TRANSMISSION ELECTRON MICROSCOPY (TEM) for Detection of Adventitious Agents in Cells
Regulatory guidelines: ICH Q5A; 1993, 1997 PTC
Electron Microscopy
MDS offers TEM services to detect viral particles in general and retroviruses in particular in test articles and bell banks. If the test article is seen to be contaminated with retroviruses by observation of the cell line in TEM, the detection of retrovirus is then confirmed by direct assays. Regardless of whether retroviruses are present in the MCB, WCB or EPCB, the unprocessed bulk supernatant should be tested to determine the retroviral titer. It is essential to quantify the retrovirus, preferably using TEM as specified in “Points to Consider in the Manufacture and Testing of Monoclonal Antibody Products for Human Use”
MDS offers the following TEM services
- Pelleted cell lines are chemically fixed, embedded in epoxy resin and thin-sections are prepared.
- At least 100 cells are observed and evaluated the presence of viral particles at 5,000X to 50,000X magnifications in a transmission electron microscope.
- Negative stain electron microscopy for the detection of retroviruses in liquid samples before purification.
- A first analysis enables retrovirus detection if the concentration exceeds 5x105 virus-like particles (VLP) per ml.
- In order to titrate the average retrovirus concentration of VLP in the unprocessed bulk, at least three different concentrations will
- If no VLP is recovered, ultracentrifugation can be performed.
- The sensitivity of this method is approximately 5x105/ml.
Additionally, MDS provides custom services in which an indicator cell line (e.g. Vero, MRC-5, BT or PPK) is inoculated with the test product. The cells are then examined by TEM, to visualize the virus particles, and identify the viral species involved
Submission of Samples: Samples for testing can be submitted as either a growing culture or a frozen cryovial.
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