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Challenges for registration of Challenges for registration of

Vaccine production: improved supply in the region through

collaborations

byDr.NoraDellepianeWorkshop:GlobalRegistra8onandVaccineShortage

Taipei,Taiwan6to10March2017

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Vaccine types √  Bacterialvaccines:

•  Killed(chemicaland/orheat),e.g.wholecellpertussis•  Toxoids,e.g.tetanusanddiphtheria•  Atenuated:livemodi@iedmicro-organismsinwhichthevirulent

propertieshavebeenmodi@ied.Theyareabletoreplicateandinfectcellsintheorganismbuttheydonotcausethedisease,e.g.BCG

•  Subunits:polysaccharidevaccines(meningococcal,pneumococcal),acellularpertussisvaccines

ü  Viralvaccines:•  Killed/inactivated,e.g.Rabies,polio(Salk)•  Atenuatede.g.YF,JE,measles,rubella,mumpsandpolio(Sabin)•  Subunits:puri@iedprotectionconferringantigens,e.g.in@luenza

vaccine

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Recombinant DNA

ü  Identi@icationofgenesü  Transferfromoneorganismtoanotherü  ExpressionvectorforproteinsyntesisUsinggeneticengineeringtechniques,agenecodifyingfortherelevantantigenisisolatedandintroducedintoanotherorganismorcellthatwillexpresstheprotein,whichfollowingtherequiredpuri@icationstepswillconstitutethevaccine.Usuallysuchtechniquewillrenderviruslikeparticles,e.g.hepatitisBandHPV

4

Recombinant HPV L1 VLP Vaccine

CourtesyofDr.UmeshShaligram,SIIPL

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Live attenuated recombinant vaccine (dengue vaccine)

ü  TheactivesubstancescontainedintheCYD-TDVdenguevaccineare4liveattenuatedrecombinantvirusesrepresentingserotypes1,2,3,and4.

ü  EachmonovalentCYDrecombinantisobtainedseparatelybyreplacingthegenesencodingtheprMandEproteinsoftheattenuatedyellowfever(YF)17Dvirusgenomewiththecorrespondinggenesofthe4wild-typedengueviruses.

ü  The@inalformulationcontains4.5–6.0log10mediancell-cultureinfectiousdoses(CCID50)ofeachoftheliveattenuateddengueserotype1,2,3and4vaccineviruses.

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Conjugate vaccines

Polysaccharidevaccinesarenotimmunogenicinyounginfants,usuallyundertheageoftwo.Themethodofconjugationhasovercomethisdif@iculty.Immuneresponseisimprovedbychemicallylinkingthepolysaccharidetoaprotein‘carrier’.Thecarrierisofteneitherhighlypuri@iedtetanustoxoid,ordiphtheriatoxoid(CRM)Examplesofconjugatevaccinesarehaemophilustypebvaccine,meningococcalA,C,W,Yandalsopneumococcalvaccines(PCV10and13)

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Vaccine combinations

Individualantigenscanbecombinedinordertoprovideprotectionagainstseveraldiseases,thusminimizingthenumberofinjectionsandinterventions.ExamplesofcombosareDTP-HepatitisBDTP-HibDTP-HepatitisB–HibDTP-HepatitisB-Hib-IPVMeasles,mumpsandrubella

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Summary type of vaccines

ü  Bacterialvaccines:Killed,attenuatedandsubunitsü  Toxoids:DandTü  Viralvaccines:Killed,attenuatedandsubunitsü  Recombinantvaccines:HepatitisBvaccine,HPVü  Liveattenuatedrecombinantvirusvaccine,dengueü  Conjugatedvaccines:Hib,pneumococcal,meningoü  Combinedvaccines

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Summary type of vaccines Source:SANOFIPasteurwebsite

Steps involved in vaccine production

Inactivation ValenceAssembly Formulation FillingGerm

culture Harvest Puri@ication

BioreactorsCellculture

Fullycharacterisedcells,controlledculturemediaControlledcultureconditions

PrecipitationCentrifugationother

Tangential@iltrationChromatography,other

Heat,Inactivatingagents(formaldehyde,B-propiolactone,etc

Mixingserotypes

MixingofActiveingredientswithexcipientsincludingadjuvants,stabilizers,etc

GMPcompliance:processvalidationforeachstep,cleaningvalidation,preventivemaintenance,environmentalmonitoring,datatrendingandanalysis,media>ills,lineclearance,etcQualityControls:IPC,controlofintermediates,etc

