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Biotherapeutic Structural Investigations of Formulation Parameters Using Vibrational Spectroscopy

Mcavan, Bethan

[Thesis]. Manchester, UK: The University of Manchester; 2019.

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Abstract

Monoclonal antibodies (mAbs) are now the most common protein therapeutics on the market. The therapeutic properties of mAbs originate from their inherent ability to specifically bind to a chosen target and the subsequent stimulation of effector functions as result of mAb glycosylation. Therefore mAbs are able to act as drug delivery systems, inhibitors or activators. Protein therapeutic characterisation possesses inherent analytical difficulties as mAbs are complex structures made up of multiple domains. The higher order structure of mAbs is essential for their specificity for targeted treatments. During the production of mAbs, the bioformulation process can induce structural perturbations that we separate into two main categories: Post-translational modifications (PTMs) and degradation. The most common PTMs are oxidation, deamidation and glycation. Degradation of mAbs covers general loss of structure through aggregation and fragmentation. Due to the wide variation and number of possible modifications to mAbs, monitoring, identification and quantification is difficult. Current analytical methods are dependent on sampling, are often destructive, and require time and expertise. Vibrational spectroscopic methods have been highlighted as possible analytical methods to overcome previous shortcomings. In particular, Raman spectroscopy has the potential to be developed into an in-line analytical tool due to being minimally invasive, non-destructive, information rich and rapid. Raman and FTIR are already utilised for in situ monitoring of pharmaceutical manufacturing, such as small molecules, but have not yet been extended into the quality control of mAbs. In this thesis we use a range of conditions to force the degradation and increase the PTM levels of three of the most common types of mAbs: IgG1, Fab and IgG4. We use circular dichroism (CD), peptide mapping, and size exclusion chromatography (SEC) to quantify and study the structural implications of the PTMs and degradation, and compare these to the information gained from Raman spectroscopy. Bioformulation parameters probed include: agitation, pH, temperature, sugar excipients, chemical stressors and UV light. Primarily, we explore the sensitivity of Raman spectroscopy to detect, quantify and provide a structural insight into the modifications. Additionally, we study the aggregation of mAbs to investigate the structural consequences on the protein, as well as identify spectral markers that could be used to monitor aggregation.

Bibliographic metadata

Type of resource:
Content type:
Form of thesis:
Type of submission:
Degree type:
Doctor of Philosophy
Degree programme:
PhD Biochemistry (48 month)
Publication date:
Location:
Manchester, UK
Total pages:
263
Abstract:
Monoclonal antibodies (mAbs) are now the most common protein therapeutics on the market. The therapeutic properties of mAbs originate from their inherent ability to specifically bind to a chosen target and the subsequent stimulation of effector functions as result of mAb glycosylation. Therefore mAbs are able to act as drug delivery systems, inhibitors or activators. Protein therapeutic characterisation possesses inherent analytical difficulties as mAbs are complex structures made up of multiple domains. The higher order structure of mAbs is essential for their specificity for targeted treatments. During the production of mAbs, the bioformulation process can induce structural perturbations that we separate into two main categories: Post-translational modifications (PTMs) and degradation. The most common PTMs are oxidation, deamidation and glycation. Degradation of mAbs covers general loss of structure through aggregation and fragmentation. Due to the wide variation and number of possible modifications to mAbs, monitoring, identification and quantification is difficult. Current analytical methods are dependent on sampling, are often destructive, and require time and expertise. Vibrational spectroscopic methods have been highlighted as possible analytical methods to overcome previous shortcomings. In particular, Raman spectroscopy has the potential to be developed into an in-line analytical tool due to being minimally invasive, non-destructive, information rich and rapid. Raman and FTIR are already utilised for in situ monitoring of pharmaceutical manufacturing, such as small molecules, but have not yet been extended into the quality control of mAbs. In this thesis we use a range of conditions to force the degradation and increase the PTM levels of three of the most common types of mAbs: IgG1, Fab and IgG4. We use circular dichroism (CD), peptide mapping, and size exclusion chromatography (SEC) to quantify and study the structural implications of the PTMs and degradation, and compare these to the information gained from Raman spectroscopy. Bioformulation parameters probed include: agitation, pH, temperature, sugar excipients, chemical stressors and UV light. Primarily, we explore the sensitivity of Raman spectroscopy to detect, quantify and provide a structural insight into the modifications. Additionally, we study the aggregation of mAbs to investigate the structural consequences on the protein, as well as identify spectral markers that could be used to monitor aggregation.
Thesis main supervisor(s):
Thesis co-supervisor(s):
Funder(s):
Language:
en

Institutional metadata

University researcher(s):

Record metadata

Manchester eScholar ID:
uk-ac-man-scw:322558
Created by:
Mcavan, Bethan
Created:
22nd November, 2019, 15:00:30
Last modified by:
Mcavan, Bethan
Last modified:
4th December, 2020, 10:14:28

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