Materials and methods for producing blood products

Abstract

To provide materials and methods for the preparation of blood products.SOLUTION: In one aspect, provided herein is a composition including platelets or platelet derivatives and an aqueous medium, wherein the aqueous medium has a protein concentration less than 50% of the protein concentration of donor apheresis plasma.SELECTED DRAWING: None

Description

Technical Field

[0001] Cross - Reference to Related Applications This application claims the benefit of U.S. Provisional Patent Application No. 62 / 843,061, filed May 3, 2019, and U.S. Provisional Patent Application No. 62 / 936,122, filed November 15, 2019, the entire contents of each application being incorporated herein by reference.

[0002] Statement Regarding Government Interests This invention was made with government support under Contract No. HHSO100201300021, authorized by the Biomedical Advanced Research and Development Authority (BARDA) of the U.S. Department of Health and Human Services. The government has certain rights in this invention.

[0003] Technical Field This disclosure generally relates to blood products, e.g., blood products with reduced HLA antibody content, and methods of producing such blood products.

Background Art

[0004] Background Blood is a complex mixture composed of a number of components. Generally, blood can be described as containing four main parts: red blood cells, white blood cells, platelets, and plasma. The first three are cells or cell - like components, while the fourth, plasma, is a liquid component containing a wide - ranging and variable mixture of salts, proteins, and other factors necessary for many body functions. The components of blood can be separated from each other in various ways. Generally, differential centrifugation is currently the most commonly used to separate the different components in blood based on size and, in some applications, based on density.

[0005] Inactivated platelets (commonly also referred to as thrombocytes) are small, often irregularly shaped (e.g., discoid or oval) blood components derived from megakaryocytes and are involved in the blood coagulation process. Platelets help protect the body from excessive blood loss not only due to trauma or injury but also from excessive blood loss during normal physiological activities. Platelets are essential for normal hemostasis and are thought to be the first line of defense against blood leakage from damaged blood vessels. Generally, platelets adhere to the inner layer of a ruptured blood vessel, change to an amorphous shape during activation, and function by interacting with components of the coagulation system that are present in plasma or released by the platelets themselves or other blood components. Purified platelets are used in the treatment of thrombocytopenia (a decrease in platelet count) and platelet dysfunction (such as thrombasthenia). Concentrated platelets are often used to control bleeding after injury or bleeding during acquired platelet function disorders or deficiencies (e.g., those occurring during surgery and those due to the presence of platelet inhibitors).

Summary of the Invention

[0006] Summary This specification is based, at least in part, on the production of a blood product (e.g., a composition comprising platelets or platelet derivatives (e.g., thrombosomes)) with reduced levels of free proteins (e.g., antibodies (e.g., human leukocyte antigen (HLA) antibodies, or human neutrophil antigen (HNA) antibodies)).

[0007] Provided herein is a composition comprising platelets and an aqueous medium, wherein the aqueous medium has a protein concentration of less than 50% of the protein concentration of donor apheresis plasma.

[0008] The implementation form may have one or more of the following features. The protein concentration of the aqueous medium may be less than 30% of the protein concentration of the donor apheresis plasma. The human leukocyte antigen (HLA) class I antibody concentration of the aqueous medium may be less than 30% of the HLA class I antibody concentration in the donor apheresis plasma. The human leukocyte antigen (HLA) class II antibody concentration of the aqueous medium may be less than 30% of the HLA class II antibody concentration in the donor apheresis plasma. The HNA antibody concentration of the aqueous medium may be less than 30% of the human neutrophil antigen (HNA) antibody concentration in the donor apheresis plasma. The protein concentration may be less than 10% of the protein concentration of the donor apheresis plasma. The human HLA class I antibody concentration of the aqueous medium may be less than 10% of the HLA class I antibody concentration in the donor apheresis plasma. The human HLA class II antibody concentration of the aqueous medium may be less than 10% of the HLA class II antibody concentration in the donor apheresis plasma. The human HNA antibody concentration of the aqueous medium may be less than 10% of the HNA antibody concentration in the donor apheresis plasma. The protein concentration may be less than 5% of the protein concentration of the donor apheresis plasma. The human HLA class I antibody concentration of the aqueous medium may be less than 5% of the HLA class I antibody concentration in the donor apheresis plasma. The human HLA class II antibody concentration of the aqueous medium may be less than 5% of the HLA class II antibody concentration in the donor apheresis plasma. The human HNA antibody concentration of the aqueous medium may be less than 5% of the HNA antibody concentration in the donor apheresis plasma. The protein concentration may be less than 3% of the protein concentration of the donor apheresis plasma. The aqueous medium may be the concentration of human HLA class I antibody that is less than 3% of the HLA class I antibody concentration in the donor apheresis plasma. The human HLA class II antibody concentration of the aqueous medium may be less than 3% of the HLA class II antibody concentration in the donor apheresis plasma. The human HNA antibody concentration of the aqueous medium may be less than 3% of the HNA antibody concentration in the donor apheresis plasma. The protein concentration may be less than 1% of the protein concentration of the donor apheresis plasma. The human HLA class I antibody concentration of the aqueous medium may be less than 1% of the HLA class I antibody concentration in the donor apheresis plasma.The concentration of human HLA class II antibodies in the aqueous medium can be less than 1% of the HLA class II antibody concentration in donor apheresis plasma. The concentration of human HNA antibodies in the aqueous medium can be less than 1% of the HNA antibody concentration in donor apheresis plasma. The protein concentration can be quantified by absorbance at 280 nanometers (nm) using a path length of 0.5 cm. In some embodiments, the absorbance at 280 nm can be less than 1.7 AU. In some embodiments, the absorbance at 280 nm can be less than 1.66 AU. In some embodiments, the absorbance at 280 nm can be less than 1.6 AU. In some embodiments, the platelet count can be at least 200×10. 3 Platelets / μL. In some embodiments, the platelet count can be at least 2250×10 3 Platelets / μL. In some embodiments, the number of red blood cells in the composition can be 0.2×10 6 Red blood cells / μL or less. In some embodiments, the composition can further contain red blood cells. In some embodiments, the number of red blood cells can be 0.2×10 6It can be less than [[X]] red blood cells / μL. The composition can be negative for HLA class I antibodies based on tests approved by the regulatory agency. The composition can be negative for HLA class II antibodies based on tests approved by the regulatory agency. The composition can be negative for HNA antibodies based on tests approved by the regulatory agency. