BILS 2015 Jesse Mc Cool Cytovance
-
Upload
gbx-summits -
Category
Engineering
-
view
103 -
download
5
Transcript of BILS 2015 Jesse Mc Cool Cytovance
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!