While polymeric membranes are widely used in protein pharmaceuticals purification, protein interactions with membrane surfaces can lead to membrane fouling and reduced membrane performance.  Using single-molecule total internal reflection fluorescence microscopy (SM-TIRFM), we directly observed the dynamics of fluorescently-labeled bovine serum albumin (BSA) and human monoclonal antibody (IgG) at the interface between aqueous buffer and polymeric membrane materials such as regenerated cellulose and polyethersulfone blended with different wetting agents.  SM-TIRFM uniquely allowed us to identify heterogeneous protein surface behavior due to both protein population (e.g. monomers and oligomers) and surface spatial heterogeneity (e.g. strong-binding vs. weak-binding surface sites).  At extremely dilute protein concentrations – where isolate protein-surface interactions can be probed – protein adsorption was found to be highly dynamic, with protein monomers desorbing with ~1s.  Protein oligomers, while rare, desorbed from the surface up to ten times slower than monomers.  By using super-resolution methods, protein adsorption sites could be localized with ~50 nm resolution and surface spatial heterogeneity could be quantified on the various surfaces.  While less common, anomalous adsorption sites, where proteins adsorbed more frequently and resided for significantly longer times, were observed on all surfaces.  Proteins resided for shorter times overall on more homogeneous surfaces, with fewer strong surface sites.  Thus, while rare, both protein oligomers and anomalously strong-binding sites disproportionally accounted for the adsorbed protein species and, we propose, may nucleate protein surface aggregation and accelerate membrane fouling.  Spatial heterogeneity also could be modulated by varying solution conditions, such as protein concentration.  At higher protein concentrations (i.e. 0.3-1 mg/ml), fewer anomalous adsorption sites were observed and overall protein desorption was enhanced, suggesting that strong-binding sites were blocked by adsorbed protein.

Role of spatial heterogeneity in protein adsorption on polymeric membrane materials at the single-molecule level

249th National Meeting (2015)

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