Method for purifying inter-alpha inhibitor protein

Abstract

Described herein is a method for purifying IαIp from biological material using an endotoxin-binding agent, which comprises applying the biological material containing IαIp to the endotoxin-binding agent, discarding the flow-through, applying a wash buffer, and eluting IαIp from the endotoxin-binding agent.

Description

[Background technology]

[0001] Inter-alpha inhibitor proteins (IαIp) are a family of naturally occurring immunomodulatory plasma proteins that circulate at high concentrations in the blood of all mammals. IαIp promotes a protective effect against inflammation caused by infection, trauma, and injury. The protective effect of IαIp is independent of the causative microorganism or trigger.

[0002] Members of this family are composed of heavy and light polypeptide subunits covalently linked to glycosaminoglycans. IαIp can be found in vivo as inter-alpha inhibitor (IαI), a 250 kDa molecule consisting of two heavy chains (H1 and H2) and one light chain (L), called bikunin, and pre-alpha inhibitor (PαI), a 125 kDa molecule consisting of one heavy chain (H3) and one light chain (L).

[0003] When inflammatory signals occur in the body, such as those induced during injury or infection, IαIp is transmitted into tissues and reaches the site of inflammation directly. The heavy chain of IαIp enhances the anti-inflammatory response by binding to proteins (damage signals) that are part of the inflammatory cascade, such as complement and extracellular histones, thereby suppressing the inflammatory process, while the light chain, bikunin, inhibits the activity of serine proteases such as trypsin, elastase, plasmin, cathepsin G, and furin.

[0004] IαIp has been shown to promote lung epithelial repair after injury in both in vitro and in vivo models, and IαIp has also been shown to downregulate proinflammatory cytokines such as TNF-α and IL-6 in multiple in vivo models.

[0005] In healthy individuals, the amount of IαIp circulating in the blood is relatively high ( IαIp levels rapidly decline during systemic inflammation / sepsis in neonatal and adult patients (Baek YW, et al. J Pediatr. 2003;143:11-15; Lim YP, et al. J Infect Dis. 2003;188:919-926 and Opal SM, et al. Crit Care Med. 2007;35:387-392), and decreased IαIp levels have been shown to strongly correlate with disease progression. IαIp therapy has been described for the treatment of sepsis and associated organ dysfunction, pneumonia, acute respiratory disease, necrotizing enterocolitis (NEC), wounds, burns, cancer, stroke, and Alzheimer's disease.

[0006] Previously, IαIp has been prepared using stepwise extraction followed by chromatographic separation. While these methods can achieve high purity (e.g., greater than 90%), they suffer from low yields of IαIp (e.g., 20-30% (w / w)). Therefore, there is a need for methods to purify or prepare IαIp in high yield and purity, for use in, for example, preparing therapeutic compositions. Summary of the Invention

[0007] The disclosure features methods for purifying IαIp (e.g., one or more of IαI, PαI, and bikunin), in which, as one step in the purification process, biological material (e.g., blood or milk) containing IαIp, such as biological material obtained from a subject (e.g., a human), is applied to an endotoxin-binding agent (e.g., a solid support such as a chromatography column containing the endotoxin-binding agent).

[0008] A first aspect of the present disclosure is characterized by purifying inter-alpha inhibitor protein (IαIp) from biological material by (a) applying the biological material containing IαIp to an endotoxin-binding agent and separating the flow-through containing biological material that does not bind to the endotoxin-binding agent, and (b) applying an elution buffer containing salt to the endotoxin-binding agent and recovering an eluate containing IαIp.

[0009] In some embodiments, the endotoxin-binding agent is immobilized on a support (e.g., a monolithic support or a particle-based support). In some embodiments, the monolithic support or particle-based support is or comprises a resin. The support can be, for example, a column, a membrane, a disk, or a chip.

[0010] In other embodiments, the endotoxin binding agent is selected from the group consisting of ETOXICLEAR™, PIERCE™ high-capacity endotoxin removal resin, TOXINERASER™ endotoxin removal resin, PURKINE™ endotoxin removal resin, DETOXI-GEL™ endotoxin removal gel, and PROMEGA™ endotoxin removal resin. In certain embodiments, the endotoxin binding agent is DETOXI-GEL™ or ETOXICLEAR™.

[0011] Biological materials include alpha-1 antitrypsin, C1-inhibitor, albumin, globulins (e.g., immunoglobulins (e.g., IgA, IgE, IgM, IgD, and IgG (e.g., intravenous Ig (IVIg), anti-D), The antibody may contain three or more proteins selected from the group consisting of: human IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, and varicella-zoster IgG), fibrinogen (factor I), prothrombin (factor II), thrombin, antithrombin III, factor III, factor V, factor VII, factor VIII, factor IX, factor X, factor XI, factor XII, factor XIII, fibronectin, alpha-2 antiplasmin, urokinase, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2, von Willebrand factor, factor H, prekallikrein, high molecular weight kinogen, and heparin cofactor II.

[0012] In some embodiments, the biological material comprises between 3 and 10, between 3 and 15, between 3 and 20, between 3 and 25, between 3 and 30, between 10 and 20, between 10 and 25, between 10 and 30, between 15 and 25, between 15 and 30, between 20 and 30, or more than 30 different proteins.

[0013] In some embodiments, the biological material comprises about 40 to about 65% albumin (w / w) and / or about 25 to about 45% globulin (w / w) and / or about 2 to about 12% fibrinogen (w / w).

[0014] In some embodiments, the method further comprises applying a first wash buffer to the endotoxin-binding agent after step (a) and before step (b). In some embodiments, the method further comprises separating the flow-through comprising the first wash buffer. In some embodiments, the first wash buffer has a pH of about 4.5 to 8.5 (e.g., about pH 5.2). In some embodiments, the first wash buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the first wash buffer comprises about 10 to about 200 mM glycine and / or about 20 to 300 mM acetic acid (e.g., 75 mM glycine and about 100 mM acetic acid). In some embodiments, the first wash buffer comprises about 200 mM NaCl or less (e.g., about 50 to about 150 mM NaCl (e.g., about 50 mM NaCl or about 100 mM NaCl)). In some embodiments, the first wash buffer comprises about 100 to about 300 mM NaCl (e.g., about 200 mM NaCl). In some embodiments, the first wash buffer comprises about 75 mM glycine, about 100 mM AcOH, and about 200 mM NaCl. In some embodiments, the method further comprises applying a second wash buffer to the endotoxin-binding agent after applying the first wash buffer. In some embodiments, the method further comprises separating the flow-through comprising the second wash buffer. In some embodiments, the second wash buffer has a pH of about 4.5 to about 8.5 (e.g., about pH 7.2). In some embodiments, the second wash buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the second wash buffer comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the second wash buffer comprises about 500 mM NaCl or less (e.g., about 100 to about 500 mM NaCl (e.g., about 300 mM NaCl)).In some embodiments, the second wash buffer comprises about 200 to about 400 mM NaCl (e.g., about 300 mM NaCl), hi some embodiments, the second wash buffer comprises about 20 mM Tris and about 300 mM NaCl.

[0015] In some embodiments, the method further comprises applying a third wash buffer to the endotoxin-binding agent after applying the second wash buffer. In some embodiments, the elution buffer has a pH of about 4.5 to about 8.5 (e.g., about pH 7.2). In some embodiments, the elution buffer comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the elution buffer comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the elution buffer comprises about 1,000 mM or less NaCl (e.g., about 500 to about 1,000 mM NaCl (e.g., about 500 mM NaCl or about 1,000 mM NaCl)). In some embodiments, the elution buffer comprises about 20 mM Tris and about 500 mM NaCl.

[0016] In some embodiments, the method further comprises applying a second elution buffer to the endotoxin-binding agent after applying the elution buffer, wherein the second elution buffer has a pH of about 4.5 to about 8.5 (e.g., about pH 7.2). In some embodiments, the second elution buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the second elution buffer comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the second elution buffer comprises about 1,000 mM or less NaCl (e.g., about 500 to about 1,000 mM NaCl (e.g., about 500 mM NaCl or about 1,000 mM NaCl)). In some embodiments, the second elution buffer comprises about 20 mM Tris and about 1,000 mM NaCl.

[0017] In some embodiments, the method further comprises applying a dilution buffer to the biological material prior to step (a). In some embodiments, the dilution buffer comprises deionized water. In some embodiments, the dilution buffer comprises purified water. In some embodiments, the dilution buffer has a pH of about 4.5 to about 8.5 (e.g., about pH 5.5, about pH 7.2, or about pH 7.3). In some embodiments, the biological material is diluted 1:1 to 1:10 (v / v) with the dilution buffer. In some embodiments, the dilution buffer comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the dilution buffer comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the dilution buffer comprises about 50 mM or less NaCl (e.g., no salt, no NaCl, or about 50 mM NaCl). In some embodiments, the dilution buffer comprises about 5 to about 100 mM phosphate (eg, 15 mM phosphate).

[0018] In some embodiments, the method further comprises adjusting the pH of the biological material prior to step (a). In some embodiments, the method comprises adjusting the pH of the biological material to about 4.5 to about 8.5 (e.g., about pH 5.5, about pH 7.2, or about pH 7.3) by adding acetic acid prior to step (a). In some embodiments, the method further comprises adjusting the electrical conductivity of the biological material prior to step (a). In some embodiments, the method comprises adjusting the electrical conductivity of the biological material to about 10 to about 30 mS / cm, about 15 to about 25 mS / cm (e.g., about 20 mS / cm) prior to step (a).

[0019] In some embodiments, the method further comprises detecting the amount of IαIp in the flow-through, hi some embodiments, the method comprises discarding the flow-through.

[0020] In some embodiments, the method further comprises detecting the amount of IαIp in the eluate.

[0021] In some embodiments, the method comprises a flow rate of about 1-10 mL / min.

[0022] In some embodiments, the method further comprises applying the biological material to a chromatography support (e.g., an anion exchange chromatography support, a size exclusion chromatography support, an ion exchange chromatography support, an affinity chromatography support, or a combination thereof). In some embodiments, the chromatography support is an anion exchange chromatography support described above.

[0023] In some embodiments, the method further comprises (i) applying the biological material to a chromatographic support and separating the flow-through of step (i) comprising the biological material that does not bind to the chromatographic support; and (ii) applying an elution buffer to the chromatographic support and collecting an eluate comprising IαIp.

[0024] In some embodiments, the chromatographic support is a monolithic support or a particle-based support. In some embodiments, the monolithic support or particle-based support comprises an immobilized anion exchange resin (e.g., diethylaminoethane (DEAE) resin or a quaternary amine (Q) resin). In some embodiments, the monolithic support or particle-based support comprises a quaternary amine (Q) resin. In some embodiments, the chromatographic support is a column, a membrane, a disk, or a chip.

[0025] In some embodiments, the method further comprises applying a first wash buffer to the chromatography support after step (i) and before step (ii). In some embodiments, before step (ii), the method further comprises separating the flow-through comprising the first wash buffer. In some embodiments, the first wash buffer applied to the chromatography support has a pH of about 4.5 to about 8.5 (e.g., 7.2). In some embodiments, the first wash buffer applied to the chromatography support comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the first wash buffer applied to the chromatography support comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the first wash buffer applied to the chromatography support comprises about 400 mM or less NaCl (e.g., about 50 to about 250 mM NaCl (e.g., about 250 mM NaCl)). In some embodiments, the first wash buffer applied to the chromatographic support comprises about 20 mM Tris and about 250 mM NaCl.

[0026] In some embodiments, the method further comprises applying a second wash buffer to the chromatographic support after applying the first wash buffer. In some embodiments, the method further comprises separating the flow-through comprising the second wash buffer. In some embodiments, the second wash buffer applied to the chromatographic support has a pH of about 4.5 to about 8.5 (e.g., about pH 5.2). In some embodiments, the second wash buffer applied to the chromatographic support comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, NaOH, and Tris-HCl. In some embodiments, the second wash buffer applied to the chromatographic support comprises about 10 to about 200 mM glycine and / or about 20 to about 300 mM acetic acid (e.g., about 50 mM glycine and about 100 mM acetic acid). In some embodiments, the second wash buffer applied to the chromatographic support comprises about 100 to about 500 mM NaCl (e.g., about 175 mM NaCl).

[0027] In some embodiments, the method further comprises applying a third wash buffer to the chromatographic support after applying the second wash buffer. In some embodiments, the method further comprises separating the flow-through comprising the third wash buffer. In some embodiments, the third wash buffer applied to the chromatographic support has a pH of about 4.5 to about 8.5 (e.g., about pH 7.2). In some embodiments, the third wash buffer applied to the chromatographic support has a pH of about 4.5 to about 8.5 (e.g., 7.2). In some embodiments, the third wash buffer applied to the chromatographic support comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the third wash buffer applied to the chromatographic support comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the first wash buffer applied to the chromatography support comprises about 400 mM or less NaCl (e.g., about 50 to about 250 mM NaCl (e.g., about 200 mM NaCl)). In some embodiments, the first wash buffer applied to the chromatography support comprises about 20 mM Tris and about 200 mM NaCl. In some embodiments, the elution buffer applied to the chromatography support has a pH of about 4.5 to about 8.5 (e.g., about pH 7.2). In some embodiments, the elution buffer applied to the chromatography support comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the elution buffer applied to the chromatography support comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the elution buffer applied to the chromatography support comprises about 1,000 mM or less NaCl (e.g., about 750 mM NaCl).

[0028] In some embodiments, the method further comprises applying a dilution buffer to the biological material prior to step (i). In some embodiments, the dilution buffer comprises deionized water. In some embodiments, the dilution buffer comprises purified water. In certain embodiments, the dilution buffer has a pH of about 4.5 to about 8.5 (e.g., about pH 7.2). In some embodiments, the biological material is diluted 1:1 to 1:10 (v / v) with the dilution buffer. In some embodiments, the dilution buffer comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the dilution buffer comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the dilution buffer comprises about 300 mM or less NaCl (e.g., no salt or about 200 mM NaCl).

[0029] In some embodiments, the method further comprises detecting the amount of IαIp in the flow-through of step (i). In some embodiments, the method comprises discarding the flow-through of step (i).

[0030] In some embodiments, the method further comprises detecting the amount of IαIp in the eluate of step (ii).

[0031] In some embodiments, the method comprises a flow rate of about 1-10 mL / min.

[0032] In some embodiments, the IαIp recovered in the eluate of step (b) has a purity of about 5% to 99% by weight or more relative to the purity of IαIp in the biological material.

[0033] In some embodiments, the yield of IαIp in the eluate collected in step (b) is greater than about 20% (w / w) of the IαIp present in the biological material (e.g., 35% to about 90% or more (e.g., about 95% or more)). In some embodiments, the yield of IαIp from the biological material is at least about 5 μg / ml, about 50 μg / ml, about 100 μg / ml, about 300 μg / ml, about 600 μg / ml, or about 900 μg / ml (e.g., about 5 μg / ml to about 900 μg / ml).

[0034] In some embodiments, the purity of IαIp is at least about 5% (w / w) (e.g., at least about 25%, about 50% (w / w), or about 75% (w / w) (e.g., about 5% (w / w) to about 75% (w / w) or more (e.g., about 90%, 95%, 97%, 99%, or 100% (w / w))).

[0035] In some embodiments, IαIp comprises two or more of inter-alpha inhibitor (IαI), pre-alpha inhibitor (PαI), and bikunin.

[0036] In some embodiments, the IαIp present in the biological material comprises 60% to 80% (w / w) IαIp and / or 20% to 40% (w / w) PαI, and / or the IαIp present in the eluate of step (b) comprises 60% to 80% (w / w) IαIp and / or 20% to 40% (w / w) PαI.

[0037] In some embodiments, IαIp comprises two or more of inter-alpha inhibitor (IαI), pre-alpha inhibitor (PαI), and bikunin. In some embodiments, IαIp is inter-alpha inhibitor (IαI). In some embodiments, IαIp is pre-alpha inhibitor (PαI). In some embodiments, IαIp is inter-alpha inhibitor (IαI) and pre-alpha inhibitor (PαI).

