Why Antibody Glycosylation is So Important

Therapeutic monoclonal antibodies have become one of the fastest-growing drug classes in modern biopharmaceuticals. As glycoproteins, antibodies carry a conserved N-linked glycan at position 297 (Asn297) of the Fc region. This seemingly tiny glycan structure profoundly influences the efficacy, safety, and pharmacokinetics of antibodies, including key effector functions such as Antibody-Dependent Cell-Mediated Cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

Recently, a study published in The AAPS Journal systematically analyzed the glycosylation characteristics of 209 FDA-approved therapeutic antibodies and Fc fusion proteins up to May 2025. The research team reviewed the Biologics License Applications (BLAs) for these products, compiling Glycan Characterization data, release criteria, and Fc effector function claims, providing the industry with a comprehensive glycosylation benchmark reference.

A massive analysis covering 209 products

The dataset analyzed includes:

• 160 innovator products and 49 biosimilars
• 147 monoclonal antibodies, 13 bispecific antibodies, 13 Antibody-Drug Conjugates (ADCs), 17 Fc fusion proteins, 9 fragment molecules, and 10 combination formulations
• Disease areas covered include oncology (83 products), autoimmune diseases (74 products), cardiovascular conditions (15 products), and infectious diseases (9 products).

In terms of expression systems, 81% of the products used Chinese hamster ovary cells (CHO), 8% used NS0 cells, and 6% used Sp2/0 cells. All approved IgG antibodies showed only N-linked glycans in their Fc regions, with no O-linked glycosylation detected.

Top Ten Predominant Fc N-Glycans Form Industry Benchmark

The study identified 10 predominant Fc N-Glycan structures in the CHO, NS0, and Sp2/0 expression systems. Six glycans are common to all three systems, including: G0F, G1F, and G2F (core fucosylated biantennary complex-type N-glycans), G0 (afucosylated biantennary glycans), M5 (high-mannose glycans), and G0F-GN (fucosylated glycans with terminal GlcNAc).

In CHO-derived IgG antibodies, the combined mean abundance of these three major glycans, G0F, G1F, and G2F, is as high as 88%. Low-abundance afucosylated glycans (such as G0 and G1) and fucosylated G0F-GN and G1F-GN are consistently detected at low levels (≤ 2.4%). The mean abundances of high-mannose glycans M5 and M6 are 2.3% and 0.3%, respectively.

Fig. 1 Fc N-glycan profiles of FDA-approved therapeutic antibodies. (Luo, et al. 2026)

Expression System Determines Glycan Characteristics; Non-Human Glycans Require Attention

Although antibodies derived from NS0 and Sp2/0 cells also primarily produce glycans of G0F, G1F, and G2F (with combined abundances of 83% and 81%, respectively), they exhibit significant differences in low-abundance glycans:

• NS0-derived antibodies primarily contain glycans with the α-1,3-galactose (α-Gal) epitope, such as G2F+αGal and G2F+2αGal
• Sp2/0 cells: Primarily produce glycans containing N-Glycolylneuraminic Acid (NGNA), such as G2F+NGNA and G1F+NGNA
• CHO cells: Primarily carry human-compatible N-acetylneuraminic acid (NANA), with abundance typically below 0.5%

α-Gal and NGNA are non-human glycan epitopes that may trigger a human immune response. It is noteworthy that many biosimilars of original NS0 or Sp2/0 derived monoclonal antibodies have switched to CHO cell expression, likely to minimize the immunogenicity risks of these non-human glycans.

Of the 29 products derived from NS0 or Sp2/0, only 10 included glycan release testing in their release criteria, and only 3 of these controlled for both α-Gal and NGNA. This reflects differences in risk control strategies for non-human glycans among different sponsors.

Fc Fusion Proteins Exhibit More Complex Glycosylation Features

Compared to traditional IgG antibodies, Fc fusion proteins exhibit significantly different glycosylation profiles:

• Glycan distribution is more dispersed, with the abundance of the top ten glycans ranging from 1.9% to 16.1%.
• Sialylation levels are significantly higher, with 6 out of the top 10 predominant glycans containing sialic acid.
• Triantennary and tetraantennary structures are more common, such as G3F+3NANA.
• Of the 13 products with fusion domain glycosylation, 8 also showed O-linked Glycans.

