Retrovirus
Meeting Your Retrovirus Testing Needs
Retroviruses are a family of viruses with an extremely complicated life cycle. They are of particular concern because 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 that is capable of generating a virus particle visible by transmission electron microscopy (TEM). This is noninfectious, but some mouse cell lines, such as NS0, will produce an infectious retrovirus.
The International Council for Harmonization of Technical Requirements for Pharmaceuticals for Human Use (ICH) Guidelines (Section 3.2.1) describe tests for retroviruses that 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 TEM studies. Further testing is only required if an 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 should be used.
Our experts design a retrovirus testing plan around your drug development or commercial product needs
Ready-to-use, quality-assured testing plans and prevalidated cGMP tests allow immediate access to specialized testing and faster results
Our experts interpret results from cell lines known to contain endogenous retrovirus, providing a path forward for product release/regulatory submission
Our cGMP-compliant assays align with European Pharmacopeia (EP) and United States Pharmacopoeia (USP) guidelines
Testing recommendations depend on the species of the cell line being used to manufacture the product. Cell lines not known to contain endogenous retrovirus (human or primate cell lines) typically must show negative TEM and product enhanced reverse transcriptase (PERT) results. Cell lines known to contain endogenous retrovirus (hamster cell lines) must show positive TEM results and negative results from a cell-based infectivity assay. Testing recommendations also depend on the intended use of the product (e.g., recombinant protein production, vaccine production, viral vaccine, viral vector gene therapy).
Virus Like Particles (VLP) may be present but must be shown to be noninfectious.
Sampled from | Transmission Electron | Product Enhanced | Extended S+L Assay |
|---|---|---|---|
Research Cell Bank | - | - | - |
Master Cell Bank | X | X | X* |
Working Cell Bank | - | - | X* |
End of Production Cell Bank | X | X | X* |
Master Virus Seed Stock | X | X | - |
Working Virus Seed Stock | - | X | - |
Unpurified Bulk Harvest | X | X | X* |
Drug Substance | - | - | - |
Drug Product | - | - | - |
X* – murine-based cell lines only (e.g., - hamster)
Transmission Electron Microscopy (TEM)
Examination of 200 Cell Profiles
In this test, mainly used for mammalian cell banks, at least 200 median cell profiles (cells with nuclei) are screened for TEM. Key parts of the cells — cell nucleus, cell organelles, Golgi bodies and the plasma membrane — are examined closely. This assay looks to see that the cell morphology remains intact. It also identifies any viruses, virus-like particles, fungi, yeast, bacteria and mycoplasmas, and records the percentage of cell profiles with which these are associated. For identified retroviruses, the type of viral particle is recorded. This assay provides quantitative and qualitative data for the product, accompanied by a TEM visual for submissions.
Thin-Section
Another method employed, mainly for bulk harvest preparations, is thin-section electron microscopy. Ultra-thin sections of the test material are prepared using a diamond knife and microtome and then stained with uranyl acetate and lead citrate. Viral particles are counted from 10 random grid squares and the titer calculated.
Negative Staining
Negative staining allows for a high-magnification TEM study of biological specimens to determine morphology and ultrastructure. An electron-opaque stain is applied directly to biological samples from suspension fluids. The stain surrounds and penetrates the specimen, creating contrast between the biological material and its background. The sample is mixed with a known concentration of latex beads as reference, which makes it possible to generate a titer of virus like particles for the sample.
FPERT
Fluorescent Product Enhanced Reverse Transcriptase (FPERT) assays are highly sensitive tests that measure the activity of reverse transcriptase, an enzyme that transcribes DNA from RNA and is present in all retroviruses.
As a qPCR-based method, results can be generated more rapidly than TEM and cell-based methods.
S+L Assay
The Extended S+L Assay is a limit test for the presence of infectious retrovirus within MCB, WCB or EoP cell bank samples where the detection of infectious murine retroviruses is required (primarily for testing of CHO cell banks or murine cell banks).
Mus dunni cells can support infection of all four classes of murine leukemia viruses (MLVs), with the exception of Moloney MLV. Using either MiCl1 and PG4 endpoint cell lines, this assay is able to detect an array of xenotropic and amphotropic infectious murine retroviruses.
