Therapeutic monoclonal antibodies (mAbs) have been increasingly used for the treatment of a number of diseases, due to their high specificity and affinity to antigen targets. As a manufactured therapeutic protein, a mAb is subjected to many modifications throughout the manufacturing process, which may result in structural changes, ultimately leading to potential biological function changes. Glycation is one of the chemical modifications widely observed and critically controlled in the production and manufacturing of mAb therapeutics. Here, Creative Biolabs presents mAb glycan analysis as part of our integrated antibody structure analytical services to global clients.
Glycation refers to the non-enzymatic addition of a monosaccharide to an amino acid residue via the Maillard reaction. It occurs when an unstable Schiff base intermediate is formed via condensation of a reducing sugar carbonyl group with a free amine group (e.g., protein N-terminus, ε-amino group of Lys or Arg) and then rearranges to form a ketoamine derivative known as an Amadori product. Protein glycation reactions can occur to recombinant mAbs during different stages of their development. Firstly, mAb glycation occurs during the fermentation process by exposing to reducing sugars that are used as an energy source for mAb-producing cells. Secondly, during the storage of therapeutic mAbs, glycation can be introduced by reducing sugars in the formulation, even in the solid state after lyophilization. Thirdly, glycation of antibodies also occurs during in vivo circulation. The level of therapeutic mAb glycation can be affected by various factors, including glucose feed strategies, temperature, pH, time, etc.
Fig.1 Glycation mechanism.
Glycation can occur during cell culture in manufacturing, storage upon shelf life, clinical administration, and circulation in the human body. Differences in these process conditions can generate structural heterogeneity in mAb glycation. Moreover, glycation has been reported to impact mAb properties, such as blocking the biologically functional site, or further degradation that induces aggregation. In this regard, characterization and control of glycation modification are of great importance to ensure product quality as well as manufacturing process consistency for recombinant mAbs in the biopharmaceutical industry.
Fig.2 Boronate affinity chromatograms of untreated and forced-glycated antibody samples. (Wei, 2017)
As an expert antibody service provider, Creative Biolabs offers comprehensive studies to characterize the structural variants of therapeutic mAb candidates during different stages. Besides, we have developed a wide range of antibody function assays that can be applied to help understand the structure and function relationships of glycation in mAbs. Moreover, forced glycation of mAbs can be conducted to detect susceptible sites for glycation in conditions such as the high concentration of reducing sugars, elevated temperatures, and low pH conditions. The characterization and analysis of glycation can be performed using charge-based techniques, boronate affinity chromatography (BAC), or mass spectrometry (MS)-based techniques.
Besides glycation, Creative Biolabs also offers analytical services of other mAb modifications, including glycosylation, C-terminal lysine variant, N-terminal cyclization, deamidation and isomerization, oxidation, etc. If you are interested in our services, please do not hesitate to contact us for more details.
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