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.

Figure 1. Fc glycans observed on antibodies. Symbol nomenclature was adopted from the Consortium for Functional Glycomics. The nomenclature used for complex and oligomannose species are NA1F (Gal-1, G1), NA2F (Gal-2, G2), NGA2F (Gal-0, G0), Mann-5, Mann-6, Mann-7 (M5, M6, M7 or Oligomannose-5,6,7).

Figure 3. Overlay of IEX profile. Weak cation exchange method was used to evaluate antigen-based affinity recovery of mAb-1 from serum. An overlay of eluted mAb-1 recovered from serum showed similar profile to mAb-1 recovered from formulation buffer. Zooming into acidic region also showed similar recovery of different species.

Figure 4. Evaluation of mannosidase activity following in vitro serum incubation studies. mAb-1 was spiked into pooled human serum and at various time points (24 and 96 hours) recovered and the glycans analyzed. As shown in Figure 4B and 4C, the level of high mannose species (M6 and M7) was reduced within 24 hours of incubation in serum. In contrast, M5 levels increased over the same time period (Fig. 4A); presumably as a result of enzymatic cleavage (mannosidases) of M6 and M7 to the M5 species.

Figure 5. Glycan profile of mAb-1 separated by normal phase-HPLC after recovery from serum of healthy volunteer. Shown in Figure 5A is the glycan profile of volunteer #5 at t=0.5 hrs (mAb-1 concentration in serum was 185.5 μg/mL) and in Figure 5B at t=168 hrs (mAb-1 concentration in serum was 41.7 μg/mL). Samples were diluted with pooled serum to 25 μg/mL prior to recovery by affinity chromatography and analysis by 2-AB oligosaccharide assay.

Figure 6. Mean percentage of glycan species of mAb-1 in serum following single dose administration in healthy individuals.

Figure 7. Average NGA2F fraction of mAb-1 in serum from 15 volunteers. The levels of NGA2F were found to remain fairly constant over 336 Hours (14 days).

Figure 8. Average M5 fraction of mAb-1 in serum from 15 volunteers. The levels of M5 were found to initially increase in the first 6 hours, presumably as a result of mannosidase activity on the high mannose species (M6-M9). After 6 hours, the levels of M5 were found to rapidly decrease. There was about a 40% decrease in M5 by day 14.

Figure 2. Overview of procedure used for assay qualification and glycan analysis of samples from the clinical study.