Enhanced Thermal Stability and Reduced Aggregation in an Antibody Fab Fragment at Elevated Concentrations

Zhang, Cheng, Bye, Jordan W., Lui, Lok H., Zhang, Hongyu, Hales, John, Brocchini, Steve, Curtis, Robin A. and Dalby, Paul A. 2023. Enhanced Thermal Stability and Reduced Aggregation in an Antibody Fab Fragment at Elevated Concentrations. Molecular Pharmaceutics 20 (5) , pp. 2650-2661. 10.1021/acs.molpharmaceut.3c00081

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Abstract

The aggregation of protein therapeutics such as antibodies remains a major challenge in the biopharmaceutical industry. The present study aimed to characterize the impact of the protein concentration on the mechanisms and potential pathways for aggregation, using the antibody Fab fragment A33 as the model protein. Aggregation kinetics were determined for 0.05 to 100 mg/mL Fab A33, at 65 °C. A surprising trend was observed whereby increasing the concentration decreased the relative aggregation rate, ln(v) (% day–1), from 8.5 at 0.05 mg/mL to 4.4 at 100 mg/mL. The absolute aggregation rate (mol L–1 h–1) increased with the concentration following a rate order of approximately 1 up to a concentration of 25 mg/mL. Above this concentration, there was a transition to an apparently negative rate order of −1.1 up to 100 mg/mL. Several potential mechanisms were examined as possible explanations. A greater apparent conformational stability at 100 mg/mL was observed from an increase in the thermal transition midpoint (Tm) by 7–9 °C, relative to those at 1–4 mg/mL. The associated change in unfolding entropy (△Svh) also increased by 14–18% at 25–100 mg/mL, relative to those at 1–4 mg/mL, indicating reduced conformational flexibility in the native ensemble. Addition of Tween or the crowding agents Ficoll and dextran, showed that neither surface adsorption, diffusion limitations nor simple volume crowding affected the aggregation rate. Fitting of kinetic data to a wide range of mechanistic models implied a reversible two-state conformational switch mechanism from aggregation-prone monomers (N*) into non-aggregating native forms (N) at higher concentrations. kD measurements from DLS data also suggested a weak self-attraction while remaining colloidally stable, consistent with macromolecular self-crowding within weakly associated reversible oligomers. Such a model is also consistent with compaction of the native ensemble observed through changes in Tm and △Svh.

Item Type:	Article
Date Type:	Publication
Status:	Published
Schools:	Schools > Biosciences
Publisher:	American Chemical Society
ISSN:	1543-8384
Date of Acceptance:	3 April 2023
Last Modified:	28 Nov 2024 14:31
URI:	https://orca.cardiff.ac.uk/id/eprint/173703

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