Antibodies used in biopharmaceuticals are usually modified with glycans. The glycan is a molecule with high structural heterogeneity, composed of complex coupled monosaccharides (such as glucose and mannose), and it is known that the complex structure plays an important role in the function regulation of proteins. Since mAbs are heterogeneous molecules in nature, it is very important to clarify the distribution and composition of various glycans when antibodies are biosynthesized as biopharmaceuticals. Mass spectrometers are now commonly used to determine binding sites and glycan structures. At Creative Biolabs, we use high-resolution MALDI-TOF MS with unique high-energy fragmentation capabilities to determine the structure and binding sites of glycopeptides bound to antibodies.
First, use a commercially available monoclonal antibody as a sample, and perform reduction and alkylation in a solution. Then, trypsin was added to the solution, and the enzymatic digestion was continued for 2 hours. Then the enzyme digested solution was added to the spin column containing Sepharose CL4B gel. The gel was equilibrated with butanol: ethanol: water (4:1:1) in advance, and then the solution was allowed to interact with the gel. The glycopeptide is adsorbed to the gel. Then, the non-glycosylated peptide is removed by washing with an equilibrium solution, and the glycopeptide is recovered using an aqueous ethanol solution. Using 2,5-dihydroxybenzoic acid (DHB) as a matrix, the recovered glycopeptides were spotted on the target plate and analyzed by MS.
The mass spectrum of the recovered glycopeptide portion is shown in Figure 1. Among the detected signals, m/z 2405.72, 2430.75, 2567.72, 2592.73, 2635.74, 2795.74 and 2957.74 were considered as glycopeptide-derived signals. Because glycans have a non-uniform structure, there are multiple different glycan structures in a peptide backbone. Since the signals in MADLI-TOF MS are related to monovalent ions, the mass differences between the peaks can be viewed. If they match the mass of the glycan moiety, these peaks are most likely glycopeptides.
Figure 1: The mass spectrum of the glycopeptide derived from the monoclonal IgG glycopeptide signal is represented by a red circle.
With extensive experience in glycan characterization, our team can work on custom solutions to meet the unique requirements of each product. With flexible global capabilities, you can provide services where you need it most. Contact our expert scientists today to discuss how our range of glycan analytical services can improve your unique process.
Creative Biolabs provides luciferase-based ADCC assay. This Jurkat cell based assay is pioneered by Creative Biolabs, and the methodology is very well accepted by the field. See attached ADCC Reporter Assay Protocol for further details.
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