Abstract
Determining protein content in biologics is an important part of the production process. An example of interest to public health is the influenza vaccine, where the amount of the major antigenic protein hemagglutinin and the amount of egg proteins from the expression system are regulated. Mass spectrometry has advantages of higher specificity, speed and permits other proteins to be analyzed simultaneously. Here, we present the use of a MRM method for quantitation of hemagglutinin and other vaccine proteins developed in our laboratory and compare this approach to a label free method (MSE) for simultaneous identification and absolute quantitation of virus and egg proteins.
Influenza vaccine samples were tryptically digested using a protocol developed in our laboratory to ensure consistent and reproducible results. Traditional IDMS measurements were made on ThermoFisher Scientific TSQ quantum triple quadrupole platform. Label free methods (LC-MSE) were performed on a Waters qTOF Premier platform and the PLGS software. Both instruments were coupled to an Identical Agilent 1200 LC platform to insure an accurate comparison.
The results of the study illustrated that IDMS remains the gold standard for absolute protein quantitation via mass spectrometry. MSE performed with comparable precision and accuracy in cases where the sample was less complex (monovalent pandemic vaccines vs. seasonal trivalent vaccines). In addition the choice of peptides made by the MSE algorithm and the choice of influenza proteins used in the database also affected the precision and accuracy of the MSE absolute quantitation results.