Figure 1. Comparison of glycosylation patterns of BG505 SOSIP.664 trimers produced in CHO and 293T cells.

HILIC-UPLC spectra of fluorescently labelled N-linked glycans isolated from (A) the entire trimeric gp120/gp41_ECTO complex (gp140), and the (B) gp120 and (C) gp41_ECTO subunits of BG505 SOSIP.664 trimers. The subunits analyzed in each case are illustrated on the left. Trimers were purified by 2G12-affinity chromatography followed by SEC. The gp140 band was extracted from a non-reducing SDS-PAGE gel, while the gp120 and gp41_ECTO bands were resolved by SDS-PAGE under reducing conditions. The glycan contents of proteins extracted from the bands were analyzed. Peaks corresponding to oligomannose glycans (M5-M9; Man_5-9GlcNAc₂) are colored in shades of green; the remaining peaks (gray) correspond to complex and hybrid-type glycans. Peak areas (as a % of total glycans) are illustrated in the associated pie charts. Glycan structures are represented according to the color scheme established by the Consortium for Functional Glycomics (http://www.functionalglycomics.org/). The SOSIP.664 construct contains the following mutations: a T332N mutation to introduce the 332 glycosylation site; cysteines at 501 and 605 form a disulphide bridge to covalently link the gp120 and gp41_ECTO subunits; replacement of the gp120 furin cleavage site (RXXR) with a hexa-arginine (R6) sequence to promote furin cleavage; an I559P mutation to stabilize the gp41_ECTO subunits in the pre-fusion form; and deletion of the MPER region at residue 664 to reduce aggregation.