Method for purifying interalpha inhibitor proteins

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 Art]

[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 bound to glycosaminoglycans. In vivo, IαIp can be found 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 an inflammatory signal, such as those triggered during injury or infection, occurs in the body, IαIp is transported into tissues and reaches the site of inflammation directly. The heavy chains of IαIp enhance the anti-inflammatory response by binding to proteins (damage signals) that are part of inflammatory cascades 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 repair of lung epithelium after injury in both in vitro and in vivo models, and IαIp has been shown to downregulate pro-inflammatory 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 ( (400-800 mg / L). In neonates and adult patients, IαIp levels rapidly decrease during systemic inflammation / sepsis (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 correlate strongly with disease progression. IαIp therapy has been described for the treatment of sepsis and related organ damage, pneumonia, acute respiratory disease, necrotizing enterocolitis (NEC), wounds, burns, cancer, stroke, and Alzheimer's disease.

[0006] Previously, IαIp was prepared using stepwise extraction followed by chromatographic separation. While these methods can achieve high purity (e.g., over 90%), they suffer from low IαIp yield (e.g., 20-30% (w / w)). Therefore, there is a need for a method to purify or prepare IαIp with high yield and high purity, for use, for example, in the preparation of therapeutic compositions. [Overview of the project]

[0007] This disclosure features a method for purifying IαIp (e.g., one or more of IαI, PαI, and bikunin). As a step in the purification process, this method applies a biological material containing IαIp, such as a biological material obtained from a subject (e.g., human) (e.g., blood or milk), to an endotoxin binder (e.g., a solid support such as a chromatography column containing an endotoxin binder).

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

[0009] In several embodiments, the endotoxin binder is immobilized on a support (e.g., a monolithic support or a particle-based support). In several embodiments, the monolithic support or particle-based support is or contains a resin. The support may be, for example, a column, a membrane, a disk, or a chip.

[0010] In other embodiments, the endotoxin binder 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 specific embodiments, the endotoxin binder is DETOXI-GEL® or ETOXICLEAR®.

[0011] The 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) It may contain three or more proteins selected from the group consisting of 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 quinogen, and heparin cofactor II.

[0012] In some embodiments, 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.

[0013] In some embodiments, the biological material contains 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 includes applying a first washing buffer to the endotoxin binder after step (a) and before step (b). In some embodiments, the method further includes separating the flow-through containing the first washing buffer. In some embodiments, the first washing buffer has a pH of about 4.5 to 8.5 (e.g., about pH 5.2). In some embodiments, the first washing buffer contains one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the first washing buffer contains 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 washing buffer contains NaCl of about 200 mM 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 washing buffer contains NaCl of about 100 to about 300 mM (e.g., about 200 mM NaCl). In some embodiments, the first washing buffer contains about 75 mM glycine, about 100 mM AcOH, and about 200 mM NaCl. In some embodiments, the method further includes applying a second washing buffer to the endotoxin binder after applying the first washing buffer. In some embodiments, the method further includes separating the flow-through containing the second washing buffer. In some embodiments, the second washing buffer has a pH of about 4.5 to about 8.5 (e.g., about pH 7.2). In some embodiments, the second washing 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 washing buffer comprises about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the second washing 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 washing buffer contains about 200 to about 400 mM NaCl (e.g., about 300 mM NaCl). In some embodiments, the second washing buffer contains about 20 mM Tris and about 300 mM NaCl.

[0015] In some embodiments, the method further includes applying a third washing buffer to the endotoxin binder after applying a second washing 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 contains one or more of glycine, acetic acid, citrate, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the elution buffer contains about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the elution buffer contains less than or equal to about 1,000 mM 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 contains about 20 mM Tris and about 500 mM NaCl.

[0016] In some embodiments, the method further comprises applying a second elution buffer to an endotoxin binder after applying an elution buffer, the second elution buffer having 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, citrate, 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 includes 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, acetic acid, 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 contains about 5 to about 100 mM phosphate (e.g., 15 mM phosphate).

[0018] In some embodiments, the method further includes adjusting the pH of the biological material before step (a). In some embodiments, the method includes 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 before step (a). In some embodiments, the method further includes adjusting the conductivity of the biological material before step (a). In some embodiments, the method includes adjusting the 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) before step (a).

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

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

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

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

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

[0024] In some embodiments, the chromatography 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 quaternary amine (Q) resin). In some embodiments, the monolithic support or particle-based support comprises a quaternary amine (Q) resin. In some embodiments, the chromatography support is a column, membrane, disk, or chip.

[0025] In some embodiments, the method further includes applying a first washing buffer to the chromatography support after step (i) and before step (ii). In some embodiments, before step (ii), the method further includes separating the flow-through containing the first washing buffer. In some embodiments, the first washing 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 washing buffer applied to the chromatography support contains one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the first washing buffer applied to the chromatography support contains about 5 to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl). In some embodiments, the first washing buffer applied to the chromatography support contains 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 washing buffer applied to the chromatography support contains about 20 mM Tris and about 250 mM NaCl.

[0026] In some embodiments, the method further includes applying a second wash buffer to the chromatographic support after applying a first wash buffer. In some embodiments, the method further includes separating the flow-through containing 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 contains one or more of glycine, acetic acid, 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 contains 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 contains 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 chromatography 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 chromatography support has a pH of from about 4.5 to about 8.5 (e.g., about pH 7.2). In some embodiments, the third wash buffer applied to the chromatography support has a pH of from about 4.5 to about 8.5 (e.g., 7.2). In some embodiments, the third wash buffer applied to the chromatography support comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the third wash buffer applied to the chromatography support comprises from about 5 mM to about 100 mM of Tris-HCl (e.g., about 20 mM of Tris-HCl). In some embodiments, the first wash buffer applied to the chromatography support comprises no more than about 400 mM of NaCl (e.g., from about 50 mM to about 250 mM of NaCl (e.g., about 200 mM of NaCl)). In some embodiments, the first wash buffer applied to the chromatography support comprises about 20 mM of Tris and about 200 mM of NaCl. In some embodiments, the elution buffer applied to the chromatography support has a pH of from 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, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the elution buffer applied to the chromatography support comprises from about 5 mM to about 100 mM of Tris-HCl (e.g., about 20 mM of Tris-HCl). In some embodiments, the elution buffer applied to the chromatography support comprises no more than about 1,000 mM of NaCl (e.g., about 750 mM of NaCl).

[0028] In some embodiments, prior to step (i), the method further comprises applying a dilution buffer to the biological material. In some embodiments, the dilution buffer comprises deionized water. In some embodiments, the dilution buffer comprises purified water. In one embodiment, the dilution buffer has a pH of from 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, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. In some embodiments, the dilution buffer comprises from about 5 mM to about 100 mM Tris-HCl (e.g., about 20 mM Tris-HCl)). In some embodiments, the dilution buffer comprises NaCl at no more than about 300 mM (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 to 10 mL / min.

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

[0033] In some embodiments, the yield of IαIp in the eluate recovered 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 inter-alpha inhibitors (IαI), pre-alpha inhibitors (PαI), and bicin.

[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 interalpha inhibitors (IαI), prealpha inhibitors (PαI), and bicin. In some embodiments, IαIp is an interalpha inhibitor (IαI). In some embodiments, IαIp is a prealpha inhibitor (PαI). In some embodiments, IαIp is an interalpha inhibitor (IαI) and a prealpha 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 IαIp present in the biological material is IαI. In some embodiments, about 90% to 99.5% (w / w) of IαIp present in the biological material is IαI. In some embodiments, about 80% to 90% (w / w) of IαIp present in the biological material is IαI. In some embodiments, about 70% to 80% (w / w) of IαIp present in the biological material is IαI. In some embodiments, about 60% to 70% (w / w) of IαIp present in the biological material is IαI. In some embodiments, about 50% to 60% (w / w) of IαIp present in the biological material is IαI. In some embodiments, about 40% to 50% (w / w) of IαIp present in the biological material is IαI. In some embodiments, approximately 30% to 40% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, approximately 20% to 30% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, approximately 10% to 20% (w / w) of the IαIp present in the biological material is IαI. In some embodiments, approximately 1% to 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 99.5% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 80% to 90% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 70% to 80% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 60% to 70% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 50% to 60% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, about 40% to 50% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, approximately 30% to 40% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, approximately 20% to 30% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, approximately 10% to 20% (w / w) of the IαIp present in the biological material is PαI. In some embodiments, approximately 1% to 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, approximately 91% to 96% (w / w) of IαIp present in the biological material are IαI and PαI. In some embodiments, approximately 90% to 95% (w / w) of IαIp present in the biological material are IαI and PαI. In some embodiments, approximately 89% to 94% (w / w) of IαIp present in the biological material are IαI and PαI. In some embodiments, approximately 88% to 93% (w / w) of IαIp present in the biological material are IαI and PαI. In some embodiments, approximately 87% to 92% (w / w) of IαIp present in the biological material are IαI and PαI. In some embodiments, approximately 86% to 91% (w / w) of IαIp present in the biological material are IαI and PαI. In some embodiments, approximately 85% to 90% (w / w) of the IαIp present in the biological material are 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, approximately 64% to 74% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 62% to 72% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 60% to 65% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 61% to 66% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 62% to 67% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 63% to 68% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 64% to 69% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 65% ​​to 70% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 66% to 71% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 67% to 72% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 68% to 73% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 69% to 74% (w / w) of the IαIp present in the eluate of step (b) is IαI.In some embodiments, approximately 59% to 64% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 58% to 63% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 57% to 62% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 56% to 61% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 55% to 60% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 54% to 59% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 54% to 59% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 53% to 58% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 52% to 57% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 51% to 56% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 50% to 55% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 49% to 54% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 48% to 53% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 47% to 52% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 46% to 51% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 45% to 50% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 60% to 70% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 58% to 68% (w / w) of the IαIp present in the eluate of step (b) is IαI.In some embodiments, approximately 56% to 66% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 54% to 64% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 52% to 62% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 50% to 60% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 48% to 58% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 46% to 56% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 44% to 54% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 42% to 52% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 40% to 50% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 30% to 40% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 20% to 30% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 10% to 20% (w / w) of the IαIp present in the eluate of step (b) is IαI. In some embodiments, approximately 1% to 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, approximately 40% to 50% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 38% to 48% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 36% to 46% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 34% to 44% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 32% to 42% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 30% to 35% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 31% to 36% (w / w) of IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 32% to 37% (w / w) of IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 33% to 38% (w / w) of IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 34% to 39% (w / w) of IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 35% to 40% (w / w) of IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 30% to 40% (w / w) of IαIp present in the eluate of step (b) is PαI.In some embodiments, approximately 28% to 38% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 26% to 36% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 24% to 34% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 22% to 32% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 30% to 35% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 29% to 34% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 28% to 33% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 27% to 32% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 26% to 31% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 25% to 30% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 24% to 29% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 23% to 28% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 22% to 27% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 21% to 26% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 20% to 25% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 20% to 30% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 10% to 20% (w / w) of the IαIp present in the eluate of step (b) is PαI. In some embodiments, approximately 1% to 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) are IαI and PαI. In some embodiments, about 90% to about 99.5% (w / w) of the IαIp present in the eluate of step (b) are IαI and PαI. In some embodiments, about 80% to about 90% (w / w) of the IαIp present in the eluate of step (b) are IαI and PαI. In some embodiments, about 95% to about 99.5% (w / w) of the IαIp present in the eluate of step (b) are IαI and PαI. In some embodiments, about 94% to about 98% (w / w) of the IαIp present in the eluate of step (b) are IαI and PαI. In some embodiments, about 93% to about 97% (w / w) of the IαIp present in the eluate of step (b) are IαI and PαI. In some embodiments, approximately 91% to 96% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, approximately 90% to 95% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, approximately 89% to 94% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, approximately 88% to 93% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, approximately 87% to 92% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, approximately 86% to 91% (w / w) of the IαIp present in the eluate of step (b) is IαI and PαI. In some embodiments, approximately 85% to 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) consists of 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) consists of 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 (e.g., cytokine inhibitor activity, chemokine inhibitor activity, or serine protease inhibitor activity).

[0048] In some embodiments, the biological material is blood product material (e.g., whole plasma, cryopreserved plasma, liquid plasma, frozen plasma (FP) (e.g., fresh frozen plasma (FFP), FFP24, FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, and their diluted or concentrated preparations), raw plasma, recovered plasma, solvent / surfactant treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, whole blood, and their diluted or concentrated preparations).

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

[0050] In some embodiments, the biological material is a blood product produced from blood product material through one or more process steps (e.g., filtration, centrifugation, sedimentation, 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 derived from mammals (e.g., humans, primates, cattle, horses, pigs, sheep, cats, or dogs).

