HighlightsDeep sequencing has potential as an improved adventitious virus screening method.15 laboratories sequenced a common reagent containing 25 target viruses.6 viruses were detected by all lab, the remainder were detected by 4–14 labs.A wide range of sample preparation and bioinformatics methods is currently used.A common reference material is essential to enable results to be compared.

There is a need for a broad and efficient testing strategy for the detection of both known and novel viral adventitious agents in vaccines and biologicals. High-throughput sequencing (HTS) is an approach for such testing; however, an optimized testing method is one with a sample-processing pipeline that can help detect any viral adventitious agent that may be present. In this study, 11 commercial methods were assessed for efficient extraction of nucleic acids from a panel of viruses. An extraction strategy with two parallel arms, consisting of both the Invitrogen PureLink™ Virus RNA/DNA kit for total nucleic acid extraction and the Wako DNA Extractor® kit with an RNase A digestion for enrichment of double-stranded nucleic acid, was selected as the strategy for the extraction of all viral nucleic acid types (ssRNA, dsRNA, and dsDNA). Downstream processes, such as double-strand DNA synthesis and whole-genome amplification (WGA), were also assessed for the retrieval of viral sequences. Double-stranded DNA synthesis yielded larger numbers of viral reads, whereas WGA exhibited a strong bias toward amplification of double-stranded DNA, including host cellular DNA. The final sample-processing strategy consisted of the dual extraction approach followed by double-stranded DNA synthesis, which yielded a viral population with increased detection of some viruses by 8600-fold. Here we describe an efficient extraction procedure to support viral adventitious agent detection in cell substrates used for biological products using HTS.

High-throughput sequencing (HTS) is capable of broad virus detection encompassing both known and unknown adventitious viruses in a variety of sample matrices. We describe the development of a general-purpose HTS-based method for the detection of adventitious viruses. Performance was evaluated using 16 viruses equivalent to well-characterized National Institutes of Health (NIH) virus stocks and another six viruses of interest. A viral vaccine crude harvest and a cell substrate matrix were spiked with 22 viruses. Specificity was demonstrated for all 22 viruses at the species level. Our method was capable of detecting and identifying adventitious viruses spiked at 10 4 genome copies per milliliter in a viral vaccine crude harvest and 0.01 viral genome copies spiked per cell in a cell substrate matrix. Moreover, 9 of the 11 NIH model viruses with published in vivo data were detected by HTS with an equivalent or better sensitivity (in a viral vaccine crude harvest). Our general-purpose HTS method is unbiased and highly sensitive for the detection of adventitious viruses, and has a large breadth of detection, which may obviate the need to perform in vivo testing.

The microplate assay with Chinese Hamster Ovary (CHO) cells is currently used as a safety test to monitor the residual pertussis toxin (PT) amount in acellular pertussis antigens prior to vaccine formulation. The assay is based on the findings that the exposure of CHO cells to PT results in a concentration-dependent clustering response which can be used to estimate the amount of PT in a sample preparation. A major challenge with the current CHO cell assay methodology is that scoring of PT-induced clustering is dependent on subjective operator visual assessment using light microscopy. In this work, we have explored the feasibility of replacing the microscopy readout for the CHO cell assay with the xCELLigence Real-Time Cell Analysis system (ACEA BioSciences, a part of Agilent). The xCELLigence equipment is designed to monitor cell adhesion and growth. The electrical impedance generated from cell attachment and proliferation is quantified via gold electrodes at the bottom of the cell culture plate wells, which is then translated into a unitless readout called cell index. Results showed significant decrease in the cell index readouts of CHO cells exposed to PT compared to the cell index of unexposed CHO cells. Similar endpoint concentrations were obtained when the PT reference standard was titrated with either xCELLigence or microscopy. Testing genetically detoxified pertussis samples unspiked or spiked with PT further supported the sensitivity and reproducibility of the xCELLigence assay in comparison with the conventional microscopy assay. In conclusion, the xCELLigence RTCA system offers an alternative automated and higher throughput method for evaluating PT-induced clustering in CHO cells.

Testing of biological products is required to ensure the safety of patients. Recently, a licensed vaccine was found to be contaminated with a virus. This contamination did not cause a safety concern to the patients; however, it highlights the need for using new testing methods to control our biological products. This paper introduces the benefits of these new tests and outlines the challenges with the current tests.