Icosagen Story

Tartu Biotechnology Park 29.04.2015

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by Mart Ustav

Curriculum vitae Mart Ustav

1. Education – Organic and Bioorganic Chemistry, 1972, Univ. Of Tartu2. Military service – 1972-19743. Institute of Cybernetics, Tallinn – researcher, enzyme kinetics 19754. Ph.D. – rRNA-protein interactions, Tartu University, 1976-19795. Post-doctoral Fellow – Uppsala University 1982-19856. Senior scientist, Head of Laboratory of Oncogenesis, UT 1985-19897. 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 1997-20019. Howard Hughes Medical Institute Fellow, USA 1995-200510. Member of the Academy of Sciences of Estonia 200111. Professor of Biomedical Technology 2007….,

Director of the Institute of Technology, University of Tartu, 2002-2007, 2012….

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The first 10-years story

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• 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

1999

•Diversification of medical services and establishment of Quattromed Cell Factory as a subsidiary company

2005

•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

2008

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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Icosagen Group

Biotech and Protein Production Company in Estonia

Icosagen Cell FactoryEerika tee 1, Ülenurme vald, 61713 Tartumaa, EstoniaTel: +372 737 7070E-mail: info@icosagen.com

Tartu, Estonia

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Icosagen Group

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Icosagen

Management, financing, QC/QA, Sales&MarketingProducts/Services: catalog products (antibodies,

proteins, ELISA kits); food safety/quality control.

Icosagen Cell FactoryR&D, Business Development

Products/Services: technology licensing,

protein production services

IcoParkEstablished 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

Technology Developer and Service Partner for Global Pharma and Biotech Industry

Business Fields

Development and production of recombinant proteinsProteins, Poly- and monoclonal

antibodies, VLPs

Quality control laboratory testing services

Food microbiology, latex allergen testing

Development and sales of catalogue products

Antibodies, proteins, ELISA kits

Collaborative research and development,

technology licensing

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Technology Developer and Service Partner for Global Pharma and Biotech Industry

Animal Cell as the Factory – Design, Engineering and

Exploitation

Mart UstavBio- and medtech business: real stories and opportunitiesTallinn, 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

Stable Cell Lines, where proteins are expressed from the chromosomes

However, there is a huge gap between them!

Transient System, where proteins are expressed from extrachromosomal plasmids

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QMCF System Consists of Two Components

CHO85 cell line that expresses factors for plasmid maintenance and replication.

EBNA1

Py LT

QMCF plasmids that carry elements for replication and mainenance.

Maintenance (EBNA1)

Origin of replication (Py LT)

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E2

TA hinge DBD

McBride, 2006

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)

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

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Southern blot analysis of hNGF and human IgG1 antibody expression vector 48 hrs and 16-18 days after transfection (doubling time ~15 h)

In transient system, transfection has to be done in a large volume, few days before protein production

1 2 4 6 8 10 12 14 16 …

Volume of the cell culture

cell cultureexpansion

Time (days)

16 L

4 L

1 L

0.25 L

60 mL

15 mL

4 mL

1 mL

Start of the production,Shift to 30 oC

Transient transfection(1 L culture, (1mg DNA)

QMCFtransfection (1 mL culture, 1mg DNA)

QMCF Technology Is Scalable and More Convenient Than Transient 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

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pQMCF-T Vectors Are Superior for Transient Production

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

pQMCF

pQMCF-T

Objective: In order to increase the productivity of QMCF system, we inserted replication enhancer into the pQMCF vectors (T-plasmids)

Results:

pQM

CF

pQM

CF-T

CDNF

Transient QMCF Stable cell lines

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.

Protein quantities(grams, IgG)

0.01 0.1 1 10 100 …

suitable

unsuitable

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.

QMCF Technology Bridges „the Gap“ in Protein Production

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Icosagen Cell Factory Provides Protein Production Services by Using QMCF Technology

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pQMCF vector construtcion

Chemical transfection

Scale-up & production at 30°C

Purification, analysis

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

Small Scale Protein Production Services (<100mg of Protein (IgG))

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Transfection Agent 007

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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

EG

FP%

0

20

40

60

80

100

Transfection effieciency is up to 95% in CHO cell lines in seerum-free conditions with an excellent cell recovery

Cell bank generation in 2 weeks after transfection

Medium Scale Protein Production (<10g of Protein (IgG)) Together with Cell Bank Generation!

pQMCF vector construtcion

Transfection/ EP

Antibiotic selection &

scale-up

Production at 30°C

Purification, analysis

Production cell bank generation

Week 1 Week 2 Week 2-3 Week 4 Week 5

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Stable Production from QMCF Cell Bank

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).

