Baolin Zhang

N-linked glycosylation is a critical quality attribute of therapeutic monoclonal antibodies (mAbs), influencing efficacy, safety, and pharmacokinetics. Accurate and high-throughput glycan profiling is therefore critical for product quality and manufacturing consistency. Here, we evaluated a novel lectin microarray platform for the detection of glycan epitopes commonly present in therapeutic antibodies. To assess specificity and performance, a panel of anti-HER2 mAbs with uniform glycan structures was generated via chemoenzymatic glycoengineering. These antibodies were analyzed in parallel by lectin microarray profiling of intact antibodies and mass spectrometry-based characterization of released glycans. Results from both approaches were highly concordant, demonstrating the lectin microarray’s semi-quantitative capability for accurate glycan profiling. Further studies confirmed the platform’s dynamic range and intermediate precision. Evaluation with the adalimumab reference product and eight biosimilars further confirmed its utility as a complementary tool for therapeutic antibody characterization. Collectively, these findings established the lectin microarray as a robust, high-throughput method for glycan analysis in therapeutic antibody development.

Purpose To qualify a mechanism-of-action (MoA)–reflective reporter gene assay (RGA) for measuring the biological activity of adalimumab (Humira) and its biosimilars, supporting assessment of product quality, comparability, and functional consistency across the product lifecycle. Methods The assay evaluates TNF-α neutralization by monitoring inhibition of NF-κB signaling in a reporter system. Qualification focused on key performance attributes, including system suitability, working range, reproducibility, and intermediate precision, to confirm fitness for routine use. Results The RGA yielded MoA-relevant readouts of NF-κB pathway inhibition in the presence of adalimumab, demonstrating strong system suitability, a broad working range, high reproducibility, and consistent intermediate precision across repeated measures. These characteristics support reliable measurement of functional activity among adalimumab products and biosimilars. Conclusions The qualified, MoA-reflective RGA provides a robust tool for lifecycle management of adalimumab products, enabling quality assessment, comparability exercises, and monitoring of functional consistency across indications in which adalimumab is broadly used (e.g., rheumatoid arthritis, Crohn’s disease, psoriasis).

Glycosylation is a critical quality attribute of certain therapeutic proteins, influencing efficacy, safety, and pharmacokinetics. This study analyzed glycan characterization data and drug substance release specifications from 209 Biologics License Applications (BLAs) approved by the U.S. Food and Drug Administration (FDA) through May 2025. Ten predominant Fc N-glycans were identified across IgG antibodies expressed by CHO, NS0, and Sp2/0 cell lines, with six glycans common to all systems. Five low-abundance afucosylated glycans (< 10%) were tightly controlled within drug substance release specifications for antibodies with Fc effector functions, although acceptance criteria varied across products. Glycan profiles were strongly dependent on the expression system: CHO-derived antibodies predominantly contained human-compatible glycan structures, whereas NS0 and Sp2/0 antibodies introduced non-human epitopes. Fc fusion proteins exhibited higher branching and sialylation compared with conventional IgG antibodies. Notably, analysis of product labels revealed that Fc effector functions were described exclusively as in vitro observations or proposed mechanisms, with limited clinical validation. Overall, these findings establish a comprehensive benchmark for glycan profiles of FDA-approved therapeutic antibodies and underscore the need for harmonized control strategies and stronger correlation between in vitro Fc assays and clinical outcomes. Graphical Abstract

Purpose: To qualify a mechanism‑of‑action (MoA)–reflective reporter gene assay (RGA) for measuring the biological activity of adalimumab (Humira) and its biosimilars, supporting assessment of product quality, comparability, and functional consistency across the product lifecycle. Methods: The assay evaluates TNF‑α neutralization by monitoring inhibition of NF‑κB signaling in a reporter system. Qualification focused on key performance attributes, including system suitability, working range, reproducibility, and intermediate precision, to confirm fitness for routine use. Results: The RGA yielded MoA‑relevant readouts of NF‑κB pathway inhibition in the presence of adalimumab, demonstrating strong system suitability, a broad working range, high reproducibility, and consistent intermediate precision across repeated measures. These characteristics support reliable measurement of functional activity among adalimumab products and biosimilars. Conclusions: The qualified, MoA‑reflective RGA provides a robust tool for lifecycle management of adalimumab products, enabling quality assessment, comparability exercises, and monitoring of functional consistency across indications in which adalimumab is broadly used (e.g., rheumatoid arthritis, Crohn’s disease, psoriasis).

The analytical comparability of biologic products and their biosimilars, including higher-order structure (HOS) assessment, ensures product quality and is required for regulatory approval. In this study, nuclear magnetic resonance (NMR) spectroscopy was used to evaluate the HOS of Humira (adalimumab) and its biosimilars under normal and photo-stressed conditions. Under normal conditions, 1D and 2D NMR spectra showed strong structural similarity among all products. However, photo-stressed samples exhibited distinct structural differences, including increased methionine oxidation, and localized conformational changes, most notably in the reference product. These changes correlated with findings from size-exclusion chromatography, capillary isoelectric focusing, and mass spectrometry (MS), which revealed size and charge heterogeneity, as well as site-specific methionine oxidation in the heavy chains. The differences in photostability were found to be influenced by container closure systems (CCSs) and formulations. In contrast, circular dichroism spectral analysis showed minimal variation in secondary structures among stressed and unstressed samples. These results underscore the utility of NMR as a sensitive tool for comparative structural analysis of monoclonal antibodies and their biosimilars, particularly under stress conditions, and highlight the impact of formulation and CCS on product stability.