[0038] In some embodiments, the IαIp present in the biological material comprises 60% to 80% (w / w) IαIp and / or 20% to 40% (w / w) PαI, and / or the IαIp present in the eluate of step (b) comprises 60% to 80% (w / w) IαIp and / or 20% to 40% (w / w) PαI.

[0039] In some embodiments, essentially all of the IαIp present in the biological material is IαI. In some embodiments, about 90% to about 99.5% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 80% to about 90% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 70% to about 80% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 60% to about 70% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 50% to about 60% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 40% to about 50% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 30% to about 40% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 20% to about 30% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 10% to about 20% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, about 1% to about 10% (w / w) of the IαIp present in the biological material is IαI.

[0040] In some embodiments, essentially all of the IαIp present in the biological material is PαI. In some embodiments, about 90% to about 99.5% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 80% to about 90% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 70% to about 80% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 60% to about 70% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 50% to about 60% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 40% to about 50% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 30% to about 40% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 20% to about 30% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 10% to about 20% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 1% to about 10% (w / w) of the IαIp present in the biological material is PαI.

[0041] In some embodiments, essentially all of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 90% to about 99.5% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 80% to about 90% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 95% to about 99.5% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 94% to about 98% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 93% to about 97% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 91% to about 96% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 90% to about 95% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 89% to about 94% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 88% to about 93% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 87% to about 92% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 86% to about 91% (w / w) of the IαIp present in the biological material is IαI and PαI. In some embodiments, about 85% to about 90% (w / w) of the IαIp present in the biological material is IαI and PαI.

[0042] In some embodiments, essentially all of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 90% to about 99.5% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 80% to about 90% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 70% to about 80% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 68% to about 78% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 66% to about 76% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 64% to about 74% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 62% to about 72% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 60% to about 65% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 61% to about 66% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 62% to about 67% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 63% to about 68% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 64% to about 69% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 65% to about 70% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 66% to about 71% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 67% to about 72% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 68% to about 73% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 69% to about 74% (w / w) of the IαIp present in the eluate of step (b) is IαI.In some embodiments, about 59% to about 64% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 58% to about 63% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 57% to about 62% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 56% to about 61% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 55% to about 60% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 54% to about 59% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 54% to about 59% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 53% to about 58% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 52% to about 57% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 51% to about 56% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 50% to about 55% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 49% to about 54% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 48% to about 53% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 47% to about 52% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 46% to about 51% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 45% to about 50% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 60% to about 70% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 58% to about 68% (w / w) of the IαIp present in the eluate of step (b) is IαI.In some embodiments, about 56% to about 66% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 54% to about 64% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 52% to about 62% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 50% to about 60% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 48% to about 58% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 46% to about 56% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 44% to about 54% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 42% to about 52% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 40% to about 50% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 30% to about 40% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 20% to about 30% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 10% to about 20% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, about 1% to about 10% (w / w) of the IαIp present in the eluate of step (b) is IαI.

[0043] In some embodiments, essentially all of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 90% to about 99.5% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 80% to about 90% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 70% to about 80% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 60% to about 70% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 50% to about 60% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 40% to about 50% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 38% to about 48% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 36% to about 46% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 34% to about 44% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 32% to about 42% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 30% to about 35% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 31% to about 36% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 32% to about 37% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 33% to about 38% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 34% to about 39% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 35% to about 40% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 30% to about 40% (w / w) of the IαIp present in the eluate of step (b) is PαI.In some embodiments, about 28% to about 38% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 26% to about 36% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 24% to about 34% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 22% to about 32% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 30% to about 35% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 29% to about 34% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 28% to about 33% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 27% to about 32% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 26% to about 31% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 25% to about 30% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 24% to about 29% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 23% to about 28% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 22% to about 27% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 21% to about 26% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 20% to about 25% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 20% to about 30% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 10% to about 20% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, about 1% to about 10% (w / w) of the IαIp present in the eluate of step (b) is PαI.

[0044] In some embodiments, essentially all of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 90% to about 99.5% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 80% to about 90% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 95% to about 99.5% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 94% to about 98% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 93% to about 97% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 91% to about 96% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 90% to about 95% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 89% to about 94% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 88% to about 93% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 87% to about 92% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 86% to about 91% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, about 85% to about 90% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI.

[0045] In some embodiments, the IαIp present in the eluate of step (b) comprises about 60% to about 70% (w / w) IαI and about 20% to about 30% (w / w) PαI. In some embodiments, the IαIp present in the eluate of step (b) comprises about 62% to about 72% (w / w) IαI and about 28% to about 30% (w / w) PαI.

[0046] In some embodiments, IαIp has an apparent molecular weight of about 60 to about 280 kDa.

[0047] In some embodiments, IαIp has biological activity (eg, cytokine inhibitor activity, chemokine inhibitor activity, or serine protease inhibitor activity).

[0048] In some embodiments, the biological material is a blood product material (e.g., whole plasma, cryo-poor plasma, liquid plasma, frozen plasma (FP) (e.g., fresh frozen plasma (FFP), FFP24, FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, and diluted or concentrated versions thereof), source plasma, collected plasma, solvent / detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, whole blood, and diluted or concentrated versions thereof).

[0049] In some embodiments, the biological material is a plasma fraction intermediate produced from a blood product material via one or more process steps (e.g., filtration, centrifugation, precipitation, chromatography, adsorption, isolation, freezing, thawing, dilution, concentration, viral clearance, etc.).

[0050] In some embodiments, the biological material is a blood product produced from a blood product material through one or more process steps (e.g., filtration, centrifugation, precipitation, chromatography, adsorption, isolation, freezing, thawing, dilution, concentration, viral clearance, etc.) In some embodiments, the biological material is milk or colostrum.

[0051] In some embodiments, the biological material is from a mammal (eg, a human, a primate, a bovine, an equine, a porcine, an ovine, a feline, or a canine).

[0052] In some embodiments, the biological material is substantially unprocessed prior to application to the endotoxin-binding agent.

[0053] In some embodiments, the method further comprises performing one or more chromatography steps (e.g., repeating the method of the first aspect and / or embodiments thereof one or more times using the eluate collected in step (b)).

[0054] In some embodiments, the elution buffer applied to the endotoxin-binding agent in step (b) has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 500 mM NaCl.

[0055] In some embodiments, the first wash buffer applied to the endotoxin-binding agent has a pH of 5.2 and comprises about 75 mM glycine, about 100 mM acetic acid, and about 150 mM NaCl.

[0056] In some embodiments, the second wash buffer applied to the endotoxin-binding agent has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 300 mM NaCl.

[0057] In some embodiments, the elution buffer applied to the chromatographic support has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 750 mM NaCl.

[0058] In some embodiments, the first wash buffer applied to the chromatographic support has a pH of 7.2 and comprises about 20 mM Tris and about 250 mM NaCl.

[0059] In some embodiments, the second wash buffer applied to the chromatographic support has a pH of 5.2 and comprises about 50 mM glycine, about 100 mM acetic acid, and about 175 mM NaCl.

[0060] A second aspect of the disclosure features a composition comprising IαIp produced by the method of the first aspect and embodiments thereof. In one embodiment, the composition is suitable for administration to a human.

[0061] A third aspect of the present disclosure features a pharmaceutical composition including the composition of the second aspect and embodiments thereof and a pharmaceutically acceptable excipient.

[0062] A fourth aspect of the present disclosure features a method of treating a disease or condition in a subject in need thereof by administering to the subject a composition of the second aspect or one of its embodiments, or a pharmaceutical composition of the third aspect.

[0063] A fifth aspect of the present disclosure features a kit including the composition of the second aspect or one of its embodiments, or the pharmaceutical composition of the third aspect. In one embodiment, the kit further includes instructions for therapeutic use.

[0064] A sixth aspect of the present disclosure provides a method for preparing diluted plasma, comprising: (a) diluting plasma with a dilution buffer comprising deionized water to form diluted plasma; (b) applying the diluted plasma to an ETOXICLEAR™ resin and separating a flow-through comprising diluted plasma that does not bind to the ETOXICLEAR™ resin; and (c) applying a first wash buffer comprising about 75 mM glycine, about 100 mM AcOH, and about 150 mM NaCl, at a pH of about 5.2, to the ETOXICLEAR™ resin to separate a flow-through comprising the first wash buffer. (d) applying a second wash buffer comprising about 20 mM Tris-HCl and about 300 mM NaCl at a pH of about 7.2 to the ETOXICLEAR™ resin and separating the flow-through comprising the second wash buffer; and (e) applying an elution buffer comprising about 20 mM Tris-HCl and about 500 mM NaCl at a pH of 7.2 to the ETOXICLEAR™ resin and collecting an eluate comprising IαIp.

[0065] A seventh aspect of the present disclosure provides a method for preparing a plasma sample, comprising: (a) diluting plasma with a dilution buffer comprising 15 mM phosphate at a pH of about 5.5 to form diluted plasma; (b) applying the diluted plasma to a DETOXI-GEL™ resin and separating a flow-through comprising diluted plasma that does not bind to the DETOXI-GEL™ resin; and (c) applying a first wash buffer comprising about 15 mM phosphate and about 50 mM NaCl, at a pH of about 5.5, to the DETOXI-GEL™ resin to form a diluted plasma sample. The method features a method for purifying IαIp from plasma by (a) separating the flow-through containing a first wash buffer; (b) applying a second wash buffer containing about 15 mM phosphate and about 100 mM NaCl at a pH of about 5.5 to the DETOXI-GEL™ resin and separating the flow-through containing the second wash buffer; and (c) applying an elution buffer containing about 15 mM phosphate and about 1,000 mM NaCl at a pH of about 5.5 to the DETOXI-GEL™ resin and collecting an eluate containing IαIp.

[0066] definition As used herein, the singular forms "a," "an," and "the" include plural references unless specifically indicated otherwise.

[0067] As used herein, the term "about" means + / - 10% of the stated value.

[0068] As used herein, "administration" refers to a method of providing a dose of a substance (e.g., IαIp) or composition to a subject. IαIp utilized in the methods described herein can be administered, for example, orally, intramuscularly, intravenously, intradermally, transdermally, intra-arterially, intraperitoneally, intralesionally, intracerebrally, intra-articularly, intraprostatically, intrapleurally, intratracheally, intranasally, intravitreally, intravaginally, intrarectally, topically, intratumorally, intraperitoneally, subcutaneously, subconjunctivally, intravascularly, intramucosally, intrapericardially, intraumbilically, intraocularly, topically, locally, by inhalation, by injection, by infusion, by continuous infusion, by local perfusion directly bathing target cells, by catheter, by lavage, in a cream or liquid composition, etc. The method of administration can vary depending on various factors (e.g., the substance or composition being administered and the severity of the condition, disease, or disorder being treated).

[0069] As used herein, the term "biological material" refers to a sample from a subject (e.g., a mammal, such as a human) containing IαIp. Examples of biological materials include blood product materials, such as whole plasma, cryopreserved plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, source plasma, collected plasma, solvent / detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, whole blood, and their diluted or concentrated formulations, milk or colostrum, urine, sputum, and cerebrospinal fluid. Examples of biological materials include plasma fraction intermediates or blood products produced through one or more process steps (e.g., filtration, centrifugation, precipitation, chromatography, adsorption, isolation, freezing, thawing, dilution, concentration, viral clearance, etc.). Biological materials may be derived from humans, primates, cattle, horses, pigs, sheep, cats, dogs, or combinations thereof. The biological material may also be an extract prepared using cells that express IαIp, or may be or contain cells that secrete IαIp, for example, cells that have been recombinantly modified to express IαIp.

[0070] The biological material can be substantially unprocessed, e.g., to the extent that no other purification steps have been applied to the material prior to the purification step using an endotoxin-binding agent described herein, or to the extent that any previous purification steps performed with the material remove less than 10% (w / w) (e.g., less than 0.1% to 10% (w / w), e.g., less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, or less than 9% (w / w)) of one or more substances from the material. The biological material can also be processed or prepared prior to the purification step, e.g., using an endotoxin-binding agent, as described herein. For example, the biological material can be processed using a decantation step, a clarification step, a chromatography step (e.g., anion exchange chromatography), a centrifugation and / or sedimentation step, a freezing step, a drying step, an evaporation step, an extraction step, a filtration step, a precipitation step, or other purification or preparation method known in the art. The process steps may remove up to, for example, 10% (w / w) or more (e.g., 10-30% (w / w) or more, e.g., 15%, 20%, 25%, or 30% (w / w)) of one or more substances from the biological material (e.g., proteins other than IαIp).

[0071] As used herein, the term "chromatography" or "chromatographic process" refers to the separation of one or more analytes in a mixture by passing the mixture in solution or suspension through a medium through which the analytes of the mixture move at different rates. For example, the chromatographic process can be size exclusion chromatography, ion exchange chromatography, affinity chromatography, or dye-ligand chromatography. More specifically, as described herein, a chromatographic process can be performed using, for example, an endotoxin-binding agent (such as a resin) to separate IαIp from biological material containing IαIp.

[0072] In this disclosure, the terms "comprises," "comprising," "containing," and "having," etc., have the meanings given them in U.S. patent law and may mean "includes," "including," etc. "Consisting essentially of" or "consists essentially" likewise have the meanings given them in U.S. patent law, and the term is open-ended, allowing for the presence of more than what is recited, but excluding prior art embodiments, so long as the basic or novel characteristics of what is recited are not changed by the presence of more than what is recited.

[0073] As used herein, the term "eluate" refers to a fraction containing analyte material (e.g., IαIp) that elutes from a medium (e.g., a support material) during a purification step (e.g., a chromatography step). The eluate can be released from the medium by applying an eluent to the medium, thereby releasing the analyte. More specifically, the eluate can refer to a fraction containing IαIp that is released from the medium (e.g., an endotoxin-binding agent) after applying an eluent (e.g., an elution buffer, such as a buffer containing salt) to the medium.

[0074] As used herein, the term "endotoxin" refers to a substance containing lipids and polysaccharides. Endotoxins, such as lipopolysaccharide (LPS), are typically found in the outer membrane of the cell wall of Gram-negative bacteria. Endotoxins can be approximately 10 kDa in size, but can readily form large aggregates up to 1,000 kDa. LPS (or endotoxin) can be found not only in E. coli but also in other Gram-negative bacteria (see, e.g., Bertani and Ruiz, EcoSal Plus doi:10.1128 / ecosalplus.ESP-0001-2018,2018, which is incorporated herein by reference in its entirety).

[0075] As used herein, "endotoxin-binding agent" refers to a molecule capable of or known to bind endotoxins, such as lipopolysaccharides. An endotoxin-binding agent may be one that specifically binds endotoxins. An endotoxin-binding agent may be, for example, a resin known to be used to remove lipopolysaccharides from biological materials or protein mixtures. Exemplary endotoxin-binding agents include ETOXICLEAR™ (www.astreabioseparations.com / product / etoxiclear), PIERCE™ High Capacity Endotoxin Removal Resin (www.thermofisher.com / order / catalog / product / 88270# / 88270), TOXINERASER™ Endotoxin Removal Resin (www.genscript.com / kit / L00402-ToxinEraser_sup_TM_sup_Endotoxin_Removal_Resin.html), PURKINE™ Endotoxin Removal Resin (www Examples of suitable endotoxin-removal resins include, but are not limited to, DETOXI-GEL™ Endotoxin Removal Gel (www.thermofisher.com / order / catalog / product / 20339# / 20339), and PROMEGA™ Endotoxin Removal Resin (www.promega.com / products / nucleic-acid-extraction / plasmid-purification / endotoxin-removal-resin / ?catNum=A2191). Endotoxin-binding agents can be molecules such as polymyxin B, polylysine or derivatives thereof, or synthetic mimetic peptides.

