RT Assays (FPERT and QPERT)
Manufacturers of viral vaccines and other biological products must ensure that biological starting materials and mammalian cell substrates are free of contaminating viruses. One of the common types of contaminating viruses that can be found in starting materials and cell substrates are retroviruses. Regulatory guidance advises the use of reverse transcriptase activity packaged into extracellular retrovirus particles as a marker for the potential presence of retrovirus contamination. These reverse transcriptase (RT) assays (also described in the literature as Amp-RT, PBRT and PERT) detect the conversion of RNA template to cDNA (due to the presence of RT enzyme when retroviruses are present in the test sample). The development of these RT-dependent assays led to the requirement of the PERT assay for cell banks and/or viral vaccine products originating from mammalian & avian cell substrates (Lovatt et al., 1999). It is well recognized that avian cells used to manufacture vaccines contain endogenous retroviral RT activity, and appropriate test systems can be applied for detection of infectious avian retroviruses.

Retrovirus testing using PERT assays should be performed on cell-free culture medium to detect retroviruses. PERT assays can detect all infectious retroviruses since they encode and contain a functional RT enzyme. The material that is tested includes, cell substrates, viral seeds, and/or harvests of all viral vaccines produced in mammalian cell substrates.

Vitrology’s PERT assays are highly sensitive since they include a cellular DNA polymerase suppressant termed calf thymus DNA. This together with an additional ultracentrifugation step for the concentration of retroviral particles ensures specific detection of retroviral RT activity, and that optimal conditions are used for each test sample.

Often, infectivity tests may be augmented with sensitive PERT assays. Under some circumstances, for example when tumorigenic cell substrates are proposed for use, it could be appropriate to pre-treat cells with chemical agents known to induce reactivation or replication of endogenous or latent viruses. Subsequent co-cultivation or transmissibility assays with cells susceptible to a wide range of retroviruses, combined with a relevant detection system (e.g., PERT assay and electron microscopy) may be used to confirm the absence of detectable virus, or to amplify viruses with a specific host cell tropism (e.g., cocultivation with HEK 293 cells, to detect retroviruses capable of infecting human cells).

Vitrology provides two versions of real time TaqMan-based PERT assays which can be interpreted in two different ways:

SP-M.8001
Quantitiation of Reverse Transcriptase (RT) by Quantitative Real Time Fluorescent Product Enhanced Reverse Transcriptase (qPERT) assay.

qPERT assay is utilised when quantitative results are required and in this case the test will contain a highly linear purified RT enzyme standard curve with a known number of copies over a 5 log dynamic range. qPERT is used to determine if there is a significant numerical elevation of RT activity in the supernatant at different time-points during co-culture assays (eg post-passage 1 compared to post-passage 5 of co-culture). If significant elevation of RT activity is detected during co-culture this can provide further evidence of infectious retrovirus contamination. In addition, the qPERT assay is used to test products manufactured in primary cells that are tested lot-by-lot, where qPERT is used to measure endogenous retrovirus level in the bulk harvest versus that of control cells.

SP-M.8002
Detection of Reverse Transcriptase (RT) by Real Time Fluorescent Product Enhanced Reverse Transcriptase (FPERT) assay.

FPERT assay is used when highly sensitive qualitative fluorescent results are required for vaccine seed or bulk produced in human cell substrates. This method is fully validated and produces either a negative test, or a “suspect” positive test. A suspect positive test requires further investigation by alternative technology, usually a co-cultivation or transmissibility assay to determine if infectious retrovirus is present in the test sample.

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