Avian
influenza viruses, such as A(H5N1) and A(H7N9), are primary
public health concerns due to their pandemic potential. Influenza
vaccines represent the most effective response to this threat especially
with timely provision. The current pandemic response timelines require
a substantial period for strain-specific reference antigen and sera
preparation for use with single-radial immunodiffusion (SRID), the
accepted vaccine potency assay. To address this time lag, the isotope
dilution mass spectrometry (IDMS) method was developed to quantify
the absolute hemagglutinin (HA, the main influenza antigen) amount
in the vaccine without the need for purified, inactivated, and calibrated
virus reference antigens. However, an additional challenge in determining
potency is to differentiate between vaccine antigens in their most
potent form from other less potent, stressed antigen forms. The limited
trypsin digestion (LTD) method has been developed and does not require
strain-specific full-length reference antigens or antibodies; instead,
stressed HA is selectively degraded, leaving the more potent form
to be measured. LTD, followed by precipitation and IDMS, allows for
efficient differentiation between potent and significantly less potent
HA for vaccine release and potency testing across the vaccine’s
shelf life. In this study, we tested the LTD-IDMS assay on A(H5N1)
vaccine material that had been stressed by low pH, heat, and multiple
freeze–thaw cycles. The results showed that the LTD-IDMS method
effectively quantified the potent HA in A(H5N1) vaccine material with
results comparable to SRID. As such, it shows great promise to complement
and potentially replace SRID in a pandemic when strain-specific reagents
may not be readily available.