The Future Vaccine Manufacturing Hub: Tools and technologies...Imperial College London University of...

TheFutureVaccineManufacturingHub:ToolsandtechnologiesMartinaMichelettim.micheletti@ucl.ac.ukDepartmentalofBiochemicalEngineeringUniversityCollegeLondon

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

PresentationOutline

•  IntroductiontoVax-Hub,platformfundingandGrandChallengeresearchoverview

•  Vaccinetechnologies

•  Adenovirusmanufacturingplatformprocess

•  VLPvaccines:Qualitybydesign

•  Novelglycoconjugatevaccinestechnologies

•  Nextsteps

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

TheGlobalVaccineActionPlan(GVAP)

In 2012, the World Health Assembly, representing 194 countries, endorsed the GVAP to ensure that no one missed out on a vital immunisation by 2020. To date, progress towards the GVAP targets is off track. In 2015, more than 19 million children missed out on basic immunisations.

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Themanufacturinglandscape–supplyisfailingdemand

VaccineaffordabilityandsupplyisoneofthekeyprioritiesidentifiedbytheWHOGlobalActionPlan

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

HubVisionandAim

Toadvancetechnologiesthatwillensurefuture,uninterruptedsupply.

Toensurethattheseadvancestranslateto

LMICmarketsandmanufacturers.

Abilitytosupportandrespondtoepidemicthreats.

TheHubsupportsanambitiousprogrammeofinnovativeresearchrelatedtothechallengesofdeveloping,scaling-upandmanufacturing

vaccinesofbenefittolowandmiddleincomecountries.

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Hub-Spokemodel

HubDirectors:ProfessorsSarahGilbertandMartinaMicheletti£7M,3years(April2018-March2021)TwoHubs:•  UCLBiochemicalEngineering•  TheJennerInstitute,UniversityofOxfordThreeUKSpokes:•  ImperialCollegeLondon•  UniversityofLeeds•  LondonSchoolofHygieneandTropical

medicine

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Hubactivities

GCManufacturingResearchonthreemaindemonstration

technologies(viralvectors,conjugatesandVLPs)

Platform Operations Interaction Vouchers,

Training and Feasibility Studies

Hub Activities

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

PlatformOperations

InteractionvouchersCall8vouchersintotal,budgetofupto£10Kpervoucher(<6monthsduration)AvailabletoUsersgroupmembersonlyMustbringtogetheranytwoorganizations(includingacademia-industrypartnerships)FeasibilityprojectsCall(early2020)Minimumof6projects,£100Keach(<12monthsduration)

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

ManagementStructure

International Advisory

Board (IAB)

Hub Management Group

Co-Directors

User Group

UKVN

EPSRC

DoH

Grand Challenges Research

Platform Activities

Total£6.9million

89%

11%

GCResearch PlatformOps

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

GrandChallengeResearchOverview

GrandChallenge2:Enhancedoperationalandeconomictoolsforuninterrupted,lowcostsupply

GC1.3.2NextGenAnalytics

GC1.2.1Advancedaffordablemanufactureofviralvectors

GC1.2.1Advanced,affordablemanufacturingofViralVectors

GC1.3.1VLPAnalytics

GC1.1.2Newanddisruptiveconjugationtechnologies

NewToolsandTechnologies(GC1.1)

PlatformManufacturing(GC1.2)

Analytics(GC1.3)

Formulation(GC1.4)

Subunits/VLPs

Conjugates

ViralVectors

GC1.2.2Rapidlyscalablesystems

Techno-economicevaluation

GC1.4Thermo-stable

formulationsystems

GC1.1.1Microscaleprocess

development

GC1.1.2Novelconjugatetechnologies

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

HubExpertise-UCL

IndustrialBiotechnologye.g.chemicalsandpharmaceuticals

MacromolecularMedicinese.g.antibodiesandvaccines

CellandGeneTherapiese.g.cellsand

engineeredtissues

HighThroughputBioprocessDesignandScale-up

SyntheticBiology(Protein,VectorandCellEngineering)

DecisionalTools(DataMining,ProcessandEconomicModelling,LCA)

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

HubExpertise-UCL

IndustrialBiotechnologye.g.chemicalsandpharmaceuticals

MacromolecularMedicinese.g.antibodiesandvaccines

CellandGeneTherapiese.g.cellsand

engineeredtissues

HighThroughputBioprocessDesignandScale-up

SyntheticBiology(Protein,VectorandCellEngineering)

DecisionalTools(DataMining,ProcessandEconomicModelling,LCA)

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Toolsforbioprocessdesignandscaling

Experimentalstudyofflowdynamics,mixingandsuspensiondynamics

§  Laboratoryscalebioreactors(rocked,shakenandstirred)§  Single-useandconventionaltechnologies(Ambr250,SartoriusCultibag,

