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J.D. Lai et al.
in BHK cells, suggesting that differences in glycan profiles may be protein-specific.40
Given the conflicting evidence relating to the role of mannosylation on FVIII immunity, and the high abun- dance of endogenous high-mannose glycans on other plas- ma proteins that do not exhibit similar immunogenic responses, we assessed sialic acid as a regulator of FVIII immunogenicity.15,16,41 N-glycans on BHK-rFVIII exhibited greater sialylation, which considering our clearance data, is in contrast to previously reported influences of sialic acid on FVIII and VWF half-life.42 The inhibition of com-
plex glycan formation has been shown to increase the spe- cific activity of FVIII, suggesting that the enhanced nega- tive charge due to increased sialic acid may alter the affini- ties between BHK-rFVIII and its interacting coagulant partners.43 While the influence of the FVIII procoagulant activity on immunogenicity remains a subject of debate, the increased negative charge associated with enhanced sialylation may modulate clearance receptor binding.44,45
Sialic acid can signal through inhibitory receptors such as Siglec-5, or the activating homolog Siglec-14, which have nearly identical binding motifs.14,46 In our study, we
A
B
C
E
D
Figure 5. Comparative N-linked glycosylation of different rFVIII products by LC-MS/MS. (A) Heat plot representing the proportion of each of the detected glycans at each N-linked consensus sequence across different rFVIII products. Sites portrayed with an X were not detected in the analyses. Glycan structures are denoted as additions to the core (Man3GlcNAc2-Asn) where H=hexose, N=GlcNAc, F=fucose, S=sialic acid. (B) High-mannose, (C) asialylated, (D) partially sialylated, and (E) fully sialylated N-linked glycans presented as a proportion of the total occupied glycans. Amino acid numbering for N-linked Asn glycosylation sites does not include the 19 amino acid signal peptide. Each rFVIII product was analyzed at least twice, including an initial assessment of sample quality. BHK: baby hamster kidney cells; CHO: Chinese hamster ovary cells; rFVIII: recombinant factor VIII; BDD: B-domain deleted.
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haematologica | 2018; 103(11)