Sunday, August 8, 2010

TESTING FOR ADVENTITIOUS AGENTS CELL PROPERTIES Presence of Residual Cells Residual Cellular DNA General Safety Test


For a vaccine it is required that the final finished product meets the requirements of freedom from adventitious agents , you may be aware of recent developments on rota virus vaccines (rotarix vaccine) was found to contain DNA fragments , but later on after a detailed study rotarix vaccine was declared safe.

We are trying to provide our readers , a topic wise information about viral vaccine manufacturing quality assurance and quality controlling requirements, and today's topic we have chosen is Testing Adventitious Agents In Viral Vaccines during production.

1. Testing for the Presence of Residual Cells
2. Testing for Residual Cellular DNA
3. General Safety Test (GST)

A.ADVENTITIOUS AGENT: A microorganism (including bacteria, fungi, mycoplasma/spiroplasma, mycobacteria, rickettsia, viruses, protozoa, parasites, TSE agent) that is inadvertently introduced into the production of a biological product.

1. In Vivo Tests
a. Adult Mice
b. Suckling Mice
c. Guinea Pigs
d. Rabbits
e. Embryonated Chicken Eggs
f. Antibody Production Tests

2. In Vitro Tests for Viruses

a. Cell Culture Safety Tests
* Human Diploid Cells
* Monkey Kidney Cell Line (usually Vero cells)
b. Transmission Electron Microscopy (TEM)
c. Biochemical Tests for Retroviruses
d. Infectivity Tests for Retroviruses
e. PCR or Other Specific In Vitro Tests
f. Sourcing and Testing of Bovine-Derived Materials
g. Testing of Porcine-Derived Reagents
3. In Vitro Tests for Non-Viral Agents
a. Mycoplasma/Spiroplasm
b. Bacterial and Fungal Sterilit
c. Mycobacteria Testing

1. Tests for Tumorigenicity
2. Testing for Oncogenicity
3. Identity Testing of Cell Substrates
4. Testing for Genetic Stability


1. Testing for the Presence of Residual Cells
2. Testing for Residual Cellular DNA
3. General Safety Test (GST)

For a vaccine to be considered free of a contaminant, your assay should demonstrate, at a predefined level of sensitivity, that a certain quantity of the substance is free of that contaminant.
Assurance that virus vaccines are free of adventitious agents is a critical component of meeting the 21 CFR 610.13 requirement for purity. Cultures must be tested for the presence of detectable microbial agents and tests necessary to assure the safety and purity of the product may be required (21 CFR 610.18).

Biological starting materials being used for producing virus vaccines should be characterized sufficiently to ensure that they do not contaminate the final viccine product with extraneous infectious organisms, such as bacteria, fungi, cultivatable and non-cultivatable mycoplasmas and spiroplasma, mycobacteria, viruses, and the agent(s) responsible for transmissible spongiform encephalopathies (TSEs).

Alternatively, a validated process that is known to remove a contaminant to a defined level may be used to demonstrate the absence of that contaminant. If an adventitious agent is known to be present in your cell substrate or viral seed, then you should demonstrate that your production process is sufficiently robust to eliminate or inactivate the agent with an appropriate margin of safety. You should avoid exposure of your product to agents that are known to be infectious for humans (other than the vaccine virus) and/or to agents for which there are no appropriately sensitive validated testing procedures (such as TSEs). You should have appropriate and sufficient controls in place to assure that such exposures do not occur.

Coming article will cover topics on testing of cell substrates, control cell cultures, viral seeds, and vaccine harvests. When cell substrates are tested, generally, a preparation at 10 rest to 7 cells/mL (or when appropriate, cell lysate from an equivalent number of cells in culture medium) is used, with the indicated volumes. When viral seeds or vaccine harvests are tested, the tests are performed either at the indicated volumes or dose-equivalents. When viral seeds and vaccine harvests are tested, it is often necessary to neutralize the virus prior to inoculation. If the vaccine virus needs to be neutralized in order to perform a valid test, the source of the antiserum and its influence on test results should be considered. In general, antiserum used for neutralization should not be of human or simian origin or from the same cell type used for production of the vaccine virus.

The most appropriate tests for adventitious agents in a given vaccine will vary depending on a variety of factors, including the origin of the cell substrate and its history. We encourage manufacturers to consult with us in selecting the appropriate tests. See Section III for additional information on factors to take into account in selecting appropriate tests.
For each of the tests for adventitious agent alternatives such as those recommended by the World Health Organization (WHO) or the European Pharmacopoeia (EP) or different inoculation strategies for testing cell bank lysates and viral harvests are also considered by US FDA when justified in the context of the entire testing program.

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