In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy

The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfatepolyacrylamide gel electrophoresis for identifying fragmentation, ion exchange chromatography and isoelectric focusing for identifying deamidation, reverse-phase high-performance liquid chromatography (HPLC) for identifying oxidation, and size-exclusion HPLC for identifying aggregation. These stability assessments require essential processes that are destructive to the product tested. All these techniques are lab based and require sample removal from a sealed storage vial, which can breach the sterility. In this work, we investigate the heat-and surfactant-induced denaturation of an in-vial stored model protein, bovine serum albumin (BSA), by analyzing its intrinsic fluorescence without removing the sample from the vial. A lab-based bespoke setup which can do the measurement in vial is used to demonstrate the change in fluorescence polarization of the protein to determine the denaturation level. The results obtained are compared to circular dichroism and size-exclusion HPLC measurements. The results prove that in-vial fluorescence measurements can be performed to monitor protein denaturation. A costeffective portable solution to provide a top-level overview of biopharmaceutical product stability from manufacture to the point of patient administration can be further developed using the same technique.

Automated summary

Conventional quality control techniques for assessing biopharmaceutical denaturation are destructive and require sample removal. In this study, a lab-based setup was used to analyze the heat- and surfactant-induced denaturation of bovine serum albumin (BSA) stored in-vial by measuring its intrinsic fluorescence. This in-vial fluorescence measurement proved to be a reliable method for monitoring protein denaturation, offering a potential cost-effective solution for assessing biopharmaceutical product stability.