Biological drugs, such as monoclonal antibodies (mAbs), have become very important due to their ability to address complex diseases, including various forms of cancer and autoimmune disorders. After translation, they undergo many post-translational modifications (PTMs) that can affect the safety and efficacy of the drug, including secretion rate, effector functions, folding, immunogenicity, solubility, and biological activity. Analytical methods for PTMs are crucial for drug development, for ensuring production consistency and for proving of biosimilarity. Glycosylation, the most common PTM, involves the sequential addition of monosaccharide residues, leading to extreme heterogeneity and the occurrence of mAb isoforms. Various analytical techniques are used for glycosylation analysis, with the most common being hydrophilic interaction liquid chromatography (HILIC), high-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD), capillary electrophoresis (CE), and mass spectrometry (MS). These techniques allow the determination of the structure, composition, and location of glycans on proteins, as well as the sialic acid content. There are many options for sample preparation for analysis, each with its advantages and disadvantages. The complexity of glycosylation presents challenges in separation and characterization, often requiring a combination of several techniques for comprehensive glycan profiling. Additionally, the analyses are expensive and complex, increasing the costs of mAb production. It is expected that future developments in analytical methods and increased automation in the biopharmaceutical industry will improve the consistency, reliability, and cost-efficiency of mAb production.
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