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Adventitious Agent Testing of Adventitious Agent Testing of Novel Cell Substrates for Vaccine Novel Cell Substrates for Vaccine

ManufactureManufacture

Arifa S. Khan, PhD.Arifa S. Khan, PhD.Division of Viral ProductsDivision of Viral Products

Office of Vaccines Research and ReviewOffice of Vaccines Research and ReviewCBER, FDACBER, FDA

November 16, 2005November 16, 2005

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OutlineOutline

Cell substrates used in U.S. licensed viral vaccinesCell substrates used in U.S. licensed viral vaccines

Safety concerns and challenges for testing novel cell Safety concerns and challenges for testing novel cell substrates, especially tumorigenic cellssubstrates, especially tumorigenic cells

FDA experience with tumorigenic cell substratesFDA experience with tumorigenic cell substrates

Testing recommendations for novel and tumorigenic cell Testing recommendations for novel and tumorigenic cell substrates, such as Madin-Darby canine kidney (MDCK) substrates, such as Madin-Darby canine kidney (MDCK) cellscells

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Types of Cell Substrates Used in Current Types of Cell Substrates Used in Current U.S. Licensed Viral VaccinesU.S. Licensed Viral Vaccines

Primary Cells or Tissues:Primary Cells or Tissues: used without passage in tissue used without passage in tissue cultureculture

Diploid Cells:Diploid Cells: cells with a finite lifespan and passage in cells with a finite lifespan and passage in tissue culturetissue culture

Continuous Cell Lines:Continuous Cell Lines: immortal, neoplastic cells with immortal, neoplastic cells with unrestricted passage in tissue cultureunrestricted passage in tissue culture Non-tumorigenicNon-tumorigenic

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U.S. Licensed Viral Vaccines:U.S. Licensed Viral Vaccines:Primary Cells or TissuesPrimary Cells or Tissues

Cell SubstrateCell Substrate Live VaccinesLive Vaccines Inactivated VaccinesInactivated Vaccines

Mouse brainMouse brain Japanese Japanese EncephalitisEncephalitis

Calf lymphCalf lymph SmallpoxSmallpox

Embryonated hens’ eggsEmbryonated hens’ eggs Yellow Fever InfluenzaYellow Fever Influenza

InfluenzaInfluenza

Monkey kidney cellsMonkey kidney cells PoliovirusPoliovirus

Chicken embryoChicken embryo Measles RabiesMeasles Rabies

fibroblasts (CEFs)fibroblasts (CEFs) MumpsMumps

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U.S. Licensed Viral Vaccines:U.S. Licensed Viral Vaccines:Diploid Cell StrainsDiploid Cell Strains

Cell SubstrateCell Substrate Live VaccinesLive Vaccines Inactivated VaccinesInactivated Vaccines

Rhesus fetal lung:Rhesus fetal lung: FRhL-2FRhL-2 Rotavirus RabiesRotavirus Rabies

Human fetal lung: Human fetal lung: WI-38WI-38 RubellaRubella AdenovirusAdenovirus

MRC-5MRC-5 Varicella PoliovirusVaricella Poliovirus Hepatitis AHepatitis A RabiesRabies

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Viral Vaccines:Viral Vaccines:Continuous Cell LinesContinuous Cell Lines

Cell Substrate Cell Substrate Live VaccinesLive Vaccines Inactivated Inactivated

VaccinesVaccines

African green monkey kidney:African green monkey kidney:

Vero Vero Poliovirus PoliovirusPoliovirus Poliovirus

(Europe) (U.S.)(Europe) (U.S.)

