Use of Raman spectroscopy and PCA for quality evaluation and out-of-specification identification in biopharmaceutical products.

ABSTRACT

Over the past three decades, there was a remarkable growth in the approval of antibody-based biopharmaceutical products. These molecules are notably susceptible to the stresses occurring during drug manufacturing, often leading to structural alterations. A key concern is thus the ability to detect and comprehend these alterations caused by processes, such as aggregation, fragmentation, oxidation levels, as well as the change in protein concentration throughout the process steps, potentially resulting in out-of-spec products. In the present study, Raman spectroscopy, coupled with Principal Component Analysis (PCA), has proven to be an excellent tool for characterizing protein-based products. Notably, it offers the advantages of being minimally invasive, rapid and relatively insensitive to water. Therefore, it was successfully employed to discriminate between various stresses impacting a monoclonal antibody (mAb). The molecule used in this study is a fully human IgG1 fusion protein. Thermal stress was induced by incubating the samples at 50 °C for one month, while oxidative stress was induced by introducing hydrogen peroxide. Additionally, dilutions were performed to explore a broader range of protein concentrations. Specific key bands were identified in the Raman spectra, which facilitated the PCA classification and allowed for their association with distinct changes in the secondary and tertiary structures of the protein. Notably, it was observed that signals corresponding to amino acids exhibited a decrease in intensity with increasing levels of thermal stress, while other alterations were noted in the amide bands. It was shown that changes in the range 2800-3000 cm-1 pertains to the dilution process, while specific peaks of C-H stretching were essential for the discrimination between the oxidative-stressed samples and the thermal and diluted counterparts. Furthermore, the model calibrated on the mAb demonstrated remarkable performance when used to evaluate a different product, e.g. a hormone.