What types of samples can I submit for Glycan Analysis?

We accept a wide range of samples, including purified glycoproteins, monoclonal antibodies, serum, plasma, cell lines, and tissues. The specific sample volume and preparation requirements may vary depending on the chosen analytical technique, so we always recommend discussing your project with our experts for detailed guidance.

How does Glycan Analysis differ from general protein characterization, and why is it so critical?

While general protein characterization focuses on amino acid sequence and basic structural integrity, Glycan Analysis specifically delves into the complex sugar modifications attached to proteins. These glycans are non-template driven, meaning they're not directly coded by genes, yet they are crucial for a protein's proper folding, stability, biological activity, and interaction with other molecules. For biopharmaceuticals, precise glycan profiling is absolutely critical to ensure optimal efficacy, safety, and consistent quality, especially when compared to protein-only characterization.

Can your service help identify specific glycan biomarkers for disease?

Absolutely. Aberrant glycosylation is a hallmark of many diseases. Our advanced Glycan Analysis can profile altered glycan patterns on cell surfaces or in biofluids, helping to identify disease-specific glycan biomarkers or changes that correlate with disease progression or therapeutic response. This supports diagnostic development and more targeted therapeutic strategies.

Glycan Analysis Service

In the face of formidable hurdles in biopharmaceutical development—from prolonged drug development cycles to intricate challenges in protein expression, purification, and clinical trials—Creative Biolabs delivers advanced Glycan Analysis services. We empower you to significantly accelerate drug discovery, secure high-quality recombinant proteins, and engineer highly specific antibodies by expertly unraveling the complex world of glycans through cutting-edge high-throughput screening platforms and innovative analytical techniques.

The Critical Role of Glycosylation in Biopharmaceutical Development and Disease

Glycosylation, the enzymatic addition of glycans (sugar chains) to proteins and lipids, is one of the most common and crucial post-translational modifications. These modifications profoundly impact the function, efficacy, stability, pharmacokinetics, and immunogenicity of therapeutic proteins, including monoclonal antibodies (mAbs) and other biopharmaceuticals. Analyzing these complex "sugar codes" is essential for understanding biological processes, developing effective drugs, and ensuring product safety. Aberrant glycosylation patterns are also linked to various diseases, including cancer and autoimmune disorders, making precise glycan analysis vital for biomarker discovery and diagnostic development. Without detailed glycan profiling, the full potential and safety profile of many advanced therapeutics, including those potentially interacting with gene therapy products, cannot be fully realized.

Fig.1 Workflow of IgG Fab and Fc glycosylation analysis. (OA Literature) Fig.1 Analyzing IgG Fab and Fc glycosylation workflow.¹

Glycan Analysis at Creative Biolabs

Addressing the complexities and variability of glycosylation, a critical challenge in biopharmaceutical development, Creative Biolabs' Glycan Analysis service offers a robust and comprehensive solution for characterizing these intricate glycan structures essential to your projects. Adhering strictly to the highest industry standards and employing high-sensitivity, high-specificity testing principles, our service provides specific deliverables, clear solutions, and key problem-solving capabilities. From meticulous sample preparation and advanced high-resolution analysis to in-depth bioinformatic interpretation, our streamlined process typically delivers results within 3 to 8 weeks, ensuring unparalleled quality, accuracy, and reproducibility to optimize your drug development pipeline.