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Transition to Novel Cell Substrates Transition to Novel Cell Substrates Continues…Continues…

Primary Cells/TissuesPrimary Cells/Tissues (21 CFR 600 series)(21 CFR 600 series)

Diploid Cell StrainsDiploid Cell Strains (Guidelines: J. Biol. Stand., 1981)(Guidelines: J. Biol. Stand., 1981)

Continuous Cell LinesContinuous Cell Lines (Points to Consider: 1984; (Points to Consider: 1984; 1987; 1993)1987; 1993)

Non tumorigenicNon tumorigenic TumorigenicTumorigenic

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Health of the tissue donorHealth of the tissue donor

Viruses in donor speciesViruses in donor species naturally-occurring: endogenous and exogenous virusesnaturally-occurring: endogenous and exogenous viruses specific exposure (e.g. animal vaccines)specific exposure (e.g. animal vaccines)

Cell growthCell growth

increased susceptibility for virus infection and replicationincreased susceptibility for virus infection and replication provide broader host range to different virusesprovide broader host range to different viruses

Cell passage historyCell passage history propagation in different labspropagation in different labs biological reagents used (sera, trypsin, others)biological reagents used (sera, trypsin, others) other cell lines or viruses grown at same time other cell lines or viruses grown at same time facilitiesfacilities

Cell phenotype (Non-tumorigenic vs. Tumorigenic) Cell phenotype (Non-tumorigenic vs. Tumorigenic) endogenous retrovirusesendogenous retroviruses latent DNA viruseslatent DNA viruses oncogenic virusesoncogenic viruses

Testing Considerations for Novel Cell Substrates

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FDA Experience with Tumorigenic Cells-IFDA Experience with Tumorigenic Cells-I

THERAPEUTIC PRODUCTS (highly purified):THERAPEUTIC PRODUCTS (highly purified): Namalwa cells Namalwa cells for interferonfor interferon

EBV genomeEBV genome

BHK BHK for recombinant Factor VIIa for recombinant Factor VIIa CHO cells CHO cells for many products including recombinant Factor VIII for many products including recombinant Factor VIII Murine Hybridoma cells Murine Hybridoma cells (myeloma, NS0, SP2/0) for monoclonal (myeloma, NS0, SP2/0) for monoclonal

antibodiesantibodies endogenous retroviruses endogenous retroviruses murine viruses: MVM,LCMV,EDIM, LDHmurine viruses: MVM,LCMV,EDIM, LDH

293 HEK cells 293 HEK cells for Drotrecogin alfa (activated) [Xigris]for Drotrecogin alfa (activated) [Xigris] Adenovirus 5 DNA sequencesAdenovirus 5 DNA sequences

INACTIVATED VACCINES (highly purified): INACTIVATED VACCINES (highly purified): CHO CHO cells for investigational protein vaccinescells for investigational protein vaccines

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Accepted for human use based upon:Accepted for human use based upon:

Advantages of using the cell line outweighs its tumorigenicity concernsAdvantages of using the cell line outweighs its tumorigenicity concerns

Extensive testing regimens for testing different stages of production: cell Extensive testing regimens for testing different stages of production: cell banks, raw materials and lotsbanks, raw materials and lots

Development / application of sensitive assays for specific agents of Development / application of sensitive assays for specific agents of concerns; e.g. in rodent cells (CHO and BHK):concerns; e.g. in rodent cells (CHO and BHK): 324K cell-based infectivity assay for Minute Virus of Mice324K cell-based infectivity assay for Minute Virus of Mice PERT assay for retrovirusesPERT assay for retroviruses

Incorporation ofIncorporation of viral validation studies viral validation studies to evaluate the effectiveness of to evaluate the effectiveness of the manufacturing process in clearing virus that may potentially be the manufacturing process in clearing virus that may potentially be present in Master Cell Bankpresent in Master Cell Bank

FDA Experience with Tumorigenic Cells-II

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ADVENTITIOUS VIRUS TESTING ADVENTITIOUS VIRUS TESTING OF MDCK CELLSOF MDCK CELLS

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Comprehensive testing regimens for detection of Comprehensive testing regimens for detection of known and unknown adventitious viruses in known and unknown adventitious viruses in novel vaccine cell substrates should be novel vaccine cell substrates should be designed to minimize the risk of virus designed to minimize the risk of virus contamination in vaccines, thereby assuring contamination in vaccines, thereby assuring product safety. product safety.

