(511d) Evaluation of Novel Quantification Approaches for Infectious Viruses

In the use of virus derived pharmaceuticals, the accurate and rapid quantification of product titers is of paramount importance. It is relevant during process development at the research level as well as during the actual manufacturing. Reliable product quantity data allows for efficient monitoring during all phases of the production, including the virus amplification in the upstream process as well as the product recovery for individual unit operations in the downstream process. The quantification is most demanding in the evaluation of infectious viruses, such as those used for live (attenuated) vaccines or viral vectors. However, the industries’ current gold standard applications, such as endpoint dilution assays, are cumbersome and do not provide true process analytical monitoring, as the manufacturing process is typically completed within a week. Other approaches commonly used at the research level include flow cytometry and quantitative polymerase chain reaction, which offer various advantages for a rapid estimation of infectious viruses. In most cases, successful methods are only suitable for a specific virus, and even minor changes in the viral genotype or structural composition require rigorous adaptation and optimization.

In this study, we compared different flow cytometric approaches for the assessment of infectious viruses to evaluate the analysis time, sensitivity, linear range and reproducibility of the methods. Two different baculovirus genotypes were applied as a model to demonstrate potential platform applications suitable for a range of different viruses. For all of the evaluations, Spodoptera frugiperda (Sf9) cells were used as the baculovirus host. Analytical targets included the direct detection of fluorophore expression due to the virus, viral envelope proteins, found on the surface of infected cells, and the viral mRNA translated inside the cells. The latter was achieved by combining a labeling by hybridization of a probe and fluorophores to the mRNA with a subsequent flow cytometric detection.

The results confirm that the use of flow cytometry greatly accelerates the viral analysis. Quantification using a reporter fluorophore requires an overnight incubation of approximately 17 hours, whereas the reference endpoint dilution assay was completed in seven days. However, for viruses used as pharmaceutical products, the expression of a reporter fluorophore is not a viable option. In this case, labeling of viral surface proteins is an efficient alternative to rapidly assess the total amount of virus. We evaluated two different fluorophores, phycoerythrin and allophycocyanin, conjugated to anti-gp64 antibodies. Since gp64 is a surface protein found on baculoviruses and on the surface of infected cells, it is a suitable target to observe cell-virus interactions. It was possible to detect positive, i.e., infected, cells as early as five minutes after infection. A robust and reproducible quantification required at least four hours and was thus significantly faster than the detection of expressed reporter fluorophores. In addition, the latter had a detection limit of 1.5E+04 infectious units (IU)/mL, whereas the labeling of viral surface proteins allowed for increased sensitivity with a detection limit of 1.0E+03 IU/mL, regardless of the fluorophore used. For both techniques, the linear range of the procedure spanned 1-2 log steps and an intra- and interassay coefficient of determination greater than 0.99 was possible. Although labeling of viral surface proteins significantly reduced analysis time while increasing sensitivity, it is only feasible for certain enveloped viruses and therefore has limited platform applicability.

Therefore, the possibility of viral mRNA labeling in infected cells was evaluated. First proof-of-concept studies showed a successful detection of viral infection, but also indicated the need for optimization. While increasing viral mRNA levels were detected within the first four hours of infection, the signal to noise ratios were rather low after when using a multiplicity of infection (MOI) of 1. Improvements of the procedure focussed on the initial incubation time of virus and cells, on the cell preparation (fixation and permeabilization) prior to probe addition, as well as on the probe hybridization conditions and the linear range identification. An optimized mRNA labeling procedure would allow a thorough analytical platform approach for many different viruses, as viral mRNA is found in infected cells for DNA and RNA viruses, with and without the presence of a viral envelope.