Division of Cellular and Gene Therapies, OCTGT/CBER/FDA, Bethesda, MD, USA.
Influenza Other Respir Viruses. 2013 Jul;7(4):521-30. doi: 10.1111/irv.12001. Epub 2012 Sep 3.
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.
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.
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.
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.
The label-free MS method presented here is ideally suited for timely preparation of reference material needed for potency testing of seasonal and pandemic vaccines.
流感疫苗接种是预防流感及其严重并发症的主要方法。一种准确快速的方法来确定血凝素（HA）浓度将有助于参考抗原的制备，从而加速季节性和大流行疫苗的供应。
本研究的目的是开发一种无标记的质谱（MS）方法，该方法能够同时鉴定和定量流感疫苗或病毒制剂中的 HA、神经氨酸酶（NA）和其他病毒蛋白和蛋白污染。
本方法基于 LC/MSE 分析疫苗或病毒制剂的胰蛋白酶消化物，其中加入了一定量的标准蛋白，由此生成通用响应因子，并应用于计算混合物中鉴定出的蛋白浓度。
我们表明，使用适当的内标，无标记 MS 基于蛋白定量方法适用于同时鉴定和绝对定量 HA 以及鉴定和相对定量其他流感蛋白以及流感疫苗和病毒制剂中的蛋白杂质。我们表明，不同亚型重组 HA 是首选的内标，可提供最准确的 HA 和其他流感蛋白绝对定量结果。我们应用该方法来测量 HA 的绝对量以及全病毒和单价疫苗制剂中其他病毒蛋白和杂质的相对量，提供数据来证明 NA 数量的菌株依赖性差异。
本文提出的无标记 MS 方法非常适合及时制备用于季节性和大流行疫苗效力测试的参考材料。
