CRYOPRESERVED PLATELET COMPOSITIONS AND METHODS FOR MAKING
20200046771 ยท 2020-02-13
Inventors
- Benjamin J. KUHN (Norfolk, VA, US)
- Stephen Edward AMOS (Buckeystown, MD, US)
- Rafael Jorda (Bethesda, MD, US)
Cpc classification
A61K47/18
HUMAN NECESSITIES
A61L24/0005
HUMAN NECESSITIES
A61K31/715
HUMAN NECESSITIES
A61L24/001
HUMAN NECESSITIES
A01N1/0221
HUMAN NECESSITIES
A61K47/10
HUMAN NECESSITIES
A61K31/336
HUMAN NECESSITIES
A61K47/26
HUMAN NECESSITIES
A61K47/20
HUMAN NECESSITIES
A61K9/19
HUMAN NECESSITIES
A01N1/00
HUMAN NECESSITIES
A61K47/36
HUMAN NECESSITIES
International classification
A61K31/715
HUMAN NECESSITIES
A01N1/00
HUMAN NECESSITIES
A61K31/336
HUMAN NECESSITIES
Abstract
This disclosure relates to a composition that, in some embodiments, includes cryopreserved platelets; and a cryoprotectant, wherein the composition comprises less than 6% (v/v) of dimethyl sulfoxide (DMSO). In some embodiments, the composition is substantially free of dimethyl sulfoxide (DMSO).
Claims
1. A composition comprising: cryopreserved platelets; and a cryoprotectant; wherein the composition comprises DMSO in the amount of about 0.1% (v/v) to about 5% (v/v).
2.-3. (canceled)
4. The composition of claim 1, wherein the cryoprotectant is present at about 1 mM to about 1 M.
5.-7. (canceled)
8. The composition of claim 1, wherein the cryoprotectant comprises a component selected from the group consisting of a saccharide, a monosaccharide, a disaccharide, and combinations thereof.
9.-11. (canceled)
12. The composition of claim 1, wherein the cryoprotectant comprises a polymer derived from sucrose and epichlorohydrin, wherein the polymer is a polysucrose.
13.-16. (canceled)
17. The composition of claim 1, wherein the cryoprotectant comprises a high molecular weight polymer, wherein the high molecular weight polymer has an average molecular weight of at least about 70 kDa.
18. (canceled)
19. The composition of claim 1, wherein the cryoprotectant is selected from the group consisting of serum albumin, dextran, polyvinyl pyrolidone (PVP), starch, hydroxyethyl starch (HES), and combinations thereof.
20. The composition of claim 1, wherein the cryoprotectant comprises a sugar alcohol, wherein the sugar alcohol is selected from the group consisting of mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, isomalt, hydrogenated starch hydrolysates, serum albumin, dextran, polyvinyl pyrolidone (PVP), starch, hydroxyethyl starch (HES), and combinations thereof.
21. (canceled)
22. (canceled)
23. The composition of claim 1, wherein the cryoprotectant is selected from the group consisting of glycerol, ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, a saccharide, hydroxypropyl--cyclodextrin, a glycerol oligomer, and combinations thereof.
24. The composition of claim 1, wherein the cryoprotectant comprises glycerol present in an amount of about 0.1% (v/v) to about 10% (v/v).
25.-36. (canceled)
37. The composition of claim 1, further comprising a buffer material, wherein the buffer is selected from the group consisting of phosphate buffered saline (PBS), bicarbonate/carbonic acid, such as a sodium-bicarbonate buffer, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), a tris-based buffer, a tris-buffered saline (TBS), and combinations thereof.
38.-39. (canceled)
40. The composition of claim 1, wherein the composition is a hemostatic composition, wherein the hemostatic composition is suitable for reducing or preventing blood loss from injured tissue.
41.-43. (canceled)
44. The composition of claim 1, wherein the composition is stable for at least about six months.
45.-61. (canceled)
62. The composition of claim 1, wherein the DMSO is in the amount of about 0.5% (v/v) to about 1% (v/v).
63. A method of making a composition comprising: cryopreserved platelets; and a cryoprotectant; wherein the composition comprises dimethyl sulfoxide (DMSO) in the amount of about 0.1% (v/v) to about 5% (v/v), the method comprising: obtaining platelets contained in a liquid medium; suspending the platelets in a suspension medium comprising a cryoprotectant, the suspension medium comprising dimethyl sulfoxide (DMSO) in the amount of about 0.1% (v/v) to about 5% (v/v); and subjecting the suspended platelets to a temperature sufficient to form the composition.
64.-66. (canceled)
67. The method of claim 63, wherein the suspension medium further comprises a salt, wherein the salt is selected from the group consisting of a phosphate salt, a sodium salt, a potassium salt, a calcium salt, a magnesium salt, and combinations thereof.
68.-71. (canceled)
72. The method of claim 62, wherein the suspension medium further comprises a buffering component, wherein the buffering component comprises a phosphate buffer, wherein the phosphate buffer is selected from the group consisting of a phosphate buffered saline (PBS), a bicarbonate/carbonic acid, a sodium-bicarbonate buffer, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), a tris-based buffer, a tris-buffered saline (TBS), and combinations thereof.
73.-76. (canceled)
77. The method of claim 63, wherein the cryoprotectant comprises a saccharide, wherein the saccharide is selected from the group consisting of a monosaccharide, a disaccharide, sucrose, maltose, trehalose, glucose, mannose, dextrose, xylose, and combination thereof.
78. (canceled)
79. The method of claim 63, wherein the cryoprotectant comprises a polymer derived from sucrose and epichlorohydrin, wherein the polymer is a polysucrose.
80. (canceled)
81. The method of claim 79, wherein the polysucrose is present in the suspension medium in an amount equal to about 3% to about 10% (w/v) of the cryopreserved platelet composition.
82.-84. (canceled)
85. The method of claim 63, wherein the cryoprotectant is in an amount of about 0.5% (v/v) to about 5% (v/v).
86. The method of claim 86, wherein the suspension medium further comprises an organic solvent, wherein the organic solvent is present in an amount of about 0.1% to about 2% (v/v) of the composition.
87. (canceled)
88. The method of claim 86, wherein the organic solvent comprises a short-chain alcohol, wherein the short-chain alcohol is selected from the group consisting of methanol, ethanol, propanol, 1-propanol, 2-propanol, and combinations thereof.
89.-93. (canceled)
94. The method of claim 63, wherein the DMSO is in the amount of about 1% (v/v) to about 3% (v/v).
95.-99. (canceled)
100. The method of any one of claims 63, wherein the temperature is adjusted at a rate of 1 C. per minute from a starting temperature of at least about 15 C.
101.-105. (canceled)
106. A method of making a composition comprising: cryopreserved platelets; and a cryoprotectant; wherein the composition comprises dimethyl sulfoxide (DMSO) in the amount of about 0.1% (v/v) to about 5% (v/v), the method comprising: obtaining a liquid medium containing platelets; suspending platelets in a suspension medium, the suspension medium comprising dimethyl sulfoxide (DMSO) in the amount of about 0.1% (v/v) to about 5% (v/v); and subjecting the suspended platelets in the suspension medium to a temperature suitable for forming the composition.
107. The method of claim 106, further comprising adding a pH modifier to the liquid medium containing the platelets until the pH is less than about 7.
108.-110. (canceled)
111. The method of claims 106, wherein the pH is about 6.6 to about 6.8.
112. The method of claim 106, further comprising isolating the platelets contained in the liquid medium, wherein the isolating comprises applying centrifugation, tangential flow filtration, or both, to the liquid medium.
113. (canceled)
114. The method of claim 106, wherein the suspending comprises suspending the platelets in the suspension medium containing a concentration of 210.sup.9 platelets per milliliter (ml).
115. The method of claim 106, further comprising incubating the suspended platelets at a temperature of about 37 C. for about 3 hours.
116. The method of claim 106, further comprising placing the suspended platelets in the suspension medium in a sealable container, wherein the sealable container comprises a fluorinated ethylene propylene (FEP) bag, a polyvinyl chloride (PVC) bag, or a borosilicate serum vial.
117. (canceled)
118. The method of claim 116, wherein the suspended platelets in the suspension medium are placed in the sealable container in an amount of about 110.sup.7 platelets to about 310.sup.11 platelets.
119.-123. (canceled)
124. The method of claim 106, wherein the temperature is about 10 C. to about 30 C.
125. (canceled)
126. The method of claim 106, further comprising thawing the cryopreserved platelet composition, wherein the thawing comprising submersing the cryopreserved platelet composition in a 37 C. water bath for about 8 minutes to about 10 minutes.
127.-128. (canceled)
129. The method of claim 106, wherein the composition contains DMSO in an amount of less 0.001% (v/v).The method of any one of claims 50 to 110, wherein the method does not comprise a lyophilization step.
130.-158. (canceled)
159. A method of making a composition comprising: cryopreserved platelets; and a cryoprotectant, wherein the composition comprises dimethyl sulfoxide (DMSO), the method comprising: obtaining platelets contained in a liquid medium; suspending the platelets in a suspension medium comprising a cryoprotectant, the suspension medium comprising dimethyl sulfoxide (DMSO) in the amount of about 0.1% (v/v) to about 5% (v/v); and subjecting the suspended platelets to a temperature sufficient to form the composition.
160. A method of making a composition comprising: cryopreserved platelets; and a cryoprotectant comprising glycerol; wherein the composition comprises dimethyl sulfoxide (DMSO), the method comprising: obtaining a liquid medium containing platelets; suspending platelets in a suspension medium comprising a cryoprotectant, the suspension medium comprising dimethyl sulfoxide (DMSO) in the amount of about 0.1% (v/v) to about 5% (v/v); and subjecting the suspended platelets in the suspension medium to a temperature suitable for forming the composition.
Description
DESCRIPTION OF DRAWINGS
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DETAILED DESCRIPTION
[0162] The present disclosure relates to the field of blood and blood products. More specifically, it relates to cryopreserved platelets and cryopreserved platelet compositions, including those containing stabilized platelets or compositions derived from platelets. In some embodiments, the present disclosure provides cryopreserved platelets and cryopreserved platelet compositions containing a small amount of DMSO (e.g., less than 2% (v/v) of DMSO), or the present disclosure provides cryopreserved platelets and cryopreserved platelet compositions containing no DMSO.
[0163] In some embodiments provided herein is a cryopreserved platelet composition made by a method comprising: [0164] a. mixing platelets with a medium comprising a cryoprotectant, such as a cryoprotectant comprising trehalose, a polysucrose, glycerol, or combinations thereof, to form a first mixture; [0165] b. optionally modifying the pH of the first mixture to a desired pH; and [0166] c. subjecting the first mixture, or the optionally pH-modified first mixture, to a temperature of about 1 C. or less to form the cryopreserved platelet composition, wherein the cryopreserved platelet composition comprises less than 6% (v/v) of dimethyl sulfoxide (DMSO).
[0167] In some embodiments provided herein, the platelets are human platelets and the cryopreserved platelet composition is cryopreserved human platelet composition.
