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


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.


Themanufacturinglandscape–supplyisfailingdemand

VaccineaffordabilityandsupplyisoneofthekeyprioritiesidentifiedbytheWHOGlobalActionPlan


HubVisionandAim

Toadvancetechnologiesthatwillensurefuture,uninterruptedsupply.

Toensurethattheseadvancestranslateto

LMICmarketsandmanufacturers.

Abilitytosupportandrespondtoepidemicthreats.

TheHubsupportsanambitiousprogrammeofinnovativeresearchrelatedtothechallengesofdeveloping,scaling-upandmanufacturing

vaccinesofbenefittolowandmiddleincomecountries.


Hub-Spokemodel

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

medicine


Hubactivities

GCManufacturingResearchonthreemaindemonstration

technologies(viralvectors,conjugatesandVLPs)

Platform Operations Interaction Vouchers,

Training and Feasibility Studies

Hub Activities


PlatformOperations

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


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


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


HubExpertise-UCL

IndustrialBiotechnologye.g.chemicalsandpharmaceuticals

MacromolecularMedicinese.g.antibodiesandvaccines

CellandGeneTherapiese.g.cellsand

engineeredtissues

HighThroughputBioprocessDesignandScale-up

SyntheticBiology(Protein,VectorandCellEngineering)

DecisionalTools(DataMining,ProcessandEconomicModelling,LCA)


Toolsforbioprocessdesignandscaling

Experimentalstudyofflowdynamics,mixingandsuspensiondynamics

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

MilliporeCellReady,DasBox)§  Impactofenvironmentfordifferentcelltypesandproducts§  Robustscalingequationsandmethodologies

MiniaturisationanddevelopmentofScale-DownTools

§  Quasi-perfusionmicroscalemethodologies§  250mlperfusionbioreactor§  Microscaletangentialflowfiltrationdevice§  Integrationofmimicswithinautomatedplatformstospeedup

bioprocessdevelopment


UltraScale-Down(USD)Technologies:Manufacturinginsightinthelab

Continuous centrifugation Chromatography Formulation/Fill-

finish

Fermentation Depth filtration Membrane filtration

Opportunitywithnewtechnologiestolinkwholebioprocesssequence

USDTechnologies


HubExpertise–UniversityofOxford

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

DesignandtestingofviralvectoredandVLPvaccinesTransitionfromresearchtoGMPGMPmanufactureofviralvectors,VLPandrecombinantproteinsAssaydevelopmentforreleaseandin-processtestingFormulationofdrugproductClinicalvaccinedevelopment(UKandoverseas)

AdenovirusManufacturingPlatform


ProcessrequirementsforPhaseI

Small≥100doses

SimpleLimitedstaff,oneteammakesallproductsLimitedcapitalequipmentLimitedcapacitytovalidatenewequipment/processesTransferabletoLMICmanufacturers

RobustTransferableacrossmultipleproducts

Qualitymeetingregulatoryrequirements

Aneffectiveadenovirusmanufacturingapproachislikelytobeapplicabletovaccinesagainstmultiplepathogens:EmergingoutbreakpathogensVeterinaryAntibody&Tcells


Adenovirusbiology

•  Non-envelopeddsDNAvirus,90nm

•  Non-replicatingduetoE1(andE3)genedeletion

•  HEK293orPERC6cellssupplyE1intrans•  Antigen-encodingtransgeneunderstrong

constitutivemammalianpromoter•  Antigenisnotastructuralpartofthevirionà

vaccinesusingasingleAdserotypearestructurallythesame,regardlessofAg

•  Antigenisexpressedinculture:canaltergrowthcharacteristics,selectionpressureforgeneticinstability


Chimpanzeeadenovirusvectors(‘ChAds’)

•  Minimalpre-existinganti-vectorimmunityinhumanpopulation

•  Multipleserotypes

•  Differenthexon/fibercapsidproteins

•  IssueofcompatibilitywithHEK293Ad5-

derivedE1:•  Manufacturingcanbeenhancedbynon-structural

genemanipulation


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


Vaccinesusedfor‘testcases’

ChAdOx2RabG(rabiesvaccine)

ChAdOx1RVFVGnGc(RiftValleyFevervaccine)

ChAd63ME-TRAP(malariavaccine)


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

2×1011

24 42 46 72 96

0

2×108

4×108

6×108

8×108

24 42 46 72 96

0

50

100

24 42 46 72 96

0

1×109

2×109

3×109

24 42 46 72 96

0

50

100

150

24 42 46 72 96

ChAdOx2 ChAdOx1ChAd63

Viru

s pa

rtic

le ti

ter

(VP/

mL

of c

ultu

re)

