Advancing forced degradation studies: Design of experiments for enhanced structure-function relationship analysis in biotherapeutics

Abstract

Understanding the relationship between structure and function is crucial in drug development. Recombinant proteins present unique challenges when establishing structure–function relationships (SFR) due to the extensive options for potential modifications across numerous residues. Even simple stress studies lack degradation selectivity as they lead to multiple, simultaneous modifications. Co-occurring modifications at amino acid residues make direct correlations with a specific stressor and resolving the causal impact on functionality difficult. The introduction of more variance into the data set would reduce the correlation structure. In this study, we demonstrate the feasibility and benefits of a multifactorial design approach for SFR establishment. Design of experiments (DoE) allows for the parallel investigation of stress factors via combined experiments, resulting in a higher variance of stress conditions and consequently a broader variation in degradation. This approach enables a more insightful correlation analysis along with model-based data evaluation strategies, thereby facilitating significantly improved data interpretation results during SFR studies.