Particles shed from syringe filters and their effects on agitation-induced protein aggregation

摘要

We tested the hypothesis that foreign particles shed from filters can accelerate the rate of protein aggregation and particle formation during agitation stress. Various types and brands of syringe filters were tested. Particle counts and size distribution (≥1 μm) in buffer alone or in solutions of keratinocyte growth factor 2 (KGF-2) were determined with a micro-flow imaging. Submicron particle populations were characterized by dynamic light scattering. Loss of soluble protein during filtration or postfiltration incubation was determined by ultraviolet spectroscopy and bicinchoninic acid protein assay. There was a wide range (from essentially none to >100,000/mL) in the counts for at least 1 μm particles shed into buffer or KGF-2 solution from the different syringe filters (with or without borosilicate glass microfibers). Filtration of KGF-2 with units containing glass microfibers above the membrane resulted in 20%-80% loss of protein due to adsorption to filter components. Filtration with systems containing a membrane alone resulted in 0%-20% loss of KGF-2. Effects of 24-h postfiltration incubation were tested on KGF-2 solution filtered with polyether sulfone membrane filters. Loss of soluble protein and formation of particles during agitation were much greater than that in control, unfiltered KGF-2 solutions. Similar acceleration of protein aggregation and particle formation was observed when unfiltered KGF-2 solution was mixed with filtered buffer and agitated. Particle shedding from syringe filters-and the resulting acceleration of protein aggregation during agitation-varied greatly among the different syringe filters and individual units of a given filter type. Our results demonstrate that nanoparticles and microparticles shed from the filters can accelerate protein aggregation and particle formation, especially during agitation.