|
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.
|
