The Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD... Show moreThe Consortium for Top-Down Proteomics (www.topdownproteomics.org) launched the present study to assess the current state of top-down mass spectrometry (TD MS) and middle-down mass spectrometry (MD MS) for characterizing monoclonal antibody (mAb) primary structures, including their modifications. To meet the needs of the rapidly growing therapeutic antibody market, it is important to develop analytical strategies to characterize the heterogeneity of a therapeutic product's primary structure accurately and reproducibly. The major objective of the present study is to determine whether current TD/MD MS technologies and protocols can add value to the more commonly employed bottom-up (BU) approaches with regard to confirming protein integrity, sequencing variable domains, avoiding artifacts, and revealing modifications and their locations. We also aim to gather information on the common TD/MD MS methods and practices in the field. A panel of three mAbs was selected and centrally provided to 20 laboratories worldwide for the analysis: Sigma mAb standard (SiLuLite), NIST mAb standard, and the therapeutic mAb Herceptin (trastuzumab). Various MS instrument platforms and ion dissociation techniques were employed. The present study confirms that TD/MD MS tools are available in laboratories worldwide and provide complementary information to the BU approach that can be crucial for comprehensive mAb characterization. The current limitations, as well as possible solutions to overcome them, are also outlined. A primary limitation revealed by the results of the present study is that the expert knowledge in both experiment and data analysis is indispensable to practice TD/MD MS. Show less
A broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current... Show moreA broad-based interlaboratory study of glycosylation profiles of a reference and modified IgG antibody involving 103 reports from 76 laboratories.Glycosylation is a topic of intense current interest in the development of biopharmaceuticals because it is related to drug safety and efficacy. This work describes results of an interlaboratory study on the glycosylation of the Primary Sample (PS) of NISTmAb, a monoclonal antibody reference material. Seventy-six laboratories from industry, university, research, government, and hospital sectors in Europe, North America, Asia, and Australia submitted a total of 103 reports on glycan distributions. The principal objective of this study was to report and compare results for the full range of analytical methods presently used in the glycosylation analysis of mAbs. Therefore, participation was unrestricted, with laboratories choosing their own measurement techniques. Protein glycosylation was determined in various ways, including at the level of intact mAb, protein fragments, glycopeptides, or released glycans, using a wide variety of methods for derivatization, separation, identification, and quantification. Consequently, the diversity of results was enormous, with the number of glycan compositions identified by each laboratory ranging from 4 to 48. In total, one hundred sixteen glycan compositions were reported, of which 57 compositions could be assigned consensus abundance values. These consensus medians provide community-derived values for NISTmAb PS. Agreement with the consensus medians did not depend on the specific method or laboratory type. The study provides a view of the current state-of-the-art for biologic glycosylation measurement and suggests a clear need for harmonization of glycosylation analysis methods. Show less
Over the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of... Show moreOver the past few years, loss of patent protection for blockbuster monoclonal antibody (mAb) drugs has caused a significant shift in the pharmaceutical industry towards the development of biosimilar products. As a result, multiple biosimilar mAbs are becoming available for a single originator drug. As opposed to small-molecular drugs, protein biopharmaceuticals do not have fully defined and reproducible structures, making it impossible to create identical copies. Therefore, regulators demand biosimilar sponsors to demonstrate similarity with the reference product to prevent safety and efficacy issues with the proposed product. Protein glycosylation is considered a crucially important quality attribute, because of its major role in immunogenicity and clinical efficacy of therapeutic proteins. However, the intrinsic biological variability of glycan structures creates a significant challenge for the current analytical platforms. In this review, we discuss the importance of glycan characterization on therapeutic proteins, with a particular focus on the analytical techniques applied for glycan profiling of biosimilar mAb products. In addition, we present a case study on infliximab biosimilars to illustrate the potential clinical implications of differences in glycan profile between originator and biosimilar mAb products. Show less
Burgt, Y.E.M. van der; Kilgour, D.P.A.; Tsybin, Y.O.; Srzentic, K.; Fornelli, L.; Beck, A.; ... ; Nicolardi, S. 2019