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General Approaches for Viral Safety of General Approaches for Viral Safety of BiologicalsBiologicals

Qualification of cell banks, virus seed and biological Qualification of cell banks, virus seed and biological raw materialsraw materials Extensive testing of cell substrate and vaccine virus seedExtensive testing of cell substrate and vaccine virus seed Use of raw materials certified or tested to be free of detectable virusUse of raw materials certified or tested to be free of detectable virus

In-process testingIn-process testing Develop a comprehensive testing plan to evaluate bulk/production Develop a comprehensive testing plan to evaluate bulk/production

lots for known and novel viruseslots for known and novel viruses

Process validation Process validation Design an efficient process Design an efficient process

to avoid risk of contaminationto avoid risk of contamination eliminate or reduce potential virus loadeliminate or reduce potential virus load inactivate potentially contaminating virusinactivate potentially contaminating virus

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Adventitious Virus TestingAdventitious Virus Testing 1993 Points to Consider in the Characterization of Cell 1993 Points to Consider in the Characterization of Cell

Lines Used to Produce BiologicalsLines Used to Produce Biologicals

GENERAL:GENERAL: In vitroIn vitro cell culture tests: Inoculation of minimum of 3 species: cell culture tests: Inoculation of minimum of 3 species:

same species and tissue type as that used in production same species and tissue type as that used in production human diploid cells human diploid cells monkey kidney cellsmonkey kidney cells

In vivoIn vivo assays assays adult miceadult mice suckling mice suckling mice embryonated hens’ eggsembryonated hens’ eggs (guinea pigs, rabbits)(guinea pigs, rabbits)

Transmission electron microscopy (TEM)Transmission electron microscopy (TEM)

PCR-based reverse transcriptase (PERT) assay for RetrovirusesPCR-based reverse transcriptase (PERT) assay for Retroviruses

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Adventitious Virus Testing1993 Points to Consider in the Characterization of Cell

Lines Used to Produce Biologicals

SPECIES-SPECIFIC: Tests for animal viruses due to raw materials (9CFR113.47 and

113.53)

Antibody production assays for rodent viruses due to extensive passage history (MAP, RAP, HAP)

Assays for known viruses based upon species PCR (specific and generic) Infectivity (in vitro and in vivo) Western blot, ELISA, IFA

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Adventitious Virus TestingAdventitious Virus TestingPotential Viruses of Safety Concern in MDCK Cells Potential Viruses of Safety Concern in MDCK Cells

(Due to Naturally-Occurring Viruses)(Due to Naturally-Occurring Viruses)

Different Families of Dog Viruses are prevalent Worldwide:Different Families of Dog Viruses are prevalent Worldwide:

Virus familyVirus family Disease Disease PersistencePersistence ParamyxoviridaeParamyxoviridae Canine distemper + Canine distemper + Parainfluenzavirus 2 infection - Parainfluenzavirus 2 infection - Parvoviridae Parvoviridae Parvovirus infection (sometimes) Parvovirus infection (sometimes) Adenoviridae Adenoviridae Infectious canine hepatitis + Infectious canine hepatitis + Canine laryngotracheitis - Canine laryngotracheitis - Rhabdoviridae Rhabdoviridae Rabies + Rabies + Coronaviridae Coronaviridae Canine coronovirus infection ? Canine coronovirus infection ? Herpesviridae Herpesviridae Hemorrhagic disease of pups + Hemorrhagic disease of pups + PapillomaviridaePapillomaviridae** Canine papillomatosis + Canine papillomatosis +

RetroviridaeRetroviridae** Canine leukemia (lentivirus) + Canine leukemia (lentivirus) + Lymphosarcomas (type C) +Lymphosarcomas (type C) +

**Oncogenic Viruses Oncogenic Viruses

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Adventitious Virus TestingAdventitious Virus TestingAdditional Viruses of Potential Concern in MDCK Cells Additional Viruses of Potential Concern in MDCK Cells

(Due to Cell Susceptibility)(Due to Cell Susceptibility)

