An in vivo platform to select and evolve aggregation-resistant proteins

Abstract

Protein biopharmaceuticals are highly successful, but their utility is compromised by their propensity to aggregate during manufacture and storage. As aggregation can be triggered by non-native states, whose population is not necessarily related to thermodynamic stability, prediction of poorly-behaving biologics is difficult, and searching for sequences with desired properties is labour-intensive and time-consuming. Here we show that an assay in the periplasm of E. coli linking aggregation directly to antibiotic resistance acts as a sensor for the innate (un-accelerated) aggregation of antibody fragments. Using this assay as a directed evolution screen, we demonstrate the generation of aggregation resistant scFv sequences when reformatted as IgGs. This powerful tool can thus screen and evolve ‘manufacturable’ biopharmaceuticals early in industrial development. By comparing the mutational profiles of three different immunoglobulin scaffolds, we show the applicability of this method to investigate protein aggregation mechanisms important to both industrial manufacture and amyloid disease.

Metadata

Item Type:	Article
Authors/Creators:	Ebo, JS https://orcid.org/0000-0002-5684-4355 Saunders, JC Devine, PWA Gordon, AM Warwick, AS Schiffrin, B https://orcid.org/0000-0002-1670-7356 Chin, SE https://orcid.org/0000-0002-9103-9028 England, E https://orcid.org/0000-0001-9581-0046 Button, JD Lloyd, C Bond, NJ https://orcid.org/0000-0002-0312-7360 Ashcroft, AE https://orcid.org/0000-0002-1676-123X Radford, SE https://orcid.org/0000-0002-3079-8039 Lowe, DC Brockwell, DJ https://orcid.org/0000-0002-0802-5937
Copyright, Publisher and Additional Information:	© The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/.
Dates:	Accepted: 19 March 2020 Published (online): 14 April 2020 Published: 14 April 2020
Institution:	The University of Leeds
Academic Units:	The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Biomolecular Mass Spectroscopy (Leeds) The University of Leeds > Faculty of Biological Sciences (Leeds) > School of Molecular and Cellular Biology (Leeds) > Structural Molecular Biology 2 (Leeds)
Funding Information:	Funder Grant number BBSRC (Biotechnology & Biological Sciences Research Council) BB/M01259X/1 BBSRC (Biotechnology & Biological Sciences Research Council) BB/N007603/1 BBSRC (Biotechnology & Biological Sciences Research Council) BB/T000635/1 Wellcome Trust 204963/Z/16/Z
Depositing User:	Symplectic Publications
Date Deposited:	20 Mar 2020 17:01
Last Modified:	17 Dec 2024 15:19
Status:	Published
Publisher:	Nature Research
Identification Number:	10.1038/s41467-020-15667-1
Related URLs:	Dataset
Open Archives Initiative ID (OAI ID):	oai:eprints.whiterose.ac.uk:158603