Qualitative and Quantitative Characterization of Therapeutic Antibodies by Native Mass Spectrometry

Abstract

This thesis describes the development of novel mass spectrometric methods for the analysis of therapeutic monoclonal antibodies. The first chapter of my thesis introduces the reader to the two main subjects discussed in this thesis: native mass spectrometry and therapeutic monoclonal antibodies. After a brief discussion on the general features and applications of native mass spectrometry, the first section of this chapter takes the reader into the details of the instrumentation used in native mass spectrometry with particular focus of the type of mass analyzers used for the realization of this work. In the second part, the discussion is focused on therapeutic monoclonal antibodies. Here a taste of the pharmacological properties and therapeutic relevance of this relatively new class of therapeutics is provided. Subsequently, in chapter II the two main subjects of the thesis, discussed separately in the introduction, are brought together. Here the discussion digs into the numerous applications of mass spectrometry for the analysis of monoclonal antibodies. The following chapters highlight some of the innovative research applications performed during my PhD studies. In chapter III native mass spectrometry was used for the qualitative and semi-quantitative analysis of composite mixtures of antibodies representing biopharmaceutical products co-expressed from single cells. This chapter shows how native mass spectrometry presents a valuable alternative to existing analytical methods for qualitative and quantitative profiling of biopharmaceutical products. It provides both the identity of each species in a mixture by mass determination and the relative abundance through comparison of relative ion signal intensities. An innovative instrumental improvement that characterized the rest of my thesis is described in chapter IV. With this work, for the first time, a modified Orbitrap Exactive PlusTM instrument is used for the analysis of monoclonal antibodies under native conditions. The improved experimental resolution at high m/z achievable using the Orbitrap analyzer is very advantageous for studying such naturally heterogeneous proteins as antibodies.