NIH Research Festival
Antibody-based vaccines are the primary focus of vaccine design for neutralizing HIV-1 virions. The strategies of the vaccine-development programs have been focused on the HIV Env surface glycoprotein (Env), which enables the virus to enter host cells. Various Env constructs have been designed as the prototypes of native-like immunogens. During the product development and process scale-up, a panel of liquid chromatography – mass spectrometry (LC-MS) peptide mapping analyses was applied to monitor Env product quality. Known as the most densely glycosylated protein with 28 glycosylation sites, Env presents a challenge for the routine biopharmaceutical MS applications. Here we demonstrated the strategy to meet these challenges, and to highlight some inevitable problems with this glycoprotein characterization in the analytical field. Using a set of orthogonal proteolytic digests, 99% sequence of the Env glycoprotein was confirmed. The furin cleavage site was characterized, with the remnants of partially uncleaved Env single chain detected, and up to 1 Arg at the gp120 C-terminus (out of possible 6), suggesting an alternative chemistry of furin cleavage. Out of total 28 glycan sites of the Env construct, percent glycan occupancy was provided for 25 sites (the exact glycan profile for 21 sites, the overall type of glycans for 4 sites). No further method optimization could lead to reporting the 3 unassigned sites, as this deficiency is a consequence of the sequence arrangement and is an intrinsic feature of the heavily glycosylated Env molecule, as opposed to a lack of method development.
Scientific Focus Area: Virology
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