1 Adventitious Agent Testing of Novel Cell Substrates for Vaccine Manufacture Arifa S. Khan, PhD. Division of Viral Products Office of Vaccines Research and Review CBER, FDA November 16, 2005

2 Outline  Cell substrates used in U.S. licensed viral vaccines  Safety concerns and challenges for testing novel cell substrates, especially tumorigenic cells  FDA experience with tumorigenic cell substrates  Testing recommendations for novel and tumorigenic cell substrates, such as Madin-Darby canine kidney (MDCK) cells

3 Types of Cell Substrates Used in Current U.S. Licensed Viral Vaccines  Primary Cells or Tissues: used without passage in tissue culture  Diploid Cells: cells with a finite lifespan and passage in tissue culture  Continuous Cell Lines: immortal, neoplastic cells with unrestricted passage in tissue culture  Non-tumorigenic

4 U.S. Licensed Viral Vaccines: Primary Cells or Tissues Cell Substrate Live Vaccines Inactivated Vaccines Cell Substrate Live Vaccines Inactivated Vaccines Mouse brain Japanese Encephalitis Mouse brain Japanese Encephalitis Calf lymph Smallpox Calf lymph Smallpox Embryonated hens’ eggs Yellow Fever Influenza Embryonated hens’ eggs Yellow Fever Influenza Influenza Influenza Monkey kidney cells Poliovirus Monkey kidney cells Poliovirus Chicken embryo Measles Rabies Chicken embryo Measles Rabies fibroblasts (CEFs) Mumps fibroblasts (CEFs) Mumps

5 U.S. Licensed Viral Vaccines: Diploid Cell Strains Cell Substrate Live Vaccines Inactivated Vaccines Cell Substrate Live Vaccines Inactivated Vaccines Rhesus fetal lung: FRhL-2 Rotavirus Rabies Human fetal lung: WI-38 Rubella Adenovirus Adenovirus MRC-5 Varicella Poliovirus Hepatitis A Hepatitis A Rabies Rabies

6 Viral Vaccines: Continuous Cell Lines Cell Substrate Live Vaccines Inactivated Vaccines Vaccines African green monkey kidney: Vero Poliovirus Poliovirus (Europe) (U.S.) (Europe) (U.S.)

7 Transition to Novel Cell Substrates Continues…  Primary Cells/Tissues (21 CFR 600 series)  Diploid Cell Strains (Guidelines: J. Biol. Stand., 1981)  Continuous Cell Lines (Points to Consider: 1984; 1987; 1993)  Non tumorigenic  Tumorigenic

8  Health of the tissue donor  Viruses in donor species  naturally-occurring: endogenous and exogenous viruses  specific exposure (e.g. animal vaccines)  Cell growth  increased susceptibility for virus infection and replication  provide broader host range to different viruses  Cell passage history  propagation in different labs  biological reagents used (sera, trypsin, others)  other cell lines or viruses grown at same time  facilities  Cell phenotype (Non-tumorigenic vs. Tumorigenic)  endogenous retroviruses  latent DNA viruses  oncogenic viruses Testing Considerations for Novel Cell Substrates

9 FDA Experience with Tumorigenic Cells-I THERAPEUTIC PRODUCTS (highly purified):  Namalwa cells for interferon  EBV genome  BHK for recombinant Factor VIIa CHO cells for many products including recombinant Factor VIII CHO cells for many products including recombinant Factor VIII Murine Hybridoma cells (myeloma, NS0, SP2/0) for monoclonal antibodies Murine Hybridoma cells (myeloma, NS0, SP2/0) for monoclonal antibodies  endogenous retroviruses  murine viruses: MVM,LCMV,EDIM, LDH  293 HEK cells for Drotrecogin alfa (activated) [Xigris]  Adenovirus 5 DNA sequences INACTIVATED VACCINES (highly purified):  CHO cells for investigational protein vaccines

10 Accepted for human use based upon:  Advantages of using the cell line outweighs its tumorigenicity concerns  Extensive testing regimens for testing different stages of production: cell banks, raw materials and lots  Development / application of sensitive assays for specific agents of concerns; e.g. in rodent cells (CHO and BHK):  324K cell-based infectivity assay for Minute Virus of Mice  PERT assay for retroviruses  Incorporation of viral validation studies to evaluate the effectiveness of the manufacturing process in clearing virus that may potentially be present in Master Cell Bank FDA Experience with Tumorigenic Cells-II