N-Glycan Analysis
N-linked oligosaccharide is covalently bonded with the nitrogen of an asparagine residue, and all N-linked proteins share a basic core structure of conserved pentasaccharide (GlcNAc2Man3) backbone. For immunoglobulins, N-linked oligosaccharides have received particular attention because changes in the attached glycans can impact immunoglobulin solubility, structural stability, and biological functions. For example, the differential of a single monosaccharide at Asn-297 glyco-site at the Fc fragment of human IgG can drastically affect IgG binding to FcγR and complement action. Technologies for N-Glycan analysis provided by Creative Biolabs, including but not limited to: HPAEC-PAD High-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) is a selective and specific glycan analysis method that separates carbohydrates with specific interactions between the hydroxyl and carboxyl groups of glycans. Generally, samples are acid hydrolyzed by peptide-N-glucosidase F (PNGaseF) to release the N-linked glycans and then analyzed by HPAEC-PAD. It has been a powerful tool to analyze the total glycosylation of a protein, the types of glycans, the amounts of specific monosaccharides, and the carbohydrate sequence of a glycan. Moreover, HPAEC-PAD can detect the aberrant differences in glycan patterns that may indicate disease conditions. The qualitative and quantitative estimation values are standardized by known N-linked glycans. HPLC High-performance liquid chromatograph (HPLC) is also a common method for the characterization of antibody glycan. HPLC is usually used in conjunction with different detection methods, such as fluorescence-based detectors (HPLC-FD) or mass spectrometers (HPLC-MS). Before HPLC-FD analysis, glycans must be labeled with some fluorescent tags [usually 2-aminobenzamide (2-AB) or 2-aminobenzoic acid (2-AA)]. Highly efficient hydrophilic interaction chromatography (HILIC) is the most widely used chromatography column for glycan analysis. The qualitative and quantitative values are standardized by known N-linked glycans. Mass Spectrometry (MS) Mass spectrometry is a versatile technique that has increasingly been used in structural elucidation of glycans due to its sensitivity, potential for high throughput, and the capability for tolerating diverse biological mixtures. Separation techniques such as liquid chromatography (LC), ion mobility are typically used in conjunction with mass spectrometry to analyze the antibody monosaccharide with more accuracy.
O-Glycan Analysis
O-acetylgalactosamine (O-GalNAc) and O-acetylglucosamine (O-GlcNAc) are the common O-glycosylation types, among which, O-GalNAc is attached to the hydroxyl group of the protein serine or threonine residues through an α-linkage, while O-GlcNAc is attached through a β-linkage. Technologies for O-Glycan analysis provided by Creative Biolabs, including but not limited to: MALDI-TOF MS Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a high-throughput method to analyze the structural characterization of glycosylated compounds with high sensitivity and robustness. Before O-glycan profiling, the permethylation of the O-glycan to transform all hydroxyl groups into methyl ethers and stabilize sialic acids by methyl esterification of their carboxyl groups is necessary, which allows MALDI-TOF MS to analyze all O-glycans in the positive mode. HPAEC-PAD HPAEC-PAD can also provide the characterization of O-linked glycans, but unlike N-Glycan analysis, O-linked glycans can be cleaved from the proteins using alkaline sodium borohydride (NaBH4) in a β-elimination reaction. Followed by the release from the proteins, O-linked glycans can be analyzed to illustrate the total glycosylation, the types of glycans, the amounts of specific monosaccharides, and the carbohydrate sequence of a glycan.

N-Glycan Analysis

N-linked oligosaccharide is covalently bonded with the nitrogen of an asparagine residue, and all N-linked proteins share a basic core structure of conserved pentasaccharide (GlcNAc2Man3) backbone. For immunoglobulins, N-linked oligosaccharides have received particular attention because changes in the attached glycans can impact immunoglobulin solubility, structural stability, and biological functions. For example, the differential of a single monosaccharide at Asn-297 glyco-site at the Fc fragment of human IgG can drastically affect IgG binding to FcγR and complement action. Technologies for N-Glycan analysis provided by Creative Biolabs, including but not limited to:

HPAEC-PAD

High-performance anion exchange chromatography coupled with pulsed amperometric detection (HPAEC-PAD) is a selective and specific glycan analysis method that separates carbohydrates with specific interactions between the hydroxyl and carboxyl groups of glycans. Generally, samples are acid hydrolyzed by peptide-N-glucosidase F (PNGaseF) to release the N-linked glycans and then analyzed by HPAEC-PAD. It has been a powerful tool to analyze the total glycosylation of a protein, the types of glycans, the amounts of specific monosaccharides, and the carbohydrate sequence of a glycan. Moreover, HPAEC-PAD can detect the aberrant differences in glycan patterns that may indicate disease conditions. The qualitative and quantitative estimation values are standardized by known N-linked glycans.

HPLC

High-performance liquid chromatograph (HPLC) is also a common method for the characterization of antibody glycan. HPLC is usually used in conjunction with different detection methods, such as fluorescence-based detectors (HPLC-FD) or mass spectrometers (HPLC-MS). Before HPLC-FD analysis, glycans must be labeled with some fluorescent tags [usually 2-aminobenzamide (2-AB) or 2-aminobenzoic acid (2-AA)]. Highly efficient hydrophilic interaction chromatography (HILIC) is the most widely used chromatography column for glycan analysis. The qualitative and quantitative values are standardized by known N-linked glycans.

Mass Spectrometry (MS)

Mass spectrometry is a versatile technique that has increasingly been used in structural elucidation of glycans due to its sensitivity, potential for high throughput, and the capability for tolerating diverse biological mixtures. Separation techniques such as liquid chromatography (LC), ion mobility are typically used in conjunction with mass spectrometry to analyze the antibody monosaccharide with more accuracy.

O-Glycan Analysis

O-acetylgalactosamine (O-GalNAc) and O-acetylglucosamine (O-GlcNAc) are the common O-glycosylation types, among which, O-GalNAc is attached to the hydroxyl group of the protein serine or threonine residues through an α-linkage, while O-GlcNAc is attached through a β-linkage. Technologies for O-Glycan analysis provided by Creative Biolabs, including but not limited to:

MALDI-TOF MS

Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) is a high-throughput method to analyze the structural characterization of glycosylated compounds with high sensitivity and robustness. Before O-glycan profiling, the permethylation of the O-glycan to transform all hydroxyl groups into methyl ethers and stabilize sialic acids by methyl esterification of their carboxyl groups is necessary, which allows MALDI-TOF MS to analyze all O-glycans in the positive mode.

