Implementation of inline Raman spectroscopy for buffer exchange monitoring in monoclonal antibody formulation.

Buffer exchange is a critical step in the formulation of monoclonal antibodies, as it ensures protein stability in an appropriate medium. Traditional offline methods used to monitor this process are slow and provide delayed feedback. In contrast, Raman spectroscopy offers a fast, inline, non-invasive alternative that aligns with the principles of Process Analytical Technology (PAT) and Quality by Design (QbD). This study aimed to evaluate the application of inline Raman spectroscopy for real-time monitoring of excipients-specifically sucrose, histidine, mannitol, and hydroxypropyl-β-cyclodextrin (HPβCD)-during the buffer exchange process of adalimumab-based formulations. Partial least squares (PLS) calibration models were developed using two approaches: conventional offline measurements and placebo (protein-free) inline process data. These models were validated under real processing conditions involving monoclonal antibodies. All PLS models demonstrated strong predictive performance (R2 > 0.91; low RMSEP and RSEP values). Offline calibration was rapid, and accurate, while placebo-based models showed greater robustness in actual processing environments, especially for sucrose and mannitol, whereas offline calibration was more suitable for histidine. The results from inline monitoring confirmed the real-time applicability of the models. In summary, Raman spectroscopy, when combined with validated chemometric models, enables the real-time quantification of multiple excipients during buffer exchange. This approach offers a fast, non-destructive and cost-effective alternative to HPLC, enhancing process control, manufacturing efficiency, and product quality in continuous biomanufacturing.