Selective clearance of monoclonal antibodies via the mannose receptor is dependent on glycan pairing

The pharmacokinetics (PK) of therapeutic monoclonal antibodies (mAbs) are influenced by N-glycosylation, a critical quality attribute (CQA) that affects serum half-life and receptor interactions. High-mannose N-glycans are known to accelerate mAb clearance, likely via the mannose receptor (MR). However, the impact of high-mannose glycan pairing - whether symmetrical or asymmetrical - on this process remains poorly understood. MAbs enriched in high-mannose N-glycans were fractionated using mannose receptor—affinity chromatography to isolate symmetrical and asymmetrical high-mannose glyco-pairs. These fractions were characterized for physicochemical properties and labeled for a cell-based internalization assay using MR-expressing SUP-B15 cells to quantify internalization rates via flow cytometry. A PK study in rats was conducted using a high-mannose enriched mAb drug product, and glyco-pair-specific clearance was analyzed by mass spectrometry following immunocapture. Symmetrical high-mannose glyco-pair exhibited the highest internalization rate in vitro, followed by asymmetrical high-mannose and symmetrical complex glyco-pairs. In vivo, symmetrical high-mannose glyco-pair showed the fastest clearance, with a half-life of 2.4 days, compared to 7.2 days for asymmetrical high-mannose and 17.4 days for symmetrical complex glyco-pairs. The area under the curve was reduced to 73% and 38% for asymmetrical and symmetrical high-mannose glyco-pairs, respectively, relative to the symmetrical complex glyco-pair. These differences were attributed solely to glycan pairing, as other physicochemical properties remained consistent across fractions. Competitive inhibition with mannan confirmed MR-mediated uptake in vitro. The findings also suggest potential implications for immunogenicity, as increased internalization by antigen-presenting cells may enhance antigen presentation and anti-drug antibody formation. This study demonstrates that glycan pairing significantly influences the PK of mAbs by correlating with differential MR interactions, providing evidence for MR-mediated faster clearance of high-mannose containing mAbs. Symmetrical high-mannose glyco-pair are cleared more rapidly than asymmetrical counterparts, underscoring the need to consider glycan pairing as a distinct CQA. Current control strategies based solely on released N-glycans may overlook clinically relevant heterogeneity. Incorporating high-mannose glyco-pair analysis into manufacturing and quality control processes could improve therapeutic consistency and reduce immunogenicity risks.