When and Why do we need Glycosylation Analysis of Proteins?

12th July 2011

Category: Bioanalytical / Biosimilars

Tags: ,

By: Dr Daniel Galbraith, CSO

Most, if not all, biologics products start life with little or no “chemistry analysis” of the product. The biological (clinical) function of the molecule ruled supreme and was the most important factor. This is clearly changing as we now recognise the importance of the information provided by chemical analysis and we have the tools to perform such analysis. There is clearly a need to understand the chemical composition since it can affect the clinical outcome of the use of the molecule and also assist in the efficient production of the compounds.

Today, when developing a new biologic, the basic studies carried out during product development such as selecting clones or bioreactor conditions for production, would use mass spectroscopy analysis to ensure the glycan composition is as expected. There are, however, a myriad of Mass spectrometry techniques which are used in such analyses which can be daunting to development scientists without the specialist knowledge of the science.

Mass spectroscopy (MS) analysis could be seen as a ladder which is ascended as necessary during the product development process and moving to clinical evaluation of the molecule. As mentioned previously MS techniques, such as GC-MS, will easily quantify the level of each monosaccharide found in a new compound and thereby the glycosylation type found. This is particularly important as there are a few sugars which are known to be critical for functionality of a compound. Where such analyses  would prove useful would be in the development of media used in the growth of cells for production or even earlier in the selection of clones as this method is fairly simple, rapid and requires little detailed analysis or interpretation.

The next rung on the ladder of analysis may be seen as determining the profile of any N-and/or O-glycans present on the sample. This is used often to determine the consistency of batch production of the product and to provide an overall characterisation of the product. Pulsed amperometric detection (HPAEC-PAD) can be seen as one of the key techniques for this type of analysis. Matrix Assisted Laser Desorption Ionization (MALDI) carried out on released and chemically derivatized (permethylated) glycans provides compositional information on the structures present. At the engineering or consistency batch level this technique would be applied providing assurance of the stability of the process and also helping with the evaluation of process changes.

For manufacturers making biosimilar products or manufacturers who require further analyses may be required to pursue more complex (and expensive) analytical techniques to fully elucidate the chemical analysis of the product. Most commonly this will determine the structural composition of the molecule. The previously discussed techniques will give us the outline of the picture of the compound of interest but to determine the full structures of the carbohydrates and how these link together requires a technique such as Electrospray Mass Spectroscopy. This interesting method ionises the sugars from the non-reducing end of the product which can then be used to determine the sugar composition. In some cases the terminal sugar residue can be critical to the biological function of the molecule.

The last rung of the ladder of analyses commonly followed is a determination of the linkages between the monosaccharides, which essentially is which carbon atom provides the link to the next sugar. The importance of these linkages cannot be down played as they have been shown to be linked not only to biological function but also to the critical aspect of immunogenicity of the molecule. Mass spectrometry is the analytical tool which we can use for this but the molecule must be prepared prior to analysis by multi-step chemical hydrolysis and derivatization process.

These techniques in part or together are clearly exciting and useful in a number of ways but caution should be applied in most if not all instances. The technology and analytical software available mean that this analysis is readily available to most manufacturers either within their own facility or using a trusted CRO partner. The interpretation of the huge amount of data provided by these techniques is where the excitement can falter. We are still at a very early stage of knowing how these carbohydrate molecules vary in products such as monoclonal antibodies and what these changes mean to the product in relation to clinical efficacy and side effects. Discovering differences or similarities between molecules at a micro or macro level is a simple method today – it’s almost at the flick of a switch. However, releasing this knowledge to allow critical and sensible decisions to be made of the fate or a product or a batch of manufactured product is a much more difficult exercise which few if any companies have truly mastered today.

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