2014 MVP Kevin Durant

ProcessDevelopment,cGMPCellBanking,

Analy:calLabServices.

CYTBreaksgroundonnewcGMPMammalian

ManufacturingFacility.

FullyCommissioned&Validated.100L&

500LMammalianBioreactor&

Purifica:onCapacity

Con:nuedgrowthinProcess&Analy:calDevelopment,Quality

Services,Manufacturing&CYT

employees.Mul:ple100L&500LPhaseI/IIClinicalGMP

Runs.

Microbialmanufacturing

capabili:eswithaPDexpansion.

Introduc:onofa100LcGMPFermenter

Suite.Announcednew1000LMammalian

Bioreactor.

New1000LMammalianBioreactorTrainfullyvalidated&placedinto

service.Asecondindependentpurifica:onsuitewithpreandpostviralsegrega:onaddedto885.Added50L&

200LSartoriusDisposableBioreactor

(SUB)Systems. MicrobialManufacturing

ExpansionwiththeIntroduc:onofanew200LFermenter.

Introduc:onofLargeScaleSingleUse

Bioreactors(250Land1000LHyclone

Systems).AnnouncedPlansfora5000L

BioreactorTrainandAutomatedFillFinish

Capabili:es.

AutomatedFillFinishServiceOfferingFully.ValidatedandBroughtOnlineSeveralClients.MoveIntoPhaseIII

Trials.ProcessValida:on(Conformance)

CampaignPerformed.

NewSantaFeFacilityComesOnline.1000LMicrobialExpansionFullyValidatedandOnline.5000LRefoldSystemsInstalled&Opera:onal.Ini:ateAdd’lPurifica:on

ExpansionsinBldg.885

14Areas

>20uniqueindica2ons43APIs>9INDs

6MajorAPIclasses

cGMPCellBankingServices

•  MammalianandMicrobial

•  cGMPMasterandWorkingCellBanks

•  StorageinCustomBioGenicsV1500liquidnitrogenfreezers

•  Managementandcoordina:onofallrequiredtes:ngperFDAandICHguidelines

•  Longtermstorageofcellbanks,withalarmandbackuppowersystems

SingleUseSystems(25L-1000LSystems)

50L & 200L Sartorius

SUB�s

250L & 1000L Hyclone SUB�s

25/50and100LWAVEs

TradiIonalStainlessSystems

1000LDakotaSystemBioreactorSuite

100L&500LSartoriusD-StatBioreactorSuites

5000LDakotaSystemBioreactorSuite(Q12016)5000LBioreactorand5000LClarifica:ontank

FermentaIonEquipment:–  200LB.BraunFermentor–  1000LB.BraunFermentor(Q32015)

CellProcessing–  Alfa-LavalDiskStackCentrifuga:onat

200Land1000LscaleAlsohavetradi:onalbucketcentrifugeop:onsfor200L

–  GEASepara:onsforHomogeniza:onat200Land1000L

–  PilotMicrofluidizerforAdapta:onStudies

DownstreamOperaIons•  SupportFunc:ons

–  WeighandDispense–  EquipmentPrepara:on–  Solu:onPrep

PurificaIon(Mammalian&Microbial)•  Extensiveuseofdisposables•  GEChromatographyskidsu:lizingUnicorn•  AktaReady,AktaProcess0.5,AktaProcess1.5•  Columnsupto80cminscale•  UF/DF•  RefoldingCapability(Microbial)•  Pegyla:on•  PreViral&PostViralSegrega:on(Mammalian)

Equipment Chase Logeman Automated Vial Line

•  100% pre / post check-weigh •  Single use filling lines / needles

Customized Dual Biosafety Cabinet System •  Watson Marlow 520 EIL pump system •  Utilizes single use filling lines / needles

Operation Up to 45 Vials Per Minute (Typically run at ~25VPM) ~2-3 vials per minute

