Bioseparations

Features and Benefits

• High (>6 logs) virus clearance - Clearance can be increased by thickening filter.
• High flux- hundreds of times greater than ultraporous membranes. Sustained flow velocity of 0.5-1 cm/sec at applied pressures of 0.3 to 1.0 bar, equivalent to 18,000 to 36,000 l/m2/hr. 
• High capacity for particulates.
• Greater resistance to clogging.
• Robust - Point defects in a depth filter are compensated by fibers randomly spaced behind such defects, while there is no such redundancy in a membrane filter.
• High capacity for nucleic acids and endotoxins.
• Certain proteins (e.g.- BSA) adsorbed even in saline and without requiring surface activation.
• Macromolecules are separable by charge rather than excluded by size.

NanoCeram® is a new form of alumina fibers that attracts and retains virus and other macromolecules by electrostatic forces.  These fibers have been incorporated into fibrous ("depth") filters and have found to have ~ 6-7 logs of virus retention even at flow rates several orders of magnitude greater than could be obtained using ultraporous membranes.  The fibers and resulting filters received a "Best 100 New Product" award by R and D magazine for 2002. 

Boehmite (AlOOH) is the main component of the fibers.  The surface of the nanofibers is positively charged and attracts and retains negatively charged particles including bacteria, virus, protozoa, organic and inorganic colloids and negatively charged macromolecules.  Data are presented showing increased processing speeds as compared to ultraporous filters, while preserving high recoveries.   NanoCeram® will prove to be a valuable tool for sample concentration and purification, crude fractionation, water and serum purification and many applications where membranes are currently used.   NanoCeram® also provides a way to separate particles on the basis of charge, rather than separation by size exclusion.

NanoCeram^® Properties

Attraction and Adhesion of Pathogens

Partition coefficients of virus and bacteria were measured between the sorbent and neutral water.  Bacteriophages MS-2 and PRD-1 were diluted in 150 mL of 0.02M imidazole/0.02M glycine buffer (pH 7.2) to give an effective concentration of approx. 8 x 10⁵ PFU/ml.  NanoCeram^® fibers (0.5 grams) were placed into a 15 mL conical centrifuge tube containing 10 mL of the bacteriophage solution.  The powder was dispersed by vortex mixing and was shaken on a rocking table for 15 minutes.  The mixture was centrifuged at low speed (2500 rpm) to collect the powder at the bottom of the tube and the supernatant was assayed for the presence of viruses.   An initial and final aliquot was taken from the buffer solution containing bacteriophage, to verify that the virus concentration remained constant throughout the experiment.  Analysis showed that 99.99% of the MS-2 and 99.5+% of PRD-1 virus were removed from the solution.

Similar testing was done with bacteria and bacillus. The microbes were of the genus Micrococcus (spherical, non-motile), bacillus (minor motility, spore-forming, rod-shaped) and Pseudemonas (rod-shaped and motile).   A suspension of the cells was prepared in a sterile 0.5% saline solution.  Initial concentration of the microorganisms was determined by the method of limiting dilutions followed by seeding on meat-peptone agar medium (MPA).  The mixture was passed through a column 0.8 cm diameter, 15 cm high, filled with the sorbent.  The non-sorbed biomass was then washed out of the column using buffered solution with a volume that was 3-4 times the volume of the column.  The sorbent was then transferred to a test tube containing 9 ml of sterile 0.5% NaCl containing 1-2 drops of TWIN-80 surfactant to desorb the microorganisms.  The sample was then agitated for 1 min and after 10 min at rest, the supernatant was sampled and inoculated onto MPA to determine the number of microorganisms that had been on the sorbent.  Table 1 shows a high capacity for the different microbes. 

Filter Development

Filters were developed by wet laying NanoCeram® along with supporting microglass (0.6µ) fibers to produce a filter that is about 1.5 mm thick and has about 90% void volume. The filters were challenged with 10⁷ PFU/mL virus at a pH of 7.5 through a 2.5 cm diameter filter.  The data in Table 2 show that there is > 6 logs retention starting at approximately 20 weight % NanoCeram®.   These data were obtained at water flow velocities of approximately 0.3 cm/sec (for the heavier loadings of NanoCeram®) and 0.5 cm/sec for lighter (<30 wt %) loadings.  This flow capability is about 2 orders of magnitude greater than ultraporous membranes could maintain.   The very high retention at high flow rate is indicative of very rapid adsorption kinetics.  The filters were also capable of excluding larger particles such as Cryptosporidium and E. coli O157H7 to >5 logs by excluding them at the filter's surface.   Filters were also challenged by 1.15x10⁶ cells/mL of Saccharomyces cerevisiae (yeast) and there were no cells detected in the effluent. 
 

Regeneration

Under a NASA program to develop a filter for recycling space cabin water, we evaluated methods of desorbing virus and regenerating filters.   We found no discernable elution of MS2 would occur up to pH 9.  Elution with 0.02 M Na₂CO₃  (pH= 9.77) resulted in some elimination of MS2 (note- the isoelectric point of the NanoCeram® fibers has been measured as ~ 9.7).  At this condition the virus were not inactivated.  EDTA was found to be an effective elutant at lower pH's.   The virus could also be displaced using a solution of 1.5% beef serum extract with 0.25% glycine at a pH of 9.3.