Background/Objectives: This study comprehensively characterized the O- and N-glycosylation profiles of bispecific antibodies (BsAbs) via advanced analytical techniques to evaluate their structural and functional implications. Methods: High-resolution MS revealed O-xylosylation at Ser468 within the (G4S)4 linker peptide, which was identified as xylose with a molecular weight of 132.042 Da. HILIC-HPLC analysis of N-glycosylation revealed glycan species engineered to eliminate Fc effector functions. O-glycosylation analysis via β-elimination followed by high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) identified xylose as the predominant glycan. Results: O-xylosylation does not affect the binding of BsAbs to either antigen Programmed Death-1 (PD-1) or Vascular Endothelial Growth Factor (VEGF). Notably, O-xylosylation interactions with mannose receptor represent the first discovery highlighting potential immunomodulatory roles. Conclusions: This study highlights the critical importance of monitoring comprehensive glycosylation characterization during the development of BsAb with (G4S)n linkers to ensure optimal therapeutic efficacy, safety, and reduced immunogenic potential.

Antibody–drug conjugates (ADCs) are a pivotal technology for precision cancer therapy, harnessing the synergistic effects of antibody targeting and toxin delivery. However, traditional ADCs encounter limitations in efficacy that stem from tumor resistance, heterogeneity, and intense target competition. Dual-payload ADCs (DP-ADCs) represent a promising solution to these challenges, as they leverage dual mechanisms of action that mitigate acquired drug resistance and enhance adaptability to tumor heterogeneity. The complex structure of DP-ADCs presents substantial quality control hurdles. In this manuscript, we review the current payload selection and conjugation strategies of DP-ADCs and examine recent advances in quality control research. Specifically, we analyze the analytical challenges related to the quantification of free toxins, the determination of the total antibody content, and the characterization of the drug-to-antibody ratio and its distribution. Ultimately, the aim of this work is to provide valuable guidance for future DP-ADC quality control analyses to facilitate their clinical translation and application.

Background/Objectives: Bispecific antibody–drug conjugates (bispecific ADCs) are among the most rapidly advancing antibody therapeutic modalities, with only one product (BL-B01D1) currently approved for marketing worldwide to date. In this study, we investigated several critical quality attributes (CQAs) of a glyco-site-specific antibody–drug conjugate (ADC) targeting two distinct epitopes of HER2. Methods: We established quality control methods for the assessment of mispaired species, conjugation site, drug-to-antibody ratios (DARs), charge variants, residual free drugs, residual β1,4-galactosyltransferase I (GalT) enzyme, relative binding activity, and cell proliferation inhibition activity. Conclusions: An analytical method suite was developed for the bispecific ADC to comprehensively characterize and appropriately control its critical quality attributes, thereby guiding the research, development, and manufacturing process optimization of the ADC product and ensuring its safety, efficacy, and quality consistency.

Antibody–drug conjugates (ADCs) are a pivotal technology for precision cancer therapy, harnessing the synergistic effects of antibody targeting and toxin delivery. However, traditional ADCs encounter limitations in efficacy that stem from tumor resistance, heterogeneity, and intense target competition. Dual-payload ADCs (DP-ADCs) represent a promising solution to these challenges, as they leverage dual mechanisms of action that mitigate acquired drug resistance and enhance adaptability to tumor heterogeneity. The complex structure of DP-ADCs presents substantial quality control hurdles. In this manuscript, we review the current payload selection and conjugation strategies of DP-ADCs and examine recent advances in quality control research. Specifically, we analyze the analytical challenges related to the quantification of free toxins, the determination of the total antibody content, and the characterization of the drug-to-antibody ratio and its distribution. Ultimately, the aim of this work is to provide valuable guidance for future DP-ADC quality control analyses to facilitate their clinical translation and application.