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Multi-approach LC-MS methods for the characterization of species-specific attributes of monoclonal antibodies from plants

TitleMulti-approach LC-MS methods for the characterization of species-specific attributes of monoclonal antibodies from plants
Publication TypeArticolo su Rivista peer-reviewed
Year of Publication2022
AuthorsTengattini, S., Rinaldi F., Perez-Fernandez V., Fabbri A., Donini Marcello, Marusic Carla, Sferrazza G., Pierimarchi P., Zonfrillo M., Calleri E., Massolini G., Pisano C., and Temporini C.
JournalJournal of Pharmaceutical and Biomedical Analysis
KeywordsAgrobacterium tumefaciens, Alkylation, Amino Acid Sequence, animal, animal cell, Animals, Antibodies, Antineoplastic Agents, article, chemistry, Chromatography, controlled study, drug structure, Female, gene expression system, glycopeptide, Glycosylation, hydrophilic interaction chromatography, Immunological, immunological antineoplastic agent, ion exchange chromatography, Liquid, liquid chromatography, liquid chromatography-mass spectrometry, mammal, Mammals, Molecular Weight, Monoclonal, Monoclonal antibody, monomer, Nicotiana benthamiana, nonhuman, peptide hydrolase, Peptide Hydrolases, Peptide Mapping, procedures, protein aggregation, protein processing, protein stability, proteinase, reversed phase liquid chromatography, rituximab, size exclusion chromatography, Tandem Mass Spectrometry, Tobacco

In this work, an analytical platform based on the use of chromatography and mass spectrometry (MS), has been applied to the characterization of Rituximab (RTX) obtained from two plant expression systems (rice and tobacco) in comparison to the mammalian cell-derived reference monoclonal antibody (mAb). Different chromatographic approaches, hyphenated to high resolution MS (HRMS), were applied to RTX structural investigation both at middle- and peptide level. In particular, cation exchange chromatography (CEX), size exclusion chromatography (SEC), reversed phase (RPLC) and hydrophilic interaction liquid chromatographic (HILIC) methods were developed and applied on intact mAbs, IdeS-, and trypsin digests in order to address critical attributes such as primary structure, glycan composition, species-related heterogeneity, glycosylation degree, charge variants, aggregation tendency and enzymatic stability. All the collected data highlight the features and criticalities of each production approach. Production in rice results in a heterogeneous but stable product over time, suggesting the absence of proteases in seeds; while tobacco expression system leads to more homogeneous glycosylation, but protein stability seems to be a critical issue probably due to the presence of proteases. This analytical strategy represents a robust support to scientists in the selection and optimization of the best plant expression system to produce recombinant humanized mAbs. © 2022 Elsevier B.V.


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Citation KeyTengattini2022