Freezedrying

(1) (2) (3)

(1)  Bulkproduction(2)  Finalbulk(3)  Finalproduct

Steps involved in vaccine production

Germculture

Bacteria,

CellbanksystemFullycharacterisedcells,controlledculturemediaControlledcultureconditionsControlledvirusmultiplicationconditions

VirusesCellculture

Fermentation

Straincerti@icationforbacteriaandviruses(historicalrecords,originSeedlotsystem

Controlledculturemedia,GPP,sterility,etcControlledcultureconditionsControlledreplication

V

Animal Cell

Infected Animal Cell

Virus Growth

12

Viruses cannot grow on their own, they require a host cell for multiplication

Courtesy: GTN Lot Release Course CDL India

13

Original source of cells

Master cell bank

Working cell bankCells for production

Cell Bank System

testing

testing

testing

Courtesy: GTN Lot Release Course CDL India

14

Production cells Infected cells

Production cells

Working virus seed lot

Master virus seed lot

Virus strain

Virus Seed Lot System

All lots start with this virus

testing

testing

Courtesy: GTN Lot Release Course CDL India

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WHO references

ü  TRSNo978,Annex3:2013.Recommendationsfortheevaluationofanimalcellculturesassubstratesforthemanufactureofbiologicalmedicinalproductsandforthecharacterizationofcellbanks.Replacementofannex1ofWHOTRS878

ü  TRS999,Annex2:2016WHOgoodmanufacturingpracticesforbiologicalproductsReplacementofAnnex1ofWHOTechnicalReportSeries,No.822

ü  Vaccinespeci@icrequirements.

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Example of Hib polysaccharide conjugated vaccine

WHOReference

17

Example of a polysaccharide conjugate and non-conjugate vaccine

Polysaccharide coat only

Polysaccharide vaccinee.g. Meningococcal A,C,W,Y vaccine

Conjugated polysaccharide vaccinee.g. Hib vaccine, conjugated pneumococcal or conjugated meningococcal vaccine

Purification

Purification and conjugation

Courtesy: GTN Lot Release Course CDL India

Bacterialgrowth

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Haemophilus influenzae type b capsular polysaccharide (PRP)

OHOH2C O

OHOP

O

OH

O

HO

OH OH

O

O

OHOH2C O

OHOP

O

O

HO

OH OH

HOH2C O

OHOP

O

HOH2C

O

HO

OH OH

OH

O

OHOP

O

OH

O

HO

OH OH

Crisel, R.M. et al. 1975. J. Biol. Chem. 260(13): 4926-4930 Branefors-Helander, P. et al. 1976. Acta Chem. Scand. B 30(3): 276-277

n

OHOH2C O

OHOP

O

O-

OHO

OH

HO

→O)-P-(O→3)-β-D-Ribf-(1→1)-D-Ribol-(5→

C10H19O11P = 346.228

C10H18NaO11P = 368.210

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Formulation of different Hib vaccines

Bulkconjugate

Finalbulk

FinalProduct

ProteinCarrier(modi>ied)Polysaccharide

Conjugation

Modi5ication

Hib-workingseedlot

Crudepolysaccharide

Puri@iedpolysaccharide

Puri5ication

fermentation

StraincharacterizationStrainhistoryMediumcompositionConsistency:Growth,pHandpolysaccharideyieldBacterialpurityIdentityMolecularsizedistributionMoisturecontentPolysaccharidecomposition:riboseandphosphoruscontentProteinNucleicacidEndotoxin

NumberoffunctionalgroupsperrepeatunitofpolysaccharideMolecularsizedistribution

StraincharacterizationMediumcompositionStrainhistoryGrowthconsistency:growthrate,pHandproteinyieldPurityandconcentration

ResidualreagentsConjugationmarkersResidualreactivefunctionalgroupsPRPcontentConjugatedandunboundPRPProteincontentPRP-to-proteinratioMolecularsizedistributionSterilitySpeci@ictoxicityofthecarrierprotein

sterility

Formulation

FillingIdentitySterilityPRPcontentResidualmoisturePyrogencontentAdjuvantcontentPreservativecontentGeneralsafetytestpH

