Creative Biolabs has years of experience in the field of antibody carbohydrate structure analysis and can execute a set of analyses. This covers not only the analysis of the total population of glycans on an antibody but also the structure of glycans at individual antibody glycosylation sites in the molecule. Our experts apply their experience to design strategic packages pertinent to your actual need and phase of development from preclinical-phase characterization studies.
Importance of Analyzing Carbohydrate Structure
For antibodies, the carbohydrate is an important part of the molecules, which has shown to affect the stability and bioactivity of antibodies. It is vital to generate structural information on carbohydrate part since a detailed knowledge of product structure is imperative for any antibody drug to be brought to market. Besides, comparability of the glycan profiles across various batches will prove that your manufacturing process is under control and for this reason, the regulatory authorities will request monosaccharide composition analysis data such as levels of fucose, mannose, galactose and N-linked (and O-linked if applicable) structural data on glycosylation from various batches.
Fig.1 Schematic diagram of carbohydrate position on an antibody.
We apply a range of techniques to obtain a complete overview of the carbohydrate structure in your antibody, including high resolution mass spectrometry (MS), high performance liquid chromatography (HPLC), electrophoretic methods and so on. A popular strategy is shown below.
Fig.2 Analytical strategy.
You can choose from the following types of analysis:
Since many antibodies are N-linked glycoproteins, a comprehensive analysis of antibody’s structure should include a detailed look at the N-glycan structure. To determine the structure of N-glycans, we will use matrix-assisted laser desorption ionization (MALDI) or liquid chromatography-electrospray ionization-tandem MS (LC-ESI-MS/MS) after labelling or permethylation. But to determine the antennary of glycans, or to quantify glycans, we’ll use high performance anion exchange chromatography with pulsed amperometric detection (HPAEC-PAD). Being a typical HPLC method, it has the advantage that you then can easily validate the assay for release testing. The Normal-Phase HPLC (NP-HPLC) complements this setup by providing an additional, highly sensitive and high-resolution analysis of your samples.
O-linked glycans are usually a lot smaller and can be attached to a much wider range of amino acids, so it’s difficult to pinpoint their exact locations. For determining the structure and heterogeneity of antibody glycosylation, either for the entire antibody or for each potential glycosylation site, we’ll commence this type of research with LC or MS. For the sites of glycan attachment, we’ll identify them by peptide mapping or MALDI-ISD (Matrix-Assisted Laser Desorption Lonization In-Source Decay).
Creative Biolabs also provides detailed glycan composition analysis for global clients.
Creative Biolabs uses Gas Chromatography with Mass Spectrometric detection (GC-MS) to identify and quantitate of the levels of the neutral sugars (commonly Fucose, Mannose and Galactose), amino sugars (N-Acetylglucosamine, N-Acetylgalactosamine) and LC with fluorescence for the detection of sialic acids (N-Acetylneuraminic acid and N-Glycolylneuraminic acid) following DMB labeling.
Antibody oligosaccharide analysis at Creative Biolabs involves the isolation and subsequent analysis of N-glycans and O-glycans using MS and chromatographic analytical processes. These are very powerful tools for the investigation of the complex set of glycan structures present on an antibody. The use of chemical and enzymic procedures for sample work up generates released glycans in a suitable condition for analysis.
Do you need to investigate carbohydrate structure to ensure your antibody structural integrity during drug development? Would you like to see if any molecular alterations have taken place during fermentation, purification or storage?
Creative Biolabs has such an in-depth understanding of carbohydrate structure that we’re already unparalleled in terms of the level of detail, such as rigorous structural information, we can provide. If you are interested in our carbohydrate structure analysis services, please feel free to contact us.
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