Effect of Arginine on Protein Aggregation Studied by Fluorescence Correlation Spectroscopy and Other Biophysical Methods

Arginine has been used extensively as an excipient in the formulation development of proteinbased biopharmaceuticals. We investigate the role of arginine in suppressing protein aggregation and its mechanism by using bovine serum albumin as a model system. By using sedimentation velocity and other analytical techniques, we show that the use of arginine inhibits temperature-induced aggregation of the protein. We use fluorescence correlation spectroscopy and other spectroscopic techniques to show that arginine inhibits accumulation of partially folded intermediates, potentially involved in the aggregation process. The hydrodynamic radii of the protein in its native, unfolded, and intermediate states have been determined using fluorescence correlation spectroscopy at single-molecule resolution. A possible mechanism of the effects of arginine and its role as an aggregation suppressor has been discussed

Protein aggregation and bioprocessing

Protein aggregation is a common issue encountered during manufacture of biotherapeutics. It is possible to influence the amount of aggregate produced during the cell culture and purification process by carefully controlling the environment (eg, media components) and implementing appro-priate strategies to minimize the extent of aggregation. Steps to remove aggregates have been successfully used at a manufacturing scale. Care should be taken when developing a process to monitor the compatibility of the equipment and process with the protein to ensure that potential aggregation is minimized

Single-step formation of degradable intracellular biomolecule microreactors

Here we present a single-step all-aqueous approach to encapsulate biomolecules such as enzymes and proteins into stable microreactors. Key In this method is the use of spray-drying of the biomolecules of interest in combination with oppositely. charged,polyelectrolytes and mannitol as the sacrificial template. Remarkably, upon spray-drying in the presence of polyelectrolyte, mannitol crystallization is suppressed and the obtained amorphous mannitol offers enhanced preservation of the biomolecules' activity. Moreover, the use of mannitol allows the formation of nanopores within the microparticles upon rehydration of the microparticles in aqueous medium and subsequent dissolution, of the mannitol. The oppositely charged polyelectrolytes provide a polymeric framework which stabilizes the microparticles upon rehydration. The versatility of this approach Is demonstrated using horseradish peroxidase as the model enzyme and ovalbumin as the model antigen