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, which might contaminant them. 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 (3 R's) 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.

Vaccine Analysis: Strategies, Principles, and Control

“…Typical verifications of the compendial methods for cell culture-based assays, for instance, might utilize only a few viruses (not unlike qualification of sterility tests), and in vivo assays have never, until recently (Gombold et al 2014), been challenged systematically to our knowledge. Typical verifications of the compendial methods for cell culture-based assays, for instance, might utilize only a few viruses (not unlike qualification of sterility tests), and in vivo assays have never, until recently (Gombold et al 2014), been challenged systematically to our knowledge.…”

“…Recent work has suggested that their sensitivity for detection of viruses may not be as good as previously believed (Gombold et al 2014), despite having been relied upon for years. Clinical observations of the effects viruses have on experimental animals formed the basis for early clinical diagnostics for viruses.…”

“…Some work (Gombold et al 2014) has been done to address this problem, but it is only a beginning and does not provide comparisons between various compendia and regulations, having only followed the US methods. The volumes, the routes of inoculation, the age of egg embryos at inoculation, length of incubation, and other differences exist.…”

“…There are numerous descriptions of reporter cell lines developed for specific viruses (a few examples, HSV, CMV, VZV, BIV, Herpes B virus, alphaviruses, influenza, rubella) usually developed for the purpose of investigating or verifying specific viral infections. For instance, Gombold et al (2014) demonstrated differences among cell lines used in conventional assays, suggesting for instance that HeLa and A549 cells were more sensitive than MRC-5 and Vero for adenovirus 41 and rhinovirus, although the A549 cell line was dramatically more sensitive than HeLa for adenovirus type 5. Others have considered recombinant cell lines overexpressing antiapoptotic genes or primary cells treated with apoptosis inhibitors as means of enabling greater viral replication, and thus enhancing the detectability of transforming viruses (Sandstron and Folks 2001 and references therein).…”

First update of the International Xenotransplantation Association consensus statement on conditions for undertaking clinical trials of porcine islet products in type 1 diabetes—Chapter 2a: source pigs—preventing xenozoonoses

“…Deep sequencing also has great potential but is currently not a feasible option for most laboratories due to the high cost associated when performing routinely, the bioinformatics burden, and the potential for sequence variations to produce false‐negative results. Currently, the ability to assess the presence of unknown and some adventitious viruses relies on time‐consuming and costly in vitro coculture/cytotoxicity assays which limits the ability to determine a complete safety profile solely on a labile islet product with a typical culture period of days to weeks and which have been shown to lack sensitivity in the case of certain viruses . In the future, if alternative methodologies are developed and validated (e.g., per CLSI, ISO, or AAVLD guidelines), they may enable the screening of semen for safe genetic introduction into DPF closed herds.…”