[0052] In some embodiments, the biological material is substantially unprocessed before application to the endotoxin binder.

[0053] In some embodiments, the method further includes performing one or more chromatographic steps (for example, repeating the method of the first embodiment and / or its embodiments one or more times using the eluate recovered in step (b)).

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

[0055] In some embodiments, the first washing buffer applied to the endotoxin binder has a pH of 5.2 and contains about 75 mM glycine, about 100 mM acetic acid, and about 150 mM NaCl.

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

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

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

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

[0060] A second aspect of this disclosure features a composition comprising IαIp prepared by the methods of the first aspect and its embodiments. In one embodiment, the composition is suitable for administration to humans.

[0061] A third aspect of this disclosure is characterized by a pharmaceutical composition comprising the composition of the second aspect and its embodiments and a pharmaceutically acceptable excipient.

[0062] A fourth aspect of this disclosure is a method for treating a disease or condition in a subject requiring treatment, characterized by administering to the subject a composition of the second aspect or one embodiment thereof, or a pharmaceutical composition of the third aspect.

[0063] A fifth aspect of the present disclosure features a kit comprising a composition of the second aspect or one embodiment thereof, or a 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 includes (a) diluting plasma with a dilution buffer containing deionized water to form diluted plasma, (b) applying the diluted plasma to an ETOXICLEAR® resin to separate the flow-through containing diluted plasma that does not bind to the ETOXICLEAR® resin, and (c) applying a first washing buffer containing about 75 mM glycine, about 100 mM AcOH, and about 150 mM NaCl, with a pH of about 5.2 to an ETOXICLEAR® resin to separate the flow-through containing the first washing buffer. The method is characterized by (d) separating the flow-through, (d) applying a second washing buffer containing approximately 20 mM Tris-HCl and approximately 300 mM NaCl at pH approximately 7.2 to an ETOXICLEAR® resin to separate the flow-through containing the second washing buffer, and (e) applying an elution buffer containing approximately 20 mM Tris-HCl and approximately 500 mM NaCl at pH 7.2 to an ETOXICLEAR® resin to recover the eluate containing IαIp, thereby purifying IαIp from plasma.

[0065] A seventh aspect of this disclosure is (a) diluting plasma with a dilution buffer containing 15 mM phosphate at pH approximately 5.5 to form diluted plasma; (b) applying the diluted plasma to a DETOXI-GEL® resin and separating the flow-through containing diluted plasma that does not bind to the DETOXI-GEL® resin; and (c) applying a first washing buffer containing approximately 15 mM phosphate and approximately 50 mM NaCl at pH approximately 5.5 to DETOXI-GEL®. The method is characterized by (d) separating the flow-through containing the first washing buffer, (d) applying a second washing buffer containing approximately 15 mM phosphate and approximately 100 mM NaCl at pH approximately 5.5 to a DETOXI-GEL® resin and separating the flow-through containing the second washing buffer, and (e) applying an elution buffer containing approximately 15 mM phosphate and approximately 1,000 mM NaCl at pH approximately 5.5 to a DETOXI-GEL® resin and recovering the eluate containing IαIp, thereby purifying IαIp from plasma.

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

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

[0068] As used herein, “administration” means a method of giving a target a dose of a substance (e.g., IαIp) or composition. IαIp used in the methods described herein can be administered, for example, orally, intramuscularly, intravenously, intradermally, transdermally, intraarterially, intraperitoneally, intrainjured, intracerebrally, intraarticularly, 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 immersing target cells, by catheter, by lavage, or in a cream or liquid composition. The method of administration may 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” means a sample from an organism containing IαIp (e.g., a mammal such as a human). Examples of biological materials include blood product materials, e.g., whole plasma, cryopreserved plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, raw plasma, recovered plasma, solvent / surfactant treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, whole blood, and their diluted or concentrated preparations, milk or colostrum, urine, sputum, and cerebrospinal fluid. Examples of biological materials include plasma fraction intermediates or blood products produced by one or more process steps (e.g., filtration, centrifugation, sedimentation, chromatography, adsorption, isolation, freezing, thawing, dilution, concentration, viral clearance, etc.). Biological materials may originate from humans, primates, cattle, horses, pigs, sheep, cats, dogs, or combinations thereof. Furthermore, the biological material may be an extract prepared using cells expressing IαIp, or it may be a cell that secretes IαIp, for example, a cell that has been recombinantly modified to express IαIp, or it may contain such a cell.

[0070] Biological materials may be substantially unprocessed to the extent that no other purification steps have been applied to the material prior to the purification step using the endotoxin binder described herein, or to the extent that any prior purification step performed using the material has removed one or more substances from the material in amounts of less than 10% (w / w) (e.g., 0.1% to less than 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)). Biological materials may also be processed or prepared prior to the purification step using the endotoxin binder, as described herein. For example, biological materials may be processed using decantation, clarification, chromatography (e.g., anion exchange chromatography), centrifugation and / or sedimentation, freezing, drying, evaporation, extraction, filtration, precipitation, or other purification or preparation methods known in the art. The process can remove one or more substances from biological material (e.g., proteins other than IαIp) in amounts of 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)).

[0071] As used herein, the terms “chromatography” or “chromatographic process” refer to the separation of one or more analytes in a mixture by passing the mixture, in solution or suspension, through a medium in which the analytes of the mixture move at different rates. For example, a chromatographic process may be size exclusion chromatography, ion exchange chromatography, affinity chromatography, or dye-ligand chromatography. More specifically, as described herein, a chromatographic process can be carried out using, for example, an endotoxin binder (such as a resin) to separate IαIp from biological materials containing IαIp.

[0072] In this disclosure, terms such as “comprises,” “comprising,” “containing,” and “having” have the meanings assigned to them under U.S. patent law and may mean “includes,” “including,” etc. Similarly, “consisting essentially of” or “consists essentially” also have the meanings assigned to them under U.S. patent law and are open-ended, allowing for the existence of things beyond what is described, as long as the basic or novel characteristics of what is described are not altered by the existence of things beyond what is described, except for embodiments of the prior art.

[0073] As used herein, the term “eluate” refers to a fraction containing analyte material (e.g., IαIp) that is eluted from a medium (e.g., a support material) during a purification process (e.g., a chromatography process). The eluate can be released from the medium by applying the eluate to the medium, thereby releasing the analyte. More specifically, the eluate may refer to a fraction containing IαIp released from the culture medium (e.g., an endotoxin binder) after applying the eluate (e.g., an elution buffer such as a salt-containing buffer) to the medium.

[0074] In this specification, the term “endotoxin” refers to substances including lipids and polysaccharides. Endotoxins, such as lipopolysaccharide (LPS), are typically found on the outer membrane of the cell walls of Gram-negative bacteria. Endotoxins can be approximately 10 kDa in size, but can readily form large aggregates of up to 1,000 kDa. LPS (or endotoxins) can be found not only in E. coli but also in other Gram-negative bacteria (see, for example, 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 binder” means a molecule that can bind or is known to bind endotoxins, such as lipopolysaccharides. Endotoxin binders may be those that specifically bind to endotoxins. Endotoxin binders may be, for example, resins known to be used to remove lipopolysaccharides from biological materials or protein mixtures. Exemplary endotoxin binders include ETOXICLEAR® (www.astreabioseparations.com / product / etoxiclear), PIERCE® High-Volume 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), and PURKINE® Endotoxin Removal Resin (ww Examples include, but are not limited to, w.abbkine.com / product / purkine-endotoxin-removal-resin-bmr21400 / , 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). The endotoxin binder may be a molecule, such as polymyxin B, polylysine or its derivatives, or a synthetic mimetic peptide.

[0076] As used herein, the term “flow-through” refers to the volume of a fraction or fluid of a substance that passes through a medium (e.g., a medium used in chromatography, such as an endotoxin binder) without binding. Additional mobile phases (e.g., fluids such as low-salt (e.g., less than 50 mM salt) or salt-free (e.g., sodium chloride) buffers) may be added to ensure that one or more components of a mixture applied to the medium are fully supported on 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 “interalpha inhibitor protein” and “IαIp” and their plural form refer to a family of structurally related serine protease inhibitors, which are multicomponent glycoproteins. IαIp has been shown to be important for the inhibition of numerous proteases, including neutrophil elastase, plasmin, trypsin, chymotrypsin, granzyme K, preprotein converters, furin, cathepsin G, and acrosin. In human plasma, IαIp is detected at relatively high concentrations (400–800 mg / L). Unlike other inhibitor molecules, this family of inhibitors typically contains a combination of polypeptide chains (light and heavy chains) 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 inhibitors (IαI) containing two heavy chains (H1 and H2) and one light chain (L), and pre-alpha inhibitors (PαI) containing one heavy chain (H3) and one light chain (L). Another IαIp is a light chain (also called bikunin, a double Kunitz inhibitor) with two Kunitz domains, which is known to broadly inhibit plasma serine proteases. Yet another IαIp is the heavy chain-related molecule H4, which circulates in the blood independently of bikunin. There is yet another IαIp, the heavy chain-related molecule H5. IαI and PαI present in the plasma fraction have apparent molecular weights ranging from approximately 60 kDa to approximately 280 kDa.

[0078] As used herein, the term “pharmaceutically acceptable excipient” means one or more suitable solid or liquid fillers, diluents, or encapsulating materials suitable for administration to humans. Excipients may contain additives such as substances that enhance isotonicity and / or chemical stability. Such materials are nontoxic to the recipient in the amount and concentration used and include buffers, e.g., phosphates, citrates, succinates, acetates, lactates, tartrates, and other organic acids or their salts; tris-hydroxymethylamine methane (Tris), bicarbonates, carbonates, 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); may include amino acids, such as glycine, glutamic acid, aspartic acid, histidine, lysine, or arginine; monosaccharides, disaccharides, and other carbohydrates including cellulose or its derivatives, sulfated carbohydrate derivatives such as glucose, mannose, sucrose, dextrin, or 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 polysorbate or poloxamer. Other additives, such as stabilizers, antimicrobial agents, inert gases, fluids, and nutritional supplements (i.e., Ringer's dextrose), and electrolyte supplements, may also be included and may be present in conventional amounts.

[0079] As used herein, the terms “prevent,” “preventing,” and “prophylactic treatment” refer to reducing the probability of developing a disease, disorder, or condition in subjects who are not currently affected but are at risk of developing or are prone to developing such a condition.

[0080] As used herein, the term “processed” means a biological material that has been modified using one or more sample preparation steps (e.g., filtration, centrifugation, sedimentation, chromatography, etc.) before contacting the material with an endotoxin binder according to the methods described herein. Other examples of processes include decantation, clarification, freezing, drying, evaporation, extraction, filtration, precipitation, or other purification or preparation methods known in the art. A process step can remove one or more substances from the biological material (e.g., proteins other than IαIp) in amounts of 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)).

[0081] As used herein, terms such as "purify," "purifying," and "purification" mean removing 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 but without 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 for producing a composition in which proteins and / or substances other than IαIp are reduced by 40% by weight or more (e.g., 50%, 60%, 70%, 80%, 85%, 90%, 95%, 97%, or 99% or more) compared to a starting mixture (e.g., a 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), This includes, but is not limited to, 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 quinogen, and heparin cofactor II.

[0082] As used herein, the terms “pure” or “purity” mean the degree 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 absence of any contaminants (e.g., one or more unrelated proteins or other substances). For example, the purity of an IαIp composition indicates what percentage of the composition is IαIp relative to the total weight of the isolated substance, which can be determined, for example, based on pure IαIp. The levels of purity found herein may 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. The “pure” IαIp compositions of this disclosure may be purer than 20% by weight, 25% by weight, 30% by weight, 35% by weight, 40% by weight, 45% by weight, 50% by weight, 55% by weight, 60% by weight, 65% by weight, or up to 70% by weight. The “substantially pure” IαIp compositions may be substantially free of contaminants or impurities and have a purity greater than, for example, 70% by weight, 75% by weight, 80% by weight, 85% by weight, 90% by weight, 95% by weight, or 99% by weight. In some embodiments, the level of contaminants or impurities is about 20% by weight, 15% by weight, 12% by weight, 10% by weight, 9% by weight, 8% by weight, 7% by weight, 6% by weight, 5% by weight, 4% by weight, 3% by weight, 2% by weight, or 1% by weight or less. Purity can be determined by detecting the level of a specific analyte (e.g., IαIp) using an immunoassay or other technique (e.g., MAb 69.26-heparin-biotin sandwich ELISA, SDS / PAGE, and / or Western blotting) and calculating the ratio of the analyte to the total protein content (w / w) (e.g., determined by a total protein assay (e.g., bicinchoninate assay (BCA), Bradford assay, Biuret test, or another assay known in the art)).