[0168] In some embodiments provided herein is a method for the preparation of a cryopreserved platelet composition comprising less than 6% (v/v) of dimethyl sulfoxide (DMSO), comprising steps a., b. and c. herein.
[0169] In some embodiments of the composition or of the method, the method does not comprise a lyophilization step.
[0170] In some embodiments provided herein is a cryopreserved platelet composition comprising: [0171] cryopreserved platelets; [0172] a cryoprotectant comprising: [0173] dimethyl sulfoxide (DMSO) in an amount of less than 6% (v/v); [0174] trehalose; and [0175] ethanol; [0176] wherein an amount of intracellular trehalose present in the cryopreserved platelets, after a predetermined time of cooling a mixture of platelets and the cryoprotectant, is greater than an amount of intracellular trehalose in the cryopreserved platelets after the same predetermined time of cooling with a cryoprotectant that is substantially free of at least one of DMSO or ethanol.
[0177] In some embodiments provided herein is a cryopreserved platelet composition comprising: [0178] cryopreserved platelets; [0179] a cryoprotectant comprising: [0180] at least one of dimethyl sulfoxide (DMSO) or glycerol wherein the sum of the amounts of dimethyl sulfoxide (DMSO) and glycerol is less than 15% (v/v); trehalose; and [0181] ethanol; [0182] wherein an amount of intracellular trehalose present in the cryopreserved platelets, after a predetermined time of cooling a mixture of platelets and the cryoprotectant, is greater than an amount of intracellular trehalose in the cryopreserved platelets after the same predetermined time of cooling with a cryoprotectant that is substantially free of at least one of DMSO, glycerol or ethanol.
[0183] Each platelet has an intracellular domain and an extracellular domain. As used herein, intracellular refers to the domain that is inside a cell membrane, e.g., within a platelet membrane, and extracellular refers to the domain that is outside of the cell membrane, e.g., outside of the platelet membrane. In some embodiments, the intracellular amount of trehalose in cryopreserved platelets correlates to an intracellular concentration of trehalose that is about 0.5 to about 20 mM, or about 0.5 to about 10 mM in the cryopreserved platelets. In some embodiments, the intracellular concentration of trehalose in cryopreserved platelets is more than about 0.5 mM (e.g., more than about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 6.0 mM, about 7.0 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or more than about 15 mM). In some embodiments, the total concentration of trehalose in the cryopreserved platelet is more than about 0.5 mM (e.g., more than about 1.0 mM, about 1.5 mM, about 2.0 mM, about 2.5 mM, about 3.0 mM, about 4.0 mM, about 4.5 mM, about 5.0 mM, about 6.0 mM, about 7.0 mM, about 8 mM, about 9 mM, about 10 mM, about 11 mM, about 12 mM, about 13 mM, about 14 mM, or more than about 15 mM). In some more particular embodiments, the intracellular amount and/or concentration of trehalose in the cryopreserved platelets as disclosed herein is obtained by mixing platelets with trehalose in an amount of about 80 mM.
[0184] In some embodiments, the predetermined time of cooling a mixture comprising platelets and a cryoprotectant is about 1 hour to about 6 hours (e.g., from about 1 hour to about 5 hours, from about 1 hour to about 4 hours, from about 1 hour to about 3 hours, from about 1 hour to about 2 hours, from about 2 hours to about 6 hours, from about 2 hours to about 5 hours, from about 2 hours to about 4 hours, from about 2 hours to about 3 hours, from about 3 hours to about 6 hours, from about 3 hours to about 5 hours, from about 3 hours to about 4 hours, from about 4 hours to about 6 hours, from about 4 hours to about 5 hours, or from about 5 hours to about 6 hours). In some embodiments, the predetermined time is less than about 6 hours (e.g., less than about 5 hours, less than about 4 hours, less than about 3 hours, less than about 2 hours, or less than about 1 hour). In some embodiments, is more than about 1 hour (e.g., more than 2 hours, more than about 3 hours, more than about 4 hours, more than about 5 hours, or more than about 6 hours). In some more particular embodiments, the intracellular concentration of trehalose in the cryopreserved platelets is as disclosed herein and the total concentration of trehalose in the cryopreserved platelets is about 80 mM. In some embodiments, the intracellular amount of trehalose in the cryopreserved platelets is about 0.5 to about 20 mM, or about 0.5 to about 10 mM, following a predetermined time of cooling a mixture comprising platelets and trehalose of about 2 hours to about 6 hours, or about 3 hours to about 5 hours, or about 4 hours.
[0185] In some embodiments provided herein is a cryopreserved platelet composition comprising: [0186] i) cryopreserved platelets comprising a first amount of trehalose; [0187] ii) dimethyl sulfoxide (DMSO) in an amount of less than 6% (v/v); [0188] iii) ethanol; [0189] and [0190] iv) a second amount of trehalose that is not comprised in the cryopreserved platelets; [0191] wherein the ratio of the first amount to the second amount of trehalose is greater than the ratio of the first amount to the second amount of trehalose when the composition is substantially free of at least one of DMSO or ethanol.
[0192] In some embodiments provided herein is a cryopreserved platelet composition comprising: [0193] i) cryopreserved platelets comprising a first amount of trehalose; [0194] ii) dimethyl sulfoxide (DMSO) in an amount of less than 6% (v/v); [0195] iii) ethanol; [0196] and [0197] iv) a second amount of trehalose that is not comprised in the cryopreserved platelets; [0198] wherein the ratio of the first amount to the second amount of trehalose is greater than the ratio of the first amount to the second amount of trehalose in a second platelet composition, wherein the second platelet composition differs from the cryopreserved platelet composition only in that the second platelet composition does not comprise at least one of DMSO or ethanol.
[0199] In some embodiments provided herein is a cryopreserved platelet composition comprising: [0200] i) cryopreserved platelets comprising a first amount of trehalose; [0201] ii) at least one of dimethyl sulfoxide (DMSO) or glycerol wherein the sum of the amounts of dimethyl sulfoxide (DMSO) and glycerol is less than 15% (v/v); [0202] iii) ethanol; [0203] and [0204] iv) a second amount of trehalose that is not comprised in the cryopreserved platelets; [0205] wherein the ratio of the first amount to the second amount of trehalose is greater than the ratio of the first amount to the second amount of trehalose when the composition is substantially free of at least one of DMSO, glycerol or ethanol.
[0206] In some embodiments provided herein is a cryopreserved platelet composition comprising: [0207] i) cryopreserved platelets comprising a first amount of trehalose; [0208] ii) at least one of dimethyl sulfoxide (DMSO) or glycerol wherein the sum of the amounts of dimethyl sulfoxide (DMSO) and glycerol is less than 15% (v/v); [0209] iii) ethanol; [0210] and [0211] iv) a second amount of trehalose that is not comprised in the cryopreserved platelets; [0212] wherein the ratio of the first amount to the second amount of trehalose is greater than the ratio of the first amount to the second amount of trehalose in a second platelet composition, wherein the second platelet composition differs from the cryopreserved platelet composition only in that the second platelet composition does not comprise at least one of DMSO, glycerol or ethanol.
[0213] In some embodiments provided herein is a cryopreserved platelet composition made by a method comprising: [0214] a. mixing platelets with a medium comprising: i) a cryoprotectant, such as a cryoprotectant comprising trehalose, a polysucrose, glycerol, or combinations thereof; ii) dimethyl sulfoxide (DMSO) in an amount of less than 6% (v/v); and iii) ethanol, to form a first mixture; [0215] b. optionally modifying the pH of the first mixture to a desired pH; and [0216] c. subjecting the first mixture, or the optionally pH-modified first mixture, to a temperature of about 1 C. or less for a predetermined time to form the cryopreserved platelet composition, [0217] wherein the cryopreserved platelet composition comprises cryopreserved platelets, wherein a first amount of trehalose is comprised in the cryopreserved platelets and a second amount of trehalose in the cryopreserved platelet composition is not comprised in the cryopreserved platelets; [0218] wherein the ratio of the first amount to the second amount of trehalose is greater than the ratio of the first amount to the second amount of trehalose when the composition is substantially free of at least one of DMSO, glycerol, or ethanol.
[0219] In some embodiments provided herein is a cryopreserved platelet composition made by a method comprising: [0220] a. mixing platelets with a medium comprising: i) a cryoprotectant, such as a cryoprotectant comprising trehalose, a polysucrose, glycerol, or combinations thereof; ii) dimethyl sulfoxide (DMSO) in an amount of less than 6% (v/v); and iii) ethanol, to form a first mixture; [0221] b. optionally modifying the pH of the first mixture to a desired pH; and [0222] c. subjecting the first mixture, or the optionally pH-modified first mixture, to a temperature of about 1 C. or less for a predetermined time to form the cryopreserved platelet composition, [0223] wherein the cryopreserved platelet composition comprises cryopreserved platelets, [0224] wherein a first amount of trehalose is comprised in the cryopreserved platelets and a second amount of trehalose in the cryopreserved platelet composition is not comprised in the cryopreserved platelets; [0225] wherein the ratio of the first amount to the second amount of trehalose is greater than the ratio of the first amount to the second amount of trehalose in a second platelet composition, wherein the second platelet composition differs from the cryopreserved platelet composition only in that the second platelet composition is made by a method in which the medium with which the platelets are mixed in step a. does not comprise at least one of dimethyl sulfoxide (DMSO), glycerol or ethanol.
[0226] In some embodiments provided herein is a cryopreserved platelet composition made by a method comprising: [0227] a. mixing platelets with a medium comprising: i) a cryoprotectant, such as a cryoprotectant comprising trehalose, a polysucrose, glycerol, dimethyl sulfoxide (DMSO), or combinations thereof; wherein the sum of the amounts of dimethyl sulfoxide (DMSO) and glycerol is less than 15% (v/v); and ii) ethanol, to form a first mixture; [0228] b. optionally modifying the pH of the first mixture to a desired pH; and [0229] c. subjecting the first mixture, or the optionally pH-modified first mixture, to a temperature of about 1 C. or less for a predetermined time to form the cryopreserved platelet composition, [0230] wherein the cryopreserved platelet composition comprises cryopreserved platelets, [0231] wherein a first amount of trehalose is comprised in the cryopreserved platelets and a second amount of trehalose in the cryopreserved platelet composition is not comprised in the cryopreserved platelets; [0232] wherein the ratio of the first amount to the second amount of trehalose is greater than the ratio of the first amount to the second amount of trehalose when the composition is substantially free of at least one of DMSO, glycerol or ethanol.
