Biological Assay: In Vitro Safety test Mycoplasma and Adventitious Agents

Transcription

1 Biological Assay: In Vitro Safety test Mycoplasma and Adventitious Agents Supaporn Phumiamorn (Ph.D, Virology) Institute of Biological Products (IBP) 11 March 2016 Supaporn Phumiamorn 11 March

2 1956, first report on mycoplasma detection in cell culture by Robinson et. Al , some extensive studies in the US samples Mycoplasma (15% infected cell cultures) -CCL, DCL and PCC Supaporn Phumiamorn 11 March

3 Mycoplasma the simplest self-replicating prokaryotes. infect a wide variety of eukaryotic hosts including humans, animals (birds, reptiles, fish, mammals), insects, plants, and bovine. resistant to penicillin and can squeeze through 0.2-µm and 0.45-µm membranes routinely used for sterile filtration. Supaporn Phumiamorn 11 March

4 frequent contaminants of cell cultures. biologic products prepared using cell culture substrates expected to be free of mycoplasma to assure safety, purity, potency, and consistency. Supaporn Phumiamorn 11 March

5 Mycoplasma contamination Altered growth rate Morphology changes Chromosome aberrations Alterations in amino acid and nucleic acid metabolism Dead Supaporn Phumiamorn 11 March

6 Uphoff C.C and Drexler H.G Prevalence 15-35% CCLs 5% early passage cell cultures 1% PCCs Most common species 20-40% M. orale (human, egg) 10-40% M.hyorhinis (swine) 20-30% M. arginini (bovine) 10-20% M. hominis (human) 10-20% M. fermentans (human, egg) 5-20% A. laidlawi (bovine) Supaporn Phumiamorn 11 March

7 Source of mycoplasma contamination Cross-contamination from infected cultures Original tissue isolate (less than 1%) Supaporn Phumiamorn 11 March

8 Mycoplasma detection Bacterial culture DNA staining (epifluorescence in cell culture) PCR PCR and microarray (DNA microarray testing) ELISA etc. Supaporn Phumiamorn 11 March

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11 Mycoplasma detection Supaporn Phumiamorn 11 March

12 Adventitious agent Manufacture of Biological Products Manufacture of a large class of biological products, including vaccines and cell and gene therapy products, often requires use of cell substrates and raw materials of animal origin. Potential risk that cell substrates and raw materials can be contaminated with adventitious agents (endogenous and exogenous) Supaporn Phumiamorn 11 March

13 Adventitious agent contaminating microorganisms of the cell culture or source materials (bacteria, fungi, mycoplasma/spiroplasmas, mycobacterium, rickettsia, protozoa, parasites, transmissible spongiform encephalopathies (TSE) agents and viruses) Supaporn Phumiamorn 11 March

14 Potential concern that adventitious agents can be unintentionally introduced into the manufacturing process Supaporn Phumiamorn 11 March

15 Current Methods of Detection of Adventitious Agents Methods evolved over time; in many ways recapitulate the history of virus discovery Infectivity assays in animals and cell culture Non-specific methods known/unknown agents - in vivo (animals) - in vitro (cell culture) - molecular/biochemical Specific methods known agents - molecular, e.g., PCR (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

16 Non-Specific Methods: In Vivo Systems Various animal models used, e.g., adult mice, suckling mice, embryonated hens eggs, guinea pigs, rabbits These tests originally used because they detected viruses not readily detected in other systems (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

17 Non-Specific Methods: In Vivo Systems Examples of viruses detected in mice include: cocksackie A & B viruses; picornaviruses (polioviruses, echoviruses); alphaviruses; bunyaviruses (phleboviruses, nairoviruses); arenaviruses; flaviviruses; rabies virus; herpesviruses; lymphocytic choriomeningitis virus; also various murine viruses Examples of viruses detected in eggs include: orthomyxoviruses; paramyxoviruses; alphaviruses; vesiculoviruses; herpesviruses; poxviruses; rhabdoviruses (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

18 Adventigious agent Extraneous agent using animals - Mice - Sucking mice -Guinea pigs -embryonated hens egg Supaporn Phumiamorn 11 March