[0076] As used herein, the term "flow-through" refers to the fraction of material or volume of fluid that passes through a medium (e.g., a medium used in chromatography, such as an endotoxin-binding agent) without binding. Additional mobile phase (e.g., a fluid such as a low (e.g., less than 50 mM salt) or salt-free (e.g., sodium chloride) buffer) can be added to ensure that one or more components of a mixture applied to the medium are fully loaded by the medium and to achieve initial or additional separation of an analyte (e.g., IαIp) from other components in the mixture.

[0077] As used herein, the terms "inter-alpha inhibitor protein" and "IαIp" and their plural forms refer to a multicomponent glycoprotein in a family of structurally related serine protease inhibitors. IαIp has been shown to be important in the inhibition of several proteases, including neutrophil elastase, plasmin, trypsin, chymotrypsin, granzyme K, preprotein convertase, furin, cathepsin G, and acrosin. In human plasma, IαIp is detected at relatively high concentrations (400-800 mg / L). Unlike other inhibitor molecules, this inhibitor family typically contains a combination of polypeptide chains (light and heavy) covalently linked by chondroitin sulfate chains. The heavy chains (H1, H2, and H3) of IαIp are also known as hyaluronic acid (HA)-binding proteins. The main forms of IαIp found in human plasma are inter-alpha inhibitor (IαI), which contains two heavy chains (H1 and H2) and one light chain (L), and pre-alpha inhibitor (PαI), which contains one heavy chain (H3) and one light chain (L). Another IαIp is the light chain (also called bikunin (double Kunitz inhibitor)) that has two Kunitz domains and is known to broadly inhibit plasma serine proteases. Another IαIp is the heavy chain-related molecule H4, which circulates in the blood independently of bikunin. Yet another IαIp is the heavy chain-related molecule H5. IαI and PαI present in the plasma fraction have apparent molecular weights of approximately 60 kDa to approximately 280 kDa.

[0078] As used herein, the term "pharmaceutically acceptable excipient" refers to one or more compatible solid or liquid fillers, diluents, or encapsulating substances suitable for human administration. Excipients may contain additives, such as substances that enhance isotonicity and / or chemical stability. Such materials are nontoxic to recipients in the amounts and concentrations employed, and include buffers, e.g., phosphate, citrate, succinate, acetate, lactate, tartrate, and other organic acids or their salts; tris-hydroxymethylamine methane (Tris), bicarbonate, carbonate, and other organic bases and their salts; antioxidants, e.g., ascorbic acid; low molecular weight (e.g., less than about 10 residues) polypeptides, e.g., polyarginine, polylysine, polyglutamic acid, and polyaspartic acid; proteins, e.g., serum albumin, gelatin, or immunoglobulins; hydrophilic polymers, e.g., polyvinylpyrrolidone (PVP), polypropylene glycol (PPG), and polyethylene glycol (PE). G); amino acids such as glycine, glutamic acid, aspartic acid, histidine, lysine, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, glucose, mannose, sucrose, dextrin, or sulfated carbohydrate derivatives such as heparin, chondroitin sulfate, and dextran sulfate; polyvalent metal ions such as divalent metal ions including calcium ions, magnesium ions, and manganese ions; chelating agents such as ethylenediaminetetraacetic acid (EDTA); sugar alcohols such as mannitol or sorbitol; counterions such as sodium or ammonium; and / or nonionic surfactants such as polysorbates or poloxamers. Other additives, such as stabilizers, antimicrobial agents, inert gases, fluid and nutrient replenishers (e.g., Ringer's dextrose), electrolyte replenishers, and the like, can also be included and can be present in conventional amounts.

[0079] As used herein, the terms "prevent," "preventing," "prophylactic treatment," and the like refer to reducing the probability of developing a disorder or condition in a subject who does not have the disease, disorder, or condition, but who is at risk of or susceptible to developing the disorder or condition.

[0080] As used herein, the term "processed" refers to biological material that has been modified using one or more sample preparation steps (e.g., filtration, centrifugation, sedimentation, chromatography, etc.) prior to contacting the material with an endotoxin-binding agent according to the methods described herein. Other example processes include decantation, clarification, freezing, drying, evaporation, extraction, filtration, precipitation, or another purification or preparation method known in the art. A process step may remove up to, for example, 10% (w / w) or more (e.g., 10-30% (w / w) or more, e.g., 15%, 20%, 25%, or 30% (w / w)) of one or more substances from the biological material (e.g., proteins other than IαIp).

[0081] As used herein, the terms "purify," "purifying," "purification," and the like refer to the removal of proteins (e.g., proteins other than IαIp) and / or non-protein substances (e.g., phospholipids and nucleic acids) from a heterogeneous mixture (e.g., a biological material, such as blood or milk) containing IαIp and other proteins and / or substances to produce a composition containing IαIp free of other proteins and / or substances present in the original mixture (e.g., the biological material), or This refers to one or more steps or processes that produce a composition in which proteins and / or substances other than IαIp are reduced by, for example, 40% by weight or more (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% or more) compared to the starting mixture (e.g., biological material). Examples of proteins that can be removed from a mixture containing IαIp include alpha-1 antitrypsin, C1-inhibitor, albumin, globulins (including immunoglobulins, e.g., IgA, IgG (e.g., intravenous Ig (IVIg)), anti-D). Antibodies include, but are not limited to, antibodies against IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, and varicella zoster IgG), IgM, IgD, and IgE, fibrinogen (Factor I), prothrombin (Factor II), thrombin, antithrombin III, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, fibronectin, alpha-2 antiplasmin, urokinase, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2, von Willebrand factor, factor H, prekallikrein, high molecular weight kinogen, and heparin cofactor II.

[0082] As used herein, the terms "pure" or "purity" refer to the extent to which an analyte is isolated and free from other components. In the context of proteins, the purity of an isolated protein can be expressed in terms of the protein being free of any contaminants (e.g., one or more unrelated proteins or other substances). For example, the purity of an IαIp composition indicates how much of the composition is IαIp relative to the total weight of the isolated material, and can be determined, for example, relative to pure IαIp. Purity levels found in the present disclosure can be 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95% or more, or 99% (w / w) or more. A "pure" IαIp composition of the present disclosure can be greater than 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or up to 70% pure by weight. A "substantially pure" IαIp composition can be substantially free of contaminants or impurities, e.g., greater than 70%, 75%, 80%, 85%, 90%, 95%, or 99% pure by weight. In some embodiments, the level of contaminants or impurities is about 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% or less by weight. Purity can be determined by detecting the level of a particular analyte (e.g., IαIp) using immunoassays or other techniques (e.g., MAb 69.26-heparin-biotin sandwich ELISA, SDS / PAGE, and / or Western blot) and calculating the ratio (w / w) of the analyte to the total protein content (e.g., as determined by a total protein assay (e.g., bicinchoninic acid assay (BCA), Bradford assay, biuret test, or another assay known in the art)).

[0083] As used herein, the term "subject" refers to a mammal, including, but not limited to, a human or non-human mammal, such as a primate, cow, horse, pig, sheep, cat, or dog. A subject can be a patient.

[0084] As used herein, the term "substantially unprocessed" refers to biological material that has been minimally, if at all, modified relative to the original source material (e.g., blood). For example, substantially unprocessed biological material can retain the original content (e.g., the same proteins and / or substances and / or the same ratio of two or more proteins or substances) and / or the original properties (e.g., one or more biological activities) of the original source material. A biological material can be substantially unprocessed such that any prior purification steps performed on the material remove less than 10% (w / w) (e.g., less than 0.1-10% (w / w), e.g., less than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% (w / w)) of one or more proteins or substances from the material. A substantially unprocessed biological material can be one that has not been modified using sample preparation steps (e.g., filtration, centrifugation, sedimentation, chromatography, etc.), particularly, for example, prior to contacting the biological material with an endotoxin-binding agent according to the methods described herein.

[0085] As used herein, the phrase "specifically binds" refers to a binding reaction between an analyte (e.g., a protein such as IαIp) and a binder (e.g., an endotoxin-binding agent). A specific binding reaction is one that occurs between an analyte and a binder even in the presence of a heterogeneous population of proteins and other biomolecules (e.g., proteins other than IαIp in a biological material). Specific binding between an analyte (e.g., IαIp) and a binder (e.g., an endotoxin-binding agent) has a K of less than about 1000 nM (e.g., 1 pM to 1000 nM). d An analyte (e.g., IαIp) that does not specifically bind to a binder (e.g., an endotoxin binder) can be characterized by a K of greater than about 1000 nM (e.g., greater than 1 μM, 100 μM, 500 μM, or 1 mM). dcan be characterized by

[0086] As used herein, the term "support" refers to any device containing an agent (e.g., an endotoxin-binding agent) that can be contacted with a material (e.g., a biological material) containing at least one analyte (e.g., IαIp). The support may be a column, membrane, disk, chip, or other device for chromatography or affinity capture, examples of which are known in the art and described herein.

[0087] As used herein, the term "treating" refers to reducing or ameliorating a disorder and / or one or more symptoms associated therewith. It will be understood that treating a disorder or condition does not require, although not excluded, complete elimination of the disorder or its associated symptoms.

[0088] As used herein, the term "yield" refers to the relative amount of an analyte (e.g., IαIp) obtained after a purification step or process compared to the amount of the analyte (w / w) in the starting material (e.g., biological material). Yield can be expressed as a percentage. In the context of the present disclosure, the amount of an analyte (e.g., IαIp) in the starting material and the analyte obtained after a purification step can be measured using an immunoassay or assay (e.g., anti-IαIp antibody (e.g., MAb 69.26)-heparin biotin sandwich ELISA, SDS / PAGE, and / or Western blot). The methods of the present disclosure can be used to provide a yield of purified IαIp of about 20% (w / w) or more relative to the amount present in the original biological material. For example, the methods can be used to provide a yield of purified IαIp of about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more. [Brief explanation of the drawings]

[0089] [Figure 1]These images show the starting material, flow-through, wash, and elution fractions obtained during chromatographic isolation of IαIp from cryo-poor plasma. The starting material, flow-through, wash, and elution fractions were separated on a 4-15% Mini-PROTEAN® TGX stain-free (BioRad) SDS-PAGE gel (Figure 1A) and transferred to a nitrocellulose membrane for Western blot analysis (Figure 1B). IαIp was detected using a monoclonal antibody against IαIp (Mab 69.26) and biotin-conjugated heparin. The elution fraction of cryo-poor plasma processed using Q anion exchange resin (arrow in Figure 1A, lane 5) and the elution fraction of cryo-poor plasma processed using an endotoxin-binding agent (ETOXICLEAR™; arrow in Figure 1A, lane 10) both showed 125 kDa and 250 kDa bands of IαIp, corresponding to PαI and IαI, respectively. Lanes 1-6 of the SDS-PAGE gel and Western blot correspond to the results of Q anion exchange chromatography using cryo-poor plasma as the starting material, while lanes 7-12 correspond to the results of purification with an endotoxin-binding agent using the Q anion exchange chromatography eluate as the starting material. Lanes are as follows: Lane 1: starting material - cryo-poor plasma; Lane 2: flow-through from the Q anion exchange resin; Lane 3: wash fraction 1 (pH 7.2, 250 mM NaCl); Lane 4: wash fraction 2 (pH 5.2, 175 mM NaCl); Lane 5: eluate from the Q anion exchange resin (pH 7.2, 750 mM NaCl); Lane 6: clear solution from the Q anion exchange resin (1 M NaOH + 2 M NaCl). Lane 7: flow-through from endotoxin binder; Lane 8: wash fraction 1 (pH 5.2, 150 mM NaCl); Lane 9: wash fraction 2 (pH 7.2, 300 mM NaCl); Lane 10: elution fraction 1 (pH 7.2, 500 mM NaCl); Lane 11: elution fraction 2 (pH 7.2, 1,000 mM NaCl); Lane 12: clear solution from endotoxin binder (1 M NaOH + 2 M NaCl). [Figure 2]Chromatogram showing the chromatographic isolation of IαIp from human plasma using an endotoxin-binding agent (DETOXI-GEL™) and 15 mM phosphate buffer (pH 7.3). IαIp was detected in the peak indicated by the arrow. The y-axis represents absorbance (A280), and the x-axis represents time (minutes). [Figure 3] Chromatogram showing the chromatographic isolation of IαIp from human plasma using an endotoxin-binding agent (DETOXI-GEL™) and 15 mM phosphate buffer (pH 5.5). IαIp was detected in the peak indicated by the arrow. The y-axis represents absorbance (A280) and the x-axis represents time (min). [Figure 4] Chromatogram showing the chromatographic isolation of IαIp from human plasma using an endotoxin-binding agent (DETOXI-GEL™) and 20 mM Tris-HCl buffer (pH 7.2). IαIp was detected in the peak indicated by the arrow. The y-axis represents absorbance (A280), and the x-axis represents time (min). [Figure 5]These images show the starting material, flow-through, wash fraction, and elution fractions obtained during chromatographic isolation of IαIp from cryo-poor plasma. The starting material, flow-through, wash fraction, and eluate were separated on a 4-20% TGX stain-free (BioRad) precast SDS-PAGE gel (Figure 5A) and transferred to a nitrocellulose membrane for Western blot analysis (Figure 5B). IαIp was detected using a biotinylated monoclonal antibody against IαIp (Mab 69.26) and HRP-conjugated streptavidin. The elution fractions of cryo-poor plasma eluted using Q anion exchange resin (arrow in Figure 5B, lane 5) and an endotoxin-binding agent (ETOXICLEAR™; arrows in Figure 5B, lanes 6-7) both showed 125 kDa and 250 kDa bands of IαIp corresponding to PαI and IαI, respectively. Lanes 1–5 of the SDS-PAGE gel and Western blot correspond to the results of Q anion exchange chromatography using cryo-poor plasma as the starting material, whereas lanes 6–7 correspond to the results of purification with an endotoxin-binding agent using the Q anion exchange chromatography eluate as the starting material. Lanes are as follows: Lane 1: starting material - cryo-poor plasma; Lane 2: flow-through from the Q anion exchange resin; Lane 3: wash fraction 1 (pH 7.2, 250 mM NaCl); Lane 4: wash fraction 2 (pH 5.2, 150 mM NaCl); Lane 5: eluate from the Q anion exchange resin (pH 7.2, 750 mM NaCl); Lane 6: elution fraction 1 (pH 7.2, 400 mM NaCl); Lane 7: elution fraction 2 (pH 7.2, 500 mM NaCl). [Figure 6]These images show the starting material, flow-through, wash, and elution fractions obtained during chromatographic isolation of IαIp from cryo-poor plasma. The starting material, flow-through, wash, and elution fractions were separated on a 4-20% TGX stain-free (BioRad) precast SDS-PAGE gel (Figure 6A) and transferred to a nitrocellulose membrane for Western blot analysis (Figure 6B). IαIp was detected using a biotinylated monoclonal antibody against IαIp (Mab 69.26) and HRP-conjugated streptavidin. The elution fractions of cryo-poor plasma eluted using Q anion exchange resin (Figure 6B, lane 5, arrowhead) and cryo-poor plasma eluted using an endotoxin-binding agent (ETOXICLEAR™; Figure 6B, lane 9, arrowhead) both showed 125 kDa and 250 kDa bands of IαIp corresponding to PαI and IαI, respectively. Lanes 1-7 of the SDS-PAGE gel and Western blot correspond to the results of Q anion exchange chromatography using cryo-poor plasma as the starting material, while lanes 8-12 correspond to the results of purification with an endotoxin-binding agent using the Q anion exchange chromatography eluate as the starting material. Lanes are as follows: Lane 1: starting material - cryo-poor plasma; Lane 2: flow-through from the Q anion exchange resin; Lane 3: wash fraction 1 (pH 7.2, 250 mM NaCl); Lane 4: wash fraction 2 (pH 5.2, 150 mM NaCl); Lane 5: elution fraction 1 from the Q anion exchange resin (pH 7.2, 750 mM NaCl); Lane 6: elution fraction 2 from the Q anion exchange resin (pH 7.2, 1,000 mM NaCl); Lane 7: Q anion exchange resin. Lane 8: Flow-through from endotoxin binder and wash fraction (pH 5.2, 150 mM NaCl); Lane 9: Elution fraction 1 (pH 7.2, 500 mM NaCl); Lane 10: Elution fraction 2 (pH 7.2, 1,000 mM NaCl); Lane 11: Clarified solution from endotoxin binder (1 M NaOH + 2 M NaCl); Lane 12: Concentrated elution fraction 1 after buffer exchange and ultrafiltration (Millipore Ultracell 30 kDa cutoff centrifugation membrane). DETAILED DESCRIPTION OF THE INVENTION

[0090] The present disclosure features methods for purifying IαIp (e.g., IαI, PαI, heavy chains (e.g., H1, H2, H3, H4, and / or H5), light chains (e.g., bikunin), or combinations thereof) from biological material (e.g., blood) using an endotoxin-binding agent. The method includes applying an IαIp-containing biological material (e.g., processed or substantially unprocessed biological material such as blood or milk) to an endotoxin-binding agent. The inventors have unexpectedly found that endotoxin-binding agents, such as endotoxin-specific chromatography resins (ETOXICLEAR™ and DETOXI-GEL™), bind IαIp. Surprisingly, when used in a process to purify IαIp, the endotoxin-binding agent reduces loss of IαIp during the purification process, thereby maintaining or improving the yield of recovered IαIp compared to other methods. Additionally, the use of endotoxin-binding agents as part of the IαIp purification process maintains or improves the purity of the recovered IαIp.

