Diversity and Complexity of Proteins
Aggregation can be a problem even for established protein products, according to Tudor Arvinte, Ph.D., chairman and CEO of Therapeomic. “Proteins aggregate, degrade, bind to cell walls, and generate fibrils,” he states. Because of their dynamic properties, the conventional analytical methods are not well suited to understand how a protein behaves when internalized.
Aggregation of protein therapeutic molecules can trigger a toxicity effect, compromising the effectiveness of the drug. Dr. Arvinte and his colleagues have monitored performance using dyes that specifically stain aggregates or other types of protein-degradation products. One method uses Nile Red, making aggregates visible for qualitative and quantitative analysis, allowing a fast screen of different solutions for their aggregation potential.
Protein aggregate analysis is demanding, requiring the use of various technologies including asymmetrical field flow fractionation, fluorescence spectroscopy, fluorescence microscopy, and transmission electron microscopy. Some procedures are especially informative under particular conditions, so the investigator may need to pick and choose in order to develop an accurate picture of the changes that the molecules undergo.
Dr. Arvinte discussed the formulation problems associated with Herceptin, which is supplied as a lyophilized powder. Dr. Arvinte says that the formulation described in the patient information leaflet is unduly complex and employs a novel chemical entity, apparently required for the stabilization of the Herceptin antibody. His team’s experience indicated that extremely subtle handling issues, down to the speed of injection, can create aggregates.
Following a warning on the label to refrain from the use of a dextrose diluent, Dr. Arvinte and his coworkers determined that dextrose-containing diluents caused the formation of aggregates and made the preparation clinically unacceptable.
Therapeomics has developed a set of guidelines that eliminate components that trigger aggregation and other sub-optimal formulation options. Because of the diversity of antibody molecules, a wide variety of individualized formulation options must be designed that take into account antibody flexibility and stability, and no two diluents solutions will be exactly the same.
Dr. Arvinte argues that companies that ignore a thorough investigation of formulation needs during product development do so at their peril. “Whereas the biology of most protein-based therapeutics may be well understood, the biophysical changes that they may undergo can constitute a profound unknown of critical significance to the success of the final product,” he concluded.