Capacity Qualified for up to 6000 vials per run. Up to

20,000 vials per day with additional qualification

1000 vials per shift

Component Size

3ml to 10ml Additional sizes with additional change parts /

qualifications. 2ml to 100ml

Automated Vial Filling

Semi-Automated Vial Filling

ReleaseandStabilityTesIng

•  Appearance•  ProteinConc.(A280,Bradford)•  HPLC(SEC,IEX,RP)•  SDS-PAGE•  FluorescentAc:vityAssays•  IEF•  ELISA•  pH•  Osmolality•  WesternBlot•  BindingAssays•  Pep:deMapping•  GlycanAnalysis•  CellBasedAssays

•  Bioburden•  Endotoxin•  TOC•  MCB/WCBReleaseTes:ng•  ResidualProteinA•  ResidualHostCellProtein•  ResidualDNA

•  ICHCompliantProtocols•  Long-TermStabilityStudies•  AcceleratedStabilityStudies•  Manageoutsourcedtes:ng

•  Mycoplasma•  Sterility•  Par:culateanalysis•  Virustes:ng

SummaryWeareaeen:vetoeachclient'sshort-andlong-termtechnicalandbusinessgoalsandcreateprocessesappropriately.•  Bioprocessdevelopmentformammalianandmicrobialfermenta:on•  Bioprocessop:miza:on•  Scale-up/Scale-down•  Bioprocesscharacteriza:onusingadvancedDoE•  Techtransfer•  Drop-in

•  Westrivetodeliverprocessesthatare:–  Costeffec:ve–  Robust–  Scaleable

OpenInnova:onProgram

“Thebestwaytopredictthefutureistocreateit” AquotefromPeterDrucker

Innova:on Cytovance ValueCrea:on

Cytovance

Ques2on:“Howtodriveinnova2on?”

Innova:on Process

Problem:Capturingideasfromendusers,evalua2ngthemandimplemen2ngthemischallenging

Sohowtofilluptheemptybox….

APracIcalIdeaManagementSystem

“Cyto”isderivedfromtheGreek“kytos”

meaning“hollow,asacell,container,or

box”….Innova:on Process

•  Ideacapturetoolanddatabase•  Steeringworkflow•  Priori:za:ontools•  Projectcharterswithbudgetsandsponsorship•  AwaytolinktoSMARTgoalsforindividuals•  LeaderStandardWorkformanagingprogress•  Close-outpresenta:ondeliverables•  Recogni:ontools•  PatentabilityQues:onnaire

Innova:on Process

CurrentState/ProblemStatement

Rootcause Idea/Solu:on

Impactandeffort

KYTOSMEMOtoCollectideawhereweaddress3“M”,Muda,Mura,Muri KYTOSIdeaTracker

istheideadatabase

Innova:on Process

Sponsor

Innomanager

R&Dmanagement

Ideaowner

EffortVersusImpact

SteeringInnovaHonWorkflowInvolvesstakeholdersof……

SoideascanbeprioriHzedaccordingly

Innova:on Process

TaskscanbeassignedtoindividualsasSMARTgoals

Chartersofferawaytocommunicateproblem,solu:on,effortandVALUEtouppermanagementtomaintainvisibilityandsponsorship

ChartersareApprovedProjectswherewe“D”efineSOW&problems,“M”easurecurrentstatus,“A”nalyzecriHcalfactors,“I”mprove&implementprocessand“C”ontrolprocessperformanceInnova:on Process

Lineitemforinnova:onprojects

Levelloadingrequireshighdegreeofinternalcoordina:on

InnovaHonProjectsshouldfitintothestandardworkflow

Innova:on Process

Background

Clientrequiredthedevelopmentofnon-affinitybasedpurifica:onprocessforanIGMmAb.

ObjecIve

Developanon-affinitybasedpurifica:onprocessforanIGMmAbthatmeetsregulatoryguidelinesforpurityandbioac:vity.

PathForward

Pilotscaleandengineeringscalebatchescomplete.

Accomplishments

AcompleteDSPprocesswasdevelopedresul:ngin98%purity,≤100ppmHCP,≤10ng/doserDNA,andbioac:vitycomparabletoreferencestandard.

NoaffinityresinswereusedintheDSPprocess.