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be less than 5% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be less than 3% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be less than 1% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for HLA class I antibodies can be less than 5% in the quantification of the composition by flow cytometry using beads coated with class I HLA. The percentage of beads positive for HLA class I antibodies can be less than 3% in the quantification of the composition by flow cytometry using beads coated with class I HLA. The percentage of beads positive for HLA class I antibodies can be less than 1% in the quantification of the composition by flow cytometry using beads coated with class I HLA. The percentage of beads positive for HLA class II antibodies can be less than 5% in the quantification of the composition by flow cytometry using beads coated with class II HLA.The percentage of beads positive for HLA class II antibodies can be less than 3% in the quantification of the composition by flow cytometry using beads coated with class II HLA. The percentage of beads positive for HLA class II antibodies can be less than 1% in the quantification of the composition by flow cytometry using beads coated with class II HLA. The percentage of beads positive for HNA antibodies can be less than 5% in the quantification of the composition by flow cytometry using beads coated with HNA. The percentage of beads positive for HNA antibodies can be less than 3% in the quantification of the composition by flow cytometry using beads coated with HNA. The percentage of beads positive for HNA antibodies can be less than 1% in the quantification of the composition by flow cytometry using beads coated with HNA. The aqueous medium can further comprise a buffer, a base, a filler, optionally a salt, and optionally at least one organic solvent. The buffer can be HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The base can be sodium bicarbonate. The filler can be a monosaccharide, a polysaccharide, or a combination thereof. The monosaccharide can be selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose. The monosaccharide can be trehalose. The polysaccharide can be polysucrose. The salt can be sodium chloride, potassium chloride, or a combination thereof. The organic solvent can be selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), and combinations thereof. The composition can be prepared by a process comprising tangential flow filtration (TFF) of a starting material containing platelets, centrifugation of a starting material containing platelets, or a combination thereof.The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be reduced by at least 50% compared to similar compositions not prepared by a process comprising tangential flow filtration of the blood product composition, centrifugation of the blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be reduced by at least 75% compared to similar compositions not prepared by a process comprising tangential flow filtration of the blood product composition, centrifugation of the blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be reduced by at least 90% compared to similar compositions not prepared by a process comprising tangential flow filtration of the blood product composition, centrifugation of the blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be reduced by at least 95% compared to similar compositions not prepared by a process comprising tangential flow filtration of the blood product composition, centrifugation of the blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.The starting material can be (a) positive for HLA class I antibodies based on tests approved by the regulatory agency, (b) positive for HLA class II antibodies based on tests approved by the regulatory agency, (c) positive for HNA antibodies based on tests approved by the regulatory agency, or (d) one or more of (a), (b), and (c). The protein concentration of the starting material can be from about 60 to about 80 mg / ml. The starting material can include donor blood products. The donor blood products can be pooled donor blood products. The starting material can include donor apheresis materials. TFF can include concentration. TFF may include diafiltration. Diafiltration may include diafiltration at at least 2 diavolumes. TFF may include buffer exchange. TFF may be performed using a membrane with a pore size of about 0.2 μm to about 1 μm. TFF may be performed using a membrane with a pore size of about 0.2 μm to about 0.45 μm. TFF can be carried out at a temperature of about 20 °C to about 37 °C. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 50% of the absorbance of the starting material at 280 nm. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 30% of the absorbance of the starting material at 280 nm. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 10% of the absorbance of the starting material at 280 nm. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 5% of the absorbance of the starting material at 280 nm. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 3% of the absorbance of the starting material at 280 nm. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 1% of the absorbance of the starting material at 280 nm. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 1.70 AU. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 1.66 AU. TFF can be performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 1.60 AU. TFF can be carried out until the platelet concentration is at least about 2000×10 3 platelets / μL. TFF can be carried out until the platelet concentration is at least about 2250×10 3It can be carried out until it reaches platelets / μL. TFF can include buffer exchange into a buffer solution containing a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent. The buffer can be HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The base can be sodium bicarbonate. The loading agent can be a monosaccharide, a polysaccharide, or a combination thereof. The monosaccharide can be selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose. The monosaccharide can be trehalose. The polysaccharide can be polysucrose. The salt can be sodium chloride, potassium chloride, or a combination thereof. The organic solvent can be selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), and combinations thereof. Centrifugation can include centrifugation at 1400×g to about 1550×g. Centrifugation can include centrifugation at 1450×g to about 1500×g. The process may lack centrifugation of the composition containing platelets. The composition can contain less than 5.0% of fine particles (by scattering intensity). The composition can contain less than 4.5% of fine particles (by scattering intensity). The composition can contain less than 4.0% of fine particles (by scattering intensity). The composition can contain less than 3.5% of fine particles (by scattering intensity). The CD41 positive percentage of platelets or platelet derivatives can be at least 55%. The CD41 positive percentage of platelets or platelet derivatives can be at least 60%. The CD41 positive percentage of platelets or platelet derivatives can be at least 65%. The CD42 positive percentage of platelets or platelet derivatives can be at least 80%. The CD42 positive percentage of platelets or platelet derivatives can be at least 85%. The CD42 positive percentage of platelets or platelet derivatives can be at least 90%. Platelets or platelet derivatives can retain at least about 10% of the lactate dehydrogenase activity of donor apheresis platelets.Platelets or platelet derivatives can retain at least about 15% of the lactate dehydrogenase activity of donor apheresis platelets. Platelets or platelet derivatives can retain at least about 20% of the lactate dehydrogenase activity of donor apheresis platelets. The percentage of annexin V positive in platelets or platelet derivatives can be at least 70%. The percentage of annexin V positive in platelets or platelet derivatives can be at least 75%. The percentage of annexin V positive in platelets or platelet derivatives can be at least 80%. The percentage of CD47 positive in platelets or platelet derivatives can be at least 8%. The percentage of CD47 positive in platelets or platelet derivatives can be at least 10%. The percentage of CD47 positive in platelets or platelet derivatives can be at least 15%. The percentage of CD47 positive in platelets or platelet derivatives can be at least 20%. The percentage of CD62 positive in platelets or platelet derivatives can be at least 80%. The percentage of CD62 positive in platelets or platelet derivatives can be at least 82%. The percentage of CD62 positive in platelets or platelet derivatives can be at least 85%. The percentage of CD62 positive in platelets or platelet derivatives can be at least 90%. Platelets or platelet derivatives can have fibrinogen associated with the cell membrane. The lactate concentration of the aqueous medium can be less than 2.0 mmol / L. The lactate concentration of the aqueous medium can be less than 1.5 mmol / L. The lactate concentration of the aqueous medium can be from about 0.4 to about 1.3 mmol / L. The lactate concentration of the aqueous medium can be from about 0.5 to about 1.0 mmol / L. The platelet derivative can contain thrombosomes.