[0233] In some embodiments provided herein is a cryopreserved platelet composition made by a method comprising: [0234] a. mixing platelets with a medium comprising: i) a cryoprotectant, such as a cryoprotectant comprising trehalose, a polysucrose, glycerol, dimethyl sulfoxide (DMSO), or combinations thereof; wherein the sum of the amounts of dimethyl sulfoxide (DMSO) and glycerol is less than 15% (v/v); and ii) ethanol, to form a first mixture; [0235] b. optionally modifying the pH of the first mixture to a desired pH; and [0236] c. subjecting the first mixture, or the optionally pH-modified first mixture, to a temperature of about 1 C. or less for a predetermined time to form the cryopreserved platelet composition, [0237] wherein the cryopreserved platelet composition comprises cryopreserved platelets, [0238] wherein a first amount of trehalose is comprised in the cryopreserved platelets and a second amount of trehalose in the cryopreserved platelet composition is not comprised in the cryopreserved platelets;
[0239] wherein the ratio of the first amount to the second amount of trehalose is greater than the ratio of the first amount to the second amount of trehalose in a second platelet composition, wherein the second platelet composition differs from the cryopreserved platelet composition only in that the second platelet composition is made by a method in which the medium with which the platelets are mixed in step a. does not comprise at least one of dimethyl sulfoxide (DMSO), glycerol, or ethanol.
[0240] In some embodiments, the predetermined time to form the cryopreserved platelet composition is about 1 hour to about 48 hours. In some embodiments, the predetermined time to form the cryopreserved platelet composition is about 3 hours to about 36 hours, about 6 hours to about 24 hours, about 12 hours to about 24 hours, about 24 hours to about 36 hours, or about 24 hours to about 48 hours. In some embodiments, the predetermined time to form the cryopreserved platelet composition is more than about 1 hour (e.g., more than about 3 hours, about 6 hours, about 12 hours, about 24 hours, about 36 hours or about 48 hours). In some embodiments, the predetermined time to form the cryopreserved platelet composition is less than about 48 hours (e.g., less than about 36 hours, about 24 hours, about 12 hours, about 6 hours, about 3 hours, or less than one hour).
[0241] In some embodiments, the cryopreserved platelet composition comprises about 3% to about 10% (w/v) of polysucrose and about 1.5% to about 5% (w/v) of trehalose. In some embodiments, the cryopreserved platelet composition comprises polysucrose and trehalose in a ratio of about 1:1 to about 5:1 (e.g., from about 1:1 to about 4:1, from about 1:1 to about 3:1, from about 1:1 to about 2:1, from about 2:1 to about 5:1, from about 3:1 to about 5:1, from about 4:1 to about 5:1, from about 3:1 to about 5:1, or from about 4:1 to about 5:1). In some embodiments, the cryopreserved platelet composition comprises polysucrose and trehalose in a ratio in a range from about 1:1 to about 10:1 (e.g., from about 1:1 to about 2:1, about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, or about 9:1; from about 2:1 to about 3:1, about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1; from about 3:1 to about 4:1, about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1; from about 4:1 to about 5:1, about 6:1, about 7:1, about 8:1, about 9:1, or about v10:1; from about 5:1 to about 6:1, about 7:1, about 8:1, about 9:1, or about 10:1; from about 6:1 to about 7:1, about 8:1, about 9:1, or about 10:1; from about 7:1 to about 8:1, about 9:1, or about 10:1; from about 8:1 to about 9:1, or about 10:1, or from about 9:1 to about 1:10). In some embodiments, the cryopreserved platelet composition comprises polysucrose and trehalose in a ratio in a range from about 1:1 to about 1:10 (e.g., (e.g., from about 1:1 to about 1:2, about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, or about 1:9; from about 1:2 to about 1:3, about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10; from about 1:3 to about 1:4, about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10; from about 1:4 to about 1:5, about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10; from about 1:5 about 1:6, about 1:7, about 1:8, about 1:9, or about 1:10; from about 1:6 to, about 1:7, about 1:8, about 1:9, or about 1:10; from about 1:7 to about 1:8, about 1:9, or about 1:10; from about 1:8 to about 1:9, or about 1:10, or from about 1:9 to about 1:10).
[0242] In some embodiments, the cryopreserved platelet composition comprises about 6% (w/v) of polysucrose and about 3% (w/v) of trehalose.
[0243] In some embodiments, the cryopreserved platelet composition is substantially free of polysucrose. As defined herein, the term substantially free in connection with a component (such as polysucrose, trehalose, or glycerol) refers to an amount of less about 0.001% (w/v) of the component in the composition.
[0244] As used herein, (w/v) refers to weight over volume and (v/v) refers to volume over volume.
[0245] In some embodiments, the present disclosure provides improved compositions for treating wounds by hemostasis and treating coagulopathy, and methods of making compositions for treating wounds and/or coagulopathy. In some embodiments, the methods provided herein are directed to treating wounds to stop blood loss that are rapid, effective, and suitable for use in multiple settings.
[0246] Various embodiments in the present disclosure include cryopreserved platelet compositions (which may also be referred to as platelet composition), derived from human platelets, that can be applied in the medical art for medical treating. The cryopreserved platelet compositions are prepared by a method that includes suspending the platelets with a cryoprotectant and other optional components, and subjecting the suspended platelets to a low temperature under controlled conditions. In embodiments, the method for preparation of the platelet compositions further includes thawing the cryopreserved platelet compositions. The cryopreserved platelet compositions can be used for numerous purposes, including, but not limited to, use as hemostatic agents to form clots at sites of injury involving bleeding, use for treating coagulopathy, and use to promote tissue regeneration and healing. The present disclosure also provides compositions and methods for preventing or treating expected or active excessive bleeding associated with anticoagulant therapy or other therapies or environmental effects that result in inhibition of the clotting cascade. The present disclosure also addresses needs in the art by providing compositions and methods that can be used as diagnostics for detection of blood clotting disorders. Accordingly, the present disclosure provides methods for making diagnostic compositions and using them in methods for diagnosing bleeding disorders.
[0247] The present disclosure further addresses needs in the medical art by providing methods for preparing human cryopreserved platelet compositions for storage and/or for use. For example, preparing the human cryopreserved platelet compositions for storage can include subjecting the compositions to a low temperature (e.g., less than 1 C.), partially freezing, and/or fully freezing the compositions. Preparing the cryopreserved platelet compositions for use can include subjecting the compositions to a high temperature (e.g., greater than 1 C.), partially thawing, and/or fully thawing the compositions. Methods of this disclosure provide cryopreserved platelets that are stable for extended periods of time at a wide range of temperatures. The methods, and the compositions, also provide platelets that, upon thawing (and optionally reconstitution), function well in the process of blood clotting, and thus can be used successfully in therapeutic applications, such as for wound healing and treatment of bleeding diseases and disorders in humans. Kits are provided to contain the cryopreserved platelet compositions.
[0248] In some embodiments, the present disclosure may address needs in the veterinary art by providing cryopreserved platelet compositions derived from animal (e.g., canine) platelets.
[0249] Cryopreserved platelet compositions provided herein include platelets, and/or platelet-derived materials. In some embodiments, the cryopreserved platelet composition comprises human platelets. In some embodiments, the cryopreserved platelet compositions provided herein are derived from human platelets. In some embodiments, the cryopreserved platelet compositions provided herein are derived from the platelets of other animals, such as canine platelets.
[0250] The cryopreserved platelet composition can be in a solid form (e.g., after freezing a cryopreserved platelet composition), a liquid form (e.g., after thawing a frozen cryopreserved platelet composition), or combinations thereof (e.g., partially solid (frozen), partially liquid). In some embodiments, the cryopreserved platelet composition contains less than about 10 percent (<10 wt. %), less than five percent (<5 wt. %), or less than two percent (<2 wt. %) residual moisture.
[0251] The cryopreserved platelet composition, when in liquid form, can include the thawed platelets. The liquid portion of the composition can also include water, an aqueous liquid, blood or a blood component or fraction (such as plasma), saline, buffered saline (e.g., phosphate buffered saline), or the like. In some embodiments, the cryopreserved platelet composition is sterile and has less than two Endotoxin Units (EU) per milliliter (ml) when in liquid form. The liquid form of the cryopreserved platelet composition provided herein can include at least about 50%, or at least about 70% of the particles in the range of about 0.5 m to about 0.9 m.
[0252] In various embodiments, the cryopreserved platelet composition (e.g., a frozen composition, a liquid composition, or combination thereof) contains a small amount of DMSO, or is substantially free of DMSO. For example, in some embodiments, the cryopreserved platelet composition contains an amount of less about 0.001% (v/v) of DMSO in the composition. In some embodiments, the cryopreserved platelet composition includes cryopreserved platelets and a cryoprotectant, wherein the cryopreserved platelet composition is substantially free of dimethyl sulfoxide (DMSO). In some embodiments, a cryopreserved platelet composition includes cryopreserved platelets, and a cryoprotectant comprising dimethyl sulfoxide (DMSO) in an amount of less 6% (v/v). In some embodiments, the cryopreserved platelet composition includes DMSO present in an amount of less than about 5%, about 4%, about 3%, about 2%, or about 1% (v/v). In some embodiments, the cryopreserved platelet composition includes DMSO present in an amount of at least about 0.001%, about 0.01%, about 0.1%, or about 0.5% (v/v). In some embodiments, the cryopreserved platelet composition includes DMSO is in the amount of about 0.01% (v/v) to about 5% (v/v), about 0.01% (v/v) to about 4% (v/v), about 0.01% (v/v) to about 3% (v/v), about 0.01% (v/v) to about 2% (v/v), or about 0.01% (v/v) to about 1% (v/v), about 0.5% (v/v) to about 5% (v/v), about 0.5% (v/v) to about 4% (v/v), about 0.5% (v/v) to about 3% (v/v), about 0.5% (v/v) to about 2% (v/v), or about 0.5% (v/v) to about 1% (v/v).
[0253] It has been surprisingly found that the cryopreserved platelet compositions containing a low amount of DMSO (such as from about 0.5% v/v to about 5% v/v) exhibit desirable platelet function (e.g., size distribution, yield, and aggregation characteristics) as compared to the untreated platelets. As used in this document, untreated platelets refers to platelets contained in their original collection liquid medium, for example, an apheresis liquid medium that may contain plasma and/or water. In some embodiments, cryopreserved platelet compositions that are substantially free of DMSO exhibit acceptable platelet function (e.g., size distribution, yield, and aggregation characteristics) as compared to the untreated platelets. Similarly, it has been surprisingly found that cryopreserved platelet compositions containing a low amount of DMSO exhibit desirable platelet function (e.g., size distribution, yield, and aggregation characteristics) as compared to the untreated platelets. In some embodiments, cryopreserved platelet compositions that are substantially free of DMSO also exhibit desirable platelet function (e.g., size distribution, yield, and aggregation characteristics) as compared to the untreated platelets. The cryopreserved platelet compositions provided herein can be highly advantageous because having a reduced amount (or eliminating the presence) of DMSO, which can be toxic in high amounts, yields compositions that can be safely administered to a patient. Furthermore, the cryopreserved platelet compositions provided herein can be highly advantageous because no additional processing, e.g., removing DMSO prior to use, is necessary for the compositions to be safely administered to a patient if they contain a sufficiently low amount of DMSO or no DMSO.
