Icosagen Story
by Mart Ustav
 Tartu Biotechnology Park
 29.04.2015
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Curriculum vitae
Mart Ustav
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3.
4.
5.
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7.
Education – Organic and Bioorganic Chemistry, 1972, Univ. Of Tartu
Military service – 1972-1974
Institute of Cybernetics, Tallinn – researcher, enzyme kinetics 1975
Ph.D. – rRNA-protein interactions, Tartu University, 1976-1979
Post-doctoral Fellow – Uppsala University 1982-1985
Senior scientist, Head of Laboratory of Oncogenesis, UT 1985-1989
Visiting scientist – Cold Spring Harbour Laboratory, Long Island, NY,
USA (1989-1992)
8. Professor of Microbiology and Virology 1992-2007, Univ. of Tartu
Director of the Institute of Molecular and Cell Biology 1997-2001
9. Howard Hughes Medical Institute Fellow, USA 1995-2005
10. Member of the Academy of Sciences of Estonia 2001
11. Professor of Biomedical Technology 2007….,
Director of the Institute of Technology, University of Tartu, 20022007, 2012….
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The first 10-years story
1999
• Quattromed was founded as a molecular diagnostic company providing
services to Estonian medical institutions. 2001 FIT Biotech Oy acquired
22.3% of the Quattromed shares and established FIT Biotech Oy Eesti
filiaal
2005
• Diversification of medical services and establishment of Quattromed Cell
Factory as a subsidiary company
2008
• A leading medical diagnostics and biotechnology group in Estonia,
80 FTE; revenue EUR 3.5M
• Activities:
• medical diagnostics: molecular diagnostics, clinical chemistry, hematology,
cytology, immunology etc
• molecular- and cell biology products and services
• immunoanalysis products and services for detection of allergic causativ
proteins in rubber products
• Q3: the medical diagnostic subsidiary along the trademark Quattromed was
sold to private equity firm. Restructuring of the group and business model.
• Establishment of Icosagen Group
www.icosagen.com
Icosagen Group
 CEO Mart Ustav, professor of biomedical technology,
University of Tartu
 49 FTE, 8 PhDs
 ISO 9001:2008, ISO 17025, GLP
 6 patent families/25 patents (EU, US, CA, JP, AU, CH, IN)
 Partners in several international collaboration projects
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Biotech and Protein Production
Company in Estonia
Icosagen Cell Factory
Eerika tee 1, Ülenurme vald, 61713
Tartumaa, Estonia
Tel: +372 737 7070
E-mail: info@icosagen.com
Tartu, Estonia
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Icosagen Group
Icosagen
Management, financing, QC/QA, Sales&Marketing
Products/Services: catalog products (antibodies,
proteins, ELISA kits);
food safety/quality control.
Icosagen Cell Factory
R&D, Business Development
Products/Services:
technology licensing,
protein production services
IcoPark
Established in 2013.
Development the
infrasturcture of Icosagen
Group
Icosahedron with 20 identical tringular facets.
Icosagen, a company of variety of options for every facet of
icosahedron
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Technology Developer and Service Partner
for Global Pharma and Biotech Industry
Development and production
of recombinant proteins
Development and sales of
catalogue products
Proteins, Poly- and monoclonal
antibodies, VLPs
Antibodies, proteins, ELISA kits
Business Fields
Quality control laboratory
testing services
Food microbiology, latex allergen
testing
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Collaborative research
and development,
technology licensing
Technology Developer and Service Partner
for Global Pharma and Biotech Industry
Animal Cell as the Factory –
Design, Engineering and
Exploitation
Mart Ustav
Bio- and medtech business: real stories and opportunities
Tallinn, February 12th, 2015
Market, drugs and money