[0009] Also provided herein is a process for preparing a composition comprising platelets and an aqueous medium, the process comprising tangential flow filtration (TFF) of a starting material comprising platelets, centrifugation of a starting material comprising platelets, or a combination thereof, wherein the aqueous medium has a protein concentration of less than 50% of the protein concentration of donor apheresis plasma.

[0010] The implementation form may include one or more of the following features. The starting material may be positive for HLA class I antibodies based on tests approved by a regulatory agency, (b) positive for HLA class II antibodies based on tests approved by a regulatory agency, (c) positive for HNA antibodies based on tests approved by a regulatory agency, or (d) one or more of a), b), and c). The protein concentration of the starting material may be from about 60 to about 80 mg / mL. The starting material may include donor blood products. The donor blood product may be a pooled donor blood product. The starting material may include donor apheresis material. TFF may include concentration. TFF may include diafiltration. Diafiltration may include diafiltration with at least 2 diavolumes. TFF may include buffer exchange. TFF may be performed using a membrane with a pore size of about 0.2 μm to about 1 μm. TFF may be performed using a membrane with a pore size of about 0.2 μm to about 0.45 μm. TFF can be carried out at a temperature of about 20°C to about 37°C. TFF can be carried out using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 50% of the absorbance of the starting material at 280 nm. TFF is carried out using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 30% of the absorbance of the starting material at 280 nm. TFF can be carried out using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 10% of the absorbance of the starting material at 280 nm. TFF can be carried out using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 5% of the absorbance of the starting material at 280 nm. TFF can be carried out using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 3% of the absorbance of the starting material at 280 nm. TFF can be carried out using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 1% of the absorbance of the starting material at 280 nm. TFF can be carried out using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm is less than 1.70 AU.TFF can be carried out using a path length of 0.5 cm until the absorbance at 280 nm of the aqueous medium is less than 1.66 AU. TFF can be carried out using a path length of 0.5 cm until the absorbance at 280 nm of the aqueous medium is less than 1.60 AU. TFF is carried out until the platelet concentration is at least about 2000×10. 3 platelets / μL. TFF is carried out until the platelet concentration is at least about 2250×10 3It can be carried out until it reaches platelets / μL. TFF can include buffer exchange into a buffer solution containing a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent. The buffer can be HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The base can be sodium bicarbonate. The loading agent can be a monosaccharide, a polysaccharide, or a combination thereof. The monosaccharide can be selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose. The monosaccharide can be trehalose. The polysaccharide can be polysucrose. The salt can be sodium chloride, potassium chloride, or a combination thereof. The organic solvent can be selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), and combinations thereof. Centrifugation can include centrifugation at 1400×g to about 1550×g. Centrifugation can include centrifugation at 1450×g to about 1500×g. The process may lack centrifugation of the composition containing platelets. The percentage of beads that are positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 50% reduced compared to a similar composition not prepared by a process including tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be reduced by at least 75% compared to similar compositions not prepared by a process comprising tangential flow filtration of the blood product composition, centrifugation of the blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be reduced by at least 90% compared to similar compositions not prepared by a process comprising tangential flow filtration of the blood product composition, centrifugation of the blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be reduced by at least 95% compared to similar compositions not prepared by a process comprising tangential flow filtration of the blood product composition, centrifugation of the blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The protein concentration can be less than 30% of the protein concentration of donor apheresis plasma. The human leukocyte antigen (HLA) class I antibody concentration in the aqueous medium can be less than 30% of the human leukocyte antigen (HLA) class I antibody concentration in donor apheresis plasma. The human leukocyte antigen (HLA) class II antibody concentration in the aqueous medium can be less than 30% of the human leukocyte antigen (HLA) class II antibody concentration in donor apheresis plasma. The HNA antibody concentration in the aqueous medium can be less than 30% of the human neutrophil antigen (HNA) antibody concentration in donor apheresis plasma. The protein concentration can be less than 10% of the protein concentration of donor apheresis plasma.The concentration of human HLA class I antibodies in the aqueous medium can be less than 10% of the HLA class I antibody concentration in the donor apheresis plasma. The concentration of human HLA class II antibodies in the aqueous medium can be less than 10% of the HLA class II antibody concentration in the donor apheresis plasma. The concentration of human HNA antibodies in the aqueous medium can be less than 10% of the HNA antibody concentration in the donor apheresis plasma. The protein concentration can be less than 5% of the protein concentration of the donor apheresis plasma. The concentration of human HLA class I antibodies in the aqueous medium can be less than 5% of the HLA class I antibody concentration in the donor apheresis plasma. The concentration of human HLA class II antibodies in the aqueous medium can be less than 5% of the HLA class II antibody concentration in the donor apheresis plasma. The concentration of human HNA antibodies in the aqueous medium can be less than 5% of the HNA antibody concentration in the donor apheresis plasma. The protein concentration can be less than 3% of the protein concentration of the donor apheresis plasma. The concentration of human HLA class I antibodies in the aqueous medium can be less than 3% of the HLA class I antibody concentration in the donor apheresis plasma. The concentration of human HLA class II antibodies in the aqueous medium can be less than 3% of the HLA class II antibody concentration in the donor apheresis plasma. The concentration of human HNA antibodies in the aqueous medium can be less than 3% of the HNA antibody concentration in the donor apheresis plasma. The protein concentration can be less than 1% of the protein concentration of the donor apheresis plasma. The concentration of human HLA class I antibodies in the aqueous medium can be less than 1% of the HLA class I antibody concentration in the donor apheresis plasma. The concentration of human HLA class II antibodies in the aqueous medium can be less than 1% of the HLA class II antibody concentration in the donor apheresis plasma. The concentration of human HNA antibodies in the aqueous medium can be less than 1% of the HNA antibody concentration in the donor apheresis plasma. The composition can be negative for HLA class I antibodies based on tests approved by the regulatory agency. The composition can be negative for HLA class II antibodies based on tests approved by the regulatory agency. The composition can be negative for HNA antibodies based on tests approved by the regulatory agency.The percentage of beads that are positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be less than 5% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be less than 3% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies can be less than 1% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. The percentage of beads positive for HLA class I antibodies can be less than 5% in the quantification of the composition by flow cytometry using beads coated with class I HLA. The percentage of beads positive for HLA class I antibodies can be less than 3% in the quantification of the composition by flow cytometry using beads coated with class I HLA. The percentage of beads positive for HLA class I antibodies can be less than 1% in the quantification of the composition by flow cytometry using beads coated with class I HLA. The percentage of beads positive for HLA class II antibodies can be less than 5% in the quantification of the composition by flow cytometry using beads coated with class II HLA. The percentage of beads positive for HLA class II antibodies can be less than 3% in the quantification of the composition by flow cytometry using beads coated with class II HLA. The percentage of beads positive for HLA class II antibodies can be less than 1% in the quantification of the composition by flow cytometry using beads coated with class II HLA. The percentage of beads positive for HNA antibodies can be less than 5% in the quantification of the composition by flow cytometry using beads coated with HNA. The percentage of beads positive for HNA antibodies can be less than 3% in the quantification of the composition by flow cytometry using beads coated with HNA.The percentage of beads that are positive for HNA antibodies can be less than 1% in the quantification of the composition by flow cytometry using beads coated with HNA. The process according to any one of claims 125 to 199, wherein the composition contains less than 5.0% (by scattered intensity) of microparticles. The composition can contain less than 4.5% (by scattered intensity) of microparticles. The composition can contain less than 4.0% (by scattered intensity) of microparticles. The composition can contain less than 3.5% (by scattered intensity) of microparticles. The CD41 positive percentage of platelets or platelet derivatives can be at least 55%. The CD41 positive percentage of platelets or platelet derivatives can be at least 60%. The CD41 positive percentage of platelets or platelet derivatives can be at least 65%. The CD42 positive percentage of platelets or platelet derivatives can be at least 80%. The CD42 positive percentage of platelets or platelet derivatives can be at least 85%. The CD42 positive percentage of platelets or platelet derivatives can be at least 90%. Platelets or platelet derivatives can retain at least about 10% of the lactate dehydrogenase activity of donor apheresis platelets. Platelets or platelet derivatives can retain at least about 15% of the lactate dehydrogenase activity of donor apheresis platelets. Platelets or platelet derivatives can retain at least about 20% of the lactate dehydrogenase activity of donor apheresis platelets. The annexin V positive percentage of platelets or platelet derivatives can be at least 70%. The annexin V positive percentage of platelets or platelet derivatives can be at least 75%. The annexin V positive percentage of platelets or platelet derivatives can be at least 80%. The CD47 positive percentage of platelets or platelet derivatives can be at least 8%. The CD47 positive percentage of platelets or platelet derivatives can be at least 10%. The CD47 positive percentage of platelets or platelet derivatives can be at least 15%. The CD47 positive percentage of platelets or platelet derivatives can be at least 20%. The CD62 positive percentage of platelets or platelet derivatives can be at least 80%. The CD62 positive percentage of platelets or platelet derivatives can be at least 82%.The CD62 positive percentage of the platelets or platelet derivatives can be at least 85%. The CD62 positive percentage of the platelets or platelet derivatives can be at least 90%. The platelets or platelet derivatives can have fibrinogen associated with the cell membrane. The lactic acid concentration of the aqueous medium can be less than 2.0 mmol / L. The lactic acid concentration of the aqueous medium can be less than 1.5 mmol / L. The lactic acid concentration of the aqueous medium can be from about 0.4 to about 1.3 mmol / L. The lactic acid concentration of the aqueous medium can be from about 0.5 to about 1.0 mmol / L. The platelet derivative can contain thrombosomes. The process can further include a pathogen reduction step. The pathogen reduction step may precede TFF. The process can further include lyophilizing a composition containing platelets or platelet derivatives. The process can further include thermally treating a composition containing platelets or platelet derivatives.

[0011] Also provided herein is a composition comprising platelets prepared by any of the processes described herein and an aqueous medium.

[0012] Also provided herein is a process for preparing freeze-dried platelets, the process comprising: (a) preparing a composition comprising platelets and an aqueous medium using any of the processes described herein; and (b) freeze-drying the composition comprising the platelets and the aqueous medium.

[0013] Also provided herein is a composition comprising freeze-dried platelets prepared by any of the processes described herein.

[0014] Also provided herein is a method for preparing a composition comprising platelets or platelet derivatives and an aqueous medium, the method comprising diluting a starting material containing platelets to form a diluted starting material, and adjusting the platelets to about 2250×10 3 cells / μL (±250×10 3Concentrating into (0) to form a concentrated platelet composition, and washing the concentrated platelet composition with at least 2 dialysate volumes (DV) of a preparation to form a tangential flow filtration (TFF)-treated composition. A method is also provided that includes:

[0015] Embodiments may include one or more of the following features. Diluting may include diluting with a preparation having approximately the same weight (±10%). The method may further include a pathogen reduction step. The pathogen reduction step may be performed before diluting the starting material. The residual plasma percentage may be less than about 15% relative plasma (quantified by plasma protein content). After washing, if the concentration of cells in the TFF-treated composition is not about 2000×10 3 cells / μL (±300×10 3 ), the method may further include diluting or concentrating the preparation to be within this range. The method may further include lyophilizing the TFF-treated composition to form a lyophilized composition. The method may further include treating the lyophilized composition at about 80°C for about 24 hours.

[0016] Also provided herein are compositions comprising platelets or platelet derivatives prepared by any of the methods described herein.

