|Institution:||Colorado School of Mines|
|Keywords:||Field-flow fractionation; Nanoparticles; Separation science; Light scattering; Biotechnology; Protein aggregation|
|Full text PDF:||http://hdl.handle.net/11124/170434|
Field-flow fractionation (FFF) is a family of analytical techniques used to characterize macromolecules and particles from ~1 nm to >1?m. 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 ?m) 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 ?m 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… Advisors/Committee Members: Williams, S. Kim R. (advisor), Ayers, Reed A. (committee member), Posewitz, Matthew C. (committee member), Trewyn, Brian (committee member).