Physical Stability of Therapeutic Proteins in Solution : Exploring Aggregation under Heat, Pumping, and Seeding Conditions
(2025)- Abstract
- This thesis aims to deepen the knowledge of physical stability and aggregation of proteins by studying two proteins under combinations of stress factors such as heat, pumping, and seeding. The work addressed questions such as how combinations of techniques can be used to study aggregation pathways, how the formulation might affect the formation of micron-sized particles during pumping, and finally, how different pre-formed aggregates can affect the stability of proteins in solution during storage.
Two different proteins, human growth hormone (hGH) and a monoclonal antibody, Antibody A, were investigated. These proteins represent different classes of therapeutic proteins, hormones, and antibodies; they differ in size and structure.... (More) - This thesis aims to deepen the knowledge of physical stability and aggregation of proteins by studying two proteins under combinations of stress factors such as heat, pumping, and seeding. The work addressed questions such as how combinations of techniques can be used to study aggregation pathways, how the formulation might affect the formation of micron-sized particles during pumping, and finally, how different pre-formed aggregates can affect the stability of proteins in solution during storage.
Two different proteins, human growth hormone (hGH) and a monoclonal antibody, Antibody A, were investigated. These proteins represent different classes of therapeutic proteins, hormones, and antibodies; they differ in size and structure. The studies included investigating their physical stability and aggregation behaviour under a combination of stresses, which included heat, pumping, and storage at different temperatures. The storage studies investigated the effect of seeding and agitation on particle formation.
The aggregation pathway of human growth hormone upon heating at neutral pH could be elucidated utilising the powerful combination of MiniTEM for imaging, dynamic light scattering, and asymmetric flow field flow fractionation for size and concentration. In the second study, it was observed that the type of salt (Na2SO4, NaCl, and NaSCN) with anions ranking in the Hofmeister series dramatically influenced the particle formation propensity for Antibody A during peristaltic pumping. The combined effect of protein adsorption and protein-protein interaction can explain how the salts affect the number of particles formed. Interestingly, the ion-specific effects of NaCl, which is supposed to have the most neutral impact on the protein, created the “perfect storm” and decreased the stability during pumping. Finally, the evolution of small aggregates and micron-sized particles during different storage conditions (temperature, static, agitation) was examined. It turns out that small particles (<100 nm) created during heating follow a different process than large, micrometre-sized particles. Small aggregates might have a seeding effect, increasing size or number, while large particles undergo concurrent formation and dissolution processes during agitated storage. Elevated storage temperature (40 °C) mainly accelerates the degradation of the already formed particles rather than aggregation.
To summarise, studying aggregation by different combinations of stress factors and formulations of two therapeutic proteins has contributed to an improved understanding of physical stability and protein aggregation. These findings will hopefully give more insights into the continued research and development of therapeutic protein formulations. (Less)
Please use this url to cite or link to this publication:
https://lup.lub.lu.se/record/16f778e8-4503-48eb-965d-82416828e42f
- author
- Västberg, Amanda LU
- supervisor
- opponent
-
- Prof. Svilenov, Hristo, Technichal University of Munich, Germany.
- organization
- publishing date
- 2025
- type
- Thesis
- publication status
- published
- subject
- pages
- 77 pages
- publisher
- Department of Process and Life Science Engineering, Lund University.
- defense location
- Lecture Hall KC:A, Kemicentrum, Naturvetarvägen 22, Faculty of Engineering LTH, Lund University, Lund. The dissertation will be live streamed, but part of the premises is to be excluded from the live stream.
- defense date
- 2025-02-25 09:00:00
- ISBN
- 978-91-8096-093-9
- 978-91-8096-092-2
- language
- English
- LU publication?
- yes
- id
- 16f778e8-4503-48eb-965d-82416828e42f
- date added to LUP
- 2025-01-24 11:25:33
- date last changed
- 2025-04-04 14:12:05
@phdthesis{16f778e8-4503-48eb-965d-82416828e42f,
abstract = {{This thesis aims to deepen the knowledge of physical stability and aggregation of proteins by studying two proteins under combinations of stress factors such as heat, pumping, and seeding. The work addressed questions such as how combinations of techniques can be used to study aggregation pathways, how the formulation might affect the formation of micron-sized particles during pumping, and finally, how different pre-formed aggregates can affect the stability of proteins in solution during storage.<br/><br/>Two different proteins, human growth hormone (hGH) and a monoclonal antibody, Antibody A, were investigated. These proteins represent different classes of therapeutic proteins, hormones, and antibodies; they differ in size and structure. The studies included investigating their physical stability and aggregation behaviour under a combination of stresses, which included heat, pumping, and storage at different temperatures. The storage studies investigated the effect of seeding and agitation on particle formation. <br/><br/>The aggregation pathway of human growth hormone upon heating at neutral pH could be elucidated utilising the powerful combination of MiniTEM for imaging, dynamic light scattering, and asymmetric flow field flow fractionation for size and concentration. In the second study, it was observed that the type of salt (Na2SO4, NaCl, and NaSCN) with anions ranking in the Hofmeister series dramatically influenced the particle formation propensity for Antibody A during peristaltic pumping. The combined effect of protein adsorption and protein-protein interaction can explain how the salts affect the number of particles formed. Interestingly, the ion-specific effects of NaCl, which is supposed to have the most neutral impact on the protein, created the “perfect storm” and decreased the stability during pumping. Finally, the evolution of small aggregates and micron-sized particles during different storage conditions (temperature, static, agitation) was examined. It turns out that small particles (<100 nm) created during heating follow a different process than large, micrometre-sized particles. Small aggregates might have a seeding effect, increasing size or number, while large particles undergo concurrent formation and dissolution processes during agitated storage. Elevated storage temperature (40 °C) mainly accelerates the degradation of the already formed particles rather than aggregation.<br/><br/>To summarise, studying aggregation by different combinations of stress factors and formulations of two therapeutic proteins has contributed to an improved understanding of physical stability and protein aggregation. These findings will hopefully give more insights into the continued research and development of therapeutic protein formulations.}},
author = {{Västberg, Amanda}},
isbn = {{978-91-8096-093-9}},
language = {{eng}},
publisher = {{Department of Process and Life Science Engineering, Lund University.}},
school = {{Lund University}},
title = {{Physical Stability of Therapeutic Proteins in Solution : Exploring Aggregation under Heat, Pumping, and Seeding Conditions}},
url = {{https://lup.lub.lu.se/search/files/206856745/e-nailing_ex_Amanda.pdf}},
year = {{2025}},
}