20.500.12556/RUL-165181 A multifaceted approach to understanding protein-buffer interactions in biopharmaceuticals The excipient selection process plays a crucial role in biopharmaceutical formulation development to ensure the long-term stability of the drug product. Though there are numerous options approved by regulatory authorities, only a subset is commonly utilized. Previous research has proposed various stabilization mechanisms, including protein-excipient interactions. However, identifying these interactions remains challenging due to their weak and transient nature. In this study, we present a comprehensive approach to identify such interactions. Using the $^1$H T$_2$ CPMG (Carr-Purcel-Meiboom-Gill) filter experiment we identified interactions of rituximab with certain buffers and amino acids, shedding light on its Fc fragment instability that manifested during the enzymatic cleavage of the antibody. Moreover, chemometric analyses of 2D NMR fingerprints revealed interactions of selected excipients with antibody fragments. Furthermore, molecular dynamics simulations revealed potential interacting hotspots without NMR spectra assignment. Our results highlight the importance of an orthogonal method approach to uncovering these critical interactions, advancing our understanding of excipient stabilization mechanisms and rational formulation design in biopharmaceutics. buffer interaction methyl fingerprinting molecular dynamics monoclonal antibody nuclear magnetic resonance medpomnilnik interakcija metilni prstni odtisi molekularna dinamika monoklonsko protitelo jedrska magnetna resonanca pomožne snovi zdravila true false true Angleški jezik Slovenski jezik Članek v reviji 2024-11-26 11:13:34 2024-11-26 11:13:38 2024-11-27 03:50:48 0000-00-00 00:00:00 2025 0 0 12 str. Vol. 206, art. 114582 2025 0000-00-00 Zaloznikova Objavljeno NiDoloceno 0000-00-00 0000-00-00 0000-00-00 54:615 0939-6411 10.1016/j.ejpb.2024.114582 216273923 1-s2.0-S0939641124004089-main.pdf 1-s2.0-S0939641124004089-main.pdf 1 086E31618B6A025D55ACBBD4740327D2 b3e814ca54f02a6bfb72d324d619fe771ab9d5284d6803e8cc7dfeeaa9b6577a fe9710c2-abde-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=196186 https://www.sciencedirect.com/science/article/pii/S0939641124004089 1 b1be997f-abde-11ef-b232-0050569b8976 https://repozitorij.uni-lj.si/Dokument.php?lang=slv&id=196185 Fakulteta za farmacijo 0 0 0