MilliporeCellReady,DasBox)§  Impactofenvironmentfordifferentcelltypesandproducts§  Robustscalingequationsandmethodologies

MiniaturisationanddevelopmentofScale-DownTools

§  Quasi-perfusionmicroscalemethodologies§  250mlperfusionbioreactor§  Microscaletangentialflowfiltrationdevice§  Integrationofmimicswithinautomatedplatformstospeedup

bioprocessdevelopment

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

UltraScale-Down(USD)Technologies:Manufacturinginsightinthelab

Continuous centrifugation Chromatography Formulation/Fill-

finish

Fermentation Depth filtration Membrane filtration

Opportunitywithnewtechnologiestolinkwholebioprocesssequence

USDTechnologies

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

HubExpertise–UniversityofOxford

TheJennerInstituteUniquemissiontodevelopinnovativevaccinesagainstmajorglobaldiseaseandfocusontranslationalresearch(rapidearlystagedevelopmentandassessmentofnewvaccinesinclinicaltrials)ClinicalBiomanufacturingFacility(CBF)TheUniversityofOxfordGMPfacility–MHRAAuthorisationforviralvectoredvaccinesandATMPs–providingalinkbetweenacademicresearchandclinicaldrugdevelopment

DesignandtestingofviralvectoredandVLPvaccinesTransitionfromresearchtoGMPGMPmanufactureofviralvectors,VLPandrecombinantproteinsAssaydevelopmentforreleaseandin-processtestingFormulationofdrugproductClinicalvaccinedevelopment(UKandoverseas)

AdenovirusManufacturingPlatform

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

ProcessrequirementsforPhaseI

Small≥100doses

SimpleLimitedstaff,oneteammakesallproductsLimitedcapitalequipmentLimitedcapacitytovalidatenewequipment/processesTransferabletoLMICmanufacturers

RobustTransferableacrossmultipleproducts

Qualitymeetingregulatoryrequirements

Aneffectiveadenovirusmanufacturingapproachislikelytobeapplicabletovaccinesagainstmultiplepathogens:EmergingoutbreakpathogensVeterinaryAntibody&Tcells

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Adenovirusbiology

•  Non-envelopeddsDNAvirus,90nm

•  Non-replicatingduetoE1(andE3)genedeletion

•  HEK293orPERC6cellssupplyE1intrans•  Antigen-encodingtransgeneunderstrong

constitutivemammalianpromoter•  Antigenisnotastructuralpartofthevirionà

vaccinesusingasingleAdserotypearestructurallythesame,regardlessofAg

•  Antigenisexpressedinculture:canaltergrowthcharacteristics,selectionpressureforgeneticinstability

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Chimpanzeeadenovirusvectors(‘ChAds’)

•  Minimalpre-existinganti-vectorimmunityinhumanpopulation

•  Multipleserotypes

•  Differenthexon/fibercapsidproteins

•  IssueofcompatibilitywithHEK293Ad5-

derivedE1:•  Manufacturingcanbeenhancedbynon-structural

genemanipulation

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Smallscaleadenovirusproductionprocess

Cells

3.Culture&infection

4.Lysis&DNAremoval5.Clarification

6.TFF17.AEX8.TFF2

Upstream

process(USP)

DSP

Tet-repressingHEK293

3LstirredbioreactorsTween20&benzonase,in

bioreactor

MerckC0SPdepthfilter

SpectrumLabs300kDahollowfibre

2.Startingmaterial 1-3Lshakeflask1.Cellseedtrain Shakeflasks

MerckPellicon2300kDa

MustangQmembrane

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Vaccinesusedfor‘testcases’

ChAdOx2RabG(rabiesvaccine)

ChAdOx1RVFVGnGc(RiftValleyFevervaccine)

ChAd63ME-TRAP(malariavaccine)

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Vaccinesusedfor‘testcases’

0

5×1010

1×1011

1.5×1011

2×1011

2.5×1011

24 42 46 72 960

5×1010

1×1011

1.5×1011

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24 42 46 72 96

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2×108

4×108

6×108

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24 42 46 72 96

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24 42 46 72 96

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2×109

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24 42 46 72 96

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ChAdOx2 ChAdOx1ChAd63

Viru

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24 42 46 72 96

Time after infection (hours)MOI3MOI10

Fedosyuketal,Vaccine2019

InAg-repressingHEKs,infectionatMOI3,harvestat~42hgivesgoodyieldsofallthreeviruses

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

3Lstirredtankbioreactor•  Goodresultswithtwodifferentvessels

•  Yieldc.1x105VPpercell

•  Simple<48hrbatchprocess•  Cellexpansioninshakeflasks

0 2 0 4 03 5

3 6

3 7

3 8

E la p s e d t im e (h rs )

