Comprehensive analysis and characterization of glycan pairing in therapeutic antibodies and Fc-containing biotherapeutics: Addressing current limitations and implications for N-glycan impact.

N-glycosylation of the Fc part is a (critical) quality attribute of therapeutic antibodies and Fc-containing biotherapeutics, that impacts their stability, immunogenicity, pharmacokinetics, and effector functions. Current glycosylation analysis methods focus on the absolute amounts of glycans, neglecting the apparent glycan distribution over the entirety of proteins. The combination of the two Fc N-glycans, herein referred to as glyco-pair, therefore remains unknown, which is a major drawback for N-glycan impact assessment. This study presents a comprehensive workflow for the analysis and characterization of Fc N-glycan pairing in biotherapeutics, addressing the limitations of current glycosylation analysis methods. The applicability of the method across various biotherapeutic proteins including antibodies, bispecific antibody formats, and a Fc-Fusion protein is demonstrated, and the impact of method conditions on glycan pairing analysis is highlighted. Moreover, the influence of the molecular format, Fc backbone, production process, and cell line on glycan pairing pattern was investigated. The results underscore the significance of comprehensive glycan pairing analysis to accurately assess the impact of N-glycans on important product quality attributes of therapeutic antibodies and Fc-containing biotherapeutics.

CE methods for charge variant analysis of mAbs and complex format biotherapeutics.

Charge heterogeneity analysis of monoclonal antibodies (mAbs) and complex formats, such as bispecifics, is crucial for therapeutic applications. In this study, we developed two capillary electrophoresis (CE)-based methods, capillary zone electrophoresis (CZE) and imaged capillary isoelectric focusing (iCIEF), for analyzing a broad spectrum of mAbs and complex mAb formats. For CZE, we introduced a new buffer system and optimized the background electrolyte (BGE) with an alternative dynamic coating agent and a superior polymeric additive. The pH of the BGE was increased, leading to enhanced resolution of high pI and complex format mAbs. In iCIEF, we identified an ampholyte combination offering a highly linear pH gradient and covering a suitable pH range. We also investigated alternatives to denaturing stabilizers and found that non-detergent sulfobetaine 195 exhibited excellent properties for iCIEF applications. These optimized methods provide a framework for the charge heterogeneity analysis of therapeutic mAbs and complex formats.