Molecular Diagnostics Services, Inc.
A Contract Research Organization


RT Assays F-PERT 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. Reverse transcriptase activity from the test article is detected by incubation of the samples with target RNA, followed by the amplification of target cDNA by quantitative real time PCR (qRT-PCR). The method is not fully quantitative but it allows detection of low amounts of reverse transcriptase.

Regulatory guidance (ICH Q5A; 1993, 1997 PTC) 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).

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. 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., co-cultivation with HEK 293 cells, to detect retroviruses capable of infecting human cells).

MDS offers these RT-dependent assays (F-PERT and QPERT) for qualification of cell banks and/or viral vaccine products originating from mammalian & avian cell substrates. The potential exists for non-retroviral polymerases, such as mammalian DNA polymerase alpha and gamma, to yield “false positive” results in this assay.  Procedures are included in the assay to eliminate these false-positive signals, thereby ensuring that the polymerase activity detected is retroviral in origin.

A positive signal from this F-PBRT assay does not necessarily indicate the presence of infectious retroviral particles.  Infectivity studies may be required to demonstrate that the source of the activity is not an infectious retrovirus.

PCR-Based RT Assay or PBRT
MDS offers  the PCR-based RT assay, or PBRT, which is approximately 105-106 times more sensitive than a conventional RT assay and is capable of detecting the activity of as few as 10-100 molecules of RT.  This technology is also frequently referred to as a PCR-enhanced reverse transcriptase (PERT) assay.  Utilizing real-time PCR, specifically TaqMan technology, the F-PBRT (fluorescent-PCR-based reverse transcription) assay maintains the same sensitivity and specificity as traditional PBRT or PERT assays while significantly decreasing the time and labor required in completing the assay.

Each test article is assayed unspiked in triplicate, undiluted and 100x diluted, and each dilution is spiked with two amounts of AMV reverse transcriptase (in triplicate) as a test for the presence of substances in the test article that can interfere with either the RT step or the PCR amplification step of the assay.  This procedure is performed both with and without activated DNA, such that each test article is assayed in 36 reactions, 18 without activated DNA (6 unspiked, 12 spiked) and 18 with activated DNA (6 unspiked, 12 spiked) in the reactions.
MDS’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.

QPCR-Based RT Assay, or qPBRT
qPERT assay is utilized 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.

Real Time Fluorescent Product Enhanced RT Assay or F-PERT
F-PERT 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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