METHODS FOR PURIFYING INTER-ALPHA INHIBITOR PROTEINS
20230416342 ยท 2023-12-28
Inventors
Cpc classification
International classification
Abstract
Described herein are methods for purifying IIp from a biological material using an endotoxin-binding agent. The method involves applying the biological material containing the IIp to an endotoxin-binding agent, discarding the flow through, applying a wash buffer(s), and eluting the IIp from the endotoxin-binding agent.
Claims
1. A method of purifying an inter-alpha inhibitor protein (IIp) from a biological material comprising: (a) applying the biological material comprising the IIp to an endotoxin-binding agent and separating a flow through comprising the biological material that does not bind to the endotoxin-binding agent; and (b) applying an elution buffer comprising a salt to the endotoxin-binding agent and collecting an eluate comprising the IIp.
2. The method of claim 1, wherein the endotoxin-binding agent is immobilized on a support.
3. The method of claim 2, wherein the support is a monolithic support or a particle-based support.
4. The method of claim 3, wherein the monolithic support or particle-based support is or comprises a resin.
5. The method of any one of claims 2-4, wherein the support comprises a column, membrane, disc, or chip.
6. The method of any one of claims 1-5, wherein the endotoxin-binding agent is selected from the group consisting of ETOXICLEAR, PIERCE High Capacity Endotoxin Removal Resin, TOXINERASER Endotoxin Removal Resin, PURKINE Endotoxin Removal Resin, DETOXI-GEL Endotoxin Removing Gel, and PROMEGA Endotoxin Removal Resin.
7. The method of claim 6, wherein the endotoxin-binding agent is DETOXI-GEL or ETOXICLEAR.
8. The method of any one of claims 1-7, wherein the biological material further comprises three or more proteins selected from the group consisting of alpha-1 antitrypsin, C1-inhibitor, albumin, a globulin, fibrinogen (factor I), prothrombin (factor II), thrombin, anti-thrombin 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.
9. The method of claim 8, wherein the globulin is an immunoglobulin (Ig).
10. The method of claim 9, wherein the immunoglobulin is selected from the group consisting of IgA, IgE, IgM, IgD, and IgG.
11. The method of claim 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.
12. The method of any one of claims 1-11, wherein the biological material comprises three to ten three to fifteen, three to twenty, three to twenty five, three to thirty, ten to twenty, ten to twenty five, ten to thirty, fifteen to twenty five, fifteen to thirty, twenty to thirty, or thirty or more different proteins.
13. The method of any one of claims 1-12, wherein the biological material comprises about 40 to about 65% albumin (w/w).
14. The method of any one of claims 1-13, wherein the biological material comprises about 25 to about 45% globulins (w/w).
15. The method of any one of claims 1-14, wherein the biological material comprises about 2 to about 12% fibrinogen (w/w).
16. The method of any one of claims 1-15, wherein the method further comprises applying a first wash buffer to the endotoxin-binding agent after step (a) and prior to step (b).
17. The method of claim 16, wherein the method further comprises separating a flow through comprising the first wash buffer.
18. The method of claim 16 or 17, wherein the first wash buffer has a pH of about 4.5 to 8.5.
19. The method of claim 18, wherein the first wash buffer has a pH of about 5.2.
20. The method of any one of claims 16-19, wherein the first wash buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl.
21. The method of any one of claims 16-20, wherein the first wash buffer comprises about 10 to about 200 mM glycine and/or about 20 to 300 mM acetic acid.
22. The method of claim 21, wherein the first wash buffer comprises about 75 mM glycine and about 100 mM acetic acid.
23. The method of any one of claims 16-22, wherein the first wash buffer comprises about 200 mM or less NaCl.
24. The method of claim 23, wherein the first wash buffer comprises about 50 to about 150 mM NaCl.
25. The method of claim 24, wherein the first wash buffer comprises about 50 mM NaCl or about 100 mM NaCl.
26. The method of any one of claims 16-25, wherein the method further comprises applying a second wash buffer to the endotoxin-binding agent after applying the first wash buffer.
27. The method of claim 26, wherein the method further comprises separating a flow through comprising the second wash buffer.
28. The method of claim 26 or 27, wherein the second wash buffer has a pH of about 4.5 to about 8.5.
29. The method of claim 28, wherein the second wash buffer has a pH of about 7.2.
30. The method of any one of claims 26-29, wherein the second wash buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl.
31. The method of any one of claims 26-30, wherein the second wash buffer comprises about 5 to about 100 mM Tris-HCl.
32. The method of claim 31, wherein the second wash buffer comprises about 20 mM Tris-HCl.
33. The method of any one of claims 26-32, wherein the second wash buffer comprises about 500 mM NaCl or less.
34. The method of claim 33, wherein the second wash buffer comprises about 100 to about 500 mM NaCl.
35. The method of claim 34, wherein the second wash buffer comprises about 300 mM NaCl.
36. The method of any one of claims 1-35, wherein the elution buffer has a pH of about 4.5 to about 8.5.
37. The method of claim 36, wherein the elution buffer has a pH of about 7.2.
38. The method of any one of claims 1-37, wherein the elution buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl.
39. The method of any one of claims 1-38, wherein the elution buffer comprises about 5 to about 100 mM Tris-HCl.
40. The method of claim 39, wherein the elution buffer comprises about 20 mM Tris-HCl.
41. The method of any one of claims 1-40, wherein the elution buffer comprises about 1,000 mM NaCl or less.
42. The method of any one of claims 1-41, wherein the elution buffer comprises about 500 to about 1,000 mM NaCl.
43. The method of claim 41 or 42, wherein the elution buffer comprises about 500 mM NaCl or about 1,000 mM NaCl.
44. The method of any one of claims 1-43, the method further comprises applying a dilution buffer to the biological material prior to step (a).
45. The method of claim 44, wherein the dilution buffer comprises deionized water.
46. The method of claim 44 or 45, wherein the dilution buffer has a pH of about 4.5 to about 8.5.
47. The method of claim 46, wherein the dilution buffer has a pH of about 5.5, about 7.2, or about 7.3.
48. The method of any one of claims 44-47, wherein the biological material is diluted 1:1 to 1:10 (v/v) with the dilution buffer.
49. The method of any one of claims 44-48, wherein the dilution buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl.
50. The method of any one of claims 44-49, wherein the dilution buffer comprises about 5 to about 100 mM Tris-HCl.
51. The method of claim 50, wherein the dilution buffer comprises about 20 mM Tris-HCl.
52. The method of any one of claims 44-51, wherein the dilution buffer comprises about 50 mM NaCl or less.
53. The method of claim 52, wherein the dilution buffer comprises no salt or about 50 mM NaCl.
54. The method of any one of claims 44-53, wherein the dilution buffer comprises about 5 to about 100 mM phosphate.
55. The method of claim 54, wherein the dilution buffer comprises about 15 mM phosphate.
56. The method of any one of claims 1-55, wherein the method further comprises detecting an amount of the IIp in the flow through.
57. The method of claim 56, wherein the method comprises discarding the flow through.
58. The method of any one of claims 1-57, wherein the method further comprises detecting an amount of IIp in the eluate.
59. The method of any one of claims 1-58, the method comprises a flow rate of about 1 to 10 mL/minute.
60. The method of any one of claims 1-58, wherein, prior to step (a), the method comprises applying the biological material to a chromatography support.
61. The method of claim 60, wherein the chromatography support comprises an anion-exchange chromatography support, a size-exclusion chromatography support, an ion-exchange chromatography support, an affinity chromatography support, or a combination thereof.
62. The method of claim 61, wherein the chromatography support is said anion-exchange chromatography support.
63. The method of any one of claims 60-62, wherein the method further comprises: (i) applying the biological material to the chromatography support and separating a flow through of step (i) comprising the biological material that does not bind to the chromatography support; and (ii) applying an elution buffer comprising a salt to the chromatography support and collecting a first eluate comprising the IIp.
64. The method of claim 63, wherein the chromatography support is a monolithic support or a particle-based support.
65. The method of claim 64, wherein the monolithic support or particle-based support comprises an immobilized anion-exchange resin.
66. The method of claim 65, wherein the immobilized anion-exchange resin is diethylaminoethane (DEAE) resin or a quaternary amine (Q) resin.
67. The method of any one of claims 60-66, wherein the chromatography support is a column, membrane, disc, or chip.
68. The method of any one of claims 60-67, wherein the method further comprises applying a first wash buffer to the chromatography support after step (i) and prior to step (ii).
69. The method of claim 68, wherein, prior to step (ii), the method further comprises separating a flow through comprising the first wash buffer.
70. The method of claim 68 or 69, wherein the first wash buffer applied to the chromatography support has a pH of about 4.5 to about 8.5.
71. The method of claim 70, wherein the first wash buffer applied to the chromatography support has a pH of about 7.2.
72. The method of claim any one of claims 68-71, wherein the first 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.
73. The method of any one of claims 68-72, wherein the first wash buffer applied to the chromatography support comprises about 5 to about 100 mM Tris-HCl.
74. The method of claim 73, wherein the first wash buffer applied to the chromatography support comprises about 20 mM Tris-HCl.
75. The method of any one of claims 68-74, wherein the first wash buffer applied to the chromatography support comprises about 400 mM or less NaCl.
76. The method of claim 75, wherein the first wash buffer applied to the chromatography support comprises about 50 to about 250 mM NaCl.
77. The method of claim 76, wherein the first wash buffer applied to the chromatography support comprises about 250 mM NaCl.
78. The method of any one of claims 68-77, wherein the method further comprises applying a second wash buffer to the chromatography support after applying the first wash buffer.
79. The method of claim 78, wherein the method further comprises separating a flow through comprising the second wash buffer.
80. The method of claim 78 or 79, wherein the second wash buffer applied to the chromatography support has a pH of about 4.5 to about 8.5.
81. The method of claim 80, wherein the second wash buffer applied to the chromatography support has a pH of about 5.2.
82. The method of any one of claims 78-81, wherein the second 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.
83. The method of any one of claims 78-82, wherein the second wash buffer applied to the chromatography support comprises about 10 to about 200 mM glycine and/or about 20 to about 300 mM acetic acid.
84. The method of claim 83, wherein the second wash buffer applied to the chromatography support comprises about 50 mM glycine and about 100 mM acetic acid.
85. The method of any one of claims 78-84, wherein the second wash buffer applied to the chromatography support comprises about 100 to about 500 mM NaCl.
86. The method of claim 85, wherein the second wash buffer applied to the chromatography support comprises about 175 mM NaCl.
87. The method of any one of claims 63-86, wherein the elution buffer applied to the chromatography support has a pH of about 4.5 to about 8.5.
88. The method of claim 87, wherein the elution buffer applied to the chromatography support has a pH of about 7.2.
89. The method of any one of claims 63-88, 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.
90. The method of any one of claims 63-89, wherein the elution buffer applied to the chromatography support comprises about 5 to about 100 mM Tris-HCl.
91. The method of claim 90, wherein the elution buffer applied to the chromatography support comprises about 20 mM Tris-HCl.
92. The method of any one of claims 63-91, wherein the elution buffer applied to the chromatography support comprises about 1,000 or less mM NaCl.
