QMCF Technology

Report
Technology Development and
Business Development at
Icosagen
Mart Ustav
CCCR meeting
Sagadi
September 5th, 2014
Biotech and Protein Production
Company in Estonia
Icosagen Cell Factory
Eerika tee 1, Ülenurme vald, 61713
Tartumaa, Estonia
Tel: +372 737 7070
E-mail: [email protected]
Tartu, Estonia
2
www.icosagen.com
Icosagen Group
 Established in 1999 as Quattromed AS
 CEO Mart Ustav, professor of biomedicine and virology,
University of Tartu
 49 FTE, 7 PhDs
 ISO 9001:2008, ISO 17025, GLP
 4 patent families/25 patents (EU, US, CA, JP, AU, CH, IN)
 Partner in several international collaboration projects
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www.icosagen.com
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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www.icosagen.com
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
QMCF Technology – The Asset for the Company
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Two Kinds of Technologies are Available for Protein Production
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 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.
Transient
QMCF Technology
For R&D and Diagostics
Stable
cell lines
suitable
unsuitable
0.01
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0.1
1
10
100
…
Protein
quantities
(grams, IgG)
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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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
…
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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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
www.icosagen.com
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 Combines the Features of Transient System
and Stable Cell Line
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Transient
QMCF Technology
Stable Cell Lines
Protein in 2-4 days after
transfection
Protein in 10-14 days after
transfection
Protein in >4 months after
transfection
Small quantities µg → mg
Scalable: µg → mg →g
Large quantities: mg → g
No cell banks
Cell banks
Cell banks
Productivity of antibodies:
average 80-100 mg/l
in shaker flask
Productivity of antibodies:
average 300-600 mg/l
in shaker flask
Productivity of antibodies
up to 10g/l
in bioreactor
www.icosagen.com
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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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
QMCF Technology Applications:
Development of Monoclonal Antibodies
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Development of MAbs using pQMCF
technology



Previously, we have worked out MAb development technology
based on enrichment of antigen specific B-cells
The technology has been succesfully utilized in Icosagen for cloning
MAbs from immunized mouse and chicken, from hybridomas
However, as also used in phage display methods, we analysed the
antibody specificity in E .coli cells
From this two limitations were apparent:
1. sometimes the VH/VL combinations were active when
produced in E. coli, but not active in context of
antibodies produced in mammalian cells;
2. Link between development and production was time
and
labour consuming: from bacterial system to
mammalian vectors .
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Confidential
E. coli system was replaced with QMCF!
Enrichment with antigen specific B-cells
Isolation of VH and VL regions → enriched QMCF library
Identification of optimal VH and VL combinations
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Confidential
Development of MAbs using QMCF
Identification of optimal VH and VL combinations by HTS
single clones
plasmid
DNA
chemical
transfection into
QMCF (CHO) cells
in 96-well format
1 day
2-3 days
Identification of antigen recognizing
scFv-Fcs by ELISA of supernatants;
sequencing of selected clones
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Confidential
#3-22
A
B
C
D
E
F
G
H
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
Advantages of MAbs development
using our technology
 Robust: no sterile work with spleen, does
not include cultivation or sorting of the Bcells neither single cell PCR
 Rapid link from identification to
production: identified scFVs can be
amplified directly
 Especially favorable when production in
mammalian cells are desired
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Confidential
Using the sequence information for
design and recombinant production of
desired final product in QMCF cells









human antibodies (e.g. IgG1, IgG2, IgG4)
mouse antibodies (e.g. IgG2a, IgG2b)
chicken antibodies (IgY)
chimeric antibodies
antibody fragments
single chain molecule
fusion proteins
bispecific antibodies (bi-scFV-Fc, DVD-Ig, Crossmab)
etc.
Usually codon optimisation step is included
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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
100
Transfection effieciency is
up to 95% in CHO cell lines
in seerum-free conditions with
an excellent cell recovery
60
40
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EB
NA
CH
O
28
LT
O
85
-S
0
CH

