A deep dive into spin-labeled polysorbate’s interaction with therapeutic antibody using 2D NMR, EPR and MD simulations

Lebar, Blaž (author), Orehova, Marija Vjačeslavovna (author), Japelj, Boštjan (author), Šprager, Ernest (author), Podlipec, Rok (author), Knaflič, Tilen (author), Urbančič, Iztok (author), Knez, Benjamin (author), Zidar, Mitja (author), Cerar, Jure (author), Mravljak, Janez (author), Žula, Aleš (author), Arčon, Denis (author), Plavec, Janez (author), Pajk, Stane (author)

Polysorbates (PS) are widely used surfactants in biopharmaceutical formulations playing a crucial role in protecting proteins against mechanical stress and interface-induced damage. However, their susceptibility to degradation can compromise their function and lead to particle formation. Recent studies suggest that monoclonal antibodies (mAbs) may mitigate PS degradation catalyzed by histidine chloride buffer, indicating the presence of protein-PS interactions. In this study, we investigated these interactions using NMR, starting with $^1$H T$_2$ CPMG filter experiments and methyl fingerprinting, which failed to detect interactions. To enhance sensitivity, we synthesized spin-labeled PS (SLPS), enabling paramagnetic relaxation enhancement (PRE) NMR experiments, specifically amide fingerprinting, which successfully revealed interactions. Complementary electron paramagnetic resonance (EPR) measurements of SLPS and mAb also detected interactions, but only in the presence of sucrose, underscoring their weak and transient nature. Additionally, molecular dynamics simulations identified potential interaction hotspots on the antibody structure, providing mechanistic insights into these interactions.

• Language: English
• Keywords: monoclonal antibody, interaction, polysorbate, nuclear magnetic resonance, electron paramagnetic resonance, molecular dynamics
• Work type: Article
• Typology: 1.01 - Original Scientific Article
• Organization: FFA - Faculty of Pharmacy
• Publication status: Published
• Publication version: Version of Record
• Year: 2025
• Number of pages: 9 str.
• Numbering: Vol. 217, art. 114892
• PID: 20.500.12556/RUL-182732
• UDC: 577:615.32
• ISSN on article: 1873-3441
• DOI: 10.1016/j.ejpb.2025.114892
• COBISS.SI-ID: 255171075
• Publication date in RUL: 21.05.2026

Record is a part of a journal

• Title: European journal of pharmaceutics and biopharmaceutics
• Publisher: Elsevier
• ISSN: 1873-3441
• COBISS.SI-ID: 23171845

Licences

• License: CC BY 4.0, Creative Commons Attribution 4.0 International
• Link: http://creativecommons.org/licenses/by/4.0/
• Description: This is the standard Creative Commons license that gives others maximum freedom to do what they want with the work as long as they credit the author.

Secondary language

• Language: Slovenian
• Keywords: monoklonsko protitelo, interakcija, polisorbat, jedrska magnetna resonanca, elektronska paramagnetna resonanca, molekularna dinamika, farmacevtska kemija, biokemija, zdravila

Projects

• Funder: Novartis Pharmaceutical Manufacturing LLC

• Funder: ARIS - Slovenian Research and Innovation Agency
• Project number: P1-0208
• Name: Farmacevtska kemija: načrtovanje, sinteza in vrednotenje učinkovin