Polymeric sorbent for removal of impurities from whole blood and blood products

Abstract

The invention concerns methods of treating blood, blood products or physiologic fluid to maximize shelf life and/or minimizing transfusion related complications such as non-hemolytic transfusion reactions such as fever, transfusion-related acute lung injury (TRALI), transfusion associated dyspnea (TAD), and allergic reactions by removing undesirable molecules in the blood, blood product or physiologic fluid milieu through use of a sorbent.

Claims

1. A storage container for storage and purification of blood, blood product or physiologic fluid prior to transfusion into a patient, the storage container comprising: (a) said storage container being a compliant container suitable for the storage of blood, blood products or physiologic fluids; (b) sorbent comprising hemocompatible material suitable for treating blood, blood product or physiologic fluid, said sorbent performing at least one of (i) increasing shelf life of the blood, blood product or physiologic fluid, (ii) maintaining freshness of new blood, blood product or physiologic fluid, and (iii) removing undesirable molecules from the blood, blood product or physiologic fluid; said sorbent comprises cross-linked polymeric material derived from the reaction of a cross-linker with one or more of the following polymerizable monomers: divinylbenzene, styrene, ethylstyrene, acrylonitrile, butyl methacrylate, octyl methacrylate, butyl acrylate, octyl acrylate, cetyl methacrylate, cetyl acrylate, ethyl methacrylate, ethyl acrylate, vinyltoluene, vinylnaphthalene, vinylbenzyl alcohol, vinylformamide, methyl methacrylate, and methyl acrylate; wherein said sorbent comprises a hemocompatible surface comprising hydroxyethyl cellulose, hydroxypopyl cellulose, poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), poly(hydroxypropyl methacrylate), poly(hydroxypropyl acrylate), poly(dimethylaminoethyl methacrylate), poly(dimethylaminoethyl acrylate), poly(diethylamimoethyl methacrylate), poly-(diethylaminoethyl acrylate), poly(vinyl alcohol), heparin, polyethylene glycol, poly(N-vinylpyrrolidinone), salts of poly(methacrylic acid), salts of poly(acrylic acid) or copoplymers of mixtures thereof, wherein said hemocompatible surface is chemically bound to the cross-linked polymeric material; said sorbent being contained within a purification device within said compliant container, said sorbent being separated from said blood, blood product or physiologic fluid by a membrane wherein said membrane allows liquid comprising said undesirable molecules to pass through the membrane and contact said sorbent but where said membrane substantially excludes white blood cells, red blood cells and platelets from passing through said membrane and contacting said sorbent; wherein said hemocompatible material is supplied as slurry, or suspension, or dry powder or other dry particulate and said hemocompatible material is non-immobilized within the purification device; and wherein said storage container is a bag.

2. The storage container of claim 1, wherein said undesirable molecules are related to non-hemolytic transfusion reactions.

3. The storage container of claim 1, wherein said hemocompatible material comprises particles having a diameter in the range for 0.1 micron meters to 2 centimeters.

4. The storage container of claim 1, wherein said hemocompatible material is porous and has a pore structure that the total pore volume of pore size in the range of 50 to 10,000 is greater than 0.5 cc/g to 3.0 cc/g dry polymer.

5. The storage container of claim 1, wherein: said hemocompatible material is characterized as having a pore structure having a total volume of pore sizes in the range of from 10 to 10,000 greater than 0.5 cc/g to 3.0 cc/g of dry polymer; wherein the ratio of pore volume between 10 to 3,000 in diameter to pore volume between 500 to 3,000 in diameter of the said hemocompatible polymer is smaller than 7:1 and wherein the ratio of pore volume between 10 to 3,000 in diameter to pore volume between 10 to 6,000 in diameter of said hemocompatible polymer is less than 2:1.

6. The storage container of claim 1, wherein: said hemocompatible material is characterized as having a pore structure having a total volume of pore sizes in the range of from 20 to 10,000 is greater than 0.5 cc/g to 3.0 cc/g dry polymer; wherein the ratio of pore volume between 20 to 3,000 in diameter to pore volume between 500 to 3,000 in diameter of the said hemocompatible polymer is smaller than 7:1 and wherein the ratio of pore volume between 20 to 3,000 in diameter to pore volume between 20 to 6,000 in diameter of said hemocompatible polymer is less than 2:1.

