In contrast to small molecules, which can be totally described with respect to their molecular structure, it is a challenge to characterise complex glycosylated proteins. Moreover, the production of recombinant proteins using mammalian cell cultures yield proteins with a mixture of glycan structures known as microheterogeneity. While some of the heterogeneity is intrinsic and of no clinical significance some heterogeneity may have an influence on the safety and/or efficacy profile of the respective therapeutic protein.
The ability to generate proteins bearing site-specific modifications like additional glycan structures has a tremendous potential for advancing biotechnology-derived proteins, as demonstrated by the creation of the second-generation anti-anaemia drug darbepoetin with new and improved therapeutic qualities.
Particularly with regard to safety the analysis of the carbohydrate structures is an important issue in the approval process. Furthermore, for the demonstration of batch to batch consistency it is necessary to evaluate the impact of glycan varieties between essentially identical molecules. Inconsistencies during any step of the process can lead to inter-batch variations and the slightest changes may have significant clinical implications.
The increasing number of marketing authorisation applications for biosimilars will pose a further challenge for the regulatory bodies. The complexity of these products, differences in the cell lines and/or in the manufacturing process must be taken into account when evaluating such marketing authorisation applications. The identification and characterisation of the proteins glycan profile including site occupancy, type of linkage, composition, antennarity, sialylation and absence of immunogenic structures is therefore indispensable for their evaluation.