Raman spectroscopy for monitoring free sulfhydryl formation during monoclonal antibody manufacturing

During production, harvested cell culture fluid (HCCF) can degrade due to reductases breaking interchain disulfide bonds, forming low molecular weight (LMW) impurities that contain free sulfhydryl and high molecular weight (HMW) impurities through disulfide shuffling. Thus, detecting and quantifying the free sulfhydryl increase in HCCF is critical. Herein, Raman spectroscopy is implemented as a process analytical technology, and multivariate data analysis is applied to characterize and quantify sulfhydryl formation in HCCF with disulfide-containing indicator molecules. Raman spectra qualitatively probe the presence or absence of disulfide bond breakage in antibodies, consistent with offline non-reduced capillary electrophoresis sodium dodecyl sulfate results. Between two antibodies studied, mAb A was identified for a higher risk of antibody reduction where sulfhydryl formation was observed within 16 h, while mAb B did not show similar concerns even after 1 week. The offline measurement of redox potential is below -100 mV in HCCF for mAb A, while the stable mAb B HCCF shows redox potentials above +20 mV. A multivariate partial least squares (PLS) model for quantification is developed using an offline free sulfhydryl assay, applying Raman spectra to predict free sulfhydryl concentration with high accuracy (R² > 0.98) and expected mean error of 0.677 mM from the offline Ellman's Assay. This work confirms the use of Raman PAT to monitor real-time disulfide reduction, enabling improvements to process understanding and product quality.