93. The method of claim 92, wherein the elution buffer applied to the chromatography support comprises about 750 mM NaCl.
94. The method of any one of claims 63-93, the method further comprises applying a dilution buffer to the biological material prior to step (i).
95. The method of claim 94, wherein the dilution buffer comprises deionized water.
96. The method of claim 94 or 95, wherein the dilution buffer has a pH of about 4.5 to about 8.5.
97. The method of claim 96, wherein the dilution buffer has a pH of about 7.2.
98. The method of any one of claims 94-97, wherein the biological material is diluted 1:1 to 1:10 (v/v) with the dilution buffer.
99. The method of any one of claims 94-98, wherein the dilution buffer comprises one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl.
100. The method of any one of claims 94-99, wherein the dilution buffer comprises about 5 to about 100 mM Tris-HCl.
101. The method of claim 100, wherein the dilution buffer comprises about 20 mM Tris-HCl.
102. The method of any one of claims 94-101, wherein the dilution buffer comprises about 300 mM NaCl or less.
103. The method of claim 102, wherein the dilution buffer comprises no salt or about 200 mM NaCl.
104. The method of any one of claims 63-103, wherein the method further comprises detecting an amount of IIp in the flow through of step (i).
105. The method of claim 104, wherein the method comprises discarding the flow through of step (i).
106. The method of any one of claims 63-105, the method further comprises detecting an amount of IIp in the eluate in step (ii).
107. The method of any one of claims 63-106, the method comprises a flow rate of about 1 to 10 mL/minute.
108. The method of any one of claims 1-107, wherein the IIp collected in the eluate of step (b) has a purity of about 5% to 99% or greater by weight relative to the purity of the IIp in the biological material.
109. The method of any one of claims 1-108, wherein the yield of IIp in the eluate collected in step (b) is greater than about 20% (w/w) relative to the IIp present in the biological material.
110. The method of claim 109, wherein the yield is about 35% to about 90% or greater (w/w) relative to the IIp present in the biological material.
111. The method of claim 110, wherein the yield IIp is about 95% or greater by (w/w) relative to the IIp present in the biological material.
112. The method of any one of claims 109-111, wherein the yield of IIp from the biological material is at least about 5 g/ml.
113. The method of claim 112, wherein the yield of IIp from the biological material is at least about 50 g/ml.
114. The method of claim 113, wherein the yield of IIp from the biological material is at least about 100 g/ml.
115. The method of claim 114, wherein the yield of IIp from the biological material is at least about 300 g/ml.
116. The method of claim 115, wherein the yield of IIp from the biological material is at least about 600 g/ml.
117. The method of claim 116, wherein the yield of IIp from the biological material is at least about 900 g/ml.
118. The method of any one of claims 108-117, wherein the purity of the IIp is at least about 5% (w/w).
119. The method of claim 118, wherein the purity of the IIp is at least about 25% (w/w).
120. The method of claim 119, wherein the purity of the IIp is at least about 50% (w/w).
121. The method of claim 120, wherein the purity of the IIp is at least about 75% (w/w).
122. The method of any one of claims 1-121, wherein the IIp comprises two or more of inter-alpha inhibitor (II), pre-alpha inhibitor (PI), and bikunin.
123. The method of any one of claims 1-122, wherein the IIp present in the biological material comprises between 60% to 80% (w/w) IIp and/or between 20% to 40% (w/w) PI; and/or wherein the IIp present in the eluate of step (b) comprises between 60% to 80% (w/w) IIp and/or between 20% to 40% (w/w) PI.
124. The method of any one of claims 1-123, wherein the IIp has an apparent molecular weight of between about 60 to about 280 kDa.
125. The method of any one of claims 1-124, wherein the IIp has biological activity.
126. The method of claim 125, wherein the biological activity comprises cytokine inhibitor activity, chemokine inhibitor activity, or serine protease inhibitor activity.
127. The method of any one of claims 1-126, wherein the biological material is a blood product material.
128. The method of claim 127, wherein the blood product material is selected from the group consisting of whole plasma, cryo-poor plasma, liquid plasma, frozen plasma (FP), source plasma, recovered plasma, solvent/detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, whole blood, and a diluted or concentrated preparation thereof.
129. The method of claim 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 a diluted or concentrated preparation thereof.
130. The method of any one of claims 1-126, wherein the biological material is milk or colostrum.
131. The method of any one of claims 1-130, wherein the biological material is from a mammal.
132. The method of claim 131, wherein the mammal is a human, primate, bovine, equine, porcine, ovine, feline, or canine.
133. The method of any one of claims 1-59, wherein the biological material is substantially unprocessed prior to application to the endotoxin-binding agent.
134. The method of any one of claims 1-133, wherein the method further comprises performing one or more chromatography steps using the eluate collected in step (b).
135. The method of claim 134, wherein the one or more additional chromatography steps comprises repeating the method of any one of claims 1-134.
136. The method of any one of claims 1-135, wherein the elution buffer applied to the endotoxin-binding agent in step (b) has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 500 mM NaCl.
137. The method of any one of claims 16-35 wherein the first wash buffer applied to the endotoxin-binding agent has a pH of 5.2 and comprises about 75 mM glycine, about 100 mM acetic acid, and about 150 mM NaCl.
138. The method of any one of claims 26-35, wherein the second wash buffer applied to the endotoxin-binding agent has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 300 mM NaCl.
139. The method of any one of claims 63-107, 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.
140. The method of any one of claims 68-86, wherein the first wash buffer applied to the chromatography support has a pH of 7.2 and comprises about 20 mM Tris-HCl and about 250 mM NaCl.
141. The method of any one of claims 78-86, wherein the second wash 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.
142. A composition comprising the IIps produced by the method of any one of claims 1-141.
143. The composition of claim 142, wherein the composition is suitable for administration to a human.
144. A pharmaceutical composition comprising the composition of claim 142 or 143 and a pharmaceutically acceptable excipient.
145. A method of treating a disease or condition in a subject in need thereof comprising administering to the subject the composition of claim 142 or 143 or the pharmaceutical composition of claim 144.
146. A kit comprising the composition of claim 142 or 143 or the pharmaceutical composition of claim 144.
147. The kit of claim 146, wherein the kit further comprises instructions for therapeutic use.
148. A method of purifying an IIp from plasma comprising: (a) diluting the plasma with a dilution buffer comprising deionized water to form diluted plasma; (b) applying the diluted plasma to an ETOXICLEAR resin and separating a flow through comprising the diluted plasma that does not bind to the ETOXICLEAR resin; (c) applying a first wash buffer comprising about 75 mM glycine, about 100 mM AcOH, and about 150 mM NaCl at a pH of about 5.2 to the ETOXICLEAR resin and separating a flow through comprising the first wash buffer; (d) applying a second wash buffer comprising about 20 mM Tris-HCl and about 300 mM NaCl at a pH of about 7.2 to the ETOXICLEAR resin and separating a flow through comprising the second wash buffer; and (e) applying an elution buffer comprising about 20 mM Tris-HCl and about 500 mM NaCl at a pH of 7.2 to the ETOXICLEAR resin and collecting an eluate comprising the IIp.
149. A method of purifying an IIp from plasma comprising: (a) diluting the plasma with a dilution buffer comprising 15 mM phosphate at a pH of about 5.5 to the plasma to form diluted plasma; (b) applying the diluted plasma to a DETOXI-GEL resin and separating a flow through comprising the diluted plasma that does not bind to the DETOXI-GEL resin; (c) applying a first wash buffer comprising about 15 mM phosphate and about 50 mM NaCl at a pH of about 5.5 to the DETOXI-GEL resin and separating a flow through comprising the first wash buffer; (d) applying a second wash buffer comprising about 15 mM phosphate and about 100 mM NaCl at a pH of about 5.5 to the DETOXI-GEL resin and separating a flow through comprising the second wash buffer; and (e) applying an elution buffer comprising about 15 mM phosphate and about 1,000 mM NaCl at a pH of about 5.5 to the DETOXI-GEL resin and collecting an eluate comprising the IIp.
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION
[0096] The disclosure features methods of purifying an IIp (e.g., II, PI, a heavy chain (e.g., H1, H2, H3, H4, and/or H5), a light chain (e.g., bikunin), or a combination thereof) from a biological material (e.g., blood) by using an endotoxin-binding agent. The methods involve applying a biological material (e.g., a processed or substantially unprocessed biological material, such as blood or milk) containing IIp(s) to an endotoxin-binding agent. We discovered that endotoxin-binding agents, such as endotoxin-specific chromatography resins (e.g., ETOXICLEAR and DETOXI-GEL), bind IaIps, which was not expected. Surprisingly, an endotoxin-binding agent, when used in the process of purifying an IIp, reduces the loss of IIp during purification process, thereby retaining or improving the yield of recovered IIp, relative to other methods. In addition, the use of an endotoxin-binding agent as part of the IIp purification process also maintains or increases the purity of the recovered IIp.
[0097] Thus, we have used such endotoxin-binding agents to purify IaIps, for example, from a biological source, such as blood. Moreover, a yield and purity of the IaIps of up to 50% or more (e.g., up to 90% or more) could be obtained when using an endotoxin-binding agent as part of the purification process. This discovery can be used to simplify the purification process and concurrently increase the yield and purity of IIps when employing the methods of the disclosure.
[0098] Also featured are pharmaceutical compositions prepared using the purified IIps obtained by the methods described herein and methods for treating and/or reducing the likelihood of developing a disease or condition in a subject in need thereof by administering a pharmaceutical composition prepared using the purified IIps obtained by the methods described herein.
Methods of Purification
[0099] An endotoxin-binding agent can be used to purify an IIp (e.g., II, PI, a heavy chain (e.g., H1, H2, H3, H4, and/or H5), a light chain (e.g., bikunin), or a combination thereof) from a biological material (e.g., blood and milk). The methods described below can be used to separate IIp from other components present in the biological material. The methods can be used to prepare IIps with a purity ranging from about 5% to about 99% or greater (e.g., 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, greater than 95%, such as 97% or 99%, or greater than 99%). In addition, the methods can be used to produce a yield of purified IIp of about 20% (w/w) or greater relative to the amount present in the original biological material. For example, the methods can be used to produce a yield of purified IIp of about 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 65%, 70%, 75%, 80%, 85%, or 90% (w/w) or greater.
[0100] Biological Material
[0101] The methods described below can be used to purify IIp from a biological material. The biological material containing IIp can be obtained from a human, primate, bovine, equine, porcine, ovine, feline, canine, or combinations thereof. The biological material can be, e.g., blood, milk (e.g., colostrum), urine, sputum, and cerebrospinal fluid. For example, the biological material can be, but is not limited to, whole plasma, cryo-poor plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, source plasma, recovered plasma, solvent/detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, blood (e.g., whole blood), and a diluted or concentrated preparation thereof. The biological material containing IIp can be, but is not limited to, a plasma fraction intermediate. The plasma fraction intermediate is produced through one or more process steps (e.g. filtration, centrifugation, sedimentation, chromatography, adsorption, isolation, freezing, thawing, dilution, concentration, S/D treatment, etc) from whole plasma, cryo-poor plasma, liquid plasma, fresh frozen plasma (FFP), FFP24, frozen plasma (FP), FP24, thawed FFP, thawed FFP24, thawed FP, thawed FP24, source plasma, recovered plasma, solvent/detergent-treated plasma (SDP), platelet-rich plasma (PRP), platelet-poor plasma (PPP), serum, blood (e.g., whole blood), and a diluted or concentrated preparation thereof.