11 ADVENTITIOUS VIRUS TESTING OF MDCK CELLS

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

13 General Approaches for Viral Safety of Biologicals  Qualification of cell banks, virus seed and biological raw materials  Extensive testing of cell substrate and vaccine virus seed  Use of raw materials certified or tested to be free of detectable virus  In-process testing  Develop a comprehensive testing plan to evaluate bulk/production lots for known and novel viruses  Process validation  Design an efficient process  to avoid risk of contamination  eliminate or reduce potential virus load  inactivate potentially contaminating virus

14 Adventitious Virus Testing 1993 Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals GENERAL:  In vitro cell culture tests: Inoculation of minimum of 3 species:  same species and tissue type as that used in production  human diploid cells  monkey kidney cells  In vivo assays  adult mice  suckling mice  embryonated hens’ eggs  (guinea pigs, rabbits)  Transmission electron microscopy (TEM)  PCR-based reverse transcriptase (PERT) assay for Retroviruses

15 Adventitious Virus Testing 1993 Points to Consider in the Characterization of Cell Lines Used to Produce Biologicals SPECIES-SPECIFIC:  Tests for animal viruses due to raw materials (9CFR and )  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

16 Adventitious Virus Testing Potential Viruses of Safety Concern in MDCK Cells (Due to Naturally-Occurring Viruses) Different Families of Dog Viruses are prevalent Worldwide: Virus family Disease Persistence  Paramyxoviridae Canine distemper + Parainfluenzavirus 2 infection - Parainfluenzavirus 2 infection -  Parvoviridae Parvovirus infection (sometimes)  Adenoviridae Infectious canine hepatitis + Canine laryngotracheitis - Canine laryngotracheitis -  Rhabdoviridae Rabies +  Coronaviridae Canine coronovirus infection ?  Herpesviridae Hemorrhagic disease of pups +  Papillomaviridae * Canine papillomatosis +  Retroviridae * Canine leukemia (lentivirus) + Lymphosarcomas (type C) + Lymphosarcomas (type C) + * Oncogenic Viruses

17 Adventitious Virus Testing Additional Viruses of Potential Concern in MDCK Cells (Due to Cell Susceptibility)  Influenza virus  Vesicular stomatitis virus (Indiana)  Vaccinia virus  Coxsackie B5 virus  Swine vesicular disease virus  Reovirus type 2 and 3 (persistence)  Bluetongue virus (persistence)  LCMV (persistence)

18 Adventitious Virus Testing Viruses of Possible Concern in MDCK Cells (Due to Novel and Tumorigenic Cell Substrate) Additional General Assays  Endogenous retroviruses and latent DNA viruses  chemical treatment of cell substrate with virus inducers  Oncogenic viruses (such as  -herpesviruses, polyomaviruses, papillomaviruses, adenoviruses, certain poxviruses, retroviruses)  inoculation of cell lysate (and DNA) into newborn rodents (3 species) Viral Clearance Studies  Potential, unknown viruses  model viruses  resistant to inactivating agents  oncogenic viruses

19 Virus Induction Assays

20 In Vitro Induction Assays for Detection of Endogenous and Latent Viruses INDUCER IUdR, AzaC NaB, TPA DETECTION ASSAYS TEM PERT (Retrovirus) Generic PCR Infectivity / Coculture

21 Chemical Inducers are Potent Virus Activators  5’-iodo-2’-deoxyuridine (IUdR) and 5-azacytidine (AzaC) are known inducers of endogenous retroviruses from cells of different species including avian and mammalian (ranging from mouse to human).  12-O-tetradecanoly phorbol-13-acetate (TPA) and sodium butyrate (NaB) can induce various latent DNA viruses including herpesviruses and some retroviruses (HIV-1)

22 Induction of Endogenous Retrovirus Induction and detection of endogenous MLV from mouse cells treated with IUdR + AzaC TEMPERT assay

23 Induction of Latent DNA Virus Induction and detection of HHV-8 from human B cells treated by TPA TEM PCR β-actin HHV TPA hrs. post treatment

24 Cell Lysate Testing

25 In Vivo Cell Lysate Assays for Detection of Oncogenic Viruses  Inoculation of cell lysates [and DNA] from 10 7 cell equivalent into <4 day-old animals:  newborn hamster  newborn nude mice  newborn rats up to 5 months