7. The blood purification device of claim 1, wherein: said hemocompatible material is characterized as having a pore structure having a total volume of pore sizes in the range of from 50 to 10,000 is greater than 0.5 cc/g to 3.0 cc/g dry polymer; wherein the ratio of pore volume between 50 to 3,000 in diameter to pore volume between 500 to 3,000 in diameter of the said hemocompatible polymer is smaller than 7:1 and wherein the ratio of pore volume between 50 to 3,000 in diameter to pore volume between 50 to 6,000 in diameter of said hemocompatible polymer is less than 2:1.

8. The blood purification device of claim 1, where the hemocompatible material can sorb molecules from about 100 Daltons to approximately 1,000 kDa.

9. The blood purification device of claim 1, wherein said hemocomaptible material is derived from a pyrolyzed composition.

10. The blood purification device of claim 1, wherein said cross-linked polymeric material is pyrolyzed.

11. The blood purification device of claim 1, wherein said sorbent is pyrolyzed.

12. The blood purification device of claim 1, wherein said bag comprises a polymeric material comprising one or more of the following types of polymers: a polyvinyl chloride (PVC), a polyolefin (PO), a poly(ethylene-co-vinyl acetate) (EVA), and a fluorinated polyethylene propylene (FEP).

13. The blood purification device of claim 12, wherein the polymeric material further comprises a biocompatible plasticizer.

14. The blood purification device of claim 1, wherein said solid form sorbent further comprises a dispersing agent.

15. A method of treating blood, blood product, or physiologic fluid within a storage container comprising a bag, prior to transfusion of the blood, blood product, or physiologic fluid into a patient, to provide at least one of (i) increasing shelf life of the blood, blood product or physiologic fluid, (ii) maintaining freshness of new blood, blood product or physiologic fluid, and (iii) removing undesirable molecules from the blood, blood product or physiologic fluid through use of a sorbent, said sorbent being contained within a compliant container suitable for the storage of blood, blood products or physiologic fluid and said sorbent being separated from said blood, blood product of physiologic fluid by a membrane wherein said membrane allows liquid comprising said undesirable molecules to pass through the membrane and contact said sorbent but where said membrane substantially excludes white blood cells, red blood cells and platelets from passing thought said membrane and contacting said sorbent, said sorbent supplied as slurry, or suspension, or dry powder or other dry particulate and said sorbent is non-immobilized within the purification device and said sorbent comprises cross-linked polymeric material derived from the reaction of cross-linker with one or more of the following polymerizable monomers: divinylbenzene, styrene, ethylstyrene, acrylonitrile, butyl methacrylate, octyl methacrylate, butyl acrylate, octyl acrylate, cetyl methacrylate, cetyl acrylate, ethyl methacrylate, ethyl acrylate, vinyltoluene, vinylnaphthalene, vinylbenzyl alcohol, vinylformamide, methyl methacrylate, and methyl acrylate; wherein said sorbent comprises a hemocompatible surface comprising hydroxyethyl cellulose, hydroxypopyl cellulose, poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), poly(hydroxypropyl methacrylate), poly(hydroxypropyl acrylate), poly(dimethylaminoethyl methacrylate), poly(dimethylaminoethyl acrylate), poly (diethylamimoethyl methacrylate), poly-(diethylaminoethyl acrylate), poly(vinyl alcohol), heparin, polyethylene glycol, poly(N-vinylpyrrolidinone), salts of poly(methacrylic acid), salts of poly(acrylic acid) or copoplymers of mixtures thereof, wherein said hemocompatible surface is chemically bound to the cross-linked polymeric material.

16. The methods of claim 15, wherein said undesirable molecules are biologically active molecules (BAMs), biological response modifiers (BRMs), products of hemolysis, products of membrane or cellular degradation, toxins, drugs, antibodies, prions and similar molecules found in stored blood and blood products.

17. The method of claim 16, wherein the biologically active molecules comprise inflammatory mediators and stimulators.

18. The method of claim 17, wherein said inflammatory mediators and stimulators comprise cytokines, nitric oxide, thromboxanes, leukotrienes, platelet, activating factor, prostaglandins, glycoproteins, kinins, kininogens, complement factors, cell-adhesion molecules, superantigens, monokines, chemokines, interferons, free radicals, proteases, arachidonic acid metabolites, prostacyclins, beta endorphins, myocardial depressant factors, anandimide, 2-arachadonylglycerol, tetrahydrobiopterin, serotonin, histamine, bradykinin, soluble CD40 ligand, bioactive lipids, oxidized lipids, hemoglobin, red cell particulates, membrane or cellular components, growth factors, glycoproteins, prions, toxins, endotoxins, drugs, vasoactive substances, foreign antigens, and antibodies.