[0102] The biological material may also be an extract prepared using cells expressing IIp or may be or contain cells that secrete IIp, e.g., recombinant cells that have been modified to express IIp.
[0103] The biological material may contain, in addition to IIp, a mixture of proteins, such as three or more proteins found in the blood or three or more proteins found in milk (e.g., colostrum). For instance, the biological material may contain alpha-1 antitrypsin, C1-inhibitor, albumin, a globulin (such as an 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, anti-thrombin 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. The biological material may be milk (e.g., colostrum), which may contain one or more 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 a globulin (e.g., an immunoglobulin, such as 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).
[0104] The biological material contacted or applied to an endotoxin-binding agent (or to a different support described herein, such as an anion-exchange chromatography support), according to the methods described below, can be substantially unprocessed (e.g., original source material) or the biological material can be processed prior to being contacted or applied to the endotoxin-binding agent, for example, by using one or more sample preparation methods or other known purification methods, such as those described herein.
[0105] A substantially unprocessed biological material is one that has been minimally modified, if at all, relative to the original source material (e.g., blood or another source, as described herein), such that the biological material maintains the original characteristics of the source material. For example, a substantially unprocessed biological material may be subjected to a sample preparation or purification step(s) that removes less than 10% (w/w) (e.g., less than 0.1%-10% (w/w), such as less than 0.1%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, or 9% (w/w)) of one or more substances from the material before the material is contacted or applied to an endotoxin-binding agent. Alternatively, a substantially unprocessed biological material can be an eluate or a fraction from a prior purification step(s), in which the prior purification step(s) removes less than 10% of impurities from the material.
[0106] The biological material may also be subjected to one or more processing steps, such as those described herein, prior to application of the biological material to an endotoxin-binding agent (or to a different support described herein, such as an anion-exchange chromatography support). For example, one or more proteins found in the biological material (e.g., blood or milk), such as, e.g., alpha-1 antitrypsin, C1-inhibitor, albumin, a globulin (such as an 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, anti-thrombin 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 can be partially removed (e.g., 1%-70% (w/w) removed) or substantially removed (e.g., greater than 70%-100% (w/w) removed) from the biological material prior to a purification step using an endotoxin-binding agent.
[0107] Methods of the disclosure can be used to prepare IaIps (e.g., II, PI, a heavy chain (e.g., H1, H2, H3, H4, and/or H5), a light chain (e.g., bikunin), or a combination thereof) from a biological material containing three to ten (or more) of the aforementioned blood proteins. The disclosed methods can also be used to separate IaIps from a biological material containing three to fifteen, three to twenty, three to twenty five, three to thirty, ten to twenty, ten to twenty five, ten to thirty, fifteen to twenty five, fifteen to thirty, twenty to thirty, or thirty or more different proteins (e.g., blood or milk proteins). The biological material applied to an endotoxin-binding agent may also contain, in addition to IIp, about 40-65% (e.g., about 55%) albumin by total weight of protein in the biological material, about 25-45% (e.g., about 38%) globulins by total weight of protein, about 2-12% (e.g., about 7%) fibrinogen by total weight of protein, or any combination thereof, by total weight of protein.
[0108] Endotoxin-Binding Agent
[0109] The methods described herein involve the use of an endotoxin-binding agent, which can be used to bind to IIp in a mixture of proteins (e.g., IIp present in a biological material, such as milk or blood). An endotoxin-binding agent is a molecule that may be known to bind to an endotoxin, such as a lipopolysaccharide. The endotoxin-binding agent may be one that specifically binds to an endotoxin. The endotoxin-binding agent may be, for example, incorporated into or immobilized on a support. The support may be a monolithic support or a particle-based support. The particle can be, for example, a resin. The endotoxin-binding agent can be packed or immobilized on any number of known supports, e.g., a column, membrane, disc, or chip. The endotoxin-binding agent can be a molecule, e.g., polymyxin B, polylysine or a derivative thereof, or a synthetic mimetic peptide.
[0110] Non-limiting examples of the endotoxin-binding agent that can be used in the methods described herein include, e.g., ETOXICLEAR, PIERCE High Capacity Endotoxin Removal Resin, TOXINERASER Endotoxin Removal Resin, PURKINE Endotoxin Removal Resin, DETOXI-GEL Endotoxin Removal Gel, or Promega Endotoxin Removal Resin. The methods can be performed using, e.g., a pre-packed column or cartridge containing the endotoxin-binding agent (e.g., a column having a volume of about 0.1 mL to about 100 mL, or a column having a larger volume).
[0111] A column containing an endotoxin-binding agent can be prepared, e.g., by applying a slurry of endotoxin-binding agent suspended in buffer (e.g., deionized water) to a filter-fritted column (e.g., a column of about 2 to about 100 mL, or larger) and allowing the endotoxin-binding agent to settle for about 30 minutes. The settled resin can then be equilibrated with about 3 to about 5 column volumes of a suitable, pyrogen-free buffer or water (e.g., deionized water) before a biological material is applied.
[0112] In some examples, DETOXI-GEL Endotoxin Removal Gel is used as the source of the endotoxin-binding agent. DETOXI-GEL uses immobilized polymyxin B to bind lipid A domains of endotoxins. In another example, ETOXICLEAR is used as the endotoxin-binding agent.
[0113] Reagents for Use in the Purification Methods
[0114] Dilution Buffer
[0115] A biological material containing IIp (e.g., II, PI) may be combined with a dilution buffer prior to a purification step, such as prior to contacting or applying the biological material to a medium, such as an endotoxin-binding agent. The dilution buffer can be added to lower a salt (e.g., NaCl) concentration of the biological material, e.g., to avoid or reduce the possibility of early elution of IIp off the endotoxin-binding agent. The biological material may be diluted, e.g., 1:1 to 1:10 (v/v) (e.g., 1:1, 1:2, 1:3, 1:4, 1:5, 1:6, 1:7, 1:8, 1:9, or 1:10 (v/v), such as 1:3 (v/v)) with the dilution buffer and then contacted or applied to the medium (e.g., an endotoxin-binding agent).
[0116] The dilution buffer can have a pH range of about 4.5 to 8.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or about 8.5). The dilution buffer can contain one or more of deionized water, glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. A dilution buffer used for loading the biological material may have a low concentration of a salt, such as NaCl (e.g., 300 mM or less salt (e.g., NaCl, such as 200 mM, 150 mM, 100 mM, 75 mM, 50 mM, 25 mM, 10 mM, 5 mM, or 0 mM salt (e.g., NaCl)). For example, a biological material containing an IIp can be diluted 1:3 (v/v) with a buffer containing 20 mM Tris-HCl and the diluted material can be contacted or applied to an endotoxin-binding column. The biological material can be, e.g., one that was prepared during a prior purification step (e.g., a chromatography step, such as a step using an anion-exchange support). The dilution buffer can be water.
[0117] Loading Buffer
[0118] A biological material containing IIp (e.g., II, PI) may be adjusted pH and conductivity prior to a purification step, such as prior to contacting or applying the biological material to a medium, such as an endotoxin-binding agent, anion exchanger. A loading buffer containing the biological material may have a pH range of about 4.5 to 8.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or about 8.5). The loading buffer can contain one or more of deionized water, glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. The loading buffer may have a low concentration of a salt, such as NaCl (e.g., 300 mM or less salt (e.g., NaCl, such as 200 mM, 150 mM, 100 mM, 75 mM, 50 mM, 25 mM, 10 mM, 5 mM, or 0 mM salt (e.g., NaCl)). The buffer may contain about 20 mM Tris-HCl and 200 mM NaCl. The loading buffer can be, e.g., one that was prepared during a prior process step (e.g., a chromatography step, such as a step using an anion-exchange support, a filtration step). The loading buffer may have the 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.
[0119] Flow Through Buffer
[0120] A flow through buffer can optionally be used to ensure that all of the biological material is loaded onto a medium (e.g., an endotoxin-binding agent or another support described herein) during a purification step. The flow through buffer can also be used to achieve initial or additional separation of the components present in the biological material following application to the medium. The flow through buffer can be the same as the dilution buffer. Alternatively, the flow through buffer can be different from the dilution buffer.
[0121] For example, a flow through buffer may have the same or a different pH (e.g., a pH within the range of about 4.5 to about 8.5 (e.g., about 4.5, 4.6, 4.7, 4.8, 4.9, 5.0, 5.1, 5.2, 5.3, 5.4, 5.5, 5.6, 5.7, 5.8, 5.9, 6.0, 6.1, 6.2, 6.3, 6.4, 6.5, 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8.0, 8.1, 8.2, 8.3, 8.4, or about 8.5)) and/or a different constitution of components (e.g., about 5 mM to about 300 mM of one or more of glycine, acetic acid, citric acid, phosphate, NaCl, calcium, magnesium, EDTA, and Tris-HCl) relative to a dilution buffer used for loading a biological material onto the medium. A flow through buffer may have a higher salt concentration than that of a dilution buffer (e.g., a salt (e.g., NaCl) concentration of 5 to about 300 mM higher than that of the dilution buffer (e.g., the method may involve the use of a flow through buffer with about 150 mM NaCl following the use of a dilution buffer with less than 150 mM NaCl, such as a dilution buffer with no NaCl). The properties of the flow through buffer can be selected to improve initial separation among the components of the biological material during a purification step (e.g., during a column chromatography step).
[0122] Wash Buffer
[0123] The purification method can also include a wash buffer that is used in one or more wash steps (e.g., 1, 2, 3, 4, or more wash steps) that occur, e.g., after contacting or applying a biological material to an endotoxin-binding agent or during one or more other purification steps, as described herein. The wash step(s) can be performed to remove components (e.g., proteins or other substances found in the biological material that are not IIp) that may present in, but less strongly bound to (e.g., weakly bound, such as with a K.sub.d of about 1 mM or greater), the endotoxin-binding agent or other medium (e.g. anion exchanger).
[0124] The wash buffer applied to a medium (e.g., an endotoxin-binding agent 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 the salt concentration of the medium. A first wash buffer applied to the medium may change the pH, while a second wash buffer applied to the medium may change the salt concentration, or vice versa. The wash step(s) using a wash buffer can facilitate the purification of IIp by promoting the release of proteins other than IIp from the medium.