Influenza virusInfluenza virus Vesicular stomatitis virus (Indiana)Vesicular stomatitis virus (Indiana) Vaccinia virusVaccinia virus Coxsackie B5 virusCoxsackie B5 virus Swine vesicular disease virusSwine vesicular disease virus Reovirus type 2 and 3 (persistence)Reovirus type 2 and 3 (persistence) Bluetongue virus (persistence)Bluetongue virus (persistence) LCMV (persistence)LCMV (persistence)

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Adventitious Virus TestingAdventitious Virus TestingViruses of Possible Concern in MDCK Cells Viruses of Possible Concern in MDCK Cells

(Due to Novel and Tumorigenic Cell Substrate)(Due to Novel and Tumorigenic Cell Substrate)

Additional General AssaysAdditional General Assays Endogenous retroviruses and latent DNA virusesEndogenous retroviruses and latent DNA viruses

chemical treatment of cell substrate with virus inducerschemical treatment of cell substrate with virus inducers

Oncogenic viruses (such as Oncogenic viruses (such as -herpesviruses, polyomaviruses, -herpesviruses, polyomaviruses, papillomaviruses, adenoviruses, certain poxviruses, papillomaviruses, adenoviruses, certain poxviruses, retroviruses)retroviruses)

inoculation of cell lysate (and DNA) into newborn rodents (3 inoculation of cell lysate (and DNA) into newborn rodents (3 species)species)

Viral Clearance StudiesViral Clearance Studies Potential, unknown virusesPotential, unknown viruses

model viruses model viruses resistant to inactivating agents resistant to inactivating agents oncogenic virusesoncogenic viruses

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Virus Induction AssaysVirus Induction Assays

2020

In VitroIn Vitro Induction Assays for Detection of Induction Assays for Detection of Endogenous and Latent VirusesEndogenous and Latent Viruses

INDUCER IUdR, AzaC

NaB, TPA

DETECTION ASSAYS

TEMPERT (Retrovirus)

Generic PCRInfectivity / Coculture

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Chemical Inducers are Potent Virus ActivatorsChemical Inducers are Potent Virus Activators

5’-iodo-2’-deoxyuridine (IUdR) and 5-azacytidine 5’-iodo-2’-deoxyuridine (IUdR) and 5-azacytidine (AzaC) are known inducers of (AzaC) are known inducers of endogenous endogenous retrovirusesretroviruses from cells of different species from cells of different species including avian and mammalian (ranging from including avian and mammalian (ranging from mouse to human). mouse to human).

12-12-OO-tetradecanoly phorbol-13-acetate (TPA) -tetradecanoly phorbol-13-acetate (TPA) and sodium butyrate (NaB) can induce various and sodium butyrate (NaB) can induce various latent DNA viruseslatent DNA viruses including herpesviruses and including herpesviruses and some retroviruses (HIV-1)some retroviruses (HIV-1)

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Induction of Endogenous RetrovirusInduction of Endogenous Retrovirus

Induction and detection of endogenous MLV from mouse Induction and detection of endogenous MLV from mouse

cells treated with IUdR + AzaCcells treated with IUdR + AzaC

0E+00

1E+05

2E+05

3E+05

4E+05

5E+05

6E+05

7E+05

8E+05

PE

RT

Act

ivit

y (p

U)

0 1 2 3 4 5

Days post Induction

IdU + AzCControl

TEM PERT assay

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Induction of Latent DNA VirusInduction of Latent DNA Virus

Induction and detection of HHV-8 from human B cells treated by TPA

TEM PCR

β-actin

HHV-8

- - + + TPA

24 72 24 72 hrs. post treatment

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Cell Lysate TestingCell Lysate Testing

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In Vivo Cell Lysate Assays for Detection of Oncogenic Viruses

Inoculation of cell lysates [and DNA] from 107 cell equivalent into <4 day-old animals:

newborn hamster newborn nude mice newborn rats

up to 5 months

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In VivoIn Vivo Detection of Oncogenic Viruses in Cell Detection of Oncogenic Viruses in Cell LysatesLysates

TISSUESTISSUES 1911, Peyton Rous discovered the first avian retrovirus (Rous 1911, Peyton Rous discovered the first avian retrovirus (Rous

sarcoma virus) by injecting filtered extract into chickens sarcoma virus) by injecting filtered extract into chickens

1950-1953, Gross discovered Gross leukemia virus and polyoma 1950-1953, Gross discovered Gross leukemia virus and polyoma virus when he obtained tumors in newborn C3H mice that were virus when he obtained tumors in newborn C3H mice that were inoculated with cell-free material from Ak mouse leukemia cells.inoculated with cell-free material from Ak mouse leukemia cells.

