Retroviral contamination

The initial emphasis in analytical biotechnology was on broad safety concerns that translated into detection of host-cell components such as DNA, endotoxins, Escherichia colt proteins, and retroviral contamination.2 The detection of these impurities requires development of high-sensitivity assays that are based primarily on antibodies [e.g., enzyme-linked immunosorbent assay (ELISA) for E. coli proteins) or radioactivity (e.g., dot-blot assays for DNA detection). New developments are focused on low-sensitivity detection, characterization, and removal of undesirable target sequence variants. Bioseparations play an important role even after a product has been isolated and shown to contain a low level of contaminants for initiation of clinical studies. The focus shifts to achievement of a reproducible, large-scale manufacturing process. At this stage, analytical methods provide essential informa-... 

According to WHO (1998), DNA from a continuous cell line can be considered a cellular contaminant, instead of a significant risk factor that requires reduction to extremely low levels. Therefore, upper limits of 10 ng per dose are acceptable for products generated from continuous cell lines. Only in specific situations that might be considered harmful, for example, when infectious retroviral pro-virion sequences are present, the acceptable limit per dose should be assigned by the regulatory authorities. Levels of cellular DNA should be measured in the supernatant harvest, in the intermediate purification steps and in the final product to determine its initial concentration and whether it was removed or concentrated. The removal efficiency must be determined based on several runs to ensure confidence in the data. Validation of the process excludes the need for residual cellular DNA testing in the bulk product (FDA, 1997 WHO, 1998). 

Cumulative experience from 106 monkey-years and 23 human-years of retroviral gene therapy had revealed no side-effects or malignancies. Recently, however, three monkeys used in clinical trials developed T cell lymphomas which were traced to a helper virus-contaminated retroviral vector preparation (Anderson, 1992a). Contamination of defective viral vector stocks by infectious viruses produced in helper cells was a serious problem with the early version of the helper cells. New helper cell lines which greatly reduce the probability of virus production have been developed (Miller, 1990). Eurther, it is also imperative to test the lots of vector virus preparations used in therapy for the presence of other infectious viruses. The probability... 

The retroviral vectors in clinical use have mainly been derived from murine leukemia virus (MLV) [14]. MLV causes leukemia in mice and replication-competent retrovirus (RCR) in a contaminated vector preparation was shown to cause leukemia in severely immunosuppressed monkeys. RCR absence therefore has to be verified before human use of retroviraUy modified cells MLV vector use in vivo has been very rare. The genome of the retroviral vectors consists of two copies of single-stranded RNA, which contains one or more coding regions flanked by the viral control elements, the so-called long terminal repeat (LTR)... 

Initially discovered in extracts of calf thymus as a contaminating enzyme during the purification of RNA polymerase (1,2), RNase H has since been found almost ubiquitously in lower and higher eukaryotes as well as in prokaryotes. A single cell type may have one or more species of RNases H residing in the cell nucleus and/or cytosol. Some RNases H have a single ribonucleolytic reaction specificity, whereas others are associated with DNA polymerase activities. Cellular RNases H, for example, from E. coli and calf thymus, are endonucleases the retroviral reverse transcriptase-associated RNases H can function as both exo- and endoribo-nucleases depending on the type of substrates available. 

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