TY - CONF A1 - Döring, Sarah A1 - Konthur, Zoltán A1 - Heiner, Zsuzsanna T1 - Optimization of surface-functionalized particles for improved antibody digestion N2 - Therapeutic monoclonal antibodies are the fastest-growing group of biological agents which generated a yearly turnover of USD 210 billion in 2022 and whose sales are expected to grow by 10% annually over the next 10 years. With steadily increasing market importance, analytical methods for reliable quantification of therapeutic antibodies also become more and more relevant. Liquid chromatography coupled with tandem mass spectrometry (LC–MS/MS) has become the main technology for antibody quantification. This approach, however, requires enzymatic digestion of the intact protein into peptides, for which a wide range of different protocols exists that often lead to different results depending on the digestion procedure or trypsin variants used. In particular, the amount and type of detergents added for protein unfolding prior to digestion is known to create significant bias in measurement results. The overall goal of the presented project is the application of novel thermostable and surface-functionalized trypsin particles for improved antibody digestion. Specifically, a trypsin-variant described in the literature exhibiting increased activity and thermal stability above 80°C, will be examined. The application of this enzyme should allow to perform digestion at elevated temperatures where the protein is naturally unfolding thereby increasing enzyme accessibility without the need for detergents. Furthermore, we will immobilize the thermostable trypsin onto the surface to further enhance enzyme stability, prevent self-digestion, and enable separation of trypsin from target peptides before LC–MS/MS analysis. As an immobilization platform, cheap and non-porous corundum particles will be used as these show high chemical stability and low levels of interaction of matrix proteins with the functionalized surface. adsorption. In a multidisciplinary collaboration with the SALSA Photonics Lab, we will investigate the characteristics of covalent enzyme binding and unspecific peptide binding using an interface-sensitive analytical tool, vibrational sum-frequency generation (VSFG) spectroscopy. The insights gained will not only lead to new competencies in peptide and enzyme surface analysis using VSFG spectroscopy in SALSA but will also significantly contribute to optimizing antibody quantification. T2 - SALSA STF24 Kick-Off Meeting CY - Berlin, Germany DA - 11.04.2024 KW - Antibody Quantification KW - Vibrational Sum-Frequency Generation Spectroscopy KW - LC-MS/MS KW - Enzyme Immobilisation KW - Corundum PY - 2024 AN - OPUS4-59864 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - GEN A1 - Döring, Sarah T1 - Vergleichende Darstellung SARS-CoV-2-spezifischer Nanobodys aus unterschiedlichen Wirtsorganismen T2 - Masterarbeit N2 - Aufgrund der anhaltenden COVID-19-Pandemie werden neutralisierende Therapeutika benötigt. Eine Möglichkeit zur Behandlung stellt die Verwendung monoklonaler Anti-SARS-CoV-2-Immun-globuline dar. Ihre Produktion in Säugetierzellen ist jedoch schwer skalierbar, um den weltweiten Bedarf zu decken. VHH-Antikörper, auch Nanobodys genannt, bieten hierfür eine Alternative, da sie eine hohe Temperaturstabilität aufweisen und eine kostengünstige Produktion in prokaryotischen Wirtsorganismen ermöglichen. KW - E. coli KW - Corona KW - Virus KW - Spike-Protein KW - Nanobody KW - Antikörper KW - Expression KW - Fingerprint KW - Vhh KW - RBD KW - COVID-19 KW - SARS-CoV-2 KW - ELISA KW - MST KW - Halomonas elongata KW - Periplasma KW - SDS-PAGE KW - ACE2-Rezeptor PY - 2021 SP - 1 EP - 111 PB - Technische Universität Berlin CY - Berlin AN - OPUS4-54624 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -