Polar vs Non-polar Excipients in BSA Aggregation
- 1Faculty of Medicine and Health, The University of Sydney, Sydney School of Pharmacy, Sydney, NSW 2006 Australia
- 2Present Address: Faculty of Health and Medical Sciences, The University of Copenhagen, Department of Pharmacy, 2100 Copenhagen, Denmark
Abstract
Purpose: In recent years, there has been a rapid expansion in the development of new protein therapeutics in the pharmaceutical industry. Problematically, proteins are conformationally and colloidally marginally stable, and thus it is necessary to formulate them using buffering and stabilizing agents.
Methods: In this study, we use experimental and computation techniques to evaluate the aggregation pathway, as well as the stabilizing properties of a range of excipients on a model protein; bovine serum albumin (BSA).
Results: Our results show that polar excipients (L-arginine and trimethylamine N-oxide) decrease BSA stability, whereas non-polar excipients (L-alanine and L-valine) enhances it. To elucidate molecular mechanisms behind these trends, we constructed a multi-step unfolding and aggregation model. Upon thermal stress, the protein system is driven to form a disrupted dimer/trimer species across a steep activation energy barrier. This acts as the primary kinetic bottleneck, as once these intermediate states form sufficiently, large aggregates in tetramers and pentamers form across a significantly lower energy barrier. High temperature molecular dynamics simulations reveal that the unfolding mode of the monomer is characterized by the detachment of its C-terminus domain, creating an extended, aggregation prone structure. Crucially, in the native structure, this flexible region contains the only prominent solvent exposed hydrophobic patch on an otherwise hydrophilic protein surface. Therefore, hydrophobic excipients are predicted to interface with this hotspot, stabilizing the folded structure and suppressing downstream aggregation formation.
Conclusions: Overall, this work demonstrates that understanding the specific protein aggregation mechanisms is key for the rational selection of stabilizers.
Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s11095-026-04204-2.
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BibTeX
@article{tien2026polar,
title = {{Polar vs Non-polar Excipients in BSA Aggregation}},
author = {Samuel Tien and Simone Nørgaard Krabbe and Annika Sollann Sørensen and Thomas Balle and Veysel Kayser},
journal = {Pharmaceutical Research},
year = {2026},
month = oct,
doi = {10.1007/s11095-026-04204-2},
url = {https://doi.org/10.1007/s11095-026-04204-2},
note = {CC BY 4.0},
}
RIS
TY - JOUR
TI - Polar vs Non-polar Excipients in BSA Aggregation
AU - Samuel Tien
AU - Simone Nørgaard Krabbe
AU - Annika Sollann Sørensen
AU - Thomas Balle
AU - Veysel Kayser
PY - 2026
DA - 2026/10/07
JO - Pharmaceutical Research
DO - 10.1007/s11095-026-04204-2
UR - https://doi.org/10.1007/s11095-026-04204-2
AB - Purpose: In recent years, there has been a rapid expansion in the development of new protein therapeutics in the pharmaceutical industry. Problematically, proteins are conformationally and colloidally marginally stable, and thus it is necessary to formulate them using buffering and stabilizing agents. Methods: In this study, we use experimental and computation techniques to evaluate the aggregation pathway, as well as the stabilizing properties of a range of excipients on a model protein; bovine serum albumin (BSA). Results: Our results show that polar excipients (L-arginine and trimethylamine N-oxide) decrease BSA stability, whereas non-polar excipients (L-alanine and L-valine) enhances it. To elucidate molecular mechanisms behind these trends, we constructed a multi-step unfolding and aggregation model. Upon thermal stress, the protein system is driven to form a disrupted dimer/trimer species across a steep activation energy barrier. This acts as the primary kinetic bottleneck, as once these intermediate states form sufficiently, large aggregates in tetramers and pentamers form across a significantly lower energy barrier. High temperature molecular dynamics simulations reveal that the unfolding mode of the monomer is characterized by the detachment of its C-terminus domain, creating an extended, aggregation prone structure. Crucially, in the native structure, this flexible region contains the only prominent solvent exposed hydrophobic patch on an otherwise hydrophilic protein surface. Therefore, hydrophobic excipients are predicted to interface with this hotspot, stabilizing the folded structure and suppressing downstream aggregation formation. Conclusions: Overall, this work demonstrates that understanding the specific protein aggregation mechanisms is key for the rational selection of stabilizers. Supplementary Information: The online version contains supplementary material available at https://doi.org/10.1007/s11095-026-04204-2.
SN - 0724-8741
SN - 1573-904X
ER -