Application of Raman spectroscopy during pharmaceutical process development for determination of critical quality...

Raman spectroscopy is considered a Process Analytical Technology (PAT) tool in biopharmaceutical downstream processes. In the past decade, researchers have shown Raman spectroscopy's feasibility in determining Critical Quality Attributes (CQAs) in bioprocessing. This study verifies the feasibility of implementing a Raman-based PAT tool in Protein A chromatography as a CQA monitoring technique, for the purpose of accelerating process development and achieving real-time release in manufacturing. A system connecting Raman to a Tecan liquid handling station enables high-throughput model calibration. One calibration experiment collects Raman spectra of 183 samples with 8 CQAs within 25 h. After applying Butterworth high-pass filters and k-nearest neighbor (KNN) regression for model training, the model showed high predictive accuracy for fragments (Q² = 0.965) and strong predictability for target protein concentration, aggregates, as well as charge variants (Q²≥ 0.922). The model's robustness was confirmed by varying the elution pH, load density, and residence time using 19 external validation preparative Protein A chromatography runs. The model can deliver elution profiles of multiple CQAs within a set point ± 0.3 pH range. The CQA readouts were presented as continuous chromatograms with a resolution of every 28 s for enhanced process understanding. In external validation datasets, the model maintained strong predictability especially for target protein concentration (Q² = 0.956) and basic charge variants (Q² = 0.943), except for overpredicted HCP (Q² = 0.539). This study demonstrates a rapid, effective method for implementing Raman spectroscopy for in-line CQA monitoring in process development and biomanufacturing, eliminating the need for labor-intensive sample pooling and handling.