Technologies to Improve Cell Line Development and Engineering

Susan Dana Jones, Ph.D.BioProcess Technology Consultants, Inc.

Cambridge Healthtech Institute PEPtalk ConferenceSan Diego, CA

January 14, 2009

From Clone to Commercial

Key Features of Initial Production Cell Line

Geneticandphenotypicstability

Maintenanceofinsertedgene(s)throughouttensofgenerations,preferablywithnoselection

Stable,reproducibleproductivity

Sufficientyieldtoenableinitiationofclinicaldevelopment

Yieldcanbeimprovedbyprocessdevelopmentbutgoodstartingcellularproductivityisessential

Decisiononacceptableyieldshouldconsiderallrequirementsformaterial,notonlyclinic

Desiredproductquality

Appropriateglycosylationorposttranslationalprocessing

Minimalaggregationormisfoldedforms

Potency

From Clone to Commercial

Cell Line Development Activities

Geneidentificationandsynthesis

Codonoptimization,mRNAsecondarystructureevaluation

Expressionvectorconstruction

Singleordoubleinsertion(ie,heavy&lightchain)

Selectablemarker

Promoterandothergeneticelements

Transfectionandselection

Selectforexpressionlevelsbutusuallyinshakeflask

Isolateclonalcellline

Adaptiontosuspensionandserumfreegrowth

PrepareMasterandWorkingCellbank

Fullcharacterization

From Clone to Commercial

Cell Line Development Timelines

Genedesignandsynthesis

Vectorconstruction

Transfectionandselection

Adapttosuspension/SFM

PrepareCellbanks

2 4weeks

2 4weeks

5 60weeks

6 12weeks

16weeks

Most time-consuming activity is TRANSFECTION & SELECTION!

From Clone to Commercial

Genetic Approaches to Shorten Timeline

Expressionvectorscanbeconstructedtoincludeelementsthatimproveexpressionlevels

Higherpercentageoftransfectedhostcellswillbehighexpressors

Easiertoidentifyhighexpressorswithlessscreening

Typesofgeneticelementsinclude:

Promoterswithhigherintrinsicactivity:CHEF1

Moreeffectiveselectivemarkers:GSSystem

Novelvectors:CatalentGPEx retroviralvectors

ChromosomalElements:SelexisGeneticElements,UCOE,Star

TranslationalEnhancers:PromosomeTEE

From Clone to Commercial

Traditional Genetic Approach: Amplification

Underselectivepressure,someessentialmetabolicgenescanbeamplifiedtoprovideincreasedproteinexpression

Genomicregionisamplified,causingnearbygenestoalsobeexpressedathigherlevels

GenomicDiHydroFolateReductase(DHFR)canbeamplifiedbyexposuretosequentiallyhigherlevelsofmethotrexate

TransfectedDHFRcDNAisalsoamplifiedunderselection

Eachroundofamplificationandselectionrequires36monthsandprovides25foldincreaseinthelinkedtransgeneexpression

Genomicstructureinamplifiedregionisnotstableandgenecopiesareoftendeletedwhenselectivepressureisremoved

Toxicandexpensiveinhibitorsoftenusedduringcellgrowthandproduction

From Clone to Commercial

Improved Selectable Marker: GS

Glutamineisessentialformammaliancellsandcanbeprovidedexogenouslyorendogenously

GlutamineSynthetase(GS)ispresentinsomemammaliancells

GScanbeinhibitedbyMethionineSulfoximine(MSX)

UseofGSasaselectablemarkerismoreefficientthanDHFRandmaynotrequireamplification

TimelineshortenedcomparedtoDHFRamplification

Highlevelexpressionoflinkedtransgenesisachievable

EngineeredCHOcelllinesthatareGSnegativehavebeendevelopedforusewithGSvectors

LonzaownscelllinesandpatentsonGSsystem

Vectorsandcelllinesavailablethroughlicensingprogram

CelllinesgeneratedusingtheGSsystem havebeenusedin5commercialproducts

From Clone to Commercial

New Promoter Technology: CHEF-1

Typicalexpressionvectorsincludeviralpromoters

Strongburstoftranscriptionalactivityassociatedwithinfection

Coexpressionofviralhelpergenesinhostcellcanimproveexpressionlevels

Promoterfromhighlyexpressedendogenoushousekeepinggene,CHOcellelongationfactor1(CHEF1),providesstablehighleveltranscriptioninCHOcells

DHFRusedforselectionbutnomethotrexateoramplification

Expressionlevelsof~250mg/Lachievablein11weeks

TechnologydevelopedandavailablefromCMCIcosBiologics

CelllinesconstructedusingCHEF1 vectorshavebeenusedinclinicalproductiontodate

From Clone to Commercial

Chromosomal Elements

SeveralcompanieshavedevelopedgeneticelementsthatimpacttheDNAstructure

MilliporeUbiquitousChromatinOpeningElements(UCOE)areavailablethroughalicensingprogram

