dc.description.abstract
Understanding and controlling antibody stability is crucial in biopharmaceutical development, as
protein misfolding, denaturation, and aggregation - caused by stress factors such as temperature
changes, pH variations, mechanical stress, or light exposure – can compromise the antibody's
biological functionality.(1,2) These structural changes may lead to a loss of therapeutic efficacy or
even trigger immune responses in patients.(2) This study investigates the potential of atomic force
microscopy coupled to infrared spectroscopy (AFM-IR)(3) for nanoscale characterization of
immunoglobulin G (IgG) antibodies in both their native and denatured states, captured by a
biosensor, thus providing high-resolution chemical and structural insights into this model protein
system. The introduction of a biosensor offers distinct selectivity for analysing the targeted
antibodies, but the AFM-IR system requires a homogeneous sensor surface with a thin layer of
covalently anchored selective capture proteins for IgG that absorb minimally in the targeted
infrared regions, all while functioning under ambient conditions.
A novel biosensor design is introduced, utilizing direct surface functionalization of silicon
substrates with covalently immobilized antibody-capturing proteins. These proteins exhibit high
yet reversible selective affinity for the Fc region of IgG(4), allowing for straightforward biosensor
regeneration through pH variation, which enhances the biosensor’s applicability. The system’s
early-stage success is demonstrated by silicon substrate functionalization with (3
Aminopropyl)triethoxysilane (APTES) and glutaraldehyde (GA), verified through AFM scans and
water contact angle analysis of the surfaces. Ongoing research is focused on model protein binding
tests, with particular emphasis on optimizing antibody-capturing protein G immobilization
strategies.
By integrating a reversible biosensor design for AFM-IR analysis, this work introduces a novel
approach for studying native and denatured antibodies at the nanoscale. This approach holds the
potential for enhancing quality control in biopharmaceutical production by enabling the
development of inline sensors for antibody stability testing during bioprocessing.
(1) Hawe, A.; Wiggenhorn, M.; Van De Weert, M.; Garbe, J. H. O.; Mahler, H.; Jiskoot, W. Forced Degradation
of Therapeutic Proteins. J. Pharm. Sci. 2012, 101 (3), 895–913. https://doi.org/10.1002/jps.22812.
(2) Pease III, L. F.; Elliott, J. T.; Tsai, D.-H.; Zachariah, M. R.; Tarlov, M. J. Determination of Protein
Aggregation with Differential Mobility Analysis: Application to IgG Antibody. Biotechnol. Bioeng.
2008, 101 (6), 1214–1222. https://doi.org/10.1002/bit.22017.
(3) Dazzi, A.; Glotin, F.; Carminati, R. Theory of Infrared Nanospectroscopy by Photothermal Induced
Resonance. J. Appl. Phys. 2010, 107 (12), 124519. https://doi.org/10.1063/1.3429214.
(4) Budde, B.; Schartner, J.; Tönges, L.; Kötting, C.; Nabers, A.; Gerwert, K. Reversible Immuno-Infrared
Sensor for the Detection of Alzheimer’s Disease Related Biomarkers. ACS Sens. 2019, 4 (7), 1851
1856. https://doi.org/10.1021/acssensors.9b00631.
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