19. The method of claim 15 where undesirable molecules are antibodies.

20. The method of claim 15, wherein said sorbent is a hemocompatible polymer.

21. The method of claim 20, wherein said biocompatible polymer comprises particles having a diameter in the range for 0.1 micron meters to 2 centimeters.

22. The method of claim 20, wherein said hemocompatible polymer is porous and has a pore structure that the total pore volume of pore size in the range of 50 to 10,000 is greater than 0.5 cc/g to 3.0 cc/g dry polymer.

23. The method of claim 20, wherein said hemocompatible polymer is characterized as having a pore structure having a total volume of pore sizes in the range of from 10 to 10,000 is greater than 0.5 cc/g to 3.0 cc/g dry polymer; wherein the ratio of pore volume between 10 to 3,000 in diameter to pore volume between 500 to 3,000 in diameter of the said hemocompatible polymer is smaller than 7:1 and wherein the ratio of pore volume between 10 to 3,000 in diameter to pore volume between 10 to 6,000 in diameter of said hemocompatible polymer is less than 2:1.

24. The method of claim 20 where said hemocompatible polymer can sorb molecules from about 100 Daltons to approximately 1,000 kDa.

25. The method of claim 20, wherein said hemocompatible polymer is modified with ligands that specifically or non-specifically bind reactive biomolecules.

26. The method of claim 15, wherein said polymer may be porous or non-porous.

27. A method of making a blood purification device comprising placing sorbent comprising hemocompatible material in a compliant container suitable for the storage of blood or blood products, said container comprising a bag; wherein said hemocompatible material suitable for treating stored blood and blood products prior to transfusion of the blood, blood product, or physiologic fluid into a patient, said sorbent performing at least one of (i) increasing shelf life of the blood, blood product or physiologic fluid, (ii) maintaining freshness of new blood, blood product or physiologic fluid, and (iii) removing undesirable molecules from the blood, blood product or physiologic fluid; and said sorbent being separated from said blood, blood product or physiologic fluid by a membrane wherein said membrane allows liquid comprising said undesirable molecules to pass through the membrane and contact said sorbent but where said membrane substantially excludes white blood cells, red blood cells and platelets from passing through said membrane and contacting said sorbent within said compliant container; said sorbent comprising a slurry, or suspension, or dry powder or other dry particulate and said sorbent is non-immobilized within the purification device; wherein said sorbent comprises cross-linked polymeric material derived from the reaction of a cross-linker with one or more of the following polymerizable monomers: divinylbenzene, styrene, ethylstyrene, acrylonitrile, butyl methacrylate, octyl methacrylate, butyl acrylate, octyl acrylate, cetyl methacrylate, cetyl acrylate, ethyl methacrylate, ethyl acrylate, vinyltoluene, vinylnaphthalene, vinylbenzyl alcohol, vinylformamide, methyl methacrylate, and methyl acrylate; wherein said sorbent comprises a hemocompatible surface comprising hydroxyethyl cellulose, hydroxypopyl cellulose, poly(hydroxyethyl methacrylate), poly(hydroxyethyl acrylate), poly(hydroxypropyl methacrylate), poly(hydroxypropyl acrylate), poly(dimethylaminoethyl methacrylate), poly(dimethylaminoethyl acrylate), poly(diethylamimoethyl methacrylate), poly-(diethylaminoethyl acrylate), poly(vinyl alcohol), heparin, polyethylene glycol, poly(N-vinylpyrrolidinone), salts of poly(methacrylic acid), salts of poly(acrylic acid) or copoplymers of mixtures thereof, wherein said hemocompatible surface is chemically bound to the cross-linked polymeric material.

28. The storage container of claim 1, wherein said storage container is capable of lowering the amount of Human Cytokine IL-8 in stored blood to a level lower than the initial Cytokine IL-8 level in said stored blood for a period of at least 41 days of storage.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1 presents a plot of pore volume as a function of the pore diameter.