Extracts from a variety of different transplantable sarcomas and Extracts from a variety of different transplantable sarcomas and carcinomas injected into newborn mice carcinomas injected into newborn mice

leukemias leukemias murine retrovirusesmurine retroviruses [Graffi, 1955, Moloney, 1960, Friend 1957, and Rauscher 1962].[Graffi, 1955, Moloney, 1960, Friend 1957, and Rauscher 1962].

CELL CULTURECELL CULTURE 1957-1958, Bernice Eddy used extracts of primary rhesus monkey 1957-1958, Bernice Eddy used extracts of primary rhesus monkey

kidney cells to induce tumors in newborn hamsters (due to SV40)kidney cells to induce tumors in newborn hamsters (due to SV40)

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In VivoIn Vivo Detection of Oncogenic Viruses Detection of Oncogenic Viruses

NB Syrian Hamster NB Mice NB RatsNB Syrian Hamster NB Mice NB Rats

MurineMurine leukemia/sarcoma leukemia/sarcoma + + ++ + +

HamsterHamster polyoma polyoma + - -+ - -

BovineBovine papilloma papilloma + + ++ + +

HumanHumanadeno 12,18,21,31 adeno 12,18,21,31 + + ++ + +adeno 3, 7,14,16,21 adeno 3, 7,14,16,21 + - -+ - -adeno 9 adeno 9 - - +- - +adeno: SA7,CELO, canine adeno: SA7,CELO, canine + - -+ - -

papilloma 16, 18 papilloma 16, 18 - - -- - -polyoma (SV40, JCV, BKV) polyoma (SV40, JCV, BKV) + - -+ - -

Herpes 1,2 Herpes 1,2 + + ++ + +HHV-8 HHV-8 - - -- - -

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Virus Clearance StudiesVirus Clearance Studies

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Influence of Influence of Viral Clearance Studies in Vaccine ManufactureViral Clearance Studies in Vaccine Manufacture

Evaluate manufacturing processes for their ability to Evaluate manufacturing processes for their ability to clear viruses that are known to be present in the cell clear viruses that are known to be present in the cell substratesubstrate

Estimate the robustness of the process for clearance Estimate the robustness of the process for clearance of of potential, unknown virusespotential, unknown viruses by using “model” by using “model” virusesviruses

Assist in the quantification of risk, but do not by Assist in the quantification of risk, but do not by themselves prove absence of riskthemselves prove absence of risk

REF.: 1998, International Conference on Harmonisation: Q5A Viral Safety Evaluation of Biotechnology Products Derived from Cell Lines of Human or Animal Origin.

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Viral Clearance Studies: Viral Clearance Studies: Selection of Model VirusesSelection of Model Viruses

Process evaluation of viral clearance using “relevant” Process evaluation of viral clearance using “relevant” or “specific” model viruses or “specific” model viruses

selected based upon what is present or expected to be presentselected based upon what is present or expected to be present

Process characterization of viral clearance using Process characterization of viral clearance using “nonspecific” model viruses“nonspecific” model viruses

generally assessed using at least 3 different viruses with generally assessed using at least 3 different viruses with differing characteristics differing characteristics

viruses that display a significant resistant to inactivating agentviruses that display a significant resistant to inactivating agent

viruses that display a wide range of physical and biological viruses that display a wide range of physical and biological properties properties

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Viral Clearance Studies: Viral Clearance Studies: Viral Safety EvaluationViral Safety Evaluation

