Protein Aggregation and Soluble Aggregate Formation Screened by a Fast Microdialysis Assay

Abstract

Protein aggregation is a major obstacle in recombinant protein production as it reduces the yield of soluble polypeptides. Also, the formation of aggregates occurring in the soluble fraction is more common than formerly expected, and the prevalence of these entities might significantly affect the average quality of the soluble protein species. Usually, the formation of soluble aggregates remains unperceived because analytical methods such as dynamic light scattering are not routinely applied as quality control procedures. The authors have developed a methodologically simple and fast procedure, based on microdialysis and image processing, that reveals the aggregation tendency of a given protein in a specific environment. Because they also show a good correlation between macroscopic aggregation and soluble aggregate formation, the microdialysis approach also permits an estimation of the occurrence of soluble aggregates.

Intracellular CXCR4+ cell targeting with T22-empowered protein-only nanoparticles

“…These features are suggestive of proteolytic stability, tight architecture, and regular organization of T22-GFP-H6 building blocks, in contrast with the random particle size and morphologies observed during amorphous protein aggregation, even in the form of soluble aggregates. [24][25][26][27] Indeed, the mean size of T22-GFP-H6, measured by dynamic light scattering, was 13.45 nm (Figure 6C), a value fully compatible with images of these nanoparticles under transmission electron microscopy ( Figure 6D) showing relatively monodispersed entities. To assess further the structural and functional stability of T22-empowered nanoparticles, we determined their size distribution after storage for one year at -80°C, and also at room temperature for an additional 24 hours, followed by one additional step of freezing and thawing.…”

High yield purification and first structural characterization of the full‐length bacterial toxin CNF1

“…A detailed and extensive biophysical characterization of CNF1 recombinant protein requires that the final protein formulation is characterized not only by a high purity level, but also by acceptable homogeneity, solubility, and stability over time. To assess these features, DLS experiments were routinely performed to define the most suitable buffers to be used during the purification and storage of the protein . Furthermore, this procedure was also useful to define the behavior of CNF1‐H8 when preserved in concentrated form (up to 10 mg/mL) for long periods of time .…”

“…To assess these features, DLS experiments were routinely performed to define the most suitable buffers to be used during the purification and storage of the protein. 47 Furthermore, this procedure was also useful to define the behavior of CNF1-H8 when preserved in concentrated form (up to 10 mg/mL) for long periods of time. 48 This feature is of pivotal importance as various structural and analytical techniques may require the use of highly concentrated and stable preparations.…”

Analytical Approaches for Assessing Aggregation of Protein Biopharmaceuticals

“…It is based on the fact that during microdialysis, protein aggregates tend to be sedimented on membrane filters, and the amount of deposited protein can be quantified by image analysis after Coomassie blue staining [108]. Interestingly, the extent of the membrane staining signal correlates not only with the occurrence of insoluble aggregates, but also with the amount of soluble supramolecular entities present in the solvent, being a good indicator of soluble protein conformational quality and the tendency of a given protein to form soluble aggregates.…”