Post-translational modifications of proteins encompass a wide variety of modifications, including glycosylation, oxidation, phosphorylation, sulphation, lipidation, disulphide bond formation and deamidation. Mass spectrometry (MS) has become the tool of choice for detecting and investigating these modifications.
In some cases, nuclear magnetic resonance spectroscopy (NMR) can also be useful. Indeed, it was NMR that identified the recently publicized issue of heparin contaminated with O-linked glycans.
The so-called higher-order structure of a protein gives it its unique 3-D shape and thus contributes to its functions. Subtle differences in such higher-order structures might explain observed biological/immunological differences between otherwise identical proteins and also serve as a basis for comparison of reference products with FOBs.
Myriad classical biophysical techniques are used to characterize higher-order structures, including circular dichroism, fluorescence, differential scanning calorimetry, isothermal calorimetry, analytical ultracentrifugation, and size-exclusion chromatography. Detecting subtle changes requires use of additional techniques such as NMR, x-ray crystallography, and MS.
Formation of undesirable protein aggregates represents a substantial problem for biopharmaceuticals. Aggregates can display adverse toxicological and immunological profiles, in addition to having an obvious detrimental impact on dosage. Characterizing aggregates is a complex undertaking. Among the numerous methods employed are size-exclusion chromatography, analytical ultracentrifugation, and asymmetric flow field flow fractionation. Detection of sub-visible particles present at very low concentration requires techniques such as dynamic or static light scattering.
Sophisticated analytical protein-characterization methods will likely have an impact, not only in establishing the similarity of a FOB to its reference product, but also in accounting for potential adverse clinical events. For example, a number of FOB versions of a human erythropoietin reference product (Eprex®) have been approved by the European Medicines Agency (EMA), and while these versions were deemed by EMA to be comparable in quality, safety, and efficacy to Eprex, clear differences in structure have been documented by analytical methods.
If any of these erythropoietin FOBs is, in the future, associated with adverse clinical events, these structural differences—and the fact that they were known in advance—may well play a role in potential litigation related to the adverse events. This, in turn, may influence similarity standards applied to FOBs by FDA and other regulatory agencies.