When expected or known viruses are present, the When expected or known viruses are present, the number of virus particles in the starting material can number of virus particles in the starting material can be estimated and a specific clearance value may be be estimated and a specific clearance value may be used to calculate a specific safety risk used to calculate a specific safety risk

(e.g., for rodent cells that produce non-infectious particles, (e.g., for rodent cells that produce non-infectious particles, a 6 loga 6 log1010 reduction of virus above the starting value is reduction of virus above the starting value is recommended)recommended)

In case of In case of unknown potential contaminantsunknown potential contaminants, the goal , the goal should be to provide sufficient virus clearance that should be to provide sufficient virus clearance that can assure that the product is free of virus can assure that the product is free of virus contamination.contamination.

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Viral Clearance Studies: Viral Clearance Studies: LimitationsLimitations

Accurate determination of virus reduction factors requires:Accurate determination of virus reduction factors requires: Use of orthogonal clearance stepsUse of orthogonal clearance steps Use of “relevant” model virusesUse of “relevant” model viruses Reduction value >1 logReduction value >1 log1010 for each individual step for each individual step

Reduction factors are normally expressed on a Reduction factors are normally expressed on a logarithmic scale, which implies that residual virus logarithmic scale, which implies that residual virus infectivity will never be reduced to zero: the absolute infectivity will never be reduced to zero: the absolute absence of virus can never be statistically provenabsence of virus can never be statistically proven

The behavior of the tissue culture grown model viruses The behavior of the tissue culture grown model viruses used in the virus clearance studies may be different from used in the virus clearance studies may be different from that of the native virus (in the case of unknown viruses, that of the native virus (in the case of unknown viruses, the model viruses are selected based upon best the model viruses are selected based upon best representation)representation)

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Adventitious Virus Testing of Adventitious Virus Testing of Novel and Tumorigenic Cell Substrates: Novel and Tumorigenic Cell Substrates: MDCK Cells for Inactivated Flu VaccineMDCK Cells for Inactivated Flu Vaccine

CELL BANK TESTINGCELL BANK TESTING Species-specific virusesSpecies-specific viruses Other viruses based upon susceptibility of cellsOther viruses based upon susceptibility of cells Rodent viruses due to extensive and unknown passage history of Rodent viruses due to extensive and unknown passage history of

cells in different laboratoriescells in different laboratories Bovine, equine, and porcine viruses based upon raw materials Bovine, equine, and porcine viruses based upon raw materials

used in history of cell propagation (serum and trypsin)used in history of cell propagation (serum and trypsin) In vitro induction assay for unknown retroviruses and DNA viruses In vitro induction assay for unknown retroviruses and DNA viruses

with subsequent generic detection assayswith subsequent generic detection assays In vivo cell lysate assay for unknown oncogenic viruses due to In vivo cell lysate assay for unknown oncogenic viruses due to

tumorigenicity of cellstumorigenicity of cells

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Adventitious Virus Testing of Adventitious Virus Testing of Novel and Tumorigenic Cell Substrates: Novel and Tumorigenic Cell Substrates: MDCK Cells for Inactivated Flu VaccineMDCK Cells for Inactivated Flu Vaccine

IN PROCESS TESTINGIN PROCESS TESTING Evaluation for presence of any potential virusesEvaluation for presence of any potential viruses

virus seedvirus seed biological raw materialsbiological raw materials

VIRAL CLEARANCE STUDIESVIRAL CLEARANCE STUDIES Evaluation of inactivation using different viruses Evaluation of inactivation using different viruses Evaluation of virus removal during the manufacturing Evaluation of virus removal during the manufacturing

process process Estimation of virus reduction using appropriate model Estimation of virus reduction using appropriate model

viruses (spiking studies)viruses (spiking studies)

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MULTI-STEP TESTING SCHEME FOR INACTIVATED MULTI-STEP TESTING SCHEME FOR INACTIVATED VACCINES MADE IN TUMORIGENIC CELLSVACCINES MADE IN TUMORIGENIC CELLS

VaccineVirus Seed

Cell Substrate

In Process Testing

Viral Clearance Studies