Fc Glycosylation Analysis Service

Q: What sample types are compatible with your Fc glycosylation analysis service?

Our service is versatile and accommodates various sample types, including monoclonal antibodies, polyclonal antibodies, Fc-fusion proteins, and serum-derived IgG. We can process samples from different species and production systems, ensuring broad applicability across research and therapeutic development projects.

Q: How do you ensure site-specific analysis of Fc glycans, especially when Fab regions may also carry N-glycosylation?

We employ IdeS digestion to cleave below the hinge region of IgG, generating defined Fc and Fab fragments. This allows exclusive profiling of the Fc N-glycan at Asn297. The released Fc is further purified before deglycosylation or direct intact mass analysis, ensuring Fab-derived glycans do not confound Fc-specific glycan quantification. This separation is critical for therapeutic antibody characterization and is validated in our standard workflow.

Q: What distinguishes Creative Biolabs’ Fc glycosylation analysis from other providers?

At Creative Biolabs, our Fc glycosylation analysis service is tailored to deliver precise, site-specific glycan profiling. We employ a combination of advanced techniques, including enzymatic digestion (e.g., IdeS), high-resolution mass spectrometry (LC-MS/MS), and specialized labeling methods, to ensure accurate characterization of Fc glycan structures. Our approach allows for the differentiation between Fc and Fab glycosylation, providing comprehensive insights into glycan heterogeneity, occupancy, and structural variations.

Introduction Workflow Sample Applications Highlights Other Solutions FAQs Products Supports

Why Fc Glycosylation Matters?

The glycosylation pattern of the Fc region determines antibody functionality because it affects mechanisms including ADCC, complement activation, and Fcγ receptor interactions. Minor modifications in fucosylation, galactosylation, or sialylation at the Fc region significantly impact therapeutic antibody function and immune responses. Physiological and pathological states such as autoimmunity, infection, and pregnancy can be monitored through glycan modifications which act as sensitive biomarkers. Fc N-glycans typically consist of a biantennary core with variable decorations:

Core fucose reduces binding to FcγRIIIa, dampens ADCC
Galactose influences complement activation (via C1q binding)
Sialic acid drives anti-inflammatory responses via Type II FcγRs
Bisecting GlcNAc alters glycan conformation and immune modulation
High-mannose forms are associated with increased clearance from serum

Fig.1 Fc Glycosylation Profiling of IgG Antibodies. (OA Literature) Fig.1 IgG Fc glycosylation.¹

At Creative Biolabs, we offer a suite of specialized services to support Fc glycosylation research and development. Our antibody glycosylation analysis services include platforms tailored for monoclonal, polyclonal, Fab, and Fc-specific glycan profiling, leveraging cutting-edge mass spectrometry, enzymatic digestion, and labeling techniques. Whether you're engineering antibodies or investigating immune regulation, our Fc glycosylation analysis service provides precise, site-specific glycan characterization to accelerate your project with high accuracy and scientific depth.

Our Fc Glycosylation Analysis Workflow

Facing with various analytical challenges, Fc glycosylation analysis is technically demanding due to:

Microheterogeneity: Over 30 glycoforms can co-exist in serum IgG.
Site-specificity: Need to distinguish Fc from Fab glycans.
Subtle changes: Small differences (e.g., partial afucosylation) have large functional consequences.

Thus, specialized analytical workflows are essential for accurate glycosylation mapping. At Creative Biolabs, we offer a comprehensive Fc glycosylation analysis platform optimized for sensitivity, resolution, and throughput. The process integrates:

1. Sample Preparation

Total IgG is purified from serum or monoclonal production.
The Fc domain is separated post-IdeS digestion, isolating it from Fab-derived glycans.

2. N-Glycan Release and Labeling

PNGase F is used to cleave N-glycans enzymatically.
Labeling enhances sensitivity for fluorescence detection and MS ionization.

3. LC-FLD/MS or MALDI-TOF-MS

HILIC-UPLC-FLD enables quantitation of labeled glycans.
MALDI-TOF-MS provides rapid, high-resolution glycan profiling for both intact Fc and released glycans.

4. Advanced Data Interpretation

Glycan quantification (% relative abundance)
Trait calculation: fucosylation, sialylation, galactosylation, bisection, high-mannose
Comparison with reference standards or across batches

Sample Requirements

Sample Type	Minimum Volume	Submission Notes
Purified IgG	≥ 20 µg	Preferably in PBS. Avoid buffers with high glycerol, Tween, or harsh detergents. Frozen at –80°C or fresh samples recommended. Clarified supernatant preferred; pre-filtration optional. Expression host and isotype information encouraged for method optimization.
Human or Mouse Serum/Plasma	≥ 5 µL
Hybridoma Culture Supernatant	≥ 100 µL
Recombinant Monoclonal Antibodies	≥ 20 µg

Key Applications

Glycoform profiling of therapeutic mAbs (e.g., rituximab, trastuzumab)
Stability and lot-to-lot consistency analysis
Biosimilar comparability
Correlating Fc glycoforms with ADCC, CDC, and anti-inflammatory activity
Fc sialylation and placental antibody transfer studies
Monitoring Fc glycosylation changes in autoimmune diseases (RA, SLE)
Pregnancy-associated modulation of Fc glycans

Advantages of Our Service

✅ Site-specific profiling (Fc vs. Fab)

✅ High-throughput capabilities (96 samples per day)

✅ Broad glycan coverage including sialylation linkage-specificity

✅ Interpretation by senior glycobiologists

Services You May Be Interested in

Custom Antibody Glycosylation Service

Creative Biolabs supplies sophisticated antibody glycosylation solutions to advance therapeutic development and analytical characterization. Through cell line modification and enzymatic or chemoenzymatic techniques our custom service allows precise engineering of N- or O-glycans which enhances ADCC, CDC, PK, or antigen binding.

Glycan Profiling Service

We deliver extensive N-/O-glycan characterization through LC-ESI-MS/MS and MALDI-TOF-MS with fluorescent labeling to generate detailed glycan trait information for quality control, biosimilar comparability studies and biomarker research.

Glycosylation Site Mapping Service

Our glycosylation site mapping service provides advanced resolution by pinpointing glycan modifications to specific residues through LC-MS/MS glycopeptide analysis which enables evaluation of site occupancy and microheterogeneity as well as noncanonical glycosylation events.

FAQs

What sample types are compatible with your Fc glycosylation analysis service?

How do you ensure site-specific analysis of Fc glycans, especially when Fab regions may also carry N-glycosylation?

What distinguishes Creative Biolabs’ Fc glycosylation analysis from other providers?

References:

Sjögren, Jonathan, Rolf Lood, and Andreas Nägeli. "On enzymatic remodeling of IgG glycosylation; unique tools with broad applications." Glycobiology 30.4 (2020): 254-267. Distributed under Open Access license CC BY 4.0, without modification. https://doi.org/10.1093/glycob/cwz085
Bondt, Albert, et al. "Immunoglobulin G (IgG) Fab glycosylation analysis using a new mass spectrometric high-throughput profiling method reveals pregnancy-associated changes." Molecular & Cellular Proteomics 13.11 (2014): 3029-3039. https://doi.org/10.1074/mcp.M114.039537
Wang, Taia T., and Jeffrey V. Ravetch. "Functional diversification of IgGs through Fc glycosylation." The Journal of clinical investigation 129.9 (2019): 3492-3498. https://doi.org/10.1172/JCI130029
Zaytseva, Olga O., et al. "Fc-linked IgG N-glycosylation in FcγR knock-out mice." Frontiers in cell and developmental biology 8 (2020): 67. https://doi.org/10.3389/fcell.2020.00067