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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
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TechnologyDevelopment and BusinessDevelopment at Icosagen Mart Ustav CCCR meeting Sagadi September 5th, 2014
Biotech and Protein Production Company in Estonia IcosagenCellFactory Eerika tee 1, Ülenurmevald, 61713 Tartumaa, Estonia Tel: +372 737 7070 E-mail: info@icosagen.com Tartu, Estonia
IcosagenGroup • Established in 1999 asQuattromed 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 severalinternationalcollaborationprojects
Icosagen Group Icosahedron with 20 identical tringular facets. Icosagen, a company of variety of options for every facet of icosahedron
Technology Developerand ServicePartnerfor GlobalPharmaand BiotechIndustry Development and production of recombinantproteins Proteins, Poly- and monoclonalantibodies, VLPs Development and sales of catalogueproducts Antibodies, proteins, ELISA kits Qualitycontrollaboratorytestingservices Foodmicrobiology, latexallergentesting Collaborativeresearch and development, technologylicensing
Technology Developerand ServicePartnerfor GlobalPharmaand BiotechIndustry
TransientSystem, whereproteinsare expressedfromextrachromosomalplasmids StableCellLines, whereproteinsare expressedfromthechromosomes TwoKinds of Technologies are AvailableforProtein Production However, thereis a hugegapbetweenthem!
Transientsystems are thebestfor a fastproduction of small-scale protein amounts. However, itisnotfeasibleiflargeamounts of proteins are required. Productionof largeamounts ofprotein demandscelllinedevelopment and stableexpression of yourfavouriteprotein. IcosagenCellFactoryhasdeveloped QMCF technologytooptimizethemid-scaleproteinproduction. QMCF TechnologyBridges „theGap“ in ProteinProduction suitable QMCFTechnology For R&D and Diagostics Stable cell lines Transient unsuitable Protein quantities (grams, IgG) 0.01 0.1 1 10 100 …
CHO85 cell line thatexpressesfactorsforplasmidmaintenance and replication. QMCF plasmids thatcarry elementsforreplication and mainenance. Origin of replication (Py LT) QMCF SystemConsists of TwoComponents Maintenance (EBNA1) EBNA1 Py LT
Conventionalplasmidsgetlost in dividingcells pQMCFplasmidsare maintained at thelevel of ~200 copies/cell QMCF Plasmids Are Maintained in DividingCells Plasmid Chromosome
Southern blot analysis of hNGF and human IgG1 antibody expression vector 48 hrs and 16-18 days aftertransfection (doublingtime ~15 h) QMCF Plasmids Are Maintained in DividingCells
Volume of the cellculture cell culture expansion Transient transfection (1 L culture, (1mg DNA) Start of theproduction, Shift to 30 oC In transientsystem, transfectionhastobedone in a largevolume, fewdaysbeforeproteinproduction In QMCF systemisscalable and transfectionisdoneconveniently in a small volume 16 L 4 L 1 L 0.25 L 60 mL 15 mL 4 mL 1mL Therefore QMCF TechnologyisalsowellsuitedfortheHigh-ThroughputScreeningapplications QMCF TechnologyIsScalable and More ConvenientThanTransientProteinProduction QMCF transfection (1 mLculture, 1mg DNA) 1 2 4 6 8 10 12 14 16 … Time(days)
IcosagenCellFactoryProvidesProteinProductionServicesbyUsing QMCF Technology
Week 1 Week 2 Week 3 SmallScaleProteinProductionServices(<100mg of Protein (IgG)) WeuseNOVELpeptide-basedTransfection Reagent 007. Transfectionefficiencyis 80-95% in CHO85 cellswithexcellent cellrecovery
Cell bank generation in 2 weeks after transfection MediumScaleProtein Production (<10g of Protein (IgG)) Togetherwith Cell BankGeneration! Production cell bank generation Week 1 Week 2 Week 2-3 Week 4 Week 5
- - + + Stable Productionfrom QMCF Cell Bank Production of the GDNF-familyneuralgrowthfactorbyusingCHOEBNALT85 suspensioncellline. Productionwerestarted from two different cell banksindependently (lanes 1and 2).