CrucellhasdevelopedStar technologywhichusesDNAelementsthatcounteractendogenousgenerepression

SelexisGeneticElementsarebasedonmatrixattachmentsequencesthatenablechromatinnearthesesequencestobeaccessibletotranscriptionfactors

Chromosomalelementsarerelativelynewtechnologyandhavebeenusedforproductionofclinicalmaterial

Nocommercialproductstodate

From Clone to Commercial

Matrix Attachment Regions (MARs)

MARsorganizethestructureofthechromatinfiber

MARscontroltheON/OFFrateofloopformation

Openchromatinenablesmorerapidtranscription Open Chromatin

From Clone to Commercial

In silico Identification of Powerful DNA Elements

~1600SelexisGeneticElementsidentifiedinhumangenome

Rangefrom24kbp;sequencesarefromnoncodingregions

Highestdensityingenerichareas

1. Identification of putative functional motifs that activate gene expression

2. Construction of bioinformatics search toolScreening for high curvature, deep DNA major grove, wide minor grove,

melting temp and transcription factor binding sites

3. Identification of high-scoring putativeSGEs in whole genomes

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 X Y

SGE distribution along chromosomes

From Clone to Commercial

Iterative transfections with SGE-containing vectors eliminates the need for gene amplification

First transfection

Amp SGE

SV40GFP

11

GFP expressionGFP gene nb (qPCR)Average on entire population of cells

High expression linked to high transgene copy

number ?

High expression linked to high transgene copy

number ?

Second transfection

Neo SGE

SV40GFP

+

21.420.1

5.13.4

GFPSGE-GFP

SGE-GFP +SGE-GFP

SGE Increase Gene Copy # And Expression

From Clone to Commercial

Expression Enhancement using SGEs

Polyclonal CHO cell populations

Clone isolation and culture without selection

GFP expression101 102 103 104

Cel

l cou

nts 512

256

0

No SGE

GFP expression101 102 103 104

Cel

l cou

nts

512

256

0

+ Human SGE

From Clone to Commercial

Evaluation of MAb Minipools in CHO Cell Line

MAbexpressionevaluatedinnonoptimizedshakeflaskcultures

Lowheterogeneityinexpressionlevelsseenindifferentminipools

0

100

200

300

400

500

600

700

0 2 4 6 8 10 12

Yiel

d (m

g/L)

Days

From Clone to Commercial

Translation and Processing Improvements

Technologiestoimprovetheglycosylationpatterntoimproveantibodyfunctionhavebeendeveloped

Improvedoralteredglycosylationcanbeachievedbymodifyingcelllinetoincludedifferentprocessingenzymes

Invitromodificationisfeasiblebuttoocostly

MicrobialoptionssuchasGlycoFisPichiatechnologycouldenableprecisedesignofglycosylation

Rateoftranslation,processing,andsecretioncanpotentiallybeimprovedthroughgeneticmodificationorelements

Promosomestechnologyisanexampleofimprovedtranslationefficiency

Promosometechnologyisindevelopmentandhasnotbeenusedinclinicalorcommercialproductiontodate

From Clone to Commercial

Translation Enhancer Elements (TEE)

Utilizeribosomeentrysitestoimprovetranslationefficiency

Ribosomalrecruitmentcanoccuratm7Gcaporinternalribosomeentrysite(IRES)

SomeIRESesarecomposedofshorterfunctionalelements(IRESmodules)

From Clone to Commercial

Synthetic IRES Improves Protein Expression

From Clone to Commercial

IRES-modules Identified from Random Libraries

IRESmodulesarecelltypespecific

CHOcellspecificsyntheticIRESmoduleswereidentified

Fivecopiesenhances

translationefficiency

515fold

From Clone to Commercial

Antibody Production using TEE Vectors

Monoclonalantibodiesaccountforalargepercentageofbiopharmaceuticalsalesin2008andcontinuetobeadominanttypeofmoleculeindevelopment

Productionrequirementsforantibodiesarehighbecausedosesarehighandtargetpopulationsarelarge

Antibodyproductionrequiressimilarexpressionlevelsfortwodifferentproteins,heavyandlightchain

From Clone to Commercial

Antibody Heavy Chain Expression Enhancement

From Clone to Commercial

New Cell Lines: General Approaches

PreadaptedCHOcelllinesimprovetimetoproductioncellline

Eliminateselectionforserumfreeandsuspensiongrowthandsave612weeks

Enablemoreeffectivecloneselectioninitially

MostcompanieswithsignificantpipelinesorserviceofferingshavedevelopedadaptedCHOcelllines

Celllinesdesignedfortargetedintegration

FlpIn singlesiteintegrationsystemenablesuseofanyhostcelllinefortargetedintegration

Targetedintegrationhasbeenusedforclinicalandcommercialproductionbuthasnotbeenwidelyadapted

Symphogenisabiotechnologycompanywithauniqueproductforwhichtargetedintegrationprovidesanexcellentsolution

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