VaxArray potency assay for rapid assessment of “pandemic” influenza vaccines

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%.

Overcoming scientific barriers in the transition fromin vivoto non-animal batch testing of human and veterinary vaccines

“…Not all quantification methods require antigen extraction or desorption from adjuvanted vaccines. With respect to emulsion adjuvants, a multiplexed sandwich immunoassay for influenza vaccines against strains with pandemic potential was shown to be compatible with the squalene-based adjuvant MF59 [112]. An in situ method based on the fluorescent nucleic acid-reactive dye SYBR Green II has been developed to determine the stability of an inactivated vaccine for footand-mouth disease and was found compatible with aluminum salt, water-in-oil and oil-in-water adjuvanted vaccines [113].…”

Antibody enhanced HPLC for serotype-specific quantitation of polysaccharides in pneumococcal conjugate vaccine

“…Conventionally, such serotype-specific antigen assays were performed on the immunoassay platforms; among them, the sandwich enzyme-linked immunosorbent assay (ELISA) is the preferred format for this assay 20 , 21 . Although immunoassays, such as ELISA have been reported to quantify antigen content in several vaccines 22 – 24 , there is only a light scattering (LS)-based nephelometry method reported for pneumococcal vaccines 25 . Due to the lack of precedent reports for a reliable antigen-specific pneumococcal immunoassay and the need for such assay for our PCV development, we sought to develop an innovative assay platform for serotype-specific polysaccharide quantitation.…”

Multiplexed VaxArray Immunoassay for Rapid Antigen Quantification in Measles and Rubella Vaccine Manufacturing

“…Infectivity measurements are widely adopted and highly predictive of vaccine efficacy [17][18][19][20] but present certain limitations, as the time to result is 10-14 days and execution requires trained personnel. In addition, CCID50 assays rely on subjective, discontinuous endpoint measurements and are notoriously error prone with observed variability of ± 0.3 log10 infectious units per mL (50-100% variability) [21][22][23], resulting in costly hold times and lot rejections that can increase manufacturing costs and delay delivery of critical vaccine doses.…”

“…Traditional cell culture assays, such as CCID50, are subject to variability of up to 0.3 log10 IFU/mL[21][22][23]. This translates to relative error of up to 50%-100%.…”