[0254] In some embodiments, the cryopreserved platelet composition provided herein includes platelets and/or platelet-derived particles having a particle size (e.g., diameter, max dimension) of at least about 0.2 m (e.g., at least about 0.3 m, at least about 0.4 m, at least about 0.5 m, at least about 0.6 m, at least about 0.7 m, at least about 0.8 m, at least about 0.9 m, at least about 1.0 m, at least about 1.0 m, at least about 1.5 m, at least about 2.0 m, at least about 2.5 m, or at least about 5.0 m). In some embodiments, the cryopreserved platelet composition provided herein includes platelets having a particle size of less than about 5.0 m (e.g., less than about 2.5 m, less than about 2.0 m, less than about 1.5 m, less than about 1.0 m, less than about 0.9 m, less than about 0.8 m, less than about 0.7 m, less than about 0.6 m, less than about 0.5 m, less than about 0.4 m, or less than about 0.3 m). In some embodiments, the cryopreserved platelet composition provided herein includes platelets having a particle size of from about 0.3 m to about 5.0 m (e.g., from about 0.4 m to about 4.0 m, from about 0.5 m to about 2.5 m, from about 0.6 m to about 2.0 m, from about 0.7 m to about 1.0 m, from about 0.5 m to about 0.9 m, or from about 0.6 m to about 0.8 m).
[0255] In some embodiments, a cryopreserved platelet composition has at least 50% (e.g., at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 95%, or at least about 99%) of platelets and/or platelet-derived particles in the range of about 0.3 m to about 5.0 m (e.g., from about 0.4 m to about 4.0 m, from about 0.5 m to about 2.5 m, from about 0.6 m to about 2.0 m, from about 0.7 m to about 1.0 m, from about 0.5 m to about 0.9 m, or from about 0.6 m to about 0.8 m). In some embodiments, the cryopreserved platelet composition has at most 99% (e.g., at most about 95%, at most about 80%, at most about 75%, at most about 70%, at most about 65%, at most about 60%, at most about 55%, or at most about 50%) of platelets and/or platelet-derived particles in the range of about 0.3 m to about 5.0 m (e.g., from about 0.4 m to about 4.0 m, from about 0.5 m to about 2.5 m, from about 0.6 m to about 2.0 m, from about 0.7 m to about 1.0 m, from about 0.5 m to about 0.9 m, or from about 0.6 m to about 0.8 m). In some embodiments, the cryopreserved platelet composition has about 50% to about 99% (e.g., about 55% to about 95%, about 60% to about 90%, about 65% to about 85, about 70% to about 80%) of platelets and/or platelet-derived particles in the range of about 0.3 m to about 5.0 m (e.g., from about 0.4 m to about 4.0 m, from about 0.5 m to about 2.5 m, from about 0.6 m to about 2.0 m, from about 0.7 m to about 1.0 m, from about 0.5 m to about 0.9 m, or from about 0.6 m to about 0.8 m).
[0256] In some embodiments, the cryopreserved platelet composition retains a sufficient level of components necessary for the blood clotting function of platelets when introduced into subjects in need of platelet functions. The cryopreserved platelet composition can comprise other blood components, and in particular can comprise blood clotting factors, such as Factor VII and Factor VIII, in their normal or activated states. These other components may be present as a result of concentrating of the platelets or they may be added as separately purified components to the platelets prior to or after subjecting the platelets to a low temperature or freezing the platelets. These other blood components may be present singly (i.e., only one is present in the composition), or multiple other blood components may be included in the composition together with the platelets and/or platelet-derived particles. Typically, the other blood components are included in amounts or concentrations that, when administered to a subject, provide a detectable change in at least one physiological process of the treated subject, or provide a known benefit.
[0257] In various embodiments, the cryopreserved platelet composition provided herein has a pH of less than about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In some embodiments, the cryopreserved platelet composition provided herein has a pH range of about 3.0 to about 9.0, about 4.0 to about 8.0, about 5.0 to about 7.0, about 6.0 to about 7.5, or about 6.5 to about 7.0, during the method of preparation. In some embodiments, a cryopreserved platelet composition has a pH that is less than about 10.0 (e.g., less than about 9.5, about 9.0, about 8.5, about 8.0, about 7.5, about 7.0, about 6.5, about 6.0, about 5.5, about 5.0, about 4.5, about 4.0, about 3.5, about 3.0, or less).
[0258] The cryopreserved platelet composition can be made by the processes described herein. In some embodiments, the method includes a first step of obtaining a composition containing platelets in a liquid medium, such as a liquid medium (e.g., water, plasma, or the like). Platelets can be obtained from pooled platelet concentrates, by separation of platelet concentrates from whole blood, by plateletphresis, or combinations thereof.
[0259] The method can include acidifying the platelets and the liquid medium to a desired pH level. For example, in some embodiments, a pH modifier can be added to the platelets and the liquid medium to lower the pH of the composition to no more than about 5.0, about 5.5, about 6.0, about 6.5, about 7.0, about 7.5, or about 8.0. In some embodiments, the pH modifier is added in an amount such that the composition has a pH range of about 6.0 to about 9.0, about 6.2 to about 8.0, about 6.6 to about 6.8, or about 6.4 to about 7.4. In some embodiments, the pH modifier includes an acid citrate dextrose (ACD) composition. In some embodiments, the ACD composition can contain sodium citrate, citric acid, dextrose, or combinations thereof. In some embodiments, the sodium citrate is present at about 0.01 M to about 0.8 M (e.g., from about 0.03 M to about 0.5 M, or from about 0.05 M to about 0.1 M) in the ACD composition. In some embodiments, the ACD composition can include sodium citrate in an amount of about 3 mg to about 250 mg (e.g., about 5 mg to about 200 mg, about 10 mg to about 100 mg, or about 15 mg to about 50 mg) per milliliter (mL) in the ACD composition. In some embodiments, the sodium citrate is present at about 0.01 M to about 0.7 M (e.g., from about 0.03 M to about 3.0 M, or from about 0.05 M to about 1.0 M) in the composition. In some embodiments, the ACD composition can include sodium citrate in an amount of about 3 mg to about 250 mg) per mL of the ACD composition. In some embodiments, the dextrose is present at about 0.01 M to about 1.5 M (e.g., from about 0.03 M to about 1.0 M, from about 0.05 M to about 0.5 M, or from about 0.8 M to about 0.3 M) in the composition. In some embodiments, the ACD composition can include sodium citrate in an amount of about 2 mg to about 200 mg (e.g., about 5 mg to about 100 mg, about 10 mg to about 50 mg, or about 15 mg to about 25 mg) per mL of the ACD composition.
[0260] The method can include isolating the platelets, for example, by purifying the platelets to a desired extent to form platelet rich plasma (PRP). The step of purifying the platelets can use any method known in the art as useful for obtaining purified platelets, including centrifugation (such as differential centrifugation) and filtration (e.g., tangential flow filtration). In some embodiments, plateletpheresis can be used to provide PRP.
[0261] The method for making the cryopreserved platelet composition provided herein can include suspending the purified platelets in a suspension medium. In various embodiments, the suspension medium includes a cryoprotectant (e.g., a non-reducing disaccharide) for a sufficient amount of time and at a suitable temperature to allow for entry of the cryoprotectant into the platelets (also referred to herein as loading the platelets). Accordingly, in some embodiments, the first mixture referred to herein comprises platelets and the suspension medium comprising the cryoprotectant. Without applicants being bound by any theory or mechanism, the cryoprotectant is thought to stabilize proteins and other biological substances in the interior of the platelets. The identity of the cryoprotectant is not limited as long as it can enter the platelets and provide a cryoprotectant property. In some embodiments, the suspension medium includes a cryoprotectant that comprises DMSO. In some embodiments, the suspension medium is substantially free of DMSO.
[0262] Other non-limiting examples of suitable cryoprotectants include saccharides, such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, xylose, and combinations thereof. In some embodiments, the saccharide for use in the method of preparing a cryopreserved platelet composition provided herein is trehalose.
[0263] In some embodiments, a suitable cryoprotectant includes one or more sugar alcohols. Non-limiting examples of sugar alcohols can include mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, and combinations thereof.
[0264] In various embodiments, one or more saccharides can be present in the composition in any suitable amount. In some embodiments, the saccharide can be present at about 1 mM to about 1 M. In embodiments, the saccharide is present at about 10 mM to about 500 mM. In some embodiments, the saccharide is present at about 20 mM to about 200 mM. In embodiments, the saccharide is present at about 40 mM to about 100 mM. In some embodiments, one or more saccharides are present in about 0.04 mg/ml to about 4 mg/ml, about 0.04 mg/ml to about 0.4 mg/ml, or about 0.4 mg/ml to about 4 mg/ml in the composition. In some embodiments, one or more saccharides are present in about 3 mg/ml to about 300 mg/ml, about 3 mg/ml to about 30 mg/ml, or about 30 mg/ml to about 300 mg/ml in the composition.
[0265] In various embodiments, the saccharide is present in different specific concentrations within the ranges recited above, and one of skill in the art can immediately understand the various concentrations without the need to specifically recite each herein. Where more than one saccharide is present in the composition, each saccharide can be present in an amount according to the ranges and particular concentrations recited above.
[0266] In some embodiments, the cryoprotectant includes one or more polyols. In some embodiments, suitable cryoprotectants can include glycerol (glycerin), ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, a saccharide, hydroxypropyl--cyclodextrin, a glycerol oligomer, or combinations thereof. In some embodiments, the cryoprotectant is no more than about 10% (v/v) (e.g., no more than about 9% (v/v), 8% (v/v), 7% (v/v), 6% (v/v), 5% (v/v), 4% (v/v), 3% (v/v), 2% (v/v), 1% (v/v), 0.5% (v/v), or 0.1% (v/v). In some embodiments, the cryoprotectant is in an amount of at least about 1% (w/v) (e.g., at least about 2% (v/v), 3% (v/v), 4% (v/v), 5% (v/v), 6% (v/v), 7% (v/v), 8% (v/v), 9% (v/v), or 10% (v/v)). In some embodiments, the cryoprotectant is in an amount of about 0.1% (v/v) to about 10% (v/v). In some embodiments, the cryoprotectant is in an amount of about 0.5% (v/v) to about 7% (v/v). In some embodiments, the cryoprotectant is in an amount of about 1% (v/v) to about 5% (v/v). In some embodiments, the glycerol is in an amount of about 0.1% (v/v) to about 1% (v/v). Glycerol provides beneficial properties as an antifreeze agent. It can also serve as a humectant, in some embodiments.
