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Test for retrovirus detection

As specified in the “Points to Consider on the Characterisation of Cell Lines Used to Produce Biologicals” and in the international “Note for guidance on Quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin” (CPMP/ICH/295/95), three different techniques should be used to test samples for retroviruses:

• Infectivity assays.
• Transmission electron microscopy (TEM),
• Product-enhanced reverse transcriptase (PERT) assays.

Infectivity assays

The major focus is on the use of murine cell lines or hybrid cell lines containing a murine component. Indeed, these cell lines are inherently capable of producing infectious mouse retroviruses.

Texcell provides very sensitive protocols for detecting a wide range of retroviruses such as ecotropic, xenotropic, amphotropic and mink cell focus-forming (MCF) retroviruses.

For the detection of retroviruses, two kinds of protocol exist:

• The direct focus assays (using one detector cell line) enable direct detection of retrovirus particles. The selected cell line will specifically detect the type of retrovirus.
• Extended assays use a initial cell line to amplify the retrovirus (with between three and five passages), following by a direct focus assay. These extensive amplifications allow more sensitive detection of retrovirus particles. Hence, if the quantity of retrovirus in the test article is expected to be low, the extended protocol will be chosen.

In the table below, the detector cell line is modified with sarcoma-positive (S+) and leukaemia-negative (L-) isotypes in order to obtain foci show the presence of the retrovirus in the cells during the focus assay. If the test articles are supernatants, the inoculation of the test article is directly carried out on the detector cell lines. If the test articles are cells, the latter are co-cultured with the detector cell lines.

Remarks:
If the test article are supernatants, the protocol number bears the suffix "S".
If the test article are cell lines, the protocols number bears the suffix "C".
*cell line uses for retrovirus amplification

Transmission electron microscopy

Transmission electron microscopy (TEM) is commonly used to detect retroviruses in test articles and to type retrovirus particles (A, B, C or D) in cell lines.
If the test article is seen to be contaminated by observation of the cell line in TEM, the detection of retrovirus is then confirmed by direct assays.

TEM can also be used to characterise cell lines and to detect other contaminants such as Mycoplasma, bacteria, fungi and other viruses.

TEM is an indispensable tool for confirming that a cell line is in good health.

Regardless of whether retroviruses are present in the MCB, WCB or EPCB, the unprocessed bulk supernatant should be tested to determine the retroviral titre. 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” note published by the FDA, if the TEM results are negative, the retrovirus titre should be considered to be equivalent to the lower detection limit (1x106/ml).
Accordingly, Texcell has developed a specific analysis involving negative stain electron microscopy for the detection of retroviruses in liquid samples. The sensitivity of this method is approximately 5x105/ml.
The purification method must be proven to eliminate retrovirus particles in cases of this type of contamination (See the virus validation section).

To obtain more information on specific contaminants, Texcell provides custom-made protocols in which an indicator cell line (e.g. Vero, MRC-5, BT or PPK) is inoculated with the test product. The cells are then observed by TEM, making it possible to visualise the production of virus particles. This protocol can identify the viral species involved.

F-PERT assay

When TEM and infectivity assay results are negative, test samples should be confirmed to be free of retrovirus particles by the absence of reverse transcriptase activity.