Challenges for design of aggregation-resistant variants of granulocyte colony-stimulating factor

Non-native protein aggregation is a long-standing issue in pharmaceutical biotechnology. A rational design approach was used in order to identify variants of recombinant human granulocyte colony-stimulating factor (rhG-CSF) with lower aggregation propensity at solution conditions that are typical of commercial formulation. The approach used aggregation-prone-region (APR) predictors to select single amino acid substitutions that were predicted to decrease intrinsic aggregation propensity (IAP). The results of static light scattering temperature-ramps and chemical unfolding experiments demonstrated that none of the selected variants exhibited improved aggregation resistance, and the apparent conformational stability of each variant was lower than that of WT. Aggregation studies under partly denaturing conditions suggested that the IAP of at least one variant remained unaltered. Overall, this study highlights a general challenge in designing aggregation resistance for proteins, due to the need to accurately predict both APRs and conformational stability.