The effectiveness of therapeutic antibodies is often associated with their Fc-mediated effector functions, such as antibody-dependent cellular cytotoxicity and antibody-dependent cellular phagocytosis. These functions rely on interactions between Fc gamma receptors (FcγRs) on immune cells and the Fc region of antibodies. Genetic variations in these receptors, known as FcγR polymorphisms, can influence therapeutic outcomes by altering receptor expression levels, affinity, and function. This review examines the impact of FcγR polymorphisms on antibody therapy, emphasizing their role in developing and optimizing functional bioassays to assess product quality. Understanding these polymorphisms is essential for refining bioassays, which are crucial for accurately characterizing antibody products and ensuring consistency in manufacturing processes.

Glycosylation plays a crucial role in determining the quality and efficacy of therapeutic antibodies. This necessitates a thorough analysis and monitoring process to ensure consistent product quality during manufacturing. In this study, we introduce a custom-designed lectin microarray featuring nine distinct lectins: rPhoSL, rOTH3, RCA120, rMan2, MAL_I, rPSL1a, PHAE, rMOA, and PHAL. These lectins have been specifically tailored to selectively bind to common N-glycan epitopes found in therapeutic IgG antibodies. By utilizing intact glycoprotein samples, our nine-lectin microarray provides a high-throughput platform for rapid glycan profiling, enabling comparative analysis of glycosylation patterns. Our results demonstrate the practical utility of this microarray in assessing glycosylation across various manufacturing batches or between biosimilar and innovator products. This capacity empowers informed decision-making in the development and production of therapeutic antibodies.

Purpose: This study aims to establish a benchmark glycan profile for commercial therapeutic monoclonal antibodies (mAbs) approved by the US Food and Drug Administration (FDA). Methods: We conducted a rigorous comparison of glycosylation data from the regulatory submissions for FDA-approved therapeutic antibodies up to May 2023. This analysis includes over 150 mAbs produced by various mammalian cell expression systems. Results: The study identified nine prevalent glycan epitopes across all FDA-approved monoclonal antibodies produced by different expression systems. These epitopes include terminal N-acetylglucosamine, core fucose, terminal galactose, high mannose, α-galactose, terminal α2,3-linked N-acetylneuraminic acid, terminal α2,6-linked N-glycolylneuraminic acid, triantennary structure, and bisecting N-acetylglucosamine, thus establishing a benchmark glycan profile. Conclusions: The findings of this study have significant implications for therapeutic antibody development, quality control, and regulatory compliance. The benchmark glycan profile enables the assessment of glycosylation consistency and comparability across a diverse range of antibody products, ensuring improved product quality within the biopharmaceutical industry.

Circulating tumor cells (CTCs) in the peripheral blood are believed to be the source of metastasis and can be used as a liquid biopsy to monitor cancer progression and therapeutic response. However, it has been challenging to accurately detect CTCs because of their low frequency and the heterogeneity of the population. In this study, we have developed an in vitro model of CTCs by using non-adherent suspension culture. We used this model to study a group of breast cancer cell lines with distinct molecular subtypes (TNBC, HER2⁺, and ER⁺/PR⁺). We found that, when these breast cancer cell lines lost their attachment to the extracellular matrix, they accumulated a subtype of cancer stem cells (CSC) that expressed the surface markers of stem cells (e.g., CD44⁺CD24⁻). These stem-like CTCs also showed high expressions of hypoxia-inducible gene products, particularly the hypoxia-inducible carbonic anhydrase IX (CAIX). Inhibition of CAIX activity was found to reduce CAIX expression and stem cell phenotypes in the targeted CTCs. Further studies are needed, using CTC samples from breast cancer patients, to determine the role of CAIX in CTC survival, CSC transition, and metastasis. CAIX may be a useful surface marker for the detection of CSCs in the blood, and a potential target for treating metastatic breast cancers.

Background: Neutralising antibodies against SARS-CoV-2 are a vital component in the fight against COVID-19 pandemic, having the potential of both therapeutic and prophylactic applications. Bispecific antibodies (BsAbs) against SARS-CoV-2 are particularly promising, given their ability to bind simultaneously to two distinct sites of the receptor-binding domain (RBD) of the viral spike protein. Such antibodies are complex molecules associated with multi-faceted mechanisms of action that require appropriate bioassays to ensure product quality and manufacturing consistency.Methods: We developed procedures for biolayer interferometry (BLI) and a cell-based virus neutralisation assay, the focus reduction neutralisation test (FRNT). Using both assays, we tested a panel of five BsAbs against different spike variants (Ancestral, Delta and Omicron) to evaluate the use of these analytical methods in assessing binding and neutralisation activities of anti-SARS-CoV-2 therapeutics.Results: We found comparable trends between BLI-derived binding affinity and FRNT-based virus neutralisation activity. Antibodies that displayed high binding affinity against a variant were often followed by potent neutralisation at lower concentrations, whereas those with low binding affinity also demonstrated reduced neutralisation activity.Conclusion: The results support the utility of BLI and FRNT assays in measuring variant-specific binding and virus neutralisation activity of anti-SARS-CoV-2 antibodies.