Multi-attribute Raman spectroscopy (MARS) for monitoring product quality attributes in formulated monoclonal antibody therapeutics.

Rapid release of biopharmaceutical products enables a more efficient drug manufacturing process. Multi-attribute methods that target several product quality attributes (PQAs) at one time are an essential pillar of the rapid-release strategy. The novel, high-throughput, and nondestructive multi-attribute Raman spectroscopy (MARS) method combines Raman spectroscopy, design of experiments, and multivariate data analysis (MVDA). MARS allows the measurement of multiple PQAs for formulated protein therapeutics without sample preparation from a single spectroscopic scan. Variable importance in projection analysis is used to associate the chemical and spectral basis of targeted PQAs, which assists in model interpretation and selection. This study shows the feasibility of MARS for the measurement of both protein purity-related and formulation-related PQAs; measurements of protein concentration, osmolality, and some formulation additives were achieved by a generic multiproduct model for various protein products containing the same formulation components. MARS demonstrates the potential to be a powerful methodology to improve the efficiency of biopharmaceutical development and manufacturing, as it features fast turnaround time, good robustness, less human intervention, and potential for automation.

Structure-Based Correlation of Light-Induced Histidine Reactivity in A Model Protein.

Light is known to induce covalently linked aggregates in proteins. These aggregates can be immunogenic and are of concern for drug product development in the biotechnology industry. Histidine (His) is proposed to be a key residue in cross-link generation ( Pattison , D. I. Photochem. Photobiol. Sci. 2012 , 11 , 38 - 53 ). However, the factors that influence the reactivity of His in proteins, especially the intrinsic factors are little known. Here, we used rhDNase, which only forms His-His covalent dimers after light treatment to determine the factors that influence the light-induced reactivity of His. This system allowed us to fully characterize the light-induced covalent dimer and rank the reactivities of the His residues in this protein. The reactivities of these His residues were correlated with solvent accessibility-related parameters both by crystal structure-based calculations of solvent-accessible surface area and by hydrogen-deuterium exchange (HDX) experiments. Through this correlation, we demonstrate that the photoreactivity of His is determined by both solvent accessibility and structural flexibility. This new insight can explain the highly complex chemistry of light-induced aggregation and help predict the aggregation propensity of protein under light treatment.