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

Abstract

The impact of asymmetrical flow field-flow fractionation (AF4) on protein aggregate species is investigated with the aid of multiangle light scattering (MALS) and dynamic light scattering (DLS). The experimental parameters probed in this study include aggregate stability in different carrier liquids, shear stress (related to sample injection), sample concentration (during AF4 focusing), and sample dilution (during separation). Two anti-streptavidin (anti-SA) IgG1 samples composed of low and high molar mass (M) aggregates are subjected to different AF4 conditions. Aggregates suspended and separated in phosphate buffer are observed to dissociate almost entirely to monomer. However, aggregates in citric acid buffer are partially stable with dissociation to 25% and 5% monomer for the low and high M samples, respectively. These results demonstrate that different carrier liquids change the aggregate stability and low M aggregates can behave differently than their larger counterparts. Increasing the duration of the AF4 focusing step showed no significant changes in the percent monomer, percent aggregates, or the average Ms in either sample. Syringe-induced shear related to sample injection resulted in an increase in hydrodynamic diameter (dh) as measured by batch mode DLS. Finally, calculations showed that dilution during AF4 separation is significantly lower than in size exclusion chromatography with dilution occurring mainly at the AF4 channel outlet and not during the separation. This has important ramifications when analyzing aggregates that rapidly dissociate (<∼2s) upon dilution as the size calculated by AF4 theory may be more accurate than that measured by online DLS. Experimentally, the dhs determined by online DLS generally agreed with AF4 theory except for the more well retained larger aggregates for which DLS showed smaller sizes. These results highlight the importance of using AF4 retention theory to understand the impacts of dilution on analytes.

Asymmetric flow field-flow fractionation as a multifunctional technique for the characterization of polymeric nanocarriers

“…In general, AF4 is not suitable for carriers whose release kinetics is on the timescale of minutes or is heavily concentration-dependent. In fact, it is known that in the AF4 channel, the samples are preconcentrated during the focusing step, which may force drug reassociation [125], and then greatly diluted by the main eluent flow when exiting the channel [126]. Since polymeric nanocarriers suffer sometimes from low incorporation stability and early drug release [127], the stability of the carrier during the separation is the first point to take into account to guarantee representative results of drug loading.…”

Recovery, overloading, and protein interactions in asymmetrical flow field-flow fractionation

“…The asymmetrical variant of flow FFF [ 5 ], abbreviated as AsFlFFF or AF4, is a mild size-based separation technique which has found various applications in biopharmaceuticals [ 6 , 7 ]. It has been demonstrated that flow FFF is able to give higher recoveries and better resolution for the large aggregates of antibodies compared to size exclusion chromatography (SEC) [ 8 – 10 ]. SEC is conventionally applied for the quantification of the protein aggregates, but these may be filtered out by the column, elute unresolved in the void volume, or adsorb on the chromatographic support due to non-specific interactions, especially the large ones [ 8 , 11 ].…”

“…SEC is conventionally applied for the quantification of the protein aggregates, but these may be filtered out by the column, elute unresolved in the void volume, or adsorb on the chromatographic support due to non-specific interactions, especially the large ones [ 8 , 11 ]. Furthermore, the sample dilution inside the AF4 channel is lower than that in the SEC column, decreasing the chances of dissociation of reversible aggregates [ 10 ].…”

Process analytical technique (PAT) miniaturization for monoclonal antibody aggregate detection in continuous downstream processing

“…This makes AF4 a powerful alternative for SEC, as long as the AF4 retention theory is used to understand the impacts of dilution on analytes. 127 Although AF4 presents many advantages as a separation technique for aggregate detection, its adoption has been limited. This is mainly due to the large footprint of available separation cartridges, extended analysis times and solvent consumption.…”