Method and device for preparing IL-1Ra enriched plasma

Abstract

A method and apparatus are disclosed for preparing plasma enriched with interleukin receptor 1 antagonist ("IL-1Ra") using glass beads or polyacrylamide microbeads and collecting the IL-1Ra-enriched plasma using a filter.

Description

[Technical field]

[0001] The present invention relates to a method and apparatus for preparing plasma enriched with interleukin receptor 1 antagonist ("IL-1Ra") using glass beads or polyacrylamide microbeads. Embodiments of the present invention relate to the preparation of platelet rich plasma (PRP) compositions enriched with IL-1Ra. [Background technology]

[0002] Nos. 9,962,480 and 10,617,812, both by the present inventor, contain embodiments directed to systems and methods for obtaining cell-enriched cell samples, such as platelet rich plasma (PRP), including separation using centrifugation of a blood sample in a centrifuge tube using a density separation medium and separation of cellular components using various filter systems, the disclosures of which are incorporated by reference.

[0003] U.S. Patent No. 8,734,373, by the present inventor, describes a device and method for preparing PRP that includes using an elongated filter device adapted to fit snugly into a test tube to separate cellular components from the plasma fraction, and the disclosure of this patent is also incorporated by reference as it relates to a filter adapted to be inserted into a collection tube.

[0004] U.S. Patent Nos. 10,167,310 and 10,519,196, also by the inventor of the present invention, contain embodiments directed to systems and methods for obtaining a plasma fraction enriched in interleukin-1 receptor antagonist (IL-1Ra). In embodiments, the disclosed methods include centrifuging a blood sample in a blood collection tube and incubating the plasma fraction to enrich the fraction for IL-1Ra. The disclosures of these patents are likewise incorporated by reference.

[0005] There remains a need in the art for methods and devices for obtaining IL-1Ra-enriched plasma samples with enhanced recovery of IL-1Ra, as well as methods for providing IL-1Ra-enriched PRP with greater efficiency. [Prior art documents] [Patent documents]

[0006] [Patent Document 1] U.S. Patent No. 9,962,480 [Patent Document 2] U.S. Pat. No. 10,617,812 [Patent Document 3] U.S. Pat. No. 8,734,373 [Patent Document 4] U.S. Patent No. 10,167,310 [Patent Document 5] U.S. Pat. No. 10,519,196 Summary of the Invention [Problem to be solved by the invention]

[0007] Some aspects of the present invention relate to simple and effective methods for obtaining IL-1Ra enriched plasma samples and methods for providing IL-1Ra enriched PRP. [Means for solving the problem]

[0008] In one aspect, the invention relates to a method for preparing a plasma composition enriched in IL-1Ra, comprising the steps of collecting a PRP fraction in a collection container or tube containing polymeric microbeads, mixing the polymeric microbeads with the PRP fraction to form a homogenous mixture of microbeads and PRP, allowing the homogenous mixture to sit for a predetermined period of time, and passing the mixture through a filter (and / or centrifuging the container or tube containing the mixture) to separate the microbeads together with cells and / or cell fractions of the plasma and obtaining the IL-1Ra enriched plasma composition.

[0009] In embodiments, recovery of IL-1Ra (defined as the mass of IL-1Ra in the final processed product divided by the mass of IL-1Ra in baseline PRP) ranges from 2.2-21 fold, with intermediate values ​​such as greater than 5-fold and greater than 10-fold. As an increase in concentration (picograms / ml), the technology of the present invention may result in a 2.8-25.8-fold increase in concentration over baseline.

[0010] In another aspect, the present invention is embodied in a method for preparing a plasma composition enriched in IL-1Ra, the method comprising the steps of collecting a whole blood sample in a centrifuge tube containing glass beads, contacting the whole blood sample with the surface of the beads for a predetermined time (e.g., i) 4 to 24 hours at a temperature of 30 to 40°C, or ii) 5 minutes to 1 hour at room temperature), centrifuging the whole blood sample to obtain IL-1Ra-enriched plasma, and passing the IL-1Ra-enriched plasma through a filter to obtain the IL-1Ra-enriched plasma composition.

