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  • ReGenesys2013

    7th Technical Meeting, Newcastle, 24 September 2013

    Pros and cons of different expansion systems for manufacturing of an

    allogeneic cell therapy

    Jef Pinxteren, Manager and Head R&D

  • 2

    MultiStem cell therapy product: a multimodal biologic product

    MultiStemProductProfile CelltherapyproductbasedonMultipotentAdultProgenitorCell(MAPC)technology Developingforofftheshelfadministration notissuematchingneeded Expandedproduct withhighproductionyield(e.g.,millionsofdosesfromeachdonor) Longstoragelife canbekeptinfrozenformforyears Consistentsafetyprofile Promoteshealingandtissuerepair throughmultiplemechanismsofaction Notapermanenttransplant cellsclearedfromthebodyovertime

    Expanded,bankedproduct Obtainedfromhealthy,consentingadult donors

    MultiStemCellTherapy

    Frozenstorage Administeredsystemically(IV)orlocally(catheter,injection,matrix/implant)

  • 2013

    MultiStemImmunological

    IBD/UlcerativeColitisHematopoieticStemCellTransplant/GVHD

    SolidOrganTransplantDiabetes

    CardiovascularAMI

    CongestiveHeartFailurePeripheralArteryDisease

    NeurologicalStroke

    TraumaticBrainInjuryMultipleSclerosisSpinalCordInjury

    OtherBoneAllograft

    5HT2cAgonists(obesity,other)

    IMMUNOLOGICALCARDIOVASCULAR

    NEUROLOGICAL

    20134data

    2013Data

    FDAapprovedP2

    FDAdiscussionsre:P2/3designGermanP1approval

    3

    Overview of the MultiStem Production Process

  • 4

    Overview of the MultiStem Production Process

    LotRelease&ProductCharacterizationTesting

    Sterility,Potency,PurityandViabilityStableCytogenetics

    Absenceoftumorgenicpotentialinvivo

  • 5

    Cell Banking Approach

    IsolatedMultiStem

    1donor

    IsolateMultiStem

    20PD

    13days

    MasterCellBank

    20million/vial(200vials)

    4.109 cells

    1vial 8PD

    6days

    WorkingCellBank

    20million/vial(200vials)

    1vial

    800.109 cells

    12PD

    9days

    ClinicalDoses

    >40doses/vial180.106 cells/bag

    288.1012 cells~1.6.106 doses

  • Requires working in a clean room Labor intensive and numerous open

    events Manual seeding, feeding and harvesting

    Limited process control Difficulties in achieving high

    reproducibility

    Includes xeno media components (e.g., serum)

    6

    Current cell culture process

  • 7

    Images from current MultiStem production process

  • High capacity mfg which will allow for manufacturing runs in the 500 billion 2 trillion cell range (decrease testing costs due to lot size)

    Removal of xeno/human source materials when possible (lowering of regulatory/safety risk)

    Determine optimal storage conditions for final product and generate stability data (no special requirements for storage at clinical site)

    Final container configuration that supports thaw deliver use (bags, vials or other)

    Automate mfg process (reduce labor costs) Economy of scale for reagents (reduction in materials costs)

    8

    Future development

  • CourtesyofJonRowley,Lonza

    Heavy Lifting Scale Up

  • Cogs in Scale Up

    CourtesyofJonRowley,Lonza

  • Stem Cell Research and Assay Development MultiStem identity / cell equivalency (distinguish between stem cells) Molecular mechanisms of MultiStem function Immunology Pre-clinical animal studies

    Evaluate novel expansion methods Quantum Cell System (Terumo) Microcarrier/Stirred bioreactor

    Development of xenobiotic free workflow

    11

    The next stage

  • 40LayerCellFactory,Hyperstack directpathtoscaleupwithexistingtechnologies Advantages

    o Scaledplatformfromcurrentindustrywideexperiencebase Disadvantages

    o 2dimensionalplatformswithlargevolumemanagementrequirementso Currentlyuntestedlargevolumereductionoptions

    TFF multiplemanufacturers(Pall,Millipore) kSep promisingbutuntested

    HollowFiberBioreactor;ATMI(closedsystemstackedpizzaplatform) Advantages

    o Providesscaleoutcapacitywithsignificantadvantages Eliminatesrequirementforlargescalevolumereduction Closedsystemwithincreasedsafetyforsterilebreach Significantlyreducedlaborcosts

    o Allowsinlinetestingformetabolites,controlofgas/glucoseinrealtime Disadvantages

    o Limitedscalabilitywithincurrentconfigurationo Bettersuitedforautologousorpatientdesignatedapproach

