Label‐free mass spectrometry‐based quantification of hemagglutinin and neuraminidase in influenza virus preparations and vaccines

Background Influenza vaccination is the primary method for preventing influenza and its severe complications. An accurate rapid method to determine hemagglutinin (HA) concentration would facilitate reference antigen preparation and consequently expedite availability of seasonal as well as pandemic vaccines. Objective The goal of this study was to develop a label-free mass spectrometry (MS) based method that enables simultaneous identification and quantification of HA, neuraminidase (NA), and other viral proteins and protein contaminations in influenza vaccine or virus preparations. Methods The method presented is based on LC ⁄MSE analysis of vaccine or virus preparations tryptic digests spiked with a known amount of protein standard from which a universal response factor is generated and applied to calculate the concentration of proteins identified in the mixture. Results We show that, with the use of an appropriate internal standard, the label-free MS-based protein quantification method is applicable for simultaneous identification and absolute quantification of HA and identification and relative quantification of other influenza proteins as well as protein impurities in influenza vaccines and virus preparations. We show that different subtype recombinant HA is preferred internal standard that provides the most accurate results in absolute quantification of HAs and other influenza proteins. We applied this method to measure the absolute quantity of HA as well as relative quantities of other viral proteins and impurities in preparations of whole virus and monovalent vaccine, providing data to demonstrate strain-dependent differences in the amount of NA.