(2) FIG. 2 presents a polymer transfer adaptor for blood experiments

(3) FIG. 3 presents a blood transfer adaptor for sample collection

(4) FIG. 4, presents a plot of adsorption of Hemoglobin from Phosphate Buffered Saline versus time

(5) FIG. 5, presents a plot of adsorption of Hemoglobin from New Human Blood versus time

(6) FIG. 6 presents a plot of adsorption of human IgG from human blood versus time.

(7) FIG. 7 presents a plot of LysoPC adsorption from human blood versus time.

(8) FIG. 8 presents a plot of IL-7 adsorption from human blood versus time.

(9) FIG. 9 presents a plot of IL-8 adsorption from human blood versus time.

(10) FIG. 10 presents a plot of TNF adsorption from human blood versus time.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

(11) As required, detailed embodiments of the present invention are disclosed herein; it is to be understood that the disclosed embodiments are merely exemplary of the invention that may be embodied in various forms. Therefore, specific structural and functional details disclosed herein are not to be interpreted as limits, but merely as a basis for teaching one skilled in the art to employ the present invention. The specific examples below will enable the invention to be better understood. However, they are given merely by way of guidance and do not imply any limitation.

(12) Three porous polymeric sorbents are characterized for their pore structures and their syntheses are described in Example 1, 2, and 3. The pore structure characterization is given in Example 3.

(13) The synthesis process consists of (1) preparing the aqueous phase, (2) preparing the organic phase, (3) carrying out the suspension polymerization, and (4) purifying the resulting porous polymeric sorbent product (work-up).

(14) Remaining examples demonstrate removal of unwanted substances from blood.

Example 1

Sorbent 1-11 Synthesis

(15) Reactor Setup, Kettle (0.5 L) is fitted with over head stirrer, Multi-level stirrer blade, water cooled condenser, thermocouple, and bubbler. A gasket was installed between the top lid and bottom kettle. All unused ports are capped with the appropriate plug. Temperature is controlled with a heating mantle regulated by a temperature controller fitted with the above thermocouple.

(16) Polymerization, The Polyvinyl Alcohol is dispersed in the water charge at room temperature (RT) and then heated to 70 C. The remaining salts (See Table 1, MSP, DSP, TSP, & Sodium Nitrite) are then dissolved in the water charge. The PVA and Salts solutions are heated to 80 C. with stirring. The pre-mixed organic phase listed in Table 2 including the initiator is poured into the reactor onto the aqueous phase with the stirring speed set at the rpm for formation of the appropriate droplet size. Once temperature reaches the specified value start reaction timer (16 hours).

(17) TABLE-US-00001 TABLE 1 Aqueous Phase Charges Item Charge, g Ultrapure Water 231.26 Polyvinyl Alcohol (PVA) 0.68 Monosodium Phosphate 0.71 (MSP) Disodium Phosphate (DSP) 2.36 Trisodium Phosphate (TSP) 1.47 Sodium Nitrite 0.01 Total 236.48

(18) Work-up, Mark solvent level. After cooling the solvent is siphoned out to bead level. Reactor is filled to mark with (RT) water and heated to 50 C. to 70 C. and stirred for 30 minutes, allowed to settle for 3 to 5 minutes and then siphoned out to bead level. Beads are washed 5 times in this manner. For polymers using cyclohexanol as a porogen 3 additional methanol in pot washes are added. Polymer 1 uses 3 in pot IPA washes. If indicated, the polymer is extracted via a Soxhlet apparatus with per Table 2 overnight. The polymer is steam stripped 6 hours and then dried in an oven overnight (100 C.). This process results in a clean, dry porous sorbent in the form of spherical, porous polymer beads, Sorbent Polymers 1 to 11.

(19) TABLE-US-00002 TABLE 2 Organic Charges, g Divinyl Divinyl Polyproplene Sorbent/ benzene benzene glycol, PPG, Polymer # (63%) (80%) Toluene Isooctane Cyclohexanol Mw 3500 1 84.70 55.78 64.07 2 83.03 151.00 3 71.3 163.15 4 129.55 77.11 25.70 5 106.38 107.80 19.02 6 106.38 114.14 12.68 7 106.38 115.73 11.10 8 94.73 130.21 8.68 9 106.38 109.07 17.76 10 124.05 88.6 11 129.55 102.82 Organic Charges, g Benzoyl Reaction Polystyrene, Peroxide Total, Conditions Work-up, Conditions Sorbent/ Mw (BPO) w/o Rxn 1st Soxhlet 2nd Soxhlet Polymer # 230,000 (97%) BPO Temp. C. Solvent Solvent 1 0.64 236.48 80 2 0.84 234.03 87 Methanol 3 0.73 234.45 80 Methanol 4 1.32 232.37 80 Acetone 5 1.08 232.20 80 Acetone 6 1.08 233.20 80 Acetone 7 1.08 233.20 80 Acetone 8 0.96 233.62 80 Acetone 9 1.08 233.20 80 Acetone 10 9.8 0.94 222.49 80 Toluene Acetone 11 1.32 232.37 80 Methanol

