A multifaceted approach to understanding protein-buffer interactions in biopharmaceuticals

Lebar, Blaž (author), Orehova, Maria (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)

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.

• Language: English
• Keywords: buffer, interaction, methyl fingerprinting, molecular dynamics, monoclonal antibody, nuclear magnetic resonance
• 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: 12 str.
• Numbering: Vol. 206, art. 114582
• PID: 20.500.12556/RUL-165181
• UDC: 54:615
• ISSN on article: 0939-6411
• DOI: 10.1016/j.ejpb.2024.114582
• COBISS.SI-ID: 216273923
• Publication date in RUL: 26.11.2024

Record is a part of a journal

• Title: European journal of pharmaceutics and biopharmaceutics
• Shortened title: Eur. j. pharm. biopharm.
• Publisher: Elsevier
• ISSN: 0939-6411
• COBISS.SI-ID: 13176839

Licences

• License: CC BY-NC 4.0, Creative Commons Attribution-NonCommercial 4.0 International
• Link: http://creativecommons.org/licenses/by-nc/4.0/
• Description: A creative commons license that bans commercial use, but the users don’t have to license their derivative works on the same terms.

Secondary language

• Language: Slovenian
• Keywords: medpomnilnik, interakcija, metilni prstni odtisi, molekularna dinamika, monoklonsko protitelo, jedrska magnetna resonanca, pomožne snovi, zdravila

Projects

• Funder: Other - Other funder or multiple funders
• Funding programme: Novartis Pharmaceutical Manufacturing LLC

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