[0267] In some embodiments, the method includes suspending the purified platelets in an aqueous suspension medium that is buffered with one or more buffering components. The buffering component can be any buffer that is non-toxic to the platelets and provides adequate buffering capacity to the solution at the temperatures at which the solution will be exposed during the method provided herein. Thus, the buffer may comprise any of the known biologically compatible buffers available commercially, such as phosphate buffers, such as phosphate buffered saline (PBS), bicarbonate/carbonic acid, such as sodium-bicarbonate buffer, N-2-hydroxyethylpiperazine-N-2-ethanesulfonic acid (HEPES), and tris-based buffers, such as tris-buffered saline (TBS). Likewise, it may comprise one or more of the following buffers: propane-1,2,3-tricarboxylic (tricarballylic); benzenepentacarboxylic; maleic; 2,2-dimethyl succinic; EDTA; 3,3-dimethylglutaric; bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS); benzenehexacarboxylic (mellitic); N-(2-acetamido)imino-diacetic acid (ADA); butane-1,2,3,4-tetracarboxylic; pyrophosphoric; 1,1-cyclopentanediacetic (3,3 tetramethylene-glutaric acid); piperazine-1,4-bis-(2-ethanesulfonic acid) (PIPES); N-(2-acetamido)-2-amnoethanesulfonic acid (ACES); 1,1-cyclohexanediacetic; 3,6-endomethylene-1,2,3,6-tetrahydrophthalic acid (EMTA; ENDCA); imidazole; 2-(aminoethyl)trimethylammonium chloride (CHOLAMINE); N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES); 2-methylpropane-1,2,3-triscarboxylic (beta-methyltricarballylic); 2-(N-morpholino)propane-sulfonic acid (MOPS); phosphoric; and N-tris(hydroxymethyl)methyl-2-amminoethane sulfonic acid (TES).
[0268] In various embodiments, one or more buffering components can be present in the composition in any suitable amount. In some embodiments, the buffering component can be present in an amount of 1 mM to 1 M. In some embodiments, one or more buffering components are present in about 0.2 to about 20 mg/ml, or about 0.2 to about 2 mg/ml, or about 2 mg/ml to about 20 mg/ml in the composition. In some embodiments, one or more salts are present in about 0.08 to about 8 mg/ml, such as about 0.08 to about 0.8 mg/ml, or about 0.8 mg/ml to about 8 mg/ml in the composition.
[0269] The method of preparing the cryopreserved platelet composition provided herein can also comprise adding to the suspension medium one or more salts, such as phosphate salts, sodium salts (e.g., NaCl), potassium salts (e.g., KCl), calcium salts, magnesium salts, and any other salt that can be found in blood or blood products, or that is known to be useful in cryopreserving platelets, or any combination of two or more of these.
[0270] In some embodiments, the salts are present in the composition at a concentration of about 1 mM to about 1000 mM, such as about 0.01 M to about 0.2 M. In some embodiments, one or more salts are present in about 0.4 to about 40 mg/ml, or about 0.4 to about 4 mg/ml, or about 4 mg/ml to about 40 mg/ml in the composition. In some embodiments, one or more salts are present in about 0.03 to about 3 mg/ml, or about 0.03 to about 0.3 mg/ml, or about 0.3 mg/ml to about 3 mg/ml in the composition.
[0271] In some embodiments, these salts are present in the composition at an amount that is about the same as is found in whole blood.
[0272] In some embodiments, the method for preparing a cryopreserved platelet composition includes adding to the suspension medium an organic solvent, such as an alcohol, such as ethanol, to the suspension medium. The organic solvent can include one or more alcohols, e.g., short-chain alcohols, such as ethanol. Short-chain alcohols are alcohols having 1 to 6 carbon atoms, in particular 2, 3 or 4 carbon atoms, such as methanol, ethanol, and propanol including 1-propanol and 2-propanol, preferably ethanol. The organic solvent may also be a mixture of different organic solvents. In such a suspension medium, the solvent can range from 0.1% to 5.0% (v/v). In some embodiments, one or more organic solvents are present in about 0.08% (v/v) to about 8% (v/v), or about 0.08% (v/v) to about 0.8% (v/v), or about 0.8% (v/v) to about 8% (v/v) in the composition. Thus, in one embodiment, the first mixture comprises platelets, a cryoprotectant, such as a cryoprotectant comprising trehalose, a polysucrose, or a combination thereof, and a solvent, such as ethanol.
[0273] The method for preparing a cryopreserved platelet composition can include transferring the first mixture, which can comprise the suspended platelets in the suspension medium, to a sealable container (e.g., a container closure system) before subjecting the platelets to a low temperature, and/or freezing the platelets. In some embodiments, the sealable container is a bag such as a fluorinated ethylene propylene (FEP) bag or a polyvinyl chloride (PVC) bag. In some embodiments, the sealable container is a borosilicate serum vial. The platelets and/or suspension medium can be placed (e.g., by aliquotting) in the container at a desired dose size. In some embodiments, the dose size of the platelets can range from about 110.sup.7 platelets to about 310.sup.11 platelets, such as about 210.sup.9 platelets.
[0274] The method further includes subjecting the first mixture in sealable container to a low temperature that forms the cryopreserved platelet composition. In some embodiments, the temperature freezes the suspended platelets in the suspension medium. In some embodiments, the suspended platelets and suspension medium is subjected to a low temperature of about or less than about 1 C. (e.g., about or less than about 5 C., about 10 C., about 20 C., about 30 C., about 40 C., about 50 C., about 60 C., about 70 C., about 80 C., or about of less than about 90 C.). In some embodiments, the first mixture is subjected to a temperature of about 70 C. to about 90, 50 C. to about 70, 30 C. to about 50, 10 C. to about 30, or about 10 C. to about 10 C.). In some embodiments, the first mixture is subjected to a temperature of about 80 C.
[0275] The method for preparing the cryopreserved platelet composition for use by thawing the frozen suspended platelets in the suspension medium. In some embodiments, the thawing includes submersing the cryopreserved platelet composition in a 37 C. water bath for a suitable amount of time, e.g., about 8 minutes to about 10 minutes.
[0276] In some embodiments, the cryopreserved platelet composition is the composition obtained from subjecting a mixture containing platelets, or platelet-derived materials, to a temperature of about 1 C., or less. In some embodiments, the cryopreserved platelet composition is either partially or fully frozen composition.
[0277] In some of the embodiments wherein a cryopreserved platelet composition is obtained by a method disclosed herein, the composition obtained from the freezing steps is thawed to form the cryopreserved platelet composition. Thus, in such embodiments, the method further comprises rehydrating the composition obtained from the freezing steps, to form the cryopreserved platelet composition.
[0278] In some embodiments, the method may optionally further include incubating the re-suspended platelets. The step of incubating the platelets to load them with a cryoprotectant includes incubating the platelets for a time suitable for loading, as long as the time, taken in conjunction with the temperature, is sufficient for the cryoprotectant to come into contact with the platelets and, preferably, be incorporated, at least to some extent, into the platelets. In embodiments, incubation is carried out for about 1 minute to about 180 minutes or longer. In some embodiments, the re-suspended platelets are incubated in an air-permeable bag (e.g., FEP bag) in an air incubator at about 37 C. for about 3 hours with gentle agitation.
[0279] The step of incubating the platelets to load them with a cryoprotectant includes incubating the platelets and the cryoprotectant at a temperature that, when selected in conjunction with the amount of time allotted for loading, is suitable for loading. In general, the composition is incubated at a temperature above freezing (e.g., >1 C.) for at least a sufficient time for the cryoprotectant to come into contact with the platelets. In embodiments, incubation is conducted at 37 C. In certain embodiments, incubation is performed at 20 C. to 42 C. For example, in embodiments, incubation is performed at 35 C. to 40 C. (e.g., 37 C.) for 110 to 130 (e.g., 120) minutes.
[0280] The method of preparing a cryopreserved platelet composition can optionally include a protectant, such as a high molecular weight polymer, into the loading composition. By high molecular weight it is meant a polymer having an average molecular weight of about or above 70 kDa.
[0281] Non-limiting examples of polymeric cryoprotectants are polymers containing one or more monosaccharide units. Non-limiting examples are polysaccharides. Non-limiting examples are polymers derived from sucrose, epichlorohydrin, or combinations thereof. In some embodiments, the polymers are made by the copolymerization of sucrose and epichlorohydrin, such as a polysucrose (e.g., Ficoll 70 and Ficoll 400 provided by GE Healthcare Bioprocess, Uppsala, Sweden). Although any amount of high molecular weight polymer can be used, it is preferred that an amount be used that achieves a final concentration of about 3% to 10% (w/v), such as 3% to 7% (w/v), for example 6% (w/v). In some embodiments, one or more polymers are present in about 6 mg/ml to about 600 mg/ml, or about 6 mg/ml to about 60 mg/ml, or about 60 mg/ml to about 600 mg/ml in the composition. Other non-limiting examples of lyoprotectants are serum albumin, dextran, polyvinyl pyrolidone (PVP), starch, and hydroxyethyl starch (HES). In some embodiments, a lyoprotectant provided herein may also, or alternatively, serve as a cryoprotectant.
[0282] The cryopreserved platelet composition provided herein is highly stable, having a shelf-life of at least six months or above, such as at least eighteen months, at room temperature or below. For example, the cryopreserved platelet composition is stable for at least six months at a temperature of about 80 C.+/10 C. For example, the cryopreserved platelet composition can show hemostatic triggers for primary coagulation properties up to one year after manufacture when stored at room temperature or below, up to 18 months at room temperature or below, or even longer. By stable it is meant that the platelets of the cryopreserved platelet composition function within the parameters mentioned above, and provide adequate hemostatic triggers for primary coagulation. These clotting functions include hemostatic function and primary coagulopathic function. This stability is of great advantage in providing platelet products to those in need, particularly those found at sites some distance from blood collection centers, those in combat theaters, and those working in disaster areas as first responders. Furthermore, because the cryopreserved platelet composition can be stored at room temperature up to 40 C. for long-term storage and up to 80 C. to 90 C. for short periods of about 24 hours, complicated, bulky, or expensive containers for storage (e.g., refrigerators) are not needed. That is, the problem of cold-chain storage is eliminated.
[0283] When needed for treatment of bleeding and for formation of clots, use as a primary hemostatic agent, or for research purposes, the cryopreserved platelet composition of this disclosure can be thawed. The thawed cryopreserved platelet composition is considered a liquid composition according to this disclosure. In some embodiments, the frozen compositions can be thawed to form a liquid composition at a temperature that is above 1 C. In some embodiments, the cryopreserved platelet composition is thawed at a temperature of about 37 C. for about 8 to about 10 minutes.
[0284] As will be recognized by those of skill in the art, the cryopreserved platelet composition of this disclosure has the ability to act as a hemostatic agent to form clots at sites of injury involving bleeding. This concept can be understood as use of the composition in a method for treating bleeding or a method for treating a subject having a bleeding wound. In general, the method comprises contacting a site of bleeding with a sufficient amount of a composition provided herein to reduce or stop the bleeding. The step of contacting can be performed in any suitable way, including, but not necessarily limited to, i) systemic administration of the composition via intravenous infusion or bolus injection and ii) topical administration directly to the site of bleeding. For intravenous administration, the composition is a liquid composition. It should be noted that intravenous administration is suitable for both treating bleeding due to a wound or other trauma and treating bleeding due to coagulopathy. For topical administration, the cryopreserved platelet composition is a liquid composition. When topically administering the liquid form, the composition can be dripped, sprayed, or poured (or the equivalent) directly onto the site of bleeding. The methods of treating, whether therapeutic or prophylactic, of this disclosure can further comprise administering a composition provided herein a second or multiple times. Therefore, the methods of this disclosure encompass treatment regimens in which administration is repeated one or more times at preselected time intervals. Successive administrations may include administration of additional components. The choice of amounts and composition components can be selected by those of skill in the art based on various parameters commonly considered by those of skill in the art, such as subject age, weight, health history, clinical presentation, ancillary presentations, and the like. It is well within the skill of those in the art to make appropriate changes and adjustments to treatment regimens without undue experimentation.