However, when only the Fc domain of the Fc fusion protein is analyzed, its glycosylation profile is highly similar to that of traditional CHO-derived IgG1 antibodies. This indicates that glycosylation features are significantly influenced by local protein conformation, and the complex glycans of the fusion domain mainly originate from its unique amino acid sequence and spatial structure.

Quality Control Strategies for Afucosylated Glycans

Although the abundance of afucosylated glycans is typically below 10%, they have a significant impact on Fc effector function. In 41 products that included Fc effector function in their mechanism of action (MOA) claims, afucosylated glycans were routinely included in DS release testing:

• The number of monitored glycan species ranged from one to all afucosylated glycans.
• The most common controlled species included G0, G1, G2, G0-GN, and M5.
• The acceptance range for release criteria was relatively wide, ranging from 0.5–1.7% to 0–12%.

In contrast, the acceptance criteria for ADCC or CDC bioactivity assays are generally more uniform, with relative potency typically set between 70–130%. This difference reflects the current lack of unified standards in the industry for controlling afucosylated glycans.

A Gap Exists Between Fc Effector Function Claims and Clinical Evidence

An analysis of the Mechanism of Action section on product labels reveals a concerning reality:

• Only 41 antibodies explicitly list Fc effector functions (ADCC, CDC, etc.) as proposed mechanisms of action in their labels.
• These descriptions are almost entirely based on in vitro experimental data, using qualifying terms such as "in vitro," "proposed mechanism of action," or "possible mechanisms."
• Almost no products provide direct clinical evidence demonstrating the precise contribution of Fc effector functions in patients.

Such phrasing reflects regulatory acceptance of in vitro mechanism hypotheses while acknowledging the current inadequacy of clinical validation. Although genetic factors such as FcγRIIIa-V158F polymorphism may influence ADCC activity, related research results are inconsistent and rarely reflected in product labels.

Industry Status of Analytical Methods

Among the 95 products that included glycan detection in their release standards, the analytical methods were distributed as follows:

• Separation Techniques: Hydrophilic Interaction Liquid Chromatography (HILIC) accounted for 67%, reversed-phase HPLC and normal-phase HPLC each accounted for 11%, and capillary electrophoresis-laser induced fluorescence (CE-LIF) accounted for 8%.
• Labeling Reagents: 2-Aminobenzamide (2-AB) was the most commonly used fluorophore (78%), with the remainder including RapiFluor-MS (8%), APTS (8%), etc.

The 2-AB HILIC method is the most predominant analytical platform, applied to 56% of the products.

For the 41 products with Fc effector functional claims, 76% simultaneously used glycan detection and bioactivity detection (ADCC, CDC, or FcγRIIIa binding assay), 12% used only glycan detection, and 12% used only bioactivity detection.

Implications for the Industry

This analysis, based on 209 FDA-approved products, provides several important references for therapeutic antibody development:

• Six prevalent Fc N-glycans can serve as benchmarks for product development, batch consistency evaluation, and biosimilar similarity assessment.
• The choice of expression system directly affects the immunogenicity risk profile of glycans; the CHO system has a clear advantage in human compatibility.
• Fc fusion proteins require independent glycosylation assessment strategies; benchmarks from traditional IgG antibodies cannot be simply applied.
• The control strategy for afucosylated glycans urgently needs industry-wide harmonization to reduce quality control differences between products.
• Future research needs to design more comprehensive clinical studies to establish a strong correlation between in vitro Fc activity detection and clinical efficacy, thereby enriching the mechanism claims in product labels.

With the continuous emergence of bispecific antibodies, antibody-drug conjugates, and biosimilars, the importance of Glycosylation as a key quality attribute will become increasingly prominent. The benchmark dataset established in this study provides a solid scientific basis for subsequent product development, regulatory review, and industry standard setting.

Related Services & Products

• Glycan Release Service
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• Glycoprotein Enrichment
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• Glyco-biomarker Detection Service
• Glycoprotein Structure Analysis
• N-Glycan
• O-Glycan

Reference

1. Luo, S., et al. (2026). Glycan Profiles of FDA-Approved Therapeutic Antibodies: Insights from Regulatory Submissions. The AAPS Journal, 28(2), 53. DOI: 1208/s12248-026-01207-0.