[0011] In embodiments, the recovery of IL-1Ra (relative to baseline) with glass beads is 1.2-fold, 1.3-fold, 1.4-fold or more greater than the recovery without glass beads. In embodiments, glass beads may be used in combination with polymeric microbeads, and in combination with a separation medium (such as a gel) with or without polymeric microbeads. [Brief description of the drawings]

[0012] The subject matter which is regarded as the invention is particularly pointed out and distinctly claimed in the documents annexed to the specification, however, the invention, together with its objects, features, and advantages, both as to organization and method of operation, may best be understood by reference to the following detailed description when read in conjunction with the accompanying drawings.

[0013] [Figure 1] FIG. 1 depicts a process sequence in which PRP is enriched for IL-1Ra by incubating the PRP on polymer microbeads, followed by collection of the IL-1Ra-enriched PRP using a sleeve filter, according to one embodiment of the present invention. [Diagram 2] FIG. 2 depicts a process sequence in which whole blood is incubated with glass beads, followed by centrifugation and recovery of IL-1Ra enriched plasma, according to an embodiment of the invention.

[0014] It will be understood that for simplicity and clarity of illustration, elements shown in the figures have not necessarily been drawn to scale. For example, the dimensions of some of the elements may be exaggerated relative to other elements for clarity. Further, where considered appropriate, reference numerals may be repeated among the figures to indicate corresponding or similar elements. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0015] Containers or tubes for use in the present invention may be of the evacuated or non-evacuated type, made of glass or plastic. In embodiments, collection tubes for use in the present invention are made from durable plastics such as polyacrylate (PA) or polyethylene terephthalate (PET), polyethylene terephthalate glycol (PETG) or polyethylene naphthalate (PEN). In other embodiments, the tubes or containers may be glass or coated glass.

[0016] In an embodiment, the container or tube is provided with a separation medium, such as a gel having a density in the range of 1.04-1.08 g / ml, in an embodiment in the range of 1.073-1.078 g / ml. In an embodiment, the tube or container is provided with an anticoagulant, such as heparin salts, citrate / citric acid (e.g., sodium citrate / citric acid), citrate / citrate-dextrose (acid-citrate-dextrose ACD), citrate-dextrose-phosphate, oxalate, and oxalate-fluoride salts. In an embodiment, the container or tube is provided without a gel. In an embodiment, the container or tube is provided without an anticoagulant.

[0017] The vessel or tube may be pre-inserted with glass beads and / or polymeric microbeads with high water absorption, such as polyacrylamide (PAA) microbeads. Alternatively, glass beads of the type conventionally used for incubation of cell samples may be used. Examples of both systems are provided below, sometimes leading to surprisingly different results depending on the combination of process steps.

[0018] The container or tube may be filled with 10-100 ml of whole blood, for example 10-50 ml of whole blood. Alternatively, the glass beads or polymer microbeads may be pre-inserted into a second container or tube, which is then used to collect the plasma or PRP fraction generated in a separate container or tube.

[0019] If the anticoagulant and glass beads are pre-inserted into the collection tube, the tube may be incubated for 4 to 24 hours or left at ambient temperature for 0 to 1 hour and then centrifuged (separated) at 1000 g to 3000 g, for example at 1500 g, in which centrifugation the fraction of plasma enriched in IL-1Ra may be separated by a sleeve filter as described in the above mentioned US Pat. No. 10,519,196, or transferred directly to a syringe or through a 0.22 to 0.45 μm disc filter to separate the cells and cell debris as well as the microbeads and allow the plasma enriched in IL-1Ra to be stored at a suitable temperature, for example -5 to -20° C., which allows the formulation of IL-1Ra to be preserved and prepared for use. The above filtration procedure may be followed by a second centrifugation, for example at 2000-4000 g for 5-10 minutes, to sediment cells, cell fractions and microbeads.

[0020] If the first container or collection tube is provided without glass beads or polymeric microbeads, the blood sample may be centrifuged at 1000-3000 g, e.g. 1500 g, for a few minutes, e.g. 5-10 minutes, to obtain separated plasma, the upper fraction being platelet poor plasma (PPP) and the lower fraction (still above the gel) being PRP enriched in monocytes and lymphocytes but depleted of red blood cells and granulocytes due to the specific properties of the gel acting as a separator. The PPP is removed, e.g. 50%-75% of the total plasma volume, and the cells are dispersed in the remaining plasma to prepare the IL-1Ra enriched composition.