Infe

ctiv

ity ti

ter

(IU/m

L of

cul

ture

)Vi

able

cel

l den

sity

(% o

f sta

rtin

g va

lue)

0

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4×108

6×108

8×108

24 42 46 72 96

0

2×1010

4×1010

6×1010

8×1010

24 42 46 72 96

0

50

100

150

24 42 46 72 96

Time after infection (hours)MOI3MOI10

Fedosyuketal,Vaccine2019

InAg-repressingHEKs,infectionatMOI3,harvestat~42hgivesgoodyieldsofallthreeviruses


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

6 0

8 0

1 0 0

0

2

4

6

8

1 0


DO

(%

)

Air flo

w ra

te (m

l/min

)

0 1 0 2 0 3 0 4 07 .0

7 .1

7 .2

7 .3

7 .4

0

1

2

3

4

5


pH

CO

2 F

low

(ml/m

in) &

Ba

se

ad

ditio

n (p

um

p m

inu

tes

)

InfectionFeed InfectionFeed InfectionFeed

Fedosyuketal,Vaccine,2019


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


VLPVaccines:UnderstandingVLPassemblyandqualityattributes

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


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

…

…

…


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


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

+


VLPVaccines:PlatformApproachesforDenguevaccine

390millionsinfectionsperyearAsymptomatictosevereacutefebriledisease

•  Controlmeasurestargetingmosquitovectorshaveverylimitedeffectiveness

•  Vaccinationisanimportantpartofanintegrateddenguepreventionandcontrolstrategy

•  Onelicenseddengue,Dengvaxia®(CYD-TDV)andonlyeffectiveinseropositiveindividuals

Developmentofacost-effectiveplatformforthedeliveryofamulticompetentDengue

vaccine


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


Preliminaryresults

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

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

0

50

100

150

200

250

0 50 100 150 200

Optic

al d

ensit

y

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


RelevanceofGlycoconjugateVaccines

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

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

BertiandAdamoChemSoc.Rev.2018


ProteinGlycanCouplingTechnology(PGCT)forlowcostglycoconjugatevaccines

•  PGCTischeap,safeandflexibleindesign

•  Itcanbeappliedtoimproveexistingvaccines(pneumococcol)

•  ordevelopnewones(Francisella)

•  Orenternewmarkets(veterinary)

PGCTreview:Kay,CuccuiandWren,npjVaccines2019


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

commensalandrespiratorypathogen•  Over95differentserotypes

•  Causespneumonia,meningitis,conjunctivitis,bacteraemiaandotitismedia

•  Estimatedthatglobally0.5millionchildrenunderfivedieofpneumococcaldiseaseeachyear(mostlyindevelopingcountries)

•  Efficaciousvaccines(eg.PCV13)areavailable,butexpensiveandoftenunaffordablefordevelopingcountries


Strategiesforglycoconjugatevaccineproduction

Chemicalorenzymaticconjugation

•  Requiresseparatepurificationoftheglycanandcarrierprotein

•  Strainusedforglycanpurificationmaybeunsafe

•  Multi-stepheterogeneouspreparation•  Expensiveandtimeconsuming

Biologicalconjugation•  EngineeredsafelaboratoryE.colistrain•  Singlepurificationstepofthe

glycoconjugate•  Homogeneousprep•  Manufacturingexpectedtobecheaper

andlesstimeconsuming


Designandongoingwork


VaccinesindevelopmentKay,CuccuiandWren,npjVaccinesreview2019


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


BenefitsforHubUsers

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

Driving the research agenda

Access to funding, outputs and skillset


PlatformOperations

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


UpcomingeventsVax-HubUsersGroupmeeting–8thNovemberVouchersInteractionSubmissiondeadline–4th

NovemberFormoreinformationandnews

BiochemicalEngineeringDepartmentWebsite@VaxHub

ForhowtobecomeamemberPleasecontactDrNavGill([email protected])


Acknowledgements/Thankyou

AdenovirusplatformmanufacturingDrSandyDouglasProfessorSarahGilbertDrFatemehVahidDastjerdiVLPVaccinesDrSaraPlacemente,DrSteffiFrankDrSalomeDeSaMalaghaes,ProfEliKeshavarz-MooreGlycoconjugatesDrMartaMauri,ProfBrendanWrenDrJasminSamaras

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

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