26 In Vivo Detection of Oncogenic Viruses in Cell Lysates  TISSUES  1911, Peyton Rous discovered the first avian retrovirus (Rous sarcoma virus) by injecting filtered extract into chickens  , Gross discovered Gross leukemia virus and polyoma virus when he obtained tumors in newborn C3H mice that were inoculated with cell-free material from Ak mouse leukemia cells.  Extracts from a variety of different transplantable sarcomas and carcinomas injected into newborn mice  leukemias  murine retroviruses  leukemias  murine retroviruses [Graffi, 1955, Moloney, 1960, Friend 1957, and Rauscher 1962]. [Graffi, 1955, Moloney, 1960, Friend 1957, and Rauscher 1962].  CELL CULTURE  , Bernice Eddy used extracts of primary rhesus monkey kidney cells to induce tumors in newborn hamsters (due to SV40)

27 In Vivo Detection of Oncogenic Viruses In Vivo Detection of Oncogenic Viruses NB Syrian Hamster NB Mice NB Rats NB Syrian Hamster NB Mice NB Rats Murine leukemia/sarcoma Hamster polyoma Bovine papilloma Human adeno 12,18,21, adeno 3, 7,14,16, adeno adeno: SA7,CELO, canine papilloma 16, polyoma (SV40, JCV, BKV) Herpes 1, HHV

28 Virus Clearance Studies

29 Influence of Viral Clearance Studies in Vaccine Manufacture  Evaluate manufacturing processes for their ability to clear viruses that are known to be present in the cell substrate  Estimate the robustness of the process for clearance of potential, unknown viruses by using “model” viruses  Assist in the quantification of risk, but do not by themselves 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.

30 Viral Clearance Studies: Selection of Model Viruses  Process evaluation of viral clearance using “relevant” or “specific” model viruses  selected based upon what is present or expected to be present  Process characterization of viral clearance using “nonspecific” model viruses  generally assessed using at least 3 different viruses with differing characteristics  viruses that display a significant resistant to inactivating agent  viruses that display a wide range of physical and biological properties

31 Viral Clearance Studies: Viral Safety Evaluation  When expected or known viruses are present, the number of virus particles in the starting material can be estimated and a specific clearance value may be used to calculate a specific safety risk (e.g., for rodent cells that produce non-infectious particles, a 6 log 10 reduction of virus above the starting value is recommended) (e.g., for rodent cells that produce non-infectious particles, a 6 log 10 reduction of virus above the starting value is recommended)  In case of unknown potential contaminants, the goal should be to provide sufficient virus clearance that can assure that the product is free of virus contamination.

32 Viral Clearance Studies: Limitations  Accurate determination of virus reduction factors requires:  Use of orthogonal clearance steps  Use of “relevant” model viruses  Reduction value >1 log 10 for each individual step  Reduction factors are normally expressed on a logarithmic scale, which implies that residual virus infectivity will never be reduced to zero: the absolute absence of virus can never be statistically proven  The behavior of the tissue culture grown model viruses used in the virus clearance studies may be different from that of the native virus (in the case of unknown viruses, the model viruses are selected based upon best representation)

33 Adventitious Virus Testing of Novel and Tumorigenic Cell Substrates: MDCK Cells for Inactivated Flu Vaccine  CELL BANK TESTING  Species-specific viruses  Other viruses based upon susceptibility of cells  Rodent viruses due to extensive and unknown passage history of cells in different laboratories  Bovine, equine, and porcine viruses based upon raw materials used in history of cell propagation (serum and trypsin)  In vitro induction assay for unknown retroviruses and DNA viruses with subsequent generic detection assays  In vivo cell lysate assay for unknown oncogenic viruses due to tumorigenicity of cells

34 Adventitious Virus Testing of Novel and Tumorigenic Cell Substrates: MDCK Cells for Inactivated Flu Vaccine  IN PROCESS TESTING  Evaluation for presence of any potential viruses  virus seed  biological raw materials  VIRAL CLEARANCE STUDIES  Evaluation of inactivation using different viruses  Evaluation of virus removal during the manufacturing process  Estimation of virus reduction using appropriate model viruses (spiking studies)

35 MULTI-STEP TESTING SCHEME FOR INACTIVATED VACCINES MADE IN TUMORIGENIC CELLS Vaccine Virus Seed Cell Substrate In Process Testing Viral Clearance Studies