[0125] The wash buffer may differ from the flow through buffer, the dilution buffer or the loading buffer in terms of the components or other properties (e.g., pH, conductivity or salt concentration). For example, a wash buffer may contain a higher concentration of a salt (e.g., NaCl) than the concentration of a salt in the flow through or dilution buffer. If the salt concentration in the wash buffer is the same as the flow through or dilution buffer, the wash buffer may differ instead in its pH or in one or more of its components. For example, a flow through buffer used in the purification method may contain 20 mM Tris-HCl+150 mM NaCl (pH 7.2), whereas a wash buffer may contain 75 mM glycine+100 mM acetic acid+150 mM NaCl (pH 5.2). For example, a loading buffer used in the purification method may contain about 18 mM Tris-HCl+about 2 mM Tris+about 200 mM NaCl (about pH 7.2), whereas a first wash buffer contains about 18 mM Tris-HCl+about 2 mM Tris+about 250 mM NaCl (pH 7.2), whereas a second wash buffer may contain about 75 mM Glycine+about 100 mM HAc+about 150 mM NaCl+about 92.5 mM NaOH (about pH 5.2). For example, a loading buffer used in the purification method may contain about about 18 mM Tris-HCl+about 2 mM Tris+about 200 mM NaCl (about pH 7.2), whereas a first wash buffer contains about 75 mM Glycine+about 100 mM HAc+about 200 mM NaCl (about pH 5.2), whereas a second wash buffer may contain (about 18 mM Tris-HCl+about 2 mM Tris+about 250 mM NaCl (about pH 7.2).
[0126] The wash 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 wash buffer may have a pH of about 5.2. The wash buffer may have a pH of about 7.2. The wash buffer can also contain about 5 mM to about 400 mM of one or more of glycine, acetic acid, citric acid, phosphate, sodium chloride (NaCl), calcium, magnesium, EDTA, and Tris-HCl. For example, the wash buffer may have a concentration of a salt (e.g., NaCl) concentration of about 50 to about 400 mM. For example, the wash buffer may have a concentration of a salt (e.g., NaCl) concentration of about 200 to about 300 mM. For example, the wash buffer may have a concentration of a salt (e.g., NaCl) concentration of about 250 mM, about 250 mM, or about 300 mM. For example, the wash buffer may have about 200 to about 300 mM NaCl. For example, the wash buffer may have about 200 mM NaCl, about 250 mM NaCl or about 300 mM NaCl. The wash buffer may also be prepared with a pH that differs from other buffers previously used in the purification process (e.g., a dilution buffer, flow through buffer, and/or prior wash buffer(s)).
[0127] Wash buffers used in the purification methods may contain different salt concentrations and may be applied to a medium, such as an endotoxin-binding agent, an anion exchange resin, starting with a low salt concentration wash buffer followed by a subsequent wash step(s) using a wash buffer(s) with an increasing salt concentration at each wash step.
[0128] For example, a first wash step may involve applying a first wash buffer with a low pH (e.g., less than pH 7.0, such as pH 5.5 or less (e.g., pH 5.2)) and/or salt concentration (e.g., a salt (e.g., NaCl) concentration of less than 150 mM) to a medium, such as an endotoxin-binding agent (e.g., a first wash buffer may contain 75 mM glycine+100 mM acetic acid+150 mM NaCl (pH 5.2)) and a second wash step may involve applying a second wash buffer with a higher pH (e.g., a pH above pH 7.0, such as pH 7.2) and/or salt concentration (e.g., a salt (e.g., NaCl) concentration of greater than 150 mM (e.g., about 300 mM)) to the medium (e.g., a second wash buffer may contain 20 mM Tris-HCl+300 mM NaCl (pH 7.2)). In another example, a first wash step may use a wash buffer containing 15 mM phosphate+50 mM NaCl (pH 5.5), whereas a subsequent wash step may use a second wash buffer containing 15 mM phosphate+100 mM NaCl (pH 5.5).
[0129] Elution Buffer
[0130] The purification method may also include the collection of an eluate containing IIp from a medium (e.g., an endotoxin-binding agent or other agent, such as an anion exchange resin). The method involves contacting or applying an eluent or elution buffer to the medium (e.g., an endotoxin-binding agent, an anion exchange resin) and collecting the eluate.
[0131] The elution buffer may be prepared with a sufficiently high salt (e.g., sodium chloride (NaCl)) concentration (e.g., greater than about 200 mM salt (e.g., NaCl) (e.g., 250 mM, 300 mM, 350 mM, 375 mM, 400 mM, 450 mM, 500 mM, 550 mM, 600 mM, 650 mM, 700 mM, 750 mM, 800 mM, 850 mM, 900 mM, 950 mM, or 1,000 mM salt (e.g., NaCl)), such that bound IIp can be released from the medium (e.g., an endotoxin-binding agent, anion exchange resin). The elution buffer may contain the same components as the buffers previously described (e.g., dilution buffer, loading buffer, flow through buffer, and wash buffer(s)) or the elution buffer may contain one or more different components (e.g., the elution buffer may contain about 5 mM to about 300 mM of one or more of glycine, acetic acid, citric acid, phosphate, and Tris-HCl). Other salts or additives can be used in place of NaCl, e.g., calcium, magnesium, or EDTA at an equivalent concentration. The pH of the elution buffer may also be the same as the buffers (e.g., dilution and/or wash buffer) previously contacted or applied to the medium (e.g., an endotoxin-binding agent) 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 wash buffer(s) previously applied to the medium (e.g., an endotoxin-binding agent). The pH of the elution buffer may be about 7.2. 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 1000 mM NaCl. In a preferred example, IIp is eluted from an endotoxin-binding agent by applying a buffer to the endotoxin-binding agent containing 20 mM Tris-HCl+500 mM NaCl (pH 7.2). Application of an elution buffer to the endotoxin-binding agent can be repeated one or more time. The elution buffer applied to the endotoxin-binding agent may be the same or different. For example, a second or subsequent elution buffer applied to the endotoxin-binding agent can contain a higher salt concentration (e.g., 1,000 mM NaCl) relative to a first or prior elution buffer (e.g., 500 mM NaCl). If desired, the fractions collected following application of the elution buffer can be analyzed separately for the presence and concentration of IaIps using, e.g., ELISA or other techniques known in the art, and then, optionally pooled. Alternatively, the fractions may be pooled and then analyzed for the presence and concentration of IaIps.
[0132] Cleaning Buffer
[0133] The purification method can also include an optional cleaning step, in which a cleaning buffer is applied to the medium in order to regenerate the medium (e.g., an endotoxin-binding agent) for use in another round of purification. The cleaning buffer can be prepared with a sufficiently high pH (e.g., about 12 to about 14). The cleaning buffer can contain about 1 M of an alkaline solute, e.g., sodium hydroxide (NaOH). Optionally, the cleaning buffer can additionally contain about 1 to about 2 M salt (e.g., NaCl). In a preferred example, the cleaning buffer contains 1 M NaOH+2 M NaCl (pH 14).
[0134] Purification of IIp Using an Endotoxin-Binding Agent
[0135] IIp can be purified from a biological material by contacting or applying the biological material containing the IIp to an endotoxin-binding agent (e.g., one or more of the endotoxin-binding agents described herein). The biological material can be applied directly to the endotoxin-binding agent without dilution or, alternatively, the biological material can be diluted with a dilution buffer (as described above) and then applied to the endotoxin-binding agent. For example, the volume of a biological material (with or without dilution) contacted or applied to the endotoxin-binding agent may be, e.g., about 0.5 to about 20 column volumes (or other suitable volume).
[0136] After application of the biological material containing IIp to the endotoxin-binding agent, the flow through can optionally be analyzed to confirm that it does not contain (or contains less than 10% (w/w)) IIp. For example, an ELISA assay (e.g., using an anti-IIp antibody, such as MAb 69.26) or other known techniques (e.g., SDS-PAGE, and/or Western Blot, or other known techniques) can be performed. Once the flow through has been confirmed to contain no or an insubstantial amount of IIp (e.g., an amount of about 30 g/mL or less, such as about 20 g/mL, 10 g/mL, 5 g/mL, or 1 g/mL or less), the flow through can be discarded. An additional flow through buffer may be applied (e.g., in a volume of about 1 to about 50 column volumes) to the endotoxin-binding agent to ensure that all of the biological material is loaded onto the endotoxin-binding agent. This flow through may then be discarded (e.g., after confirming the absence (or an insubstantial amount) of IIp in the flow through, if desired).
[0137] Next, one or more wash steps (e.g., 2, 3, 4, or more wash steps) may be performed to remove non-IIps present in the biological material that are weakly bound to the endotoxin-binding agent. About 0.5 to about 10 column volumes of a wash buffer (or other appropriate volume) can be applied in a given wash step. The resulting wash fractions can be analyzed (e.g., using ELISA or other known techniques), if desired, to confirm that it does not contain (or contains less than, e.g., 10% (w/w)) IIp. If a substantial amount of IIp (e.g., 10% (w/w) or greater) is detected in the wash buffer flow through, the flow through can, if desired, be processed to collect and purify the IIp present in the flow through (e.g., using an endotoxin-binding agent or other medium described herein (e.g., an anion-exchange support)).
[0138] After the wash step(s) is performed, an eluate containing IIp can be collected by applying an elution buffer (e.g., about 0.4 to about 5 column volumes or other suitable volume) to the endotoxin-binding agent. If desired, more than one (e.g., 2, 3, 4, or more) elution buffers may be applied to the endotoxin-binding agent to elute or to ensure elution of all IaIps. The eluate can be analyzed for the presence of IaIps using, e.g., ELISA or other techniques known in the art. The fractions from different elution buffers may then be pooled, if desired.
[0139] After an eluate is collected, the endotoxin-binding agent can optionally be cleaned by applying a cleaning buffer (e.g., about 0.5 to about 5 column volumes or other suitable volume) to the endotoxin-binding agent to regenerate the endotoxin-binding agent for a future use. The resulting cleaning fraction(s) can be analyzed (e.g., using ELISA or other known techniques), if desired, for the presence of IIp.
[0140] Each step of the purification process (e.g., the application of the biological material, the wash step(s), and the elution step(s)) can be performed using either gravity flow or with low pressure (e.g., 0-15 psi) in order to produce a flow rate of about 1 to about 10 mL per minute (e.g., about 1.0, 1.5, 2.0, 2.5, 3.0, 3.5, 4.0, 4.5, 5.0, 5.5, 6.0, 6.5, 7.0, 7.5, 8.0, 8.5, 9.0, 9.5, or 10.0 mL per minute). In some examples, the flow rate is about 2 mL per minute.
[0141] This process can be repeated one or more times, if desired. Alternatively, the IIp 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 IIp may be subjected to one or more additional purification steps, if desired, as is discussed below.
[0142] Purification of IIp Following the Use of an Endotoxin-Binding Agent
[0143] An eluate fraction containing IIp collected following elution from an endotoxin-binding agent can be further purified using other known purification steps (e.g., one or more of the steps described below under Additional purification steps). For example, the eluate containing IIp can be subjected to one or more purification step(s), such as those described in, e.g., US 2003/0190732, US 2011/0190194, US 2012/0053113, and US 2014/0206844, each of which is incorporated herein by reference. Furthermore, the eluate containing IIp can be further purified using an anion-exchange chromatography as described above.
[0144] Purification Step(s) Prior to the Use of an Endotoxin-Binding Agent
[0145] A biological material containing IIp may be processed prior to contacting or applying the biological material to an endotoxin-binding agent. For example, the biological material may be processed using a sample preparation or purification step(s) that removes up to, e.g., 10% or more (w/w) (e.g., 10-30% (w/w), such as 15%, 20%, 25%, or 30% (w/w), or more) of one or more substances from the biological material (e.g., a protein or substance other than an IIp).