[0017] The materials and methods described herein can provide several advantages. First, it enables the collection of donors who would otherwise have their collection postponed and reduces competition for apheresis materials. [Invention 1001] A process for preparing a composition comprising a platelet or platelet derivative and an aqueous medium, comprising preparing the composition by tangential flow filtration (TFF) of a starting material comprising platelets, a diluted starting material comprising platelets, a concentrated platelet composition, or a combination thereof, wherein the aqueous medium has a protein concentration that is 50% or less of the protein concentration of donor apheresis plasma. Process. [Invention 1002] The process of Invention 1001, further comprising a pathogen reduction step. [Invention 1003] The process of Invention 1002, wherein the pathogen reduction step precedes TFF. [Invention 1004] The process of any one of Inventions 1001 to 1003, wherein the starting material has a protein concentration of about 60 to about 80 mg / mL. [Invention 1005] The process of any one of Inventions 1001 to 1004, wherein TFF includes diafiltration at at least 2 diavolumes. [Invention 1006] The process of any one of Inventions 1001 to 1005, wherein TFF includes diafiltration using a preparation containing a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent. [Invention 1007] The process of any one of Inventions 1001 to 1006, wherein TFF includes buffer exchange with a preparation containing a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent. [Invention 1008] The process of any one of Inventions 1006 to 1007, wherein the preparation includes a buffer containing HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), a base containing sodium bicarbonate, and a loading agent containing trehalose, polysucrose, or a combination thereof. [Invention 1009] The process of any one of Inventions 1006 to 1008, wherein the preparation includes an organic solvent containing ethanol, DMSO, or a combination thereof. [Invention 1010] The process of any one of Inventions 1001 to 1009, wherein the protein concentration of the aqueous medium is 30% or less of the protein concentration of donor apheresis plasma. [Invention 1011] Any of the processes of the present invention 1001 to 1010, wherein the protein concentration of the aqueous medium is 10% or less of the protein concentration of donor apheresis plasma. [The present invention 1012] Any of the processes of the present invention 1001 to 1010, wherein the protein concentration of the aqueous medium is about 5% to about 15% of the protein concentration of donor apheresis plasma. [The present invention 1013] Any of the processes of the present invention 1001 to 1012, wherein the protein concentration of the aqueous medium is about 7% to about 10% of the protein concentration of donor apheresis plasma. [The present invention 1014] Any of the processes of the present invention 1001 to 1013, wherein the composition contains less than 5.0% (by scattering intensity) of fine particles. [The present invention 1015] Any of the processes of the present invention 1001 to 1013, wherein the composition contains less than 4.0% (by scattering intensity) of fine particles. [The present invention 1016] Any of the processes of the present invention 1001 to 1015, further comprising freeze-drying and / or cryopreserving the composition containing platelets or platelet derivatives. [The present invention 1017] Any of the processes of the present invention 1001 to 1016, further comprising thermally treating the composition containing platelets or platelet derivatives. [The present invention 1018] Any of the processes of the present invention 1001 to 1017, wherein the platelets or platelet derivatives have at least 55% CD41 positive percentage. [The present invention 1019] Any of the processes of the present invention 1001 to 1018, wherein the platelets or platelet derivatives have at least 80% CD42 positive percentage. [The present invention 1020] Any of the processes of the present invention 1001 to 1019, wherein the platelets or platelet derivatives have at least 70% annexin V positive percentage. [The present invention 1021] Any process of the present invention 1001 - 1020, wherein the platelet or platelet derivative has at least 8% CD47 positive percentage. [The present invention 1022] Any process of the present invention 1001 - 1021, wherein the platelet or platelet derivative has at least 80% CD62 positive percentage. [The present invention 1023] Any process of the present invention 1001 - 1022, wherein the platelet or platelet derivative has fibrinogen associated with the cell membrane. [The present invention 1024] The platelet or platelet derivative, when at a concentration of about 4.8×10 3 particles / μL, generates a thrombin peak height (TPH) of at least 25 nM in the presence of a reagent containing tissue factor and phospholipid, in any process of the present invention 1001 - 1023. [The present invention 1025] The platelet or platelet derivative 6 has a titer of at least 1.5 thrombin generating potency units (TGPU) per particle, in any process of the present invention 1001 - 1024. [The present invention 1026] The platelet or platelet derivative, when at a concentration of at least about 70×10 3 particles / μL, results in an occlusion time of less than 14 minutes in a total thrombosis analysis system (T - TAS) assay, in any process of the present invention 1001 - 1025. [The present invention 1027] Any process of the present invention 1001 - 1026, wherein the platelet derivative contains thrombosomes. [The present invention 1028] The starting material is (a) positive for HLA class I antibodies based on tests approved by a regulatory agency, (b) positive for HLA class II antibodies based on tests approved by a regulatory agency, (c) positive for HNA antibodies based on tests approved by a regulatory agency, or (d) one or more of (a), (b), and (c). The process according to any one of 1001 to 1027 of the present invention. [The present invention 1029] wherein the composition is (a) negative for HLA class I antibodies based on tests approved by the regulatory authority, (b) negative for HLA class II antibodies based on tests approved by the regulatory authority, (c) negative for HNA antibodies based on tests approved by the regulatory authority, or (d) one or more of (a), (b), and (c), The process according to any one of 1001 to 1028 of the present invention. [The present invention 1030] A composition comprising platelets or platelet derivatives and an aqueous medium, prepared by the process according to any one of 1001 to 1029 of the present invention. [The present invention 1031] A composition comprising platelets or platelet derivatives and an aqueous medium, wherein the aqueous medium has a protein concentration of 50% or less of the protein concentration of donor apheresis plasma. [The present invention 1032] The composition of the present invention 1031, wherein the protein concentration of the aqueous medium is 30% or less of the protein concentration of donor apheresis plasma. [The present invention 1033] The composition of the present invention 1031 or 1032, wherein the protein concentration of the aqueous medium is 10% or less of the protein concentration of donor apheresis plasma. [The present invention 1034] The composition of the present invention 1031 or 1032, wherein the protein concentration of the aqueous medium is about 5% to about 15% of the protein concentration of donor apheresis plasma. [The present invention 1035] The composition according to any one of 1031 to 1034 of the present invention, wherein the protein concentration of the aqueous medium is about 8% to about 10% of the protein concentration of donor apheresis plasma. [The present invention 1036] The composition according to any one of the present inventions 1031 to 1035, wherein the aqueous medium further comprises a buffer, a base, a filler, optionally a salt, and optionally at least one organic solvent. [The present invention 1037] The process according to any one of the present inventions 1031 to 1036, wherein the aqueous medium comprises a buffer containing HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), a base containing sodium bicarbonate, and a filler containing trehalose, polysucrose, or a combination thereof. [The present invention 1038] The process according to any one of the present inventions 1031 to 1037, wherein the aqueous medium comprises an organic solvent containing ethanol, DMSO, or a combination thereof. [The present invention 1039] The composition according to any one of the present inventions 1031 to 1038, containing less than 5.0% of microparticles (by scattering intensity). [The present invention 1040] The composition according to any one of the present inventions 1031 to 1038, containing less than 4.0% of microparticles (by scattering intensity). [The present invention 1041] The composition according to any one of the present inventions 1031 to 1040, wherein the platelet or platelet derivative has at least 55% CD41 positive percentage. [The present invention 1042] The composition according to any one of the present inventions 1031 to 1041, wherein the platelet or platelet derivative has at least 80% CD42 positive percentage. [The present invention 1043] The composition according to any one of the present inventions 1031 to 1042, wherein the platelet or platelet derivative has at least 70% annexin V positive percentage. [The present invention 1044] The composition according to any one of the present inventions 1031 to 1043, wherein the platelet or platelet derivative has at least 8% CD47 positive percentage. [The present invention 1045] The composition according to any one of the present inventions 1031 to 1044, wherein the platelet or platelet derivative has at least 80% CD62 positive percentage. [Invention 1046] Any composition of Inventions 1031 - 1045, wherein the platelet or platelet derivative has fibrinogen associated with the cell membrane. [Invention 1047] Any composition of Inventions 1031 - 1046, wherein the platelet or platelet derivative generates a thrombin peak height (TPH) of at least 50 nM in the presence of a reagent containing tissue factor and phospholipid at a concentration of about 4.8×10 3 particles / μL. [Invention 1048] Any composition of Inventions 1031 - 1047, wherein the platelet or platelet derivative has a titer of at least 1.5 thrombin generating potency units (TPGU) per particle. 6 [Invention 1049] Any composition of Inventions 1031 - 1048, wherein the platelet or platelet derivative results in an occlusion time of less than 14 minutes in a total thrombosis analysis system (T - TAS) assay at a concentration of at least about 70×10 3 particles / μL. [Invention 1050] Any composition of Inventions 1031 - 1049, wherein the platelet derivative contains thrombosomes. [Invention 1051] (a) Negative for HLA class I antibodies based on a regulatory - approved test, (b) Negative for HLA class II antibodies based on a regulatory - approved test, (c) Negative for HNA antibodies based on a regulatory - approved test, or (d) One or more of (a), (b), and (c), Any composition of Inventions 1031 - 1050. [Invention 1052] A method for treating a blood - coagulation - related disease or condition in a subject in need thereof, the method comprising administering a therapeutically effective amount of any composition of Inventions 1030 - 1051 to the subject. [Invention 1053] The method of the present invention 1052, wherein the blood coagulation-related disease or condition is selected from the group consisting of von Willebrand disease, hemophilia, thrombasthenia, thrombocytopenia, thrombocytopenic purpura, trauma, or combinations thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0018]

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DETAILED DESCRIPTION OF THE INVENTION

[0019] As used herein and in the appended claims, the term "platelets" may include whole platelets, fragmented platelets, and platelet derivatives.