[0021] The PRP fraction may be inserted into a second collection tube together with glass beads and / or plastic microbeads. If only glass beads are pre-inserted into the second tube, incubation may proceed for 1-24 hours, for example 8-16 hours, for example at 37° C., with or without 5% CO2, and the IL1Ra enriched plasma is collected, for example through a disk filter having a pore size of 0.22-0.45 microns (0.22-0.45 μm), to exclude cells, providing plasma enriched for IL-1Ra at the time of processing, or 3-12 months after the time of processing when stored at an appropriate temperature, such as −5 to −20° C.

[0022] If only polymeric microbeads or only polymeric microbeads and glass beads are pre-inserted into the collection tube, collection of IL-1Ra enriched plasma can be performed immediately or up to 1 hour, and the product may be withdrawn after 15 to 30 minutes, for example, through a sleeve filter having a pore size of 0.22 (0.22 μm) to 40 microns (40 μm) and / or through a disk filter having a preferred pore size of 0.22 microns to 0.45 microns (0.22 to 45 μm), or directly into a syringe.

[0023] In one embodiment, whole blood is collected in a glass collection vessel containing density separation gel and glass microbeads in the absence of an anticoagulant, followed by incubation of the PRP fraction for 30 minutes to 24 hours. After incubation, the mixture may be centrifuged at 900 to 2500 g to separate the fractions. To collect the IL-1Ra concentrate, the IL-1Ra enriched fraction may be passed through a 0.2 to 1.0 micron (0.2 to 1.0 μm) filter that separates the microbeads, cells and cellular fractions. Alternatively, the mixture may be centrifuged at 3000 to 5000 g to remove these components. The resulting IL-1Ra enriched plasma composition may be used for injection into a patient.

[0024] Infusion of IL-1Ra enriched plasma can be performed immediately after the above procedure, or even 1 to 12 months later if the IL-1Ra enriched plasma is stored at an appropriate temperature, for example -5 to -20°C.

[0025] In embodiments, IL-1Ra enriched plasma products prepared according to the methods described herein may be effective as treatments, such as by injection, for both early and late orthopedic pathological conditions and / or to slow down the natural progression of chronic orthopedic diseases. IL-1Ra enriched plasma products may be effective in patients with early symptoms and in patients with a family history seeking preventative therapy. In embodiments, the products may be used as preventative therapy in orthopedic surgery or as a symptom modifier option after injury. Injection intervals may be made depending on the severity and reappearance of symptoms.

[0026] Autologous IL-1Ra therapy provides a non-surgical, minimally invasive therapy without immunological response and without pharmacological treatment. The resulting IL-1Ra is highly concentrated and virtually free of contamination by red blood cells and pro-inflammatory white blood cells.

[0027] As shown in Figure 1, a tube 10 may contain polymeric microbeads 12 and PRP may be provided to the tube 10 from a syringe 14. The PRP 20 may be mixed using a blunt needle connected to a syringe 18 or using a vortex device 22. In step 24, the mixture is allowed to sit for about 15 minutes. A sleeve filter 26 may then be inserted towards the bottom of the tube to collect the IL-1Ra enriched plasma which may be removed using a syringe 30 for direct use with a subject.

[0028] As shown in FIG. 2, in another embodiment, a glass or plastic tube 40 may be provided with glass or polymer microbeads 42 and a separation gel 43. Whole blood may be added in step 44 and the tube may be incubated for 4-24 hours or left at ambient conditions for up to 1 hour in step 46. Incubation may be followed by centrifugation, for example at 1000 g to 3000 g, to separate the plasma fraction 50 from the red blood cells 56 and gel 52. A desired amount of IL-1Ra-enriched plasma may be removed from the separated fraction 58 using a syringe 60 and a 0.2-1.0 micron disc filter. An aliquot 70 of IL-1Ra may be collected for administration to one or more subjects 74 on-site or at a later date if appropriate storage 72 is provided (−5° C. to −20° C.). EXAMPLES

