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Updated: Jun 6, 2025

Analyzing DNA-Protein Interactions with Streptavidin-Based Biolayer Interferometry
Published on: January 17, 2025
A multifaceted approach to understanding protein-buffer interactions in biopharmaceuticals
Blaž Lebar1, Maria Orehova2, Boštjan Japelj3
1University of Ljubljana, Faculty of Pharmacy, Department of Pharmaceutical Chemistry, Aškerčeva 7, SI-1000 Ljubljana, Slovenia; Novartis Pharmaceutical Manufacturing LLC, Kolodvorska 27, SI-1234 Menges, Slovenia.
Abstract:
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 1HT2 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 methods approach to uncovering these critical interactions, advancing our understanding of excipient stabilization mechanisms and rational formulation design in biopharmaceutics.
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