Techtransfertomanufacturingsuccessfullyachieved.

InnovaHonProjectsClose-outtheoutputisSOPdocumentthegoalistoaddvalue

Innova:on ProcessValueCreate

Month #Ideas Accepted Rejected Parked Closed

May 33 23 0 10 13

June 2 2 0 0 0

July 7 4 0 3 0

August 8 6 0 3 0

September 1 1 0 0 0

October 3 3 0 0 0

TotalKYTOS 54

Innova:on Process Output

Wecollected54ideasin2014

Innova:on Process Output

Sponsor Lead Func:onalGroup

Closed(C) CHARTER# KYTOS# INNOPROJECTTITLE Phase COMMENT Finish

date Budget(MD) Actualcosts(MD) %Complete Re-source Pro-gress Coststodate

JesseMcCool James DSP 001 KYTOS-030 MethodDevelopmentandQualifica:onofqPCRforCHOResidualDNAReleaseTes:ng D M A I C Includeintheclientproposalandqualifica:onwillbeperformedinQC tdb 89 60 67% R P C

JesseMcCool Aaron AD 002 KYTOS-033 BiaCoreEvalua:onandIntegra:onintoOurOffering D M A I C 14may-kick-offwithAaron,DavidandElizatowriteandapprovedcharter. tdb 40 5 13% R P C

JesseMcCool James DSP 003 KYTOS-031 MethodDevelopment&Qualifica:onofqPCRforE.coliResidualDNAReleaseTes:ng D M A I C 15may-kick-offwithJimandElizatowriteandfinalizedcharter. tdb 58 19 33% R P C

JesseMcCool James DSP 004 KYTOS-032 MethodDevelopment&Qualifica:onofqPCRforMurineMinuteVirusReleaseTes:ng D M A I C 15may-kick-offwithJimandElizatowriteandfinalizedcharter. tdb 58 19 33% R P C

JesseMcCool April CL&SD 005 KYTOS-028 StrainDevelopmentToolbox D M A I C PresentedinBioProcessInterna:onalandLaunchedKeystoneExpressionSystemTM tdb 180 93 52% R P C

JesseMcCool Frank FER 006 KYTOS-029-035 Fermenta:onToolbox D M A I C 20may-kick-offwithApril,FrankandElizatowriteandapprovedcharter. tdb 595 135 23% R P C

JesseMcCool Bethany AD 007 KYTOS-013 RightFirstTime(RFT)Development&Integra:onforSD,USP,andDSP D M A I C 02SEP14-thetoolshavebeencreatedandequipmentlistsareentered,workingwithteamtodeveloptheunit

opera:onsspecificforeachdepartment. tbd 30 7 23% R P C

JesseMcCool Brandy AD 008 KYTOS-006 BioassayTrainingModule-Crosstrainingtool D M A I C 30July-kick-offwithBethanyandElizatowriteandapprovedcharter. tbd 34 4 12% R P C

JesseMcCool Mike AD 009 N/A Createassayforheterogeneityofstabletransfectedpools/clones D M A I C PendingCharter tbd #DIV/0! R P C

JesseMcCool Rachel AD 010 KYTOS-006 CrosstrainingMatrix D M A I C PendingCharter tbd #DIV/0! R P C

JesseMcCool Caitlin DSP 011 N/A CreateKanbansystemforDSP D M A I C Waitonspaceavailability tbd 20 10 50% R P C

JesseMcCool Birgit DSP 012 N/A Toleadthecrea:onofaplauormapproachforpre-formula:onstudies D M A I C DoEProtocolapproved,experimentson-going(60-days:mepoints) tbd 52 18 35% R P C

JesseMcCool Samantha FER 013 N/A CreateKanbansystemforFER D M A I C PendingCharter tbd #DIV/0! R P C

JesseMcCool Varvara AD 014 N/A Monosaccharideassayqualifica:on D M A I C Reportisdoneforfinalreview.Workontheslidedeck tbd 98% R P C

JesseMcCool Yifeng DSP 015 KYTOS-041 UsingTEVproteasetodevelopanefficienttagremovalmethodforaffinitypurifica:on D M A I C Preliminaryexperimentsdoneandneedsometrouble-shoo:ng tbd 70% R P C

