Fluorescence Correlation Spectroscopy for Particle Sizing in Highly Concentrated Protein Solutions

Area of Science:

• Biopharmaceutical drug development
• Analytical chemistry
• Physical chemistry

Background:

• Highly concentrated biomolecule solutions are crucial in biopharmaceutical development.
• Reversible aggregate formation poses analytical challenges for traditional methods like HPLC, often requiring sample dilution.
• There's a need for analytical techniques that can assess these aggregates under native formulation conditions.

Purpose of the Study:

• To present a state-of-the-art protocol for analyzing protein self-assembly in highly concentrated solutions.
• To demonstrate the utility of Fluorescence Correlation Spectroscopy (FCS) for determining particle size distributions.
• To address the analytical challenges posed by reversible aggregates in biopharmaceutical formulations.

Main Methods:

• Utilizing Fluorescence Correlation Spectroscopy (FCS) to measure the diffusion of fluorescently labeled particles.
• Applying FCS to analyze protein or peptide self-assembly in highly concentrated solutions.
• Determining particle size distributions of reversible aggregates under formulation conditions.

Main Results:

• FCS provides a flexible and powerful method for measuring particle diffusion.
• The protocol enables the analysis of reversible aggregates in concentrated protein solutions.
• FCS overcomes limitations of dilution-dependent methods for aggregate analysis.

Conclusions:

• FCS is well-suited for characterizing the size distribution of reversible protein aggregates in concentrated formulations.
• This approach facilitates the analysis of biomolecular assemblies under relevant biopharmaceutical conditions.
• The described protocol offers a valuable tool for biopharmaceutical drug development and quality control.