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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 2016

1956, first report on mycoplasma detection in cell culture by Robinson et. Al.

1960-1980, some extensive studies in the US

- 1000 samples

(15% infected cell cultures)

-CCL, DCL and PCC

Supaporn Phumiamorn 11 March 2016 2

Mycoplasma

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 2016 3

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.


Mycoplasma contamination

Altered growth rate

Morphology changes

Chromosome aberrations

Alterations in amino acid and nucleic acid metabolism

Dead



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)

Uphoff C.C and Drexler H.G


Source of mycoplasma contamination

Cross-contamination from infected cultures Original tissue isolate (less than 1%)

Mycoplasma detection

• Bacterial culture

• DNA staining (epifluorescence in cell culture)

• PCR

• PCR and microarray (DNA microarray testing)

• ELISA etc.


Mycoplasma detection


• 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)


Adventitious agent

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)


Potential concern that adventitious agents can be unintentionally introduced into the manufacturing process


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)



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



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

Non-Specific Methods: In Vivo Systems


Adventigious agent

Extraneous agent using animals - Mice - Sucking mice -Guinea pigs -embryonated hens’ egg



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.



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



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



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)


Adventigious agent

Extraneous agent using cell lines

- sensitive cell lines (Vero and MRC-5)

- induce Cytopathic Effect (CPE) or haemadsorbing activity



Adventigious agent

Extraneous agent: Any viral contaminants


Positive viruses -Adenovirus-5 -Reovirus-3 -parainfluenza type 3 -HSV-1

Adventitious agent


Adventitious agent


Extraneous agent: Any viral contaminants

Heamagglutination



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.

Cell-Culture Method: Readout



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)

Limitations of In Vitro Tests



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 106 particles per mL to be detected

Qualitative assay; a positive result would require additional tests (e.g., PCR, infectivity)


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


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


Novel Molecular Methods 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

Strategy for Adventitious Agent Detection


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


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

Adventitious Agents: A Continuing Challenge

Composite of all adventitious agent testing

Test Note

Sterility (Bacteria, fungi)

Mycoplasma Both culture and non-culture

Sucking mice

Adult mice

Guinea pigs (some cases)

Rabbits (some cases)

Embryonated eggs

Monkeys



Test Note

TEM

Reverse Transcriptase testing (test for retroviruses)

Retrovirus testing (Simian virus)

Porcine parvovirus

lymphocyctic

choriomeningitis virus (LCM)

EBV



Test Note

Human CMV

HAV, HBV, HCV

Papilloma

Adeno- associated virus

HHV-6, HHV-7

Mycobacterium

HSV-1.-2


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


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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

Arifa S. Khan, PhD. Division of Viral Products Office of Vaccines Research and Review CBER, FDA, 2005

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


Tumourigenicity:

performed in hamsters, immunosuppressed newborn rats, and nude mice at several passage levels of Vero cells

at 106 or 107 cells/animal.

Tumourigenicity:



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 2010 2. Testing for the detection of mycoplasma contamination. VICH GL34 (Biologicals:Mycoplasma) Feb.2013.p.1-15 3. 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

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; 2014. 2232


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