Tools for characterization
BioPharm: What techniques are used to compare the structure of biosimilars and biologics?
NIBRT: LC-MS is an extremely valuable tool for the analysis of biosimilars and biologics due to its ability to separate many components in complex mixtures and determine the mass of those components in both a qualitative and quantitative manner. LC-MS is routinely used for the characterization of biologics using top-down approaches wherein the intact mass of the molecule is determined, middle-down approaches wherein subunits of domains of the molecule are separated and independently analyzed, and bottom-up approaches following digestion of the therapeutic protein into its constituent peptides through the action of a suitable protease. Such approaches facilitate verification of the primary sequence of the molecule, identification and characterization of post-translation modifications, especially wherein the combined use of
collisional-induced dissociation (CID) and/or electron-transfer dissociation (ETD) fragmentation strategies are employed. As mentioned previously, LC, MS, and LC-MS approaches are widely used for the characterization of the glycosylation present.
The combination of generated data from the analysis of the primary sequence with additional analytical platforms is often required to provide an insight into the impact of alterations in the primary sequence or post-translation modifications on the secondary, tertiary, or quaternary structure of the protein. Traditional techniques such as X-ray crystallography and nuclear magnetic resonance (NMR) spectroscopy can be used for protein structure determination; however, the application to comparability studies can be complicated. Other methods for studying higher order protein structure such as calorimetric methods, analytical ultracentrifugation, circular dichroism, and fluorescence are capable of providing information regarding overall alterations or differences in the protein structure, but generally, are incapable of localizing the area within the sequence of structural difference. Newer methods such as hydrogen-deuterium exchange (HDX) MS are valuable tools for detecting small changes in specific regions of the protein structure. HDX-MS is also more sensitive that other technologies requiring much smaller sample amounts and is also automated.
Ion-mobility spectrometry-mass spectrometry is also emerging as a new and powerful technology to allow for the elucidation of protein structure based upon comparison of the molecules collisional cross-
sectional (CCS) area in the gas phase. If the overall protein structure, say between innovator and biosimilar, is different, the molecules may have differences in drift time and associated CCS value, which may be indicative of a conformational change between the molecules.
With regard to aggregation analysis, size-exclusion chromatography (SEC), often with multi-angle light-scattering detection, is widely used for the determination of aggregates. Other methods employed include asymmetric flow field flow fractionation (A4F) or analytical ultracentrifugation (AUC), often as an orthogonal technique to SEC to increase overall analytical confidence and interpretation.
All of the aforementioned methods provide information regarding the molecules structural similarity; however, to determine similarity of function or efficacious effect bioequivalence assays must
be performed.