There is an emerging interest in developing biosimilar monoclonal antibodies (MAbs). To avoid expensive clinical trials and
shorten time to market, the biosimilar industry must establish that a developing product is, as much as possible, similar
to a marketed innovator product through comprehensive analysis. Here, we demonstrate that ultra high pressure liquid chromatography
(UHPLC) and mass spectrometry (MS) can be used routinely to characterize minor differences between a candidate biosimilar
and an innovator IgG1 MAb. A two amino acid residue variance in the heavy chain sequence was detected by LC–MS intact protein
mass measurement and located by tryptic peptide mapping with data independent acquisition LC–MS. Microheterogeneities due
to N-linked glycosylation and chemical degradation were comprehensively catalogued and compared. The results show that complementary
LC–MS methods can be used as a set of routine tools for rapid comparison of molecular similarity between a candidate biosimilar
and a commercially available MAb.
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