[0091] Thus, the present inventors have used such endotoxin-binding agents to purify IαIp from biological sources, such as blood. Furthermore, when endotoxin-binding agents are used as part of the purification process, yields and purities of IαIp of up to 50% or more (e.g., up to 90% or more) can be obtained. This finding can be used to simplify the purification process and simultaneously increase the yield and purity of IαIp when utilizing the methods of the present disclosure.

[0092] Also featured are pharmaceutical compositions prepared using purified IαIp obtained by the methods described herein, as well as methods for treating and / or reducing the likelihood of developing a disease or condition in a subject in need thereof by administering a pharmaceutical composition prepared using purified IαIp obtained by the methods described herein.

[0093] Purification method Endotoxin-binding agents can be used to purify IαIp (e.g., IαI, PαI, heavy chains (e.g., H1, H2, H3, H4, and / or H5), light chains (e.g., bikunin), or combinations thereof) from biological materials (e.g., blood). The methods described below can be used to separate IαIp from other components present in the biological material. The methods can be used to prepare IαIp with purities ranging from about 5% to about 99% or greater (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, greater than 95%, e.g., 97% or 99%, or greater than 99%). Additionally, the methods can be used to provide a yield of purified IαIp of about 20% (w / w) or more relative to the amount present in the original biological material, e.g., the methods can be used to provide a yield of purified IαIp of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more.

[0094] biological material The method described below can be used to purify IαIp from biological materials. Biological materials containing IαIp can be obtained from humans, primates, cattle, horses, pigs, sheep, cats, dogs, or combinations thereof. Examples of biological materials include blood, milk (e.g., colostrum), urine, sputum, and cerebrospinal fluid. For example, biological materials can include, but are not limited to, whole plasma, cryo-poor plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, source plasma, recovered plasma, solvent / detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, blood (e.g., whole blood), and diluted or concentrated preparations thereof. Biological materials containing IαIp can also be, but are not limited to, plasma fraction intermediates. Plasma fraction intermediates are produced from whole plasma, cryo-poor plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, source plasma, collected plasma, solvent / detergent treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, whole blood, and their diluted or concentrated preparations through one or more process steps (e.g., filtration, centrifugation, precipitation, chromatography, adsorption, isolation, freezing, thawing, dilution, concentration, S / D processing, etc.).

[0095] The biological material may also be an extract prepared using cells that express IαIp, or may be or contain cells that secrete IαIp, for example, cells that have been recombinantly modified to express IαIp.

[0096] The biological material may contain, in addition to IαIp, a mixture of proteins, such as three or more proteins found in blood or three or more proteins found in milk (such as colostrum). For example, the biological material may contain alpha-1 antitrypsin, C1-inhibitor, albumin, globulins (e.g., immunoglobulins, e.g., IgA, IgG (e.g., intravenous Ig (IVIg)), anti-D (anti-D)). The antibody may contain antibodies against IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, and varicella zoster IgG), IgM, IgD, and IgE), fibrinogen (Factor I), prothrombin (Factor II), thrombin, antithrombin III, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, fibronectin, alpha-2 antiplasmin, urokinase, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2, von Willebrand factor, factor H, prekallikrein, high molecular weight kinogen, and heparin cofactor II. The biological material may be milk (e.g., colostrum), which may contain one or more whey (e.g., up to about 50-80% (w / w); e.g., beta-lactoglobulin (e.g., about 1-5% (w / w)), alpha-lactalbumin (e.g., about 0.5-2% (w / w)), albumin, ovalbumin, and globulins (e.g., immunoglobulins, e.g., IgA, IgG (e.g., IVIg), IgM, IgD, and IgE, e.g., about 0.01-1% (w / w)), casein (e.g., alpha-casein and / or beta-casein; e.g., up to about 3-35% (w / w)), lactoferrin (e.g., about 0.01-0.2% (w / w)), lactose, alpha-1 antitrypsin, antichymotrypsin, plasminogen, fibrinogen, growth factors, and cytokines).

[0097] In accordance with the methods described below, the biological material that is contacted or applied to the endotoxin-binding agent (or a different support described herein, such as an anion exchange chromatography support) can be substantially unprocessed (e.g., the original source material), or the biological material can be treated using one or more sample preparation methods or other known purification methods, such as, for example, the methods described herein, prior to contacting or applying to the endotoxin-binding agent.

[0098] A substantially unprocessed biological material is a biological material that has been minimally, if at all, modified relative to its original source material (e.g., blood or another source described herein) such that the biological material maintains the original characteristics of the source material. For example, a substantially unprocessed biological material can be subjected to a sample preparation or purification step that removes less than 10% (w / w) (e.g., between 0.1% and 10% (w / w), e.g., less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 3%, less than 4%, less than 5%, less than 6%, less than 7%, less than 8%, or less than 9% (w / w)) of one or more substances from the material before the material is contacted with or applied to an endotoxin-binding agent. Alternatively, the substantially unprocessed biological material can be an eluate or fraction from a prior purification step, where the prior purification step removes less than 10% of impurities from the material.

[0099] The biological material may also be subjected to one or more process steps, such as those described herein, before application to the endotoxin-binding agent (or a different support described herein, such as an anion exchange chromatography support). For example, one or more proteins found in the biological material (e.g., blood or milk), such as alpha-1 antitrypsin, C1-inhibitor, albumin, globulins (e.g., immunoglobulins, e.g., IgA, IgG (e.g., intravenous Ig (IVIg), anti-D), etc.) may be bound to the endotoxin-binding agent. IgG, Hepatitis B IgG, Measles IgG, Rabies IgG, Tetanus IgG, and Varicella-Zoster IgG), IgM, IgD, and IgE), Fibrinogen (Factor I), Prothrombin (Factor II), Thrombin, Antithrombin III, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, Fibronectin, Alpha-2 Antiplasmin, Urokinase, Protein C, Protein S, Protein Z, Protein Z-related Protease In some embodiments, endotoxin-binding agents, such as endotoxin inhibitor, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2, von Willebrand factor, factor H, prekallikrein, high molecular weight kinogen, and heparin cofactor II, can be partially removed (e.g., 1% to 70% (w / w) removed) or substantially removed (e.g., 70% to 100% (w / w) removed) from the biological material prior to the purification step using an endotoxin-binding agent.

[0100] The disclosed methods can be used to purify IαIp (e.g., IαI, PαI, heavy chains (e.g., H1, H2, H3, H4, and / or H5), light chains (e.g., bikunin), or combinations thereof) from biological materials containing 3-10 (or more) of the above-mentioned blood proteins. The disclosed methods can be used to isolate IαIp from biological materials containing 3-15, 3-20, 3-25, 3-30, 10-20, 10-25, 10-30, 15-25, 15-30, 20-30, or 30 or more different proteins (e.g., blood or milk proteins). In addition to IαIp, the biological material to which the endotoxin-binding agent is applied may contain, by total weight, about 40-65% (e.g., about 55%) albumin, about 25-45% (e.g., about 38%) globulin, about 2-12% (e.g., about 7%) fibrinogen, or any combination thereof.

[0101] Endotoxin-binding agents The methods described herein involve the use of endotoxin-binding agents, which can be used to bind IαIp in protein mixtures (e.g., IαIp present in biological materials such as milk or blood). Endotoxin-binding agents are molecules that may be known to bind endotoxins, such as lipopolysaccharides. Endotoxin-binding agents may specifically bind to endotoxins. Endotoxin-binding agents can be, for example, incorporated into or immobilized on a support. Supports can be monolithic or particle-based supports. Particles can be, for example, resins. Endotoxin-binding agents can be packed or immobilized on any number of known supports, such as columns, membranes, disks, or chips. Endotoxin-binding agents can be molecules, such as polymyxin B, polylysine or derivatives thereof, or synthetic mimetic peptides.

[0102] Non-limiting examples of endotoxin-binding agents that can be used in the methods described herein include, for example, ETOXICLEAR™, PIERCE™ high-capacity endotoxin removal resin, TOXINERASER™ endotoxin removal resin, PURKINE™ endotoxin removal resin, DETOXI-GEL™ endotoxin removal gel, or Promega™ endotoxin removal resin. The methods can be performed, for example, using a pre-packed column or cartridge containing the endotoxin-binding agent (e.g., a column having a capacity of about 0.1 mL to about 100 mL, or a column with a larger capacity).

[0103] A column containing an endotoxin-binding agent can be prepared, for example, by applying a slurry of the endotoxin-binding agent suspended in a buffer (e.g., deionized water) to a filter frit column (e.g., about 2 to about 100 mL, or more) and allowing the endotoxin-binding agent to settle for about 30 minutes. The settled resin can then be equilibrated with about 3 to about 5 column volumes of an appropriate pyrogen-free buffer or water (e.g., deionized water) before applying the biological material.

[0104] In some instances, DETOXI-GEL™ endotoxin removal gel is used as a source of endotoxin-binding agent. DETOXI-GEL™ uses immobilized polymyxin B to bind the lipid A domain of endotoxin. In another instance, ETOXICLEAR™ is used as an endotoxin-binding agent.

[0105] Reagents for use in purification methods Dilution Buffer Biological material containing IαIp (e.g., IαI, PαI) may be combined with a dilution buffer prior to a purification step, e.g., before contacting or applying the biological material to a medium such as an endotoxin-binding agent. The dilution buffer may be added to reduce the salt (e.g., NaCl) concentration of the biological material, e.g., to avoid or reduce the possibility of premature elution of IαIp from the endotoxin-binding agent. The biological material may be diluted with the dilution buffer, e.g., 1:1 to 1:10 (v / v) (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (v / v), e.g., 1:3 (v / v)), and then contacted or applied to a medium (e.g., an endotoxin-binding agent).

[0106] The dilution buffer can have a pH range of about 4.5 to 8.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or about 8.5). The dilution buffer can contain one or more of deionized water, glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. The dilution buffer used to load the biological material may have a low concentration of a salt such as NaCl (e.g., 300 mM or less of salt (e.g., 200 mM, 150 mM, 100 mM, 75 mM, 50 mM, 25 mM, 10 mM, 5 mM, or 0 mM of salt (e.g., NaCl)). For example, biological material containing IαIp can be diluted 1:3 (v / v) with a buffer containing 20 mM Tris-HCl, and the diluted sample can then be contacted or applied to an endotoxin-binding column. The biological material may, for example, have been prepared during a prior purification step (e.g., a chromatography step using an anion exchange support). The dilution buffer may be water.

[0107] Loading buffer Biological materials containing IαIp (e.g., IαI, PαI) may have their pH and conductivity adjusted prior to a purification step, e.g., before contacting or applying the biological material to a medium such as an endotoxin-binding agent, an anion exchanger, etc. The loading buffer containing the biological material may have a pH range of about 4.5 to 8.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or about 8.5). The loading buffer may contain one or more of deionized water, glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. The loading buffer may have a low concentration of salt such as NaCl (e.g., 300 mM or less salt (e.g., 200 mM, 150 mM, 100 mM, 75 mM, 50 mM, 25 mM, 10 mM, 5 mM, or 0 mM salt (e.g., NaCl))). The buffer may contain about 20 mM Tris-HCl and 200 mM NaCl. The loading buffer may be, for example, one prepared during a previous process step (e.g., a chromatography step using an anion exchange support, a filtration step). The loading buffer may have a conductivity of about 10 mS / cm to about 30 mS / cm, about 15 mS / cm to about 25 mS / cm, or about 20 mS / cm.

[0108] Flow-through buffer The flow-through buffer can optionally be used to ensure that all of the biological material is loaded into the medium (e.g., the endotoxin-binding agent or another support described herein) during the purification process. The flow-through buffer can also be used to achieve initial or additional separation of components present in the biological material after application to the medium. The flow-through buffer can be the same as the dilution buffer. Alternatively, the flow-through buffer can be different from the dilution buffer.

[0109] For example, the flow-through buffers may be of the same or different pH (e.g., pH within the range of about 4.5 to about 8.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5) The flow-through buffer may have a pH of about 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or about 8.5) and may have a different composition of components (e.g., about 5 mM to about 300 mM of one or more of glycine, acetate, citrate, phosphate, NaCl, calcium, magnesium, EDTA, and Tris-HCl) relative to the dilution buffer used to load the biological material into the medium. The flow-through buffer may have a higher salt concentration (e.g., a salt (e.g., NaCl) concentration 5 to about 300 mM higher than the concentration of the dilution buffer) than the dilution buffer (e.g., a dilution buffer with less than 150 mM NaCl, such as a dilution buffer without NaCl, followed by a flow-through buffer with about 150 mM NaCl). The properties of the flow-through buffer may be selected to improve initial separation between components of the biological material during the purification process (e.g., during a column chromatography step).

[0110] Washing buffer The purification method can also include a wash buffer used in one or more washing steps (e.g., 1, 2, 3, 4, or more washing steps) that occur, for example, after contacting or applying the biological material to an endotoxin-binding agent, or during one or more other purification steps, as described herein. The wash steps may be performed using an endotoxin-binding agent or other medium (e.g., an anion exchanger) that does not bind strongly (e.g., a K of about 1 mM or greater), but may be present in the endotoxin-binding agent or other medium (e.g., an anion exchanger). d This can be done to remove weakly binding components (e.g., proteins or other substances found in biological materials that are not IαIp) that have a high affinity for IαIp.

[0111] Wash buffers applied to media (e.g., endotoxin-binding agents or other types of supports described herein) can be used to change the pH of the media, change the salt concentration of the media, or change both the pH and salt concentration of the media. A first wash buffer applied to the media may change the pH, and a second wash buffer applied to the media may change the salt concentration, or vice versa. Washing steps with wash buffers can facilitate the purification of IαIp by promoting the release of proteins other than IαIp from the media.

[0112] The wash buffer may differ from the flow-through buffer, dilution buffer, or loading buffer in terms of components or other properties (e.g., pH, conductivity, or salt concentration). For example, the wash buffer may contain a higher concentration of salt (e.g., NaCl) than the concentration of salt in the flow-through or dilution buffer. If the salt concentration in the wash buffer is the same as the flow-through or dilution buffer, the wash buffer may instead differ in its pH or one or more of its components. For example, the flow-through buffer used in the purification method may contain 20 mM Tris-HCl + 150 mM NaCl (pH 7.2), while the wash buffer may contain 75 mM glycine + 100 mM acetic acid + 150 mM NaCl (pH 5.2). For example, the loading buffer used in the purification method may contain about 18 mM Tris-HCl + about 2 mM Tris + about 200 mM NaCl (pH 7.2), while the first wash buffer may contain about 18 mM Tris-HCl + about 2 mM Tris + about 250 mM NaCl (pH 7.2), and the second wash buffer may contain about 75 mM glycine + about 100 mM HAc + about 150 mM NaCl + about 92.5 mM NaOH (pH 5.2). For example, the loading buffer used in the purification method may contain about 18 mM Tris-HCl+about 2 mM Tris+about 200 mM NaCl (pH 7.2), while the first wash buffer may contain about 75 mM glycine+about 100 mM HAc+about 200 mM NaCl (pH 5.2), and the second wash buffer may contain about 18 mM Tris-HCl+about 2 mM Tris+about 250 mM NaCl (pH 7.2).

