Site-Specific Glycan Microheterogeneity Evaluation of Aflibercept Fusion Protein by Glycopeptide-Based LC-MSMS Mapping

Abstract

The evaluation of the protein glycosylation states of samples of aflibercept obtained from three different regions was conducted by site-specific N-linked glycan microheterogeneity profiling. Glycopeptide-based nano-LC MSMS mapping of tryptic-digested samples of each aflibercept lot provided site-specific information about glycan microheterogeneity on each of the five N-glycosylation sites (two sites in the VEGFR-1 region, two sites in the VEGFR-2 region, and one site in the human IgG Fc region). Next, the glycopeptide-mapping results obtained from the three different aflibercept lots were compared to evaluate the similarity between the samples. Three aflibercept lots showed a high degree of similarity in glycan composition, fucosylation level, sialylation level, and branching, when all five N-glycosylation sites were assessed together as a group. On the other hand, noticeable variations between lots in the glycan types and sialylation levels on the two sites of the VEGFR-2 domain were observed when each of the five N-glycosylation sites were assessed using the glycopeptide-based method. The presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics, were also detected on the sites of VEGFR1 and VEGFR2 domains, but not on the IgG Fc domain site. These results imply that analyses of the glycosylation profiles of fusion proteins containing multiple N-glycosylation sites, such as aflibercept, being done as a part of quality control for the therapeutics manufacturing process or for biosimilar development, can be done with a more applicable outcome by assessing each site separately.

Efficient Expression of Functionally Active Aflibercept with Designed N-glycans

“…This opens a possibility for more detailed investigation on the impact of this dominant PTM in future studies. Our results are in stark contrast to mammalian-cell-produced counterparts that show significant glycan-associated microheterogeneity due to the large endogenous glycosylation repertoire of mammalian cells [ 10 , 11 ]. In addition, the presence of N-glycolylneuraminic acid (NeuGc) glycans, which may mediate adverse immune reactions in antibody therapeutics [ 11 ], have also been detected on the sites of the VEGFR1 and VEGFR2 domains.…”

“…High amounts of mannosidic structures (>50%) were reported on other Fc fusions expressed in plants [ 43 ] and might be the consequence of insufficient secretion. Such structures were detected on CHO-produced aflibercept, albeit to a much lesser extent [ 11 , 44 ]. Our results show that mannosidic structures of aflibercept do not impact on VEGF binding; however, some recombinant fusion proteins containing high mannose-type glycans exhibit reduced serum half-life [ 45 ].…”

“…It was shown that the glycosylation pattern of recombinant aflibercept produced in mammalian cells is diverse, with up to 18 different N-glycan species [ 10 ]. Also, significant glycan heterogeneity between different batches was detected [ 11 ]. Both observations not only encumber its use due to the high standards required in therapeutic protein expression, but also hamper investigation of its glycan-associated features.…”

Immunomodulation of Antibody Glycosylation through the Placental Transfer

“…In IgG molecules, each Fc domain contains a glycosylation site at asparagine 297 (Asn297), which maintains structural stability and regulates protein spatial conformation [35][36][37][38]. In addition to the conserved glycosylation site Asn297 in the CH2 domain found in all IgG subclasses, IgG3 has an additional N-glycosylation site in the CH3 domain at Asn392 [39]. The core structure of IgG glycan is a heptasaccharide composed of four N-acetylglucosamine (GlcNAc) and three mannose residues that can be extended with galactose, sialic acid, core fucose, and bisected GlcNAc (Figure 1) [40,41].…”