Overview

As a core post-translational modification in eukaryotic proteins, glycosylation profoundly influences protein functionality. The structure and abundance of glycans affect antibody stability, clearance rate, immunogenicity, and activities such as antibody-dependent cellular cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC).

As a critical quality attribute (CQA) of antibody drugs, accurate identification and analysis of N-glycosylation are indispensable for ensuring drug quality and clinical efficacy.

Limitations of Conventional N-Glycan Analysis

As a high-tech enterprise providing core raw materials for biopharmaceuticals, Hzymes Biotech is honored to showcase its innovative solutions at this exhibition!

N-glycosylation refers to the attachment of sugar chains to the amide nitrogen of asparagine (Asn) residues within the Asn-X-Ser/Thr (X≠Pro) motif. The conserved core structure is Manα1-3[Manα1-6] Manβ1-4GlcNAcβ1-4GlcNAcβ1-N-Asn-X-Ser/Thr, which can be categorized into high-mannose, complex, and hybrid types depending on structural modifications.

The traditional workflow for N-glycan analysis includes:

1. Enzymatic cleavage of glycans using PNGase F to release N-glycans;
2. Derivatization labeling of the reducing ends;
3. Purification to remove unreacted glycans and residual reagents;
4. UPLC or LC-MS for separation and identification;
5. Quantification based on comparison with standards or glycan databases.

However, traditional N-glycan analysis suffers from long processing time, low throughput, and poor reproducibility.
Typical issues include:

• Enzymatic digestion requiring several hours or overnight;
• Low labeling efficiency and weak signal response of 2-AB;
• Sample concentration and purification steps leading to glycan loss and inconsistent recovery.

Principles of Different Derivatization Labeling Methods

As a high-tech enterprise providing core biopharmaceutical raw materials, Hzymes Biotech once again presents innovative analytical solutions at this exhibition!

01. RFMS Labeling

RFMS (RapiFluor-MS) is an NHS-carbamate reagent that reacts rapidly with the primary amine group of N-glycans to form a stable urea linkage (NH–CO–NH). It contains both a quinoline fluorophore for high-sensitivity fluorescence detection and a tertiary amine for enhanced MS ionization efficiency (ESI+).

• Hydrazide group: Couples efficiently with the aldehyde group of glycans released by PNGase F.

• Quinoline fluorophore: Provides strong fluorescence for UPLC-FLD quantitative detection.

• Tertiary amine group: Improves protonation and MS detection sensitivity in positive ion mode.

02. 2-AB Labeling

2-Aminobenzamide (2-AB) contains an aromatic amine (-NH₂) that reacts with the aldehyde group at the glycan reducing end to form a Schiff base (C=N) intermediate, which is then reduced into a stable secondary amine (C–N) linkage.

• The glycan aldehyde reacts with the amine of 2-AB to form an unstable Schiff base (C=N).
• The intermediate is reduced by agents such as sodium cyanoborohydride or 2-picoline borane.
• Since 2-AB lacks fluorescence-enhancing groups, MS signal response is weak.

03. ProA Labeling

Procainamide (ProA) builds upon the 2-AB structure by adding a tertiary amine group, enabling a reductive amination reaction with the glycan aldehyde via its primary amine. The tertiary amine contributes to stronger fluorescence and improved MS ionization sensitivity.

• ProA reacts with the glycan aldehyde through reductive amination.
• The tertiary amine carries a proton in MS analysis, yielding high ionization efficiency.
• Its small molecular weight ensures minimal retention time shift in reverse-phase chromatography.

04. InstantPC Labeling

InstantPC is an activated form of procainamide that reacts rapidly with the primary amine of glycans to form a stable urea linkage (NH–CO–NH). It also contains a tertiary amine, enabling strong ionization and high MS signal intensity in positive ion mode.

• Under electrospray ionization (ESI), InstantPC-labeled glycans instantly generate a permanently charged cation, leading to high sensitivity.

Summary of Four Labeling Methods

Hzymes’ Newly Upgraded N-Glycan Analysis Kits

To overcome the limitations of traditional N-glycan workflows, Hzymes Biotech has launched a comprehensive N-glycan analysis solution:

• Covers four derivatization labeling methods in a single product line.
• 5-minute digestion and 5-minute labeling — total sample prep in just 30 minutes.
• High enzymatic efficiency, enabling complete digestion of mAbs, bispecifics, ADCs, and fusion proteins.
• Safety first — ProA and 2-AB kits are free of toxic sodium cyanoborohydride.
• Automation-ready — compatible with high-throughput sample processing systems.
• Pharmacopoeia-grade method validation, including digestion efficiency, specificity, repeatability, intermediate precision, linearity, durability, detection limit, batch consistency, and stability.

01. High Enzymatic Digestion Efficiency

IgG1, IgG4, and fusion proteins show complete deglycosylation by PNGase F as verified by CE-SDS analysis.

02. Excellent Repeatability and Precision

Two operators processed six replicates each following kit instructions. The CV values of G0F for repeatability and intermediate precision were <10%.

03. Wide Linearity Range

Sample dilutions (20%, 40%, 100%, 200%, 400%) were processed and fitted, yielding a linear correlation of R² > 0.99.

04. High Batch-to-Batch Consistency

The same sample processed with three different kit batches (three replicates each) showed highly consistent detection results.

05. Accurate Real Sample Performance

Analyzed Vedicitumab (HER2-ADC), Trastuzumab (HER2), Cadonilimab (PD-1/CTLA-4), and EGFR×HER3 bispecific antibody — the peak patterns and areas matched those of leading international brands.

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