[0083] As used herein, the term “subject” refers to human or non-human mammals, including but not limited to primates, cattle, horses, pigs, sheep, cats, or dogs. A subject may be a patient.

[0084] As used herein, the term “substantially raw” refers to biological material that has been modified, if any, minimally, relative to the original source material (e.g., blood). For example, substantially raw biological material may retain the original contents of the original source material (e.g., the same proteins and / or substances, and / or the same ratios of two or more proteins or substances), and / or the original properties (e.g., one or more biological activities). Biological material may be substantially raw, and as a result, any prior purification steps performed using that material will remove less than 10% (w / w) of one or more proteins or substances from that material (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)). Substantially raw biological material may, in particular, not have been modified by sample preparation steps (e.g., filtration, centrifugation, sedimentation, chromatography, etc.) before contacting the biological material with an endotoxin binder, for example, according to the methods described herein.

[0085] As used herein, the term "specifically binds" refers to the binding reaction between the analyte (e.g., a protein such as IαIp) and the binder (e.g., an endotoxin binder). Specific binding is a reaction that occurs between the analyte and the binder, even in the presence of a heterogeneous population of proteins and other biomolecules (e.g., proteins other than IαIp in biological materials). Specific binding between the analyte (e.g., IαIp) and the binder (e.g., an endotoxin binder) occurs at K levels less than approximately 1000 nM (e.g., 1 pM to 1000 nM). d Analytes that do not specifically bind to the binder (e.g., endotoxin binders) (e.g., IαIp) are characterized by a K content greater than approximately 1000 nM (e.g., greater than 1 μM, 100 μM, 500 μM, or 1 mM). dIt can be characterized by the following.

[0086] As used herein, the term “support” means any apparatus containing a drug (e.g., an endotoxin binder) that can come into contact with a material (e.g., a biological material) containing at least one analyte (e.g., IαIp). The support may be a column, membrane, disk, tip, or other apparatus for chromatography or affinity capture, examples of which are known in the art and described herein.

[0087] As used herein, the term “treat” means to reduce or improve one or more symptoms of a disorder and / or related thereto. It will be understood, though not excluded, that treating a disorder or condition does not require the complete elimination of the disorder or related symptoms.

[0088] As used herein, the term “yield” refers to the relative amount of analyte (e.g., IαIp) obtained after a purification step or process compared to the amount (w / w) of analyte in the starting material (e.g., biological material). Yield can be expressed as a percentage. In the context of this disclosure, the amounts of analyte (e.g., IαIp) in the starting material and the analyte obtained after the purification step can be measured using immunoassays or assays (e.g., anti-IαIp antibody (e.g., MAb 69.26)-heparin biotin sandwich ELISA, SDS / PAGE, and / or Western blotting). The methods of this disclosure may be used to yield purified IαIp at a yield of about 20% (w / w) or higher relative to the amount present in the original biological material. For example, the method can be used to yield purified IαIp yields of approximately 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or higher. [Brief explanation of the drawing]

[0089] [Figure 1]These images show the starting material, flow-through, wash fraction, and elution fraction obtained when IαIp was isolated from cryopreserved plasma by chromatography. The starting material, flow-through, wash fraction, and eluate 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. In the elution fraction of cryopreserved plasma using Q anion exchange resin (Figure 1A, arrow in lane 5) and the elution fraction of processed cryopreserved plasma using an endotoxin binder (ETOXICLEAR®; Figure 1A, arrow in lane 10), both 125kDa and 250kDa bands of IαIp corresponding to PαI and IαI were detected, respectively. Lanes 1-6 of the SDS-PAGE gel and Western blot correspond to the results of Q anion exchange chromatography using cryopreserved plasma as the starting material, while lanes 7-12 correspond to the results of purification with an endotoxin binder using the Q anion exchange chromatography eluate as the starting material. The lanes are as follows: Lane 1: Starting material - cryopreserved plasma; Lane 2: Flow-through from Q anion exchange resin; Lane 3: Washing fraction 1 (pH 7.2, 250 mM NaCl); Lane 4: Washing fraction 2 (pH 5.2, 175 mM NaCl); Lane 5: Eluate from Q anion exchange resin (pH 7.2, 750 mM NaCl); Lane 6: Clarification from Q anion exchange resin (1 M NaOH + 2 M NaC l); Lane 7: Flow-through from endotoxin binder; Lane 8: Washing fraction 1 (pH 5.2, 150 mM NaCl); Lane 9: Washing 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: Clarification from endotoxin binder (1 M NaOH + 2 M NaCl). [Figure 2]This chromatogram shows the chromatographic isolation of IαIp from human plasma using an endotoxin binder (DETOXI-GEL®) and 15 mM phosphate buffer (pH 7.3). IαIp was detected by the peak indicated by the arrow. The y-axis represents absorbance (A280), and the x-axis represents time (minutes). [Figure 3] This chromatogram shows the chromatographic isolation of IαIp from human plasma using an endotoxin binder (DETOXI-GEL®) and 15 mM phosphate buffer (pH 5.5). IαIp was detected by the peak indicated by the arrow. The y-axis represents absorbance (A280), and the x-axis represents time (minutes). [Figure 4] This chromatogram shows the chromatographic isolation of IαIp from human plasma using an endotoxin binder (DETOXI-GEL®) and 20 mM Tris-HCl buffer (pH 7.2). IαIp was detected by the peak indicated by the arrow. The y-axis represents absorbance (A280), and the x-axis represents time (minutes). [Figure 5]These images show the starting material, flow-through, wash fraction, and elution fraction obtained when IαIp was isolated by chromatography from cryopreserved plasma. The starting material, flow-through, wash fraction, and eluate were separated on 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. In the elution fraction of cryopreserved plasma using Q anion exchange resin (Figure 5B, arrow in lane 5) and the elution fraction of cryopreserved plasma using an endotoxin binder (ETOXICLEAR®; Figure 5B, arrows in lanes 6-7), 125kDa and 250kDa bands of IαIp corresponding to PαI and IαI were both detected, respectively. Lanes 1-5 of the SDS-PAGE gel and Western blot correspond to the results of Q anion exchange chromatography using cryopreserved plasma as the starting material, while lanes 6-7 correspond to the results of purification with an endotoxin binder using the Q anion exchange chromatography eluate as the starting material. The lanes are as follows: Lane 1: Starting material - cryopreserved plasma; Lane 2: Flow-through from Q anion exchange resin; Lane 3: Washing fraction 1 (pH 7.2, 250 mM NaCl); Lane 4: Washing fraction 2 (pH 5.2, 150 mM NaCl); Lane 5: Elutate from 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 fraction, and elution fraction obtained when IαIp was isolated by chromatography from cryopreserved plasma. The starting material, flow-through, wash fraction, and eluate were separated on 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. In the elution fraction of cryopreserved plasma using Q anion exchange resin (Figure 6B, arrow in lane 5) and the elution fraction of cryopreserved plasma using an endotoxin binder (ETOXICLEAR®; Figure 6B, arrow in lane 9), both 125kDa and 250kDa bands of IαIp corresponding to PαI and IαI were detected, respectively. Lanes 1-7 of the SDS-PAGE gel and Western blot correspond to the results of Q anion exchange chromatography using cryopreserved plasma as the starting material, while lanes 8-12 correspond to the results of purification with an endotoxin binder using the Q anion exchange chromatography eluate as the starting material. The lanes are as follows: Lane 1: Starting material - cryopreserved plasma; Lane 2: Flow-through from Q anion exchange resin; Lane 3: Washing fraction 1 (pH 7.2, 250 mM NaCl); Lane 4: Washing fraction 2 (pH 5.2, 150 mM NaCl); Lane 5: Elution fraction 1 from Q anion exchange resin (pH 7.2, 750 mM NaCl); Lane 6: Elution fraction 2 from Q anion exchange resin (pH 7.2, 1,000 mM NaCl); Lane 7: Q anion Lane 8: Clarification from exchange resin (1M NaOH + 2M NaCl); Lane 9: Flow-through from endotoxin binder and washing fraction (pH 5.2, 150mM NaCl); Lane 10: Elution fraction 2 (pH 7.2, 1,000mM NaCl); Lane 11: Clarification from endotoxin binder (1M NaOH + 2M NaCl); Lane 12: Concentrated elution fraction 1 after buffer exchange and ultrafiltration (Millipore Ultracell 30kDa cutoff centrifugation membrane). [Modes for carrying out the invention]

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

[0091] Therefore, the inventors purified IαIp from biological sources such as blood using such endotoxin binders. Furthermore, when using endotoxin binders as part of the purification process, a yield and purity 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 using the method of this disclosure.

[0092] The invention also features a pharmaceutical composition prepared using purified IαIp obtained by the method described herein, and a method for treating and / or reducing the likelihood of developing a disease or condition in subjects requiring such treatment, which involves administering a pharmaceutical composition prepared using purified IαIp obtained by the method described herein.

[0093] Purification method Endotoxin binders 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). IαIp can be separated from other components present in the biological material using the method described below. This method can be used to prepare IαIp with a purity ranging from about 5% to about 99% or more (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%). In addition, this method can be used to obtain a yield of purified IαIp of approximately 20% (w / w) or more relative to the amount present in the original biological material. For example, the method can be used to obtain a yield of purified IαIp of approximately 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w / w) or more.

[0094] biological material IαIp can be purified from biological materials using the methods described below. 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 may be, but are not limited to, whole plasma, cryoprema, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, raw plasma, recovered plasma, solvent / surfactant treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, blood (e.g., whole blood), and their diluted or concentrated preparations. Biological materials containing IαIp may also be, but are not limited to, plasma fraction intermediates. Plasma fraction intermediates are produced from whole plasma, cryopreserved plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, raw plasma, recovered plasma, solvent / surfactant 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, sedimentation, chromatography, adsorption, isolation, freezing, thawing, dilution, concentration, S / D treatment, etc.).

[0095] Furthermore, the biological material may be an extract prepared using cells expressing IαIp, or it may be a cell that secretes IαIp, for example, a cell that has been recombinantly modified to express IαIp, or it may contain such a cell.

[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, globulin (e.g., immunoglobulin, e.g., IgA, IgG (e.g., intravenous Ig (IVIg), anti-D), It may also contain 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 quinogen, and heparin cofactor II. The biological material may be milk (e.g., colostrum), which may contain one or more types of 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, anti-chymotrypsin, plasminogen, fibrinogen, growth factors, and cytokines).

[0097] The biological material that is contacted with or applied to the endotoxin binder (or other supports described herein, such as anion exchange chromatography supports) according to the methods described below is substantially raw (e.g., original source material), or the biological material may be treated with one or more sample preparation methods or other known purification methods, such as those described herein, before contact with or application to the endotoxin binder.

[0098] Substantially raw biological material is biological material that has been modified, if any, minimally with respect to the original source material (e.g., blood or another source as described herein) so that the biological material retains the original characteristics of the source material. For example, substantially raw biological material can be subjected to a sample preparation or purification process to remove one or more substances from the material in amounts of less than 10% (w / w) (e.g., 0.1% to less than 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)) before the material comes into contact with or is applied to an endotoxin binder. Alternatively, substantially raw biological material may be an eluate or fraction from a prior purification process, in which case the prior purification process 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 an endotoxin binder (or different supports as described herein, such as anion exchange chromatography supports). For example, one or more proteins found in the biological material (e.g., blood or milk), e.g., alpha-1 antitrypsin, C1 inhibitor, albumin, globulin (e.g., immunoglobulin, e.g., IgA, IgG (e.g., intravenous Ig (IVIg), anti-D), 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 Inhibitors, plasminogen, tissue plasminogen activator, plasminogen activator inhibitor 1, plasminogen activator inhibitor 2, von Willebrand factor, factor H, prekallikrein, high molecular weight quinogen, 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 before the purification step using endotoxin binders.

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

[0101] Endotoxin binder The methods described herein involve the use of an endotoxin binder, which can be used to bind to IαIp in a mixture of proteins (e.g., IαIp present in biological materials such as milk or blood). An endotoxin binder is a molecule that may be known to bind to endotoxins, such as lipopolysaccharides. The endotoxin binder may bind specifically to endotoxins. The endotoxin binder can be incorporated into or immobilized on a support, for example. The support may be a monolithic support or a particle-based support. The particles may be, for example, a resin. The endotoxin binder can be packed into or immobilized on any number of known supports, such as columns, membranes, disks, or chips. The endotoxin binder may be a molecule, such as polymyxin B, polylysine or its derivatives, or a synthetic mimetic peptide.