[0113] The wash buffer may have a pH range of about 4.5 to 8.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or about 8.5). The wash buffer may have a pH of about 5.2. The wash buffer may have a pH of about 7.2. The wash buffer may contain one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl at about 5 mM to about 400 mM. For example, the wash buffer may have a salt (e.g., NaCl) concentration of about 50 to about 400 mM. For example, the wash buffer may have a salt (e.g., NaCl) concentration of about 200 to about 300 mM. For example, the wash buffer may have a salt (e.g., NaCl) concentration of about 250 mM, about 250 mM, or about 300 mM. For example, the wash buffer may have about 200 to about 300 mM NaCl. For example, the wash buffer may have about 200 mM NaCl, about 250 mM NaCl, or about 300 mM NaCl. The wash buffer may also be prepared at a different pH than other buffers previously used in the purification process (eg, dilution buffer, flow-through buffer, and / or pre-wash buffer).

[0114] The wash buffers used in the purification method may contain different salt concentrations, and media such as endotoxin-binding agents, anion exchange resins, etc. may be subjected to wash steps starting with wash buffers with low salt concentrations followed by wash steps with wash buffers of increasing salt concentrations with each wash step.

[0115] For example, a first wash step may involve applying a first wash buffer having a low pH (e.g., below pH 7.0, e.g., pH 5.5 or lower (e.g., pH 5.2)) and / or salt concentration (e.g., a salt (e.g., NaCl) concentration of less than 150 mM) to the medium, such as an endotoxin-binding agent (e.g., the first wash buffer may contain 75 mM glycine + 100 mM acetic acid + 150 mM NaCl (pH 5.2)), and a second wash step may involve applying a second wash buffer having a higher pH (e.g., a pH above pH 7.0, e.g., pH 7.2) and / or salt concentration (e.g., a salt (e.g., NaCl) concentration of more than 150 mM (e.g., about 300 mM)) to the medium (e.g., the second wash buffer may contain 20 mM Tris-HCl + 300 mM NaCl (pH 7.2)). In another example, a first wash step may use a wash buffer containing 15 mM phosphate + 50 mM NaCl, pH 5.5, followed by a second wash step using a second wash buffer containing 15 mM phosphate + 100 mM NaCl, pH 5.5.

[0116] Elution buffer The purification method can also include recovering an eluate containing IαIp from the medium (e.g., an endotoxin-binding agent or other agent, e.g., an anion exchange resin). The method includes contacting or applying an eluate or elution buffer to the medium (e.g., an endotoxin-binding agent, an anion exchange resin) and recovering the eluate.

[0117] The elution buffer may be prepared with a sufficiently high salt (e.g., sodium chloride (NaCl)) concentration (e.g., greater than about 200 mM salt (e.g., NaCl)) (e.g., 250 mM, 300 mM, 350 mM, 375 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, or 1000 mM salt (e.g., NaCl)) to allow release of bound IαIp from the medium (e.g., endotoxin-binding agent, anion exchange resin). The elution buffer may contain the same components as the previously described buffers (e.g., dilution buffer, loading buffer, flow-through buffer, and wash buffer), or the elution buffer may contain one or more different components (e.g., the elution buffer may contain one or more of glycine, acetate, citrate, phosphate, and Tris-HCl at about 5 mM to about 300 mM). Other salts or additives, such as calcium, magnesium, and EDTA, can be used in place of NaCl at equivalent concentrations. The pH of the elution buffer can also be the same as the buffer (e.g., dilution and / or wash buffer) previously contacted or applied to the medium (e.g., endotoxin-binding agent), or the pH of the elution buffer can be different (e.g., the elution buffer can have a pH ranging from about 4.5 to about 8.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or about 8.5)). The salt (e.g., NaCl) concentration in the elution buffer may be higher than the salt (e.g., NaCl) concentration in the wash buffer previously applied to the medium (e.g., endotoxin-binding agent). The pH of the elution buffer may be about 7.2.

[0118] For example, the elution buffer may contain about 400 mM to about 1,000 mM NaCl. For example, the elution buffer may contain about 500 mM NaCl, about 750 mM NaCl, or about 1,000 mM NaCl. In a preferred embodiment, IαIp is eluted from the endotoxin-binder by applying a buffer containing 20 mM Tris-HCl + 500 mM NaCl (pH 7.2) to the endotoxin-binder. The application of the elution buffer to the endotoxin-binder can be repeated one or more times. The elution buffers applied to the endotoxin-binder may be the same or different. For example, a second or subsequent elution buffer applied to the endotoxin-binder may contain a higher salt concentration (e.g., 1,000 mM NaCl) than the first or preceding elution buffer (e.g., 500 mM NaCl). If desired, the fractions collected after application of the elution buffer can be analyzed separately for the presence and concentration of IαIp, for example, using ELISA or other techniques known in the art, and then optionally pooled. Alternatively, the fractions can be pooled and analyzed for the presence and concentration of IαIp.

[0119] Clarification Buffer The purification method can also include an optional clarification step in which a clarification buffer is applied to the medium to regenerate the medium (e.g., endotoxin-binding agent) for use in another round of purification. The clarification buffer can be prepared at a sufficiently high pH (e.g., about 12 to about 14). The clarification buffer can contain about 1 M of an alkaline solute, such as sodium hydroxide (NaOH). Optionally, the clarification buffer can additionally contain about 1 to about 2 M of a salt (e.g., NaCl). In one preferred example, the clarification buffer contains 1 M NaOH + 2 M NaCl (pH 14).

[0120] Purification of IαIp using endotoxin-binding agents IαIp can be purified from biological material by contacting or applying the biological material containing IαIp to an endotoxin-binding agent (e.g., one or more of the endotoxin-binding agents described herein). The biological material can be applied directly to the endotoxin-binding agent without dilution, or the biological material can be diluted with a dilution buffer (as described above) before being applied to the endotoxin-binding agent. For example, the volume of biological material (diluted or undiluted) contacted or applied to the endotoxin-binding agent can be, for example, about 0.5 to about 20 column volumes (or other suitable volumes).

[0121] After applying the IαIp-containing biological material to the endotoxin-binding agent, the flow-through can optionally be analyzed to confirm that it contains no IαIp (or less than 10% (w / w)). For example, an ELISA assay (e.g., using an anti-IαIp antibody such as MAb 69.26) or other known techniques (e.g., SDS-PAGE and / or Western blot, or other known techniques) can be performed. After confirming that the flow-through contains no IαIp or only a substantial amount (e.g., about 30 μg / mL or less, e.g., about 20 μg / mL, 10 μg / mL, 5 μg / mL, or 1 μg / mL or less), the flow-through can be discarded. Additional flow-through buffer can be applied to the endotoxin-binding agent (e.g., in a volume of about 1 to about 50 column volumes) to ensure that all of the biological material is loaded onto the endotoxin-binding agent. This flow-through may then be discarded (eg, optionally after verifying that there is no (or no substantial amount of) IαIp in the flow-through).

[0122] One or more wash steps (e.g., 2, 3, 4, or more wash steps) can then be performed to remove non-IαIp present in the biological material that is weakly bound to the endotoxin-binding agent. Approximately 0.5 to about 10 column volumes of wash buffer (or other suitable volume) can be applied in a given wash step. The resulting wash fractions can optionally be analyzed (e.g., using ELISA or other known techniques) to confirm that they contain no IαIp (or contain less than 10% (w / w)). If substantial amounts of IαIp (e.g., 10% (w / w) or more) are detected in the wash buffer flow-through, the flow-through can be optionally treated to recover and purify the IαIp present in the flow-through (e.g., using an endotoxin-binding agent or other media described herein, such as an anion exchange support).

[0123] After the wash step, an eluate containing IαIp can be collected by applying an elution buffer (e.g., about 0.4 to about 5 column volumes or other suitable volume) to the endotoxin-binding agent. Optionally, two or more (e.g., 2, 3, 4, or more) elution buffers can be applied to the endotoxin-binding agent to elute or ensure the elution of all IαIp. The eluate can be analyzed for the presence of IαIp, for example, using ELISA or other techniques known in the art. Fractions from different elution buffers can then be pooled, if desired.

[0124] After the eluate is collected, the endotoxin-binding agent can optionally be clarified by applying a clarification buffer (e.g., about 0.5 to about 5 column volumes or other suitable volume) to the endotoxin-binding agent to regenerate the endotoxin-binding agent for future use. The resulting clarified fractions can, if desired, be analyzed for the presence of IαIp (e.g., using ELISA or other known techniques).

[0125] Each step of the purification process (e.g., application of biological material, wash steps, and elution steps) can be carried out using either gravity flow or low pressure (e.g., 0-15 psi) to generate a flow rate of about 1 to about 10 mL per minute (e.g., a flow rate of about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mL per minute). In some examples, the flow rate is about 2 mL / min.

[0126] This process can be repeated one or more times, if desired. Alternatively, IαIp can be further processed from the eluate fraction using techniques known in the art (e.g., concentration, dialysis, and / or lyophilization, among other techniques). Alternatively, the elution fraction containing IαIp may be subjected to one or more additional purification steps, if desired, as described below.

[0127] Purification of IαIp after use of endotoxin-binding agents The elution fraction containing IαIp collected after elution from the endotoxin-binding agent can be further purified using other known purification steps (e.g., one or more of the steps described in the "Additional Purification Steps" section below). For example, the eluate containing IαIp can be subjected to one or more purification steps, such as those described in US2003 / 0190732, US2011 / 0190194, US2012 / 0053113, and US2014 / 0206844, each of which is incorporated herein by reference. Additionally, the eluate containing IαIp can be further purified using anion exchange chromatography as described above.

[0128] Purification steps before use of endotoxin-binding agents Biological materials containing IαIp can be treated before contacting or applying the biological material to an endotoxin-binding agent. For example, the biological material may be treated using a sample preparation or purification step that removes up to, for example, 10% (w / w) or more (e.g., 10-30% (w / w) or more, e.g., 15%, 20%, 25%, or 30% (w / w)) of one or more substances (e.g., proteins or substances other than IαIp) from the biological material.

[0129] For example, processing steps may include filtration, centrifugation, sedimentation, chromatography, decantation steps, clarification, freezing, drying, evaporation, extraction, filtration, precipitation, or other purification or preparation methods known in the art. In one preferred example, the method involves performing anion exchange chromatography (described below) with a biological material containing IαIp, and then contacting or applying the eluate containing one or more IαIp prepared from the anion exchange chromatography to an endotoxin-binding agent to further purify the IαIp as discussed above and herein.

[0130] The prior purification or treatment steps can include, for example, one or more of the purification methods described in US2003 / 0190732, US2011 / 0190194, US2012 / 0053113, and US2014 / 0206844, each of which is incorporated herein by reference.

[0131] Additional purification steps Biological material containing IαIp (e.g., IαI, PαI, heavy chain (e.g., H1, H2, H3, H4, and / or H5), light chain (e.g., bikunin), or a combination thereof) can be applied to one or more chromatographic supports other than those containing the endotoxin-binding agent before or after purification of IαIp using the endotoxin-binding agent (e.g., by using an anion exchange chromatography support). The chromatographic support can be a monolithic support or a particle-based support. The chromatographic support can be a column, membrane, disc, or chip. Furthermore, the chromatographic support can be, for example, a size-exclusion chromatography support, an ion-exchange chromatography support, an affinity chromatography support, or a combination thereof.

[0132] For example, the monolithic or particle-based support may contain an immobilized anion exchange resin, which can be, for example, diethylaminoethane (DEAE) or a quaternary amine (Q) (e.g., Tosoh TOYOPEARL® GigaCap Q650M).

[0133] Biological material containing IαIp can be applied directly to a chromatographic support (e.g., an anion exchange chromatographic support). Alternatively, the biological material can be diluted with a dilution buffer as described above and then applied to the support. For example, cryopreserved plasma can be diluted 1:3 (v / v) with a dilution buffer containing, for example, 20 mM Tris-HCl + 200 mM NaCl (pH 7.2) and applied to the chromatographic support (e.g., about 0.5 to about 25 column volumes or other appropriate volume).

[0134] After the biological material is applied to the chromatographic support, the flow-through can be separated and, optionally, analyzed to confirm that it contains no IαIp (or contains less than 10% (w / w)). For example, an ELISA assay or other known technique (e.g., SDS-PAGE, and / or Western blot, or other known technique) can be performed. If a substantial amount of IαIp (e.g., 10% (w / w) or more) is detected in the flow-through, the flow-through can be optionally treated to recover and purify the IαIp present in the flow-through (e.g., using an endotoxin-binding agent or other medium described herein (e.g., an anion exchange support)). After it has been confirmed that the flow-through contains no IαIp or only an insubstantial amount (e.g., about 30 μg / mL or less, e.g., about 20 μg / mL, 10 μg / mL, 5 μg / mL, or 1 μg / mL or less), the flow-through can be discarded.

[0135] Optionally, a flow-through buffer can be prepared and applied to the chromatographic support (e.g., in a volume of about 1 to about 50 column volumes, or other suitable volume) to ensure that all of the biological material is loaded onto the support. This flow-through can then be discarded (e.g., optionally after verifying that there is no (or not a substantial amount of) IαIp in the flow-through).

[0136] Next, one or more wash steps (e.g., 2, 3, 4, or more) can be performed to remove non-IαIp in the biological material that is weakly bound to the chromatographic support. The wash buffer may be prepared as described above. In a given wash step, about 0.4 to about 10 column volumes of wash buffer (or other appropriate volume) can be applied to the chromatographic support. The resulting wash fractions can optionally be analyzed (e.g., using ELISA or other known techniques) to confirm that they contain no IαIp (or contain less than 10% (w / w)). If substantial amounts of IαIp (e.g., 10% (w / w) or more) are detected in the wash buffer flow-through, the flow-through can be optionally treated to recover and purify the IαIp present in the flow-through (e.g., using an endotoxin-binding agent or other media described herein (e.g., an anion exchange support)).

[0137] In one example, a first washing step is performed by applying a buffer containing 20 mM Tris-HCl + 250 mM NaCl (pH 7.2) to a chromatographic support (e.g., a Tosoh TOYOPEARL® GigaCap Q650M column), for example, in a volume of about 5 to 10, e.g., about 8 or 9, column volumes, and then a second washing step is performed by applying a buffer containing 50 mM glycine + 100 mM acetic acid + 175 mM NaCl (pH 5.2) to the support, for example, in a volume of about 2 to 6, e.g., about 4 or 5, column volumes.

[0138] The processed material containing IαIp can then be obtained by applying an elution solution or elution buffer (e.g., about 0.4 to about 10 column volumes, or other suitable volume) to the chromatographic support and collecting the eluate containing IαIp. The elution buffer can be prepared and applied to the chromatographic support in a manner similar to that described above in connection with the endotoxin-binding agent purification process. If desired, two or more elution buffers (e.g., 2, 3, 4, or more) can be applied to the chromatographic support to elute or ensure the elution of all IαIp. The eluate can be analyzed for the presence of IαIp, for example, using ELISA (or other techniques known in the art). Fractions from different elution buffers can then be pooled, if desired.

[0139] In one example, an elution buffer containing 20 mM Tris-HCl + 750 mM NaCl (pH 7.2) is applied to the chromatographic support, e.g., in a volume of about 2-5 column volumes, e.g., 3-4 column volumes. The eluate containing IαIp can then be further treated by application to an endotoxin-binding agent, as described above, or the eluate can be further processed by repeating the chromatographic step using the same or a different chromatographic support, or by using one or more different purification or preparation steps, e.g., filtration, centrifugation, sedimentation, chromatography, decantation steps, clarification, freezing, drying, evaporation, extraction, filtration, precipitation, or another purification or preparation method known in the art.