[0146] Processing steps may include, for example, filtration, centrifugation, sedimentation, chromatography, decanting step, clarification, freezing, drying, evaporation, extraction, filtration, precipitation, or another purification or preparatory method known in the art. In a preferred example, the method involves performing anion-exchange chromatography (discussed below) using a biological material containing IIp and, subsequently, contacting or applying an eluate containing one or more IaIps prepared from the anion-exchange chromatography to an endotoxin-binding agent for further purification of the IaIps as discussed above and herein.
[0147] A prior purification step or processing step can involve one or more of the purification methods described in, e.g., US 2003/0190732, US 2011/0190194, US 2012/0053113, and US 2014/0206844, each of which is incorporated herein by reference.
[0148] Additional Purification Steps
[0149] A biological material (e.g., cryo-poor plasma, plasma fraction intermediate, blood product) containing IIp (e.g., II, PI, a heavy chain (e.g., H1, H2, H3, H4, and/or H5), a light chain (e.g., bikunin), or a combination thereof) can be applied to one or more chromatography supports other than one containing an endotoxin-binding agent, either before or after purification of IIp using an endotoxin-binding agent (e.g., by using an anion-exchange chromatography support). The chromatography support can be a monolithic or particle-based support. The chromatography support can be a column, membrane, disc, or chip. Additionally, the chromatography support can be, e.g., a size-exclusion chromatography support, an ion-exchange chromatography support, an affinity chromatography support, or a combination thereof.
[0150] For example, the monolithic support or particle-based support may contain an immobilized anion-exchange resin. The immobilized anion-exchange resin can be, e.g., a diethylaminoethane (DEAE) or a quaternary amine (Q) (e.g., Tosoh TOYOPEARL GigaCap Q650M).
[0151] The biological material containing IIp can be applied directly to the chromatography support (e.g., an anion-exchange chromatography support). Alternatively, the biological material may be diluted with a dilution buffer, as described above, and then applied to the support. For example, cryo-poor plasma can be diluted 1:3 (v/v) with dilution buffer containing, e.g., 20 mM Tris-HCl+200 mM NaCl (pH 7.2), and applied to the chromatography support (e.g., in a volume of about 0.5 to about 25 column volumes or other appropriate volume).
[0152] After applying the biological material to the chromatography support, the flow through can be separated and, optionally, analyzed to confirm that it does not contain (or contains less than 10% (w/w)) IIp. For example, an ELISA assay or other known techniques (e.g., SDS-PAGE, and/or Western Blot, or other known techniques) can be performed. If a substantial amount of IIp (e.g., 10% (w/w) or greater) is detected in the flow through, the flow through can, if desired, be processed to collect and purify the IIp present in the flow through (e.g., using an endotoxin-binding agent or other medium described herein (e.g., an anion-exchange support)). Once the flow through has been confirmed to contain no or an insubstantial amount of IIp (e.g., an amount of about 30 g/mL or less, such as about 20 g/mL, 10 g/mL, 5 g/mL, or 1 g/mL or less), the flow through can be discarded.
[0153] Optionally, a flow through buffer can be prepared and applied (e.g., in a volume of about 1 to about 50 column volumes or other appropriate volume) to the chromatography support to ensure that all of the biological material is loaded onto the support. This flow through may then be discarded (e.g., after confirming the absence (or an insubstantial amount) of IIp in the flow through, if desired).
[0154] Next, one or more wash steps (e.g., 2, 3, 4, or more) may be performed to remove non-IIps of the biological material that are weakly bound to the chromatography support. The wash buffers may be prepared as described above. About 0.4 to about 10 column volumes of a wash buffer (or other appropriate volume) can be applied to the chromatography support in a given wash step. The resulting wash fractions can be analyzed (e.g., using ELISA or other known techniques), if desired, to confirm that it does not contain (or contains less than, e.g., 10% (w/w)) IIp. If a substantial amount of IIp (e.g., 10% (w/w) or greater) is detected in the wash buffer flow through, the flow through can, if desired, be processed to collect and purify the IIp present in the flow through (e.g., using an endotoxin-binding agent or other medium described herein (e.g., an anion-exchange support)).
[0155] In an example, a first wash step is performed by applying a buffer containing 20 mM Tris-HCl+250 mM NaCl (pH 7.2) to the chromatography support (e.g., a Tosoh TOYOPEARL GigaCap Q650M column), e.g., in a volume of about 5-10, such as about 8 or 9, column volumes, followed by a second wash step that is performed by applying a buffer containing 50 mM glycine+100 mM acetic acid+175 mM NaCl (pH 5.2) to the support, e.g., in a volume of about 2-6, such as about 4 or 5, column volumes.
[0156] A processed material containing IaIps can then be obtained by applying an eluent or elution buffer (e.g., in a volume of about 0.4 to about 10 column volumes or other appropriate volume) to the chromatography support and collecting the eluate containing IaIps. The elution buffer can be prepared and applied to the chromatography support in an analogous fashion as described above in connection with the endotoxin-binding agent purification process. If desired, more than one (e.g., 2, 3, 4, or more) elution buffers may be applied to the chromatography support to elute or to ensure elution of all IaIps. The eluate can be analyzed for the presence of IaIps using, e.g., ELISA (or other techniques known in the art). The fractions from different elution buffers may then be pooled, if desired.
[0157] In an example, an elution buffer containing 20 mM Tris-HCl+750 mM NaCl (pH 7.2) is applied to the chromatography support in a volume of, e.g., about 2-5 column volumes, such as, e.g., 3-4 column volumes. The eluate containing IaIps can then be further processed by application to an endotoxin-binding agent, as described above, or the eluate can be further processed by repeating the chromatography step using the same or a different chromatography support, or by using one or more different purification or preparation steps, for example, filtration, centrifugation, sedimentation, chromatography, decanting step, clarification, freezing, drying, evaporation, extraction, filtration, precipitation, or another purification or preparatory method known in the art.
[0158] If desired, the chromatography support can be washed with a cleaning buffer to regenerate the chromatography support for a future use. The resulting cleaning fractions can be analyzed (e.g., using ELISA or other known techniques), if desired, for the presence of IIp.
[0159] In an example, a cleaning buffer containing 1 M NaOH+2 M NaCl (pH 14) is applied to the chromatography support in a volume of, e.g., about 1-5 column volumes, such as, e.g., 1.5-2 column volumes.
[0160] Detection of IIp
[0161] IIp isolated using the purification methods described herein can be quantified using one or more assays known in the art, such as those described in, e.g., WO 2009/154695, US 2020/0057077, and WO 2020/086879, which are incorporated herein by reference.
[0162] For example, an IIp quantification assay may involve contacting a sample containing IIp to an IIp binding agent (e.g., an antibody that specifically binds to IIp (such as, e.g., MAb 69.26 (see, e.g., Sha et al., J Pediatr. 180:135-140, 2017), MAb 69.31 (see, e.g., Lim et al., J Infect Dis. 188(6):919-926, 2003), or PAb R22C (see, e.g., WO 2020/086879), each of which is herein incorporated by reference in its entirety) or an IIp ligand (e.g., heparin, LPS, and/or hyaluronic acid (HA)) and detecting the amount of bound IIp (e.g., using a detection agent). The IIp binding agent may be labeled with, e.g., biotin, and detection can be by use of, e.g., horseradish peroxidase-labeled streptavidin, which can then be detected using known detection methods. In another method, the IIp binding agent is labeled with, e.g., a fluorophore, which can be detected by spectrophotometry. Alternatively, the IIp detection agent can be directly detected without the use of a label (e.g., by surface plasmon resonance (SPR)). After the addition of the IIp detection agent, an additional wash step (e.g., one or more) can be performed to remove unbound IIp detection agent.
[0163] IIp can then be measured based on signal from the conjugated label or the bound detection agent (e.g., enzyme activity or fluorescence) using standard techniques known in the art. If an enzyme is used as the label, substrate can be added to produce the signal (for example, a color change) and can be read by a device suitable for detecting the signal, such as a spectrophotometer. The signal (for example, absorbance or fluorescence) can be plotted against a standard with known concentration of IIp to establish a standard curve or can be compared against a known reference concentration. The unknown concentration in the samples can be calculated and determined based on the established standard curve or reference concentration value.
[0164] Isolated IIp The purification methods described herein (e.g., purification using an endotoxin-binding agent, either alone or in combination with one or more additional pre- or post-purification steps, such as anion-exchange chromatography) can be used to purify IIp (e.g., II, PI, a heavy chain (e.g., H1, H2, H3, H4, and/or H5), a light chain (e.g., bikunin), or a combination thereof) from a biological material. A purity of the IIps can be determined by detecting the total amount of IIp obtained after the purification (e.g., using an assay or immunoassay such as MAb 69.26heparin-biotin sandwich ELISA, SDS/PAGE, and/or Western blot) and calculating a percentage (w/w) relative to the total protein content determined by a total protein assay (e.g., bicinchoninic acid assay (BCA), Bradford assay, Biuret test, or another assay known in the art). After the purification step(s), the IIp may have a purity of at least about 5% (w/w), e.g., at least about 5%, 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, or >99% (w/w).
[0165] Isolated IaIps have an apparent molecular weight of between about 60 kDa to about 280 kDa, which can be determined by any appropriate method known in the art, e.g., by sodium dodecyl sulfate polyacrylamide gel electrophoresis. The isolated IaIps can also be tested for biological activity, such as, e.g., an activity selected from the group consisting of a cytokine inhibitor activity, chemokine inhibitor activity, protease inhibitor activity (e.g., serine protease inhibitor activity), chondroitin sulfate binding, glycosaminoglygan binding activity, hyaluronic acid binding activity, complement binding activity, histone binding activity, Arg-Gly-Asp (RGD) domain binding activity, coagulation factor binding activity, cellular repair activity, and extracellular matrix protein binding activity. The IIp can also be tested for trypsin inhibitory specific activity, e.g., trypsin inhibitory specific activity between about 1000 IU/mg to about 2000 IU/mg (e.g., 1000, 1100, 1200, 1300, 1400, 1500, 1600, 1700, 1800, 1900, or 2000 IU/mg).
[0166] The proportion or concentration of IaIps (e.g., II and/or PI) present in a final purified fraction can vary. Preferably the purified IaIps (e.g., II and/or PI) are present in the final purified fraction in a physiological proportion. Physiological proportions may be, for example, the proportions found in a person or animal that is healthy and/or the ratio of II and PI that appears naturally in human plasma. Physiological proportions are typically from between about 60% to about 80% II and between about 20% to about 40% PI.
[0167] The purification methods also produce a yield of isolated IIp of about 20% (w/w) or greater (e.g., about 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, or 90% (w/w) or greater) relative to the IIp present in the biological material (e.g., blood or milk). In some examples, the yield can be greater than or equal to about 90% or 95% by weight.
[0168] The disclosed purification methods can be used to produce a composition containing IIp. The composition can also be prepared with a higher concentration of IIp relative to the concentration of IIp that was present in the original biological material (e.g., blood or milk). Thus, the methods can be used to prepare a composition containing IIp in an amount of at least about 5 g/mL, e.g., about 5 g, 50 g, 100 g, 300 g, 600 g, 900 g, 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 IIp per mL. The IIp present in the composition may make up greater than about 5 to about 99% or greater (w/w) of the composition (e.g., greater than 5, 10, 20, 30, 40, 50, 60, 70, 80, 90, 95, 97, 99% or greater (w/w)).