19 Limitations of In Vivo Tests Sensitivity is unknown for wild-type strains, as methods were usually established with laboratory-adapted strains Many viruses pathogenic for humans do not infect or replicate in rodents or eggs. (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

20 Antibody-Production Tests Test article is inoculated into animals, and an adventitious agent is detected by the presence of antibodies to that agent Mainly performed on cell substrates when there is a possibility of exposure to rodent agents (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

21 Antibody-Production Tests Viruses detected include: ectromelia virus, mouse rotavirus, Hantaan virus, Lymphocytic choriomeningitis virus, lactate dehydrogenase virus, minute virus of mice; mouse adenovirus, murine cytomegalovirus, mouse encephalomyelitis virus, mouse hepatitis virus, pneumonia virus of mice, polyoma virus, reovirus type 3, Sendai virus, thymic virus, K virus, simian virus 5, mouse encephalomyelitis virus, Hantaan virus, Kilham rat virus, mouse adenovirus, rat coronavirus, Toolan virus, sialdacryoadenitis virus (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

22 Non-Specific Tests: In Vitro Systems Methods are based on the ability of cell cultures to grow a wide array of pathogens; also based on their extensive use in diagnostic laboratories to detect human pathogens Large amount of inocula can be applied, thus increasing their sensitivity Cell-culture tests can detect a variety of adventitious viruses, including cytopathic viruses, hemadsorbing viruses, and hemagglutinating viruses Selection of the cell line depends upon the potential exposure to agents (species and tissue type of cell substrate; human diploid cells; monkey kidney cells) (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

23 Adventigious agent Extraneous agent using cell lines - sensitive cell lines (Vero and MRC-5) - induce Cytopathic Effect (CPE) or haemadsorbing activity (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

24 Adventigious agent Extraneous agent: Any viral contaminants Positive viruses -Adenovirus-5 -Reovirus-3 -parainfluenza type 3 -HSV-1 Supaporn Phumiamorn 11 March

25 Adventitious agent Supaporn Phumiamorn 11 March

26 Adventitious agent Extraneous agent: Any viral contaminants Supaporn Phumiamorn 11 March

27 Heamagglutination Supaporn Phumiamorn 11 March

28 Cell-Culture Method: Readout Evidence of virus contamination indicated by: Cytopathic effects in culture For non-cytopathic viruses, test at the end of the observation period for: hemadsorption (binding of red blood cells from human, rhesus macaque, guinea pig, chicken) hemagglutination (agglutinate red blood cells) Other readouts can be used, such as antibody staining, PCR, etc. (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

29 Limitations of In Vitro Tests Only can detect agents that can infect and propagate in indicator cells Sensitivity is unknown for wild-type strains, as methods were usually established with laboratory-adapted strains Many viruses pathogenic for humans do not infect or replicate readily in culture (e.g., HPV, HCV) (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

30 Other Non-Specific Tests Transmission Electron Microscopy Can detect virus particles in a cell substrate, including those from endogenous viruses Morphology provides indication of the type of viral contaminant Insensitive assay generally considered to require 10 6 particles per ml to be detected Qualitative assay; a positive result would require additional tests (e.g., PCR, infectivity) Supaporn Phumiamorn 11 March

31 Reverse Transcriptase (RT) Assay for Retrovirus Detection All retroviruses have RT in their virions; therefore, these assays can detect all retroviruses Quantitative PERT assays are now recommended Some cell substrates express non-infectious endogenous retroviral particles, e.g., eggs, chick embryo fibroblasts, CHO cells Because of high assay sensitivity, false positive signals can be obtained from cell lysates Positive result may require infectivity assays Supaporn Phumiamorn 11 March

32 Specific Tests for Viruses PCR Tests When viruses are of concern in a specific product, additional testing is recommended Such tests are based on the virus sequence Conventional PCR and qpcr Partially degenerate primer PCR that detects members of a virus family Examples: - Human pathogens in certain human cells: HIV, HCV, etc. When warranted, PCR tests for various animal viruses are recommended Supaporn Phumiamorn 11 March

