Combining Structural Aggregation Propensity and Stability Predictions To Redesign Protein Solubility

Abstract

The aggregation propensity of each particular protein seems to be shaped by evolution according to its natural abundance in the cell. The production and downstream processing of recombinant polypeptides implies attaining concentrations that are orders of magnitude above their natural levels, often resulting in their aggregation; a phenomenon that precludes the marketing of many globular proteins for biomedical or biotechnological applications. Therefore, there is a huge interest in methods aimed to increase the proteins solubility above their natural limits. Here, we demonstrate that an updated version of our AGGRESCAN 3D structural aggregation predictor, that now takes into account protein stability, allows for designing mutations at specific positions in the structure that improve the solubility of proteins without compromising their conformation. Using this approach, we have designed a highly soluble variant of the green fluorescent protein and a human single-domain VH antibody displaying significantly reduced aggregation propensity. Overall, our data indicate that the solubility of unrelated proteins can be easily tuned by in silico-designed nondestabilizing amino acid changes at their surfaces.

Models for Antibody Behavior in Hydrophobic Interaction Chromatography and in Self-Association

“…Previous work from our group has focussed on the identification of hydrophobic and charged surface patches using electrostatic calculations 17,18 . Other theoretical approaches to improving antibody solution behaviour have invoked the role of hydrophobicity in the self-association of antibodies 12,19 . However, theoretical approaches for understanding and predicting antibody self-association have been limited by a lack of publically available datasets.…”

Protocols for rational design of protein solubility and aggregation properties using Aggrescan3D standalone

“…These predictions can be assessed using A3D with user-indicated mutations (-m option). Such exercises, were presented in our previous work 26 , where the effects of cumulative mutations for aggregation-prone residues detected by A3D were analyzed and confirmed experimentally, resulting in the triple mutant GFP/KKK as folded, stable and the most soluble variant (PDB: 6FWW).…”

Current Advancements in Addressing Key Challenges of Therapeutic Antibody Design, Manufacture, and Formulation

“…In particular, surface exposed hydrophobicity is sought as the lead mechanism for protein self-association driving aggregation propensity, in which aggregation-prone regions (APRs) are identified in the mAb structure. The substitution of identified APRs to gatekeeper (i.e., polar) amino acids has experimentally demonstrated resistance to aggregation and improved solubility, which validates this strategy for improving mAb stability [36,37,38,39,40,166,167,168,169,170]. The majority of APRs identified are located in biologically relevant mAb regions—those being the CDRs and the Fc.…”