications covering these discoveries, including methods for design of inhibitors of this protease. We believe that these discoveries opens up therapeutic possibilities stemming from this proprietary knowledge of the DPPI target.

PROZYMEX already has established links to research groups with expertise in computer aided drug design, determination of X-ray crystal structures and cell biology. Furthermore we are now working on establishing

  • contacts to individuals and research institutions with expertise and interest in drug development and immune/inflammatory diseases
  • in-house expertise in structure-based drug design
  • a joint-venture with a company with expertise and IPR in the areas of synthesis of cysteine protease inhibitors and medcinal chemistry
  • collaborations with experienced biotechnology and pharmaceutical companies for further pre-clinical and clinical development of inhibitors against DPPI.