Validation of retroviral detection for rodent cell-derived products and gene therapy applications.

JV Hughes, K Messner, M Burnham… - Developments in …, 1996 - europepmc.org
JV Hughes, K Messner, M Burnham, D Patel, EM White
Developments in biological standardization, 1996europepmc.org
The availability of sensitive assays for detecting infectious murine retroviruses has become
critical for the development and acceptance of a number of biopharmaceuticals, including
monoclonal antibody-derived products and gene therapy vectors. Comparative studies
demonstrated that the PG4 S+ L-retrovirus infectivity test routinely yields higher titres than
the mink cell test for xenotropic, amphotrophic and MCF murine retroviruses. A validation
study for the PG4 S+ L-assay demonstrated very good linearity (r2 of 0.95 to 0.99) …
The availability of sensitive assays for detecting infectious murine retroviruses has become critical for the development and acceptance of a number of biopharmaceuticals, including monoclonal antibody-derived products and gene therapy vectors. Comparative studies demonstrated that the PG4 S+ L-retrovirus infectivity test routinely yields higher titres than the mink cell test for xenotropic, amphotrophic and MCF murine retroviruses. A validation study for the PG4 S+ L-assay demonstrated very good linearity (r2 of 0.95 to 0.99), reproducibility within a study (+/-0.35 log10 units), and precision between tests (+/-0.45 log10 units). Interference (or selectivity) in the presence of a non-specific antibody was insignificant (less than 0.2 log10 units). Sensitivity levels established from measurements as virus titres approach zero demonstrated a threshold value of 2-3 focus forming units (FFU)/ml. Two methods for increasing assay sensitivity were used including:(i) increased product samplings combined with a Poisson distribution analysis, and (ii) a 14-day co-cultivation with Mus dunni cells. Each of these methods was shown to increase sensitivity by at least one log10 unit. Murine retroviruses may also be detected by a less sensitive immunofluorescence assay (IFA) using specific monoclonal antibodies; this assay is essential for detecting certain recombinant ecotropic MuLVs. In summary, murine retroviral detection ranked by sensitivity is mink S+ L-< IFA with monoclonal antibodies< PG4 S+ L-< Mus dunni co-cultivation followed by PG4 S+ L-.
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