Assessment of Protein Aggregation in Biotherapeutic Development and Biomarker Detection

Embargoed until 2024-03-10
Author
Date
2021Type
- Doctoral Thesis
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Abstract
Proteins are complex macromolecules which have been developed by Nature to
perform defined biological functions. Their sequences confer them unique biochemical
and biophysical properties and allows proteins to interact with desired
targets or self-assemble into functional assemblies. These complex interactions
can however also lead to the formation of aberrant protein aggregates. Aggregation
is challenging to describe and predict because of the extensively broad time
and length scales associated with the process, the heterogeneity of the species involved,
for instance in terms of size and reactivity, and the complex environment
in which proteins typically evolve.
Today, there is therefore still a strong need for in silico tools and analytical
methods that can provide informative data on protein biophysical properties and
aggregation in a high-throughput manner and with higher quality.
In this context, the present thesis aims at developing new biophysical tools
compatible with high-throughput development to inform on protein state in solution
and help rationalizing protein aggregation, with applications in biologics
development and for biomarker detection.
The first part of the thesis proposes microfluidic and nanotechnology-based
tools for the development of safe biopharmaceuticals. We present a microfluidic
diffusion sizing platform which is able to size protein aggregates in the submicron
range, and probe for viscosity and interactions directly in highly concentrated
protein solutions, which represent key quality attributes of the biopharmaceutical product. Then, we describe a nanoparticle-based assay to quantitatively assess
the effect of surfaces on protein stability, which has been identified as one of the
major causes of aggregation but is still very poorly characterized.We demonstrate
applications of this platform for “developability” studies to screen for optimal
protein or formulation candidates.
The second part of the thesis focuses on the detection of biomarkers. We describe
a generic approach to detect biomarkers with undefined biochemical and
biophysical properties, for instance protein aggregates or extracellular vesicles.
The platform is based on a microfluidic shrinking droplet concentrator device
that locally upconcentrates the analyte to enable its detection. The device can
also be applied to generate protein phase diagrams and mimic a type of cellular
compartmentalization under stress. Finally, we present a new bioseparation
platform which is able to isolate ppm amounts of target compounds directly from
complex mixtures. The tool is based on synthetic microdroplets that are designed
to preferentially exclude a wide range of compounds, and functionalized with a
binding moiety which allows to specifically recruit and locally upconcentrate desired
biomarkers.
Overall, we propose a range of new analytical platforms which can complement
the pool of existing biophysical characterization tools and inform in a quantitative
manner on protein aggregation events in solution. These new analytical
methods can contribute to a more efficient development of biopharmaceuticals by
filling the current severe need for high-throughput, quantitative and informative
experimental techniques. The collected experimental information may be used
as input for advanced computational tools which could use this higher quality
data to better describe protein behavior in solution and predict potential liabilities,
ultimately guiding protein candidate selection and rationalizing the impact
of formulation conditions. In the context of pathological protein aggregation in
vivo, the platforms provide initial steps to isolate key players involved in the
aggregation process, with potential implications in both the fundamental understanding of the aggregation process and the improvement of diagnostic biomarker
detection. Show more
Permanent link
https://doi.org/10.3929/ethz-b-000473751Publication status
publishedExternal links
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Publisher
ETH ZurichSubject
Bioprocessing; Microfluidics; Method development; Monoclonal antibody; DRUG DEVELOPMENT + DRUG DESIGN + DRUG DISCOVERY (PHARMACY); Analytical methods; PROTEIN AGGREGATION (PROTEIN MISFOLDING); Biomarker; liquid liquid phase separation; Developability; Protein stabilityOrganisational unit
09572 - Arosio, Paolo / Arosio, Paolo
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ETH Bibliography
yes
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