33 Novel Molecular Methods for Adventitious Agent Detection Supaporn Phumiamorn 11 March

34 Strategy for Adventitious Agent Detection Sample selection Cellular genome (DNA) Transcriptome (RNA) Virus particle Amplification schemes Family-specific primers Degenerate primers, etc. Detection methodologies Mass spectrometry Microarray High-throughput sequencing (Arifa S. Khan, CBER, USA 2005) Supaporn Phumiamorn 11 March

35 Considerations with New Generation Molecular Methods Sensitivities usually not determined Do not indicate whether virus is infectious Each different method may require different types of standardization and standards to be used in a regulatory context Breadth of detection not studied Reproducibility or robustness generally not known Many techniques are not commercially available Many results will require follow up Supaporn Phumiamorn 11 March

36 Adventitious Agents: A Continuing Challenge Novel viruses are being discovered and will continue to be discovered Many of these could be present as adventitious agents of cell substrates or biological products New cell substrates from insects, plants, fungi, etc. will bring additional issues Supaporn Phumiamorn 11 March

37 Composite of all adventitious agent testing Test Sterility (Bacteria, fungi) Mycoplasma Sucking mice Adult mice Guinea pigs Rabbits Embryonated eggs Monkeys Note Both culture and non-culture (some cases) (some cases) Supaporn Phumiamorn 11 March

38 Composite of all adventitious agent testing TEM Test Reverse Transcriptase testing (test for retroviruses) Retrovirus testing (Simian virus) Porcine parvovirus lymphocyctic choriomeningitis virus (LCM) EBV Note Supaporn Phumiamorn 11 March

39 Composite of all adventitious agent testing Human CMV Test HAV, HBV, HCV Papilloma Adeno- associated virus HHV-6, HHV-7 Mycobacterium HSV-1.-2 Note Supaporn Phumiamorn 11 March

40 Oncogenicity: the capacity of an acellular agent ( chemical, virus, viral nucleic acid, viral genes or a subcellular element to cause normal cells of an animal to form tumours. In vitro: PERT assay (retrovirus detection) TEM Supaporn Phumiamorn 11 March

41 In Vitro Induction Assays for Detection of Endogenous and Latent Viruses INDUCER IUdR, AzaC NaB, TPA Arifa S. Khan, PhD. Division of Viral Products Office of Vaccines Research and Review CBER, FDA, 2005 DETECTION ASSAYS TEM PERT (Retrovirus) Generic PCR Infectivity / Coculture 41

42 Tumourigenicity: the capacity of a cell population inoculated into an animal model to produce a tumour by proliferation at the site of inoculation and/ or at a distant site by metastasis. - the tumours are derived from the inoculated cells. Cells inoculated Supaporn Phumiamorn 11 March

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44 Tumourigenicity: performed in hamsters, immunosuppressed newborn rats, and nude mice at several passage levels of Vero cells at 10 6 or 10 7 cells/animal. Supaporn Phumiamorn 11 March

45 Tumourigenicity: Supaporn Phumiamorn 11 March

46 References 1. Guidance for Industry: Characterization and qualification of cell substrates and other biological materials used in the production of viral vaccines for infectious disease indications. U.S. Department of Health and Human Services, Food and Drug Administration, Center for Biologics Evaluation and Research. Feb Testing for the detection of mycoplasma contamination. VICH GL34 (Biologicals:Mycoplasma) Feb.2013.p Uphoff C.C and Drexler H.G. Cell culture mycoplasmas. DSMZ-German Collection of Microorganisms & Cell Cultures, Department of human and Animal cell cultures; Braunschweig, Germany Supaporn Phumiamorn 11 March

47 3.Keith Peden, Division of Viral Products OVRR, CBER, FDA: May 7, 2010 FDA s Approach to Adventitious-Agent Testing of Cell Substrates and Viral Vaccines: Traditional and Novel Methods 4. Regulatory risk evaluation on finding an adventitious agent in a marketed vaccine. WHO; Supaporn Phumiamorn 11 March

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