Reducing Insoluble Protein Aggregation with CLEAR: A Minimalist, Charge-Laden Peptide Bristle for scFv-Based Proteins

Abstract Protein aggregation limits the manufacturability and long-term storage of biologics. Here, we evaluated Charge-Laden Elastin-like Polypeptide-based Aggregation Reduction (CLEAR) tags in two aggregation-prone scFv-based Albubody proteins. Negatively charged variants, particularly CLEAR-E9, maintained low aggregation indices for up to 28 days and showed smaller particle sizes than the corresponding untagged proteins after 7 days at 37 °C. After 7 days, A-E9 and T-E9 retained approximately 100% and 95% of their day-0 soluble protein, compared with 87% and 40% for the corresponding untagged proteins. SEC showed persistent soluble higher-order association, indicating that CLEAR-E9 primarily limits progression toward large insoluble aggregates rather than preventing intermolecular association altogether. In Ate_Albu, CLEAR-E3 (1.34 kDa) retained a low aggregation index, and CLEAR-E9 showed higher soluble yield and a lower aggregation index than polyE9 and SynIDP3 controls. These results support CLEAR as a short, sequence-programmable tag for reducing insoluble protein loss in the tested scFv-based systems.