    Suspensionbioreactorusingmicrocarriers Advantages

    o 3Dplatformwithminimalmediaexposure,likelymosteconomicalo Multiplemicrocarrierswithcontrolledsurfacecoatingcomingintomarketo Corning,BD,GEenteringspace

    Disadvantageso Macroaggregatesleadtocomplicationsinharvest;maybeovercomewithcarriersize(Pluristem)o Difficultyincontrollingoxygenation,shearparametersagainstparticlesize

    Scale Up Manufacturing Options

  • Phase III Registration trials must be run using commercial production process

    COGS a very important element of long term competitiveness 12 18 month period required to lock down process

    Pre-clinical equivalency and biosafety Process qualification and stability studies Clinical equivalency

    New manufacturing platforms developing with 3 to 6 month generation time!

    13

    Sobering thoughts on Manufacturing

  • Cell Factories, Hyperstack

  • Large Scale Volume Reduction

  • 16

    Microcarriers

    12 microcarriers were tested for cell attachment in dishes The best 3 were tested in small 125 ml spinner flasks The best one was used for expansion in 3 liter bioreactor

    12,000 cm culture surface 2.4 liter of medium used

    PrimaryScreen:12 SecondaryScreen:3 Proofofconcept:1

  • Quantum System An automated integrated cell culture system

    Bioreactor, incubator, media and waste management

    Controlled cell expansion All steps can be programmed and monitored

    Decrease in labor Closed system

    Reduction in contamination potential

    CE marked and FDA listed device

    17

    Quantum

  • 18

    Hollow fiber technology

    Self-contained bench top unit using a hollow fiber bioreactor Small device footprint (48.3 cm x 58.4 cm x 50 cm) Contains 10,000 fibers/bioreactor Total surface is 2.1 m (3255 in) Equivalent of 280 T75 flasks or one 40-stack Smaller surface to volume ratio requires media

    perfusion Metabolites and gas easily diffuse across fiber

  • 19

    Quantum cell expansion set

    Bioreactor

    Tubing organizer

    Waste bag

    Harvest bagGas transfer module

  • 20

    Graphical user interface

  • Graphical user interface makes operation very straightforward Reduced risk of errors Reduced labor

    Release and harvest of adherent cells takes 20 minutes Potential to run more cell expansion processes simultaneously

    Key steps in the process are automated e.g. Cell loading, feeding,... Possibility to adjust procedure and save as a custom task

    21

    Automation

  • MultiStem cells were seeded in parallel on the Quantum and in standard culture conditions on cell culture plastic

    22

    Cell equivalency: Quantum vs plastic

    Growth on the Quantum is monitored and feed rates may beadjusted based on glucose and lactate levels

    Harvest populations were tested on a set of QC assays

  • 23

    Cell equivalency: Quantum vs plastic

    Flow cytometryCytogenetic analysis on CGH arrays

    DifferentiationInhibition of T cell proliferation

    Marker Criterium Standardculture Quantum

    CD13 >90% 97.51% 99.45%

    CD34 90% 99.91% 99.92%

    CD105 >90% 99.53% 95.76%

    HLAI

  • 24

    Reproducibility

    #cellsseeded(x106) #cellshavested(x106) Run time(h)

    8.45 752 146

    7.28 782 148

    8.57 753 144

    3 independent runs from the same donor generate the same harvest yields

  • 25

    MultiStem isolation from bone marrow

    With prior density centrifugation Whole bone marrow load

    WholeBMloadonQuantumasgoodasseedingBMMNCon

    cellcultureplastic

  • 26

    Cell equivalency of MultiStem isolation: Quantum vs plastic

    Angiogenesis assay

    Differentiation

    Flow cytometry

    Inhibition of T cell proliferation

    Marker Criterion Plastic Quantum

    CD13 >90% 99.65% 98.93%

    CD34 90% 98.19% 99.17%

    CD105 >90% 98.82% 99.11%

    HLAI

  • Cells from 3 donors tested

    Donor 1 3 consecutive runs on the Quantum starting from bone marrow in run 1 Cells cultured on regular plastic

    Donor 2 2 runs starting from the same seeding stock (run 1 & 2) Run with different type of coating which was not successful (run 3) Cells cultured on regular plastic

