Retrovirus Assays

Retroviruses are a family of viruses with an extremely complicated life cycle. They are of particular concern as they are often present in an endogenous form as a pro-virus in many, if not all production cell lines.

CHO cells, for example, have an endogenous virus which is capable of generating a virus particle visible by Transmission Electron Microscopy (TEM). This is non-infectious but some mouse cell lines, such as NS0, will produce an infectious retrovirus.

The ICH Guidelines (Section 3.2.1) describe tests for Retroviruses which are applied to three stages of the manufacturing process: the Master Cell Bank (MCB), the End of Production (EOP) cell banks and the Bulk Harvest. The tests described in the guidelines include infectivity assays in sensitive cell cultures and Transmission Electron Microscopy (TEM) studies. Further testing is only required if infectious retrovirus is not detected and no retrovirus or retrovirus-like particles have been observed by TEM. In this case an assay for the detection of retroviral reverse transcriptase enzyme should be used to look for retrovirus. BioOutsource has developed a sensitive Fluorescent Product Enhanced Reverse Transcriptase (PERT) method which can detect less than 100 virus particles.

Retrovirus Assays for CHO Cell Banks

To detect infectious retrovirus in CHO cell lines the most commonly used assay is the Mink or Feline S+L- assay. This assay detects xenotropic or amphotropic retroviruses and can be performed as a direct or extended assay to give a quantitative estimation of infectious retrovirus.

Retrovirus Assays for Human Cell Banks

To detect infectious retrovirus in Human cell lines BioOutsource has developed a cell based assay using HEK293 cells with an end point using a highly sensitive Fluorescent Product Enhanced Reverse Transcriptase (FPERT) method.

Test for Retrovirus in CHO Bulk Harvest

Testing for retrovirus in Bulk Harvest samples can be difficult due to the heterogenous nature of the samples. Testing for infectious retrovirus can be performed using an S+L- assay and quantitation of retrovirus particles by TEM. Samples can also be tested using PERT or PCR methodologies however these assays should be used with caution as these assays may yield low level false positive results.

Contact our biosafety experts to discuss your retrovirus assay requirements.

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