Abstract
In asymmetrical flow field-flow fractionation (AF4), similar to other separation techniques, mass recovery and overloading require special attention in order to obtain quantitative results. We conducted a systematic study with five globular proteins of different molecular weight (36.7–669 kDa) and isoelectric point (4.0–6.5), and ultrafiltration membranes that are commonly used in aqueous AF4, regenerated cellulose (RC) and polyethersulfone (PES). Phosphate-buffered saline (PBS) with ionic strength 0.15 M and pH 7.2 was used as the carrier liquid in this study. The actual molecular weight cutoff (MWCO) was found to be higher than the nominal value and varied between membranes of different chemistry but the same nominal MWCO. Adsorption on the membrane was found to be dependent on the membrane chemistry (RC had lower adsorption compared to PES), and independent of the protein standard for the examined proteins. On the other hand, the mass overloading effects (i.e., higher retention times, peak broadening, and fronting peaks) were significantly more pronounced for γ-globulin than for the other proteins. The overloading effects could be rationalized with the increase of the local viscosity close to the membrane, depending on the properties of the proteins, and we derived theoretical equations that related the dependency of the migration velocity on the protein concentration through this non-ideal viscosity effect.
Highlights
Devised by Giddings half a century ago [1], field-flow fractionation (FFF) has emerged as a powerful tool for the characterization of macromolecules and nanoparticles [2,3,4]
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) [8,9,10]
The AF4 system was an Eclipse DualTec system (Wyatt Technology Europe, Dernbach, Germany) which was connected to an Agilent HPLC system 1200 (Agilent Technologies, Waldbronn, Germany) that consisted of a degasser, an isocratic pump, an inline PVDF filter 0.1 μm (Millipore, MA, USA), a UV detector, and an autosampler equipped with a thermostat and an injection loop of 100 μL
Summary
Devised by Giddings half a century ago [1], field-flow fractionation (FFF) has emerged as a powerful tool for the characterization of macromolecules and nanoparticles [2,3,4]. 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) [8,9,10]. To obtain accurate results with AF4, a careful method optimization is required with respect to recovery, resolution, sensitivity, and reproducibility [12,13,14,15,16,17,18,19]. High recoveries can be achieved (> 90%) for proteins with a proper method optimization but they rarely reach 100% [9, 15, 20,21,22] as there are several factors that may contribute to sample loss (Fig. 1a):
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