Studying secondary structural changes and aggregation in monoclonal antibodies using ATR-FTIR spectroscopic imaging
File(s)
Author(s)
Van Haaren, Céline
Type
Thesis
Abstract
Protein aggregation is a major challenge in the production of therapeutic monoclonal antibodies (mAbs), resulting in reduced efficacy, safety concerns, and considerable economic losses. During bioprocessing, transport and storage, mAbs are exposed to various chemical and physical stresses, including temperature fluctuations, pH shifts and interfacial stresses. These stresses can induce chemical modifications and conformational changes to the protein, ultimately leading to protein unfolding and aggregation. Since these phenomena directly impact the efficacy and safety of the drug, it is of vital importance to monitor the structural integrity of mAbs during manufacturing, and to develop a comprehensive understanding of their stability under bioprocessing-relevant conditions.
In this thesis, ATR-FTIR spectroscopic imaging was used to investigate the behaviour of mAbs under such conditions. This technique enables the characterisation of protein secondary structure through the absorption of infrared light, while at the same time providing spatially resolved information across the sample. Unlike many other analytical techniques, ATR-FTIR spectroscopic imaging is non-destructive, label-free and has no strict requirements on sample form or concentration, thereby allowing for the direct monitoring of protein stability, aggregation and associated structural changes in mAb samples under a range of conditions, with opportunities for in-situ and in-line analysis.
Specifically, this thesis focused on the development of microfluidic flow setups for the study of air-liquid interfacial stress, as well as the effect of low pH buffers used during downstream purification, on the structural stability of mAbs. Furthermore, the protective effect of three different excipients on long-term storage stability was assessed using this technique in combination with DLS. As such, this research aims to demonstrate the usefulness of ATR-FTIR spectroscopic imaging for mAb structural stability testing and monitoring, particularly through the presentation of microfluidic set-ups, offering unique insights into mAb behaviour and secondary structural stability.
In this thesis, ATR-FTIR spectroscopic imaging was used to investigate the behaviour of mAbs under such conditions. This technique enables the characterisation of protein secondary structure through the absorption of infrared light, while at the same time providing spatially resolved information across the sample. Unlike many other analytical techniques, ATR-FTIR spectroscopic imaging is non-destructive, label-free and has no strict requirements on sample form or concentration, thereby allowing for the direct monitoring of protein stability, aggregation and associated structural changes in mAb samples under a range of conditions, with opportunities for in-situ and in-line analysis.
Specifically, this thesis focused on the development of microfluidic flow setups for the study of air-liquid interfacial stress, as well as the effect of low pH buffers used during downstream purification, on the structural stability of mAbs. Furthermore, the protective effect of three different excipients on long-term storage stability was assessed using this technique in combination with DLS. As such, this research aims to demonstrate the usefulness of ATR-FTIR spectroscopic imaging for mAb structural stability testing and monitoring, particularly through the presentation of microfluidic set-ups, offering unique insights into mAb behaviour and secondary structural stability.
Version
Open Access
Date Issued
2025-10-03
Date Awarded
2026-03-01
Copyright Statement
Attribution-NonCommercial 4.0 International Licence (CC BY-NC)
License URL
Advisor
Kazarian, Sergei
Sponsor
The Mohan Westlake Foundation
Publisher Department
Department of Chemical Engineering
Publisher Institution
Imperial College London
Qualification Level
Doctoral
Qualification Name
Doctor of Philosophy (PhD)