High-mannose glycans on the Fc region of therapeutic IgG antibodies increase serum clearance in humans

Abstract

Glycan structures attached to the C(H)2 domain of the Fc region of immunoglobulin G (IgG) are essential for specific effector functions but their role in modulating clearance is less clear. Clearance is of obvious importance for therapeutic monoclonal antibodies (Mabs) as it directly impacts efficacy. Here, we study the impact of Fc glycan structure on the clearance of four therapeutic human IgGs (one IgG1 and three IgG2s) in humans. The therapeutic IgGs were affinity purified from serum samples from human pharmacokinetic studies, and changes to the glycan profile over time were determined by peptide mapping employing high-resolution mass spectrometry. Relative levels of high-mannose 5 (M5) glycan decreased as a function of circulation time, whereas other glycans remained constant. These results demonstrate that therapeutic IgGs containing Fc high-mannose glycans are cleared more rapidly in humans than other glycan forms. The quantitative effect of this on pharmacokinetic area under the curve was calculated and shown to be relatively minor for three of the four molecules studied, but, depending on the dosing regimen and the relative level of the high-mannose glycan, this can also have significant impact. High-mannose content of therapeutic Mabs should be considered an important product quality attribute which may affect pharmacokinetic properties of therapeutic antibodies.

Glycation of antibodies: Modification, methods and potential effects on biological functions

“…Both in-vivo mouse model and FcRn binding studies indicated glycation at the levels observed for mAb does not affect antibody pharmacokinetics (PK). 42,68 In terms of immunogenicity and safety, the constant regions of canonical therapeutic mAbs and endogenous antibodies are typically very similar, so glycation in the constant region of mAbs can be considered similar to endogenous mAbs, and therefore less of a concern. It has been hypothesized that lysine residues in the constant region of antibodies have evolved to be resistant to glycation.…”

“…It has been hypothesized that lysine residues in the constant region of antibodies have evolved to be resistant to glycation. 8,68 One of the concerns of glycation of therapeutic antibody, especially the AGEs, is in the development of immunogenicity. The AGEs are more immunogenic than normal antibody in animal models.…”

In vivo characterization of therapeutic monoclonal antibodies

“…The two major N-linked glycan classes are the fucosylated biantennary oligosaccharide and the high-mannose oligosaccharide. The high-mannose species are cleared much faster in human blood than the fucosylated biantennary oligosaccharides [21,22]. Oxidation of methionine is another common post-translational modification.…”

Monitoring Glycosylation Profile and Protein Titer in Cell Culture Samples Using ZipChip CE-MS

“…Glycosylation is one of very important post-translational modifications because it can have significant impact on antibody-dependent cell-mediated cytotoxicity (ADCC) activity [3,4], complement-dependent cytotoxicity (CDCC) activity [5], clearance [6] and immunogenicity [7,8]. Protein glycosylation can be significantly impacted by the host cell line, clone, media composition, feeding strategy and downstream processing conditions [9][10][11].…”