[0029] Example 1 Tropokine™ IL-1Ra Enriched Plasma Using Microbeads (Suggested Protocol) 1. Purpose Inducing the production of IL-1Ra derived from blood separation platforms such as TropoCells® and fused in Tropokine™ P using polyacrylamide (PAA) microbeads. 2. Equipment 2.1. 22 mL TropoCells® tube containing gel with anticoagulant 2.2. TropoCells® Vent Needle 2.3. VACU20S 2.4. 10mL syringe x 3 2.5. Disk Filter 40 2.6. Syringe filter 0.22μm 2.7. Sharp needle 16G / 90mm 2.8. 2 Blunt needles 100mm 2.9. Tropokine™ P Glass Tubes with PAA Microbeads 2.10. Sleeve filter 2.11. Centrifuge capable of reaching at least 1500g 2.12. Vortex Machine (Optional) 3. Procedure Blood collection 3.1.1. Blood is drawn into a 22 mL TropoCells® tube. 3.1.2. Invert the tube 2-4 times to mix the blood with the anticoagulant. 3.1.3. Insert the tube into the centrifuge. Always use a balance tube. Centrifugation 3.2.1. Spin at 1,500 RCF(g) for 20 minutes. 3.2.2. Gently remove the tube from the centrifuge and place it on the stand. 3.3. PRP preparation 3.3.1. Insert the vent needle into the tube rubber stopper. 3.3.2. Insert a long sharp needle into the rubber stopper of the tube. 3.3.3. Attach the disk filter to a 10 ml syringe and connect it to the long sharp needle previously inserted into the tubing (step 3.3.2). 3.3.4. Remove the PPP, leaving only 5 ml of plasma. NOTE: Do not touch the gel with the needle. The tip of the needle should be above the gel. 3.3.5. Keep the needle inserted into the tube and remove only the syringe containing the PPP. 3.3.6. Attach a new 10 ml syringe to the disk filter connected to a long sharp needle. 3.3.7. Suspend the cells on top of the gel in the remaining plasma by inverting the tube 10 times. 3.3.8. Harvest PRP. NOTE: Do not touch the gel with the needle. The tip of the needle should be above the gel. 3.4. IL-1Ra enrichment 3.4.1. Open the rubber stopper of the 10 mL Tropokine™ P® glass tube with PAA microbeads and slowly transfer the PRP by gently tilting the tube so that all the microbeads are immersed in the PRP liquid, using a blunt needle connected to a syringe to mix the microbeads well with the PRP. NOTE: Ensure that all microbeads are well mixed with the PRP and there is no white powder residue. 3.4.2. Close the tube with a rubber stopper and allow the tube to stand with the PAA microbeads for 15 minutes at room temperature. 3.4.3. Remove the rubber stopper and insert the sleeve filter into the tube. 3.4.4. Push the sleeve filter along the tube. NOTE: Ensure that all remaining plasma has been extracted and the microbead gel precipitant has turned whitish. 3.4.5. Attach a blunt needle to a new syringe equipped with a 0.22 μm syringe filter and insert it into the bottom of the sleeve filter to collect the concentrated enriched plasma. Option 1: Mix the PAA microbeads thoroughly with the PRP using a vortex. Option 2: Centrifuge the tube for 5-10 minutes to pellet the cells, cell fractions, and microbeads. Aliquot samples and store at -20°C for up to 7 months until analysis by ELISA. Avoid repeated freeze-thaw cycles. ELISA evaluation If IL-1Ra concentration values ​​are required, proceed as follows: 3.5.1. Perform ELISA using Quantikine Human IL-1ra / IL-1F3 Immunoassay number DRA00B according to the supplier's manual. Note: In preliminary studies, the OD wavelength of the ELISA used was 450 nm, and the OD at 540 nm or 570 nm was not subtracted.

[0030] As shown below in Table 1, ELISA evaluation of one sample demonstrated an almost 10-fold increase in IL-1Ra concentration obtained following the procedure described above using PAA beads. The "fold" calculation is obtained by dividing the mass of IL-1Ra in the final product after processing by the mass of IL-1Ra in the baseline PRP.

[0031] [Table 1]