[0102] Non-limiting examples of endotoxin binders that can be used in the methods described herein include, for example, ETOXICLEAR®, PIERCE® high-volume endotoxin removal resin, TOXINERASER® endotoxin removal resin, PURKINE® endotoxin removal resin, DETOXI-GEL® endotoxin removal gel, or Promega® endotoxin removal resin. The method can be carried out, for example, using a pre-filled column or cartridge containing an endotoxin binder (for example, a column with a volume of about 0.1 mL to about 100 mL, or a column with a larger volume).

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

[0104] In some cases, DETOXI-GEL® endotoxin removal gel is used as the source of the endotoxin binder. DETOXI-GEL® binds to the lipid A domain of endotoxin using immobilized polymyxin B. In other cases, ETOXICLEAR® is used as the endotoxin binder.

[0105] Reagents for use in purification methods Dilution buffer Biological materials containing IαIp (e.g., IαI, PαI) may be combined with a dilution buffer before the purification process, for example, before contacting or applying the biological material to a medium such as an endotoxin binder. The dilution buffer may be added to lower the salt (e.g., NaCl) concentration of the biological material, for example, to avoid or reduce the possibility of premature elution of IαIp from the endotoxin binder. The biological material may be diluted with the dilution buffer to a ratio of, for example, 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), for example, 1:3 (v / v)), and then contacted or applied to a medium (e.g., an endotoxin binder).

[0106] The dilution buffer may have a pH range of approximately 4.5 to 8.5 (for example, approximately 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 approximately 8.5). The dilution buffer may contain one or more of the following: 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 contain a low concentration of salt such as NaCl (e.g., a salt of 300 mM or less (e.g., a salt of 200 mM, 150 mM, 100 mM, 75 mM, 50 mM, 25 mM, 10 mM, 5 mM, or 0 mM (e.g., NaCl))). For example, a biological material containing IαIp can be diluted 1:3 (v / v) with a buffer containing 20 mM Tris-HCl, and then the diluted sample can be contacted or applied to an endotoxin-binding column. The biological material may have been prepared, for example, during a prior purification process (e.g., a chromatography process such as a process 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 before the purification process, for example, before contacting or applying the biological material to a medium such as an endotoxin binder or anion exchanger. Loading buffers containing biological materials may have a pH range of approximately 4.5 to 8.5 (for example, approximately 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 approximately 8.5). Loading buffers may contain one or more of the following: deionized water, glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. The loading buffer may contain a salt such as NaCl at a low concentration (for example, a salt of 300 mM or less (e.g., a salt of 200 mM, 150 mM, 100 mM, 75 mM, 50 mM, 25 mM, 10 mM, 5 mM, or 0 mM (e.g., NaCl))). The buffer may contain about 20 mM Tris-HCl and 200 mM NaCl. The loading buffer may be prepared, for example, during a prior process step (e.g., a chromatography step such as a step using an anion exchange support, or 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 A flow-through buffer can optionally be used to ensure that all of the biological material is loaded onto the culture medium (e.g., an endotoxin binder or another support as described herein) during the purification process. After application to the culture medium, the flow-through buffer can also be used to achieve initial or additional separation of components present in the biological material. The flow-through buffer may be the same as the dilution buffer. Alternatively, the flow-through buffer may be different from the dilution buffer.

[0109] For example, the flow-through buffer may have the same or different pH (for example, within the range of approximately 4.5 to approximately 8.5 (for example, approximately 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 dilution buffer may have concentrations of 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 components of different compositions (e.g., one or more of glycine, acetic acid, citrate, phosphate, NaCl, calcium, magnesium, EDTA, and Tris-HCl in concentrations of about 5 mM to about 300 mM) compared to the dilution buffer used to load the biological material into the medium. The flow-through buffer may have a higher salt concentration than the dilution buffer (e.g., a salt (e.g., NaCl) concentration 5 to about 300 mM higher than the dilution buffer concentration) (e.g., this method may involve using a flow-through buffer of about 150 mM NaCl after using a dilution buffer of less than 150 mM NaCl, such as a dilution buffer without NaCl). The properties of the flow-through buffer may be selected to improve the initial separation between components of the biological material during the purification process (e.g., during the column chromatography process).

[0110] Washing buffer The purification method may also include, as described herein, a washing buffer used in one or more washing steps (e.g., 1, 2, 3, 4, or more washing steps) that occur after contacting or applying a biological material to an endotoxin binder, or during one or more other purification steps. The washing steps may be present in the endotoxin binder or other media (e.g., an anion exchanger), but do not bind strongly (e.g., K2 above about 1 mM). d This can be carried out to remove weakly bound components (for example, proteins or other substances found in biological materials that are not IαIp).

[0111] The washing buffer applied to the culture medium (e.g., the endotoxin binder or other type of support described herein) can be used to change the pH of the medium, to change the salt concentration of the medium, or to change both the pH and salt concentration of the medium. The first washing buffer applied to the culture medium may change the pH, the second washing buffer applied to the culture medium may change the salt concentration, or vice versa. The washing step using the washing buffer can facilitate the purification of IαIp by promoting the release of proteins other than IαIp from the culture medium.

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

[0113] The washing buffer may have a pH range of approximately 4.5 to 8.5 (for example, approximately 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 approximately 8.5). The washing buffer may have a pH of approximately 0.5.2. The washing buffer may have a pH of approximately 0.7.2. The washing buffer may contain one or more of the following in concentrations of about 5 mM to about 400 mM: glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. For example, the washing buffer may have a salt (e.g., NaCl) concentration of about 50 to about 400 mM. For example, the washing buffer may have a salt (e.g., NaCl) concentration of about 200 to about 300 mM. For example, the washing buffer may have a salt (e.g., NaCl) concentration of about 250 mM, about 250 mM, or about 300 mM. For example, the washing buffer may have about 200 mM NaCl, about 250 mM NaCl, or about 300 mM NaCl. The washing buffer may also be prepared at a different pH than other buffers previously used in the purification process (e.g., dilution buffer, flow-through buffer, and / or pre-wash buffer).

[0114] The washing buffers used in the purification method may contain different salt concentrations. The process can begin with a low-salt washing buffer in a medium such as an endotoxin binder or anion exchange resin, and then proceed to washing steps using washing buffers with increasing salt concentrations in each subsequent washing step.

[0115] For example, a first washing step may include applying a first washing buffer having a low pH (e.g., less than pH 7.0, e.g., pH 5.5 or less (e.g., pH 5.2)) and / or a salt concentration (e.g., a salt (e.g., NaCl) concentration of less than 150 mM) to a medium such as an endotoxin binder (for example, the first washing buffer may contain 75 mM glycine + 100 mM acetic acid + 150 mM NaCl (pH 5.2)), and a second washing step may include applying a second washing buffer having a higher pH (e.g., a pH greater than pH 7.0, e.g., pH 7.2) and / or a salt concentration (e.g., a salt (e.g., NaCl) concentration greater than 150 mM (e.g., about 300 mM)) to a medium (for example, the second washing buffer may contain 20 mM Tris-HCl + 300 mM NaCl (pH 7.2)). In another example, the first washing step may use a washing buffer containing 15 mM phosphate + 50 mM NaCl (pH 5.5), and the subsequent washing step may use a second washing buffer containing 15 mM phosphate + 100 mM NaCl (pH 5.5).

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

[0117] The elution buffer may be prepared using a sufficiently high salt concentration (e.g., sodium chloride (NaCl)) (e.g., a salt greater than about 200 mM (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 (e.g., NaCl)) so that the bound IαIp can be released from the medium (e.g., endotoxin binder, anion exchange resin). The elution buffer may contain the same components as the buffers described above (e.g., dilution buffer, loading buffer, flow-through buffer, and wash buffer), or it may contain one or more different components (e.g., the elution buffer may contain one or more of glycine, acetic acid, citric acid, phosphoric acid, and Tris-HCl in concentrations of approximately 5 mM to approximately 300 mM). Other salts or additives, such as calcium, magnesium, and EDTA, can be used instead of NaCl at equivalent concentrations. The pH of the elution buffer may also be the same as the buffer previously in contact with or applied to the medium (e.g., endotoxin binder) (e.g., dilution and / or washing buffer), or the pH of the elution buffer may be different (e.g., the elution buffer may have a pH in 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, 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 washing buffer previously applied to the medium (e.g., endotoxin binder). The pH of the elution buffer may be approximately 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 once or multiple 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 recovered after application of the elution buffer may be separately analyzed for the presence and concentration of IαIp using, for example, ELISA or other techniques known in the art, and then optionally pooled. Alternatively, the fractions may be pooled and analyzed for the presence and concentration of IαIp.

[0119] Clarification buffer Furthermore, the purification method may include an optional clarification step of applying the clarification buffer to a medium (e.g., an endotoxin binder) to regenerate the medium 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 may contain about 1 M of an alkaline solute, such as sodium hydroxide (NaOH). Optionally, the clarification buffer may additionally contain about 1 to about 2 M of a salt (e.g., NaCl). In a preferred embodiment, the clarification buffer contains 1 M NaOH + 2 M NaCl (pH 14).

[0120] Purification of IαIp using an endotoxin binder IαIp can be purified from biological materials by contacting or applying the biological material containing IαIp to an endotoxin binder (e.g., one or more of the endotoxin binders described herein). The biological material can be applied directly to the endotoxin binder without dilution, or it can be applied to the endotoxin binder after diluting the biological material with a dilution buffer (as described above). For example, the volume of biological material (with or without dilution) that is contacted or applied to the endotoxin binder may be, for example, about 0.5 to about 20 column volumes (or other suitable volume).

[0121] After applying IαIp-containing biological material to the endotoxin conjugate, the flow-through can be optionally analyzed to confirm that it does not contain IαIp (or contains 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 blotting, or other known techniques) can be performed. After confirming that the flow-through contains no IαIp at all or only a negligible amount (e.g., less than or equal to about 30 μg / mL, e.g., about 20 μg / mL, 10 μg / mL, 5 μg / mL, or 1 μg / mL), the flow-through can be discarded. Additional flow-through buffer can be applied to the endotoxin conjugate to ensure that all of the biological material is loaded onto the endotoxin conjugate (e.g., in a volume of about 1 to about 50 column volumes). This flow-through may then be discarded (for example, after confirming, if desired, that IαIp is not present (or present in substantial amounts) in the flow-through).

[0122] Next, one or more washing steps (e.g., two, three, four, or more washing steps) can be performed to remove non-IαIp present in the biological material that is weakly bound to the endotoxin binder. In a given washing step, about 0.5 to about 10 column volumes of washing buffer (or other suitable volume) can be applied. The resulting washing fraction can be analyzed as desired (e.g., using ELISA or other known techniques) to confirm that it does not contain IαIp (or contains less than 10% (w / w)). If a substantial amount of IαIp (e.g., 10% (w / w) or more) is detected in the washing buffer flow-through, the flow-through can be processed as desired to recover and purify the IαIp present in the flow-through (e.g., using the endotoxin binder or other media described herein (e.g., anion exchange support)).

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

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

[0125] Each step of the purification process (e.g., application of the biological material, washing step, and elution step) can be carried out using either gravity flow or low pressure (e.g., 0-15 psi) to generate a flow rate of approximately 1 to 10 mL per minute (e.g., flow rates of approximately 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 embodiments, the flow rate is approximately 2 mL / min.

[0126] This process can be repeated one or more times as 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 eluate fraction containing IαIp can be subjected to one or more additional purification steps as desired, as described later.

[0127] Purification of IαIp after use of endotoxin binder The eluate fraction containing IαIp recovered after elution from the endotoxin binder can be further purified using other known purification steps (e.g., one or more of the steps described in "Additional Purification Steps" 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. Furthermore, the eluate containing IαIp can be further purified using anion exchange chromatography as described above.

[0128] Purification process before use of endotoxin binder Biological materials containing IαIp may be treated before contact with or application of an endotoxin binder. For example, the biological material may be treated using a sample preparation or purification process to remove one or more substances (e.g., proteins or substances other than IαIp) from the biological material in amounts of 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)).

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

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

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

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

[0133] Biological materials containing IαIp can be applied directly to a chromatography support (e.g., anion exchange chromatography support). Alternatively, the biological material can be diluted with a dilution buffer as described above before being 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 a chromatography support (e.g., in approximately 0.5 to 25 column volumes or other suitable volumes).

[0134] After applying the biological material to the chromatographic support, the flow-through can be separated and optionally analyzed to confirm that it does not contain IαIp (or contains less than 10% (w / w)). For example, an ELISA assay or other known techniques (e.g., SDS-PAGE, and / or Western blotting, or other known techniques) 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 processed as desired to recover and purify the IαIp present in the flow-through (e.g., using an endotoxin binder or other media described herein (e.g., anion exchange support)). After it is confirmed that the flow-through contains no IαIp or only a substantial amount (e.g., less than or equal to about 30 μg / mL, e.g., less than or equal to about 20 μg / mL, 10 μg / mL, 5 μg / mL, or 1 μg / mL), the flow-through can be discarded.

