Recombinant monoclonal antibodies (MAbs) represent a class of efficient, but expensive, biotherapeutics. Developing less costly generic "biosimilar" MAbs is of great interest to both drug companies and consumers. Biosimilar drugs can be defined as compounds that are as similar as possible, structurally and functionally, to an innovator drug. The driving force for the interest in biosimilars for generic drug manufacturers is the upcoming patent expiration for marketed products.1 However, developing biosimilar products is challenging. Currently, there are no registered biosimilars in the US, and only recently has a mechanism for their approval been created.2 Biosimilar manufacturers will be under pressure to ensure that their products conform as closely as possible to existing products if they wish to avoid repeating expensive clinical trials and thus reach the market faster. Therefore they have a vested interest in comprehensive product analysis at all stages of development and manufacturing.The key criteria for approval of biosimilars include quality, efficacy, and safety. Therefore, the generic biopharmaceuticals industry must try to demonstrate consistency of a biosimilar to the innovator reference product in every aspect. A number of physicochemical and biological tests are required by regulatory authorities for the characterization of MAbs.3,4 European Union regulations specify that state-of-the art characterization studies should be applied to the "similar biological product" (Section 5.2)5 and that physicochemical characterization should include a determination of the primary structure (section 5.2.2) while post-translational modifications (PTMs) such as glycosylation exhibit only minor levels of microheterogeneity.
In the work presented here, we demonstrate the application of state-of-the-art liquid chromatography (LC) and mass spectrometry (MS) to perform a comprehensive comparison of innovator and biosimilar MAbs. LC–MS analysis was carried out at the intact protein level, providing the information about molecular weight and glycan heterogeneity. Middle-down analysis of the MAb after reduction provided mass information on the antibody light chain and heavy chain separately, whereas the bottom-up approach (analysis of the MAb tryptic digest) allowed us to locate and identify the differences in primary MAb sequence and PTMs.
For peptide mapping, we used a novel, data-independent acquisition method that allows for simultaneous collection of both MS and fragment ion data for every peptide in the data set independent of their mass, intensity, and retention time.6,7 The data-independent MS acquisition method permits reliable acquisition of both quantitative (peptide MS signal) and qualitative (peptide sequence) data in a single LC–MS analysis.
The goal of this manuscript is to demonstrate the utility of a comprehensive set of advanced UHPLC–MS methods and their suitability for comprehensive comparison of innovator products and biosimilar MAb drug candidates, using a biosimilar of trastuzumab as an example.