Protein Drug Glycosylation Analysis Service

Glycosylation is one of the most common and complex forms of post-translational modification in proteins, widely present in therapeutic protein drugs. Studies have shown that glycosylation significantly influences protein conformation, thermal stability, and in vivo half-life, as well as modulates immunogenicity, bioactivity, and pharmacokinetics. For biologics such as monoclonal antibodies, fusion proteins, and recombinant vaccines, glycosylation has become a critical quality attribute (CQA).

 



Rocamora, F. et al. Biotechnol Adv. 2023.

Figure 1. Most Protein-Based Drugs Undergo N-Linked or O-Linked Glycosylation

 

Throughout various stages of drug development—including discovery, process optimization, quality consistency evaluation, biosimilarity assessment, and regulatory filing—glycosylation analysis plays a vital role. To meet both regulatory and R&D needs, MtoZ Biolabs offers mass spectrometry-based Protein Drug Glycosylation Analysis Service, supporting comprehensive characterization of glycosylation profiles to enhance product quality and development efficiency.

 

Technical Principle

Protein glycosylation mainly includes two types: N-glycosylation and O-glycosylation. N-glycans typically attach to asparagine (Asn) residues within the consensus sequence (Asn-X-Ser/Thr), and are highly conserved. O-glycans are usually linked to serine (Ser) or threonine (Thr), exhibiting greater structural diversity and complexity. Glycan structures are classified into high-mannose, hybrid, and complex types. Variations in glycan composition and terminal modifications (e.g., sialylation, fucosylation) can significantly impact drug efficacy and immune response.

 



Wu, L. et al. ACS Omega. 2023.

Figure 2. N-Glycosylation Types and O-Glycosylation Types of Glycoproteins

 

MtoZ Biolabs utilizes high-resolution mass spectrometry (e.g., Orbitrap series) in combination with advanced separation techniques such as HILIC, PGC, and RP-LC. Coupled with optimized data processing algorithms, our platform enables tiered and traceable analysis of protein glycosylation.

 

Analysis Workflow

MtoZ Biolabs’ Protein Drug Glycosylation Analysis Service typically includes the following steps:

1. Sample Preparation

Protein quantification, buffer exchange, reduction and alkylation are performed to ensure sample compatibility with mass spectrometry.

 

2. Glycan Release and Labeling

PNGase F or O-glycosidases are used to enzymatically release glycans. Optional fluorescent labeling (e.g., 2-AB, 2-AA) enhances detection sensitivity.

 

3. Released Glycan Analysis (Glycan Profiling)

Glycan composition, distribution, and terminal modifications (such as sialic acid) are profiled using HILIC-UPLC and MALDI-TOF-MS.

 

4. Glycopeptide Analysis

Enzymatic digestion followed by LC-MS/MS enables identification and semi-quantitative analysis of glycosylation sites and glycan heterogeneity.

 

5. Intact Glycoprotein Analysis

Intact mass analysis is used to evaluate overall glycosylation profiles, suitable for assessing structural consistency and batch comparison.

 

6. Data Interpretation and Reporting

Specialized software is used for spectral annotation, structural interpretation, and visual presentation. Clients receive standardized and interpretable reports.

 



Ohyama, Y. et al. Expert Rev Proteomics. 2020. 

Figure 3. The Overall Strategy for the Analysis of Protein Glycosylation

 

Service Advantages

1. End-to-End Glycosylation Analysis

Covers released glycans, glycopeptides, and intact glycoprotein levels, delivering a comprehensive view of glycosylation features.

 

2. High Sensitivity and Established Methodology

Based on high-resolution Orbitrap platforms, optimized enrichment protocols, and chromatography conditions, enabling accurate detection of low-abundance glycoforms.

 

3. Standardized Workflow Ensures Data Quality

Validated protocols and robust quality control measures ensure reproducibility and comparability across samples and platforms.

 

4. Flexible Solutions Across Development Stages

Tailored analysis plans support early candidate screening, process development, batch-to-batch variation analysis, and post-marketing quality monitoring.