Te

mp

era

ture

(°C

)

0 1 0 2 0 3 0 4 00

2 0

4 0

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)

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pH

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(ml/m

in) &

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p m

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)

InfectionFeed InfectionFeed InfectionFeed

Fedosyuketal,Vaccine,2019

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Separationofadenoviralproductvariant

Total Virus Particles Lowry Protein Assay Dynamic Light Scattering SPR UV Measurement Full Particles Genome Quantitation Assay (GQA) Reverse-Phase HPLC Assay CsCl Gradient Analysis (% full)UV Absorbance Assay (UV-SDS) Infectious Particles TCID50 Q-PCR Based Potency Assay Antigen Expressing Particles Western blot In vitro Antigen Expression Assay

TotalVirusParticle

InfectiousParticle

EmptyParticleFullParticle

Non-InfectiousParticle

AntigenExpressingParticle

VLPVaccines:QualitybyDesign

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

VLPVaccines:UnderstandingVLPassemblyandqualityattributes

DesignandProductionofhighqualityFoot-and-MouthDisease(FMD)virus-likeparticles(VLP)

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

VLPVaccines:Design(I)

Pull-down

E. coli P. pastoris

a.

Ex vivo assembly

b.

Codon optimised precursor for E. coli and P. pastoris

method 1 method 2

E. coli P. pastoris

H

H

H

H

H

H

…

…

…

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

VLPVaccines:Cloning/Expression(II)

3C pLysSpRha-3CL127P

pET3a-P1-2A

BL21(DE3)

= T7 promoter

= Rha promoter

= rbs

= T7 terminator

=Vp0 =Vp3 =Vp1 =2A

* non-specificbinding

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

VLPVaccines:Cloning/Expression(II)

* non-specificbinding

His6

+ c.

His6

+ a.

His6

+ b.

= T7 promoter = rbs = T7 terminator

=Vp0 =Vp3 =Vp1

BL21(DE3) 37°C

cont

rol

a. b. c.

100

50 37

150 M

18°C

αTypO-FMDV

75

a. b. c. - + - + - + - + - + - +

*

BL21 CodonPlus(DE3)-RIL

100

50 37

20

150

25

75

αTypO-FMDV

37°C

a. b. c. M

18°C

a. b. c. - + - + - + - + - + - +

*

*

25 20

Vp1his6~ 25 kDa Vp3his6 ~ 27 kDa Vp0his6 ~ 35 kDa

+

cont

rol

+

cont

rol

+

cont

rol

+

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

VLPVaccines:PlatformApproachesforDenguevaccine

390millionsinfectionsperyearAsymptomatictosevereacutefebriledisease

•  Controlmeasurestargetingmosquitovectorshaveverylimitedeffectiveness

•  Vaccinationisanimportantpartofanintegrateddenguepreventionandcontrolstrategy

•  Onelicenseddengue,Dengvaxia®(CYD-TDV)andonlyeffectiveinseropositiveindividuals

Developmentofacost-effectiveplatformforthedeliveryofamulticompetentDengue

vaccine

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

ProposedPlatform

Upstreamproduction

VaccinePrototypePurification

VaccinePrototypeQualityAssessment

VectorDesignandStrainSelection

TobeperformedatBiofarma(Indonesia)

Development of a representative upstream dengue vaccine, VLP form,productionplatforminPichiaPastoriso  Studyingoptimalstrategyinmedium,feeding,inductiontechnologiesand

VLPeffectiverecoveryo  Developmentofrobustanalyticsforproductcharacterisationandquality

evaluation

Developmentofastrategyforscale-upandtechnologytransfer

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Preliminaryresults

GrowthprofileofPichiapastorisexpressingthedenguevaccinevector(DENVprM/E).Significantimprovementswereachievedinbiomassyieldbyfermentationprotocoloptimisation.However,higherdensitiesaredesiredtoachievedesiredtiters.

PurificationandpreliminarycharacterizationofrecombinantprM/Edengueserotypeusinganion-exchangechromatography(AEC)andhydrophobicinteractionchromatography(HIC).

0

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0 50 100 150 200

Optic

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Time (h)

HostImprovement

0.0

25.0

50.0

75.0

100.0

Conc.(µg/mL)

Volume(mL)

Mass (mg) Purity (%)

Rela

tive

valu

es

Goal

Virus-LikeParticle(VLP)prM/Edengueserotype1(A),2(B),3(C)and4(D)usingtransmissionelectronmicroscopy(TEM).Theparticlediametersvariedbetween29-35nmdependingontheserotypeandaresmallerthanthenaturalvirus(40-50nm).

