Increased serum clearance of oligomannose species present on a human IgG1 molecule

Abstract

The role of Fc glycans on clearance of IgG molecule has been examined by various groups in experiments where specific glycans have been enriched or the entire spectrum of glycans was studied after administration in pre-clinical or clinical pharmacokinetic (PK) studies. The overall conclusions from these studies are inconsistent, which may result from differences in antibody structure or experimental design. In the present study a well-characterized recombinant monoclonal IgG1 molecule (mAb-1) was analyzed from serum samples obtained from a human PK study. mAb-1 was recovered from serum using its ligand cross-linked to Sepharose beads. The overall purity and recovery of all isoforms were carefully evaluated using a variety of methods. Glycans were then enzymatically cleaved, labeled using 2-aminobenzamide and analyzed by normal phase high performance liquid chromatography. The assays for recovering mAb-1 from serum and subsequent glycan analysis were rigorously qualified at a lower limit of quantitation of 15 μg/mL, thus permitting analysis to day 14 of the clinical PK study. Eight glycans were monitored and classified into two groups: (1) the oligomannose type structures (M5, M6 and M7) and (2) fucosylated biantennary oligosaccharides (FBO) structures (NGA2F, NA1F, NA2F, NA1F-GlcNAc and NGA2F-GlcNAc). We observed that the oligomannose species were cleared at a much faster rate (40%) than FBOs and conclude that high mannose species should be carefully monitored and controlled as they may affect PK of the therapeutic; they should thus be considered an important quality attribute. These observations were only possible through the application of rigorous analytical methods that we believe will need to be employed when comparing innovator and biosimilar molecules.

The Emerging Importance of IgG Fab Glycosylation in Immunity

“…Structural variation in conventional biantennary Fc glycans does not significantly influence the Ab half-life (62), probably because of the inaccessibility of these glycans to glycoreceptors (see "Structure of IgG Fab Glycans" above), although "nonnatural" high-mannose glycans can increase the clearance rate (63). In contrast, Fab glycan sialylation can enhance the serum half-life of mAbs, as shown for the anti-EGFR Ab cetuximab and for the anti-TA-MUC1 Ab PankoMab (64).…”

Comparison of methods for the analysis of therapeutic immunoglobulin G Fc-glycosylation profiles—Part 1: Separation-based methods

“…Recent studies described clear evidence for selective clearance of oligomannose species (high mannose-type) of Fc glycans. 14,15 Sialylation may induce anti-inflammatory effects via Th2 signaling and decrease ADCC via reduced interaction with Fcg receptors. 16,17 Additionally, some IgG glycan structures such as a1,3-bound galactose and N-glycolylneuraminic acid may be involved in adverse immune reactions.…”

Consistency of quality attributes for the glycosylated monoclonal antibody Humira® (adalimumab)

“…19,20 Characterized by the degree of galactose sugar molecules, one of the major glycoforms of mAbs, including adalimumab, is the agalactosyl fucosylated biantennary oligosaccharide (G0F) species that contains fucose but no terminal galactosylation. 21 Evidence that glycosylation patterns affect protein function is accumulating, and differential glycosylation has been shown to influence serum clearance in human studies of therapeutic antibodies with oligomannose sugars, [21][22][23] antibodydependent cell-mediated cytotoxicity or complement-dependent cytotoxicity, 18,21,24 and serum half-life due to differences in binding to the Fc receptors. 10,12,13,15 In the case of adalimumab, manufacturing changes have included scale increases and site transfers, specification changes, or process control improvements across manufacturing sites.…”