JesseMcCool Erica AD 016 KYTOS-013 RightFirstTime(RFT)Development&Integra:onforAD D M A I C PendingCharter tbd #DIV/0! R P C

JesseMcCool Clark DSP 017 KYTOS-020 CreateR&DSafetyTeam D M A I C PendingCharter tbd #DIV/0! R P C

JesseMcCool David AD 018 N/A TrackingADspecificgoals D M A I C PendingCharter tbd #DIV/0! R P C

JesseMcCool Caitlin DSP 019 KYTOS-034 Repligenresinassessment D M A I C 18Aug-kick-offtowriteandapprovedcharter. tbd 10% R P C

JesseMcCool Christa USP 020 KYTOS-048 NutrientDeple:onAssay D M A I C PendingCharter tbd #DIV/0! R P C

JesseMcCool Srividya AD 021 N/A DoEforicIEFdevelopment D M A I C Sept-kick-offtowriteandapprovedcharter.WillstartinJan. tbd #DIV/0! R P C

JesseMcCool James DSP 022 KYTOS-026 ImproveTTprocessforDSP D M A I C 50%asofSept29 tbd 50% R P C

JesseMcCool Eliza AD 023 KYTOS-040-042 Analy:calDevelopmentcapabili:esimprovements D M A I C 02Sept-kick-offtoapprovecharter. tbd 90 35 39% R P C

JesseMcCool Andy CL&SD 024 N/A DevelopRedSwaptechnology D M A I C PendingCharter tbd #DIV/0! R P C

JesseMcCool Andy CL&SD 025 KYTOS-047-049 ReconstructpCHO1.0vectorandincorporateGFPasareporter D M A I C PriminaryStudyonProof-of-Concept tbd 25% R P C

Innova:on Process Output

Charter# Owner %Complete Charter# Owner %Complete 2014 2014001_qPCR-CHO Jim 67% 012_DoE-formula:on Birgit 35%002_BiaCore Aaron 13% 014_Monosaccharide Varvara 98%003_qPCR-Ecoli Jim 33% 015_TEVprotease Yifeng 70%004_qPCR-MMV Jim 33% 017_Safety Clark 005_Strain-toolbox April 52% 019_Repligen Caitlin 10%006_FermentaIon Frank 23% 021_DoEicIEF Vidya 007_RFT Bethany 23% 022_ImproveTT Jesse 50%008_Bioassaymodule David 12% 023_ADcapabili:es Eliza 39%010_Cross-trainingmatrix Rachel 025_Mammaliantoolbox Andy 25%

011_Kanban-DSP Caitlin 50%

MainProblem-movingforwardwiththewrongstrain•  Inadequatecharacteriza:onofresearchcellbanks(viablecellcount,purity,

gene:cstability,genotypesandphenotypes)

•  PhagesheddinglysogenstosupporttheT7expressionsystem

•  Ampicillinresistance

•  Leakypromoters

•  Lackofclearcelllinehistoryforfiling

MainProblem-processnotreadyformanufacturing=INDdelayed•  Complexmul:-stageseedcultures,likelyduetolegacyschedulingar:facts

•  Complexfeedingstrategies

•  Undefinedan:foamaddi:onschedule

•  RawmaterialshavenoGMPcompa:blesource

•  Processcontrol:some:mesnotenough,some:mestoomuch,some:mesnottheright

•  Lackofcleardevelopmenthistoryforfiling

•  E.coliisamust•  IP-freeexpressiontoolboxapproachisIPfree•  Leveragescuwngedgegenedesignandop:miza:on•  Rapidstrainconstruc:onworkflows•  2cellularcompartmentscreening•  Allowsforscreeningofsolubleandinsolubleop:ons•  Nomediasecre:on•  2complementaryinduc:onkine:cs•  Useswellcharacterizedhosts•  Hostengineeringtoolbox(toenablegeneknockouts)•  Noprophage

