Multifactorial experimental design approach in forced degradation studies with a focus on the intensity of stress...

A robust understanding of structure-function relationships (SFRs) is fundamental for tailored and robust chemistry, manufacturing, and control (CMC) activities. Yet, conventional forced degradation studies (FDS) often employ a one-factor-at-a-time (OFAT) methodology, which commonly leads to correlated modifications and therefore limited reliability of SFR determination missing statistical certainty. By applying a forced-degradation-study design of experiments (FDS-DoE) framework to trastuzumab, methodological advantages are underscored, including improved data interpretation and consistency with literature findings, while adding deeper insights to the antibodies SFRs. As the choice of stress conditions in the design of FDS studies is critically important to establish meaningful SFRs, we present a detailed experimental comparison of harsh vs. moderate CMC development relevant stress conditions applying the FDS-DoE workflow. While harsh conditions mainly reflect typical FDS protocols and test the boundaries of a molecule's resilience, the moderate conditions were designed to simulate CMC-relevant environments better. Furthermore, we introduce a critical quality attribute (CQA) assessment framework that uses partial least square regression to systematically categorize modifications according to their statistical significance (via permutation testing) and predicted potency loss. The combination of multifactorial FDS-DoE with this CQA assessment framework increased statistical power over traditional OFAT methods, establishing a predictive, automated and scalable strategy for identifying relevant SFRs and CQAs, a capability especially advantageous for macromolecules in biopharmaceutical development.