[0032] Example 2 Tropokine™ IL-1Ra Enriched Plasma using Glass Beads (Suggested Protocol) 4. Purpose Inducing the production of IL-1ra by incubating whole blood in Tropokine™ G tubes with glass beads. 5. Equipment 5.1. 22 mL or 11 mL TropoCells® tubes containing gel 5.2. 22mL or 11mL Tropokine™ G Tubes with Glass Beads (Optional Blood with Gel and Glass Beads) 5.3. Bent needle 5.4. VACU20S 5.5. 10mL Syringe 5.6. Disc filter 40μm 5.7. Syringe filter 0.22μm 5.8. Sharp needle 16G / 90mm or 16G / 65mm 5.9. 22mL or 11mL TropoCells® Tubes Containing Gel (Optional) 5.10. Centrifuge capable of reaching at least 2000g 5.11. Incubator capable of reaching 37°C 6. Procedure 6.1. Blood collection and IL-1Ra enrichment 6.1.1. Blood is drawn into a 22 mL or 11 mL Tropokine™ G tube. 6.1.2. Invert the tube 2-4 times to mix the blood with the glass beads. 6.1.3. Incubate the tubes at 37°C overnight. 6.2. Plasma blood separation Option 1 6.2.1. After incubation, spin the tubes at 1500-2000 RCF(g) for 10 minutes. 6.2.2. Gently remove the tube from the centrifuge and place it on the stand. 6.2.3. Insert the vent needle into the tube rubber stopper. 6.2.4. Insert the sharp needle into the tube rubber stopper. 6.2.5. Attach a 0.22 μm disk filter to a 10 mL syringe and connect it to the sharpened needle previously inserted into the tubing (3.2.4). 6.2.6. Draw in plasma. Aliquot samples and store at -20°C for a maximum of 7 months until analysis by ELISA. Avoid repeated freeze-thaw cycles. Option 2 - relevant when working with tubes containing only glass beads 6.2.8. After incubation, the blood is transferred to TropoCells® tubes containing anticoagulant-free gel. 6.2.9. Spin the tube at 1,500 RCF(g) for 10 minutes. 6.2.10. Insert the vent needle into the tube rubber stopper. 6.2.11. Insert the sharp needle into the rubber tube stopper. 6.2.12. Attach a 0.22 μm disk filter to a 10 mL syringe and connect it to the sharpened needle previously inserted into the tubing (3.2.11). 6.2.13. Draw in plasma. Aliquot samples and store at -20°C for a maximum of 7 months until analysis by ELISA. Avoid repeated freeze-thaw cycles. 6.3. ELISA evaluation If IL-1Ra concentration values ​​are required, proceed as follows: 6.3.1. Perform ELISA using Quantikine Human IL-1Ra / IL-1F3 Immunoassay number DRA00B according to the supplier's instructions. NOTE: In preliminary studies, the OD wavelength for ELISA was 450 nm, and OD at 540 nm or 570 nm was not subtracted.

[0033] [Table 2]

[0034] The use of glass beads was confirmed by ELISA evaluation. "Fold" was calculated by dividing the baseline IL-1Ra mass after treatment with beads by the baseline IL-1Ra mass. Whole blood with glass beads - blood in glass tubes with glass beads. Whole blood without glass beads - blood in glass tubes without glass beads.

[0035] Example 3 - Comparative Effects of Using Glass and Plastic Collection Containers; Polymer Microbeads; and Glass Beads Whole blood samples were obtained from four donors and IL-1Ra was measured and reported as concentration (pg / ml) and total mass (pg) at baseline and after processing according to the techniques described above using glass tubes containing only microbeads, plastic syringes containing only microbeads, glass collection tubes only; glass tubes containing glass beads and microbeads; and glass tubes with glass beads + separation gel + microbeads, with the following results:

[0036] [Table 3]

[0037] [Table 4]

[0038] The sample volumes are reported in Table 5.

[0039] [Table 5]

[0040] The increase factors (based on total mass) are reported in Table 6.

[0041] [Table 6]

[0042] Example 4 In this example, whole blood was collected in glass tubes containing density separation gel and glass microbeads in the absence of anticoagulant. The vessels were incubated for 30 minutes to 24 hours (to prepare different amounts of IL-1Ra in the final product), after which the tubes were centrifuged at 1500 g for 10 minutes. IL-1Ra enriched plasma compositions were obtained directly from the plasma fraction via a syringe, and the mixture was passed through a 0.2 to 1.0 micron (0.2 to 1.0 μm) disk filter. Table 7 shows the average mass of IL-1Ra in picograms obtained in the samples after different incubation periods.

[0043] [Table 7]

[0044] Although the present invention has been described with respect to a limited number of embodiments, these embodiments should not be construed as limitations on the scope of the present invention, but rather as illustrations of some of the preferred embodiments. Other possible variations, modifications, and applications are also within the scope of the present invention. Thus, the scope of the present invention should be limited not by what has been described above, but by the appended claims and their legal equivalents.

