There is growing interest in the development of new vaccines based on liveattenuated viruses (LAVs) and virus-like particles. The large size of these vaccines, typically 100-400 nm, significantly complicates the use of sterile filtration. The objectives of this study are to examine the performance of several commercial sterile filters for filtration of a cytomegalovirus vaccine candidate (referred to as the LAV) and to develop and evaluate the use of a model nanoparticle suspension to perform a more quantitative assessment. Data obtained with a mixture of 200-and 300-nm fluorescent particles provided yield and pressure profiles that captured the behavior of the viral vaccine. This included the excellent performance of the Sartorius Sartobran P filter, which provided greater than 80% yield of both the vaccine and model particles even though the average particle size was more than 250 nm. The particle yield for the Sartobran P was independent of filtrate flux above 200 L/m 2 /h, but increased with increasing particle concentration, varying from less than 10% at concentrations around 10 7 particles/ml to more than 80% at concentrations above 10 10 particles/ml due to saturation of particle capture/binding sites within the filter. These results provide important insights into the factors controlling transmission and fouling during sterile filtration of large vaccine products.

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Nanoparticle hydrophobicity is a key factor controlling the stability, adhesion, and transport of nanoparticle suspensions. Although a number of approaches have been presented for evaluating nanoparticle hydrophobicity, these methods are difficult to apply to larger nanoparticles and viruses (>100 nm in size) that are of increasing importance in drug delivery and gene therapy. This study investigated the use of a new analytical hydrophobic interaction chromatography method employing a 5.0 μm pore size polyvinylidene fluoride membrane as the stationary-phase in membrane hydrophobic interaction chromatography (MHIC). Experimental data obtained using a series of model proteins were in good agreement with literature values for the hydrophobicity (both experimental and computational). MHIC was then used to evaluate the hydrophobicity of a variety of nanoparticles, including a live attenuated viral vaccine, both in water and in the presence of different surfactants. This new method can be implemented on any liquid chromatography system, run times are typically <20 min, and the experiments avoid the use of organic solvents that could alter the structure of many biological nanoparticles.

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