Analytical tools for characterizing biopharmaceuticals and the implications for biosimilars

Abstract

Biologics such as monoclonal antibodies are much more complex than small-molecule drugs, which raises challenging questions for the development and regulatory evaluation of follow-on versions of such biopharmaceutical products (also known as biosimilars) and their clinical use once patent protection for the pioneering biologic has expired. With the recent introduction of regulatory pathways for follow-on versions of complex biologics, the role of analytical technologies in comparing biosimilars with the corresponding reference product is attracting substantial interest in establishing the development requirements for biosimilars. Here, we discuss the current state of the art in analytical technologies to assess three characteristics of protein biopharmaceuticals that regulatory authorities have identified as being important in development strategies for biosimilars: post-translational modifications, three-dimensional structures and protein aggregation.

Figure 1. Uses of analytical tools in the characterization of biopharmaceuticals

a | Post-translational modifications are observed as mass increases in peptides, and the location of the modifications can be deciphered by monitoring smaller fragments through tandem mass spectrometry (MS–MS) experiments. In this example, the addition of fucose is monitored in a specific peptide, and its location in the whole protein is known based on its mass and fragmentation pattern. The plasma-derived version of the protein (bottom spectra) contains the fucose but the recombinant form (top spectra) does not. b | Higher-order structures can be studied by hydrogen deuterium exchange MS (HDX–MS). In this example, the changes in protein conformation and dynamics in a monoclonal antibody were probed for fully glycosylated immunoglobulin G (IgG) versus deglycosylated IgG. Removal of the glycan affects hydrogen deuterium exchange and therefore conformation in the blue and red regions. c | Aggregation can be monitored by ion mobility spectrometry (IMS), in which different species have different drift times in the ion mobility separation. Here, dimeric insulin has a collisional cross-section and therefore a longer drift time than monomeric insulin. This figure is based on REFS ,,. CID, collision-induced dissociation; ECD, electron capture dissociation; ETD, electron transfer dissociation; m/z, mass-to-charge ratio.