Intact Glycoprotein Profiling Service
The glycan moieties attached to the monoclonal antibodies (mAbs) can directly affect their stability, bioactivity, and immunogenicity. Glycan analysis can profile the glycan structure and monitor the relative quantities of a particular set of glycans to ensure product quality. As a forward-looking company as well as a market leader in the field of antibody glycan analysis, Creative Biolabs has successfully developed a versatile technology platform to illustrate the structure and relative quantities of glycan in mAbs and screen the changes in glycosylation. Intact glycoprotein profiling is a good way to ascertain general glycan patterns and glycan heterogeneity.
Background of Intact Glycoprotein Profiling
Glycosylation is considered a critical quality attribute of therapeutic mAbs, which is vital in the determination of physicochemical properties (solubility, viscosity, protein folding, antigen binding, secretion, antigenicity, etc.) and biological functions [antibody-dependent-cell mediated cytotoxicity (ADCC) and complement-dependent cytotoxicity (CDC)]. Analysis of released glycans and glycopeptides provides information about the composition and/or position of the glycan, while intact glycoprotein analysis offers complementary structural information and allows assignment of individual proteoforms. The analysis of intact glycoproteins is the most straightforward approach to providing a general overview on the glycome, the total glycosylation pattern of glycoproteins, glycolipids, or other types of biomolecules, and then to revealing the complexity of a glycoprotein sample.
With the development in mass spectrometry technology, intact glycoprotein analysis is more advanced, which is fast, robust, does not require enzymatic digestion (preventing undesired modifications induced by enzymatic treatments), and enables high-throughput analysis.
Fig.1 A representative workflow for mass spectrometry-based glycoproteomic analyses.1
Technologies for Intact Glycoprotein Profiling
Intact glycoproteins can be directly analyzed for general glycan patterns and glycan heterogeneity, which is widely used in the analysis of the entire repertoire of glycans attached to proteins. Moreover, it has the diagnostic potential for different stages of cancers and various other disease states, helping to discover new biomarkers. At Creative Biolabs, we can provide diverse technologies to perform intact glycoprotein analysis, including but not limited to:
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ESI-MS
Electrospray ionization mass spectrometry (ESI-MS) is the most common method for intact glycoprotein analysis, which enables the precise mass determination and characterization of intact glycoproteins. The ability of ESI-MS to place multiple charges on particular species enables large intact glycoprotein analysis via conventional mass ranges. Particularly, it has been commonly used coupled with reversed-phase (RP) HPLC or size-exclusion chromatography (SEC). RPLC-ESI-MS provides better chromatographic separation of protein variants and has shown ability in the detection of intact mAb with 10 ppm accuracy, while SEC-MS using non-denaturing or denaturing mobile phase is operated at room temperature and produces good quality of mass spectra. -
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. It can identify the entire set of glycans in small intact proteins.
Fig.2 Glycomics workflow by MALDI-TOF MS. (Jansen, 2016)
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HILIC-MS
Hydrophilic interaction chromatography-mass spectrometry (HILIC-MS) provides detailed glycoform profiles of intact pharmaceutical proteins, including metabolites and peptides. In the context of glycoprotein characterization, HILIC-MS has been widely used for the determination of released glycans and glycopeptides.
Workflow
Proteins are extracted and, if necessary, enriched using proprietary methods to isolate glycoproteins, remove interfering substances, and improve detection sensitivity. The expected outcome is a highly purified glycoprotein fraction ready for analysis.
Depending on the project goals, samples may undergo enzymatic digestion (e.g., with proteases) to generate glycopeptides, or intact glycoproteins are directly subjected to advanced chromatographic separation techniques. This step aims to reduce sample complexity and facilitate subsequent mass spectrometry analysis.
Enriched samples are analyzed using state-of-the-art HR-MS platforms. Our advanced instruments provide highly accurate mass measurements of intact glycoproteins or glycopeptides, enabling the identification of different glycoforms.
Raw MS data is processed using specialized bioinformatics software. This involves deconvolution of spectra, identification of glycan structures, and assignment of glycosylation sites, providing a quantitative profile of each glycoprotein.
We generate comprehensive reports detailing identified glycoproteins, their glycoforms, relative abundances, and site-specific information. Our expert scientists are available for consultation to help interpret findings and guide your subsequent research.
Featured Highlights
Enhanced Accuracy & Biological Relevance
Preserves protein-glycan linkage for a complete, biologically relevant picture, aiding therapeutic protein characterization, biomarker discovery, and disease mechanism elucidation.
High Sensitivity
Detects even low-abundance glycoproteins.
High Throughput
Efficiently processes multiple samples, saving time and resources.
Minimized Data Loss
Offers a complete picture, crucial for critical project decisions.
Customer Reviews
Comprehensive Glycosylation Insight: Creative Biolabs' Intact Glycoprotein Profiling significantly improved our understanding of vaccine antigen glycosylation, revealing key modifications for enhanced immunogenicity—a level of detail previously unattainable.
- (2 months ago), Dr. K*thryn L
Accelerated Therapeutic Protein Development: Their Intact Glycoprotein Profiling rapidly characterized our recombinant enzyme's glycoforms, optimizing stability and activity. The service delivered high-quality data much faster than our in-house capabilities.
- (6 months ago), Prof. M*rk J
Unveiling Disease Biomarkers: Creative Biolabs' Intact Glycoprotein Profiling was instrumental in our biomarker discovery, enabling identification of novel glycan-based signatures in serum glycoproteins for early cancer detection.
- (1 year ago), Dr. S*rah P
FAQs
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Q1: What types of samples are suitable for intact glycoprotein profiling?
A1: Our intact glycoprotein profiling service suits diverse biological samples, including purified proteins (e.g., monoclonal antibodies), cell lysates, and biofluids (e.g., serum) from various expression systems.
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Q2: How does Intact Glycoprotein Profiling compare to traditional glycan analysis methods?
A2: Traditional methods release glycans, losing site-specific and contextual information. Our Intact Glycoprotein Profiling preserves this by directly analyzing the protein-glycan conjugate, offering a more complete and biologically relevant view.
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Q3: What is the typical resolution for glycan identification using your platform?
A3: Our high-resolution mass spectrometry platform offers exceptional resolution, precisely identifying and quantifying subtle glycan variations, enabling differentiation of isoforms and accurate glycosylation site assignment.
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Q4: What precautions should I take when preparing samples for this service?
A4: General precautions include ensuring sample purity, avoiding harsh denaturing agents, and proper storage. We recommend discussing your specific samples with our experts for tailored preparation advice. Inquire for detailed submission instructions!
Why Choose Us
Creative Biolabs stands as your premier partner for Intact Glycoprotein Profiling. Leveraging our unparalleled expertise and advanced technological capabilities, we deliver precise, comprehensive data that profoundly empowers your drug discovery and development efforts. For detailed information, project consultation, or to request a quote, we invite you to reach out to our expert team today.
References
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Tabang, Dylan Nicholas, Megan Ford, and Lingjun Li. "Recent advances in mass spectrometry-based glycomic and glycoproteomic studies of pancreatic diseases." Frontiers in chemistry 9 (2021): 707387.
Distributed under Open Access License CC BY 4.0, without modification. -
Jansen, Bas C., et al. "Pregnancy-associated serum N-glycome changes studied by high-throughput MALDI-TOF-MS." Scientific Reports 6.1 (2016): 23296.
Distributed under Open Access License CC BY 4.0, without modification.
For Research Use Only.