[0169] Additional steps (e.g., lyophilization, concentration in-vacuo) can be performed to increase the concentration of IIp in a composition that is prepared using the disclosed methods. These known methods can be used, for example, to produce a composition containing IIp in an amount of, e.g., about 1-50 mg/mL (e.g., about 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 mg or more IIp per mL) from the IIp purified according to the methods described herein.
Pharmaceutical Compositions
[0170] An IIp (e.g., II, PI, a heavy chain (e.g., H1, H2, H3, H4, and/or H5), a light chain (e.g., bikunin), or a combination thereof) obtained by purification using the purification methods disclosed herein (e.g., using an endotoxin-binding agent, as described above) can be used to prepare a pharmaceutical composition. The IIp can be combined, for example, with a pharmaceutically acceptable excipient. The pharmaceutical composition containing IIp would be suitable for administration to a human.
[0171] Examples, of pharmaceutical compositions containing IIp are described in, e.g., US 2007/0297982, US 2015/0238578, US 2019/0269765, and WO 2020/086879, which are herein incorporated by reference.
Methods of Treatment
[0172] IIp prepared by the methods described herein can be used (e.g., when prepared as a pharmaceutical composition) to treat or prevent various diseases, conditions, or symptoms thereof, e.g., diseases or conditions characterized by inflammation and/or low levels of an IIp. Methods of treatment and methods of identifying a subject suitable for treatment with a pharmaceutical composition of the disclosure can be found in, e.g., US 2007/0297982, US 2015/0238578, US 2019/0269765, US 2020/0057077, and WO 2020/086879, which are herein incorporated by reference.
Kits
[0173] IIp prepared by the methods described herein can be used to prepare a kit containing the IIp (e.g., II, PI, a heavy chain (e.g., H1, H2, H3, H4, and/or H5), a light chain (e.g., bikunin), or a combination thereof). For example, the IIp can be placed in a vial in an amount of about 0.5 to about 500 mg/mL (e.g., about 1-50 mg/mL) for distribution in the kit. Exemplary kits can be prepared as described in, e.g., US 2007/0297982, US 2015/0238578, US 2019/0269765, US 2020/0057077, and WO 2020/086879, which are herein incorporated by reference.
[0174] The following examples are intended to illustrate, rather than limit, the invention.
Examples
Example 1. Purification of an IIp from Cryo-Poor Plasma Using ETOXICLEAR
[0175] A sample of cryo-poor plasma was diluted (1:3 (v/v) dilution in 20 mM Tris-HCl+200 mM NaCl, pH 7.2) and the diluted cryo-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 per minute. The flow through was collected for analysis. The flow through, which did not contain (or contained an insubstantial amount of) IIp 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 allow the starting material to pass through the column completely. The additional flow through was collected for analysis and was determined to contain no (or an insubstantial amount of) IIp and was discarded.
[0176] The column was washed with a first wash buffer with approximately 8 CV (43.5 mL: 20 mM Tris-HCl+250 mM NaCl, pH 7.2) and the resulting fractions were collected. The combined fractions were analyzed for total protein (bicinchoninic acid assay (BCA)), IIp (MAb 69.26heparin-biotin sandwich ELISA), and trypsin inhibition activity as shown in Table 1. The combined wash fractions contained a small amount of impure IIp (0.1% pure, 61.727 g, 0.8761% yield of IIp (w/w)) were discarded.
[0177] After the wash at pH 7.2, the column was further washed (approximately 5CV) with a second wash buffer with a lower pH (23.7 mL: 50 mM Gly+100 mM AcOH+175 mM NaCl, pH 5.2) and the resulting fractions were collected, combined, and analyzed as shown in Table 1. The combined wash fractions contained a small amount of impure IIp (0.4% pure, 72.806 g, 1.0339% yield of IIp (w/w)) were discarded.
[0178] After the wash 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 (e.g., total protein, IIp, and trypsin inhibition activity) as shown in Table 1. The collected fractions contained enriched IIp (56.43% pure, 98.537% yield of IIp (w/w)).
[0179] A cleaning buffer with a high pH/high salt content (8.3 mL: 1 M NaOH+2 M NaCl) was next applied to the column and the resulting fractions were combined and analyzed for IIp as described in Table 1. The combined fractions were determined to contain no (or an insubstantial amount of) IIp and were discarded.
[0180] Table 1 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, elution, and cleaning). Furthermore,
TABLE-US-00001 TABLE 1 Capture step using Q anion-exchange resin. IIp ELISA Protein Assay (Heparin- Biological Activity Vol. (BCA) Sandwich) Trypsin Inhibition Activity Purity Fraction (mL) (g/mL) (g) (g/mL) (g) (IU/mL) (IU) (IU/mg) (%) Diluted 114.4 7965.63 911268.30 61.59 7045.38 1889.14 216117.85 237.16 0.77 cryo-poor plasma Flow 172.7 4501.87 777472.77 0 0 1129.68 195096.60 250.94 0 through Wash 1 43.5 839.17 36503.89 1.42 61.72 132.74 5774.02 158.17 0.1 (pH 7.2) Wash 2 23.7 695.23 16477.05 3.07 72.81 126.38 2995.11 181.78 0.4 (pH 5.2) Eluate 16.1 764.15 12302.88 431.20 6942.32 617.66 9944.39 808.29 56.43 Cleaning 8.3 108.87 903.64 0 0 0 0 0 0 Mass 92.58% Mass 100.45% Mass 103.68% Balance Balance Balance Step Yield: 98.537%
[0181] The eluate from the Q anion-exchange column (16 mL) containing IIp was then diluted (1:3 (v/v)) in dH.sub.2O to a volume of 64.0 mL. This fraction was applied to a commercially available 32 mL column containing an endotoxin-binding agent (ETOXICLEAR, Astrea Bioseparations) at a flow rate of 3.5 mL per minute. The flow through was collected for analysis and then discarded because no (or an insubstantial amount of) IIp was detected. An additional buffer volume (26.7 mL: 20 mM Tris-HCl+150 mM NaCl, pH 7.2) was applied to the column. The total flow through volume was collected (90.7 mL) for analysis and then discarded as no (or an insubstantial amount of) IIp was detected.
[0182] The column was washed with a first wash buffer with approximately 1.5 CV (47.0 mL: 75 mM Gly+100 mM AcOH+150 mM NaCl, pH 5.2) and the resulting fractions were collected. The fractions were combined and analyzed for total protein (bicinchoninic acid assay (BCA)), IIp (69.26 Mabheparin-biotin sandwich ELISA), and trypsin inhibition activity as shown in Table 2. The combined wash fractions were then discarded as no (or an insubstantial amount of) IIp was detected.
[0183] The column was further washed with a second wash 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 wash fractions contained a small amount of impure IIp (0.53% pure, 39.045 g, 0.5624% yield of IIp (w/w) from the Q eluate) were discarded.
[0184] The bound protein was then 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 eluate fractions were determined to contain highly pure IIp (>99.9% pure, 99.5% yield (for the current step), 98.0% overall yield for two steps).
[0185] 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 remaining IIp from the endotoxin-binding agent. No additional (or an insubstantial amount of) IIp was detected in this eluate (see Table 2).
[0186] The column was cleaned by applying a cleaning buffer (18.2 mL: 1 M NaOH+2M NaCl); no additional (or an insubstantial amount of) IIp was detected in the flow through.
[0187] Table 2 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, eluate, second eluate, and cleaning). Furthermore,
TABLE-US-00002 TABLE 2 Purification using ETOXICLEAR, pH 7.2. IIp ELISA Protein Assay (Heparin- Biological Activity Vol. (BCA) Sandwich) Trypsin Inhibition Activity Purity Fraction (mL) (g/mL) (g) (g/mL) (g) (IU/mL) (IU) (IU/mg) (%) Diluted 64.0 192.22 12302.88 108.47 6942.32 155.381 9944.390 808.298 56.43 fraction from prior step Flow 90.7 32.49 2947.29 0.000 0.000 0.000 0.000 0.000 0 through Wash 1 47.0 49.65 2333.74 0.000 0.000 0.000 0.000 0.000 0 (pH 5.2) Wash 2 47.5 53.45 2538.97 0.82 39.04 0.000 0.000 0.000 0.53 (pH 7.2) Eluate 1 32.3 211.89 6844.27 213.79 6905.39 308.718 9971.591 1456.925 >99.9 Eluate 2 28.9 151.36 4374.51 0.000 0.000 0.000 0.000 0.000 0 Cleaning 18.2 71.95 1309.44 0.000 0.000 0.000 0.000 0.000 0.000 Mass 92.58% Mass 100.45% Mass 103.676% Balance Balance Balance Step Yield: 99.468% Overall Yield: 98.013%
[0188] This example demonstrates that an endotoxin-binding agent can be used to selectively bind IIps. This binding property can be used to purify IIps from a biological material, such as blood (e.g., cryo-poor plasma).
Example 2. Purification of an IIp from Human Plasma Using DETOXI-GEL, pH 7.3
[0189] A sample of human plasma (fresh frozen plasma, 2.5 mL) was diluted (1:3 (v/v) dilution in 15 mM phosphate, pH 7.3), and the diluted plasma (10.0 mL) was applied to a commercially available 4 mL column containing an endotoxin-binding agent (DETOXI-GEL, Pierce) at a flow rate of 2 mL per minute.
[0190] 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 allow the starting material to pass through the column completely. The additional flow through was collected for analysis (see Table 3) and was determined to contain IIp (0.159% pure, 108.281 g, 14.87% yield of IIp (w/w)).
[0191] The column was washed with a first wash buffer (24.6 mL: 15 mM phosphate+50 mM NaCl, pH 7.3) and the fractions were collected, combined, and analyzed for total protein (BCA assay) and IIp (MAb 69.26heparin-biotin sandwich ELISA). The combined fractions were determined to contain IIp (1.390% pure, 179.186 g, 24.61% yield of IIp (w/w)).
[0192] After the 50 mM NaCl wash, the column was further washed with a second wash buffer having a higher concentration of NaCl (16.7 mL: 15 mM phosphate+100 mM NaCl, pH 7.3) and the resulting fractions were collected, combined and analyzed (see Table 3, Wash 2). The combined fractions were determined to contain a higher amount of IIp than the previous wash step (4.969% pure, 235.353 g, 32.33% yield of IIp (w/w)).
[0193] After the 100 mM NaCl second wash, an elution buffer having a higher concentration of NaCl (7.3 mL: 15 mM phosphate+1,000 mM NaCl, pH 7.3) was applied to the endotoxin-binding agent. The resulting fractions were collected, combined, and analyzed (see Table 3, Wash 3). The combined fractions were determined to contain a lower amount of IIp than the previous first or second wash steps (2.763% pure, 40.632 g, 5.58% yield of IIp (w/w)).
[0194] The column was further cleaned by washing with a high pH buffer (6.7 mL: 1 M NaOH) and the resulting fractions were collected, combined, and analyzed for IIp (65.292 g, 8.97% yield of IIp (w/w)).
