QMCF Technology - Energiatalgud.ee

Customized and Flexible Protein Production
using QMCF Technology.
Application for the industrial cell line
development.
Mart Ustav, CEO
Tallinn, Arengufond
5. november 2014
Icosagen Group:
Biotechology company and
Protein Production Service Company
in Estonia
Icosagen Cell Factory
Eerika tee 1, Ülenurme vald, 61713
Tartumaa, Estonia
Tel: +372 737 7070
E-mail: [email protected]
Tartu, Estonia
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Icosagen Group
 Established in 1999
 CEO Mart Ustav, professor of biomedicine and virology
at 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)
 Partners in several international collaboration projects
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Technology Developer and Service Partner
for Global Pharma and Biotech Industry
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
QMCF Technology
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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.
Stable
cell lines
QMCF
Transient
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
Conventional plasmids get
lost in dividing cells
pQMCF plasmids are maintained
at the level of ~200 copies/cell
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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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 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
…
QMCF Technology Applications:
Designing New CHO Cell Lines
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CHO is Great, but not Perfect! Innovation needed!
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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A
TALE/ZF and viral DNA replication protein fusions
E2-TA +
E1
B Viral helicase-mediated gene knock out (KO) or Amplification
dsDNA Fragments
TALE-E2
+E1 mRNA
Target gene intact
or
Frame shift (KO)
+
+
Target gene
dsDNA break
or
Amplification
C Site-specific DNA integration induced by recombination-dependent DNA replication
TALE-E2
+E1 mRNA
+
Expression vector
dsDNA Fragments
DNA
Integration site
remains intact
Replication
or
Frame shift (KO)
+
Integration site
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+
dsDNA break
or
Site-specific integration
of foreign DNA
A. Development of the parental cell line
Transfection
genomic loci
Unwinding
Transfection
E1
E1 and E2 mRNA
Integration into an active
Gene of
interest
E
1
Genomic DNA
B. Components of the recombinant DNA
lox
P
pA
Selection
markers
Intro
n
Promot
er
ORI
Promot
er
Intro
n
CDS
p
A
loxP
C. Recombination-dependent amplification of the recombinant DN
E1 and E2 mRNA
E1
genomic loci
Transfection
integration
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E
1
unwinding
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Amplification
Linear dsDNA fragments
Re-
Current State of the Art in cell line development
State of the Art
Expression vector transfection
Random integration
Cell line development by using IcoCell chassis
IcoCell project
Screening of tens of thousands of clones to
generate cell chassis with predefined
integration platform
Beyond the Project:
Expression vector transfection
Indirect amplification of recombinant DNA
accompanied with MTX or MSX treatment
Targeted integration
Specific amplification of recombinant DNA
Screening of tens of thousands of clones
to get a cell line with >5g/L productivity
Duration: up to 12 months
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Screening of hundreds of clones to get
a cell line with >5g/L productivity
Duration: 4 months
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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Acknowledgements
Andres Männik
Urve Toots
Radi Tegova
Margit Ool
Andres Tover
Anne Kalling
Gaily Kivi
Tiiu Männik
Kristiina Karro
Kadri Kangro
Kerttu Murumets
Mart Ustav
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