•  Proceduralizedstandardscreeningworkflows•  Fed-batchfermenta:ondata•  Screeningdonewithin8weeksàDecisionPoint•  Standardfed-batchfermenta:onprocesseswithsimpleprocesscontrol

strategies•  Designspaceengineeringofplauormprocess

•  Logicalholdpoints(frozenpaste,frozenwashedIBs)•  HPLCbased:teranalysis•  Rightthroughputanaly:cstosupport•  Compa:blerawmaterials•  ProcessdevelopmentwithMCB

Vector/PromoterKitCustomBuiltandCompa:blewith

DNA2.0’sTechnologyPlauorms

StandardWorkflow1Primary

Screeningin24-welldish

HostKitE.coliCustomStrainCollec:on+commerciallyavailablestrains

StandardWorkflow2SecondaryScreeninginBenchScaleFermenters 8weeksun:lfed-

bacthdata

•  WeleverageastrongcorporatealliancewithDNA2.0toconstantlyimproveouroffering(speedandmanufacturability)

•  One-stepElectra™cloning(DNA2.0)ofsynthe:cgenesintoKeystone™Vectors

•  ProprietaryCodonOp:miza:on

GeneGPS™(DNA2.0)offersprovenpredic:vity

•  24-welland125mLscreening

0

0.5

1

1.5

2

2.5

3

-0.5 0 0.5 1 1.5 2 2.5 3

Mea

sure

d E

xpre

ssio

n

Model-Predicted Expression

FP

scFv

Phi29Pol

R2 = 0.67R2(CV) = 0.59

0

10000

20000

30000

40000

50000

60000

70000

80000

STRAINA STRAINB STRAINC STRAIND STRAINERe

laIv

eFluo

rescen

ceUnits(5

25nm)

StrainsExpressingGFP

Expressionpadernsin24-welldishscalestoshakeflask

7hr(SF) 22.75hr(SF) 4.5hr(Dish) 22hr(Dish)

Therankorderof5GFPsequencevariantsconfirmedin24-welland125mLshakeflask--->

0

20

40

60

80

100

120

0

2

4

6

8

10

12

0 5 10 15 20 25 30

Percen

tofM

axim

alExpression

OD6

00

ElapsedCultureTime(hr)

KineIcsofT7andphoApromotersareverydifferent

ODT7 ODphoA T7-GFP phoA-LacZ

•  KeystoneTMVectorswerecustom-designed

•  Complementarypromotersoffersexpressionversa:lity

•  phoAissensi:vetoDNAsequenceop:miza:on(tunable)

•  T7isNOTsensi:vetoDNAsequence(bruteforceapproach)

•  Signalpep:delibraryforperiplasmicop:ons

50

T7phoA

25

I S I S

50

25

T7phoANeg I S I S

50

25

T7phoANeg I S I S

• Differentexpressionkine:cscrossedwithdifferenthostsincreaselikelihoodoffindingaproduc:veclone

Host1 Host2 Host3

•  Efficientprimaryscreenisperformedina24-welldish(3mLculturevolume)followedby1roundof125mLshakeflask

•  T7isinducedwithIPTG;phoAisautoinduced

•  125mLshakeflaskscreendemonstratesreproducibilitybeforecommiwngtoafermentor

•  Plauormfermenta:onprocessesfiteachpromoter

•  Bothprocessesyieldhighcelldensity(>200g/Lwetcellweight)underavarietyofcondi:ons,withdifferenthosts,anddifferentproducts

•  Processescanbetunedtofittheuniqueaspectsofeachproduct

•  Asinglecampaignof4x5LservesasabasisforProcessDevelopmentusingDoE

•  Providesfed-batchdataBEFOREchoosingastrainforProcessDevelopment

•  Enablesini:a:onofMCBproduc:onatanypointduringProcessDevelopment

0

50

100

150

200

250

0 5 10 15 20 25 30OD6

00

EFT(hr)

PlaeormT7Process

0

50

100

150

200

250

300

350

400

0 5 10 15 20 25 30 35 40

OD6

00

EFT(hr)

PlaeormPhoAProcess

DifferentStrainsinthePhoAProcess DifferentStrainsintheT7Process

•  Keystonescreeningevalua:onwasperformedin24welldishes:–  10plasmidswereconstructed–  HighandlowcopyversionsofpBR322(pET)–  BothT7andphoApromoters–  Foursequencevariantsofthegene–  Periplasmicandcytoplasmicexpression–  KandaBstrains–  Commerciallyavailablestrains,CYTin-housemodifica:onsofstrains,andATCC

strains.