[0195] Table 3 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, eluate, and cleaning). Furthermore,
TABLE-US-00003 TABLE 3 Purification using DETOXI-GEL, pH 7.3. IIp ELISA (Heparin- IIp Vol. Protein Assay (BCA) Sandwich) Recovery Fraction (mL) (g/mL) (g) (g/mL) (g) (%) Purity (%) Diluted 10.0 12,172.382 121,723.820 72.797 727.970 0.598 human plasma Flow 50.2 1,355.227 68,032.395 2.157 108.281 14.87 0.159 through Wash 1 24.6 523.940 12,888.924 7.284 179.186 24.61 1.390 (50 mM NaCl) Wash 2 16.7 283.641 4,736.806 14.093 235.353 32.33 4.969 (100 mM NaCl) Eluate 7.3 201.462 1,470.673 5.566 40.632 5.58 2.763 (1,000 mM NaCl) Cleaning 6.7 9.745 65.292 8.97 (1.0M NaOH) Mass 71.6% Mass 86.370% Balance Balance
[0196] IIps eluted in the second wash step, with 32.33% yield and 4.969% purity (for the chromatogram, please see
Example 3. Purification of an IIp from Human Plasma Using DETOXI-GEL, pH 5.5
[0197] A sample of human plasma (fresh frozen plasma, 2.5 mL) was diluted (1:3 (v/v) dilution in 15 mM phosphate, pH 5.5) and the diluted plasma (10.0 mL) was applied to a 4 mL endotoxin-binding agent column (DETOXI-GEL, Pierce) at a flow rate of 2 mL per minute. The flow through was collected for analysis and was then discarded. Additional dilution buffer (63.6 mL: 15 mM phosphate, pH 5.5) was applied to the column to allow the starting material to pass through the column completely. The additional flow through was collected for analysis and was discarded because no (or an insubstantial amount of) IIp was detected.
[0198] The column was washed with a first wash buffer (26.4 mL: 15 mM phosphate+50 mM NaCl, pH 5.5) and the resulting fractions were collected, combined, and analyzed for total protein (BCA assay) and IIp (MAb 69.26heparin-biotin sandwich ELISA). The combined fractions were discarded because no (or an insubstantial amount of) IIp was detected.
[0199] After the 50 mM NaCl first wash, the column was further washed with a second wash buffer having a higher concentration of NaCl (25.6 mL: 15 mM phosphate+100 mM NaCl, pH 5.5) and the resulting fractions were collected, combined, and analyzed (see Table 4). The combined fractions were determined to contain IIp (3.691% pure, 281.958 g, 38.73% yield of IIp (w/w)).
[0200] After the 100 mM NaCl second wash, an elution buffer having a higher concentration of NaCl (14.5 mL: 15 mM phosphate+1,000 mM NaCl, pH 5.5) was applied to the endotoxin-binding agent and the resulting fractions were collected, combined and analyzed for IIp. The combined eluate fractions were determined to contain a majority IIp (20.578% pure, 566.979 g, 77.88% yield of IIp (w/w)) as shown in Table 4.
[0201] The column was further cleaned by washing with a high pH buffer (6.8 mL: 1 M NaOH) and the resulting fractions were collected, combined, and analyzed for IIp. These combined fractions were determined to contain IIp (43.255 g, 5.94% yield of IIp (w/w)) as shown in Table 4.
[0202] Table 4 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, eluate, and cleaning). Furthermore,
TABLE-US-00004 TABLE 4 Purification using DETOXI-GEL, pH 5.5. IIp ELISA (Heparin- IIp Vol. Protein Assay (BCA) Sandwich) Recovery Fraction (mL) (g/mL) (g) (g/mL) (g) (%) Purity (%) Diluted 10.0 12,172.382 121,723.820 72.797 727.970 0.598 human plasma Flow 63.6 1,045.157 66,471.985 0.000 0.000 0.000 0.000 through Wash 1 26.4 451.008 11,906.611 0.000 0.000 0.000 0.000 (50 mM NaCl) Wash 2 25.6 298.441 7,640.090 11.014 281.958 38.73 3.691 (100 mM NaCl) Eluate 14.5 190.020 2,755.290 39.102 566.979 77.88 20.578 (1,000 mM NaCl) Cleaning 6.8 6.361 43.255 5.94 (1.0M NaOH) Mass 72.9% Mass 122.56% Balance Balance
[0203] The majority of the IIps eluted in the third wash step (1,000 mM NaCl), with 77.88% recovery of IIp and 20.578% purity (for the chromatogram, please see
Example 4. Purification of an IIp from Human Plasma Using DETOXI-GEL, pH 7.2
[0204] An eluate from a Q anion-exchange column (from fresh frozen human plasma) containing enriched IIp (ca. 40% w/w) was applied (30 mL) to a 4 mL endotoxin-binding agent column (DETOXI-GEL, Pierce) at a flow rate of 2 mL per minute. The flow through was collected for analysis for IIp (MAb 69.26-heparin-biotin sandwich ELISA) and was discarded because no IIp was detected.
[0205] The column was washed with a first wash buffer (14.6 mL: 20 mM Tris-HCl+150 mM NaCl, pH 7.2) and the resulting fractions were collected, combined, and analyzed for IIp. It was determined that the combined fractions contained the majority of IIp from the starting material (653.5982 g, 75.3% recovery of IIp (w/w)).
[0206] After the 150 mM NaCl wash, the column was further washed with a second wash buffer having a higher concentration of NaCl (5.8 mL: 20 mM Tris-HCl+250 mM NaCl, pH 7.2) and the resulting fractions were collected, combined, and analyzed (see Table 5, Wash 1). The combined wash fractions were determined to contain a smaller amount of IIp from the previous wash step (44.1728 g, 5.09% recovery of IIp (w/w)).
[0207] After the 250 mM NaCl wash, the endotoxin-binding agent was further washed with an elution buffer having a higher concentration of NaCl (6.8 mL, 20 mM Tris-HCl+500 mM NaCl, pH 7.2). The resulting fractions were collected, combined, and analyzed for IIp. These combined fractions contained more IIp than the second wash step fractions (143.7316 g, 16.56% recovery of IIp (w/w)).
[0208] The column was further washed with another elution buffer having a higher concentration of NaCl (3.7 mL, 20 mM Tris-HCl+1,000 mM NaCl) and the resulting fractions were combined and analyzed for IIp. These combined fractions contained less IIp than the first eluate fractions (42.14 g, 4.86% recovery of IIp (w/w)).
[0209] Table 5 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, first eluate, and second eluate). Furthermore,
TABLE-US-00005 TABLE 5 Purification using DETOXI-GEL, pH 7.2. IIp ELISA IIp Vol. (Heparin-Sandwich) Recovery Fraction (mL) (g/mL) (g) (%) Q eluate of 30 28.934 868.02 human plasma Flow through 30.5 0.000 0.000 0.00 Wash 1 (150 14.6 44.767 653.5982 75.3 mM NaCl) Wash 2 (250 5.8 7.616 44.1728 5.09 mM NaCl) Eluate 1 (500 6.8 21.137 143.7316 16.56 mM NaCl) Eluate 2 (1,000 3.7 11.39 42.14 4.86 mM NaCl) Mass balance: 101.80%
[0210] The majority of the IaIps eluted in the first wash step, along with most of the remaining proteins from the Q eluate (75.30% recovery of IIp). Furthermore, IaIps were detected in the other wash fraction and the two eluate fractions.
Example 5. Purification of an IIp from Cryo-Poor Plasma Using ETOXICLEAR
[0211] A sample of cryo-poor plasma (11.5 mL) was diluted (1:4 (v/v): in 20 mM Tris-HCl pH 7.2+200 mM NaCl) and the diluted cryo-poor plasma was applied to a commercially available 5 mL Q anion-exchange resin (Tosoh TOYOPEARL GigaCap Q650M) at a flow rate of 5 mL per minute (120 cm/hr). The flow through was collected for analysis. The flow through, which did not contain (or contained an insubstantial amount of) IIp was discarded. Additional plasma dilution buffer (20 mM Tris-HCl pH 7.2+200 mM NaCl) was applied to the column to allow the starting material to pass through the column completely. The additional flow through was collected for analysis and was determined to contain no (or an insubstantial amount of) IIp and was discarded.
[0212] The column was washed with a first wash buffer (20 mM Tris-HCl pH 7.2+250 mM NaCl) and the resulting fractions were collected. The combined fractions were analyzed for total protein (bicinchoninic acid assay (BCA)), IIp (MAb 69.26heparin-biotin sandwich ELISA), and trypsin inhibition activity as shown in Table 6. The combined wash fractions contained a small amount of impure IIp and were discarded.
[0213] After the wash at pH 7.2, the column was further washed with a second wash buffer with a lower pH (50 mM Gly 100 mM AcOH 150 mM NaCl pH 5.2) and the resulting fractions were collected, combined, and analyzed as shown in Table 6. The combined wash fractions contained a small amount of impure IIp and were discarded.
[0214] After the wash 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 (e.g., total protein, IIp, and trypsin inhibition activity) as shown in Table 6. The collected fractions contained enriched IIp.
[0215] A cleaning buffer with a high pH/high salt content (1 M NaOH+2 M NaCl) was next applied to the column and the resulting fractions were combined and analyzed for IIp as described in Table 6. The combined fractions were determined to contain no (or an insubstantial amount of) IIp and were discarded.
[0216] Table 6 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, elution, and cleaning). Furthermore,
TABLE-US-00006 TABLE 6 Capture step using Q anion-exchange resin. IIp ELISA (Heparin- Biological Activity Vol. Protein Assay (BCA) Sandwich) Trypsin Inhibition Activity Purity Fraction (mL) (g/mL) (g) (g/mL) (g) (IU/mL) (IU) (IU/mg) (%) Starting 45.6 15466.979 705294.242 119.651 5456.063 638.850 29131.560 41.304 0.77% Material Flow 110.0 6037.915 664170.650 0 0 n/d n/d n/d 0% through Wash 1 38.2 480.22 18343.716 4.676 178.623 89.062 3402.168 185.468 0.97% (pH 7.2) Wash 2 22.2 306.345 6800.859 15.799 350.738 n/d n/d n/d 5.15% (pH 5.2) Eluate 19.8 477.329 9451.114 259.038 5128.952 323.840 6412.032 678.442 54.2% Cleaning 10.5 n/d 0.069 0.725 n/d n/d n/d Mass 99.07% Mass 103.72% Mass Balance Balance Balance Step Yield: 94%
[0217] The eluate from the Q anion-exchange column containing IIp was then diluted (1:5 (v/v)) in dH.sub.2O. This fraction was applied to a commercially available 8 mL column containing an endotoxin-binding agent (ETOXICLEAR, Astrea Bioseparations) at a flow rate of 3.5 mL per minute (120 cm/hr). The flow through was collected for analysis and then discarded because no (or an insubstantial amount of) IIp was detected. 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 (or an insubstantial amount of) IIp was detected).
