Dr Shepherd: Cell lines used for production of vaccines contain retroviral elements that may be infectious

A. J. Shepherd1, 2, N. J. Wilson1 and K. T. Smith, , 1:

Characterisation of endogenous retrovirus in rodent cell lines used for production of biologicals


1 Q-One Biotech Ltd, Todd Campus, West of Scotland Science Park, Glasgow G20 0XA, UK

2 Biogen Inc., Cambridge, MA 02142, USA

Received 5 November 2002; revised 9 May 2003; accepted 18 July 2003. ; Available online 11 September 2003.

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. There was no evidence of contamination of cell banks with exogenous retrovirus. The results of retroviral characterisation of the parental mouse cell lines NS0, NS-1 and Sp2/0Ag14 by the above assays were consistent with the results of the survey. Co-cultivation of the above parental mouse cell lines with mouse and human cell lines suggested that the ability to infect human cells was related to threshold susceptibility of cell types and the levels of expression of infectious xenotropic retrovirus by mouse cells.

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.