Systematic evaluation of in vitro and in vivo adventitious virus assays for the detection of viral contamination of cell banks and biological products

Abstract

Viral vaccines and the cell substrates used to manufacture them are subjected to tests for adventitious agents, including viruses, contaminate. Some of the compendial methods (in vivo and in vitro in cell culture) were established in the mid-20th century. These methods have not been subjected to current assay validation, as new methods would need to be. This study was undertaken to provide insight into the breadth (selectivity) and sensitivity (limit of detection) of the routine methods, two such validation parameters. Sixteen viral stocks were prepared and characterized. These stocks were tested in serial dilutions by the routine methods to establish which viruses were detected by which methods and above what limit of detection. Sixteen out of sixteen viruses were detected in vitro, though one (bovine viral diarrhea virus) required special conditions to detect and another (rubella virus) was detected with low sensitivity. Many were detected at levels below 1 TCID50 or PFU (titers were established on the production cell line in most cases). In contrast, in vivo, only 6/11 viruses were detected, and 4 of these were detected only at amounts one or more logs above 1 TCID50 or PFU. Only influenza virus and vesicular stomatitis virus were detected at lower amounts in vivo than in vitro. Given the call to reduce, refine, or replace (3Rs) the use of animals in product safety testing and the emergence of new technologies for the detection of viruses, a re-examination of the current adventitious virus testing strategies seems warranted. Suggested pathways forward are offered.

Keywords: 3Rs; Adventitious agents; In vitro; In vivo; Vaccines; Viral safety.

Fig. 1. Comparison of Limits of Detection for In Vivo and In Vitro Assays for Model Adventitious Viral Agents

LOD values observed during in vivo (left panel) and in vitro (right panel) testing of each of the viruses used in this study. Shorter bars indicate lower sensitivity (i.e., higher LOD). In those cases where additional cell lines were used for in vitro tests and they provided greater sensitivity (Ad41, BVDV, and rubella virus), the LODs observed using the standard three cell lines and also using the additional cell line are represented as a stacked bar. UN = undiluted, ND = not detected, NT = not tested.