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Improving the Developability of an Antigen Binding Fragment by Aspartate Substitutions
Laila I Sakhnini1,2, Per J Greisen1, Charlotte Wiberg1
1Global Research Technologies , Novo Nordisk A/S , 2760 Måløv , Denmark.
Biochemistry
|May 24, 2019
Summary
Engineering antibody fragments with aspartate substitutions significantly enhances developability by improving stability and reducing aggregation without impacting binding affinity. This strategy offers a rapid method for developing better antibody-based therapeutics.
Area of Science:
- Biotechnology
- Pharmaceutical Science
- Protein Engineering
Background:
- Antibody aggregation poses a significant challenge in pharmaceutical development, affecting product quality during manufacturing and administration.
- Early developability assessment is crucial for identifying and removing problematic antibody molecules, but it is resource-intensive.
- Strategies for engineering enhanced developability are highly sought after to streamline drug discovery.
Purpose of the Study:
- To investigate if aspartate (Asp) substitutions can improve the developability of a murine antigen-binding fragment (Fab).
- To assess the impact of Asp substitutions on thermodynamic stability, aggregation propensity, nonspecificity, and binding affinity of the Fab.
Main Methods:
- A combinatorial library of 393 Fab variants with varying Asp substitutions was created.
- In silico screening using Rosetta predicted thermodynamic stability for variant selection.
- Selected variants underwent production and rigorous developability studies, including stability, aggregation, and binding assays.
Main Results:
- Most engineered Fab variants demonstrated thermodynamic stability comparable to or better than the wild type.
- Twenty-five variants showed improved nonspecificity, and half exhibited enhanced aggregation resistance.
- Crucially, Asp substitutions improved developability without substantially affecting the antigen-binding affinity.
Conclusions:
- Combining negative charge insertion (Asp substitutions) with in silico screening rapidly enhances Fab developability.
- This approach successfully improved aggregation resistance and stability while maintaining binding affinity.
- The findings provide a valuable strategy for engineering antibody-based therapeutics with improved developability profiles.
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