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2023 FDA Science Forum

Isotope Dilution Mass Spectrometry is an accurate method to determine hemagglutinin concentration of influenza virus standards

Download the Poster (PDF; 1.74 MB)
Authors:
Poster Author(s)
Lin, Jing, FDA/CBER; Joshi, Manju, FDA/CBER; Baichoo, Noel, FDA/CBER; Gao, Yamei, FDA/CBER; Pan, Tao, FDA/CBER; Sultana, Ishrat, FDA/CBER; Jelinek, Christine, FDA/CBER; Shahabuddin, Muhammad, FDA/CBER; Eichelberger, Maryna, FDA/CBER
Center:
Contributing Office
Center for Biologics Evaluation and Research

Abstract

Poster Abstract

Hemagglutinin (HA) is currently used as the marker for influenza vaccine potency. The amount of HA is measured using a single radial immunodiffusion assay (SRID) that provides an indirect measurement of antigenically intact HA relative to a reference that is calibrated against a primary liquid standard (PLS). The HA concentration of PLS is traditionally determined from densitometry of SDS-PAGE bands and total protein concentration. More advanced methods are now available to measure protein concentration, including mass spectrometry (MS)-based methods. In this report, we use an isotope dilution mass spectrometry (IDMS) method to quantify the HA in PLSs. IDMS combines the sensitivity and selectivity of mass spectrometry (MS) with the precision and accuracy associated with the use of isotopically labeled internal standards. We optimized the IDMS method by treating samples with detergent prior to digestion with trypsin. HA concentrations of PLSs prepared using egg and cell production platforms were measured by IDMS and SDS-PAGE methods. The data showed excellent reproducibility of the IDMS method and comparable concentrations measured by IDMS and SDS-PAGE methods for egg PLSs, but not for cell grown PLSs. Intact mass analysis indicated the presence of host cell proteins in cell grown PLSs which were not taken into account during SDS-PAGE analysis. Electron microscopy data further confirmed the differences in quality of PLS from different sources. In conclusion, IDMS is suitable for accurate determination of HA concentration in PLSs from all platforms. This advanced technique expedites the calibration of reference standards for influenza vaccines and ensures potency is correctly measured.

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Download the Poster (PDF; 1.74 MB)

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