Example 2

Pore Structure Characterization

(20) The pore structures of the sorbent polymers were analyzed with a either Micromeritics AutoPore IV 9500 V1.09 a Mercury Penetrometer (Hg Intrusion instrument). The results are provided in FIG. 1 where the pore volume is plotted as a function of the pore diameter.

Example 3

Pore Structure Comparison to Biomolecule Adsorbtion

(21) The pore structures of the sorbent polymers were compared to Cytochrome C, Human Serum Albumin, and Immunoglobulin G (IgG). Cytochrome C, 12 kDa, was used as a surrogate for middle molecular weight proteins such as cytokines, Human Serum Albumin (67 kDa) as a surrogate for Hemoglobin (64 kDa) and IgG representing antibodies. The comparisons are shown in Table 3.

(22) TABLE-US-00003 TABLE 3 Pore Diameter Based off Log Differential Pore Volume Pore Volume Percent of Percent of Plot between between total Pore Pore Volume total Pore Sorbent/ maximum 50-10000 500-3000 Volume between Between Volume between Polymer# () (cc/g) (cc/g) 500-3000 50-6000 (cc/g) 50-6000 1 1395 1.72 0.93 54 1.68 98 2 2,594 2.31 1.17 51 2.11 91 3 1,830 2.47 1.71 69 2.29 93 4 3,498 0.94 0.74 79 0.93 99 5 10,483 1.01 0.13 13 0.42 42 6 2,591 1.46 1.24 85 1.44 98 7 1,510 1.63 1.28 79 1.59 97 8 2,839 1.84 1.46 79 1.77 96 9 4,337 1.32 0.52 39 1.29 97 Ratio of Ratio of pore volume pore volume between 50-10000 between 50-10000 3 hour 3 hour Human 3 hour to pore to pore Cytochrome Serum Albumin Immunoglobulin Sorbent/ volume between volume between C Removal Removal G Removal Polymer# 500-3000 50-6000 (mg/g) (mg/g) (mg/g) 1 1.85 1.02 53 313.5 152.1 2 1.97 1.09 109.5 204.8 385.5 3 1.44 1.08 91.7 176.1 289.2 4 1.27 1.01 9.7 107 103.9 5 7.77 2.39 0.4 101.3 38.8 6 1.18 1.02 34.4 77.4 128.1 7 1.27 1.03 62.4 228.3 266.8 8 1.26 1.04 49.4 102.8 140 9 2.54 1.03 11.4 90.6 89.3

Example 4

Adsorbtion Experiments

(23) Setup and Initial Sampling

(24) Blood and Polymer Preparation

(25) Each polymer tested was initially prepared as a 50% slurry in 0.9% Saline. Blood was prepared by pooling 8 bags of non-Leukoreduced packed red blood cells 400 mL (human, <4 days old type AB+) each to an empty 3 L saline bag (PVC, NDC 0409-7983-03) and gently mixed by gently rocking 10 times. The pooled blood was aliquot to 8 empty500 mL saline bags, Part Number ND0409-7972-08, weighed and recorded for future reference. The test utilized 3 polymer and a no bead control: Sorbent 1 (Polymer 1) Sorbent 2 (Polymer 2) Sorbent 3 (Polymer 3) Control (No beads)
Blood/Polymer Charge

(26) 80 mL of polymer slurry (50%) was charged to the empty 500 mL saline bags with a Modified Polystyrene 25 mL pipette FIG. 2 and the weight recorded. The pooled packed red blood cells were then transferred into emptied 500 mL saline bags that had been charged with the bead slurry (beads 10% of RBC volume) to be studied or no beads for the control and bag weights were recorded. Each bag was gently rocked back and forth 10 times to mix thoroughly. All blood stored at 5 C. for the duration of the experiment.