[0020] As used in this specification and the appended claims, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a platelet" includes a plurality of such platelets. Further, the use of terms that can be described using equivalent terms includes the use of those equivalent terms. Thus, for example, the use of the term "subject" is to be understood to include "patient", "individual", and other terms used in the art to denote a person to be treated.

[0021] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this terminology belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present disclosure, the preferred methods and materials are described herein. All publications mentioned herein are hereby incorporated by reference for the purpose of disclosing and describing the methods and / or materials associated with the citation of these publications. In case of conflict with any incorporated publication, the present disclosure prevails.

[0022] Detailed Description It is to be understood that the terms used herein are for the purpose of describing particular embodiments only and are not intended to be limiting. Further, when a range of values is disclosed, one of ordinary skill in the art will understand that all other specific values within the disclosed range are inherently disclosed by these values and the ranges represented by these values without the need to individually disclose each specific value or range herein. For example, the disclosed range of 1 to 10 includes 1 to 9, 1 to 5, 2 to 10, 3.1 to 6, 1, 2, 3, 4, 5, etc. Further, each disclosed range includes a value that is at most 5% lower than the lower value within the range and a value that is at most 5% higher than the higher value within the range. For example, the disclosed range of 4 to 10 includes 3.8 to 10.5. This concept is incorporated herein by the term "about".

[0023] Unless otherwise defined, all technical and scientific terms used in this specification have the same meaning as commonly understood by one of ordinary skill in the art to which this term belongs. In carrying out the implementations or tests of the present disclosure, any methods and materials similar to or equivalent to the methods and materials described herein can also be used, but the preferred methods and materials are described herein. All publications mentioned in this specification are hereby incorporated by reference into this specification to disclose and explain the methods and / or materials related to the citation of these publications. In case of any conflict with the incorporated publications, the present disclosure shall prevail.

[0024] As used in this specification and the appended claims, the term "platelet" may include whole platelets, fragmented platelets, platelet derivatives, or thrombosomes. The "platelets" within the above definition include, for example, platelets in whole blood, platelets in plasma, platelets in a buffer optionally supplemented with selected plasma proteins, cryopreserved platelets, dried platelets, frozen-preserved platelets, thawed frozen-preserved platelets, rehydrated dried platelets, rehydrated frozen-preserved platelets, lyophilized platelets, thawed lyophilized platelets, or rehydrated lyophilized platelets. The "platelets" can be mammalian "platelets", for example, in the case of human platelets or non-human mammalian platelets.

[0025] As used in this specification, a "thrombosome" (also referred to as a T-some) is a platelet derivative that has been treated with a preparation (e.g., any of the preparations described herein) and lyophilized (e.g., freeze-dried). In some cases, thrombosomes can be prepared from pooled platelets. Thrombosomes can have a shelf life of 2 - 3 years in a dry form at room temperature and can be rehydrated with sterile water within minutes and immediately injected. An example of a thrombosome is THROMBOSOME (registered trademark), which is being clinically tested for the treatment of acute bleeding in thrombocytopenic patients.

[0026] Transfusion-related acute lung injury (TRALI) is a condition thought to be caused by the presence of antibodies (e.g., human leukocyte antigen (HLA), human neutrophil antigen (HNA), or granulocyte antibodies) in transfused blood products, and such antibodies may react with antigens in the transfusion recipient.

[0027] Plasma-based blood products from donors considered to be at high risk, or donors positive for human leukocyte antigen (HLA) class I, class II, and neutrophil-specific antibodies, are prohibited from use in the transfusion or production of human-derived platelet products (e.g., compositions containing platelets and / or platelet derivatives (e.g., thrombosomes)) and are therefore excluded from the donor pool.

[0028] The amount of antibodies in a blood product can be reduced, for example, to a limit where they cannot be detected by current FDA-approved testing methods, by using tangential flow filtration (TFF) or multipass centrifugation. In some cases, reducing certain plasma components (e.g., HLA antibodies) may make it possible to accept this donor population for the production of blood products (e.g., compositions containing platelets and / or platelet derivatives (e.g., thrombosomes)). In some embodiments described herein, the blood product can be a composition comprising platelets and an aqueous medium.

[0029] The production of thrombosomes or cryopreserved platelets is limited by the availability of apheresis collections performed at blood donor centers in the United States. Competition for these products is intense, and distribution to blood product manufacturing needs is typically of lower priority than patient care needs. Blood product manufacturing (e.g., scale-up) can be aided by apheresis collections from donors whose collections would otherwise be deferred. One way this can be accomplished is by reducing the level of free antibodies in donor plasma using tangential flow filtration (TFF) or centrifugation and plasma removal to meet the current FDA-approved test thresholds. Centrifugation of raw materials (e.g., donor plasma), while typically taking longer than TFF, can have a similar effect on the raw materials. In some cases, by removing donor plasma and replacing it with buffer, the inventors were able to produce and characterize a final product (e.g., a composition comprising platelets and / or platelet derivatives (e.g., thrombosomes)) with a reduced content of protein (e.g., antibodies (e.g., HLA antibodies or HNA antibodies)) (e.g., as determined by absorbance at 280 nm). Such products can enhance the safety of the product for the recipient by reducing the transfusion-related cause of TRALI.

[0030] In some embodiments, the materials and methods provided herein can enable previously deferred donors (e.g., those who screen positive for HLA antibodies or those who show a risk of positive HLA from donor history) to be included in the donor pool of raw materials used in the manufacture of blood products (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)). In some embodiments described herein, the blood product can be a composition comprising platelets and an aqueous medium. Additionally, reducing HLA antibodies from raw materials (e.g., donor apheresis material (e.g., platelets or pooled platelets)) enables the final product (e.g., a composition comprising platelets and / or platelet derivatives (e.g., thrombosomes)) to be labeled as HLA-reduced, enhancing the safety of the product for the recipient.

[0031] In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have undetectable levels of HLA antibodies. In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have undetectable levels of antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have undetectable levels of HLA class I antibodies. In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have undetectable levels of HLA class II antibodies. In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have undetectable levels of HNA antibodies. In some embodiments, the detection of antibodies can be performed using an assay approved by a regulatory agency (e.g., FDA-approved). The assay approved by a regulatory agency can be any suitable assay approved by a regulatory agency. In some embodiments, the assay approved by a regulatory agency can be LABSCREEN™ Mixed from One Lambda. In some embodiments, the assay approved by a regulatory agency can be performed using LUMINEX® 100 / 200 or LUMINEX® XY and HLA FUSION™ software. In some embodiments described herein, the blood product can be a composition comprising platelets and an aqueous medium.

[0032] In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies at levels below a reference level. In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have HLA class I antibodies at levels below a reference level. In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have HLA class II antibodies at levels below a reference level. In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) can have HNA antibodies at levels below a reference level. The reference level can be any suitable reference level. In some embodiments described herein, the blood product can be a composition comprising platelets and an aqueous medium.

[0033] In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) show a negative reaction to antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) may show a negative reaction to HLA class I antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) may show a negative reaction to HLA class II antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the blood products provided herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombosomes)) may show a negative reaction to HNA antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments described herein, the blood product can be a composition comprising platelets and an aqueous medium. The assay approved by a regulatory agency can be any suitable assay approved by a regulatory agency. In some embodiments, the assay approved by a regulatory agency can be LABSCREEN™ Mixed manufactured by One Lambda. In some implementation forms, the assay approved by a regulatory agency can be performed using LUMINEX® 100 / 200 or LUMINEX® XY and HLA FUSION™ software.

