A multifaceted approach to understanding protein-buffer interactions in biopharmaceuticals

Area of Science:

• Biopharmaceutical Formulation
• Protein Chemistry
• Analytical Chemistry

Background:

• Excipient selection is critical for biopharmaceutical drug product stability.
• Protein-excipient interactions are proposed stabilization mechanisms but are difficult to identify.
• Weak and transient nature of these interactions poses a significant challenge.

Purpose of the Study:

• To present a comprehensive approach for identifying protein-excipient interactions.
• To investigate interactions between rituximab and various excipients.
• To enhance understanding of stabilization mechanisms for rational formulation design.

Main Methods:

• Utilized ¹H T₂ CPMG (Carr-Purcel-Meiboom-Gill) filter experiments.
• Employed chemometric analyses of 2D NMR fingerprints.
• Performed molecular dynamics simulations to identify potential interaction sites.

Main Results:

• Identified specific interactions between rituximab and certain buffers/amino acids.
• Revealed interactions between selected excipients and antibody fragments using NMR.
• Discovered potential interacting hotspots via molecular dynamics simulations, even without NMR assignment.

Conclusions:

• An orthogonal methods approach is vital for uncovering critical protein-excipient interactions.
• The study advances the understanding of excipient stabilization mechanisms.
• Findings support rational formulation design in biopharmaceutical development.