Abstract
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.
Highlights
Biopharmaceuticals are an important part of the drug portfolio of most major pharmaceutical companies
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 close agreement indicates a similar secondary structure for all mutants, with only a small difference observed in the circular dichroism (CD) spectra of the DSV mutant versus the wild type (WT)
Summary
Biopharmaceuticals are an important part of the drug portfolio of most major pharmaceutical companies. Biologic drug candidates are used to treat metabolic, cardiovascular, cancer, autoimmune and infectious diseases, amongst others. Proteinaceous products include peptides, enzymes, monoclonal antibodies (mAbs), antibody-like proteins and other scaffolds and fusions [91,92]; all of which may suffer physical and chemical instability. Physical instability involves adsorption, unfolding and aggregation, all of which may occur cooperatively rather than in isolation [86]. The long term loss of monomer is generally predicted through quantitative monitoring of aggregation under accelerated and stress conditions over weeks to months. Recent progress has been made in: (i) the development of detailed kinetic models [2,4,5,34,42,49,60,96]; (ii) correlating aggregation kinetics with protein structure and folding [11,15,16,32,35,52,53,54,67,88]; (iii) and with native-state protein-protein interaction measurements [36,46,57,74,75,79,82]
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