title: Lysine conjugation properties in human IgGs studied by integrating high-resolution native mass spectrometry and bottom-up proteomics.
abstract: Antibody-drug conjugates (ADCs) are a novel class of biopharmaceuticals several of which are now being investigated in clinical studies. In ADCs, potent cytotoxic drugs are coupled via a linker to reactive residues in IgG monoclonal antibodies. Linkage to lysine residues in the IgGs, using N-hydroxysuccinimide ester based chemistry, is one of the possible options. To control drug load and specificity, proper knowledge is required about which lysine residues are most accessible and reactive. Here, we combine native MS and bottom-up proteomics to monitor the overall drug load and site-specific lysine reactivity, using N-hydroxysuccinimide-based tandem mass tags. High-resolution Orbitrap native MS enables us to monitor and quantify, due to the achieved baseline resolution, the sequential incorporation of up to 69 tandem mass tag molecules into human IgGs. Complementary, bottom-up proteomics facilitates the identification of some very reactive "hot-spot" conjugation sites. However, we also identify lysine residues that are highly resistant to chemical labeling. Our integrated approach gives insight into the conjugation properties of IgGs at both the intact protein and residue levels, providing fundamental information for controlling drug load and specificity in lysine-linked ADCs.
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands.|Netherlands Proteomics Center, University of Utrecht, Utrecht, The Netherlands.
authors: Violette
authors: V
authors: Gautier
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands.|AbLab, University of Utrecht, Utrecht, The Netherlands.
authors: Anja J
authors: AJ
authors: Boumeester
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands.|Netherlands Proteomics Center, University of Utrecht, Utrecht, The Netherlands.
authors: Philip
authors: P
authors: Lössl
authors: Biomolecular Mass Spectrometry and Proteomics, Bijvoet Centre for Biomolecular Research, Utrecht Institute for Pharmaceutical Sciences, University of Utrecht, Utrecht, The Netherlands.|Netherlands Proteomics Center, University of Utrecht, Utrecht, The Netherlands.
authors: Albert J R
authors: AJ
authors: Heck
mesh_terms: D000595:Amino Acid Sequence; D006801:Humans; D018796:Immunoconjugates / Q000737:chemistry*; D007074:Immunoglobulin G / Q000737:chemistry*; D008239:Lysine / Q000737:chemistry*; D013058:Mass Spectrometry / Q000379:methods*; D008958:Models, Molecular; D008969:Molecular Sequence Data; D040901:Proteomics / Q000379:methods*
chemical_list: D018796:Immunoconjugates; D007074:Immunoglobulin G; D008239:Lysine
keywords: Antibody-drug conjugates; Covalent chemical labeling; IgGs; Lysine conjugation; Native mass spectrometry; Technology