    Donor 3 Isolation on the Quantum Isolation on plastic MSC isolated on plastic

    HUVEC as control

    27

    Microarray analysis

  • 28

    Microarray analysis - Dendrogram

    HU

    VEC

    Don

    or 3

    MSC

    Don

    or 2

    run

    3

    Don

    or 3

    pla

    stic

    Don

    or 2

    pla

    stic

    Don

    or 1

    pla

    stic

    Don

    or 3

    run

    1

    Don

    or 1

    run

    1

    Don

    or 1

    run

    3

    Don

    or 1

    run

    2

    Don

    or 2

    run

    1

    Don

    or 2

    run

    2

  • Hollow fibers large surface area maximizes cell expansion in a minimal amount of space

    MultiStem cells can be isolated and expanded on the Quantum MultiStem cells show the same growth kinetics and retain all cell

    characteristics Automation: ease of use and better control Decrease in labor: 2 hours of lab work to generate an average of

    800 million cells Expensive clean rooms not required

    29

    Conclusions

  • MultiStem and MSC derived from same donor bone marrow Differences are of epigenetic origin Further describing MultiStem identity

    Transcriptome miRnome Methylome Proteome

    30

    Omics screenings : MultiStem vs MSC

    3.8x109

    3.3x1014

    30

  • PCA to monitor cell equivalency of different cell culture methods

    31

    Transcriptome : comparability after culture manipulation

    PlasticexpandedQuantumexpanded

    MSC

    HUVEC

    FirstPrincipalComponent

    Second

    Prin

    cipalCom

    pone

    nt

  • 32

    MultiStem versus MSC : Gene Set Enrichment analysis

    MultiStem

    MSC

    MSC more mature?

    Telomerase activitymaintenance

    Proliferation

  • Telomerase activity is preserved in XF-MS

    33

    Telomerase activity

    y i o y i o y i o0

    1.0107

    2.0107

    3.0107

    MSC MultiStem XenofreeMultiStem

    Telo

    mer

    ase

    activ

    ity (c

    opy

    n)

  • Identify markers by comparing differential expression between Multistemand MSC Extract key miRNAs Consistent effect in 3 donors Develop multiplex qPCR as xeno-free equivalency assays

    Gene Set Enrichment Analysis Identify mRNA targets of miRNA Gene Set enrichment / Pathway analysis

    34

    Application of miRNA expression data

  • miRNA screening:

    Positive MultiStem markers Negative MultiStem markers

    Using two different platforms, array and PCR-based, we are currently exploring the possibility of using miRNA profiles to confirm the uniqueness of our product.

    35

    omics screening

  • 36

    miRNA marker screening strategy

    qPCR Array

    Totaltargets 755 1400

    Detectedtargets 207 847

    Differentialexpression 160 140

    Donorindependent,FC>1.5 67 32

    18 510

    MS>MSC

    33 116

    MSC>MS

    qPCR array qPCR array

    2: MultiStem marker identification

    Positive markers Negative markers

    1: miRNome + data analysis

    4 : Use panel of top markers in cell comparability testing

    3 : Validate 16 miRs by qPCR in independent donors top markers

  • miRNA marker expression is maintained during : Regular expansion Xeno-free expansion Bioreactor expansion

    37

    miRNA expression qPCR

    Rel

    ativ

    e ex

    pres

    sion

    0.0

    0.1

    0.2

    0.3

    0.4

    0.5

    Y O Y O

    MS XF

    CES MSC

    donor A donor B

    Y O Y O

    MS XF

    CES MSC Y O Y O

    MS XF

    CES MSCR

    elat

    ive

    expr

    essi

    on0.0

    0.2

    0.4

    0.6

    0.8

    1.0

    Y O Y O

    MS XF

    CES MSCY O Y O

    MS XF

    CES MSC Y O Y O

    MS XF

    CES MSC

    donor A donor B

  • Genome-wide DNA methylation analysis

    Find epigenetic markers for distinguishing MultiStem from competing productsand proof epigenetic stability during expansions

    38

    omics screening

  • 39

    Methylome : Multi-layered epigenetic regulation of MultiStem expansion capacity

  • 40

    Application of omics data in product development

  • 41

  • Acknowledgments

    ReGenesys: BartVaes,DavidCraeye,AnneliesBogaert,AlineVisser,SaartjeWalbers,PeterSterkendries,MarianCrabb,LiesbethVandenpoel,KristelGijbels,EllenVanHoutven,LienTimmerman

    Athersys: BobDeans,TonyTing,JonRowley,WillieMays

    www.regenesys.eu