Influence of Protein Formulation and Carrier Solution on Asymmetrical Flow Field-Flow Fractionation: A Case Study of the Plant-Produced Recombinant Anthrax Protective Antigen pp-PA83

Limitations of Nanoparticles Size Characterization by Asymmetric Flow Field‑Fractionation Coupled with Online Dynamic Light Scattering

“…The fractogram can be divided in three zones according to the absorbance exhibited by the suspension of particles during the elution process. Zones 1 and 3 are associated with low particle concentration, while zone 2 is associated with high particle concentrations hypothesis could explain the trend towards an increase in diameter at the right tail of the concentration distribution but contradicts the operating principle of the AF4 for the left tail, because smaller particles are eluted before larger ones [10,11]. Other authors suggested that deviations between the real physical properties of the particles and the ones detected by the AF4-DLS-UV system are linked with a change in the surface shape of the particle during the separation and elution processes, but no experimental investigations support this hypothesis [8].…”

“…It can be noted that for low concentrations of particles in suspension (zones 1 and 3) the DLS detected an apparent and progressive increase in the size of the particles in suspension, returning a U-shaped distribution for the diameters as function of the elution time and of the concentration of particles in suspension. This phenomenon can be found in a significant number of studies in the literature describing the application of asymmetric flow field flow fractionation to separate and characterise particles of various sizes and materials [3,4,[7][8][9][10]; however, a clear understanding of the factors regulating its appearance is still missing. Some studies in the literature suggest that the apparent progressive size increase detected by the DLS at low concentrations of particles (at the beginning and at the end of the absorbance distribution curve) can be related to the presence of larger aggregates in suspension.…”

Impact of asymmetrical flow field-flow fractionation on protein aggregates stability

“…[10] As shown in Figure 1, the AF4 channel has a trapezoidal shape with one wall formed by a semi- where is the channel thickness. [12] In many cases, AF4 also allows greater flexibility in carrier fluid choice compared to SEC [13] and is able to separate over a wide size range (0.01 to >1 µm) with good selectivity (>0.5) [14]. These advantages are at the heart of many publications that provide insights into the formation and dissociation of biological and synthetic complexes and aggregates.…”

“…AF4 without focusing and crossflow has been used to study loosely bound aggregates and showed that these species remained intact during analysis. [13,17,49,50] Therefore, the influence of inchannel shear rate is not specifically addressed in this work. The flow velocity not only determines the shear rate, but also sample dilution during separations.…”

“…The effect of different carrier liquids on AF4 performance in analyzing proteins have been reported [13,52] but, there are no published studies that systematically investigate the impacts of injection shear stress, concentration, and dilution on protein aggregate stability during AF4.…”