•  TotalNumberofstrainsevaluated:19

•  Results:–  Publishedvalue:200mg/LasanIBinHCDfermenta:on–  Cytovanceoutcome:>2.3g/LasIBinplauormHCDfermenta:on

Strain# Plasmid# Promoter expectedcompartment Host Source

001 1 T7 periplasm Commercial-mod CYT 002 2 PhoA periplasm Commercial-mod CYT 003 3 T7 cytoplasm Commercial-mod NEB 004 4 PhoA cytoplasm BL21SHuffle NEB 005 3 T7 cytoplasm BL21 NEB 007 2 PhoA periplasm W3110degP CYT 009 3 T7 cytoplasm BL21T7LysYdegP CYT 010 4+partner phoA/Rha cytoplasm BL21SHuffle NEB 011 3+partner T7/Rha cytoplasm Commercial-mod CYT 013 8 phoA cytoplasm W3110degP CYT 014 9 phoA cytoplasm W3110degP CYT 015 10 phoA cytoplasm W3110degP CYT 016 5 T7 cytoplasm BL21T7LysYIQ NEB 017 6 T7 cytoplasm BL21T7LysYIQ NEB 018 7 T7 cytoplasm BL21T7LysYIQ NEB 019 5 T7 cytoplasm BL21SHuffleT7 NEB 020 6 T7 cytoplasm BL21SHuffleT7 NEB 021 7 T7 cytoplasm BL21SHuffleT7 NEB

NullClone

Refstd

Strain4

Strain15

Strain16

Strain17

Strain21

•  Examplegelassummarizedintheprevioustable.

•  Strain15waschosentoscaleupto5Lfermenters.

•  Differenthostshaveimpactofexpressionlevel

•  8weeksfromDNAtoFed-Batchdata

•  Differentcondi:onsweretestedforthiscampaignwith1strain.Condi:on“A”istheplauorm.

•  Yield:•  22.1%solids•  2-4g/L(PAGE,WIBes:mates)

•  ProcessDevelopmentwillfocusonrobustnesstes:ng.

•  cGMPMCBcampaignwascompletedwithin6weeksa~er5Lcampaign

PARAMETER FERMA FERMB FERMC FERMD

HarvestOD600 134.6 108.4 56.8 66.8

HarvestBiomass(g/L) 221 211 117 120

TotalEFT(hr) 48 72 48 72

Keystoneproducts

Product Type Promoter expectedcompartment Sol/insol Host Source

1 Enzyme phoA cytoplasm Sol In-house CYT2 Enzyme phoA cytoplasm Sol In-house CYT3 Enzyme phoA periplasm Sol Commercial NEB4 Cytokine phoA cytoplasm Insol Inhouse CYT5 cytokine phoA cytoplasm Insol In-house CYT

*5moleculesthatmetclientneeds*1MCBproduced*7moreKeystoneprojectshavebeenini:atedsincelate2014

AchievingGoalslike…

•  Value-addcorporateAlliances•  Newinstrumenta:oninAD•  NewSOPs•  Rou:neandnovelapplica:onofDOE•  UsingHTPequipmentinour

processes

CollecIngdifferentIdeaslike…•  Toolsfordrivingprocessexcellence•  At-scaleprocessingimprovements•  NewPATmethods•  Crea:ngmicrobialtechnologies•  Tes:ngnewefficientDoEtoenable

QbD•  Businessprocesstools

Innova:on ProcessValueCreate

KYTOSmemo

IdeaPriori:za:on

ApprovedCharter

Tac:calSMARTGoals

Deliverables

ThankyouforyouraZenHon!