Characterisation of endogenous retrovirus in rodent cell lines used for production of biologicals J. Shepherd et al, Biologicals, Volume 31, Issue 4, December 2003, Pages 251-260 Abstract Rodent cells are used widely to manufacture recombinant proteins for pharmaceutical use in humans and animals. However, all rodent cell lines express endogenous retroviruses that require appropriate testing regimes for identification and characterisation. In this communication we report the results of transmission electron microscopy, reverse transcriptase assay and infectious virus assays for retrovirus in 185 manufacturer cell banks of mouse, rat or hamster origin. The results indicated considerable variability of retroviral expression levels by transmission electron microscopy and reverse transcriptase assay, but nevertheless characteristic features of each cell type were observed. Infectious retrovirus was detected in mouse myeloma and hybridoma cell lines, but not in cell lines of hamster or rat origin…. Rodent cell lines have for many years been used as substrates for production of biological therapeutics such as monoclonal antibodies, recombinant proteins, vaccines and gene therapy virus vectors. It has long been recognised that such cell lines contain retrovirus elements that may be expressed as particles detectable by electron microscopy. Such particles may be infectious, as in the case of Murine leukaemia virus (MLV), or defective and non-infectious, as in the case of the Chinese hamster ovary (CHO) cell retrovirus.]. Despite the lack of evidence for an association between murine retrovirus and disease in man, the potential contamination of therapeutics with agents associated with oncogenicity and immunosuppression in therapeutic products is a cause of regulatory concern. Detection and characterisation of retrovirus in manufacturer‘s master and end of production cell banks is recommended by regulatory agencies using techniques such as electron microscopy, reverse transcriptase assay and appropriate infectivity or co-cultivation assays. In addition, determination of retrovirus particle load and experimental demonstration of appropriate removal or inactivation of retrovirus during purification is required for each product [ref: Committee for Proprietary Medicinal Products. Notes for guidance on quality of biotechnological products: viral safety evaluation of biotechnology products derived from cell lines of human or animal origin (CPMP/ICH/295/95), European Commission, Brussels (1997) – see below …The study indicated characteristic features of retroviral expression in each cell type tested. All RT-positive cell lines demonstrated preference for manganese-dependent RT, characteristic of the Gammaretroviridae. NOTE FOR GUIDANCE ON QUALITY OF BIOTECHNOLOGICAL PRODUCTS: VIRAL SAFETY EVALUATION OF BIOTECHNOLOGY PRODUCTS DERIVED FROM CELL LINES OF HUMAN OR ANIMAL ORIGIN (CPMP/ICH/295/95) http://www.ema.europa.eu/pdfs/human/ich/029595en.pdf 3.2.1 Tests for Retroviruses For the MCB and for cells cultured up to or beyond the limit of in vitro cell age used for production, tests for retroviruses, including infectivity assays in sensitive cell cultures and electron microscopy (EM) studies, should be carried out. If infectivity is not detected and no retrovirus or retrovirus-like particles have been observed by EM, reverse transcriptase (RT) or other appropriate assays should be performed to detect retroviruses which may be noninfectious. Induction studies have not been found to be useful. ..... Case B: Where only a rodent retrovirus (or a retrovirus-like particle which is believed to be non-pathogenic, such as rodent A- and R-type particles) is present, process evaluation using a`specific "model" virus, such as a murine leukemia virus, should be performed. Purified bulk`should be tested using suitable methods having high specificity and sensitivity for the`detection of the virus in question. For marketing authorisation, data from at least 3 lots of`purified bulk at pilot-plant scale or commercial scale should be provided. Cell lines such as`CHO, C127, BHK and murine hybridoma cell lines have frequently been used as substrates`for drug production with no reported safety problems related to viral contamination of the`products. For these cell lines in which the endogenous particles have been extensively`characterised and clearance has been demonstrated (note limitations below), it is not usually necessary to assay for the`presence of the non-infectious particles in purified bulk. Studies with non-specific "model"`viruses, as in Case A, are appropriate. … Cell lines derived from rodents usually contain endogenous retrovirus particles or retrovirus-like particles, which may be infectious (C-type particles) or non-infectious (cytoplasmic A- and R-type particles). The capacity of the manufacturing process to remove and/or inactivate rodent retroviruses from products obtained from such cells should be determined. This may be accomplished by using a murine leukemia virus, a specific "model'' virus in the case of cells of murine origin 6.4 Limitations of Viral Clearance Studies Viral clearance studies are useful for contributing to the assurance that an acceptable level of safety in the final product is achieved but do not by themselves establish safety. number of factors in the design and execution of viral clearance studies may lead to an incorrect estimate of the ability of the process to remove virus infectivity These factors include the following: 1. Virus preparations used in clearance studies for a production process are likely to be produced in tissue culture. The behaviour of a tissue culture virus in a production step may be different from that of the native virus; for example, if native and cultured viruses differ in purity or degree of aggregation. 2. Inactivation of virus infectivity frequently follows a biphasic curve in which a rapid initial phase is followed by a slower phase. It is possible that virus escaping a first inactivation step may be more resistant to subsequent steps. For example, if the resistant fraction takes the form of virus aggregates, infectivity may be resistant to a range of different chemical treatments and to heating. 3. The ability of the overall process to remove infectivity is expressed as the sum of the logarithm of the reductions at each step. The summation of the reduction factors of multiple steps, particularly of steps with little reduction (e.g., below 1 log10), may overestimate the true potential for virus elimination. Furthermore, reduction values achieved by repetition of identical or near identical procedures should not be included unless justified. 4. The expression of reduction factors as logarithmic reductions in titer implies that, while residual virus infectivity may be greatly reduced, it will never be reduced to zero. For example, a reduction in the infectivity of a preparation containing 8 log10 infectious units per ml by a factor of 8 log10 leaves zero log10 per ml or one infectious unit per ml, taking into consideration the limit of detection of the assay. 5. Pilot-plant scale processing may differ from commercial-scale processing despite care taken to design the scaled-down process. 6. Addition of individual virus reduction factors resulting from similar inactivation mechanisms along the manufacturing process may overestimate overall viral clearance. Considering that XMRV has proven to be very difficult to detect and requires repeated and various approaches for successful detection, does the above sound reassuring?? Another later document (2004) from European Medicines Agency’s CONCEPT PAPER ON THE DEVELOPMENT OF A GUIDELINE ON VIRAL SAFETY EVALUATION OF BIOTECHNOLOGICAL PRODUCTS TO BE USED IN CLINICAL TRIALS states the following: … Validation studies are complex.and expensive and a clear position on this is important. The extent to which manufacturers are able to refer to in-house experience concerning virus safety evaluation. For such cases, the virus safety of a given cell culture system and/or the capacity of established manufacturing process steps to inactivate/remove potential virus contaminants may have been demonstrated with several previously developed products. Such a database may serve as supportive data to justify a reduced virus safety evaluation program for new products that enter development.