HPAEC-PAD

HPAEC-PAD can also provide the characterization of O-linked glycans, but unlike N-Glycan analysis, O-linked glycans can be cleaved from the proteins using alkaline sodium borohydride (NaBH4) in a β-elimination reaction. Followed by the release from the proteins, O-linked glycans can be analyzed to illustrate the total glycosylation, the types of glycans, the amounts of specific monosaccharides, and the carbohydrate sequence of a glycan.

Required Starting Materials

Purified Glycoprotein Samples: Preferably ≥ 20 μg of purified IgG or other glycoproteins, in PBS buffer, avoiding high glycerol or detergent concentrations.
Biological Samples: Such as ≥ 5 μL of serum/plasma (human/mouse) or ≥ 100 μL of hybridoma supernatant.
Cell Lines/Tissues: For specific cell-surface glycan or tissue glycoprofiling projects.

Our Glycan Analysis Workflow

Step1. Sample Preparation & Glycan Release

Samples are prepared, denatured, and glycans enzymatically released (e.g., N-glycans with PNGase F; O-glycans via β-elimination) to preserve integrity.

Step2. Glycan Labeling (Optional)

Released glycans can be fluorescently labeled for enhanced detection.

Step3. Glycan Purification & Enrichment

Techniques like HILIC or SPE enrich and desalt glycans, removing contaminants for high-resolution analysis.

Step4. High-Resolution Glycan Analysis

Advanced platforms (HPAEC-PAD, HPLC-FLD/MS, MALDI-TOF MS, LC-MS/MS) provide detailed structural elucidation and quantitative profiling.

Step5. Data Acquisition & Bioinformatic Analysis

Raw data is processed via bioinformatic analysis, including automated scripts, comparative plots, and heatmaps, for glycoform identification and quantification.

Service Advantages

High Sensitivity & Specificity: Detects even low-abundance glycans with unparalleled precision.
Comprehensive Structural Elucidation: Provides detailed information on glycan composition, linkage, and branching.
Quantitative Profiling: Accurately quantifies different glycoforms, enabling robust comparative studies.
High Throughput & Speed: Optimized workflows for rapid analysis of multiple samples, reducing project timelines.
Reduced Sample Consumption: Designed for micro-scale input, preserving your valuable biological materials.

Customer Reviews

"The precision and detail using Creative Biolabs' Glycan Analysis in our mAb research has significantly improved our understanding of immunogenicity, directly guiding our lead candidate selection."

— 2 months ago, An***rew M.

"We found the high-throughput capability of Creative Biolabs' Glycan Analysis invaluable for screening our glycoengineered cell lines, greatly facilitating our bioproduction optimization efforts."

— 4 weeks ago, Dr. S***ara J.

"Creative Biolabs' team provided invaluable insights into O-glycan structures, which were crucial for validating the stability and activity of our therapeutic glycoprotein. Their expertise truly made a difference."

— 3 months ago, Pr***sor L.

FAQs

Q1: What types of samples can I submit for Glycan Analysis?

A1: We accept a wide range of samples, including purified glycoproteins, monoclonal antibodies, serum, plasma, cell lines, and tissues. The specific sample volume and preparation requirements may vary depending on the chosen analytical technique, so we always recommend discussing your project with our experts for detailed guidance.
Q2: How does Glycan Analysis differ from general protein characterization, and why is it so critical?

A2: While general protein characterization focuses on amino acid sequence and basic structural integrity, Glycan Analysis specifically delves into the complex sugar modifications attached to proteins. These glycans are non-template driven, meaning they're not directly coded by genes, yet they are crucial for a protein's proper folding, stability, biological activity, and interaction with other molecules. For biopharmaceuticals, precise glycan profiling is absolutely critical to ensure optimal efficacy, safety, and consistent quality, especially when compared to protein-only characterization.
Q3: Can your service help identify specific glycan biomarkers for disease?

A3: Absolutely. Aberrant glycosylation is a hallmark of many diseases. Our advanced Glycan Analysis can profile altered glycan patterns on cell surfaces or in biofluids, helping to identify disease-specific glycan biomarkers or changes that correlate with disease progression or therapeutic response. This supports diagnostic development and more targeted therapeutic strategies.

Why Choose Us

Creative Biolabs is your trusted partner for cutting-edge Glycan Analysis, driving innovation in biopharmaceutical research and development. Our comprehensive services, backed by extensive expertise and advanced platforms, provide the detailed insights you need to understand, characterize, and optimize the glycosylation of your therapeutic molecules. Please feel free to get in touch with us.

Reference

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: MCP vol. 13,11 (2014): 3029-39. doi:10.1074/mcp.M114.039537. Distributed under an Open Access License CC BY 4.0, without modification.

For Research Use Only.

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