ABSTRACT
Neuraminidase (NA) immunity leads to decreased viral shedding and reduced severity of influenza disease; however, NA content in influenza vaccines is currently not regulated, resulting in inconsistent quality and quantity of NA that can vary from manufacturer to manufacturer, from year to year, and from lot to lot. To address this problem, we have developed an assay for NA quantification that could be used by the industry to move toward developing influenza vaccines that induce a predictable immune response to NA. The VaxArray Influenza Seasonal NA Potency Assay (VXI-sNA) is a multiplexed sandwich immunoassay that relies on six subtype-specific monoclonal antibodies printed in microarray format and a suite of fluor-conjugated "label" antibodies. The performance of the assay as applied to a wide range of influenza vaccines is described herein. The assay demonstrated high NA subtype specificity and high sensitivity, with quantification limits ranging from 1 to 60 ng/mL and linear dynamic ranges of 24-500-fold. When compared to an enzymatic activity assay for samples exposed to thermal degradation conditions, the assay was able to track changes in protein stability over time and exhibited good correlation with enzyme activity. The assay also demonstrated excellent analytical precision with relative error ranging from 6 to 12% over day-to-day, user-to-user, and lot-to-lot variation. The high sensitivity and reproducibility of the assay enabled robust detection and quantification of NA in crude in-process samples and low-dose, adjuvanted vaccines with an accuracy of 100 ± 10%.
ABSTRACT
The VaxArray Influenza Pandemic HA (VXI-pHA) potency assay is a multiplexed sandwich immunoassay that consists of nine broadly reactive yet subtype-specific monoclonal capture antibodies printed in microarray format and a suite of fluor-labeled secondary antibodies that were selected to probe conserved HA epitopes. VXI-pHA was designed to optimize the probability that the ready-to-use assay would work for the most concerning, emergent influenza A strains, eliminating the need for the time-consuming process of reference reagents production. The performance of this new potency test was evaluated using a panel of 48 potentially pandemic strains of influenza viruses and vaccines spanning 16 years of antigenic drift, including the most recent pre-pandemic vaccine being developed against the "5th wave" A/H7N9 virus. The VXI-pHA assay demonstrated coverage of 93%, 92%, and 100% for H5, H7, and H9 antigens, respectively. The assay demonstrated high sensitivity with linear dynamic ranges of more than 150-fold and quantification limits ranging from 1 to 5 ng/mL. For three production lots of H7N9 monobulk drug substance, the assay exhibited excellent accuracy (100 ± 6%) and analytical precision (CV 6 ± 2%). The high assay sensitivity enabled robust detection and quantification of hemagglutinin in crude in-process samples and low-dose, adjuvanted vaccines with an accuracy of 100 ± 10%.