HIBTESTIN

G

Control of the Polysaccharide Specifications Summary

Process step “Component” Assay Specification

Hib fermentation Strain NMR Type b

Seedlot system x Consistency

Culture media x No human blood-group antigen-like material and no high-molecular-weight polysaccharide

Harvest pH, OD, polysaccharide Consistency

Contamination Gram-smear Pure

Polysaccharide purification

Identity test NMR PRP

Molecular size distribution HP-GPC Consistency

Moisture content Karl Fisher X

Ribose Orcinol >32% dry weight

Phosphorus Ames 6.8%-9% dry weight

Protein Lowry <1% dry weight

Nucleic acid UV260 <1% dry weight

Endotoxin LAL Rabbit test

<10 IU/ µg PRP 1 µg PRP / Kg

Polysaccharide modification

Degree of activation TNBS Consistency

Molecular size distribution HP-GPC Consistency

Control of the carrier protein Specifications Summary

Process step “Component” Assay Specification

Fermentation Seedlot system x Consistency

Culture media x Free from substances likely to cause toxic or allergic reactions in humans

Harvest pH, OD, Protein Consistency

Contamination Gram-smear Pure

Protein purification Purity LF test, HPLC or SDS-PAGE D&T-toxoid: >1500 LF/mg protein N CRM197: >90% Outer-membrane complex of MengB: <8% lipopolysaccharide/weight + rabbit test

Protein modification Extent of derivatization x Consistency

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ControlofbulkconjugateSpeci>icationsSummary

Process step “Component” Assay Specification

Polysaccharide-protein conjugation

Residual reagents x Removal to be confirmed

Conjugation markers PRP:Protein consistency

Residual reactive functional groups x No residual reactive group

PRP content Orcinol X

Conjugated and unbound PRP Orcinol, sample pretreatment

<40% free PRP

Protein content BCA X

Polysaccharide-protein ratio To be calculated Diphteria & tetanus toxoid: 0.3-0.6 CRM197: 0.3-0.7 OMC: 0.05-0.1

Molecular size distribution HP-GPC Consistency

Sterility Bacterial & mycotic Pass

Specific toxicity guinea-pig test Absence of specific toxicity

Controlof>inalproductSpeci>icationsSummary

Process step “Component” Assay Specification

Polysaccharide-protein conjugation

Identity Immunological test PRP

Sterility Bacterial & mycotic Pass

PRP content Orcinol and/or chromatographic

±20% of stated PRP content

Residual moisture Karl Fisher <2.5%

Pyrogen content LAL or rabbit test Acceptable

Adjuvant content Spectroscopy <1.25 mg aluminium or 1.3 mg calcium per s.h.d.

Preservative content UV Pass

General safety General safety test Animals should survive for at least 7 days

pH pH test Pass

Inspection visual No clumping, lack of integrity and/ or particles

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Summary type of vaccines Source:SANOFIPasteurwebsite

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Key factors to consider before launching vaccine production

•  Costofdevelopment•  Timefordevelopment•  Costanddif@icultiesoftechnologicalknowhow,commercialscale,consistencyofproduction,GMPcompliance

•  Costanddif@icultiesfortesting•  Technicaldif@icultiestogetappropriatelycharacterizedproductionstrains

•  IPrelatedmatters•  Costandtimeframefornon-clinicalandclinicaldevelopment

•  Registrationrelatedissuesandtimelines•  Sizeofmarketforcostrecoveryandfurtherpro@it

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Fostering collaborations between DCVMN members

•  Informationexchange•  Supporttoacquirespeci@ictechnologies(freezedrying,cellculture,

other)•  Supporttoacquiretestingmethodologies•  Sourcesofstrainsforvaccineproduction•  Sourcesofformulatedbulkreadyfor@illing,labellingandpackaging•  Sourcesofconcentratedbulkmaterialforformulation,@illing,labelling

andpackaging•  Fulltransferoftechnologyfromseed

USETHENETWORKFORMUTUALBENEFITS

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THANK YOU