1st batch from transfection (mg/L)

2nd batch from WCB (mg/L)

Storage period of cell bank

h-IgG1 144 21 186 12 12 months +- +-

Feasibility License

Technology evaluation in 6 month period

Research License or Limited Research LicenseIn-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 Licensing

QMCF Technology Applications:

Designing New CHO Cell Lines

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CHO is Great, but it is not Perfect!

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.

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

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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

orProtein of interest

Expression Factor

Protein of interest

shRNA

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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

hFurin

pro-protein

pro-protein

mature protein

+hFur

in

-hFur

in

Results:

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CHO85 hFurin Cell Line (3A5) for Complex Protein Production

Cntrl

M CHO85

3A5

Furin

Furin hFurin does not affect cell growth

0 1 2 3 4 5 6 7 8 91.00E+06

1.00E+07

1.00E+08

1.00E+09

1.00E+10

Cell Growth

3A5 pQ3 CHO85 pQ3

3A5 pQ3T CHO85 pQ3T

days

Cell #

Based on the positive results from pQMCF plasmid, we have generated CHO85 cell line (3A5) with stable expression of hFurin.

pro-GDNF

GDNF

GDNF

3A5

Target protein (48h)

CHO85

pro-

GDNF

pro-

GDNF

31 2

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Production of Glycoproteins in CHO85 Cells with Downregulated Slc35A2

Case study: Transient production of EPO in CHO85 cells

Slc35A2 transports UDP-galactose from the cytosol into Golgi vesicles where glycosyltransferases function.

EPO

Objective: To increase the homogenicity of glycosylated proteins by downregulation of Slc35A2

no shR

NA

with shR

NA

EPO

Isoelectric focusing

EPO

QMCF Technology Applications:

Development of Monoclonal Antibodies

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Immunization

+ information about

antigen binding site

B-cells

Hybridoma based

methods

mAbs

Two Approaches Are Used for the Development of Monoclonal Antibodies (mAbs)

Recombinant methods

mAbs

Isolation of B-cell population (e.g. from

spleen or blood)

Panning: Enrichment of antigen

specific B-cells

VH VL FcVH VL Fc

VH VL FcVH VL Fc

VH VL FcVH VL Fc

VH VL FcVH VL Fc

VH VL Fc

Isolation of VH and VL coding regions and generation of enriched scFv-Fc library

Generation of single clones plasmid DNA

Cloning and Expression in mammalian system

mAbsmay not work

since identified in E.coli system

#3-22 1 2 3 4 5 6 7 8 9 10 11 12A 0,97 0,93 0,73 1,42 0,65 1 0,57 0,84 0,84 0,41 0,26 0,26B 0,91 0,92 1,01 1,06 1,3 0,34 0,95 1 0,44 1,25 1,82 1,82C 0,78 0,94 0,92 0,48 0,89 0,59 0,42 0,65 0,62 0,39 0,59 0,59D 0,36 0,83 0,44 0,88 1,25 1,32 0,73 1,17 1 0,98 0,72 0,72E 0,39 0,71 0,63 0,67 1 1,2 1,05 0,97 0,68 0,38 0,74 0,74F 0,23 0,86 0,74 0,38 1,01 0,76 1,4 0,93 1 0,88 1,69 1,69G 0,2 0,83 0,62 0,81 0,89 0,86 1,1 0,51 0,85 0,71 0,73 0,1H 0,52 0,19 0,22 0,57 2,31 1,14 0,68 0,93 0,72 0,6 0,53 0,1

Identification of antigen specific scFv-Fcs

by ELISA

Antibodies produced in E. coli

in pQMCF plasmids

Antibodies produced in CHO85 cells

mAbs are identified and produced

in mammalian cells !

Development of Recombinant mAbs by Using QMCF Technology

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We can redesign the mAb to: 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)

Example of purified human antibody

Reduc

ed Ig

G1

Unred

uced

IgG1

M

1 2 3

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.

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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Summary

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Icosagen ASIcosagen Cell Factory OÜIcoPark OÜwww.icosagen.cominfo@icosagen.com

Thank you!