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Development of asymmetrical flow field-flow fractionation for the characterization of proteins, protein aggregation, and nanoparticles
Bria, Carmen R. M.
Bria, Carmen R. M.
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2016
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2017-03-27
Abstract
Field-flow fractionation (FFF) is a family of analytical techniques used to characterize macromolecules and particles from ~1 nm to >1µm. Its versatility has allowed a number of analytical challenges to be addressed, but several limitations still exist. Advances in asymmetrical flow field-flow fractionation (AF4) coupled with UV-Vis, multiangle light scattering (MALS), and/or dynamic light scattering (DLS) were made to overcome current limitations in the characterization of proteins, protein aggregates, and nanoparticles. Formation of protein aggregates in protein therapeutics is a major concern due to reduced drug efficacy and potential immunogenicity. A lack of reliable analytical methods that cover the submicron (0.1-1 µm) size range has been a major challenge for protein aggregate characterization. The development of a simple AF4 method with good size selectivity (>0.5) from 1 nm to 1 µm allowed the formation of submicron aggregates to be fitted by the Lumry-Eyring Nucleated Polymerization (LENP) model for the first time. A comparison of aggregation kinetics determined by AF4 and the LENP model before and after centrifugation of aggregate samples showed that this common sample preparation step might influence the experimentally observed kinetic mechanism. The results suggest that AF4 is able to provide more reliable kinetic data for aggregates up to and larger than 100 nm that may not be easily characterized SEC. AF4 provides a wealth of information including analyte size distributions, but this information can be influenced by the inherent dilution that occurs during separations, especially for weakly bound protein aggregates. Protein aggregate stability at each stage of the AF4 analysis was studied. The choice of carrier fluid played a significant role in aggregate stability while sample concentration during AF4 focusing did not have significant impact on aggregate populations. Calculations showed that sample dilution is significantly lower in AF4 than in SEC and that dilution occurred primarily at the channel outlet (not during the separation). This suggests that sizes from AF4 theory may be more accurate than those from online light scattering detectors because rapidly dissociating species may be altered upon dilution at the channel outlet. Understanding aggregate behavior during AF4 is critically important, not only for protein aggregates, but also for polymer and nanoparticle supramolecular complexes that may be altered by analysis. A major challenge that impacts AF4 analyses of samples from nanoparticles to polymers to proteins is unwanted analyte-membrane interactions. These interactions can potentially be reduced by modifying the membrane surface, but modification of AF4 membranes has been restricted by two key challenges: 1) large membrane areas (~90 cm2) must be modified and 2) the membrane surface must remain flat and semi-permeable. Development of a method to modify membranes for AF4 analysis has provided the foundation to overcome these challenges. The novel channel reactor developed in this work allows large, flat membrane surfaces to be grafted with polymer brushes while simultaneously reducing the reagent amounts required. Poly(N-isopropylacrylamide) brushes were successfully grafted from membranes resulting in thermo-responsive behavior to provide control over the membrane hydrophobicity. The modified membranes were then used for AF4 analysis of IVIg and BSA proteins. This method provides the necessary groundwork for addressing perhaps the most significant limitation of AF4, analyte-membrane interactions. Finally, the design and optimization of semi-preparative scale AF4 channels has pushed the boundaries of the sample amounts that can be separated in a single run. Separations of protein aggregates, biological particles, and other nanoparticles are currently limited to small analytical scale quantities. However, fractionation of large analyte quantities is critical for understanding their fundamental properties by further characterization and for subsequent applications. AF4 channels capable of handling milligram sample quantities in aqueous and, for the first time, organic carrier fluids were designed, optimized, and their performance was investigated. Channel breadth, shape, focusing position, and sample loading were studied to provide the best separation performance. Good sample resolution (>1.0) for >10 mg of silica nanoparticles suspended in water was demonstrated and fractionation of >0.5 mg inorganic hybrid nanoparticles suspended in tetrahydrofuran was possible. This represents important advances in the understanding of sP-AF4 channel design and performance, and extends its capabilities to larger sample quantities and organic solvents.
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