[0140] Optionally, the chromatographic support can be washed with clarification buffer to regenerate the chromatographic support for future use. The resulting clarified fractions can optionally be analyzed for the presence of IαIp (e.g., using ELISA or other known techniques). In one example, a clarification buffer containing 1 M NaOH + 2 M NaCl (pH 14) is applied to the chromatographic support in a volume of, for example, about 1 to 5 column volumes, such as 1.5 to 2 column volumes.

[0141] Detection of IαIp IαIp isolated using the purification methods described herein can be quantified using one or more assays known in the art, for example, as described in WO2009 / 154695, US2020 / 0057077, and WO2020 / 086879, which are incorporated herein by reference.

[0142] For example, an IαIp quantification assay involves binding a sample containing IαIp to an IαIp binder (e.g., an antibody that specifically binds to IαIp (e.g., MAb 69.26 (see, e.g., Sha et al., J Pediatr. 180:135-140, 2017), MAb 69.31 (see, e.g., Lim et al., J Infect Dis. 188(6):919-926, 2003), or PAb R22C (see, e.g., WO2020 / 086879), each of which is incorporated herein by reference in its entirety), or an IαIp ligand (e.g., heparin, LPS, and / or hyaluronic acid (HA)), and detecting the amount of bound IαIp (e.g., using a detection agent). The IαIp binding agent can be labeled, for example, with biotin, and detection can be performed using, for example, horseradish peroxidase-labeled streptavidin, followed by known detection methods. Alternatively, the IαIp binding agent can be labeled, for example, with a fluorophore and detected spectrophotometrically. Alternatively, the IαIp detection agent can be detected directly without a label (e.g., by surface plasmon resonance (SPR)). After addition of the IαIp detection agent, an additional washing step (e.g., one or more times) can be performed to remove unbound IαIp detection agent.

[0143] IαIp can then be measured based on the signal from the attached label or attached detection agent (e.g., enzyme activity or fluorescence) using standard techniques known in the art. When an enzyme is used as the label, a substrate is added to generate a signal (e.g., a color change) that can be read with an appropriate device for detecting the signal, such as a spectrophotometer. The signal (e.g., absorbance or fluorescence) can be plotted against standards with known concentrations of IαIp to establish a standard curve, or compared against known reference concentrations. Based on the established standard curve or reference concentration values, the unknown concentration in the sample can be calculated and determined.

[0144] Isolated IαIp The purification methods described herein (e.g., purification using an endotoxin-binding agent alone or in combination with one or more additional pre- or post-purification steps, such as anion exchange chromatography) can be used to purify IαIp (e.g., IαI, PαI, heavy chain (e.g., H1, H2, H3, H4, and / or H5), light chain (e.g., bikunin), or combinations thereof) from biological material. The purity of IαIp can be determined by detecting the total amount of IαIp obtained after purification (e.g., using an assay or immunoassay, such as MAb 69.26-heparin-biotin sandwich ELISA, SDS / PAGE, and / or Western blot) and calculating the percentage (w / w) of the total protein content determined by a total protein assay (e.g., bicinchoninic acid assay (BCA), Bradford assay, Biuret test, or another assay known in the art). After the purification step, IαIp may have a purity of at least about 5% (w / w), for example, at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or greater than 99% (w / w).

[0145] The isolated IαIp has an apparent molecular weight of between about 60 kDa and about 280 kDa, which can be determined by any suitable method known in the art, such as sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isolated IαIp can also be tested for biological activity, such as cytokine inhibitory activity, chemokine inhibitory activity, protease inhibitory activity (e.g., serine protease inhibitory activity), chondroitin sulfate binding, glycosaminoglycan binding activity, hyaluronic acid binding activity, complement binding activity, histone binding activity, Arg-Gly-Asp (RGD) domain binding activity, coagulation factor binding activity, cell repair activity, and extracellular matrix protein binding activity. IαIp can also be tested for trypsin inhibitory specific activity, for example, a trypsin inhibitory specific activity of about 1000 IU / mg to about 2000 IU / mg (e.g., 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 IU / mg).

[0146] The proportion or concentration of IαIp (e.g., IαI and / or PαI) present in the final purified fraction can vary. Preferably, the purified IαIp (e.g., IαI and / or PαI) is present in the final purified fraction in physiological proportions. Physiological proportions can be, for example, the proportions found in healthy humans or animals and / or the ratio of IαI to PαI that occurs naturally in human plasma. Physiological proportions are typically about 60% to about 80% IαI and about 20% to about 40% PαI.

[0147] The purification method also results in a yield of isolated IαIp of about 20% (w / w) or more (e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more) relative to the IαIp present in the biological material (e.g., blood or milk). In some examples, the yield can be about 90% or more or 95% or more by weight.

[0148] The disclosed purification methods can be used to produce compositions containing IαIp. Additionally, compositions can be prepared using higher concentrations of IαIp relative to the concentration of IαIp present in the original biological material (e.g., blood or milk). Thus, the methods can be used to prepare compositions containing IαIp in amounts of at least about 5 μg / mL, e.g., about 5 μg, 50 μg, 100 μg, 300 μg, 600 μg, 900 μg, 1 mg, 2 mg, 3 mg, 4 mg, 5 mg, 6 mg, 7 mg, 8 mg, 9 mg, 10 mg, 15 mg, 20 mg, 25 mg, 30 mg, 35 mg, 40 mg, 45 mg, or 50 mg or more per mL. The IαIp present in the composition can comprise from about 5 to about 99% (w / w) or more of the composition (e.g., greater than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99% (w / w) or more).

[0149] To increase the concentration of IαIp in compositions prepared using the disclosed methods, additional steps (e.g., lyophilization, vacuum concentration) can be performed. These known methods can be used, for example, to produce compositions containing IαIp in amounts of about 1 to 50 mg / mL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg or more of IαIp per mL) from IαIp purified according to the methods described herein.

[0150] Pharmaceutical Compositions IαIp (e.g., IαI, PαI, heavy chain (e.g., H1, H2, H3, H4, and / or H5), light chain (e.g., bikunin), or a combination thereof) obtained by purification using the purification methods disclosed herein (e.g., using an endotoxin-binding agent as described above) can be used to prepare a pharmaceutical composition. IαIp can be combined, for example, with a pharmaceutically acceptable excipient. Pharmaceutical compositions containing IαIp are suitable for administration to humans.

[0151] Examples of pharmaceutical compositions containing IαIp are described, for example, in US2007 / 0297982, US2015 / 0238578, US2019 / 0269765, and WO2020 / 086879, which are incorporated herein by reference.

[0152] Treatment methods IαIp prepared by the methods described herein can be used (e.g., when formulated as a pharmaceutical composition) to treat or prevent various diseases, conditions, or symptoms thereof, such as diseases or conditions characterized by inflammation and / or low levels of IαIp. Methods of treatment and methods for identifying subjects suitable for treatment with the pharmaceutical compositions of the present disclosure can be found, for example, in US2007 / 0297982, US2015 / 0238578, US2019 / 0269765, US2020 / 0057077, and WO2020 / 086879, which are incorporated herein by reference.

[0153] kit IαIp prepared by the methods described herein can be used to prepare kits containing IαIp (e.g., IαI, PαI, heavy chains (e.g., H1, H2, H3, H4, and / or H5), light chains (e.g., bikunin), or combinations thereof). For example, IαIp can be placed in vials in an amount of about 0.5 to about 500 mg / mL (e.g., about 1 to 50 mg / mL) for distribution in the kit. Exemplary kits can be prepared, for example, as described in US2007 / 0297982, US2015 / 0238578, US2019 / 0269765, and WO2020 / 086879, which are incorporated herein by reference.

[0154] The following examples are intended to illustrate, but not limit, the present invention. [Example]

[0155] Example 1: Purification of IαIp from cryo-poor plasma using ETOXICLEAR™ A sample of cryo-poor plasma was diluted (1:3 (v / v) with 20 mM Tris-HCl + 200 mM NaCl, pH 7.2), and the diluted cryo-poor plasma (114.4 mL) was applied to a 5 mL commercial Q anion exchange resin (Tosoh TOYOPEARL® GigaCap Q650M) at a flow rate of 3.5 mL / min. The flow-through was collected for analysis. Flow-through that did not contain (or did not contain a substantial amount of) IαIp was discarded. Additional plasma dilution buffer (172.7 mL: 20 mM Tris-HCl + 200 mM NaCl, pH 7.2) was applied to the column to ensure that the starting material passed through the column completely. Additional flow-through was collected for analysis and, determined to contain (or not contain a substantial amount of) IαIp, was discarded.

[0156] The column was washed with approximately 8 CV of the first wash buffer (43.5 mL: 20 mM Tris-HCl + 250 mM NaCl, pH 7.2), and the resulting fractions were collected. The combined fractions were analyzed for total protein (bicinchoninic acid assay (BCA)), IαIp (MAb 69.26-heparin-biotin sandwich ELISA), and trypsin inhibitor activity as shown in Table 1. The combined wash fractions were discarded because they contained a small amount of impurity IαIp (0.1% purity, 61.727 μg, 0.8761% (w / w) yield of IαIp).

[0157] After the pH 7.2 wash, the column was further washed (approximately 5 CV) with a second wash buffer at a lower pH (23.7 mL: 50 mM Gly + 100 mM AcOH + 175 mM NaCl, pH 5.2), and the resulting fractions were collected, combined, and analyzed as shown in Table 1. The combined wash fractions contained a small amount of impurity IαIp (0.4% purity, 72.806 μg, yield of IαIp 1.0339% (w / w)) and were discarded.

[0158] After washing at pH 5.2, bound proteins were eluted with high-salt elution buffer (16.1 mL: 20 mM Tris-HCl + 750 mM NaCl, pH 7.2). Fractions were collected, combined, and analyzed (e.g., total protein, IαIp, trypsin inhibitor activity) as shown in Table 1. The collected fractions contained concentrated IαIp (purity 56.43%, yield of IαIp 98.537% (w / w)).

[0159] Next, a high pH / high salt wash buffer (8.3 mL: 1 M NaOH + 2 M NaCl) was applied to the column, and the resulting fractions were combined and analyzed for IαIp as described in Table 1. The combined fractions were determined to contain no (or no substantial amount of) IαIp and were discarded.

[0160] Table 1 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, elution, and clarification). Additionally, Figures 1A-1B show SDS-PAGE and Western blots, respectively, containing the combined fractions from each step collected from a Tosoh TOYOPEARL® GigaCap Q650M column. The first step of purification removed 98.6% of the original plasma proteins. [Table 1]

[0161] The eluate (16 mL) from the Q anion exchange column containing IαIp was then diluted (1:3 (v / v)) with dH2O to a volume of 64.0 mL. This fraction was applied to a commercially available 32 mL column containing an endotoxin-binding agent (ETOXICLEAR™, Astrea Bioseparations) at a flow rate of 3.5 mL per minute. The flow-through was collected for analysis and then discarded because no (or substantial) IαIp was detected. An additional buffer volume (26.7 mL: 20 mM Tris-HCl + 150 mM NaCl, pH 7.2) was applied to the column. The total flow-through volume (90.7 mL) was collected for analysis and then discarded because it was found to contain no (or substantial) IαIp.

[0162] The column was washed with approximately 1.5 CV of first wash buffer (47.0 mL: 75 mM Gly + 100 mM AcOH + 150 mM NaCl, pH 5.2), and the resulting fractions were collected. The fractions were combined and analyzed for total protein (bicinchoninic acid assay (BCA)), IαIp (69.26 Mab-heparin-biotin sandwich ELISA), and trypsin inhibitor activity as shown in Table 2. The combined wash fractions were then discarded, as no (or substantial) IαIp was detected.

[0163] The column was further washed with a second wash buffer with a higher pH (47.5 mL: 20 mM Tris-HCl + 300 mM NaCl, pH 7.2), and the resulting fractions were collected and combined. The combined wash fractions contained a small amount of impurity IαIp (0.53% purity, 39.045 μg, yield of IαIp from Q eluate: 0.5624% (w / w)).

[0164] The bound protein was then eluted from the column by applying high-salt elution buffer (32.3 mL: 20 mM Tris-HCl + 500 mM NaCl, pH 7.2). The resulting fractions were collected, combined, and analyzed as shown in Table 2. The combined elution fractions were determined to contain highly pure IαIp (purity >99.9%, yield 99.5% (currently), total yield over two steps 98.0%).

[0165] A second elution buffer (28.9 mL: 20 mM Tris-HCl + 1000 mM NaCl, pH 7.2) was applied to the column to remove any residual IαIp from the endotoxin binder. No additional (or substantial amounts of) IαIp were detected in this eluate (see Table 2).

[0166] The column was clarified by applying clarification buffer (18.2 mL: 1 M NaOH + 2 M NaCl). No additional (or substantial amounts of) IαIp were detected in the flow-through.

[0167] Table 2 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, elution, second elution, and clarification). Additionally, Figures 1A-1B show the results of SDS-PAGE and Western blot examination of the combined fractions from each step recovered from the ETOXICLEAR™ column. [Table 2]

[0168] This example demonstrates that endotoxin-binding agents can be used to selectively bind IαIp, and this binding property can be used to purify IαIp from biological materials such as blood (e.g., cryopreserved plasma).

[0169] Example 2: Purification of IαIp from human plasma using DETOXI-GEL™, pH 7.3 A human plasma sample (2.5 mL of fresh frozen plasma) was diluted (1:3 (v / v) with 15 mM phosphate, pH 7.3), and the diluted plasma (10.0 mL) was applied to a commercially available 4 mL column containing an endotoxin-binding agent (DETOXI-GEL™, Pierce) at a flow rate of 2 mL / min. The flow-through was collected for analysis. Additional dilution buffer (50.2 mL: 15 mM phosphate, pH 7.3) was applied to the column to ensure that the starting material passed through the column completely. Additional flow-through was collected for analysis (see Table 3) and determined to contain IαIp (0.159% purity, 108.281 μg, 14.87% (w / w) yield of IαIp).

[0170] The column was washed with the first wash buffer (24.6 mL: 15 mM phosphate + 50 mM NaCl, pH 7.3), and fractions were collected, combined, and analyzed for total protein (BCA assay) and IαIp (MAb 69.26-heparin-biotin sandwich ELISA). The combined wash fractions were determined to contain IαIp (1.390% purity, 179.186 μg, 24.61% (w / w) yield of IαIp).

[0171] After the 50 mM NaCl wash, the column was washed with a second wash buffer containing an even higher concentration of NaCl (16.7 mL: 15 mM phosphate + 100 mM NaCl, pH 7.3), and the resulting fractions were collected, combined, and analyzed (see Table 3, Wash 2). The combined fractions were determined to contain a higher amount of IαIp than the previous wash step (4.969% purity, 235.353 μg, 32.33% (w / w) yield of IαIp).

[0172] After a second wash with 100 mM NaCl, an elution buffer with a higher NaCl concentration (7.3 mL: 15 mM phosphate + 1,000 mM NaCl, pH 7.3) was applied to the endotoxin-binding agent. The resulting fractions were collected, combined, and analyzed (see Table 3, Wash 3). The combined fractions were determined to contain less IαIp than the previous first or second wash steps (purity 2.763%, 40.632 μg, yield of IαIp 5.58% (w / w)).

[0173] The column was further clarified by washing with high pH buffer (6.7 mL: 1 M NaOH) and the resulting fractions were collected, combined and analyzed for IαIp (65.292 μg, 8.97% (w / w) yield of IαIp).