[0034] This specification provides a composition comprising platelets and / or platelet derivatives (e.g., thrombosomes) and an aqueous medium. In some embodiments, the aqueous medium may comprise a formulation (e.g., any of the formulations described herein). In some embodiments, the aqueous medium provided herein may have antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies at levels below a reference level. In some embodiments, the aqueous medium provided herein may have HLA class I antibodies at levels below a reference level. In some embodiments, the aqueous medium provided herein may have HLA class II antibodies at levels below a reference level. In some embodiments, the aqueous medium provided herein may have HNA antibodies at levels below a reference level. The reference level can be any suitable reference level. In some embodiments, the aqueous medium provided herein may show a negative reaction to antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the aqueous medium provided herein may show a negative reaction to HLA class I antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the aqueous medium provided herein may show a negative reaction to HLA class II antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the aqueous medium provided herein may show a negative reaction to HNA antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). The assay approved by a regulatory agency can be any suitable assay approved by a regulatory agency. In some embodiments, the assay approved by a regulatory agency can be LABSCREEN™ Mixed manufactured by One Lambda. In some embodiments, the assay approved by a regulatory agency can be performed using LUMINEX® 100 / 200 or LUMINEX® XY and HLA FUSION™ software.

[0035] In some embodiments, the amount of residual plasma in the aqueous medium can be reduced compared to donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma), and the percentage of residual plasma (e.g., about 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less of the residual plasma) can be present. In some embodiments, the amount of residual plasma in the aqueous medium can be reduced compared to donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma), and the percentage range of residual plasma (e.g., about 5% to about 50%, about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 20%, about 7% to about 15%, about 7% to about 10%, about 8% to about 15%, about 8% to about 10%, about 0.1% to about 5%, about 0.1% to about 3%, about 0.1% to about 1%, about 0.5% to about 3%, about 0.5% to about 1%, or about 1% to about 3%) can be present. In some embodiments, the protein concentration of the aqueous medium can be about 50% or less (e.g., about 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of the protein concentration of donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma). In some embodiments, the protein concentration of the aqueous medium can be about 5% to about 50% (e.g., about 5% to about 40%, about 5% to about 30%, about 5% to about 20%, about 5% to about 15%, about 5% to about 10%, about 10% to about 20%, about 7% to about 15%, about 7% to about 10%, about 8% to about 15%, or about 8% to about 10%) of the protein concentration of donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma).In some embodiments, the protein concentration of the aqueous medium can be about 0.1% to about 5% (e.g., about 0.1% to about 3%, about 0.1% to about 1%, about 0.5% to about 3%, about 0.5% to about 1%, about 1% to about 2%, or about 1% to about 3%) of the protein concentration of donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma). The protein concentration can be measured by any suitable method. In some embodiments, the protein concentration can be measured by absorbance at 280 nm (A280). In some embodiments, the A280 of the aqueous medium is less than 1.70 AU (e.g., less than 1.66 AU, less than 1.6 AU, less than 1.5 AU, less than 1.4 AU, less than 1.3 AU, less than 1.2 AU, less than 1.1 AU, less than 1.0 AU, less than 0.9 AU, less than 0.8 AU, less than 0.7 AU, less than 0.6 AU, less than 0.5 AU, less than 0.4 AU, less than 0.3 AU, less than 0.2 AU, or less than 0.1 AU) using a 0.5 cm path length.

[0036] In some embodiments, the HLA class I antibody concentration of the aqueous medium can be less than about 70% (e.g., less than about 60%, less than 50%, less than 40%, less than 30%, less than 20%, less than 15%, less than 10%, less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) of the HLA class I antibody concentration of donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma). The concentration of the HLA class I antibody can be measured by any suitable method.

[0037] In some embodiments, the HLA class II antibody concentration in the aqueous medium can be less than about 50% (e.g., less than about 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of the HLA class II antibody concentration in donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma). The concentration of the HLA class II antibody can be measured by any suitable method.

[0038] In some embodiments, the HNA antibody concentration in the aqueous medium can be less than about 50% (e.g., less than about 40%, 30%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%) of the HNA antibody concentration in donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma). The concentration of the HNA antibody can be measured by any suitable method.

[0039] In some embodiments, the platelet count of the compositions described herein is at least 10 6 (e.g., at least 5×10 6 , 10 7 , 5×10 7 , 10 8 , 5×10 8 , 10 9 , 5×10 9 , or 10 10 ) and can be. In some embodiments, the platelet count of the compositions described herein is at least about 200×10 3 platelets / μL (e.g., at least about 300×10 3 , 400×10 3 , 500×10 3 , 750×103 , 1000 × 10 3 , 1500 × 10 3 , 2000 × 10 3 , or 2500 × 10 3 platelets / μL. In some embodiments, the platelet count of the compositions described herein is at least about 2000 × 10 3 platelets / μL (e.g., at least about 2050 × 10 3 , 2100 × 10 3 , 2150 × 10 3 , 2200 × 10 3 , 2250 × 10 3 , 2300 × 10 3 , 2350 × 10 3 , 2400 × 10 3 , 2450 × 10 3 , or 2500 × 10 3 platelets / μL. In some embodiments, the platelet count of the compositions described herein is 1000 × 10 4 platelets / μL or less.

[0040] In some embodiments, the compositions provided herein may contain red blood cells. In some embodiments, the red blood cell count of the compositions provided herein is less than about 10 10 (e.g., less than 5 × 10 9 , less than 10 9 , less than 5 × 10 8 , less than 10 8 , less than 5 × 10 7 , less than 10 7 , less than 5 × 10 6 , less than, or 10 6 ). In some embodiments, the red blood cell count is less than 0.2 × 10 6 / μL (e.g., less than 0.1 × 10 6 / μL, less than 0.5 × 10 5 / μL, or less than 0.1 × 10 5 / μL).

[0041] In some cases, flow cytometry can be used to evaluate the compositions described herein. In some embodiments, an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) in the quantification of a composition comprising platelets and an aqueous medium by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. In some embodiments, the percentage of beads positive for an HLA class I antibody is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) in the quantification of a composition comprising platelets and an aqueous medium by flow cytometry using beads coated with class I HLA. In some embodiments, the percentage of beads positive for an HLA class II antibody is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) in the quantification of a composition comprising platelets and an aqueous medium by flow cytometry using beads coated with class II HLA. In some embodiments, the percentage of beads positive for an HNA antibody is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) in the quantification of a composition comprising platelets and an aqueous medium by flow cytometry using beads coated with HNA.

[0042] In some embodiments, the antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) in the quantification of an aqueous medium by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. In some embodiments, the percentage of beads positive for an HLA class I antibody is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) in the quantification of an aqueous medium by flow cytometry using beads coated with class I HLA. In some embodiments, the percentage of beads positive for an HLA class II antibody is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) in the quantification of an aqueous medium by flow cytometry using beads coated with class II HLA. In some embodiments, the percentage of beads positive for an HNA antibody is less than 10% (e.g., less than 9%, less than 8%, less than 7%, less than 6%, less than 5%, less than 4%, less than 3%, less than 2%, less than 1%, less than 0.9%, less than 0.8%, less than 0.7%, less than 0.6%, less than 0.5%, less than 0.4%, less than 0.3%, less than 0.2%, or less than 0.1%) in the quantification of an aqueous medium by flow cytometry using beads coated with HNA.

[0043] In some embodiments, the compositions provided herein may include one or more additional components. In some embodiments, the compositions provided herein may include a formulation (e.g., any of the formulations described herein). In some embodiments, the composition may include a buffer, a base, a filler, optionally a salt, and optionally at least one organic solvent. The buffer can be any suitable buffer. In some embodiments, the buffer can be HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid). The base can be any suitable base. In some embodiments, the base can be sodium bicarbonate. The filler can be any suitable filler. In some embodiments, the filler can be a monosaccharide, a polysaccharide, or a combination thereof. In some embodiments, the filler can be selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose. In some embodiments, the filler can be trehalose. In some embodiments, the polysaccharide can be polysucrose. The salt can be any suitable salt. In some embodiments, the salt can be sodium chloride, potassium chloride, or a combination thereof. The organic solvent can be any suitable organic solvent. In some embodiments, the organic solvent can be selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), and combinations thereof.

