Abstract
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 104 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.
Highlights
The potential contamination of biopharmaceuticals with adventitious viruses poses a serious safety risk and threatens public confidence in the use of biopharmaceuticals.This is true in the case of vaccines administered to large numbers of healthy people, including children[1,2]
Various reviews of High-throughput sequencing (HTS) for adventitious agent detection have shown that there is a need for unbiased extraction methods, relevant controls, the use of spike recovery experiments, and quality control measures during library preparation[7,14,15,16,17]
The limits of detection (LOD) for a panel of 22 model adventitious viruses was assessed in a viral vaccine crude harvest matrix and a cell substrate matrix
Summary
The potential contamination of biopharmaceuticals with adventitious (unintentionally introduced) viruses poses a serious safety risk and threatens public confidence in the use of biopharmaceuticals.This is true in the case of vaccines administered to large numbers of healthy people, including children[1,2]. Number of sequence reads detected for each of the spiked viruses at the different spiking levels in the viral vaccine crude harvest.
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