In-Vial Detection of Protein Denaturation Using Intrinsic Fluorescence Anisotropy.

The conventional quality control techniques for identifying the denaturation of biopharmaceuticals includes sodium dodecyl sulfate-polyacrylamide 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 cost-effective 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.

Fluorescence spectroscopy for the determination of reconstitution time of an in-vial lyophilised product.

Lyophilisation is a prominent technique used to create stabilised, dried forms of biopharmaceutical formulations. Reconstitution of lyophilised parenteral formulations is a key step prior to patient administration. The accurate determination of reconstitution time is a necessity to aid formulation development and support product quality control. Traditional methods for quantifying reconstitution time involve the visual identification of the endpoint, which has led to variable values reported across studies. In this work, the use of ultra-violet (UV) excited fluorescence spectroscopy as an alternative to the visual quantification of the reconstitution time was investigated. Spectrographic information was collected via a bespoke setup that allowed the measurement of the reconstitution time in a standard sealed lyophilisation vial. The spectra were analysed via principal component analysis (PCA) to obtain a time-based representation of the changes in a reconstituting formulation. The analysis was followed by the identification of an endpoint using three techniques ranging from fully automated to manual with regards to the required level of user input. At high protein concentration, the variability of the reconstitution time measurements was reduced from 80.4% relative standard deviation obtained via the traditional method to 8.2% for the instrumental method presented in.