NovelGlycoconjugatesTechnologies

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

RelevanceofGlycoconjugateVaccines

GlycoconjugatesfavourT-cellsdependentresponse(memory)Polysaccharides-basedvaccines(Glycanonly)TcellsindependentimmuneresponseExamplesofsuccessfulhumanglycoconjugate:

1. Haemophilusinfluenzae2. Neisseriameningitidis(excepttypeB)3. Streptococcuspneumoniae(someserotypes)

BertiandAdamoChemSoc.Rev.2018

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

ProteinGlycanCouplingTechnology(PGCT)forlowcostglycoconjugatevaccines

•  PGCTischeap,safeandflexibleindesign

•  Itcanbeappliedtoimproveexistingvaccines(pneumococcol)

•  ordevelopnewones(Francisella)

•  Orenternewmarkets(veterinary)

PGCTreview:Kay,CuccuiandWren,npjVaccines2019

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

DevelopmentofavaccineagainstS.pneumoniaStreptococcuspneumoniae:•  Gram+,alpha-haemolyticdiplococcus,

commensalandrespiratorypathogen•  Over95differentserotypes

•  Causespneumonia,meningitis,conjunctivitis,bacteraemiaandotitismedia

•  Estimatedthatglobally0.5millionchildrenunderfivedieofpneumococcaldiseaseeachyear(mostlyindevelopingcountries)

•  Efficaciousvaccines(eg.PCV13)areavailable,butexpensiveandoftenunaffordablefordevelopingcountries

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

Strategiesforglycoconjugatevaccineproduction

Chemicalorenzymaticconjugation

•  Requiresseparatepurificationoftheglycanandcarrierprotein

•  Strainusedforglycanpurificationmaybeunsafe

•  Multi-stepheterogeneouspreparation•  Expensiveandtimeconsuming

Biologicalconjugation•  EngineeredsafelaboratoryE.colistrain•  Singlepurificationstepofthe

glycoconjugate•  Homogeneousprep•  Manufacturingexpectedtobecheaper

andlesstimeconsuming

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Designandongoingwork

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

VaccinesindevelopmentKay,CuccuiandWren,npjVaccinesreview2019

University College London The Jenner Institute London School of Hygiene & Tropical Medicine Imperial College London University of Leeds

AutomatedMicroscaleProcessdevelopment

Conditionsforscreening:

•  Mediacomposition•  DO,pH,temperature•  Harvesttimes/cultureduration•  E.colistrain•  Carrierproteinandenzymealternatives

AIM:Toscaleoriginal20mLcultureto2mL

Operating conditions at themicroscale baseduponengineeringfundamentals.

•  Sca l ing based upon match ing m ix ingcharacteristicsatbothscales

•  Scalingbaseduponmatchingtheoxygentransfercoefficient,kLa

24conditionsinparallel

Highthroughputcharacterisation(e.g.ELISA)

Nextsteps

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BenefitsforHubUsers

Accesstointernationally-leadingacademicsandtopresearcherswithexpertiseinprocessdevelopment,vaccinology,analyticaldevelopment,GMPmanufacturinganddecisionaltoolsAbilitytosteertheresearchagendaoverthenext2years,alignedtoyourorganization’sprioritiesandthehubvisionandremitEarlyaccesstoHuboutputs(newmethodologiesandtechnologies)viatheCollaborationAgreementParticipationinvouchersorfeasibilitystudiestoevaluateHuboutputsusingyoursystemsandprocessesLeveragefundingforgreaterimpactviaindustry-ledInnovateUKprojectsOpportunityforwidercollaborationviatheEngineeringDoctorate(EngD)studentshipsAccesstohighlyskilledgraduatingdoctorateandresearchers

Driving the research agenda

Access to funding, outputs and skillset

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PlatformOperations

InteractionvouchersCall8vouchersintotal,budgetofupto£10Kpervoucher(<6monthsduration)AvailabletoUsersgroupmembersonlyMustbringtogetheranytwoorganizations(includingacademia-industrypartnerships)FeasibilityprojectsCall(early2020)Minimumof6projects,£100Keach(<12monthsduration)

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UpcomingeventsVax-HubUsersGroupmeeting–8thNovemberVouchersInteractionSubmissiondeadline–4th

NovemberFormoreinformationandnews

BiochemicalEngineeringDepartmentWebsite@VaxHub

ForhowtobecomeamemberPleasecontactDrNavGill(n.gill@ucl.ac.uk)

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Acknowledgements/Thankyou

AdenovirusplatformmanufacturingDrSandyDouglasProfessorSarahGilbertDrFatemehVahidDastjerdiVLPVaccinesDrSaraPlacemente,DrSteffiFrankDrSalomeDeSaMalaghaes,ProfEliKeshavarz-MooreGlycoconjugatesDrMartaMauri,ProfBrendanWrenDrJasminSamaras