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

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.

Elevated cleavage of human immunoglobulin gamma molecules containing a lambda light chain mediated by iron and histidine.

Monoclonal antibodies in liquid formulation undergo nonenzymatic hydrolysis when stored at 5 degrees C for extended periods. This hydrolysis is enhanced at extreme pH and high temperature. In this study we discover that iron in the presence of histidine also enhanced cleavage of human immunoglobulin gamma (IgG) molecules containing a lambda light chain when incubated at 40 degrees C. The level of cleavage was concentration dependent on both iron and histidine levels. Enhanced cleavage with iron and histidine was not observed on IgG molecules containing a kappa light chain. Using CE-SDS to quantify levels of Fab+Fc, the Fab arm, and free light chain (LC) and heavy chain (HC) fragments, we show that cleavage resulted in elevated levels of free light and heavy chain fragments. Using MS we find elevated scission between residues E/C on the LC resulting in LC fragment 1-215. We also observed enhanced cleavage between S/C residues of the HC resulting in HC fragment 1-217. The corresponding Fab+Fc fragment beginning with cys-218 was not found. Instead, we find elevation of a Fab+Fc fragment that began with aspartic acid (cleavage between C/D). Further studies to understand how iron and histidine enhance cleavage of lambda light chain IgG molecules are ongoing.