Rapid detection and characterization of protein aggregates for improved drug formulation

Proteins, such as monoclonal antibodies, represent an increasingly important class of therapeutic drugs. Unfortunately, the formation of protein aggregates and other sub-visible particles during manufacturing, shipping and storage is a major concern as they can have a negative impact on drug activity and cause unwanted immunogenicity in patients. In view of this, particle characterization and monitoring remains a crucial step for the pharmaceutical industry and regulatory agencies. Biochemical assays for monitoring protein aggregates often rely upon ultracentrifugation, size-exclusion chromatography, gel electrophoresis, dynamic light scattering, or turbidity measurements. These techniques are not applicable for every protein, nor are the assays ideal for addressing wide range of aggregation problems encountered during formulation development and the manufacture of protein pharmaceuticals. There is, currently, no accepted method to effectively and simultaneously size, enumerate and classify particles within the critical size range of 1-100 µm. To address this need, scientists at Enzo Life sciences developed the ProteoStat® reagent, a fluorescent dye that facilitates detection and quantification of aggregated proteins in solution. This dye is an example of a molecular rotor-type fluorochrome in which exhibits a significant increase in fluorescence quantum yield in the presence of protein aggregates. As silicone oil micro-droplets are a common contaminant in protein-based therapeutics, our scientists developed a flow cytometry protocol relying on the use of the ProteoStat® detection reagent together with the Bodipy dye to simultaneously examine protein aggregates and discriminate them from other particles. More recently, Dr. Christine Probst and colleagues from Amnis (an EMD Millipore company) demonstrated the labeling specificity of the ProteoStat® dye for protein aggregates using their Amnis ImageStreamX MKII® (ISX) imaging flow cytometer. Using this specific platform, they were able to directly measure aggregate size, classify different particle types by incorporating additional fluorescent stains, collect significant and reliable data sets via rapid image acquisition, and measure absolute particle concentrations, even in a volume as small as 20 µl. This high-throughput screening capability allows for the optimization of different protein drug formulations while ensuring the production of the most stable and particle-free formulations.

Enzo Life Sciences offers a comprehensive bioprocess portfolio to optimize and monitor product integrity and efficiency including protein aggregation and thermal shift stability kits; some of which are described below:

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