Model Virus Used for the Nanofiltration Study
The model virus used for the assay was bacteriophage PP7, a small 25-nm, nonenveloped ss-RNA Pseudomonas phage from the Leviviridae family. Leviviridae have a simple construction and are not tailed. They consist of a capsid and
are not enveloped. The capsid appears round, is 25 nm in diameter, and has an icosahedral symmetry. The genome consists of
linear, nonsegmented, positive sense ssRNA of about 3.4-4.3 kb. Nucleic acid takes up 30% of the total phage weight. PP7 is
a model virus used by several filter manufacturers for the evaluation of retention characteristics of 20-nm virus filters.
The principal feasibility of PP7 as a model virus for mammalian viruses has been demonstrated in a previous study.8
The PP7 Assay
A plaque assay used Pseudomonas aeruginosa as the target and indicator bacterial cells to be infected by bacteriophage PP7. P. aeruginosa and the bacteriophage PP7 used were from the American Type Culture Collection, USA (ATCC-No. 15692 and ATCC-No. 15692-B2
P. aeruginosa was grown as an overnight culture at 37 °C in nutrient broth supplemented with 0.5% NaCl. PP7 was harvested from suspensions
of infected P. aeruginosa by low speed centrifugation (2000 ± 100 rev/min, 10 ± 1 min). The supernatant was pooled and filtered through a 0.2-µm PESU
membrane filter, aliquoted, shock-frozen in liquid nitrogen, and stored at –66 ± 5 °C until further use.
To determine the PP7 titer, experimental samples (150-µL aliquots) were incubated with bacteria as a 50-µL aliquot of an overnight
culture, diluted 1:100 in nutrient broth (Nutrient Broth Difco 23400, Becton Dickinson) for 10–20 min at room temperature.
Subsequently, 3 mL of 0.5% agar (Difco Nutrient Agar, Becton Dickinson) were added. The entire volume was spread on a Petri
dish with 1.5% solid nutrient agar. After an incubation period of 20–24 hr at 37 °C the plaques induced by bacteriophages
Calculation and Statistical Analysis
All experimental samples are assayed for bacteriophages in fourfold replicate (150-µL each) at undiluted (100) and three different
dilutions (101, 102, and 103). After an incubation period of 20–24 hr, the number of plaques on a confluent layer of P. aeruginosa were counted. Three dilutions with countable numbers of plaques were used for the calculation of the PP7 titer.
The Log10 reduction factor in the nanofiltrate was calculated using the following equation:
in which R is the final log10 reduction factor, A
0 is the phage titer/mL upstream of test item, and A
n is the phage titer/mL downstream of the test item.
Flow decay is defined as the decrease of flow during the nanofiltration when compared to the initial buffer flow during flushing of
the filter. At 100% buffer flow, flow decay is 0%. As an example, a nanofiltration flow of 50% compared to the initial buffer
flow is described as 50% flow decay. During the nanofiltration of the protein solution, instantaneous samples were taken at
various levels of flow decays from 5% to around 90% and the LRF was calculated.