Claims

1. 1. A method for preparing an IL-1Ra enriched plasma composition, comprising: collecting the PRP fraction in a collection container or collection tube containing polymer microbeads; mixing the polymeric microbeads with the PRP fraction to form a homogenous mixture of microbeads and PRP; allowing the homogeneous mixture to stand for a predetermined period of time; filtering the mixture and / or centrifuging the container or tube containing the mixture to separate the microbeads, cells and cell fractions and obtain the IL-1Ra-enriched plasma composition; A method comprising:

2. The method of claim 1, wherein the polymeric microbeads are micron-scale hydrophilic polyacrylamide beads.

3. 3. The method of claim 2, wherein the polymer microbeads are mixed with the PRP fraction in a collection tube, and the step of filtering the mixture comprises allowing the homogenous mixture to stand for about 15 minutes and then forcing a sleeve filter into the homogenous mixture to obtain an IL-1Ra-enriched plasma fraction from which the beads have been removed in the sleeve filter.

4. The method of claim 3, further comprising the step of removing the IL-1Ra-enriched plasma fraction from the sleeve filter with a syringe while the sleeve filter remains inserted in the centrifuge tube.

5. 10. The method of claim 1, wherein the step of mixing the polymer microbeads with the PRP comprises mixing in a vortex device or stirring with a needle attached to a syringe.

6. 10. The method of claim 1, wherein the IL-1Ra concentration, measured in picograms per milliliter, is increased by 2.8 to 25.8 fold in the IL-1Ra-enriched plasma composition compared to baseline PRP as determined by an ELISA assay.

7. 10. The method of claim 1, wherein the total IL-1Ra obtained in said plasma composition is increased by 2.2 to 21 fold compared to baseline, as measured in picograms.

8. 1. A method for preparing an IL-1Ra enriched plasma composition, comprising: collecting a whole blood sample into a centrifuge tube containing glass beads, polymer microbeads, or a combination of glass beads and polymer microbeads; contacting the whole blood sample with the surface of the beads for a predetermined period of time; centrifuging the whole blood sample to obtain IL-1Ra-enriched plasma; filtering the IL-1Ra-enriched plasma to obtain said IL-1Ra-enriched plasma composition; A method comprising:

9. 9. The method of claim 8, wherein the whole blood sample is collected in a centrifuge tube containing an anticoagulant, a separating gel, and the glass beads.

10. 9. The method of claim 8, wherein the whole blood sample is contacted with the surface of the beads i) at a temperature of 30-40°C for 4-24 hours, or ii) at room temperature for 5 minutes to 1 hour, followed by centrifugation at 1000g to 3000g.

11. 9. The method of claim 8, wherein the IL-1Ra-enriched plasma is removed from the centrifuge tube through a syringe having a disc filter, and the IL-1Ra-enriched plasma is passed through the disc filter to obtain a dose aliquot of IL-1Ra-enriched plasma.

12. 1. A method for preparing an IL-1Ra enriched plasma composition, comprising: collecting whole blood and glass microbeads in a glass collection container or tube in the absence of an anticoagulant; Incubating the vessel or tube for 30 minutes to 24 hours; After incubation, centrifuging the container or tube at 900-2500 g; separating the microbeads, cells and cell fractions by passing the mixture through a 0.2-1.0 micron (0.2-1.0 μm) filter or by a second centrifugation step at 3000-5000 g; collecting said IL-1Ra-enriched plasma composition; A method comprising:

13. Incubation was carried out at 5% CO ２ The method of claim 12 carried out in an atmosphere comprising

14. 13. The method of claim 12, wherein the step of passing the mixture through a 0.2 to 1.0 micron (0.2-1.0 μm) disk filter comprises passing the mixture directly into a syringe.

15. A method for preparing an IL-1Ra-enriched plasma composition, comprising: collecting a whole blood sample in a glass centrifuge tube, the glass centrifuge tube being uncoated and containing density separation gel but no glass or polymer beads; contacting the whole blood sample with the surface of the centrifuge tube and incubating it at 30 to 40°C for a predetermined time period of 30 minutes to 24 hours; centrifuging the whole blood sample to obtain IL-1Ra-enriched plasma; filtering the IL-1Ra-enriched plasma to obtain said IL-1Ra-enriched plasma composition; A method comprising:

Images