Viral safety is a key concern for monoclonal antibodies (mAb’s) and other biologics intended for human use. MAb-producing rodent cell cultures (such as murine hybridomas and transfected CHO cells) produce type C retrovirus particles (up to 109/mL), because the rodent genome contains multiple copies of endogenous retrovirus-like sequences. Although most of the viral particles produced in cell culture are defective, a fraction can infect nonrodent cells, and some replication-competent retroviral vectors derived from murine leukemia virus components are tumorigenic in primates. Therefore, regulatory agencies require that a wide margin of safety is demonstrated for mAb’s and other biologics intended to be marketed or used in clinical trials.
Regulatory guidance advises the use of reverse transcriptase activity as a marker for the detection of extracellular retrovirus particles and the presence of retrovirus contamination. These reverse transcriptase (RT) assays (also described as Amp-RT, PERT, and PBRT) 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 for QF-PERT testing on cell banks and/or viral vaccine products originating from mammalian & avian cell substrates. QVirus™ platform offers a wide range of adventitious agent tests, including transmission electron microscopy (TEM), infectivity and PERT assays, in combination with multiple detector cell lines, Q-PCR virus detection packages, etc.
➢ Infectivity Assays
The major focus of infectivity assays is on the use of murine cell lines or hybrid cell lines containing a murine component. These cell lines are inherently capable of producing infectious mouse retroviruses. QVirus™ platform provides sensitive protocols for detecting a wide range of retroviruses such as xenotropic, ecotropic, amphotropic and Mink Cell Focus-forming (MCF) retroviruses.
For the detection of retroviruses, two kinds of protocol exist:
1. The direct focus assays use one detector cell line to enable direct detection of retrovirus particles.
2. Extended assays use an initial cell line to amplify the retrovirus with three to five passages, followed by a direct focus assay.
➢ Product-enhanced Reverse Transcriptase (PERT) or Fluorescent Product Enhanced Reverse Transcriptase (FPERT assays).
QVirus™ platform offers these RT-dependent assays (F-PERT and QPERT) for the 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.
➢ TEM Assay
TEM is commonly used to detect retroviruses in test articles and to type retrovirus particles (A, B, C or D) in cell lines. TEM can also be used to characterize cell lines and to detect other contaminants such as bacteria, mycoplasma, yeast, fungi, and other viruses.
If you have any special requirements, please feel free to contact us. Our biosafety experts will be pleased to discuss your retrovirus assay requirements.