QMCF Technology Combinesthe Featuresof TransientSystemand Stable Cell Line
QMCF TechnologyLicensing • FeasibilityLicense Technologyevaluation in 6 monthperiod • ResearchLicenseorLimitedResearchLicenseIn-house activities • Commercial License Production of catalog products, or diagnostickit, or customproductionservicesforthirdparties, etc
Based on the positive results from pQMCF plasmid, we have generated CHO85 cell line (3A5) with stable expression of hFurin. hFurindoesnot affectcellgrowth Furin Targetprotein (48h) Cell # CHO85 hFurinCellLine(3A5) forComplexProteinProduction 3A5 pro-GDNF GDNF pro-GDNF Furin CHO85 Cntrl pro-GDNF M days GDNF 1 2 3 3A5 CHO85
QMCF TechnologyApplications:Development of MonoclonalAntibodies
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 wereapparent: sometimes the VH/VL combinations were active when produced in E. coli, but not active in context of antibodies produced inmammalian cells; Link between development and production was time and labour consuming: from bacterial system to mammalian vectors .
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
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
Advantages of MAbs development using our technology • Robust: no sterile work with spleen, does not include cultivation or sorting of the B-cells 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
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
Transfection Agent 007 • New generation peptide-based vehicle for efficient delivery of nucleicacidsforthetransfection of mammalian and insectcells Arukuusk, P. et. al.. Biochim Biophys Acta. 2013 May;1828(5):1365-73 • Transfectioneffieciencyis • upto 95% in CHO celllines • in seerum-free conditions with • an excellent cell recovery
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
StudyoftheReplicationMechanismsoftheHumanPapillomavirusGenomes – thewaytoidentifythedrugsagainst HPVs
U2OS cellbasedmodelsystemtostudy HPV replication U2OS + HPV genome (HPV-16, -18, -6b, -11, -5, -8) Southernblot, qPCR, analysisofthetranscriptome 24;48;72;96h + selection; 2-3 weeks U2OS/HPV+ subclones Primarymaintenanceofthe HPV genome Fluorescencein situhybridization – FISH, immunofluorescence Growthofthesubclonesintheconfluentculture Continuouspassagingofthesubclones Amplificationofthe HPV genome HPV stablemaintenance
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
Cellbasedassayformeasuring HPV replication • Wedevelopedcell-basedassayformeasuringtheamplificational and stablereplicationofthe HPV genomes. • Wevalidatedthe HPV replicationcellbasedassayforthescreeningofthereplicationinhibitors. • Wescreenedthechemicallibrarytoidentifythesmallmoleculescapableofinhibiting HPV replication and validatedtheactivityofthesecompounds.
Compoundstoinhibitthe HPV genomereplication • Weidentified 6 compoundscapableofinhibiting HPV replication. • Fiveoutofsixweretargetingthesameactivityinthecell and allowedtoidentifythetargettoinhibit HPV genomereplication • Sixthcompungwastargetingtheactivity at thesamesignaltransductionpathway, whichwasnotessentialforsurvivalofthenormalcell • Wehaveusedthisinformationincollaborationwithchemiststodefine a newsetofcompounds, whichcouldinhibit HPV genomereplicationmoreeffectively
Conclusions • Modelsystemforthe HPV genomereplicationhasbeendeveloped • The uniquemechanismhasbeenrevealed • The cell-basedassayhasbeendevelopedtomeausrequantitatively HPV replication • Sixactiveinhibitorcompoundsidetified – targetingtwotargetsinthesamepathway • Furtherstudiestoidentifytheleadcompound(s) asinhibitorsof HPV replication • EAS inputisappreciatedverymuchinsupportingouractivitiesindrugdevelopment!
Conclusions • HPV replicationisinitiatedfromthereplicationorigin and runsbidirectionally • Initialreplicationoccursviaclassicalthetastructures • Accumulationof a specificreplicationintermediate, • However, overtimedifferentreplicationintermediatesaccumulate • Indicationofrecombination-associatedreplication
BusinessDevelopment – isitpossiblefromhere? Biotech and Protein Production Company in Estonia IcosagenCellFactory Eerika tee 1, Ülenurmevald, 61713 Tartumaa, Estonia Tel: +372 737 7070 E-mail: info@icosagen.com Tartu, Estonia
Technology Developerand ServicePartnerfor GlobalPharmaand BiotechIndustry
BusinessDevelopmentPrinciples. • We are always at homewhensomeonecomeswiththemoney – sales and BD has 4 people and heywork 24/7. • Ourgeographicpositionisnotgood! Travelto major marketsispainful, time and moneyconsuming! • WehavestartedactivitiesforincorporatingdaughtercompaniesinBelgium and USA. • Wehavehiredconsultantstoguideus on thatrockypathtothe “real markets”. Wehopethat “The Market” isnot on thehorizon, likeitwaswiththeCommunism!
Ourteam: Icosagen InstituteofTechnology 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 Andres Männik Urve Toots Radi Tegova Margit Ool Andres Tover Anne Kalling Gaily Kivi Tiiu Männik Kristiina Karro Kadri Kangro Kerttu Murumets Meelis Kadaja