5 must-know techniques for analyzing protein aggregation
At APC, our biggest driver is to provide our clients with in-depth insights which empower them to get through their development and scale-up challenges as fast as possible. Thus, it's important not to simply rely on just one analytical approach to examine complex phenomena. Protein aggregation is one such complex phenomenon which when not fully understood or addressed can slow down the development of biologics. With this in mind, we've developed a number of orthogonal approaches to ensure that we can provide our clients with a full picture of the key processes underpinning aggregation of proteins. These are described below
1. Size exclusion chromatography
A widely accepted technique; using a sub-2 micron column we can resolve monomer, dimers and higher order aggregates within just 5 minutes. Couple this with UV detection, intrinsic tryptophan fluorescence detection and multi-angle laser light scattering detection and HPLC determination of aggregation could not be more comprehensive!
2. Intrinsic tryptophan fluorescence detection
We can examine the very early stages of protein misfolding (which often escalates to full aggregation) by simply studying subtle changes in the localised environment of tryptophan residues when the protein is exposed to certain stressors resulting in a bathochromic shift, and/or change in fluorescence intensity
3. Aggregation Index
A simple but powerful UV-based method where we examine the ratio of protein absorbance at 280 nm versus 350 nm. The presence of light-scattering aggregates (at 350 nm) results in easily detectable changes in the "Aggregation Index" ratio.
4. Nanoparticle tracking analysis
With this technique, we monitor the Brownian motion of protein aggregate nanoparticles using a powerful microscope mounted with a high frame-rate CCD camera.
5. Extrinsic dye-binding fluorescence assays
Here we exploit the binding of selected fluorescent dyes to hydrophobic protein domains which have become exposed as a result of aggregation. The increase in fluorescence is linear and highly reproducible. Careful selection of the dye type helps us to determine exactly how a given protein undergoes aggregation events.