[0285] Certain embodiments of this disclosure provide frozen and liquid cryopreserved platelet compositions for the treatment of drug-induced coagulopathy and for the accelerated efficacy of procoagulant drugs. For example, the cryopreserved platelet compositions of the invention overcome the deficiencies seen in anticoagulant therapy subjects and other subjects showing delayed or absent clotting by providing at least one component in the clotting cascade that is downstream of the component that is lacking in these subjects.
[0286] In some embodiments, the cryopreserved platelet composition provided herein is administered to a subject in an amount sufficient to raise the hemostatic or coagulation properties of that subject's blood to a level that is detectably higher than it was before administration. The method can further comprise administering other biologically active agents, such as clotting factors and/or chemotherapeutic agents for treatment of cancer.
[0287] In some embodiments, the cryopreserved platelet composition provided herein provides methods of monitoring the progression of a disease or disorder of the blood clotting system. The methods generally comprise combining a composition provided herein with platelets and/or plasma removed from a subject suffering from the disease or disorder to make a mixture, and determining the blood clotting ability of the mixture. Typically, determining the blood clotting ability of the mixture indicates the blood clotting ability of the subject's blood, and comprises assaying clotting time of the mixture. Furthermore, typically, multiple assays are performed over time to give an indication of progression over time.
[0288] A further aspect of this disclosure provides kits. In general, a kit of this disclosure comprises any one of the cryopreserved platelet compositions provided herein. The container can be any material suitable for containing the composition, such as a vial, an ampule, or a bag. In embodiments, the container comprises a sufficient amount of composition to perform at least one embodiment of at least one method provided herein. Thus, the kits can be, among other things, diagnostic kits, blood clotting monitoring kits for coagulation proteins or platelets, or drug treatment monitoring kits. In embodiments, the container is provided as a component of a larger kit, which includes suitable packaging and, optionally, instructions and other information relating to use of the compositions. In embodiments, the container or kit comprises other components, such as purified components of the clotting cascade. The kit can be configured to supply the composition for use in in vivo treatments, for use in in vitro diagnostics, or for use in in vitro or in vivo research. Often, the kits will comprise some or all of the supplies and reagents to perform one or more control reactions to ensure the kits are performing properly and to provide baseline results against which test samples can be compared. In embodiments, the composition is provided in a sufficient amount to treat a subject in need of platelet function, such as a subject undergoing surgery or having a bleeding wound. In other embodiments, a sufficient amount of the composition is provided to perform studies on platelets or the blood clotting system of humans, or other animals.
EXAMPLES
[0289] A further aspect of this disclosure provides kits. In general, a kit of this disclosure comprises any one of the cryopreserved platelet compositions provided herein. The container can be any material suitable for containing the composition, such as a vial, an ampule, or a bag. In embodiments, the container comprises a sufficient amount of composition to perform at least one embodiment of at least one method provided herein. Thus, the kits can be, among other things, diagnostic kits, blood clotting monitoring kits for coagulation proteins or platelets, or drug treatment monitoring kits. In embodiments, the container is provided as a component of a larger kit, which includes suitable packaging and, optionally, instructions and other information relating to use of the compositions. In embodiments, the container or kit comprises other components, such as purified components of the clotting cascade. The kit can be configured to supply the composition for use in in vivo treatments, for use in in vitro diagnostics, or for use in in vitro or in vivo research. Often, the kits will comprise some or all of the supplies and reagents to perform one or more control reactions to ensure the kits are performing properly and to provide baseline results against which test samples can be compared. In embodiments, the composition is provided in a sufficient amount to treat a subject in need of platelet function, such as a subject undergoing surgery or having a bleeding wound. In other embodiments, a sufficient amount of the composition is provided to perform studies on platelets or the blood clotting system of humans, or other animals.
Example 1: Platelet Function Metrics of Low DMSO and DMSO-Free Platelet Compositions
[0290] A. Sample Preparation
[0291] Exemplary cryopreserved platelet compositions were prepared as described herein.
[0292] Platelets, contained in a liquid medium, obtained were untreated, human apheresis platelet material (hIDSP). The untreated, apheresis platelets were subjected to processing, including isolating platelets by applying centrifugation and tangential flow filtration. Centrifugation was carried out at 1500 g for 20 minutes, with slow acceleration and braking. Following the centrifugation process, supernatant plasma from the apheresis platelets was aspirated and disposed. Tangential flow filtration was carried out with a 0.45 m filter for >3 diavolumes.
[0293] The liquid medium was replaced with a buffer Formulation A or Formulation B (see Tables 1 and 2, respectively) to form exemplary platelet compositions Std LB and Std LB w/1% DMSO. The platelet composition (Std LB w/1% DMSO) in Formulation A contained DMSO in an amount of 1.0% (v/v).The platelet composition (Std LB) in Formulation B contained no DMSO. Each of the platelet compositions contained a concentration of 210.sup.9 platelets/ml.
[0294] The platelet composition was incubated in an air-permeable bag, made of FEP material, in an air incubator at 37 C. for 3 hours with gentle agitation.
[0295] Following incubation, the platelet composition was transferred to a bag appropriate for freezing. The platelets were aliquoted into the bag in a platelet dose size of about 310.sup.11 platelets per bag.
TABLE-US-00001 TABLE 1 Components of Buffer Formulation A Excipient Concentration NaCl 3.51 mg/ml Kcl 0.29 mg/ml HEPES 1.81 mg/ml NaHCO3 0.81 mg/ml Dextrose 0.43 mg/ml Trehalose 30.27 mg/ml Ficoll PM 400 60.00 mg/ml Polysucrose Ethanol 0.8% (v/v) DMSO 1.0% (v/v) Water Balance
TABLE-US-00002 TABLE 2 Components of Buffer Formulation B Excipient Concentration NaCl 3.51 mg/ml Kcl 0.29 mg/ml HEPES 1.81 mg/ml NaHCO3 0.81 mg/ml Dextrose 0.43 mg/ml Trehalose 30.27 mg/ml Ficoll PM 400 60.00 mg/ml Polysucrose Ethanol 0.8% (v/v) Water Balance
[0296] The bags containing the platelet compositions (Std LB, Std LB w/1% DMSO) and untreated platelets (hIDSP Thawed) were loaded into freezer at a temperature of 80 C. to form cryopreserved platelet compositions. The bags were loaded directly onto the freezer shelf such that the bags made full contact with the shelf and were allowed to equilibrate for >24 hours.
[0297] Prior to testing, the cryopreserved platelet compositions were thawed in a 37 C. water bath for 8-10 minutes.
[0298] B. Platelet Count
[0299] The platelet compositions suspended in buffer Formulation A (with DMSO) or Formulation B (without DMSO) were tested for platelet count (AcT Diff Count) using an automated Coulter AcT Diff system. These platelet compositions were compared against the untreated platelet (hIDSP).
TABLE-US-00003 TABLE 3 Platelet AcT Count AcT Count (10.sup.3 platelets/l) Product Post-Processing Thawed Yield hIDSP 1613 1360 84% Std LB 1677 1537 92% Std LB w/ 1735 1680 97% 1% DMSO
[0300] Table 3 provides platelet counts obtained using the AcT Diff cell counter for the untreated platelets (hIDSP), the platelet composition without DMSO (Std LB), and platelet composition 1% DMSO (Std LB w/1% DMSO) before freezing (Post-Processing), and after being thawing (Thawed). Post-processing refers to platelets and platelet compositions that have been subjected to processing associated with isolating platelets through centrifugation and/or tangential flow filtration.
[0301] The untreated platelets (hIDSP) yielded the lowest platelet count after being thawed. Buffer formulation A (Std LB w/1% DMSO) had >90% platelet recovery after thawing.
[0302] C. Particle Size Distribution By Flow Cytometry Forward Scattering
[0303] Flow Cytometry Forward Scattering was performed on the samples at both the post-processing and thawed states to determine their size distributions. The tested samples included the (i) untreated platelets (hIDSP); (ii) the untreated apheresis platelets (hIDSP Thawed); the exemplary DMSO-free platelet composition (Std LB), and (iii) the exemplary platelet composition with 1% DMSO (Std LB w/1% DMSO).
[0304]
[0305] The data demonstrated that the untreated platelets after thawing (hIDSP Thawed) exhibited the largest size deviation, as compared to the initial platelet size distribution (hIDSP). The data demonstrated that the cryopreserved compositions, after being thawed, exhibited similar results, when compared to the platelet size distribution of the untreated platelets (hIDSP). The platelet composition with 1% DMSO (Std LB w/1% DMSO) appeared to exhibit platelet size distributions that were most similar to the untreated platelets (hIDSP).
[0306] D. Particle Size Distribution By Thrombolux Dynamic Light Scattering
[0307] Thrombolux dynamic light scattering testing was conducted to determine size distributions for the untreated platelets (hIDSP), the thawed, untreated apheresis platelets (hIDSP Thawed), and the thawed cryopreserved platelet compositions with 1% DMSO (Std LB w/1% DMSO).
[0308]
TABLE-US-00004 TABLE 4 Thrombolux Dynamic Light Scattering Test Data Blue Blue peak Cyan Cyanpeak intensity radius Blue intensity radius Product [%] [nm] PDI [%] [nm] hIDSP 94% 1455 0.1 6% 52 hIDSP 69% 1045 0.5 28% 93 Thawed Std LB 97% 937 0.5 2% 52 Std LB w/ 98% 1109 0.2 2% 46 1% DMSO
[0309] Table 4 provides the Thrombolux size data obtained for the untreated platelets (hIDSP) and the thawed cryopreserved platelet formulations. Blue intensity represents the percent of measured particles that fall within the platelet size region; Blue peak radius gives the mean size of that population; and Blue PDI gives the polydispersity, or spread, of the population. The Cyan intensity represents the percent of measured particles that fall below the platelet size; and Cyan peak radius provides the mean size of that population.
[0310] The data in Table 4 demonstrated the thawed hIDSP showed the most significant size differences, as compared to the initial platelets, with only 69% of the particles falling in the platelet region (vs. an initial 94%) and a significantly smaller mean radius. The other thawed formulations were similar their size distributions to the initial platelets. The exemplary platelet composition with 1% DMSO (Std LB w/1% DMSO) was the most similar to the untreated platelet size distribution. The exemplary platelet composition with 1% DMSO had the greatest proportion of platelet sized particles and a smaller Blue PDI than the other compositions.