[0218] The column was washed with a first wash buffer (50 mM Gly 100 mM AcOH+200 mM NaCl pH 5.2) and the resulting fractions were collected. The fractions were combined and analyzed for total protein (bicinchoninic acid assay (BCA)), IIp (69.26 Mabheparin-biotin sandwich ELISA), and trypsin inhibition activity as shown in Table 7. The combined wash fractions were then discarded (no (or an insubstantial amount of) IIp was detected).
[0219] The column was further washed with a second wash 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 wash fractions contained a small amount of impure IIp and were discarded.
[0220] The bound protein was then eluted from the column by applying a first high salt elution buffer (20 mM Tris-HCl pH 7.2+400 mM NaCl). The bound protein was then eluted from the column by applying a second high salt elution buffer (20 mM Tris-HCl pH 7.2+500 mM NaCl). The bound protein was then eluted from the column by applying a third high salt elution buffer (20 mM Tris-HCl pH 7.2+1000 mM NaCl). The resulting fractions were collected, combined, and analyzed as shown in Table 7.
[0221] The column was cleaned by applying a cleaning buffer (1 M NaOH, 2M NaCl); no additional (or an insubstantial amount of) IIp was detected in the flow through.
[0222] Table 7 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, eluate, second eluate, and cleaning). Furthermore,
TABLE-US-00007 TABLE 7 Purification using ETOXICLEAR IIp ELISA Protein Assay (Heparin- Biological Activity Vol. (BCA) Sandwich) Trypsin Inhibition Activity Purity Fraction (mL) (g/mL) (g) (g/mL) (g) (IU/mL) (IU) (IU/mg) (%) Starting 100.0 94.511 9451.114 51.290 5128.952 64.120 6412.032 678.442 54.2% Material Flow 113.3 0.027 3.059 0.317 35.916 through + Wash 1 (pH 5.2) Wash 2 n/d n/d n/d n/d n/d n/d n/d n/d (300 mM NaCl) Eluate 1 26.3 185.658 4882.805 183.849 4835.216 187.812 4939.456 1011.602 99.02% (400 mM NaCl) Eluate 2 14.3 45.512 650.822 17.130 244.959 57.450 821.535 1262.304 37% (500 mM NaCl) Eluate 3 17.2 46.044 791.957 1.429 24.579 30.311 521.349 658.305 3.1% (1000 mM NaCl) Cleaning 12.1 38.895 470.630 0.140 1.694 n/d n/d n/d Mass 71.91% Mass 99.62% Mass 98.54% Balance Balance Balance Step Yield: 94.27% Overall Yield: 88.6%
Example 6. Purification of an IIp from Cryo-Poor Plasma Using ETOXICLEAR
[0223] A sample of cryo-poor plasma (50 mL) was diluted (1:4 (v/v): in 20 mM Tris-HCl pH 7.2+200 mM NaCl) and the diluted cryo-poor plasma was applied to a commercially available 100 mL Q anion-exchange resin (Tosoh TOYOPEARL GigaCap Q650M) at a flow rate of 12 mL per minute (120 cm/hr). The flow through was collected for analysis. The flow through, which did not contain (or contained an insubstantial amount of) IIp was discarded. Additional plasma dilution buffer (20 mM Tris-HCl pH 7.2+200 mM NaCl) was applied to the column to allow the starting material to pass through the column completely. The additional flow through was collected for analysis and was determined to contain no (or an insubstantial amount of) IIp and was discarded.
[0224] The column was washed with a first wash buffer (20 mM Tris-HCl pH 7.2+250 mM NaCl) and the resulting fractions were collected. The combined fractions were analyzed for total protein (bicinchoninic acid assay (BCA)), IIp (MAb 69.26heparin-biotin sandwich ELISA), and trypsin inhibition activity as shown in Table 8. The combined wash fractions contained a small amount of impure IIp and were discarded.
[0225] After the wash at pH 7.2, the column was further washed with a second wash buffer with a lower pH (50 mM Gly 100 mM AcOH 150 mM NaCl pH 5.2) and the resulting fractions were collected, combined, and analyzed as shown in Table 8. The combined wash fractions contained a small amount of impure IIp and were discarded.
[0226] After the wash at 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 (e.g., total protein, IIp, and trypsin inhibition activity) as shown in Table 8. The collected fractions contained enriched IIp.
[0227] A cleaning buffer with a high pH/high salt content (1 M NaOH+2 M NaCl) was next applied to the column and the resulting fractions were combined and analyzed for IIp as described in Table 8. The combined fractions were determined to contain no (or an insubstantial amount of) IIp and were discarded.
[0228] Table 8 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, elution, and cleaning). Furthermore,
TABLE-US-00008 TABLE 8 Capture step using Q anion-exchange resin. IIp ELISA (Heparin- Biological Activity Vol. Protein Assay (BCA) Sandwich) Trypsin Inhibition Activity Purity Fraction (mL) (g/mL) (g) (g/mL) (g) (IU/mL) (IU) (IU/mg) (%) Starting 200 18606.907 3721381.400 105.477 21095.300 5414.742 1082948.40 291.007 0.56% Material Flow 481.2 6964.328 3351234.634 n/d n/d 1979.453 952512.784 284.227 through Wash 1 117.3 928.419 108903.549 0.816 95.717 448.775 52641.308 483.376 0.08% (pH 7.2) Wash 2 141.6 476.973 67539.377 0.545 77.172 n/d n/d n/d 0.114% (pH 5.2) Eluate 1 126.3 1196.612 151132.096 161.332 20376.232 591.013 74644.942 493.905 13.48% (750 mM) Eluate 1 28.2 385.681 10876.204 25.627 722.681 n/d n/d n/d 6.6% (1000 mM) Cleaning 59.6 503.206 29991.078 n/d n/d n/d n/d n/d Mass 99.95% Mass 100.84% Mass 99.71% Balance Balance Balance Step Yield: 96%
[0229] The eluate from the Q anion-exchange column containing IIp was then diluted (1:5 (v/v)) in dH.sub.2O to 125 mL. This fraction was applied to a commercially available 74 mL column containing an endotoxin-binding agent (ETOXICLEAR, Astrea Bioseparations) at a flow rate of 3.5 mL per minute (120 cm/hr). The flow through was collected for analysis and then discarded because no (or an insubstantial amount of) IIp was detected. 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 (or an insubstantial amount of) IIp was detected).
[0230] The column was washed with a first wash buffer (50 mM Gly 100 mM AcOH+200 mM NaCl pH 5.2) and the resulting fractions were collected. The fractions were combined and analyzed for total protein (bicinchoninic acid assay (BCA)), IIp (69.26 Mabheparin-biotin sandwich ELISA), and trypsin inhibition activity as shown in Table 9. The combined wash fractions were then discarded (no (or an insubstantial amount of) IIp was detected).
[0231] The column was further washed with a second wash 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 wash fractions contained a small amount of impure IIp and were discarded.
[0232] The bound protein was then eluted from the column by applying a first high salt elution buffer (20 mM Tris-HCl pH 7.2+500 mM NaCl). The bound protein was then eluted from the column by applying a second high salt elution buffer (20 mM Tris-HCl pH 7.2+1000 mM NaCl). The resulting fractions were collected, combined, and analyzed as shown in Table 9.
[0233] The column was cleaned by applying a cleaning buffer (1 M NaOH, 2M NaCl); no additional (or an insubstantial amount of) IIp was detected in the flow through.
[0234] Table 9 provides a summary of the combined fractions and quantities of protein observed in each step (e.g., flow through, first wash, second wash, eluate, second eluate, and cleaning). Furthermore,
TABLE-US-00009 TABLE 9 Purification using ETOXICLEAR IIp ELISA Protein Assay (Heparin- Biological Activity Vol. (BCA) Sandwich) Trypsin Inhibition Activity Purity Fraction (mL) (g/mL) (g) (g/mL) (g) (IU/mL) (IU) (IU/mg) (%) Starting 625.0 239.322 149576.500 32.266 20166.500 118.203 73876.625 493.90 13.48% Material Flow 778.5 125.926 98033.391 0.180 140.130 n/d n/d n/d 0.14% through + Wash 1 (pH 5.2) Wash 2 (300 mM NaCl) Eluate 1 115.4 196.871 22718.913 168.163 19406.049 282.1573256 0.918 1433.208 85.41% (500 mM NaCl) Eluate 2 88.4 59.450 5255.380 n/d n/d 80.039 7075.448 1346.325 (1000 mM NaCl) Cleaning 61.8 44.808 2769.134 n/d n/d n/d n/d n/d Mass 86.09% Mass 96.92% Mass 56.23% Balance Balance Balance Step Yield: 96.23% Overall Yield: 91.99%
Example 7. Analysis of Copurified Impurity: Factor II (Prothrombin)
[0235] Factor II (Prothrombin) copurified with IIp in Example 5-6 was analyzed by ELISA (Table 10). Sandwich ELISA were performed in a 96 well-plate (NUNC Immuno MAXISORP F96) with paired commercially available polyclonal antibodies applying a peroxidase-labelled detection antibody and reaction detection by relative absorbance signal at 450 nm. ELISA plates were coated overnight with a polyclonal sheep anti-human prothrombin (1:1000 in coat buffer), washed 3 times with wash buffer, blocked with 0.1% Milch, 2 mmol/L Benzamidine in wash buffer (block buffer), and washed with wash buffer. Test material and standard calibration samples (both at a chosen dilution range with block buffer) were incubated on the ELISA coated plates during 1 h at room temperature. Standard calibration typically performed on a five-point calibration curve using a commercially available reference plasma preparation (CRYOcheck, PrecisionBioLogic), regularly checked against the secondary international standard ISTH/SSC with a certified FII activity.
[0236] Incubated ELISA plates were washed three times and incubated 1 h at room temperature with sheep anti human prothrombin-peroxidase (HRP)-labelled detection antibody. Washed 3 times with wash buffer, and the HRP chromogenic reaction was triggered by TMB reagent (3,3,5,5 tetramethylbenzidine dihydrochloride) and prothrombin level measured by the chromogenic detection signal at 450 nm
TABLE-US-00010 TABLE 10 ELISA Factor II (ProThrombin) Cryo-poor plasma Cryo-poor plasma (example 5) (example 6) FII ELISA FII reduction FII ELISA FII reduction Fraction [mU/mL] (%) [mU/mL] (%) Starting Material 716 785 of anion- exchange chromatography Eluate 1 (500 4.58 99.4% 2.9 99.6% mM NaCl) of endotoxin binding agent chromatography
OTHER EMBODIMENTS
[0237] All publications, patents, and patent applications mentioned in the above specification are hereby incorporated by reference to the same extent as if each individual publication, patent or patent application was specifically and individually indicated to be incorporated by reference in its entirety. Various modifications and variations of the described methods, pharmaceutical compositions, and kits of the invention will be apparent to those skilled in the art without departing from the scope and spirit of the invention. Although the invention has been described in connection with specific embodiments, it will be understood that it is capable of further modifications and that the invention as claimed should not be unduly limited to such specific embodiments. Indeed, various modifications of the described modes for carrying out the invention that are obvious to those skilled in the art are intended to be within the scope of the invention. This application is intended to cover any variations, uses, or adaptations of the invention following, in general, the principles of the invention and including such departures from the present disclosure come within known customary practice within the art to which the invention pertains and may be applied to the essential features herein before set forth.