(27) Sampling Day 1

(28) On Day One Hematocrit for each sample bag was taken to account for dilution due to polymer slurry charged into each sample bag. Approximately 5 mls of blood were sampled into a red top vacutainer (BD 366430) for Cytokine/IgG analysis and a second polystyrene tube (BD 352099) was sampled using 5 mls of blood for Lysophosphatidylcholine analysis (LPC). Both the red top and polystyrene collection tubes were spun for 20 minutes and the supernatant separated into polypropylene and polystyrene cryo tubes, respectively and frozen at 25 C.

(29) Sample Collection, Days 7, 14, 21, & 41

(30) Sample bags were removed from the refrigerator and gently mixed by inverting 10 times and sampled for hematocrit. Sampling for Cytokine/IgG and LPC was performed identical to Day 1 sampling. All samples collected were stored at 25 C. until analyzed.

(31) Hemoglobin Absorption from Phosphate Buffered Saline

(32) Solution of Hemoglobin in Phosphate Buffered Saline was prepared at a concentration of approximately 11.00 mg/mL. Individual 50 mL polypropylene centrifuge tubes were used for each time point sampled, excluding the t=0 time point (the t=0 samples were taken directly from the Hemoglobin stock solution). 2.5 g of wet polymer with the interstitial saline removed and 22.5 mL of Hemoglobin solution were added to each centrifuge tube. The tubes were then placed on a platform rocker in a 4-8 C. refrigerator. At the appropriate time points, the applicable centrifuge tubes were removed from the refrigerator. Four samples were removed from each centrifuge tube, labeled, and frozen at 20 C. until analysis was performed.

(33) Hemoglobin Absorption from New Human Blood14 Day Aging Study

(34) Three bags of freshly drawn blood were purchased. Upon receipt, the contents of the bags were pooled and approximately 350-400 mL of blood was distributed into two separate blood bags. 30 mL of 0.9% saline containing polymer beads (50% solids) was added to one of the bags (experiment), and 30 mL of 0.9% saline was added to the other (control). One blood sample was taken from the control bag, the result being used as the t=0 (initial) sample value. At each time point, one hematocrit sample and one blood sample was taken. The hematocrit value was measured and recorded, the blood sample was appropriately centrifuged and plasma samples were removed and stored in polypropylene sample vials at 20 C. until analysis was performed. The blood bags were placed on a platform rocker in a 4-8 C. refrigerator. At the appropriate time points, the blood bags were removed from the refrigerator and sampled as previously described. Once sampled, the bags were returned to the refrigerator.

(35) Analysis of Samples from the Hemoglobin in Phosphate Buffered Saline, Hemoglobin in New Human Blood (14 Day Aging Study), and Real Time Aging (41-day) Study of Human Blood (IgG & LPC)

(36) Human Hemoglobin Analysis; Analysis of Human Hemoglobin was conducted on the collected blood samples using Bethyl Laboratories Incorporated's Human Hemoglobin ELISA Kit, Catalog #E88-135. Analysis procedures were conducted according to the manual provided with the kit. See Table 4 & 5 for the resultant data and FIGS. 4 & 5 for a graphical representation.

(37) This experiment represented a hemoglobin adsorption experiment under controlled conditions with known starting concentrations of hemoglobin.

(38) TABLE-US-00004 TABLE 4 Hemoglobin Data (Phosphate Buffered Saline) Polymer ID Sample Description [Hb] (mg/mL) Polymer 1 t = 0 hours 11.00 t = 1 hour 9.79 t = 4 hours 6.06 t = 6 hours 6.41 t = 24 hours 1.68 Polymer 2 t = 0 hours 11.00 t = 1 hour 8.35 t = 4 hours 5.24 t = 6 hours 4.46 t = 24 hours 4.03 Control t = 0 hours 11.00 t = 1 hour 15.50 t = 4 hours 14.96 t = 6 hours 13.30 t = 24 hours 16.33

(39) This test was designed to show the dynamic removal of hemoglobin by the test polymers in a model system where hemoglobin is constantly generated by gently rocking the blood in a bag causing red blood cell lysis and release of hemoglobin.