[0311] E. Platelet Aggregation Testing
[0312] The untreated platelets and platelet compositions were subjected to aggregometry testing. Platelet aggregation is a fundamental hemostatic function that results from platelet activation by various agonists. In fresh platelets, aggregation can be used to screen for functional activation and adhesion receptors on the platelet surface.
[0313] In turbidimetric aggregometry, light is passed through a stirred suspension of platelets. A certain degree of light is scattered by the platelets, which reduces the amount of light passing through the sample. This allows the amount of light that passes through the sample, when measured, to provide an optical density measurement associated with the sample. A high optical density (i.e., low light transmission) indicates the presence of many individual platelets. Aggregates form when the platelets clump together. Aggregates generally reduce the amount of light that is scattered by the platelets, such that, in some cases, the light can pass mostly unobstructed through the suspension. This causes a decrease in the optical density of the sample, and yields a high light transmission. The percent of light transmission relative to a blank (solution without platelets) is reported as the percent aggregation.
[0314] In the Platelet Aggregation Test, the samples were prepared by diluting the thawed platelet compositions in HEPES Modified Tyrodes Albumin Buffer (HMTA). The proportion of thawed platelet compositions was 16% (v/v), having a count of about 250-300,000 platelets per L50,000 platelets per L. The diluted samples were allowed to incubate in glass cuvettes with magnetic stir bars at 37 C. with stirring for >2 minutes. The optical density (light transmission) of each sample was then measured continuously using a Helena AggRAM machine with HemoRAM software (version 1.3). After 2 minutes, in which a baseline was established, various agonists were added to induce aggregation. Agonists used included: 2.5 units of thrombin, 10 g/ml of collagen, 50 g/ml of arachidonic acid, or 20 M Thrombin Receptor Activating Peptide (TRAP-6). Samples were measured for about 10 minutes, and the highest percent aggregation during the 10 minutes of measurement was reported in the aggregation test results.
[0315]
[0316] The test results showed that the thawed, untreated platelets (hIDSP Thawed) provided the lowest response in every category and was most different from the untreated platelets (hIDSP). The platelet composition with 1% DMSO (Std LB w/1% DMSO) gave the highest response of the thawed products and was most similar to the untreated platelets (hIDSP).
[0317] F. Summary
[0318] Based on the test results provided in Sections B-E (supra), the compositions containing DMSO exhibited more desirable platelet function, based on the size distribution, yield, and aggregation test data, than the other test groups (hIDSP and hIDSP Thawed).
Example 2: Platelet Function Metrics of DMSO-Free Platelet Compositions
[0319] This example provides data of platelet function (e.g., size distribution, yield, and aggregation characteristics) of exemplary compositions containing no DMSO when subjected to different low temperature conditions.
[0320] A. Sample Preparation
[0321] Exemplary platelet compositions with no DMSO are prepared as described below.
[0322] Platelets contained in a liquid medium were isolated by applying centrifugation filtration. The liquid medium was replaced with the buffer Formulation B (see Table 2), such that the platelets were suspended in the buffer Formulation B.
[0323] The platelet composition were subjected to different low temperature for 12 hours to form the cryopreserved platelets. The different temperatures included: (i) 80 C. following a gradual 1 C./minute temperature rate of adjustment to 70 C. (Thawed), (ii) 80 C. (80C Thawed), or (iii) 20 C. (20 C Thawed). The 80 C Thawed and 20 C Thawed samples were placed immediately in the low temperature condition without any gradual temperature adjustment.
[0324] After the freezing process, the platelet compositions were stored at 80 C for at least 24 hours prior to testing.
[0325] Prior to testing, the cryopreserved platelet compositions were thawed in a 37 C. water bath.
[0326] B. Platelet Count
[0327] The DMSO-free platelet compositions suspended in Buffer Formulation B were tested for a platelet count (AcT Diff Counts) using an automated Coulter AcT Diff system.
TABLE-US-00005 TABLE 5 Platelet AcT Count AcT Count (10.sup.3 platelets/l) Product Post-Processing Thawed Yield Thawed 1677 1625 97% 80 C. Thawed 1677 1675 100% 20 C. Thawed 1677 1300 78%
[0328] Table 5 provides platelet counts by AcT Diff cell counter of the exemplary compositions that were subject to the different temperature conditions.
[0329] The platelet composition subjected to 20 C. (20 C Thawed) exhibited the lowest platelet yield after thawing. The remaining compositions (Thawed, 80 C Thawed) were comparable close in yield, each having a >90% platelet recovery after thawing.
[0330] C. Particle Size Distribution By Flow Cytometry Forward Scattering
[0331] Flow Cytometry Forward Scattering was performed on the exemplary DMSO-free platelet composition (i) prior to freezing (Pretreat); and on the thawed exemplary DMSO-free platelet compositions that were frozen at (ii) 80 C. following a 1 C./minute temperature adjustment to 70 C. (Thawed), (iii) 80 C. (80 C Thawed), or (iv) 20 C. (20 C Thawed).
[0332]
[0333] The exemplary platelet composition that was subjected to a temperature 20 C. (20 C Thawed) exhibited the largest size deviation, as compared to the initial platelet size distribution (Pretreat). The 20 C. Thawed platelet composition had a greater proportion of micro-particles than any of the other test groups (Pretreat, Thawed, 80 C Thawed).
[0334] D. Particle Size Distribution By Thrombolux Dynamic Light Scattering
[0335] Thrombolux dynamic light scattering testing determines size distributions for the DMSO-free platelet compositions (i) prior to freezing (Pretreat); and for the thawed exemplary DMSO-free platelet compositions that were frozen at (ii) 80 C. following a 1 C./minute temperature adjustment to 70 C. (Thawed), (iii) 80 C. (80 C Thawed), or (iv) 20 C. (20 C Thawed).
[0336]
TABLE-US-00006 TABLE 6 Thrombolux Dynamic Light Scattering Test Data Blue Blue peak Cyan Cyan peak intensity radius Blue intensity radius Product [%] [nm] PDI [%] [nm] Pretreat 100% 1198 0.3 0% 10 Thawed 85% 1158 0.5 14% 210 80 C. Thawed 91% 1101 0.5 8% 118 20 C. Thawed 80% 847 0.5 19% 132
[0337] Table 6 provides the Thrombolux size data for the exemplary DMSO-free platelet composition (i) prior to freezing (Pretreat); and on the thawed exemplary DMSO-free platelet compositions that were frozen at (ii) 80 C. following a 1 C./minute temperature adjustment to 70 C. (Thawed), (iii) 80 C. (80 C Thawed), or (iv) 20 C. (20 C Thawed). As discussed previously, Blue intensity represents the percent of measured particles that fall within the platelet size region; Blue peak radius gives the mean size of that population; and Blue PDI gives the polydispersity, or spread, of the population. The Cyan intensity represents the percent of measured particles that fall below the platelet size; and Cyan peak radius provides the mean size of that population.
[0338] As shown in Table 6, the platelet composition that was frozen at 20 C. (20 C Thawed) exhibited the most significant size difference, as compared to the initial platelets (Pretreat). Only 80% of the particles were in the platelet region (vs. an initial 100%), and the particles had a significantly smaller mean radius. The other platelet compositions subjected to different freezing conditions (80 C Thawed, Thawed) exhibit results similar to the initial platelets (Pretreat).
[0339] E. Platelet Aggregation Testing
[0340] Aggregometry testing was performed on the exemplary DMSO-free platelet composition (i) prior to freezing (Pretreat); and on the thawed exemplary DMSO-free platelet compositions that were frozen at (ii) 80 C. following a 1 C./minute temperature adjustment to 70 C. (Thawed), (iii) 80 C. (80 C Thawed), or (iv) 20 C. (20 C Thawed).
[0341]
[0342] The platelet composition that was frozen at 20 C. (20 C Thawed) exhibited the lowest response in every category (thrombin, collagen, and arachidonic acid) and deviated the most from the initial platelets (Pretreat). The remaining platelet compositions (80 C Thawed, Thawed) exhibited similar results as the initial platelets (Pretreat) when responding to thrombin, but exhibited a lower response as compared to the initial platelets (Pretreat) when responding to collagen and arachidonic acid.
[0343] F. Summary
[0344] Gradual and rapid freezing of the exemplary DMSO-free platelet compositions at 80 C. yielded better platelet function as measured by metrics: size distribution, yield, and aggregation, as compared to the rapid freezing of DMSO-free platelet compositions at 20 C.
Example 3: Platelet Function Metrics of DMSO-Free Platelet Compositions
[0345] This example provides exemplary compositions containing no DMSO when subjected to a low temperature condition in different sealed containers.
[0346] A. Sample Preparation
[0347] Exemplary platelet compositions were prepared as described below. Platelets contained in a liquid medium were isolated by applying centrifugation filtration. The liquid medium was replaced with buffer Formulation B (see Table 2), and platelets were suspended in the buffer Formulation B. The exemplary platelet compositions with Formulation B contained no DMSO.
[0348] The platelet compositions were frozen at 80 C. in fluorinated ethylene propylene (FEP) bags, polyvinyl chloride (PVC) bags, or in borosilicate serum vials to form cryopreserved platelet compositions.
[0349] Prior to testing, the cryopreserved platelet compositions were thawed in a 37 C. water bath.
[0350] B. Platelet Count
[0351] Each of the platelet compositions frozen in a FEP bag, a PVC bag, or a borosilicate vial were tested for platelet count (AcT Diff Counts) using an automated Coulter AcT Diff system.
TABLE-US-00007 TABLE 7 Platelet AcT Count AcT Count (10{circumflex over ()}3 platelets/l) Product Post-Processing Thawed Yield Prelyo 1850 80 C. St Gobain Thawed 1850 1883 102% 80 C. Terumo Thawed 1850 1857 100% 80 C. Vial Thawed 1850 1857 101%
[0352] In table 7, St Gobain and Terumo refer to the type of container, such as a bag or vial, used to store the samples at the desired temperature. Examples are described at https://www.terumobet.com/blood-center-services/blood-bags, and at https://cellgenix.com/wp-content/uploads/2017/09Kryosure_Kryovue_Cryopreservation_Bochure_CGX.pdf, both of which are incorporated by reference herein in their entirety.
[0353] Table 7 provides the platelet counts obtained by the AcT Diff cell counter for the exemplary compositions frozen in the different sealed containers (the FEP bag, the PVC bag, or the borosilicate vial).
[0354] The different sealed containers provided very similar yields, which were nearly identical to the pre-freezing (post-processing) yields.
[0355] C. Particle Size Distribution By Flow Cytometry Forward Scattering
[0356] Flow Cytometry Forward Scattering was performed on the platelet compositions prior to freezing (Pretreat) and for each of the platelet compositions frozen in the FEP bag, the PVC bag, or the borosilicate vial.