[0174] Table 3 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, elution, and clarification). Additionally, Figure 2 provides a chromatogram of the purification. [Table 3]

[0175] The IαIp eluted in the second wash step had a yield of 32.33% and a purity of 4.969% (see Figure 2A for the chromatogram). Furthermore, at pH 7.3, IαIp was detected in the flow-through and all wash fractions. DETOXI-GEL™ is an endotoxin-binding agent that employs polymyxin B resin as an endotoxin-binding agent. Although this method has not been optimized, the following example demonstrates that DETOXI-GEL™ binds to IαIp with improved affinity at different pH levels. Furthermore, this and subsequent examples demonstrate that DETOXI-GEL™ and other similar endotoxin-binding agents can unexpectedly selectively bind IαIp.

[0176] Example 3: Purification of IαIp from human plasma using DETOXI-GEL™, pH 5.5 A human plasma sample (2.5 mL of fresh frozen plasma) was diluted (1:3 (v / v) with 15 mM phosphate, pH 5.5), and the diluted plasma (10.0 mL) was applied to a 4 mL endotoxin-binding column (DETOXI-GEL™, Pierce) at a flow rate of 2 mL / min. The flow-through was collected for analysis and then discarded. Additional dilution buffer (63.6 mL: 15 mM phosphate, pH 5.5) was applied to the column to ensure that the starting material passed through the column completely. The additional flow-through was collected for analysis and discarded because no IαIp (or no substantial amounts) was detected.

[0177] The column was washed with the first wash buffer (26.4 mL: 15 mM phosphate + 50 mM NaCl, pH 5.5), and the resulting fractions were collected, combined, and analyzed for total protein (BCA assay) and IαIp (MAb 69.26-heparin-biotin sandwich ELISA). The combined fractions were discarded because no (or substantial amounts of) IαIp were detected.

[0178] After the first wash with 50 mM NaCl, the column was washed with a second wash buffer containing a higher concentration of NaCl (25.6 mL: 15 mM phosphate + 100 mM NaCl, pH 5.5), and the resulting fractions were collected, combined, and analyzed (see Table 4). The combined wash fractions were determined to contain IαIp (3.691% purity, 281.958 μg, 38.73% (w / w) yield of IαIp).

[0179] After a second wash with 100 mM NaCl, an elution buffer with a higher concentration of NaCl (14.5 mL: 15 mM phosphate + 1,000 mM NaCl, pH 5.5) was applied to the endotoxin-binding agent, and the resulting fractions were collected, combined, and analyzed for IαIp. The combined elution fractions were determined to contain the majority of IαIp (20.578% purity, 566.979 μg, 77.88% (w / w) yield of IαIp), as shown in Table 4.

[0180] The column was further clarified by washing with a high pH buffer (6.8 mL: 1 M NaOH), and the resulting fractions were collected, combined, and analyzed for IαIp. The combined fractions were determined to contain IαIp (43.255 μg, 5.94% (w / w) yield of IαIp), as shown in Table 4.

[0181] Table 4 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, elution, and clarification). Additionally, Figure 3 provides a chromatogram of the purification. [Table 4]

[0182] The majority of IαIp eluted in the third wash step (1,000 mM NaCl), resulting in a recovery of 77.88% and a purity of 20.578% (see Figure 2B for the chromatogram). Furthermore, at pH 5.5, IαIp was not detected in the flow-through or the first wash fraction, demonstrating that DETOXI-GEL™ selectively binds IαIp. This example confirmed that IαIp can be purified from biological materials using an endotoxin-binding agent.

[0183] Example 4: Purification of IαIp from human plasma using DETOXI-GEL™, pH 7.2 The eluate from the Q anion-exchange column (derived from fresh-frozen human plasma) containing concentrated IαIp (approximately 40% w / w) was applied (30 mL) to a 4 mL endotoxin-binding column (DETOXI-GEL™, Pierce) at a flow rate of 2 mL / min. The flow-through was collected for analysis of IαIp (MAb 69.26-heparin-biotin sandwich ELISA) and discarded because no IαIp was detected.

[0184] The column was washed with the first wash buffer (14.6 mL: 20 mM Tris-HCl + 150 mM NaCl, pH 7.2), and the resulting fractions were collected, combined, and analyzed for IαIp. The combined fractions were determined to contain the majority of IαIp from the starting material (653.5982 μg, 75.3% (w / w) recovery of IαIp).

[0185] After the 150 mM NaCl wash, the column was washed with a second wash buffer containing an even higher concentration of NaCl (5.8 mL: 20 mM Tris-HCl + 250 mM NaCl, pH 7.2), and the resulting fractions were collected, combined, and analyzed (see Table 5, Wash 1). The combined wash fractions were determined to contain less IαIp from the previous wash step (44.1728 μg, 5.09% (w / w) recovery of IαIp).

[0186] After washing with 250 mM NaCl, the endotoxin-binding agent was further washed with elution buffer containing a higher concentration of NaCl (6.8 mL, 20 mM Tris-HCl + 500 mM NaCl, pH 7.2). The resulting fractions were collected, combined, and analyzed for IαIp. These combined fractions contained more IαIp than the second wash step fractions (143.7316 μg, 16.56% (w / w) recovery of IαIp).

[0187] The column was further washed with another elution buffer containing a higher concentration of NaCl (3.7 mL, 20 mM Tris-HCl + 1,000 mM NaCl), and the resulting fractions were combined and analyzed for IαIp. These combined fractions contained less IαIp than the first elution fraction (42.14 μg, 4.86% (w / w) recovery of IαIp).

[0188] Table 5 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, first elution, and second elution). Additionally, Figure 4 provides a chromatogram of the purification. [Table 5]

[0189] The majority of IαIp was eluted in the first wash step along with most of the remaining protein from the Q eluate (75.30% recovery of IαIp). Furthermore, IαIp was detected in the other wash fraction and two elution fractions.

[0190] Example 5: Purification of IαIp from cryo-poor plasma using ETOXICLEAR™ A sample of cryo-poor plasma (11.5 mL) was diluted 1:4 (v / v) with 20 mM Tris-HCl, pH 7.2 + 200 mM NaCl. The diluted cryo-poor plasma was applied to a 5 mL commercial Q anion exchange resin (Tosoh TOYOPEARL® GigaCap Q650M) at a flow rate of 5 mL per minute (120 cm / h). The flow-through was collected for analysis. Flow-through that did not contain (or did not contain substantial amounts of) IαIp was discarded. Additional plasma dilution buffer (20 mM Tris-HCl pH 7.2 + 200 mM NaCl) was applied to the column to ensure that the starting material passed through the column completely. Additional flow-through was collected for analysis, determined to contain (or not contain substantial amounts of) IαIp, and discarded.

[0191] The column was washed with the first wash buffer (20 mM Tris-HCl pH 7.2 + 250 mM NaCl) and the resulting fractions were collected. The combined fractions were analyzed for total protein (bicinchoninic acid assay (BCA)), IαIp (MAb 69.26-heparin-biotin sandwich ELISA), and trypsin inhibitor activity as shown in Table 6. The combined wash fractions contained small amounts of impurity IαIp and were discarded.

[0192] After the pH 7.2 wash, the column was further washed with a second wash buffer at a lower pH (50 mM Gly 100 mM AcOH 150 mM NaCl pH 5.2) and the resulting fractions were collected, combined, and analyzed as shown in Table 6. The combined wash fractions contained a small amount of the impurity IαIp and were discarded.

[0193] After washing at pH 5.2, bound proteins were eluted with high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 750 mM NaCl). Fractions were collected, combined, and analyzed (e.g., total protein, IαIp, trypsin inhibitor activity) as shown in Table 6. The collected fractions contained concentrated IαIp.

[0194] A clarification buffer with high pH / high salt content (1 M NaOH + 2 M NaCl) was then applied to the column and the resulting fractions were combined and analyzed for IαIp as described in Table 6. The combined fractions were determined to contain no (or no substantial amount of) IαIp and were discarded.

[0195] Table 6 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, elution, and clarification). Additionally, Figures 5A-5B show an SDS-Page gel and Western blot, respectively, containing the combined fractions from each step collected from a Tosoh TOYOPEARL® GigaCap Q650M column. [Table 6]

[0196] The eluate from the Q anion exchange column containing IαIp was then diluted 1:5 (v / v) with dH2O. This fraction was applied to a commercially available 8 mL column containing an endotoxin-binding agent (ETOXICLEAR™, Astrea Bioseparations) at a flow rate of 3.5 mL per minute (120 cm / h). The flow-through was collected for analysis and then discarded, as no IαIp (or no substantial amounts) were detected. Flow-through buffer (20 mM Tris-HCl pH 7.2 + 200 mM NaCl) was applied to the column. The flow-through was collected for analysis and then discarded (no IαIp (or no substantial amounts) were detected).

[0197] The column was washed with the first wash buffer (50 mM Gly, 100 mM AcOH, and 200 mM NaCl, pH 5.2), and the resulting fractions were collected. The fractions were combined and analyzed for total protein (bicinchoninic acid assay (BCA)), IαIp (69.26 Mab-heparin-biotin sandwich ELISA), and trypsin inhibitor activity, as shown in Table 7. The combined wash fractions were then discarded (no (or substantial amounts of) IαIp were detected).

[0198] The column was further washed with a second wash buffer with a higher pH (20 mM Tris-HCl pH 7.2 + 300 mM NaCl), and the resulting fractions were collected and combined. The combined wash fractions contained small amounts of the impurity IαIp and were discarded.

[0199] The bound protein was then eluted from the column by applying a first high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 400 mM NaCl). The bound protein was then eluted from the column by applying a second high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 500 mM NaCl). The bound protein was then eluted from the column by applying a third high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 1000 mM NaCl). The resulting fractions were collected, combined, and analyzed as shown in Table 7.

[0200] The column was clarified by applying clarification buffer (1 M NaOH + 2 M NaCl). No additional IαIp was detected (or in substantial amounts) in the flow-through.

[0201] Table 7 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, elution, second elution, and clarification). Additionally, Figures 5A-5B show the results of SDS-PAGE and Western blot examination of the combined fractions from each step recovered from the ETOXICLEAR™ column. [Table 7]

[0202] Example 6: Purification of IαIp from cryo-poor plasma using ETOXICLEAR™ A sample of cryo-poor plasma (50 mL) was diluted 1:4 (v / v) with 20 mM Tris-HCl, pH 7.2 + 200 mM NaCl. The diluted cryo-poor plasma was applied to a 100 mL commercial Q anion exchange resin (Tosoh TOYOPEARL® GigaCap Q650M) at a flow rate of 12 mL per minute (120 cm / h). The flow-through was collected for analysis. Flow-through that did not contain (or did not contain substantial amounts of) IαIp was discarded. Additional plasma dilution buffer (20 mM Tris-HCl pH 7.2 + 200 mM NaCl) was applied to the column to ensure that the starting material passed through the column completely. Additional flow-through was collected for analysis, determined to contain (or not contain substantial amounts of) IαIp, and discarded.

[0203] The column was washed with the first wash buffer (20 mM Tris-HCl pH 7.2 + 250 mM NaCl) and the resulting fractions were collected. The combined fractions were analyzed for total protein (bicinchoninic acid assay (BCA)), IαIp (MAb 69.26-heparin-biotin sandwich ELISA), and trypsin inhibitor activity as shown in Table 8. The combined wash fractions contained small amounts of impurity IαIp and were discarded.

[0204] After the pH 7.2 wash, the column was further washed with a second wash buffer at a lower pH (50 mM Gly 100 mM AcOH 150 mM NaCl pH 5.2) and the resulting fractions were collected, combined, and analyzed as shown in Table 8. The combined wash fractions contained a small amount of the impurity IαIp and were discarded.

[0205] After washing at pH 5.2, bound proteins were eluted with high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 750 mM NaCl). Fractions were collected, combined, and analyzed (e.g., total protein, IαIp, trypsin inhibitor activity) as shown in Table 8. The collected fractions contained concentrated IαIp.

[0206] A clarification buffer with high pH / high salt content (1 M NaOH + 2 M NaCl) was then applied to the column and the resulting fractions were combined and analyzed for IαIp as described in Table 8. The combined fractions were determined to contain no (or no substantial amount of) IαIp and were discarded.

[0207] Table 8 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, elution, and clarification). Additionally, Figures 6A-6B show an SDS-Page gel and Western blot, respectively, containing the combined fractions from each step collected from a Tosoh TOYOPEARL® GigaCap Q650M column. [Table 8]

[0208] The eluate from the Q anion exchange column containing IαIp was then diluted 1:5 (v / v) with dH2O to 125 mL. This fraction was applied to a 74 mL commercial column containing an endotoxin-binding agent (ETOXICLEAR™, Astrea Bioseparations) at a flow rate of 3.5 mL per minute (120 cm / h). The flow-through was collected for analysis and then discarded, as no IαIp (or no substantial amounts) were detected. Flow-through buffer (20 mM Tris-HCl pH 7.2 + 200 mM NaCl) was applied to the column. The flow-through was collected for analysis and then discarded (no IαIp (or no substantial amounts) were detected).

[0209] The column was washed with the first wash buffer (50 mM Gly, 100 mM AcOH, and 200 mM NaCl, pH 5.2), and the resulting fractions were collected. The fractions were combined and analyzed for total protein (bicinchoninic acid assay (BCA)), IαIp (69.26 Mab-heparin-biotin sandwich ELISA), and trypsin inhibitor activity, as shown in Table 9. The combined wash fractions were then discarded (no (or substantial amounts of) IαIp were detected).

[0210] The column was further washed with a second wash buffer with a higher pH (20 mM Tris-HCl pH 7.2 + 300 mM NaCl), and the resulting fractions were collected and combined. The combined wash fractions contained small amounts of the impurity IαIp and were discarded.

[0211] The bound protein was then eluted from the column by applying a first high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 500 mM NaCl). The bound protein was then eluted from the column by applying a second high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 1000 mM NaCl). The resulting fractions were collected, combined, and analyzed as shown in Table 9.

[0212] The column was clarified by applying clarification buffer (1 M NaOH + 2 M NaCl). No additional IαIp was detected (or in substantial amounts) in the flow-through.

[0213] Table 9 provides a summary of the combined fractions and amounts of protein observed at each step (e.g., flow-through, first wash, second wash, elution, second elution, and clarification). Additionally, Figures 6A-6B show the results of SDS-PAGE and Western blot examination of the combined fractions from each step recovered from the ETOXICLEAR™ column. [Table 9]

[0214] Example 7: Analysis of co-purified impurities: Factor II (prothrombin) Factor II (prothrombin), purified simultaneously with IαIp in Examples 5 and 6, was analyzed by ELISA (Table 10). Sandwich ELISA was performed in a 96-well plate (NUNC Immuno MAXISORP F96) using a pair of commercially available polyclonal antibodies with a peroxidase-conjugated detection antibody, and the reaction was detected by the relative absorbance signal at 450 nm. The ELISA plate was coated overnight with polyclonal sheep anti-human prothrombin (1:1000 in coating buffer), washed three times with wash buffer, blocked with 0.1% Milch, 2 mmol / L benzamidine in wash buffer (blocking buffer), and washed with wash buffer. Test materials and standard calibration curve samples (all at selected dilutions in blocking buffer) were incubated on the ELISA-coated plate for 1 hour at room temperature. Standard calibration curves were typically constructed as a five-point calibration curve using a commercially available reference plasma preparation (CRYOcheck, PrecisionBioLogic), and FII activity was routinely checked against the certified secondary international standard ISTH / SSC.

[0215] The incubated ELISA plate was washed three times and incubated with sheep anti-human prothrombin peroxidase (HRP)-conjugated detection antibody for 1 hour at room temperature. After washing three times with wash buffer, the plate was incubated with TMB reagent (3,3',5,5'-tetramethylbenzidine dihydrochloride) to develop a color reaction with HRP, and the prothrombin level was measured by detecting the color signal at 450 nm. [Table 10]

[0216] Other embodiments All publications, patents, and patent applications mentioned in the above specification are herein incorporated by reference to the same extent as if each individual publication, patent, or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. While the invention has been described in conjunction with specific embodiments, it should be understood that further variations are possible, and that the claimed invention should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application generally covers any variations, uses, or adaptations of the invention in accordance with the principles of the invention, and is intended to include such departures from the present disclosure as are within known practice in the art to which this invention pertains and as applicable to the essential features set forth hereinabove.

