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Eur J Pharm Biopharm. 2017 Jun;115:18-30. doi: 10.1016/j.ejpb.2017.01.019. Epub 2017 Feb 1.

The effect of charge mutations on the stability and aggregation of a human single chain Fv fragment.

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Faculty of Biology, Medicine and Health, University of Manchester, Manchester M13 9PT, United Kingdom.
Faculty of Science and Engineering, University of Manchester, Manchester M1 7DN, United Kingdom.
Drug Product Services, Lonza, Basel 4002, Switzerland.
School of Chemistry, University of Manchester, Manchester M1 7DN, United Kingdom.
Manchester Pharmacy School, University of Manchester, M13 9PL, United Kingdom.
Forumulation Sciences, MedImmune Ltd, Granta Park, Cambridge CB21 6GH, United Kingdom.
School of Chemical Engineering and Analytical Science, University of Manchester, M1 7DN, United Kingdom. Electronic address:


The aggregation propensities for a series of single-chain variable fragment (scFv) mutant proteins containing supercharged sequences, salt bridges and lysine/arginine-enriched motifs were characterised as a function of pH and ionic strength to isolate the electrostatic contributions. Recent improvements in aggregation predictors rely on using knowledge of native-state protein-protein interactions. Consistent with previous findings, electrostatic contributions to native protein-protein interactions correlate with aggregate growth pathway and rates. However, strong reversible self-association observed for selected mutants under native conditions did not correlate with aggregate growth, indicating 'sticky' surfaces that are exposed in the native monomeric state are inaccessible when aggregates grow. We find that even though similar native-state protein-protein interactions occur for the arginine and lysine-enriched mutants, aggregation propensity is increased for the former and decreased for the latter, providing evidence that lysine suppresses interactions between partially folded states under these conditions. The supercharged mutants follow the behaviour observed for basic proteins under acidic conditions; where excess net charge decreases conformational stability and increases nucleation rates, but conversely reduces aggregate growth rates due to increased intermolecular electrostatic repulsion. The results highlight the limitations of using conformational stability and native-state protein-protein interactions as predictors for aggregation propensity and provide guidance on how to engineer stabilizing charged mutations.


Aggregation; Charged mutations; Protein stability; Protein-protein interactions; ScFv

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