 

Applications

1. Glycoform screening and optimization in early-stage drug discovery

2. Glycosylation evaluation in different expression systems during process development

3. Quality consistency studies across multiple production batches

4. Glyco-comparability assessment between biosimilars and reference products

5. Monitoring glycosylation shifts in stability studies

 

Case Study

1. Decoding Protein Glycosylation by an Integrative Mass Spectrometry-Based De Novo Sequencing Strategy

This study presents an integrated mass spectrometry-based de novo sequencing strategy that combines enzymatic removal of N- and O-glycans with EThcD fragmentation to achieve high sequence coverage and precise glycosylation site identification. The method was successfully applied to sequence the highly glycosylated therapeutic fusion protein Etanercept and several TNFR-Fc fusion biologics with previously unknown sequences. It enabled simultaneous characterization of N- and O-glycosylation at the subunit, glycopeptide, and glycan levels. This approach effectively bridges the gap between primary structure determination and glycosylation profiling, offering a powerful tool for comprehensive glycoprotein analysis. Protein Drug Glycosylation Analysis Service employs multi-level mass spectrometry techniques to characterize glycosylation features of protein drugs, providing complete information on backbone sequence, glycosylation sites, and glycan structures. It supports in-depth analysis and structural comparison of highly glycosylated proteins with high accuracy and resolution.

 



Gao, J. et al. JACS Au. 2025.

Figure 4. De Novo Sequencing of TNFR: Fc-Fusion Biopharmaceuticals

 

2. Glycan Analysis for Protein Therapeutics

This review highlights the role of glycosylation as a critical quality attribute influencing the safety and efficacy of protein therapeutics. It discusses the impact of glycosylation on protein stability and activity, and emphasizes its importance throughout drug development—from early-stage candidate selection to regulatory submission and biosimilarity evaluation. The article focuses on mass spectrometry-compatible separation techniques for glycan characterization, including hydrophilic interaction liquid chromatography (HILIC), reversed-phase liquid chromatography, capillary electrophoresis, porous graphitic carbon chromatography, and two-dimensional liquid chromatography. Advances, challenges, and limitations of these methods are discussed across different analytical levels: released glycans, glycopeptides, glycoprotein subunits, and intact glycoproteins. Protein Drug Glycosylation Analysis Service utilizes a range of separation and mass spectrometry techniques to comprehensively analyze glycosylation features of protein therapeutics. It enables structural evaluation across multiple levels, including released glycans, glycopeptides, and intact glycoproteins, and supports various stages of glycan profiling and structural consistency assessment.

 



Yang X, et al. J Chromatogr B Analyt Technol Biomed Life Sci. 2019

Figure 5. Middle-Up HILIC-MS Analysis of Cetuximab (Erbitux) and Cetuximab

 

FAQ

Q1: What types of proteins are suitable for glycosylation analysis at MtoZ Biolabs?

A1: Our service is compatible with a wide range of biologics, including recombinant proteins, monoclonal antibodies, Fc-fusion proteins, and vaccine antigens.

 

Q2: Can both N-glycans and O-glycans be analyzed simultaneously?

A2: Yes, we offer comprehensive analysis of both N- and O-glycosylation, including glycan release, structural profiling, and site-specific analysis.

 

Q3: Is it possible to detect low-abundance glycoforms?

A3: Yes. Our platform provides high sensitivity and is well-suited for detecting low-abundance glycans in complex backgrounds.

 

Q4: Do you offer structural consistency comparison services?

A4: Yes. We provide glycan comparability reports for cross-batch or cross-product evaluation to support consistency studies.

 

Q5: Are there specific sample requirements?

A5: We recommend providing protein samples with >90% purity and minimal interference from salts or detergents. Our team can provide additional guidance for sample preparation.

 

For more information or to request a customized glycosylation analysis plan, please contact the technical team at MtoZ Biolabs. We are committed to delivering high-quality Protein Drug Glycosylation Analysis Service to support biologics development and product lifecycle management.