In silico high throughput mutagenesis and screening of signal peptides to mitigate N-terminal heterogeneity of recombinant monoclonal antibodies.

This study focuses on in silico high throughput mutagenesis and screening of signal peptides to mitigate n-terminal heterogeneity of recombinant monoclonal antibodies.. The research employs high-performance liquid chromatography (HPLC) techniques to address analytical challenges in the biopharmaceutical field. N-terminal heterogeneity resulting from non-uniform signal peptide (SP) cleavage can potentially affect biologics property attributes and result in extended product development timelines. Few studies are available on engineering SPs systematically to address miscleavage issues. Herein, we developed a novel high throughput computational pipeline capable of generating millions of SP mutant... Research Background and Significance Recombinant monoclonal antibodies (mAbs) represent a cornerstone of modern biopharmaceutical therapeutics due to their specificity and clinical efficacy. However, a persistent challenge in mAb production is the presence of N-terminal heterogeneity, primarily caused by non-uniform cleavage of signal peptides (SPs). This heterogeneity can significantly impact the physicochemical properties, biological activity, and immunogenicity of mAbs, ultimately complicating product characterization and regulatory approval processes. Engineering signal peptides to improve cleavage precision is therefore critical to ensuring consistent product quality and reducing development timelines. Despite its importance, systematic approaches to optimize SP sequences have been limited, partly due to the complexity of screening vast mutant libraries. The study by Yu et al. (2022) addresses this gap by employing an innovative in silico high throughput mutagenesis and screening pipeline. Coupling computational prediction with experimental validation via high-performance