 Market for prescription drugs in 2013 was
559 billion USD.
 Market for prescription drugs in 2020 will be
793 billion USD.
 32% of all drugs approved by FDA during
last 10 years are produced from cultured
cells – it means that these are developed
also using cultured cells
 60% of all new drugs will be biologics
(proteins, antibodies etc.)
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QMCF Technology
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Two Kinds of Technologies are Available for Protein Production
for drug development
Transient System, where
proteins are expressed from
extrachromosomal plasmids
Stable Cell Lines, where proteins
are expressed from the chromosomes
However, there is a huge gap between them!
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QMCF System Consists of Two Components
CHO85 cell line that expresses
factors for plasmid maintenance
and replication.
QMCF plasmids that carry elements
for replication and mainenance.
Origin of replication
(Py LT)
Maintenance
(EBNA1)
EBNA1
Py LT
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Chromatin attachment
N-terminal TA domain of E2 is
responsible for chromatin attachment
Sufficient number of E2 binding sites, defined as
minichromosome maintenance element (MME)
McBride, 2006
E2
TA
hinge
DBD
QMCF Plasmids Are Maintained in Dividing Cells
pQMCF plasmids are maintained
at the level of ~200 copies/cell
Conventional plasmids get
lost in dividing cells
Plasmid
Chromosome
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QMCF Plasmids Are Maintained in Dividing Cells
Southern blot analysis of hNGF and human IgG1 antibody expression
vector 48 hrs and 16-18 days after transfection (doubling time ~15 h)
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QMCF Technology Is Scalable and More Convenient Than Transient
Protein Production
In transient system, transfection has to be done in a large volume, few days before protein production
In QMCF system is scalable and transfection is done conveniently in a small volume
Therefore QMCF Technology is also well suited for the High-Throughput Screening applications
Volume of the
cell culture
Transient
transfection
(1 L culture,
(1mg DNA)
16 L
Start of the production,
Shift to 30 oC
cell culture
expansion
4L
1L
0.25 L
60 mL
15 mL
QMCF
transfection (1 mL culture, 1mg DNA)
4 mL
1 mL
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1 2
4
6
8 10 12
Time (days)
14
16
…
pQMCF-T Vectors Are Superior for Transient Production
Objective: In order to increase the productivity of QMCF system,
we inserted replication enhancer into the pQMCF vectors (T-plasmids)
pQMCF
pQMCF-T
Results:
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Relative Plasmid
Copy Number
Relative
Productivity
Antibody
CDNF
Antibody
CDNF
pQMCF
1.0
1.0
1.0
1.0
pQMCF-T
3.0
3.2
1.5
2.6
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CDNF
QMCF Technology Bridges „the Gap“ in Protein Production
Transient systems are the best for a fast production of small-scale protein amounts. However, it
is not feasible if large amounts of proteins are required.
Production of large amounts of protein demands cell line development and stable expression of
your favourite protein.
Icosagen Cell Factory has developed QMCF technology to optimize the mid-scale protein
production.
Stable
cell lines
QMCF
Transient
suitable
unsuitable
0.01
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0.1
1
10
100
…
Protein
quantities
(grams, IgG)
Icosagen Cell Factory
Provides Protein Production Services
by Using QMCF Technology
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Small Scale Protein Production Services (<100mg of Protein (IgG))
Week 1
Week 2
Week 3
We use NOVEL peptide-based Transfection Reagent 007.
Transfection efficiency is 80-95% in CHO85 cells with excellent cell recovery
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Transfection Agent 007