[0135] Optionally, a flow-through buffer can be prepared and applied to the chromatographic support to ensure that all of the biological material is loaded onto the support (e.g., in a volume of about 1 to about 50 column volumes, or other appropriate volume). This flow-through may then be discarded (e.g., after confirming, if desired, that IαIp is not present (or present in substantial amounts) in the flow-through).

[0136] Next, one or more washing steps (e.g., two, three, four, or more) can be performed to remove non-IαIp in the biological material that is weakly bound to the chromatographic support. The washing buffer may be prepared as described above. In a given washing step, about 0.4 to about 10 column volumes of washing buffer (or other suitable volume) can be applied to the chromatographic support. The resulting washing fraction can be analyzed as desired (e.g., using ELISA or other known techniques) to confirm that it does not contain IαIp (or contains less than 10% (w / w)). If a substantial amount of IαIp (e.g., 10% (w / w) or more) is detected in the washing buffer flow-through, the flow-through can be processed as desired to recover and purify the IαIp present in the flow-through (e.g., using an endotoxin binder or other media described herein (e.g., anion exchange support)).

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

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

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

[0140] If desired, the chromatography support can be washed with a clarification buffer and regenerated for future use. The resulting clarified fraction can, if desired, be analyzed for the presence of IαIp (e.g., using ELISA or other known techniques). In one embodiment, a clarification buffer containing 1 M NaOH + 2 M NaCl (pH 14) is applied to a chromatography support in a volume of, for example, about 1 to 5 column volumes, or 1.5 to 2 column volumes.

[0141] IαIp detection IαIp isolated using the purification method described herein can be quantified using one or more assays known in the art, such as those 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 conjugate (e.g., an antibody that specifically binds to IαIp (e.g., MAb 69.26 (e.g., Sha et al, J Pediatr. 180:135-140, 2017), MAb 69.31 (e.g., Lim et al., J Infect Dis. 188(6):919-926, 2003) or PAb) The method may include contacting an IαIp ligand (e.g., heparin, LPS, and / or hyaluronic acid (HA)) with R22C (see, for example, WO2020 / 086879, each of which is incorporated herein by reference in whole) and detecting the amount of bound IαIp (e.g., using a detection agent). The IαIp conjugate may be labeled with, for example, biotin, and detection may be performed using, for example, streptavidin labeled with horseradish peroxidase, and then detection may be performed using a known detection method. Alternatively, the IαIp conjugate may be labeled with, for example, a phosphor and detected by a spectrophotometer. Or, the IαIp detection agent may be detected directly without labeling (e.g., by surface plasmon resonance (SPR)). After the addition of the IαIp detection agent, an additional washing step (e.g., one or more times) may be performed to remove unbound IαIp detection agent.

[0143] Subsequently, IαIp can be measured based on a signal from a bound label or bound detector (e.g., enzyme activity or fluorescence) using standard techniques known in the art. When an enzyme is used as a label, a substrate is added to generate a signal (e.g., a color change), which can be read using a device suitable for signal detection, such as a spectrophotometer. The signal (e.g., absorbance or fluorescence) can be plotted against a standard using a known concentration of IαIp, or compared against a known reference concentration, in order to establish a standard curve. Based on the established standard curve or reference concentration value, 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 binder 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 chains (e.g., H1, H2, H3, H4, and / or H5), light chains (e.g., bicinnamin), or combinations thereof) from biological materials. The purity of IαIp can be determined by detecting the total amount of IαIp obtained after purification (e.g., using an assay or immunoassay, e.g., MAb 69.26-heparin-biotin sandwich ELISA, SDS / PAGE, and / or Western blotting) and by calculating the ratio (w / w) to the total protein content determined by a whole protein assay (e.g., bicinchoninate assay (BCA), Bradford assay, Biuret test, or another assay known in the art). After the purification process, 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 more than 99% (w / w).

[0145] The isolated IαIp has an apparent molecular weight between approximately 60 kDa and approximately 280 kDa, which can be determined by any suitable method known in the art, for example, by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isolated IαIp can also be tested for biological activity, for example, for activity selected from the group consisting of cytokine inhibitory activity, chemokine inhibitory activity, protease inhibitory activity (e.g., serine protease inhibitory activity), chondroitin sulfate binding activity, 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-specific inhibitory activity, such as trypsin-specific inhibitory activity at approximately 1000 IU / mg to approximately 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 may vary. Preferably, the purified IαIp (e.g., IαI and / or PαI) is present in the final purified fraction in physiological ratios. Physiological ratios may be, for example, ratios found in healthy humans or animals, and / or ratios of IαI to PαI that naturally appear in human plasma. Typical physiological ratios are about 60% to 80% IαI and about 20% to 40% PαI.

[0147] The purification method also yields 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 may be about 90% by weight or 95% by weight or more.

[0148] The disclosed purification method can be used to produce compositions containing IαIp. Furthermore, the compositions can be prepared using a higher concentration of IαIp than that present in the original biological material (e.g., blood or milk). Therefore, this method can be used to prepare compositions containing IαIp in amounts of at least about 5 μg / mL per mL, for example, 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. IαIp present in the composition can account for approximately 5% to approximately 99% (w / w) or more of the composition (for example, 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, and greater than 99% (w / w)).

[0149] Additional steps (e.g., lyophilization, vacuum concentration) can be performed to increase the concentration of IαIp in the compositions prepared using the disclosed methods. 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 composition Pharmaceutical compositions can be prepared using IαIp (e.g., IαI, PαI, heavy chains (e.g., H1, H2, H3, H4, and / or H5), light chains (e.g., bikunin), or combinations thereof) obtained by purification using the purification methods disclosed herein (e.g., using endotoxin binders as described above). IαIp can be combined with, for example, pharmaceutically acceptable excipients. 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 to treat or prevent various diseases, conditions, or symptoms thereof, such as inflammation and / or diseases or conditions characterized by low levels of IαIp (for example, when prepared as a pharmaceutical composition). Methods of treatment and methods for identifying subjects suitable for treatment with the pharmaceutical compositions of this 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 chain (e.g., H1, H2, H3, H4, and / or H5), light chain (e.g., bikunin), or combinations thereof). For example, IαIp can be placed in vials in amounts 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 as described, for example, in US2007 / 0297982, US2015 / 0238578, US2019 / 0269765, and WO2020 / 086879, which are incorporated herein by reference.

[0154] The following examples are intended to illustrate the present invention and are not limiting. [Examples]

[0155] Example 1: Purification of IαIp from cryopreserved plasma using ETOXICLEAR® Cryoprea-poor plasma samples were diluted (1:3 (v / v) dilution with 20 mM Tris-HCl + 200 mM NaCl, pH 7.2), and the diluted cryoprea-poor plasma (114.4 mL) was applied to a commercially available 5 mL 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 contained in substantial amounts 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 completely through the column. Additional flow-through was collected for analysis and discarded if it was determined that it did not contain (or contained in substantial amounts of) IαIp.

[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 fraction was collected. The combined fraction was analyzed for total protein (bicinchoninic acid assay (BCA)), IαIp (MAb 69.26-heparin-biotin sandwich ELISA), and trypsin inhibitory activity, as shown in Table 1. The combined washed fraction contained a small amount of impurity IαIp (purity 0.1%, 61.727 μg, IαIp yield 0.8761% (w / w)) and was therefore discarded.

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

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

[0159] Next, a high pH / high salt content washing 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 shown in Table 1. The combined fraction was determined to be free of IαIp (or contain virtually no IαIp) and was discarded.

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

[0161] Next, 16 mL of eluate from a Q anion exchange column containing IαIp was diluted with dH2O (1:3 (v / v)) to a volume of 64.0 mL. This fraction was applied to a commercially available 32 mL column containing an endotoxin binder (ETOXICLEAR®, Astrea Bioseparations) at a flow rate of 3.5 mL / min. The flow-through was collected for analysis and then discarded because no IαIp was detected (or present in substantial amounts). An additional volume of buffer (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 detected to contain no IαIp (or present in substantial amounts).

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

[0163] The column was further washed with a second washing buffer having 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 washing fraction contained a small amount of impurity IαIp (purity 0.53%, 39.045 μg, yield of IαIp from Q eluate 0.5624% (w / w)) and was therefore discarded.

[0164] Subsequently, the bound protein was eluted from the column by applying a 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 eluted fraction was determined to contain high-purity IαIp (purity 99.9% or higher, yield 99.5% (current stage), total yield 98.0% in two steps).

[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 IαIp was detected (or present in substantial amounts) in this eluate (see Table 2).

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

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

[0168] This example demonstrates that IαIp can be selectively bound using an endotoxin binder. This binding property can be used to purify IαIp from biological materials such as blood (e.g., cryoprema).

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

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

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

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

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

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

[0175] IαIp eluted in the second washing 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 both the flow-through and all washing fractions. DETOXI-GEL® is an endotoxin binder that employs polymyxin B resin as the endotoxin binder. Although this method is not optimized, the following examples demonstrate that DETOXI-GEL® binds to IαIp with improved affinity at different pH levels. Furthermore, these examples and subsequent examples demonstrate that DETOXI-GEL® and other similar endotoxin binders can selectively bind to IαIp, unexpectedly.

[0176] Example 3: Purification of human plasma-derived IαIp using DETOXI-GEL (trademark), pH 5.5 A human plasma sample (fresh frozen plasma, 2.5 mL) was diluted (1:3 (v / v) dilution 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 complete passage of the starting material. The additional flow-through was collected for analysis and discarded as no IαIp was detected (or present in substantial amounts).

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

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

[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 as an endotoxin binder. The resulting fraction was collected and combined for analysis of IαIp. The combined eluted fraction was determined to contain the majority of IαIp (purity 20.578%, 566.979 μg, IαIp yield 77.88% (w / w)), 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 and combined for analysis of IαIp. The combined fraction was determined to contain IαIp (43.255 μg, IαIp yield 5.94% (w / w)) 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). Furthermore, Figure 3 provides a chromatogram of the purification process. [Table 4]

[0182] The majority of IαIp was eluted in the third washing step (1,000 mM NaCl), with an IαIp recovery rate 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 either the flow-through or the first washing fraction, demonstrating that DETOXI-GEL™ selectively binds to IαIp. This example confirms that IαIp can be purified from biological materials using an endotoxin binder.

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

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

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

[0186] After washing with 250 mM NaCl, the endotoxin binder was further washed with an 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 and combined, and analyzed for IαIp. These combined fractions contained more IαIp than the fraction from the second washing step (143.7316 μg, IαIp recovery rate 16.56% (w / w)).

[0187] The column was further washed with another elution buffer containing a high 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, IαIp recovery rate 4.86% (w / w)).

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

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

[0190] Example 5: Purification of IαIp from cryopreserved plasma using ETOXICLEAR® A cryopreserved plasma sample (11.5 mL) was diluted 1:4 (v / v) with 20 mM Tris-HCl (pH 7.2 + 200 mM NaCl). The diluted cryopreserved plasma was applied to a commercially available 5 mL Q anion exchange resin (Tosoh TOYOPEARL® GigaCap Q650M) at a flow rate of 5 mL / min (120 cm / hour). The flow-through was collected for analysis. Flow-through that did not contain (or contained in 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 complete passage of the starting material through the column. Additional flow-through was collected for analysis and discarded if it was determined that it did not contain (or contained in substantial amounts of) IαIp.

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

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

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

[0194] Next, a clarification buffer with a high pH / high salt content (1M NaOH + 2M NaCl) was applied to the column, and the resulting fractions were combined and analyzed for IαIp as shown in Table 6. The combined fraction was determined to be free of IαIp (or contain virtually no IαIp) and was discarded.

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

[0196] Next, the eluate from the Q anion exchange column containing IαIp was diluted with dH2O (1:5 (v / v)). This fraction was applied to a commercially available 8 mL column containing an endotoxin binder (ETOXICLEAR®, Astrea Bioseparations) at a flow rate of 3.5 mL per minute (120 cm / hour). The flow-through was collected for analysis and then discarded because no IαIp was detected (or present in substantial amounts). A 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 was detected (or present in substantial amounts)).

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

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

[0199] Subsequently, the bound protein was eluted from the column by applying a first high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 400 mM NaCl). Then, the bound protein was eluted from the column by applying a second high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 500 mM NaCl). Finally, the bound protein was eluted 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 a clarification buffer (1M NaOH + 2M NaCl). No additional IαIp was detected (or present in substantial amounts) during the flow-through.

