Simultaneous quantification of hemagglutinin and neuraminidase of influenza virus using isotope dilution mass spectrometry

• Published in: Vaccine Vol. 30; no. 14; pp. 2475 - 2482
• Main Authors: Williams, Tracie L., Pirkle, James L., Barr, John R.
• Format: Journal Article
• Language: English
• Published: Netherlands Elsevier Ltd 23.03.2012; Elsevier Limited
• Subjects: Allantoic fluid; Allergy and Immunology; Amino Acid Sequence; Amino acids; Animals; antigens; Cell Line; Chromatography, Liquid; Competition; Digestion; Dilution; Exo- alpha -sialidase; Hemagglutinin; Hemagglutinin Glycoproteins, Influenza Virus - analysis; Hemagglutinin Glycoproteins, Influenza Virus - chemistry; Hemagglutinins; Humans; inactivated vaccines; Influenza; Influenza Vaccines - chemistry; Influenza Vaccines - immunology; Influenza virus; isotope dilution technique; Isotopes; liquid chromatography; Mass spectrometry; Mass spectroscopy; Molecular Sequence Data; Neuraminidase; Neuraminidase - analysis; Neuraminidase - chemistry; Orthomyxoviridae; pandemic; pandemics; Peptides; Peptides - analysis; Peptides - chemistry; Proteins; Public health; sialidase; Tandem Mass Spectrometry; vaccination; Vaccine; Vaccines; viral proteins; Viral Proteins - analysis; Viral Proteins - chemistry; viruses; Proteins; Vaccine; Hemagglutinin; Mass spectrometry; Influenza; Neuraminidase
• ISSN: 0264-410X; 1873-2518; 1873-2518
• DOI: 10.1016/j.vaccine.2011.12.056

► HA and NA of seasonal subtypes are quantified simultaneously in one analytical run. ► Prior knowledge of the strain of virus being analyzed is not required. ► Standards are prepared within two weeks allowing rapid response to new strains. Influenza vaccination is the primary method for preventing influenza and its severe complications. Licensed inactivated vaccines for seasonal or pandemic influenza are formulated to contain a preset amount of hemagglutinin (HA), the critical antigen to elicit protection. There is currently no regulatory method that quantifies neuraminidase (NA), the other major membrane-bound protein thought to have protective capability. This is primarily due to the limitations both in sensitivity and in selectivity of current means to quantify these antigens. Current methods to establish the HA concentration of vaccines rely on indirect measurements that are subject to considerable experimental variability. We present a liquid chromatography-tandem mass spectrometry (LC/MS/MS) method for the absolute quantification of viral proteins in a complex mixture. Through use of an isotope dilution approach, HA and NA from viral subtypes H1N1, H3N2, and B were determined both directly and rapidly. Three peptides of each subtype were used in the analysis of HA to ensure complete digestion of the protein and accuracy of the measurement. This method has been applied to purified virus preparations, to monovalent bulk concentrates, to trivalent inactivated influenza vaccines, and even crude allantoic fluid with improved speed, sensitivity, precision, and accuracy. Detection of 1μg/mL of protein is easily obtained using this method. The sensitivity of the method covers the range expected in vaccine preparations, including adjuvant-based vaccine. This LC/MS/MS approach substantially increases the selectivity, accuracy and precision used to quantify the amount of viral proteins in seasonal and pandemic influenza vaccines and reduce the time and effort to deliver influenza vaccines for public health use during the next influenza pandemic.