(40) TABLE-US-00005 TABLE 5 Hemoglobin Data (New Human Blood - 14 day Aging Study) [Hb] (mg/mL) Corrected for Polymer ID Sample Description Hematocrit Polymer 1 t = 0 days 1.25 t = 1 day 1.05 t = 4 days 1.18 t = 14 days 1.06 Control t = 0 days 1.25 t = 1 day 1.33 t = 4 days 3.87 t = 14 days 5.41

(41) Human Immunoglobulin G Analysis; Analysis of Human Immunoglobulin G was conducted on the collected blood samples using ZeptoMetrix Corporation's Immunotek Quantitative Human IgG ELISA Kit, Catalog #0801182. Analysis procedures were conducted according to the manual provided with the kit. See Table 6 for the resultant data and FIG. 6 for a graphical representation.

(42) TABLE-US-00006 TABLE 6 [IgG] (g/mL) Polymer ID Sample Description Corrected for Hematocrit Polymer 1 t = 0 93.54 t = 7 days 60.30 t = 14 days 44.12 t = 20 days 41.81 t = 41 days 25.06 Polymer 2 t = 0 93.54 t = 7 days 55.44 t = 14 days 53.25 t = 20 days 72.27 t = 41 days 58.79 Polymer 3 t = 0 93.54 t = 7 days 77.75 t = 14 days 83.39 t = 20 days 83.39 t = 41 days 78.30 Control t = 0 93.54 t = 7 days 76.68 t = 14 days 96.82 t = 20 days 86.86 t = 41 days 103.10

(43) Human Lysophosphatidylcholine Analysis; Analysis of Human Lysophosphatidylcholine was conducted on the collected blood samples using Cosmo Bio Company's AZWELL LPC Assay Kit, Catalog #ALF-274729843. Analysis procedures were conducted according to the manual provided with the kit (translated into English), with one exception: 700 nm filters are not currently available for the microplate reader in our facility (BioTek EL800). Therefore, we were unable to measure absorbance at this wavelength for avoidance of interference as recommended by the kit manufacturer. See Table 7 for the resultant data and FIG. 7 for a graphical representation.

(44) TABLE-US-00007 TABLE 7 [LysoPC] (mol/L) Polymer ID Sample Description Corrected for Hematocrit Polymer 1 t = 0 65.85 t = 7 days 25.23 t = 14 days 19.18 t = 20 days 19.53 t = 41 days 9.40 Polymer 2 t = 0 65.85 t = 7 days 19.28 t = 14 days 16.57 t = 20 days 10.52 t = 41 days 11.84 Polymer 3 t = 0 65.85 t = 7 days 19.11 t = 14 days 14.73 t = 20 days 6.02 t = 41 days 5.33 Control t = 0 65.85 t = 7 days 58.41 t = 14 days 55.97 t = 20 days 47.63 t = 41 days 62.87

(45) Human Cytokine Analysis; Analysis of thirteen Human Cytokines (IL-1, IL-2, IL-4, IL-5, IL-6, IL-7, IL-8, IL-10, IL-12(p70), IL-13, IFN, GM-CSF, and TNF) was conducted on the collected blood samples using Millipore's Milliplex MAP High Sensitivity Human Cytokine Magnetic Bead Kit, Catalog #HSCYTMAG-60SK. Analysis procedures were conducted according to the manual provided with the kit. Several analytes returned values below the lower limit of quantitation for the assay, and thus are not reported. See Table 8 for the resultant data and FIGS. 8, 9, & 10 for a graphical representation of each reported cytokine.

(46) TABLE-US-00008 TABLE 8 [IL-7] [TNF] (pg/mL) [IL-8] (pg/mL) (pg/mL) Sample Corrected for Corrected for Corrected for Polymer ID Description Hematocrit Hematocrit Hematocrit Polymer 1 t = 0 0.89 7.17 3.03 t = 7 days 0.53 3.14 0.98 t = 14 days 0.56 2.54 0.51 t = 20 days 0.25 2.11 0.32 t = 41 days 0.00 3.44 0.09 Polymer 2 t = 0 0.89 7.17 3.03 t = 7 days 0.56 2.59 1.25 t = 14 days 0.37 1.74 0.38 t = 20 days 0.07 2.92 0.04 t = 41 days 0.15 1.46 0.06 Polymer 3 t = 0 0.89 7.17 3.03 t = 7 days 0.48 3.37 1.16 t = 14 days 0.53 3.21 0.60 t = 20 days 0.44 4.44 0.22 t = 41 days 0.51 3.79 0.07 Control t = 0 0.89 7.17 3.03 t = 7 days 0.99 12.04 3.70 t = 14 days 1.34 13.76 3.94 t = 20 days 1.52 16.89 4.11 t = 41 days 1.98 40.70 5.49