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

[0202] Example 6: Purification of IαIp from cryopreserved plasma using ETOXICLEAR® A 50 mL cryopreserved plasma sample was diluted 1:4 (v / v) with 20 mM Tris-HCl (pH 7.2 + 200 mM NaCl). The diluted cryopreserved plasma was applied to a commercially available 100 mL Q anion exchange resin (Tosoh TOYOPEARL® GigaCap Q650M) at a flow rate of 12 mL / min (120 cm / hour). The flow-through was collected for analysis. Flow-through that did not contain (or contained in 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 complete passage of the starting material through the column. Additional flow-through was collected for analysis and discarded if it was determined that it did not contain (or contained in substantial amounts of) IαIp.

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

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

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

[0206] Next, a clarification buffer with a high pH / high salt content (1M NaOH + 2M NaCl) was applied to the column, and the resulting fractions were combined and analyzed for IαIp as shown in Table 8. The combined fraction was determined to be free of IαIp (or contain virtually no IαIp) and was discarded.

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

[0208] Next, the eluate from the Q anion exchange column containing IαIp was diluted with dH2O (1:5 (v / v)) to 125 mL. This fraction was applied to a commercially available 74 mL column containing an endotoxin binder (ETOXICLEAR®, Astrea Bioseparations) at a flow rate of 3.5 mL per minute (120 cm / hour). The flow-through was collected for analysis and then discarded because no IαIp was detected (or present in substantial amounts). A 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 was detected (or present in substantial amounts)).

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

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

[0211] Subsequently, the bound protein was eluted from the column by applying a first high-salt elution buffer (20 mM Tris-HCl pH 7.2 + 500 mM NaCl). Then, the bound protein was eluted from the column again 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 a clarification buffer (1M NaOH + 2M NaCl). No additional IαIp was detected (or present in substantial amounts) during the flow-through.

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

[0214] Example 7: Analysis of impurities purified simultaneously: Factor II (prothrombin) Factor II (prothrombin) purified simultaneously with IαIp in Examples 5-6 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 peroxidase-labeled detection antibodies, 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 coat buffer), washed three times with wash buffer, blocked with 0.1% Milch and 2 mmol / L benzamidine in wash buffer (block buffer), and washed with wash buffer. Test materials and standard calibration curve samples (both within the dilution range selected using block buffer) were incubated on the ELISA coated plate at room temperature for 1 hour. Standard calibration curves were typically created using commercially available reference plasma preparations (CRYOcheck, PrecisionBioLogic) and regularly checked against the secondary international standard ISTH / SSC, which has been certified for FII activity.

