Challenges in the cGMP Manufacturing of hESCs: Lessons Learned from Monoclonal Antibodies

ISCT 2011 Annual Meeting Rotterdam, The Netherlands May 18 21, 2011

BioProcess Technology Consultants www.bptc.com

Important Considerations in cGMP Manufacturing

Capacity Quality/ Regulatory Performance/ Productivity Scalability Speed

Flexibility

Cost

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Growth in Monoclonal Antibody Product Sales

45 40 35

AnnualSales($Billions)

30 25 20 15 10 5 0 2002 2003

Largestclassof biologic productstoday

2004

2005

2006

2007

2008

2009

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Hurdles in Monoclonal Antibody Development

Productivity Lowtitersandpoor purificationyields causedinitialhighCOGS

Immunogenicity HAMAlimitedearly productdevelopment

ProductConsistency Reproducible glycosylationwith scaleandprocess

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Evolution of Monoclonal Antibody Products

Centoxin approvedbut withdrawn frommarket

FirstmurineMAb approval

FirstIgGfusion proteinapproval

1976

1986

1991

1997

2000

2002

Khler &Milstein discoverMAbs FirstChimeric MAb approval

Zenapax
Firsthumanized MAb approval Firstfullyhuman MAb approval

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Approval of Monoclonal Antibody Products by Year

6

No.ProductsApproved

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Standardized Production Host and Culture Conditions

NS0 SP2/0 CHO

Chemicallydefined, serum(orprotein) freemedia Operatingparameters improveproductivity to50pg/cell/day

Fedbatchculturewith specificnutrientfeeds Increasedpeakcell densitiesandrun duration

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CHOadaptedfor suspensionculture

Typical Monoclonal Antibody Cell Culture Process

10 100mL vial

50L

200L

1,000L

7,500L
11000 10000 9000 8000 7000

20,000L

Highcelldensityandhighgrowthrate

mediaenable maximalseeddensityin inoculumtrainandproductionbioreactor Celldensitiesatharvest3x1010 cells/L Titers5g/Lin12 15dayculture Culturetimefromvialtoharvest ~22 25days

Titer (mg/L)

6000 5000 4000 3000 2000 1000 0 0 2 4 6 8 10 12 14 16 18 20 22

Days

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Innovation has Increased Titers and Yields

10,000

1,000

Titerinmg/L

100

Newtechnologiestoimprovecelllinedevelopmentand
10

expressionlevelscoupledwithimprovedandoptimizedmedia, supplements,andbioreactorconditionshaveincreasedtiters Over3logincreaseintiteroverthelast20years Currentcommercialproductsrangefrom0.2 3.0g/L

1985 1990 1995 2000 2005 2010 2015 FcFusion
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1 1980

2020

AntibodiesRecombinant

AntibodiesHybridoma

Ref: W.Noe(2011),T.Charlebois (2006),M.Smith,(2005),F.Wurm (2004)

Platform Processes Enable Large Scale Purification

Ref: B.Kelley,ACSConference,(2006)

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Cost of Manufacturing Monoclonal Antibodies

Phaseof Development
1 2 3 Commercial

Quantity Required(Kg)
0.5 2 1 10 5 25 10 1,000 (peryear)

Relative ProductionScale
1 1 3 3 5 5 10

EstimatedCost
$0.75 1.2MM Perbatch $1.8 2.5MM Perbatch $2.5 5.0MM Perbatch
$1,000/gram Typically$150

Earlystageproductiontypicallypricedonperbatchbasisduetosmallscale

andincompleteprocessdevelopment BatchpricessimilarateachscalebutlargerbatchsizelowersCOGS Commercialproductionat2,000 20,000L Highertitersloweringaveragecommercialscalebioreactorrequirements

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Large Scale Monoclonal Antibody Production Costs

$500
9% 13% 52% 19% 7%

Depreciation RawMaterials Consumables Labor Misc.

$400

Cost$/gram

$300

$200

$100

$ 0 3 6 MAb Titerg/L 9 12

Basedonannualproductionof100Kg/year; bioreactorsizerangingfrom1,000 10,000L

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Issues in the Manufacture of Stem Cells

Productcomplexitygreaterthanmonoclonalantibodies

Finalproductnothomogeneous Maycontainpartiallydifferentiatedorundifferentiatedcells Inducedpluripotentstemcellsaregeneticallymodified Manufacturingchallenges Anchoragedependentgrowthrequiresmicrocarriers Productionscalelimitedduetometabolicconcerns,lowdensity propagation,andlimitedexperience Widevarietyofculturesystemscurrentlyinusewhichmayhave limitedscalability Uniquesupplychainissues Fragilityofcellsmayrequirefrozenstorageandshipping Limitedshelflifeofproduct Shippingprotocolsmustensureproductviabilityandsterility

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Allogeneic Cell Therapy Products in Development