New generation peptide-based
vehicle for efficient delivery of
nucleic acids for the transfection
of mammalian and insect cells
Arukuusk, P. et. al.. Biochim Biophys Acta. 2013 May;1828(5):1365-73
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Transfection effieciency is
up to 95% in CHO cell lines
in seerum-free conditions with
an excellent cell recovery
60
40
20
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EB
NA
LT
85
CH
O
-S
0
CH
O

EGFP%
80
Medium Scale Protein Production (<10g of Protein (IgG))
Together with Cell Bank Generation!
 Cell bank generation in 2 weeks after transfection
Production cell
bank generation
Week 1
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Week 2
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Week 2-3
Week 4
Week 5
Stable Production from QMCF Cell Bank
1st batch from
transfection (mg/L)
h-IgG1
-
144 + 21
Production of the GDNF-family neural
growth factor by using CHOEBNALT85
suspension cell line. Production were
started from two different cell banks
independently (lanes 1 and 2).
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2nd batch from
WCB (mg/L)
-
186 +12
Storage period of
cell bank
12 months
QMCF Technology Licensing
 Feasibility License
Technology evaluation in 6 month period
 Research License or Limited Research License
In-house activities
 Commercial License
Production of catalog products, or diagnostic kit, or custom
production services for third parties, etc
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QMCF Technology Applications:
Designing New CHO Cell Lines
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CHO is Great, but it is not Perfect!
Modifications can be introduced into CHO cell lines to improve their production
properties.
These modifications include introduction, upregulation or downregulation of
certain cellular factors:
• Components of post-translational modification pathways (e.g. protease
cleavage, glycosylation)
• Factors related to cellular growth and/or metabolism
• Components of secretion machinery
Many combinations have to be analysed in order to determine the effects and
side effects. Usually it is done by genome editing, which is time-consuming and
expensive.
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Designing New CHO Cell Lines by Using QMCF System
QMCF system is a useful tool for designing novel CHO cell lines:
 Modifications are tested first in QMCF system and then the most optimal
configurations are used for engineering new CHO cell lines.
For this purpose we designed pQMCF vectors with two expression cassettes
or
Protein of
interest
Expression
Factor
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shRNA
Protein of
interest
Production of Mature Proteins by Protease Co-expression
• Furin is an endopeptidase responsible for the proteolytic maturation of many
precursor proteins in mammalian cells.
• The levels of furin are very low in most cells (including CHO cells).
Objective: To achieve pro-protein maturation by the co-expression of hFurin from
pQMCF plasmid
Results:
pro-protein
pro-protein
hFurin
mature protein
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CHO85 hFurin Cell Line (3A5) for Complex Protein Production
Based on the positive results from pQMCF plasmid, we have generated CHO85 cell
line (3A5) with stable expression of hFurin.
Target protein (48h)
Furin
M
hFurin does not
affect cell growth
Cell #
Cell Growth
1.00E+10
Furin
pro-GDNF
1.00E+09
1.00E+08
GDNF
1 2 3
CHO85 3A5
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1.00E+07
1.00E+06
0 1 2 3 4 5 6 7 8 9 days
3A5 pQ3
CHO85 pQ3
3A5 pQ3T
CHO85 pQ3T
Production of Glycoproteins in CHO85 Cells with
Downregulated Slc35A2
Slc35A2 transports UDP-galactose from the cytosol into Golgi vesicles where
glycosyltransferases function.
Objective: To increase the homogenicity of glycosylated proteins
by downregulation of Slc35A2
Case study:
Transient production of EPO in CHO85 cells
EPO
Isoelectric focusing
EPO
EPO
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QMCF Technology Applications:
Development of Monoclonal Antibodies
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Two Approaches Are Used for the Development of Monoclonal
Antibodies (mAbs)
Immunization
B-cells
Hybridoma
based methods
Recombinant
methods
mAbs
mAbs
+
information about
antigen binding site
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Development of Recombinant mAbs
by Using QMCF Technology
Isolation of VH and VL coding regions
and generation of enriched scFv-Fc
library in pQMCF plasmids
Panning:
Enrichment of antigen
specific B-cells
VH
VL
Fc
VH
VL
Fc
VH
VL
Fc
VH
VL
Fc
VH
VL
Fc
VH
VL
Fc
VH
VL
Fc
VH
VL
Fc
VH
VL
Fc
Isolation of B-cell
population (e.g. from
spleen or blood)
Generation of Antibodies produced in
single clones
E. coli
CHO85
cells
plasmid DNA
Identification of antigen
specific scFv-Fcs
by ELISA
#3-22
A
B
C
D
E
F
G
H
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1
0,97
0,91
0,78
0,36
0,39
0,23
0,2
0,52
2
0,93
0,92
0,94
0,83
0,71
0,86
0,83
0,19
3
0,73
1,01
0,92
0,44
0,63
0,74
0,62
0,22
4
1,42
1,06
0,48
0,88
0,67
0,38
0,81
0,57
5
0,65
1,3
0,89
1,25
1
1,01
0,89
2,31
6
1
0,34
0,59
1,32
1,2
0,76
0,86
1,14
7
0,57
0,95
0,42
0,73
1,05
1,4
1,1
0,68
8
0,84
1
0,65
1,17
0,97
0,93
0,51
0,93
9
0,84
0,44
0,62
1
0,68
1
0,85
0,72
10
0,41
1,25
0,39
0,98
0,38
0,88
0,71
0,6
11
0,26
1,82
0,59
0,72
0,74
1,69
0,73
0,53
12
0,26
1,82
0,59
0,72
0,74
1,69
0,1
0,1
Cloning and Expression
in mammalian system
mAbs
mAbs
are
mayidentified
not work
and
sinceproduced
identified
in
mammalian
in E.coli system
cells !
Design and Production of Desired Final Product
Icosagen Cell Factory can generate recombinant
mAbs from mouse and chicken B-cells or
hybridomas. Production protocols for recombinant
rabbit mAb are under development.
Example of purified human
antibody
We can redesign the mAb to:
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
human antibodies (IgG1, IgG2, IgG4)

mouse antibodies (IgG1, IgG2a, IgG2b)

chicken antibodies (IgY)

chimeric antibodies

antibody fragments

single chain molecule

fusion proteins

bispecific antibodies (bi-scFV-Fc, DVD-Ig,
Crossmab)
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M
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2
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Summary
 Proprietary QMCF Technology for fast, scalable and cost-effective
production of proteins, antibodies and VLPs
 QMCF Technology can be used also for the design of new cell lines
and for the generation of monoclonal antibodies.
 Strong scientific team: principal scientists with 20+ year of
experience in the field of molecular/cell biology
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Thank you!
Icosagen AS
Icosagen Cell Factory OÜ
IcoPark OÜ
www.icosagen.com
info@icosagen.com
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www.icosagen.ee
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