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

[0216] Other Embodiments All publications, patents, and patent applications described in the above specification are incorporated herein by reference to the same extent as each individual publication, patent, or patent application is specifically and individually indicated to be incorporated by reference in whole. Various modifications and variations of the methods, pharmaceutical compositions, and kits of the present invention described herein will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in conjunction with specific embodiments, it should be understood that further modifications are possible and that the inventions of the claims should not be unduly limited to such specific embodiments. In fact, various modifications of the embodiments described for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the invention. This application generally encompasses any modifications, uses, or adaptations of the invention in accordance with the principles of the invention, and such deviations from this disclosure are intended to include those that are within known practice in the art to which the invention belongs and that are applicable to the essential features set forth herein. This application provides the invention in the following embodiments. (Aspect 1) A method for purifying interalpha inhibitor proteins (IαIp) from biological materials, (a) Applying the biological material containing IαIp to an endotoxin binder and separating the flow-through containing the biological material that does not bind to the endotoxin binder, (b) A method comprising applying an elution buffer containing a salt to the endotoxin binder and recovering the eluate containing IαIp. (Aspect 2) The method according to embodiment 1, wherein the endotoxin binder is immobilized on a support. (Aspect 3) The method according to embodiment 2, wherein the support is a monolithic support or a particle-based support. (Aspect 4) The method according to embodiment 3, wherein the monolithic support or particle-based support is a resin or contains a resin. (Appendix 5) The method according to any one of embodiments 2 to 4, wherein the support comprises a column, membrane, disk, or chip. (Aspect 6) The method according to any one of embodiments 1 to 5, wherein the endotoxin binder 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. (Aspect 7) The method according to embodiment 6, wherein the endotoxin binder is DETOXI-GEL® or ETOXICLEAR®. (Pattern 8) The method according to any one of embodiments 1 to 7, wherein 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 quinogen, and heparin cofactor II. (Aspect 9) The method according to embodiment 8, wherein the globulin is immunoglobulin (Ig). (Aspect 10) The method according to embodiment 9, wherein the immunoglobulin is selected from the group consisting of IgA, IgE, IgM, IgD, and IgG. (Aspect 11) The method according to embodiment 10, wherein 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. (Aspect 12) The method according to any one of embodiments 1 to 11, wherein the biological material comprises 3 to 10, 3 to 15, 3 to 20, 3 to 25, 3 to 30, 10 to 20, 10 to 25, 10 to 30, 15 to 25, 15 to 30, 20 to 30, or 30 or more different proteins. (Aspect 13) The method according to any one of embodiments 1 to 12, wherein the biological material contains about 40 to about 65% (w / w) albumin. (Aspect 14) The method according to any one of embodiments 1 to 13, wherein the biological material contains about 25 to about 45% (w / w) of globulin. (Aspect 15) The method according to any one of embodiments 1 to 14, wherein the biological material contains about 2 to about 12% (w / w) of fibrinogen. (Aspect 16) The method according to any one of embodiments 1 to 15, further comprising applying the first washing buffer to the endotoxin binder after step (a) and before step (b). (Aspect 17) The method according to embodiment 16, further comprising separating the flow-through containing the first wash buffer. (Aspect 18) The method according to embodiment 16 or 17, wherein the first washing buffer has a pH of approximately 4.5 to 8.5. (Aspect 19) The method according to embodiment 18, wherein the first washing buffer has a pH of approximately 5.2. (Aspect 20) The method according to any one of embodiments 16 to 19, wherein the first washing buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (Aspect 21) The method according to any one of embodiments 16 to 20, wherein the first washing buffer comprises about 10 to about 200 mM glycine and / or about 20 to about 300 mM acetic acid. (Aspect 22) The method according to embodiment 21, wherein the first washing buffer comprises about 75 mM glycine and about 100 mM acetic acid. (Aspect 23) The method according to any one of embodiments 16 to 22, wherein the first washing buffer contains NaCl in a concentration of approximately 200 mM or less. (Aspect 24) The method according to embodiment 23, wherein the first washing buffer contains about 50 to about 150 mM NaCl. (Aspect 25) The method according to embodiment 24, wherein the first washing buffer contains about 50 mM NaCl or about 100 mM NaCl. (Aspect 26) The method according to any one of embodiments 16 to 25, further comprising applying a second washing buffer to the endotoxin binder after applying the first washing buffer. (Aspect 27) The method according to embodiment 26, further comprising separating the flow-through containing the second wash buffer. (Aspect 28) The method according to embodiment 26 or 27, wherein the second washing buffer has a pH of about 4.5 to about 8.5. (Aspect 29) The method according to embodiment 28, wherein the second washing buffer has a pH of approximately 7.2. (Aspect 30) The method according to any one of embodiments 26 to 29, wherein the second washing buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (Aspect 31) The method according to any one of embodiments 26 to 30, wherein the second washing buffer contains about 5 to about 100 mM Tris-HCl. (Aspect 32) The method according to embodiment 31, wherein the second washing buffer contains approximately 20 mM Tris-HCl. (Aspect 33) The method according to any one of embodiments 26 to 32, wherein the second washing buffer contains NaCl in a concentration of approximately 500 mM or less. (Aspect 34) The method according to embodiment 33, wherein the second washing buffer contains about 100 to about 500 mM NaCl. (Aspect 35) The method according to embodiment 34, wherein the second washing buffer contains approximately 300 mM NaCl. (Aspect 36) The method according to any one of embodiments 1 to 35, wherein the elution buffer has a pH of about 4.5 to about 8.5. (Aspect 37) The method according to embodiment 36, wherein the elution buffer has a pH of approximately 7.2. (Aspect 38) The method according to any one of embodiments 1 to 37, wherein the elution buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (Aspect 39) The method according to any one of embodiments 1 to 38, wherein the elution buffer contains approximately 5 to 100 mM Tris-HCl. (Approach 40) The method according to embodiment 39, wherein the elution buffer contains approximately 20 mM Tris-HCl. (Aspect 41) The method according to any one of embodiments 1 to 40, wherein the elution buffer contains NaCl in a concentration of approximately 1,000 mM or less. (Aspect 42) The method according to any one of embodiments 1 to 41, wherein the elution buffer contains approximately 500 to approximately 1000 mM NaCl. (Aspect 43) The method according to embodiment 41 or 42, wherein the elution buffer contains approximately 500 mM NaCl or approximately 1,000 mM NaCl. (Aspect 44) The method according to any one of embodiments 1 to 43, further comprising applying a dilution buffer to the biological material prior to step (a). (Aspect 45) The method according to embodiment 44, wherein the dilution buffer contains deionized water. (Aspect 46) The method according to embodiment 44 or 45, wherein the dilution buffer has a pH of approximately 4.5 to approximately 8.5. (Aspect 47) The method according to embodiment 46, wherein the dilution buffer has a pH of about 5.5, about 7.2, or 7.3. (Aspect 48) The method according to any one of embodiments 44 to 47, wherein the biological material is diluted with the dilution buffer in a ratio of 1:1 to 1:10 (v / v). (Aspect 49) The method according to any one of embodiments 44 to 48, wherein the dilution buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (Appearance 50) The method according to any one of embodiments 44 to 49, wherein the dilution buffer contains about 5 to about 100 mM Tris-HCl. (Aspect 51) The method according to embodiment 50, wherein the dilution buffer contains about 20 mM Tris-HCl. (Appearance 52) The method according to any one of embodiments 44 to 51, wherein the dilution buffer contains approximately 50 mM or less of NaCl. (Aspect 53) The method according to embodiment 52, wherein the dilution buffer does not contain salt or contains about 50 mM NaCl. (Aspect 54) The method according to any one of embodiments 44 to 53, wherein the dilution buffer contains about 5 to about 100 mM of phosphate. (Aspect 55) The method according to embodiment 54, wherein the dilution buffer contains about 15 mM phosphate. (Aspect 56) The method according to any one of embodiments 1 to 55, further comprising detecting the amount of IαIp in the flow-through. (Aspect 57) The method according to embodiment 56, which includes discarding the flow-through. (Aspect 58) The method according to any one of aspects 1 to 57, further comprising detecting the amount of IαIp in the eluate. (Aspect 59) The method according to any one of embodiments 1 to 58, including a flow rate of approximately 1 to 10 mL / min. (Appendix 60) The method according to any one of embodiments 1 to 58, comprising applying the biological material to a chromatographic support prior to step (a). (Aspect 61) The method according to embodiment 60, wherein the chromatography support includes an anion exchange chromatography support, a size exclusion chromatography support, an ion exchange chromatography support, an affinity chromatography support, or a combination thereof. (Aspect 62) The method according to embodiment 61, wherein the chromatography support is the anion exchange chromatography support. (Aspect 63) (i) Applying the biological material to the chromatographic support and separating the flow-through of step (i) which includes the biological material that does not bind to the chromatographic support, (ii) The method according to any one of embodiments 60 to 62, further comprising applying an elution buffer containing a salt to the chromatographic support and recovering a first eluate containing the IαIp. (Personal aspect 64) The method according to embodiment 63, wherein the chromatography support is a monolithic support or a particle-based support. (Patent 65) The method according to embodiment 64, wherein the monolithic support or particle-based support comprises an immobilized anion exchange resin. (Aspect 66) The method according to embodiment 65, wherein the immobilized anion exchange resin is a diethylaminoethane (DEAE) resin or a quaternary amine (Q) resin. (Patent 67) The method according to any one of embodiments 60 to 66, wherein the chromatography support comprises a column, membrane, disk, or chip. (Pattern 68) The method according to any one of embodiments 60 to 67, further comprising applying a first washing buffer to the chromatography support after step (i) and before step (ii). (Patent 69) The method according to embodiment 68, further comprising separating the flow-through containing the first wash buffer before step (ii). (Aspect 70) The method according to embodiment 68 or 69, wherein the first washing buffer applied to the chromatographic support has a pH of about 4.5 to about 8.5. (Aspect 71) The method according to embodiment 70, wherein the first washing buffer applied to the chromatography support has a pH of about 7.2. (Aspect 72) The method according to any one of embodiments 68 to 71, wherein the first washing buffer applied to the chromatography support comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (Aspect 73) The method according to any one of embodiments 68 to 72, wherein the first washing buffer applied to the chromatography support contains about 5 to about 100 mM Tris-HCl. (Aspect 74) The method according to embodiment 73, wherein the first washing buffer applied to the chromatography support contains about 20 mM Tris-HCl. (Aspect 75) The method according to any one of embodiments 68 to 74, wherein the first washing buffer applied to the chromatography support contains approximately 400 mM or less of NaCl. (Aspect 76) The method according to embodiment 75, wherein the first washing buffer applied to the chromatographic support contains about 50 to about 250 mM NaCl. (Aspect 77) The method according to embodiment 76, wherein the first washing buffer applied to the chromatography support contains about 250 mM NaCl. (Pattern 78) The method according to any one of embodiments 68 to 77, further comprising applying a second wash buffer to the chromatography support after applying the first wash buffer. (Aspect 79) The method according to embodiment 78, further comprising separating the flow-through containing the second wash buffer. (Appendix 80) The method according to embodiment 78 or 79, wherein the second washing buffer applied to the chromatographic support has a pH of about 4.5 to about 8.5. (Aspect 81) The method according to embodiment 80, wherein the second washing buffer applied to the chromatography support has a pH of about 5.2. (Aspect 82) The method according to any one of embodiments 78 to 81, wherein the second washing buffer applied to the chromatography support comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (Aspect 83) The method according to any one of embodiments 78 to 82, wherein the second washing buffer applied to the chromatographic support comprises about 10 to about 200 mM glycine and / or about 20 to about 300 mM acetic acid. (Pattern 84) The method according to embodiment 83, wherein the second washing buffer applied to the chromatography support contains about 50 mM glycine and about 100 mM acetic acid. (Pattern 85) The method according to any one of embodiments 78 to 84, wherein the second washing buffer applied to the chromatography support contains about 100 to about 500 mM NaCl. (Pattern 86) The method according to embodiment 85, wherein the second washing buffer applied to the chromatography support contains approximately 175 mM NaCl. (Aspect 87) The method according to any one of embodiments 63 to 86, wherein the elution buffer applied to the chromatography support has a pH of about 4.5 to about 8.5. (Pattern 88) The method according to embodiment 87, wherein the elution buffer applied to the chromatography support has a pH of approximately 7.2. (Pattern 89) The method according to any one of embodiments 63 to 88, wherein the elution buffer applied to the chromatography support comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (Aspect 90) The method according to any one of embodiments 63 to 89, wherein the elution buffer applied to the chromatographic support contains about 5 to about 100 mM Tris-HCl. (Aspect 91) The method according to embodiment 90, wherein the elution buffer applied to the chromatography support contains about 20 mM Tris-HCl. (Patent 92) The method according to any one of embodiments 63 to 91, wherein the elution buffer applied to the chromatography support contains approximately 1000 mM or less of NaCl. (Aspect 93) The method according to embodiment 92, wherein the elution buffer applied to the chromatography support contains approximately 750 mM NaCl. (Aspect 94) The method according to any one of embodiments 63 to 93, further comprising applying a dilution buffer to the biological material prior to step (i). (Aspect 95) The method according to embodiment 94, wherein the dilution buffer contains deionized water. (Personal aspect 96) The method according to embodiment 94 or 95, wherein the dilution buffer has a pH of about 4.5 to about 8.5. (Patent 97) The method according to embodiment 96, wherein the dilution buffer has a pH of approximately 7.2. (Pattern 98) The method according to any one of embodiments 94 to 97, wherein the biological material is diluted in the dilution buffer to a ratio of 1:1 to 1:10 (v / v). (Pattern 99) The method according to any one of embodiments 94 to 98, wherein the dilution buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (Aspect 100) The method according to any one of embodiments 94 to 99, wherein the dilution buffer contains about 5 to about 100 mM Tris-HCl. (Aspect 101) The method according to embodiment 100, wherein the dilution buffer contains about 20 mM Tris-HCl. (Aspect 102) The method according to any one of embodiments 94 to 101, wherein the dilution buffer contains approximately 300 mM or less of NaCl. (Aspect 103) The method according to embodiment 102, wherein the dilution buffer does not contain salt or contains about 200 mM NaCl. (Aspect 104) The method according to any one of embodiments 63 to 103, further comprising detecting the amount of IαIp in the flow-through of step (i). (Aspect 105) The method according to embodiment 104, comprising discarding the flow-through of step (i). (Aspect 106) The method according to any one of embodiments 63 to 105, further comprising detecting the amount of IαIp in the eluate of step (ii). (Aspect 107) The method according to any one of embodiments 63 to 106, including a flow rate of approximately 1 to 10 mL / min. (Aspect 108) The method according to any one of embodiments 1 to 107, wherein the IαIp recovered in the eluate of step (b) has a purity of about 5% to 99% or more by weight relative to the purity of the IαIp in the biological material. (Aspect 109) The method according to any one of embodiments 1 to 108, wherein the yield of IαIp in the eluate recovered in step (b) is greater than about 20% (w / w) of the IαIp present in the biological material. (Aspect 110) The method according to embodiment 109, wherein the yield is approximately 35% to approximately 90% (w / w) or more relative to the IαIp present in the biological material. (Aspect 111) The method according to embodiment 110, wherein the yield of the IαIp is approximately 95% (w / w) or more relative to the IαIp present in the biological material. (Aspect 112) The method according to any one of embodiments 109 to 111, wherein the yield of the IαIp from the biological material is at least about 5 μg / ml. (Aspect 113) The method according to embodiment 112, wherein the yield of IαIp from the biological material is at least about 50 μg / ml. (Aspect 114) The method according to embodiment 113, wherein the yield of IαIp from the biological material is at least about 100 μg / ml. (Aspect 115) The method according to embodiment 114, wherein the yield of IαIp from the biological material is at least about 300 μg / ml. (Aspect 116) The method according to embodiment 115, wherein the yield of IαIp from the biological material is at least about 600 μg / ml. (Aspect 117) The method according to embodiment 116, wherein the yield of IαIp from the biological material is at least about 900 μg / ml. (Aspect 118) The method according to any one of embodiments 108 to 117, wherein the purity of IαIp is at least about 5% (w / w). (Aspect 119) The method according to embodiment 118, wherein the purity of IαIp is at least about 25% (w / w). (Aspect 120) The method according to embodiment 119, wherein the purity of IαIp is at least about 50% (w / w). (Aspect 121) The method according to embodiment 120, wherein the purity of IαIp is at least about 75% (w / w). (Aspect 122) The method according to any one of embodiments 1 to 121, wherein the IαIp comprises two or more of the inter-alpha inhibitor (IαI), pre-alpha inhibitor (PαI), and bikunin. (Aspect 123) The method according to any one of embodiments 1 to 122, wherein 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. (Aspect 124) The method according to any one of embodiments 1 to 123, wherein the IαIp has an apparent molecular weight of about 60 to about 280 kDa. (Aspect 125) The method according to any one of embodiments 1 to 124, wherein the IαIp has biological activity. (Aspect 126) The method according to embodiment 125, wherein the biological activity includes cytokine inhibitory activity, chemokine inhibitory activity, or serine protease inhibitory activity. (Aspect 127) The method according to any one of embodiments 1 to 126, wherein the biological material is a blood product material. (Aspect 128) The method according to embodiment 127, wherein the blood product material is selected from the group consisting of whole plasma, cryopreserved plasma, liquid plasma, frozen plasma (FP), raw plasma, recovered plasma, solvent / surfactant treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, whole blood, and diluted or concentrated preparations thereof. (Aspect 129) The method according to embodiment 128, 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 preparations thereof. (Aspect 130) The method according to any one of embodiments 1 to 126, wherein the biological material is milk or colostrum. (Aspect 131) The method according to any one of embodiments 1 to 130, wherein the biological material is derived from a mammal. (Aspect 132) The method according to embodiment 131, wherein the mammal is a human, a primate, a cattle, a horse, a pig, a sheep, a cat, or a dog. (Aspect 133) The method according to any one of embodiments 1 to 59, wherein the biological material is substantially unprocessed before application to the endotoxin binder. (Aspect 134) The method according to any one of embodiments 1 to 133, further comprising performing one or more chromatography steps using the eluate recovered in step (b). (Aspect 135) The method according to embodiment 134, wherein the one or more additional chromatography steps include repeating the method according to any one of embodiments 1 to 134. (Aspect 136) The method according to any one of embodiments 1 to 135, wherein the elution buffer applied to the endotoxin binder in step (b) has a pH of 7.2 and contains about 20 mM Tris-HCl and about 500 mM NaCl. (Aspect 137) The method according to any one of embodiments 16 to 35, wherein the first washing buffer applied to the endotoxin binder has a pH of 5.2 and comprises about 75 mM glycine, about 100 mM acetic acid, and about 150 mM NaCl. (Aspect 138) The method according to any one of embodiments 26 to 35, wherein the second washing buffer applied to the endotoxin binder has a pH of 7.2 and contains about 20 mM Tris-HCl and about 300 mM NaCl. (Aspect 139) The method according to any one of embodiments 63 to 107, wherein the elution buffer applied to the chromatography support has a pH of 7.2 and contains about 20 mM Tris-HCl and about 750 mM NaCl. (Aspect 140) The method according to any one of embodiments 68 to 86, wherein the first washing buffer applied to the chromatography support has a pH of 7.2 and contains about 20 mM Tris-HCl and about 250 mM NaCl. (Aspect 141) The method according to any one of embodiments 78 to 86, wherein the second washing 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. (Aspect 142) A composition comprising IαIp prepared by the method described in any one of embodiments 1 to 141. (Aspect 143) The composition according to embodiment 142, wherein the composition is suitable for administration to humans. (Aspect 144) A pharmaceutical composition comprising the composition described in embodiment 142 or 143 and a pharmaceutically acceptable excipient. (Aspect 145) A method for treating a disease or condition in a subject requiring treatment, comprising administering to the subject a composition described in embodiment 142 or 143 or a pharmaceutical composition described in embodiment 144. (Aspect 146) A kit comprising the composition described in embodiment 142 or 143, or the pharmaceutical composition described in embodiment 144. (Aspect 147) The kit according to embodiment 146, wherein the kit further includes instructions for therapeutic use. (Aspect 148) A method for purifying IαIp from plasma, (a) Diluting the plasma with a dilution buffer containing deionized water to form diluted plasma, (b) Applying the diluted plasma to an ETOXICLEAR® resin and separating the flow-through containing the diluted plasma that does not bind to the ETOXICLEAR® resin, (c) Apply a first washing buffer with a pH of approximately 5.2 containing approximately 75 mM glycine, approximately 100 mM AcOH, and approximately 150 mM NaCl to the ETOXICLEAR® resin and separate the flow-through containing the first washing buffer. (d) Apply a second washing buffer with a pH of approximately 7.2 containing approximately 20 mM Tris-HCl and approximately 300 mM NaCl to the ETOXICLEAR® resin and separate the flow-through containing the second washing buffer. (e) A method comprising applying an elution buffer with a pH of 7.2 containing approximately 20 mM Tris-HCl and approximately 500 mM NaCl to the ETOXICLEAR® resin and recovering the eluate containing the IαIp. (Aspect 149) A method for purifying IαIp from plasma, (a) Diluting the plasma with a dilution buffer containing 15 mM phosphate at approximately pH 5.5 to form diluted plasma, (b) Applying the diluted plasma to a DETOXI-GEL® resin and separating the flow-through containing the diluted plasma that does not bind to the DETOXI-GEL® resin, (c) Apply a first washing buffer containing approximately 15 mM phosphate and approximately 50 mM NaCl at pH approximately 5.5 to the DETOXI-GEL® resin and separate the flow-through containing the first washing buffer, (d) Apply a second washing buffer containing approximately 15 mM phosphate and approximately 100 mM NaCl at pH approximately 5.5 to the DETOXI-GEL® resin and separate the flow-through containing the second washing buffer. (e) A method comprising applying an elution buffer containing approximately 15 mM phosphate and approximately 1,000 mM NaCl at pH approximately 5.5 to the DETOXI-GEL® resin and recovering the eluate containing the IαIp.

Claims

1. A method for purifying interalpha inhibitor protein (IαIp) from biological materials, (a) Applying the biological material containing the IαIp to an endotoxin binder, and separating the flow-through containing the biological material that does not bind to the endotoxin binder, (b) The method comprising applying an elution buffer containing the salt to the endotoxin binder and recovering the eluate containing the IαIp.