60 50

NumberofProducts

40 30 20 10 0 Market Phase3 Phase2 Embryonic Somatic Phase1 Preclinical

Includescompanysponsoredproductsonly. Severalinvestigatorsponsoredtrialsinprogress
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As With Early Biologics, Process = Product

Product Complexity Willrequireextensivetesting,especiallyearlyin development,toensureproductqualityandconsistency Potentialfordifferentadviceorrequirementsbyindividual regulatoryauthorities Lackofproductandprocessknowledgelimitabilitytomake changesduringdevelopment ManufacturingprocessshouldbefullycGMPcompliantand relativelyfinalpriortoPhase1

Process Changes

Product Quality

Conceptverydifferentthanmonoclonalantibodies Identitytestingrequiresuseofexpressionmarkers

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Critical Factors in Cell Therapy Manufacturing

Qualityofallproductcontactsurfacesandmedia/buffers

Productcontactsurfacescanimpactdifferentiation,changingproduct composition Differentmicrocarriers havedifferentimpactoncellgrowthand differentiation Viralinactivationorremovalnotpossible Terminalsterilizationnotpossible Operatortrainingessential Oftenseveralfactorsusedtoinitiatedifferentiation Balanceofactivitiesessentialtoinitiatecorrectdifferentiation

Controlofproducttopreventcontamination

Potencyofgrowthfactorsusedtoinitiatedifferentiation

LimitedmaterialavailableforQCtesting

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Cell Therapy Manufacturing Process

WCB(undifferentiatedhECs) CellExpansion Differentiation

CellHarvest

FormulationandFill

Cryopreservation
Ref: www.geron.com FromClonetoClinic

Cell Therapy Process Parameters

TypicalHarvestDensities

2DcultureinTflasks 8.0x105 cells/cmsquare 3Dcultureinbioreactorsunoptimized 2.0x109 cells/L 3Dcultureinbioreactorsoptimized 2.0x1010 cells/L 50%overallrecoveryyieldfrombioreactortovial

Lowdoseapplications,e.g.,spinalcordinjury

2.0x107 cells/dose;singledose Highdoseapplications,e.g.,congestiveheartfailure 5.0x108 cells/dose;singledose Targetpatientpopulationof10,000 550,000patientsatmarketpeak Marketpenetrationof30 50%atmarketpeak

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Very Small Production Volumes Required

Lowdose/Smallpatientpopulation/ Lowmarketpenetration Highdose/Highpatientpopulation/ Highmarketpenetration

Totalannualproductionrequirement Totalannualproductionrequirement

6.6x1010 cells Totalannualproductionvolume 6.6L ProductioninTflasksorspinners

1.4x1014 cells Totalannualproductionvolume 14,000L Productioninsmallbioreactors 15batches/yr @1,000L/batch

19

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Potential Commercial COGS for hESC Products

Case1 Bioreactorsize(L) Celldensityatharvest(cells/L) Overallyield Totalcellsper batch(cells) Dosesperbatch(5x108 cells/dose) Batchesperyear Dosesproducedperyear Costperbatch($million) Costperdose($/dose) 500 2.0x1010 50% 5.0x1012 10,000 30 300,000 11.74 1,174 Case2 1,000 2.0x1010 50% 1.0x1013 20,000 15 300,000 8.41 420 Case3 2,000 2.0x1010 50% 2.0x1013 40,000 8 320,000 11.53 288

Case1&2=Singleusebioreactor;Case3=Stainlesssteelbioreactor Costsbasedonhypotheticalmanufacturingprocessanddonotinclude storageorshippingofproduct Costpredominantlydrivenbydepreciationandrawmaterials

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Single Use Bioreactors for hESC Manufacturing

Xcellerex XDRTMBioreactor

SartoriusStedim BiostatCultibag

ThermoFisher(Hyclone) SingleuseBioreactor

GEHealthcare WaveBioreactor

ATMI NucleoTM Bioreactor

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hESC Manufacturing Facility

SupplyCorridor Inoculum UpstreamProcessing Wash Media&Buffer Preparation

ClassCAreas:

MaterialsCorridor StagingArea

Fill/Finish

Cellprocessing PersonnelCorridor

Cellrecovery

Inoculumpreparation Fill/finish ClassDAreas: Cellculture(Singleusebioreactor) Downstreamprocessing Mediaandbufferpreparation Cleanglasswareunloadingand storage Mediaandbufferstoragein controlled,nonclassifiedareas
TotalArea ClassD TotalCapitalCost 13,014ft2 3,315ft2 $15MM
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ProcessArea ClassC ProcessEquipment

6,781ft2 667ft2 $5.0MM

Thank You!
Howard L. Levine, Ph.D. hlevine@bptc.com +1.781.281.2703 BioProcess Technology Consultants, Inc. 12 Gill Street, Suite 5450 Woburn, MA 01801

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