[0044] The preparation can contain any suitable ingredients. In some embodiments, the preparation can contain a liquid medium. In some embodiments, the preparation can contain one or more salts selected from phosphates, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts that can be found in blood or blood products or are known to be useful for platelet drying, or any combination of two or more of these.

[0045] In some embodiments, the preparation contains one or more salts, such as phosphates, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts that can be found in blood or blood products. Exemplary salts include sodium chloride (NaCl), potassium chloride (KCl), and combinations thereof. In some embodiments, the preparation contains one or more salts at a concentration of about 0.5 mM to about 100 mM. In some embodiments, the preparation contains one or more salts at a concentration of about 0.5 mM to about 100 mM (e.g., about 0.5 to about 2 mM, about 2 mM to about 90 mM, about 2 mM to about 6 mM, about 50 mM to about 100 mM, about 60 mM to about 90 mM, about 70 to about 85 mM). In some embodiments, the preparation contains one or more salts at about 5 mM, about 75 mM, or about 80 mM. In some embodiments, the preparation contains one or more salts selected from calcium salts, magnesium salts, and combinations of these two at a concentration of about 0.5 mM to about 2 mM.

[0046] Preferably, these salts are present in a composition containing platelets or platelet derivatives (e.g., freeze-dried platelets) in an amount approximately the same as that found in whole blood.

[0047] In some embodiments, the preparation further contains a carrier protein. In some embodiments, the carrier protein contains human serum albumin, bovine serum albumin, or a combination thereof. In some embodiments, the carrier protein is present in an amount of about 0.05% to about 1.0% (w / v).

[0048] The formulation can be any buffer that is non-toxic to platelets and provides reasonable buffering capacity at the temperature to which the solution is exposed during the processes provided herein. Thus, the buffer can be any commercially available known biologically compatible buffer, such as phosphate buffers (e.g., phosphate buffered saline (PBS)), bicarbonate / carbonic acid (e.g., sodium bicarbonate buffer), N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and Tris-based buffers (e.g., Tris buffered saline (TBS)). Similarly, the buffer can include one or more of the following buffers: propane-1,2,3-tricarboxylic acid (tricarballylic acid); benzene pentacarboxylic acid; maleic acid; 2,2-dimethylsuccinic acid; EDTA; 3,3-dimethylglutaric acid; bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS); benzene hexacarboxylic acid (mesitylenic acid); N-(2-acetamido)iminodiacetic acid (ADA); butane-1,2,3,4-tetracarboxylic acid; pyrophosphoric acid; 1,1-cyclopentanediacetic acid (3,3-tetramethyleneglutaric acid); piperazine-1,4-bis-(2-ethanesulfonic acid) (PIPES); N-(2-acetamido)-2-aminoethanesulfonic acid (ACES); 1,1-cyclohexanediacetic acid; 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-tricarboxylic acid (β-methyltricarballylic acid); 2-(N-morpholino)propanesulfonic acid (MOPS); phosphoric acid; and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES). In some embodiments, the formulation is one or more buffers, such as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), or sodium bicarbonate (NaHCO 3) is included. In some embodiments, the formulation comprises one or more buffers at about 5 to about 100 mM. In some embodiments, the formulation comprises one or more buffers at about 5 to about 50 mM (e.g., about 5 mM to about 40 mM, about 8 mM to about 30 mM, about 10 mM to about 25 mM). In some embodiments, the formulation comprises one or more buffers at about 10 mM, about 20 mM, about 25 mM, or about 30 mM.

[0049] In some embodiments, the formulation comprises one or more sugars such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, and xylose. In some embodiments, the sugar is a monosaccharide. In some embodiments, the sugar is a disaccharide. In some embodiments, the sugar is a monosaccharide, a disaccharide, or a combination thereof. In some embodiments, the sugar is a non-reducing disaccharide. In some embodiments, the sugar is sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, the sugar includes trehalose. In some embodiments, the formulation comprises starch. In some embodiments, the formulation comprises polysucrose (a polymer of sucrose and epichlorohydrin). In some embodiments, the formulation comprises one or more sugars at about 10 mM to about 1,000 mM. In some embodiments, the formulation comprises one or more sugars at about 50 to about 500 mM. In some embodiments, one or more sugars are present in an amount of 10 mM to 10 - 500 mM. In some embodiments, one or more sugars are present in an amount of 50 mM to 200 mM. In some embodiments, one or more sugars are present in an amount of 100 mM to 150 mM. In some embodiments, one or more sugars are lyoprotectants. For example, in some embodiments, the lyoprotectant comprises trehalose, polysucrose, or a combination thereof.

[0050] In some embodiments, a composition comprising platelets or platelet derivatives (e.g., thrombosomes) may comprise one or more of water or saline. In some embodiments, a composition comprising platelets or platelet derivatives (e.g., freeze-dried platelets) may comprise DMSO.

[0051] In some embodiments, the preparation comprises an organic solvent such as alcohol (e.g., ethanol). In such a preparation, the amount of the solvent can range from 0.1% to 5.0% (v / v). In some embodiments, the organic solvent can range from about 0.1% (v / v) to about 5.0% (v / v), such as from about 0.3% (v / v) to about 3.0% (v / v), or from about 0.5% (v / v) to about 2% (v / v).

[0052] In some embodiments, suitable organic solvents include, but are not limited to, alcohols, esters, ketones, ethers, halogenated solvents, hydrocarbons, nitriles, glycols, alkyl nitrates, water, or mixtures thereof. In some embodiments, suitable organic solvents include, but are not limited to, methanol, ethanol, n-propanol, isopropanol, acetic acid, acetone, methyl ethyl ketone, methyl isobutyl ketone, methyl acetate, ethyl acetate, isopropyl acetate, tetrahydrofuran, isopropyl ether (IPE), tert-butyl methyl ether, dioxane (e.g., 1,4-dioxane), acetonitrile, propionitrile, methylene chloride, chloroform, toluene, anisole, cyclohexane, hexane, heptane, ethylene glycol, nitromethane, dimethylformamide, dimethyl sulfoxide, N-methylpyrrolidone, dimethylacetamide, and combinations thereof. In some embodiments, the organic solvent is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide (DMSO), dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), or combinations thereof. In some embodiments, the organic solvent comprises ethanol, DMSO, or combinations thereof. The presence of an organic solvent such as ethanol can be beneficial for the treatment of platelets, platelet derivatives, or thrombosomes (e.g., freeze-dried platelet derivatives).

[0053] In some embodiments, the formulation does not contain an organic solvent. In some embodiments, the formulation contains an organic solvent. In some embodiments, the formulation contains DMSO.

[0054] The pH of the formulation can be any suitable pH. For example, in some embodiments, the pH of the formulation can be from about 6.0 to about 7.4 (e.g., from about 6.5 to about 6.9, or from about 6.6 to about 6.8).

[0055] In some embodiments, one or more other components may be combined with platelets (e.g., as part of a formulation). Exemplary components can include prostaglandin E1 or prostacyclin and / or EDTA / EGTA to prevent platelet aggregation and activation.

[0056] In some embodiments, the formulation can be Buffer A as shown in Example 1. In some embodiments, the formulation can include Buffer A as shown in Example 1, where one or more components (e.g., ethanol) are present in an amount up to 3 times the amount shown in Example 1. Non-limiting examples of formulation compositions that can be used are shown in Tables P1 - P6.

[0057] (Table P1) TIFF2025081548000001.tif66128

[0058] (Table P2) TIFF2025081548000002.tif51128

[0059] (Table P3) TIFF2025081548000003.tif64128

[0060] (Table P4) TIFF2025081548000004.tif70128

[0061] (Table P5) TIFF2025081548000005.tif50128

[0062] Table P5 shows the concentrations of HEPES and salts in Buffer B. The pH can be adjusted to 7.4 with NaOH. Albumin is an optional component of Buffer B.

[0063] (Table P6) TIFF2025081548000006.tif59128

[0064] Table P6 is another exemplary formulation.

[0065] In some embodiments, rehydrating a composition comprising platelets or platelet derivatives comprises adding an aqueous liquid to the platelets. In some embodiments, the aqueous liquid is water. In some embodiments, the aqueous liquid is an aqueous solution (e.g., a buffer). In some embodiments, the aqueous liquid is saline. In some embodiments, the aqueous liquid is a suspension.

