Prion Removal from Human Plasma Using Surface- Modified Membranes. PV Gurgel, Y Zheng, and RG Carbonell

Transcription

1 Prion Removal from Human Plasma Using Surface- Modified Membranes PV Gurgel, Y Zheng, and RG Carbonell

2 Nonwoven Fabrics High-speed, low cost processes 300-1,000 meters/minute, 5-6 meters wide Inexpensive materials (PP, PET, PBT, Nylon) Engineered fiber diameters, basis weight, thickness Spunbonding: d f > 200 nm; Kg/m/hour Meltblowing: d f > 500 nm; Kg/m/hour Electrospinning: d f 50 to 200 nm; gm/m/hr Bicomponent fibers, particle-nonwoven composites Fiber splitting for smaller diameters Large number of 3-D formation options Hydroentanglement, calendering Multilayer, corrugated structures

3 Nonwoven Fabrics in Bioseparations Controlled fiber diameters Variable surface areas for protein adsorption Large porosities Low pressure drops Low cost Disposables, single use Controlled pore sizes Can allow passage of debris, including cells Large molecule capture Surfaces can be modified Grafting, surface coatings Filtration, ion exchange, affinity ligand attachment Interconnected pores Convective mass transfer Negligible diffusion limitations

4 Fiber Diameter and Surface Area for Adsorption Intrinsic Surface Area Area / Mass 4 d f p Fiber Diameter Area/Mass (m 2 /g) 5 µm µm nm nm 57 Product Capture or Contaminant Capture

5 Nonwoven Properties Property PP PBT Fiber diameter (µm) 8 ± 4 3 ± 0.7 Pore diameter (µm) 11 ± 4 8 ± 3 Mat thickness (µm) Basis weight (g/m 2 ) Apparent density (g/cm 3 ) Porosity Specific area (m 2 /g)

6 UV-Induced GMA Grafts on PBT Nonwovens Blank PBT UV Polymerization PBT-GMA Epoxy groups Grafting Solution GMA in 1-butanol with BP (benzophenone) as initiator, UV wavelength (365 nm) Grafted Polymer Brush

7 GMA Grafting Controlled by Reaction Time GMA Concentration Degree of GMA grafting (%) W 1 W W BP:GMA 2:100

8 Grafting Confirmed by FTIR Analysis (Glycidyl methacrylate) GMA Polybutylene terephthalate (PBT) (a) blank PBT; (b) PBT exposed to UV without GMA and BP in 1-butanol (c) PBT after GMA grafting; (d) polygma

9 Uniform, Conformal GMA Grafts Blank PBT Grafting conditions: 2.0 M GMA - 15min BP:GMA =0.5:100 BP:GMA= 1:100 BP:GMA= 2:100

10 Flow Permeability Unchanged by Grafting Darcy s law: p u 0 k L u 0 k p L : superficial velocity : permeability : pressure drop : column length : dynamic viscosity k = 2 Х 10-8 cm 2 Bed density: 0.47 g/ml Bed porosity = 0.67

11 Average Grafted Layer Thickness For a 12% weight change (R ) 2 R 2 R R R 3 m; 180 nm 1800 Å R Consistent with SEM ( nm)

12 Capacities vs Graft Thickness 115 nm 40 nm 35 nm 40 nm 900 nm 3000 nm 900 nm 3000 nm Same Relative Weight Gain Achieve similar relative binding capacities Large fiber requires longer time to reach equilibrium (thicker layer) Same Grafting Thickness Both reach equilibrium fast Overall binding capacity better for the small fiber

13 Origin of vcjd Late 1970 s Changes to preparation of meat and bone meal used in animal feed First cases of BSE identified; feed ban introduced 1988 Government assures the public British beef safe for human consumption First case of vcjd identified Link between BSE and vcjd made; 30 month cull initiated First vcjd transfusion transmission case identified

14 Distribution of TSE Infectivity in Blood

15 Approaches to Increasing Prion Safety of Blood and Plasma Products Prion detection Prion inactivation Donor deferral Prion removal? No blood screening assay exists for vcjd; Issues surrounding false-positives. PrP res is significantly more stable than cellular components and plasma proteins Option for countries with low incidence of BSE/vCJD; not viable where large % of population has potentially been exposed to TSEs. Viable approach with appropriate affinity ligands; protection against known and future TSEs.

16 Prion Removal Effective at very low concentrations: Still active below Limits of Detection (most of the disease incubation period); Discrimination of normal vs abnormal PrP is irrelevant; There are no false positive issues: No notification, counseling, retesting, or trace-backs, No notification, testing, counseling of trace-backs themselves, No product withdrawals; No alienation of donor population from fear of positive result; Protection against a broad spectrum of TSEs (eg. new prion diseases). Synergistic with prion testing for robust protection

17 P-Capt Prion Reduction Filter Sterile, single-use prion-reduction device. Incorporates selected PRDT prion-binding affinity resin trapped between supporting membrane layers. Dockable filter - designed for use with human leuko-reduced rbcc. Device is CE Marked in Europe since Sept Efficacy conforms to UK NBS requirements: 3 log 10 reduction by brain spike study 1 log 10 reduction by endogenous study

18 Binding of Prions from Plasma Spiked with Hamster Brain Homogenate Tested with 0.01% Infectious hamster brain homogenate in plasma. Total load of about 10 7 ID About 0.4 grams of membrane were used (0.1 g membrane/ column)

19 Binding of Prions from Plasma Spiked with Normal HaBH Tested columns with membranes and columns in series 5 columns of membrane are roughly equivalent to 4 columns of packed resin Specific surface area of resin is 50 times higher than membrane Grafted layers provide additional surface area for binding

20 Binding of Prion from Plasma spiked with Infectious HaBH Tested with 0.01% Infectious hamster brain homogenate in plasma. Total load of about 10 7 ID Derivatized membrane containing a ligand bound infectious prion protein Four columns of membrane removed all detectable infectious prion About 0.4 grams of membrane were used (0.1 g membrane/ column)

21 Binding of Prions from Human Plasma Spiked with Infectious HaBH Bound protein profiles show higher binding of proteins from resin than from nonwoven Differences might be due to ligand density Molecular Marker Reference Columns 1-5 Nonwoven Columns 6 Resin Columns 1-5 L13 Resin Molecular Marker

22 Binding of Prion from Plasma Spiked with normal HaBH Col-1 to 4 Nonwoven direct attachment Col-5 L13 Resin Col-1 to 4 Nonwoven mediated attachment Col-5 L13 Resin Ligand attachment methodology shows similar prion binding capacities

23 Binding of Infectious Prion from Infectious HaBH Col-1 to 4 Nonwoven direct attachment Col-5 L13 Resin Col-1 to 4 Nonwoven mediated attachment Col-5 L13 Resin Ligand attachment methodology influences non-specific binding capacity Mediated attachment offers better results

24 Conclusions Nonwoven fabrics have interesting properties, and are suitable for specific uses in bioseparations Targets at low concentration (requiring low capacities, such as pathogens) might be specially interesting Prion removal capacities of modified nonwovens are comparable (v/v) to that of resins