EGFP%
80
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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Study of the Replication
Mechanisms of the Human
Papillomavirus Genomes – the way
to identify the drugs against HPVs
Saage tuttavaks –
papilloomiviiruste perekonnapuu
U2OS cell based model system to study
HPV replication
U2OS
+ HPV genome (HPV-16, -18, -6b, -11, -5, -8)
Southern blot, qPCR,
analysis of the
transcriptome
24;48;72;96h
+ selection; 2-3 weeks
Primary maintenance
of the HPV genome
 U2OS/HPV+ subclones
Fluorescence in situ hybridization – FISH,
immunofluorescence
Growth of the subclones
Continuous passaging
in the confluent culture
of the subclones
HPV stable
maintenance
Amplification of the
HPV genome
RESULTS – CHARACTERIZATION OF THE HPV DNA IN
TRANSIENT ASSAY
*
• 24/48h – HPV DNA signal in many cells – input
and replicating HPV DNA
• 96h – + CELL COLONIES, where intensive HPV
replication takes place
• untransfected cells should be removed by
selection
Cell based assay for measuring HPV
replication
 We developed cell-based assay for
measuring the amplificational and stable
replication of the HPV genomes.
 We validated the HPV replication cell based
assay for the screening of the replication
inhibitors.
 We screened the chemical library to identify
the small molecules capable of inhibiting
HPV replication and validated the activity of
these compounds.
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Compounds to inhibit the HPV
genome replication
 We identified 6 compounds capable of inhibiting HPV
replication.
 Five out of six were targeting the same activity in the cell
and allowed to identify the target to inhibit HPV genome
replication
 Sixth compung was targeting the activity at the same
signal transduction pathway, which was not essential for
survival of the normal cell
 We have used this information in collaboration with
chemists to define a new set of compounds, which could
inhibit HPV genome replication more effectively
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Conclusions
 Model system for the HPV genome replication has been
developed
 The unique mechanism has been revealed
 The cell-based assay has been developed to meausre
quantitatively HPV replication
 Six active inhibitor compounds idetified – targeting two
targets in the same pathway
 Further studies to identify the lead compound(s) as
inhibitors of HPV replication
 EAS input is appreciated very much in supporting
our activities in drug development!
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Conclusions
 HPV replication is initiated from the replication origin and
runs bidirectionally
 Initial replication occurs via classical theta structures
 Accumulation of a specific replication intermediate,
 However, over time different replication intermediates
accumulate
 Indication of recombination-associated replication
Business Development – is it possible from here?
Biotech and Protein Production
Company in Estonia
Icosagen Cell Factory
Eerika tee 1, Ülenurme vald, 61713
Tartumaa, Estonia
Tel: +372 737 7070
E-mail: [email protected]
Tartu, Estonia
2
www.icosagen.com
Technology Developer and Service Partner
for Global Pharma and Biotech Industry
Business Development Principles.
 We are always at home when someone comes with the
money – sales and BD has 4 people and hey work 24/7.
 Our geographic position is not good! Travel to major
markets is painful, time and money consuming!
 We have started activities for incorporating daughter
companies in Belgium and USA.
 We have hired consultants to guide us on that rocky path
to the “real markets”. We hope that “The Market” is not
on the horizon, like it was with the Communism!
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Our team:
Icosagen
Institute of Technology
Andres Männik
Jelizaveta Geimanen
Liisi Henno
Helen Isok-Paas
Airiin Laaneväli
Andres Männik
Liis Noodla
Regina Pipitch
Tormi Reinson
Fernando Rodriguez – Castaneda
Eve Sankovski
Eva – Maria Sepp
Mart Ustav Jr
Mart Toots
Liisi Võsa
Reet Kurg
Ene Ustav
Urve Toots
Radi Tegova
Margit Ool
Andres Tover
Anne Kalling
Gaily Kivi
Tiiu Männik
Kristiina Karro
Kadri Kangro
Kerttu Murumets
Meelis Kadaja
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