Claims

1. 1. A method for purifying Inter-alpha inhibitor protein (IαIp) from a biological material, comprising: (a) applying the biological material containing the IαIp to an endotoxin-binding agent and separating the flow-through containing the biological material that does not bind to the endotoxin-binding agent; and (b) applying an elution buffer comprising a salt to the endotoxin-binding agent, and recovering an eluate comprising the IαIp.

2. (i) the endotoxin-binding agent is immobilized on a support; (ii) the endotoxin-binding agent is selected from the group consisting of ETOXICLEAR™, PIERCE™ high capacity endotoxin removal resin, TOXINERASER™ endotoxin removal resin, PURKINE™ endotoxin removal resin, DETOXI-GEL™ endotoxin removal gel, and PROMEGA™ endotoxin removal resin; (iii) the biological material further comprises three or more proteins selected from the group consisting of alpha-1 antitrypsin, C1 inhibitor, albumin, globulin, fibrinogen (Factor I), prothrombin (Factor II), thrombin, antithrombin III, Factor III, Factor V, Factor VII, Factor VIII, Factor IX, Factor X, Factor XI, Factor XII, Factor XIII, fibronectin, alpha-2 antiplasmin, urokinase, protein C, protein S, protein Z, protein Z-related protease inhibitor, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2, von Willebrand factor, Factor H, prekallikrein, high molecular weight kininogen, and heparin cofactor II; (iv) the biological material comprises 3-10, 3-15, 3-20, 3-25, 3-30, 10-20, 10-25, 10-30, 15-25, 15-30, 20-30, or 30 or more different proteins; (v) the biological material comprises about 40 to about 65% (w / w) albumin; (vi) the biological material comprises about 25 to about 45% (w / w) globulin; (vii) the biological material comprises about 2 to about 12% (w / w) fibrinogen; (viii) after step (a) and before step (b), further comprising applying a first wash buffer to the endotoxin-binding agent; (ix) the elution buffer has a pH of about 4.5 to about 8.5; (x) the elution buffer comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl; (xi) prior to step (a), further comprising applying a dilution buffer to the biological material; (xii) detecting the amount of IαIp in the flow-through; (xiii) detecting the amount of IαIp in the eluate; and / or (xiv) The method of claim 1, wherein each of steps (a) and (b) comprises using a flow rate of about 1-10 mL / min.

3. (i) the support is a monolithic support or a particle-based support; (ii) the support comprises a column, a membrane, a disk, or a chip; (iii) the endotoxin-binding agent is DETOXI-GEL™ or ETOXICLEAR™; (iv) the globulin is an immunoglobulin (Ig); (v) further comprising separating the flow-through comprising the first wash buffer; (vi) the first wash buffer has a pH of about 4.5 to 8.5; (vii) the first wash buffer comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl; (viii) after applying the first wash buffer, further comprising applying a second wash buffer to the endotoxin-binding agent. (ix) the elution buffer has a pH of about 7.2; (x) the elution buffer comprises about 5 to about 100 mM Tris-HCl; (xi) the elution buffer comprises about 1,000 mM NaCl or less; (xii) the dilution buffer comprises deionized water; (xiii) the dilution buffer has a pH of about 4.5 to about 8.5; (xiv) the biological material is diluted 1:1 to 1:10 (v / v) with the dilution buffer; (xv) the dilution buffer comprises one or more of glycine, acetate, citrate, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl; or 3. The method of claim 2, comprising: (xvi) discarding the flow-through.

4. (i) the monolithic or particle-based support is or comprises a resin; (ii) the immunoglobulin is selected from the group consisting of IgA, IgE, IgM, IgD, and IgG; (iii) the first wash buffer has a pH of about 5.2; (iv) the first wash buffer comprises about 10 to about 200 mM glycine and / or about 20 to 300 mM acetic acid; (v) the first wash buffer comprises about 200 mM NaCl or less; (vi) further comprising separating the flow-through comprising the second wash buffer; (vii) the second wash buffer has a pH of about 4.5 to about 8.5; (viii) the second wash buffer comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl; (ix) the elution buffer comprises about 20 mM Tris-HCl; (x) the elution buffer comprises about 500 to about 1,000 mM NaCl; (xi) the dilution buffer has a pH of about 5.5, about 7.2, or about 7.3; (xii) the dilution buffer comprises about 5 to about 100 mM Tris-HCl; (xiii) the dilution buffer contains about 50 mM NaCl or less; or (xiv) The method of claim 3, wherein the dilution buffer comprises about 5 to about 100 mM phosphate.

5. (i) the IgG is selected from the group consisting of intravenous Ig (IVIg), anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, and varicella zoster IgG; (ii) the first wash buffer comprises about 75 mM glycine and about 100 mM acetic acid; (iii) the first wash buffer comprises about 50 to about 150 mM NaCl; (iv) the second wash buffer has a pH of about 7.2; (v) the second wash buffer comprises about 5 to about 100 mM Tris-HCl; (vi) the second wash buffer comprises about 500 mM NaCl or less; (vii) the elution buffer comprises about 500 mM NaCl or about 1,000 mM NaCl; (viii) the dilution buffer comprises about 20 mM Tris-HCl; (ix) the dilution buffer is salt-free or contains about 50 mM NaCl; or 5. The method of claim 4, wherein (x) the dilution buffer comprises about 15 mM phosphate.

6. (i) the first wash buffer comprises about 50 mM NaCl or about 100 mM NaCl; (ii) the second wash buffer comprises about 20 mM Tris-HCl; (iii) the second wash buffer comprises about 100 to about 500 mM NaCl; (iv) the elution buffer applied to the endotoxin-binding agent in step (b) has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 500 mM NaCl; or 6. The method of claim 5, wherein (v) the first wash buffer applied to the endotoxin-binding agent has a pH of 5.2 and comprises about 75 mM glycine, about 100 mM acetic acid, and about 150 mM NaCl.

7. (i) the second wash buffer comprises about 300 mM NaCl; and / or 7. The method of claim 6, wherein (ii) the second wash buffer applied to the endotoxin-binding agent has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 300 mM NaCl.

8. (i) prior to step (a), applying the biological material to a chromatographic support; or 8. The method of any one of claims 1 to 7, wherein (ii) the biological material is substantially unprocessed prior to application to the endotoxin-binding agent.

9. (i) the chromatography support comprises an anion exchange chromatography support, a size exclusion chromatography support, an ion exchange chromatography support, an affinity chromatography support, or a combination thereof; (ii) (A) applying the biological material to the chromatographic support and separating the flow-through of step (A) comprising the biological material that is not bound to the chromatographic support; and (B) applying an elution buffer comprising a salt to the chromatographic support and collecting a first eluate comprising the IαIp; or (iii) The method of claim 8, wherein the chromatographic support comprises a column, a membrane, a disc, or a chip.

10. (i) the chromatographic support is an anion exchange chromatographic support; (ii) the chromatographic support is a monolithic support or a particle-based support; (iii) after step (A) and before step (B), further comprising applying a first wash buffer to the chromatographic support; (iv) the elution buffer applied to the chromatographic support has a pH of about 4.5 to about 8.5; (v) the elution buffer applied to the chromatographic support comprises one or more of glycine, acetate, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl; (vi) prior to step (A), further comprising applying a dilution buffer to the biological material; (vii) detecting the amount of IαIp in the flow-through of step (A); (viii) detecting the amount of IαIp in the eluate of step (B); or (ix) The method of claim 9, wherein each of steps (A) and (B) comprises using a flow rate of about 1 to 10 mL / min.

11. (i) the monolithic or particle-based support comprises an immobilized anion exchange resin; (ii) prior to step (B), further comprising separating the flow-through comprising the first wash buffer; (iii) the first wash buffer applied to the chromatographic support has a pH of about 4.5 to about 8.5; (iv) the first wash buffer applied to the chromatographic support comprises one or more of glycine, acetate, citrate, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl; (v) after applying the first wash buffer, applying a second wash buffer to the chromatographic support; (vi) the elution buffer applied to the chromatographic support has a pH of about 7.2; (vii) the elution buffer applied to the chromatographic support comprises about 5 to about 100 mM Tris-HCl; (viii) the elution buffer applied to the chromatographic support comprises about 1,000 mM NaCl or less; (ix) the dilution buffer comprises deionized water; (x) the dilution buffer has a pH of about 4.5 to about 8.5; (xi) the biological material is diluted 1:1 to 1:10 (v / v) with the dilution buffer; (xii) the dilution buffer comprises one or more of glycine, acetate, citrate, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl; or 11. The method of claim 10, comprising: (xiii) discarding the flow-through of step (A).

12. (i) the immobilized anion exchange resin is a diethylaminoethane (DEAE) resin or a quaternary amine (Q) resin; (ii) the first wash buffer applied to the chromatographic support has a pH of about 7.2; (iii) the first wash buffer applied to the chromatographic support comprises about 5 to about 100 mM Tris-HCl; (iv) the first wash buffer applied to the chromatographic support comprises about 400 mM NaCl or less; (v) further comprising separating the flow-through comprising the second wash buffer; (vi) the second wash buffer applied to the chromatographic support has a pH of about 4.5 to about 8.5; (vii) the second wash buffer applied to the chromatographic support comprises one or more of glycine, acetate, citrate, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl; (viii) the elution buffer applied to the chromatographic support comprises about 20 mM Tris-HCl; (viii) the elution buffer applied to the chromatographic support comprises about 750 mM NaCl; (ix) the dilution buffer has a pH of about 7.2; (x) the dilution buffer comprises about 5 to about 100 mM Tris-HCl; or 12. The method of claim 11, wherein (xi) the dilution buffer comprises about 300 mM or less NaCl.

13. (i) the first wash buffer applied to the chromatographic support comprises about 20 mM Tris-HCl; (ii) the first wash buffer applied to the chromatographic support comprises about 50 to about 250 mM NaCl; (iii) the second wash buffer applied to the chromatographic support has a pH of about 5.2; (iv) the second wash buffer applied to the chromatographic support comprises about 10 to about 200 mM glycine and / or about 20 to about 300 mM acetic acid; (v) the second wash buffer applied to the chromatographic support comprises about 100 to about 500 mM NaCl; (vi) the dilution buffer comprises about 20 mM Tris-HCl; (vii) the dilution buffer is salt-free or contains about 200 mM NaCl; or (viii) The method of claim 12, wherein the elution buffer applied to the chromatographic support has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 750 mM NaCl.

14. (i) the first wash buffer applied to the chromatographic support comprises about 250 mM NaCl; (ii) the second wash buffer applied to the chromatographic support comprises about 50 mM glycine and about 100 mM acetic acid; (iii) the second wash buffer applied to the chromatographic support comprises about 175 mM NaCl; (vi) the first wash buffer applied to the chromatographic support has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 250 mM NaCl; or 14. The method of claim 13, wherein (v) the second wash buffer applied to the chromatographic support has a pH of 5.2 and comprises about 50 mM glycine, about 100 mM acetic acid, and about 175 mM NaCl.

15. (i) the IαIp recovered in the eluate of step (b) has a purity of about 5% to 99% by weight or more relative to the purity of the IαIp in the biological material; (ii) the yield of IαIp in the eluate recovered in step (b) is greater than about 20% (w / w) relative to IαIp present in the biological material; (iii) the IαIp comprises two or more of inter-alpha inhibitor (IαI), pre-alpha inhibitor (PαI), and bikunin; (iv) the IαIp present in the biological material comprises 60% to 80% (w / w) of IαI and / or 20% to 40% (w / w) of PαI and / or the IαIp present in the eluate of step (b) comprises 60% to 80% (w / w) of IαI and / or 20% to 40% (w / w) of PαI, (v) said IαIp has an apparent molecular weight of about 60 to about 280 kDa; (vi) the IαIp has biological activity; (vii) the biological material is a blood product material; (viii) the biological material is milk or colostrum; (ix) the biological material is of mammalian origin; or 15. The method of any one of claims 1 to 14, further comprising: (x) performing one or more chromatography steps using the eluate collected in step (b).

16. (i) the yield is about 35% to about 90% (w / w) or more based on the IαIp present in the biological material; (ii) the yield of said IαIp from said biological material is at least about 5 μg / ml; (iii) the purity of the IαIp is at least about 5% (w / w); (iv) the biological activity comprises a cytokine inhibitory activity, a chemokine inhibitory activity, or a serine protease inhibitory activity; (v) the blood product material is selected from the group consisting of whole plasma, cryo-poor plasma, liquid plasma, frozen plasma (FP), source plasma, harvested plasma, solvent / detergent treated plasma (SDP), platelet rich plasma (PRP), platelet poor plasma (PPP), serum, whole blood, and dilutions or concentrates thereof; (vi) the mammal is a human, a primate, a bovine, an equine, a porcine, an ovine, a feline, or a canine; or (vii) The method according to claim 15, wherein the one or more additional chromatography steps comprise repeating the method according to any one of claims 1 to 14.

17. (i) the yield of IαIp is about 95% (w / w) or more based on the IαIp present in the biological material; (ii) the yield of said IαIp from said biological material is at least about 50 μg / ml; (iii) the purity of the IαIp is at least about 25% (w / w); or (iv) The method of claim 16, wherein the FP is selected from the group consisting of fresh frozen plasma (FFP), FFP24, FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, and diluted or concentrated formulations thereof.

18. (i) the yield of said IαIp from said biological material is at least about 100 μg / ml; or The method of claim 17, wherein (ii) the purity of the IαIp is at least about 50% (w / w).

19. (i) the yield of said IαIp from said biological material is at least about 300 μg / ml, at least about 600 μg / ml, or at least about 900 μg / ml; or (ii) the purity of the IαIp is at least about 75% (w / w); 20. The method of claim 18.

20. A composition comprising said IαIp produced by the method according to any one of claims 1 to 19.

21. 21. The composition of claim 20, wherein the composition is suitable for administration to a human.

22. 22. A pharmaceutical composition comprising the composition of claim 20 or 21 and a pharma- ceutically acceptable excipient.

23. 23. The pharmaceutical composition of claim 22 for treating a disease or condition in a subject in need thereof.

24. 22. A kit comprising the composition of claim 20 or 21.

25. 1. A method for purifying IαIp from plasma, comprising: (a) diluting the plasma with a dilution buffer comprising deionized water to form diluted plasma; (b) applying the diluted plasma to an ETOXICLEAR™ resin and separating the flow-through comprising the diluted plasma that does not bind to the ETOXICLEAR™ resin; (c) applying a first wash buffer comprising about 75 mM glycine, about 100 mM AcOH, and about 150 mM NaCl at a pH of about 5.2 to the ETOXICLEAR™ resin and separating the flow-through comprising the first wash buffer; (d) applying a second wash buffer comprising about 20 mM Tris-HCl and about 300 mM NaCl at a pH of about 7.2 to the ETOXICLEAR™ resin and separating the flow-through comprising the second wash buffer; and (e) applying an elution buffer comprising about 20 mM Tris-HCl and about 500 mM NaCl at pH 7.2 to the ETOXICLEAR™ resin, and collecting an eluate comprising the IαIp.

26. 1. A method for purifying IαIp from plasma, comprising: (a) diluting the plasma with a dilution buffer containing 15 mM phosphate at a pH of about 5.5 to form diluted plasma; (b) applying the diluted plasma to a DETOXI-GEL™ resin and separating the flow-through comprising the diluted plasma that does not bind to the DETOXI-GEL™ resin; (c) applying a first wash buffer comprising about 15 mM phosphate and about 50 mM NaCl, at a pH of about 5.5, to the DETOXI-GEL™ resin and separating the flow-through comprising the first wash buffer; (d) applying a second wash buffer comprising about 15 mM phosphate and about 100 mM NaCl at a pH of about 5.5 to the DETOXI-GEL™ resin and separating the flow-through comprising the second wash buffer; and (e) applying an elution buffer comprising about 15 mM phosphate and about 1,000 mM NaCl at a pH of about 5.5 to the DETOXI-GEL™ resin, and collecting an eluate comprising the IαIp.