Quantitative Proteomics and CRISPR/Cas9 Editing Reveal UPR‐Mediated Control of Immunoglobulin Homeostasis in Hybridomas

ABSTRACT Despite their tremendous economic value, monoclonal antibodies are often compromised by the loss of immunoglobulin (Ig) chains, which disrupts antibody homeostasis and quality control. Through subclone screening and characterization, we identified that the loss of Ig production impaired the recognition ability of hapten‐specific hybridomas. Proteomic analysis further highlighted the critical role of the unfolded protein response (UPR) pathway in regulating aberrant Ig chain production. Using CRISPR/Cas9‐mediated knockout and rescue experiments, we revealed the importance of the UPR pathway in facilitating hybridoma antibody production by targeting Xbp1 s, an active transcription factor downstream of UPR signaling. With CRISPR/HDR, we inserted a fluorescent mGFP tag into the endogenous Hspa5 gene (encoding BiP, the master regulator of the UPR pathway), enabling in situ and real‐time monitoring of UPR activation. A strong negative correlation (R ² = 0.86) was observed between intracellular mGFP signals and IgG levels in the engineered system, indicating a close relationship between UPR activation and Ig production. Fluorescence‐activated cell sorting of high‐mGFP populations identified two dysfunctional subclones that failed to secret Ig, validating the system’s effectiveness in tracing Ig homeostasis. In summary, this study provides new insights into UPR‐mediated regulation of Ig synthesis and offers a novel UPR‐based reporter system for monitoring antibody stability.