2. (i) The endotoxin binder is fixed to the support, (ii) The endotoxin binder is selected from the group consisting of ETOXICLEAR (trademark), PIERCE (trademark) high-capacity endotoxin removal resin, TOXINERASER (trademark) endotoxin removal resin, PURKINE (trademark) endotoxin removal resin, DETOXI-GEL (trademark) endotoxin removal gel, and PROMEGA (trademark) 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 contains 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 contains approximately 40 to approximately 65% ​​(w / w) albumin, (vi) The biological material contains approximately 25 to approximately 45% (w / w) globulin, (vii) The biological material contains approximately 2 to approximately 12% (w / w) of fibrinogen, (viiii) Further comprising applying the first washing buffer to the endotoxin binder after step (a) and before step (b), (ix) The elution buffer has a pH of about 4.5 to about 8.

5. (x) The elution buffer contains one or more of the following: glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (xi) Further comprising applying a dilution buffer to the biological material prior to step (a), (xi) Further comprising detecting the amount of IαIp in the flow-through, (xiiii) further comprising detecting the amount of IαIp in the eluate, and / or (xiv) The method according to claim 1, wherein each of steps (a) and (b) is performed using a flow rate of about 1 to 10 mL / min.

3. (i) The support is a monolithic support or a particle-based support, (ii) The support includes a column, membrane, disk, or chip, (iii) The endotoxin binder is DETOXI-GEL (trademark) or ETOXICLEAR (trademark), (iv) The globulin is immunoglobulin (Ig), (v) further comprising separating the flow-through containing the first wash buffer, (vi) The first washing buffer has a pH of about 4.5 to 8.

5. (vii) The first washing buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (viiii) After applying the first washing buffer, the second washing buffer is applied to the endotoxin binder, (ix) The elution buffer has a pH of approximately 7.

2. (x) The elution buffer contains about 5 to about 100 mM Tris-HCl, (xi) The elution buffer contains NaCl of approximately 1,000 mM or less. (xi) The dilution buffer contains deionized water, (xiiii) The dilution buffer has a pH of about 4.5 to about 8.

5. (xiv) The biological material is diluted with the dilution buffer to a ratio of 1:1 to 1:10 (v / v). (xv) The dilution buffer contains one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl, or (xvi) The method according to claim 2, comprising discarding the flow-through.

4. (i) The monolithic support or particle-based support is made of resin or contains resin, (ii) The immunoglobulin is selected from the group consisting of IgA, IgE, IgM, IgD, and IgG. (iii) The first washing buffer has a pH of about 5.

2. (iv) The first washing buffer comprises about 10 to about 200 mM glycine and / or about 20 to 300 mM acetic acid. (v) The first washing buffer contains NaCl of about 200 mM or less, (vi) further comprising separating the flow-through containing the second wash buffer, (vii) The second washing buffer has a pH of about 4.5 to about 8.

5. (viiii) The second washing buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (ix) The elution buffer contains approximately 20 mM Tris-HCl, (x) The elution buffer contains approximately 500 to approximately 1,000 mM NaCl, (xi) The dilution buffer has a pH of approximately 5.5, approximately 7.2, or approximately 7.

3. (xi) The dilution buffer contains about 5 to about 100 mM Tris-HCl, (xiii) The dilution buffer contains approximately 50 mM or less of NaCl, or (xiv) The method according to claim 3, wherein the dilution buffer contains about 5 to about 100 mM of phosphate.

5. (i) The IgG is selected from the group consisting of intravenous IgG (IVIg), anti-D IgG, hepatitis B IgG, measles IgG, rabies IgG, tetanus IgG, and varicella-zoster IgG. (ii) The first washing buffer contains about 75 mM glycine and about 100 mM acetic acid. (iii) The first washing buffer contains approximately 50 to approximately 150 mM NaCl, (iv) The second washing buffer has a pH of about 7.

2. (v) The second washing buffer contains about 5 to about 100 mM Tris-HCl, (vi) The second washing buffer contains NaCl of approximately 500 mM or less. (vii) The elution buffer contains approximately 500 mM NaCl or approximately 1,000 mM NaCl. (viiii) The dilution buffer contains approximately 20 mM Tris-HCl, (ix) The dilution buffer either does not contain salt or contains about 50 mM NaCl, (x) The method according to claim 4, wherein the dilution buffer contains about 15 mM phosphate.

6. (i) The first washing buffer contains about 50 mM NaCl or about 100 mM NaCl, (ii) The second washing buffer contains approximately 20 mM Tris-HCl, (iii) The second washing buffer contains approximately 100 to approximately 500 mM NaCl, (iv) The elution buffer applied to the endotoxin binder in step (b) has a pH of 7.2 and contains about 20 mM Tris-HCl and about 500 mM NaCl, or (v) The method according to claim 5, wherein the first washing buffer applied to the endotoxin binder 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 washing buffer contains about 300 mM NaCl, and / or (ii) The method according to claim 6, wherein the second washing buffer applied to the endotoxin binder has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 300 mM NaCl.

8. (i) The process includes applying the biological material to a chromatographic support prior to step (a), or (ii) The method according to any one of claims 1 to 7, wherein the biological material is unprocessed before application to the endotoxin binder.

9. (i) The chromatography support includes 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) which includes the biological material that does not bind to the chromatographic support, (B) Further comprising applying an elution buffer containing the salt to the chromatographic support and recovering the first eluate containing the IαIp, or (iii) The method according to claim 8, wherein the chromatography support comprises a column, a membrane, a disk, or a chip.

10. (i) The chromatography support is the anion exchange chromatography support, (ii) The chromatography support is a monolithic support or a particle-based support. (iii) Further comprising applying the first washing buffer to the chromatography support after step (A) and before step (B), (iv) The elution buffer applied to the chromatography support has a pH of about 4.5 to about 8.

5. (v) The elution buffer applied to the chromatography support comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (vi) Further comprising applying a dilution buffer to the biological material prior to step (A), (vii) Further comprising detecting the amount of IαIp in the flow-through of step (A), (viiii) Further comprising detecting the amount of IαIp in the eluate of step (B), or The method according to claim 9, wherein each of steps (A) and (B) is performed using a flow rate of about 1 to 10 mL / min.

11. (i) The monolithic support or particle-based support comprises an immobilized anion exchange resin, (ii) Further comprising separating the flow-through containing the first wash buffer before step (B), (iii) The first washing buffer applied to the chromatography support has a pH of about 4.5 to about 8.

5. (iv) The first washing buffer applied to the chromatography support comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (v) After applying the first wash buffer, the second wash buffer is applied to the chromatography support, (vi) The elution buffer applied to the chromatography support has a pH of about 7.

2. (vii) The elution buffer applied to the chromatography support contains about 5 to about 100 mM Tris-HCl, (viiii) The elution buffer applied to the chromatography support contains NaCl of about 1,000 mM or less, (ix) The dilution buffer contains deionized water, (x) The dilution buffer has a pH of about 4.5 to about 8.

5. (xi) The biological material is diluted with the dilution buffer to a ratio of 1:1 to 1:10 (v / v), (xi) The dilution buffer contains one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl, or (xiiii) The method according to claim 10, comprising discarding the flow-through of step (A).

12. (i) The fixed anion exchange resin is a diethylaminoethane (DEAE) resin or a quaternary amine (Q) resin. (ii) The first washing buffer applied to the chromatography support has a pH of about 7.

2. (iii) The first washing buffer applied to the chromatography support contains about 5 to about 100 mM Tris-HCl, (iv) The first washing buffer applied to the chromatography support contains NaCl of about 400 mM or less, (v) further comprising separating the flow-through containing the second wash buffer, (vi) The second washing buffer applied to the chromatography support has a pH of about 4.5 to about 8.

5. (vii) The second washing buffer applied to the chromatography support comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. (viiii) The elution buffer applied to the chromatography support contains about 20 mM Tris-HCl, (ix) The elution buffer applied to the chromatography support contains approximately 750 mM NaCl, (x) The dilution buffer has a pH of about 7.

2. (xi) The dilution buffer contains about 5 to about 100 mM Tris-HCl, or (xi) The method according to claim 11, wherein the dilution buffer contains NaCl in a concentration of about 300 mM or less.

13. (i) The first washing buffer applied to the chromatography support contains about 20 mM Tris-HCl, (ii) The first washing buffer applied to the chromatography support contains about 50 to about 250 mM NaCl, (iii) The second washing buffer applied to the chromatography support has a pH of about 5.

2. (iv) The second washing buffer applied to the chromatography support comprises about 10 to about 200 mM glycine and / or about 20 to about 300 mM acetic acid. (v) The second washing buffer applied to the chromatography support contains about 100 to about 500 mM NaCl, (vi) The dilution buffer contains approximately 20 mM Tris-HCl, (vii) The dilution buffer does not contain salt or contains about 200 mM NaCl, (viiii) The method according to claim 12, wherein the elution buffer applied to the chromatography support has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 750 mM NaCl.

14. (i) The first washing buffer applied to the chromatography support contains about 250 mM NaCl, (ii) The second washing buffer applied to the chromatography support comprises about 50 mM glycine and about 100 mM acetic acid. (iii) The second washing buffer applied to the chromatography support contains approximately 175 mM NaCl, (vi) The first washing buffer applied to the chromatography support has a pH of 7.2 and contains about 20 mM Tris-HCl and about 250 mM NaCl, or (v) The method according to claim 13, wherein the second washing buffer applied to the chromatography 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% or more by weight 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 approximately 20% (w / w) of the IαIp present in the biological material. (iii) The IαIp comprises two or more of the interalpha inhibitor (IαI), prealpha inhibitor (PαI), and bicinine. (iv) The IαIp present in the biological material comprises 60% to 80% (w / w) IαI 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) IαI and / or 20% to 40% (w / w) PαI, (v) The 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. (viiii) The biological material is milk or colostrum, (ix) The biological material is of mammalian origin, or The method according to any one of claims 1 to 14, further comprising (x) performing one or more chromatography steps using the eluate recovered in step (b).

16. (i) The yield is approximately 35% to approximately 90% (w / w) or more relative to the IαIp present in the biological material. (ii) The yield of IαIp from the 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 includes cytokine inhibitory activity, chemokine inhibitory activity, or serine protease inhibitory activity, (v) The blood product material is selected from the group consisting of whole plasma, decryoplasma, liquid plasma, frozen plasma (FP), raw plasma, recovered plasma, solvent / surfactant treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, whole blood, and diluted or concentrated preparations thereof. (vi) The mammal is a human, primate, cattle, horse, pig, sheep, cattle, or dog, (vii) The method of claim 15, wherein the one or more additional chromatography steps include repeating the method of any one of claims 1 to 14.

17. (i) The yield of IαIp is approximately 95% (w / w) or more relative to the IαIp present in the biological material. (ii) The yield of IαIp from the biological material is at least about 50 μg / ml. (iii) The purity of IαIp is at least about 25% (w / w), or (iv) The method according to 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 preparations thereof.

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

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

20. A method for purifying IαIp from plasma, (a) Diluting the plasma with a dilution buffer containing deionized water to form diluted plasma. (b) Applying the diluted plasma to an ETOXICLEAR® resin, and separating the flow-through containing the diluted plasma that does not bind to the ETOXICLEAR® resin. (c) Apply a first washing buffer with a pH of approximately 5.2 containing approximately 75 mM glycine, approximately 100 mM AcOH, and approximately 150 mM NaCl to the ETOXICLEAR® resin, and separate the flow-through containing the first washing buffer. (d) Applying a second washing buffer with a pH of approximately 7.2 containing approximately 20 mM Tris-HCl and approximately 300 mM NaCl to the ETOXICLEAR® resin, and separating the flow-through containing the second washing buffer, (e) The method comprising applying a pH 7.2 elution buffer containing about 20 mM Tris-HCl and about 500 mM NaCl to the ETOXICLEAR® resin, and recovering the eluate containing IαIp.

21. A method for purifying IαIp from plasma, (a) Dilute the plasma with a pH of approximately 5.5 dilution buffer containing 15 mM phosphate to form diluted plasma. (b) Applying the diluted plasma to a DETOXI-GEL® resin, and separating the flow-through containing the diluted plasma that does not bind to the DETOXI-GEL® resin. (c) Apply a first washing buffer with a pH of approximately 5.5 containing approximately 15 mM phosphate and approximately 50 mM NaCl to the DETOXI-GEL™ resin, and separate the flow-through containing the first washing buffer. (d) Applying a second washing buffer with a pH of approximately 5.5 containing approximately 15 mM phosphate and approximately 100 mM NaCl to the DETOXI-GEL™ resin, and separating the flow-through containing the second washing buffer, (e) The method comprising applying an elution buffer with a pH of approximately 5.5 containing approximately 15 mM phosphate and approximately 1,000 mM NaCl to the DETOXI-GEL™ resin, and recovering the eluate containing the IαIp.

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