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Glycoprofiling of antibodies – what, how and why?

15 October 2020

Proteins go through various post-translational modifications (PTM) while produced by the cells. Glycosylation is one of the most common PTM and it can have tremendous effects on the physicochemical properties, structure, and function of proteins. Failures in the physiological glycosylation process can lead to serious health conditions, which describes its importance. This blog post elucidates why it is crucial to know and characterise glycoprofile of glycoprotein-based biologics and how this can be done.

Glycosylation means in practise that glycans (i.e. carbohydrates) are covalently bound to protein structure at specific amino acids. As you might be aware, in contrast to small molecules, biological drugs are produced by cells and glycosylation process is affected by several factors. Therefore, batch-to-batch variability is a specific challenge for biologics and need to be carefully monitored. There are a good number of possible glycoprofile variations, since glycans can vary from both their structure and the site of glycosylation in the protein. Glycoprofile can affect the ADME properties and biosimilarity of the glycoprotein-based drugs, e.g. by altering PK profile and receptor binding. Therefore, thorough glycoprofile characterisation is important during development and later in quality control.

Due to the complicated structure of glycoproteins, complementary techniques are required for their characterisation. Intact glycoprotein can be analysed with MS to obtain molecular weight and primary structure, including pattern of major glycoforms. In case of antibodies, the light and heavy chains of the antibody can be separated to facilitate more precise identification of the PTMs and major glycoforms. With peptide mapping approach, the site of the glycosylation can be identified. N-glycans are bound to the protein through asparagine side chain and O-glycans either through serine or threonine residues. To monitor and characterise glycans the parent protein can be deglycosylated, either enzymatically or chemically, and the released glycans can be analysed. As a benefit, analysis of released glycans does not only provide more detailed information but is also more straightforward approach in comparison to the analysis of an intact glycoprotein.

The regulatory bodies highlight the importance of characterising the carbohydrate content and glycan structures of glycoprotein-based biologics. Monoclonal antibodies (mAb) are one of the most common type of glycoproteins already on the market and being under development. mAbs typically contain N-glycosylation site on both Fc regions and additional ones may also exist.

Since mAbs contain N-glycans, a common applied approach to investigate their glycoprofile is to release N-glycans from the denaturated protein enzymatically by PNGaseF. To improve the detection and analytical sensitivity, the released N-glycans are typically fluorophore labelled. The labelled N-glycans are separated using chromatography and detected based on the sensitive fluorescence signal. Glycan structures are identified using a glycan library and the obtained retention times. Especially, if MS compatible solvents and labels are used and MS is connected on-line to an LC-instrument the glycan identification can be confirmed with MS, which increases assurance.

I hope this gave you an overview about the options how to characterise glycoproteins. However, as each project is unique with its own requirements the optimal way for high quality and extensive characterisation should be tailored individually for each compound. If you are wondering from where to start, there are experts on the field available for building a suitable approach for your project together with you. Perhaps it would be a fruitful solution to contact an expert without hesitation for advancing your project?

Written by Miia Kovalainen