[0357] As shown in
[0358] D. Particle Size Distribution By Thrombolux Dynamic Light Scattering
[0359] Thrombolux dynamic light scattering testing determined the particle size distributions for the formulated product prior to freezing (Pretreat) and for product frozen in the FEP bag, the PVC bag, or the borosilicate vial.
[0360]
TABLE-US-00008 TABLE 8 Thrombolux Dynamic Light Scattering Test Data Blue Blue peak Cyan Cyan peak intensity radius Blue intensity radius Product [%] [nm] PDI [%] [nm] Pretreat 99% 1312 0.3 1% 21 FEP Bag 95% 1288 0.4 4% 105 PVC Bag 94% 1231 0.4 5% 118 Borosilicate 94% 1225 0.5 5% 93 Vial
showing the size distributions of the different test groups. The size distribution data, as shown, was consistent with the flow cytometry data (see Section B, supra). All of the compositions were similar in size distribution.
[0361] Table 8 provides the Thrombolux size data for the exemplary platelet compositions prior to freezing (Pretreat) and for product frozen in the FEP bag, the PVC bag, or the borosilicate vial. As discussed previously, Blue intensity represents the percent of measured particles that fall within the platelet size region; Blue peak radius gives the mean size of that population; and Blue PDI gives the polydispersity, or spread, of the population. The Cyan intensity represents the percent of measured particles that fall below the platelet size; and Cyan peak radius provides the mean size of that population.
[0362] The results showed that the different containers had a minimal effect on platelet particle size.
[0363] E. Platelet Aggregation Testing
[0364] The platelet and platelet compositions were subjected to aggregometry testing.
[0365]
[0366] The results showed that the different sealed containers did not impact the aggregation response of the final product.
[0367] F. Summary
[0368] The type of sealed container used for containing compositions during freezing did not significantly impact the characteristics of the exemplary DMSO-free platelet compositions.
Example 4: Platelet Function Metrics of Platelet Compositions Containing Low Amounts of DMSO and/or Glycerol
[0369] A. Sample Preparation
[0370] Exemplary cryopreserved platelet compositions were prepared as described herein.
[0371] Platelets, contained in a liquid medium, obtained were untreated, human apheresis platelet material (hIDSP). For all tested compositions, the apheresis platelets were isolated by applying centrifugation and tangential flow filtration. Centrifugation was carried out at 1500 g for 20 minutes, with slow acceleration and braking. Following the centrifugation process, supernatant plasma from the apheresis platelets was aspirated and disposed. Tangential flow filtration was carried out with a 0.45 m filter for >3 diavolumes.
[0372] For the cryopreserved platelet compositions, the original liquid medium was replaced with a buffer Formulations A and B (see Example 1, Tables 1 and 2 of Section A, supra), Formulation C (see Table 9 below), or Formulation D (see Table 10 below) to form the following exemplary platelet compositions: [0373] a) hIDSP ThawedA platelet composition containing isolated, untreated platelets. This composition serves as a control in this experiment. [0374] b) Std LBA platelet composition with Formulation B, which contains no DMSO. [0375] c) LB+1% DMSOA platelet composition with Formulation A, which contains DMSO, 1.0% (v/v). [0376] d) LB+1% DMSO/GlycerolA platelet composition (LB+1% DMSO/Glycerol) with Formulation C, which contains DMSO, 1.0% (v/v) and glycerol, 1.0% (v/v). [0377] e) LB+1% GlycerolA platelet composition (LB+1% Glycerol) with Formulation D, which contains glycerol, 1.0% (v/v).
TABLE-US-00009 TABLE 9 Components of Buffer Formulation C Excipient Concentration NaCl 3.51 mg/ml Kcl 0.29 mg/ml HEPES 1.81 mg/ml NaHCO3 0.81 mg/ml Dextrose 0.43 mg/ml Trehalose 30.27 mg/ml Ficoll PM 400 60.00 mg/ml Polysucrose Ethanol 0.8% (v/v) DMSO 1.0% (v/v) Glycerol 1.0% (v/v) Water Balance
TABLE-US-00010 TABLE 10 Components of Buffer Formulation D Excipient Concentration NaCl 3.51 mg/ml Kcl 0.29 mg/ml HEPES 1.81 mg/ml NaHCO3 0.81 mg/ml Dextrose 0.43 mg/ml Trehalose 30.27 mg/ml Ficoll PM 400 60.00 mg/ml Polysucrose Ethanol 0.8% (v/v) Glycerol 1.0% (v/v) Water Balance
[0378] Each of the platelet compositions contained a concentration of 210.sup.9 platelets/ml.
[0379] Each of the platelet compositions, with exception of the untreated platelets (hIDSP), were incubated during recirculation in plastic bottles and tubing at room temperature.
[0380] Following incubation, the platelet compositions (except the untreated platelets (hIDSP)) were transferred to a bag appropriate for freezing. The platelets compositions were aliquoted into each bag such that the platelet dose size was about 310.sup.11 platelets per bag.
[0381] The bags containing each of the platelet compositions, with exception of the untreated platelets (hIDSP), were loaded into freezer at a temperature of 80 C. The bags were loaded directly onto the freezer shelf such that the bags made full contact with the shelf and were allowed to equilibrate for >24 hours.
[0382] Prior to testing, the platelet compositions, except for the untreated platelets (hIDSP), were thawed in a 37 C. water bath for 8-10 minutes.
[0383] B. Platelet Count
[0384] The untreated platelets and the cryopreserved platelet compositions were tested for platelet count (AcT Diff Count) using an automated Coulter AcT Diff system.
TABLE-US-00011 TABLE 11 Platelet AcT Count AcT Count (10.sup.3 platelets/l) Product Post-Processing Thawed Yield Std LB 1930 1837 95% LB + 1% DMSO 1997 1827 91% LB + 1% Glycerol 1957 1830 94% LB + 1% DMSO/ 1920 1890 98% Glycerol
[0385] Table 11 provides platelet counts obtained using the AcT Diff cell counter for each of the exemplary platelet compositions of isolated platelets before freezing (Post-Processing) and after being thawing (Thawed).
[0386] The platelet compositions containing buffer Formulation A (w/1% DMSO), Formulation C (w/1% DMSO and glycerol), and Formulation D (w/1% glycerol), each had >90% platelet recovery after thawing, with the platelet composition containing buffer Formulation C having the highest platelet recovery (98%).
[0387] C. Particle Size Distribution By Flow Cytometry Forward Scattering
[0388] Flow Cytometry Forward Scattering was performed on exemplary platelet compositions to determine platelet size distributions. The tested compositions included the (i) untreated platelets (hIDSP); and the cryopreserved platelet compositions, including: (ii) the DMSO-free platelet composition (Std LB); (iii) the platelet composition with 1% DMSO (LB+1% DMSO); (iv) the platelet composition with 1% DMSO and 1% glycerol (LB+1% DMSO/Glycerol); and (v) the platelet composition with 1% glycerol (LB+1% glycerol).
[0389]
[0390] The data demonstrated that the cryopreserved compositions, after being thawed, generally exhibited similar results to another, when compared to the initial platelet size distribution (hIDSP). The platelet compositions containing 1% DMSO (LB+1% DMSO, LB+1% DMSO/Glycerol) showed platelet size distributions that were slightly more similar to the untreated platelets (hIDSP).
[0391] D. Particle Size Distribution By Thrombolux Dynamic Light Scattering
[0392] Thrombolux dynamic light scattering testing was conducted to determine size distributions for the untreated platelets (hIDSP), and each of the cryopreserved platelet compositions.
[0393]
TABLE-US-00012 TABLE 12 Thrombolux Dynamic Light Scattering Test Data Blue Blue peak Cyan Cyan peak intensity radius Blue intensity radius Product [%] [nm] PDI [%] [nm] hIDSP 98% 1285 0.2 2% 52 Std LB 97% 1160 0.5 3% 83 LB + 1% DMSO 98% 990 0.4 1% 52 LB + 1% glycerol 97% 829 0.4 2% 52 LB w/1% DMSO/ 98% 1017 0.4 2% 59 glycerol
[0395] Table 12 provides the Thrombolux size data obtained for the untreated platelets (hIDSP) and the thawed cryopreserved platelet formulations. Blue intensity represents the percent of measured particles that fall within the platelet size region; Blue peak radius gives the mean size of that population; and Blue PDI gives the polydispersity, or spread, of the population. The Cyan intensity represents the percent of measured particles that fall below the platelet size; and Cyan peak radius provides the mean size of that population.
[0396] The results provided in Table 12 demonstrated that each of the cryopreserved platelet compositions exhibited similar size distribution data.
[0397] It can be noted that the size distribution data, as provided in
[0398] E. Activation Assessment (CD62P Expression)
[0399] The untreated platelets and cryopreserved platelet compositions were subjected to a platelet activation test to determine whether the compositions showed observable reactivity to a CD62P antibody, when using flow cytometry. In particular, the platelet compositions were observed for their reactivity to the CD62P antibody when assayed by fluorescence using a BD Accuri C6 Plus, using BD Accuri C6 Plus Software version 1.0.23.1.
[0400]
[0401] The results indicate that each of the cryopreserved platelet compositions exhibited a similar percent of activation for CD62P expression. Thus the presence of cryoprotectants such DMSO and/or glycerol do not affect the platelet activation percentage of CD62P expression.
[0402] F. Platelet Aggregation Testing
[0403] The untreated platelets and cryopreserved platelet compositions were subjected to aggregometry testing, as described in previous sections (see e.g., Example 1, Section E), when using thrombin, collagen, or arachidonic acid as stimulating substances to induce the platelet aggregation.
[0404]
[0405] The test results showed no significant difference in percent aggregation in thrombin between the different cryopreserved platelet compositions. The test results, however, showed that platelet composition containing no DMSO (Std LB) exhibited the least amount of percent aggregation in collagen and arachidonic acid, as compared to the other cryopreserved platelet compositions, which contained DMSO (only), glycerol (only) or both DMSO and glycerol. The platelet compositions containing DMSO (LB+1% DMSO, LB+1% DMSO/glycerol) exhibited the highest percent of platelet aggregation by collagen and arachidonic acid.
[0406] G. Summary
[0407] Based on the results provided in Sections B-F (supra), the composition containing DMSO with glycerol (LB+1% DMSO/glycerol), exhibited slightly better platelet function, based on the size distribution, yield, and aggregation test data, as compared to untreated platelets (hIDSP) and all of the other platelet compositions (STD LB, LB+1% DMSO, LB+1% glycerol). The composition containing DMSO only (LB+1% DMSO) performed slightly better than the composition containing glycerol only (LB+1% glycerol) and the standard loading buffer (STD LB). The composition containing glycerol only (LB+1% glycerol) exhibited size distribution and platelet yields that were most similar to the standard loading buffer (STD LB).
[0408] While the embodiments of the invention are amenable to various modifications and alternative forms, specific embodiments have been shown by way of example in the drawings and are described in detail below. The intention, however, is not to limit the invention to the particular embodiments described. On the contrary, the invention is intended to cover all modifications, equivalents, and alternatives falling within the scope of the invention as defined by the appended claims.