[0066] In some embodiments, platelets or platelet derivatives (e.g., thrombosomes) have less than about 10% (e.g., less than about 8%, e.g., less than about 6%, e.g., less than about 4%, e.g., less than about 2%, e.g., less than about 0.5%) crosslinking of the platelet membrane via proteins and / or lipids present on the membrane. In some embodiments, rehydrated platelets or platelet derivatives (e.g., thrombosomes) have less than about 10% (e.g., less than about 8%, e.g., less than about 6%, e.g., less than about 4%, e.g., less than about 2%, e.g., less than about 0.5%) crosslinking of the platelet membrane via proteins and / or lipids present on the membrane.

[0067] In some embodiments, platelets or pooled platelets can be acidified to a pH of about 6.0 to about 7.4 before TFF or before dilution in a formulation. In some embodiments, the method comprises acidifying the platelets to a pH of about 6.5 to about 6.9. In some embodiments, the method comprises acidifying the platelets to a pH of about 6.6 to about 6.8. In some embodiments, acidification comprises adding a solution comprising acid citrate dextrose (ACD) to the pooled platelets.

[0068] In some embodiments, platelets are isolated before TFF or before being diluted with a formulation. In some embodiments, the method further includes isolating platelets using centrifugation. In some embodiments, the centrifugation is performed at a relative centrifugal force (RCF) of about 1000×g to about 2000×g. In some embodiments, the centrifugation is performed at a relative centrifugal force (RCF) of about 1300×g to about 1800×g. In some embodiments, the centrifugation is performed at a relative centrifugal force (RCF) of about 1500×g. In some embodiments, the centrifugation is performed for about 1 minute to about 60 minutes. In some embodiments, the centrifugation is performed for about 10 minutes to about 30 minutes. In some embodiments, the centrifugation is performed for about 30 minutes.

[0069] In some embodiments, platelets are isolated, for example, in a liquid medium, before treating a subject.

[0070] In some embodiments, the platelets are donor-derived platelets. In some embodiments, the platelets are obtained by a process that includes an apheresis step. In some embodiments, the platelets are pooled platelets.

[0071] In some embodiments, platelets are pooled from multiple donors. Platelets pooled from such multiple donors may also be referred to herein as pooled platelets. In some embodiments, the donors are more than 5 donors, such as more than 10 donors, such as more than 20 donors, such as more than 50 donors, such as up to about 100 donors. In some embodiments, the donors are about 5 to about 100 donors, such as about 10 to about 50 donors, such as about 20 to about 40 donors, such as about 25 to about 35 donors. The pooled platelets can be used to make any of the compositions described herein.

[0072] In some embodiments, platelets are induced in vitro. In some embodiments, platelets are induced or prepared in culture. In some embodiments, preparing platelets includes inducing or growing platelets from a culture of megakaryocytes. In some embodiments, preparing platelets includes inducing or growing platelets (or megakaryocytes) from a culture of human pluripotent stem cells (PSCs) (including embryonic stem cells (ESCs) and / or induced pluripotent stem cells (iPSCs)).

[0073] Thus, in some embodiments, platelets or platelet derivatives (e.g., thrombosomes) are prepared prior to treating a subject as described herein. In some embodiments, platelets or platelet derivatives (e.g., thrombosomes) are lyophilized. In some embodiments, platelets or platelet derivatives (e.g., thrombosomes) are cryopreserved. For example, in some embodiments, platelets or platelet derivatives can be cryopreserved in plasma and DMSO (e.g., 3-9% DMSO (e.g., 6% DMSO)). In some embodiments, platelets or platelet derivatives are cryopreserved as described in U.S. Patent Application Publication No. 2020 / 0046771A1, published February 13, 2020, which is hereby incorporated by reference in its entirety.

[0074] In some embodiments, platelets (e.g., apheresis platelets, platelets isolated from whole blood, pooled platelets, or combinations thereof) are suspended in a preparation containing a liquid medium at a concentration of 10,000 platelets / μL to 10,000,000 platelets / μL, such as 50,000 platelets / μL to 2,000,000 platelets / μL, such as 100,000 platelets / μL to 500,000 platelets / μL, such as 150,000 platelets / μL to 300,000 platelets / μL, such as 200,000 platelets / μL, to form a suspension.

[0075] In some embodiments, the method further includes drying a platelet or platelet derivative (e.g., a thrombosome). In some embodiments, the drying step includes lyophilizing the platelet or platelet derivative (e.g., a thrombosome). In some embodiments, the drying step includes freeze-drying the platelet or platelet derivative (e.g., a thrombosome). In some embodiments, the method further includes rehydrating the platelet or platelet derivative (e.g., a thrombosome) obtained from the drying step.

[0076] In some embodiments, the platelet or platelet derivative (e.g., a thrombosome) is cryopreserved, frozen, or lyophilized (e.g., to produce a thrombosome) prior to use in a therapeutic or functional assay.

[0077] Any known technique for drying platelets can be used in accordance with the present disclosure as long as the technique can achieve a final residual water content of less than 5%. Preferably, the technique achieves a final residual water content of less than 2%, such as 1%, 0.5%, or 0.1%. Non-limiting examples of suitable techniques include freeze-drying (lyophilization) and spray-drying. A suitable freeze-drying method is presented in Table LA. Further exemplary freeze-drying methods can be found in U.S. Patent No. 7,811,558, U.S. Patent No. 8,486,617, and U.S. Patent No. 8,097,403. An exemplary spray-drying method involves combining nitrogen as the drying gas with a formulation according to the present disclosure, then introducing the mixture into a GEA Mobile Minor spray dryer from GEA Processing Engineering, Inc. (Columbia MD, USA) having a two-fluid nozzle configuration, and spray-drying the mixture at an inlet temperature in the range of 150°C to 190°C, an outlet temperature in the range of 65°C to 100°C, an atomization rate in the range of 0.5 to 2.0 bar, an atomization rate in the range of 5 to 13 kg / hour, a nitrogen usage in the range of 60 to 100 kg / hour, and a running time of 10 to 35 minutes. The final step of spray-drying is to preferentially collect the dried mixture. The dried composition in some embodiments is stable for at least 6 months at temperatures ranging from -20°C or lower to 90°C or higher.

[0078] (Table LA) Exemplary Freeze-Drying Protocol TIFF2025081548000007.tif105151

[0079] In some embodiments, the step of drying the platelets or platelet derivatives (e.g., thrombosomes) obtained as disclosed herein, e.g., the step of freeze-drying the platelets and / or platelet derivatives obtained as disclosed herein, includes incubating the platelets and / or platelet derivatives with a lyoprotectant (e.g., a non-reducing disaccharide). Thus, in some embodiments, the method for preparing platelets and / or platelet derivatives further includes incubating the platelets with a lyoprotectant. In some embodiments, the lyoprotectant is a sugar. In some embodiments, the sugar is a disaccharide such as a non-reducing disaccharide.

[0080] In some embodiments, the platelets and / or platelet derivatives are incubated with the lyoprotectant for a time and at a temperature suitable for incubating the platelets with the lyoprotectant. Non-limiting examples of suitable lyoprotectants include sugars such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, and xylose. In some embodiments, non-limiting examples of lyoprotectants include serum albumin, dextran, polyvinylpyrrolidone (PVP), starch, and hydroxyethyl starch (HES). In some embodiments, exemplary lyoprotectants may include high molecular weight polymers. "High molecular weight" means a polymer having an average molecular weight of about 70 kDa or more and up to 1,000,000 kDa. Non-limiting examples include polymers of sucrose and epichlorohydrin (e.g., polysucrose). In some embodiments, the lyoprotectant is polysucrose. Any amount of high molecular weight polymer can be used as the lyoprotectant, but it is preferred to use an amount such that the final concentration is about 3% - 10% (w / v), e.g., 3% - 7%, e.g., 6%.

[0081] An exemplary sugar for use in the compositions disclosed herein is trehalose. Regardless of the content of the sugar, the sugar can be present in the composition in any suitable amount. For example, the sugar can be present in an amount from 1 mM to 1 M. In various embodiments, the sugar is present in an amount from 10 mM to 10 - 500 mM. In some embodiments, the sugar is present in an amount from 20 mM to 200 mM. In various embodiments, the sugar is present at different specific concentrations within the ranges recited above, and one of ordinary skill in the art can immediately understand the various concentrations without the need for each to be specifically described herein. If two or more saccharides are present in the composition, each sugar can be present in an amount according to the ranges and specific concentrations recited above.

[0082] In some cases, the preparation of thrombosomes further includes one or more of the procedures described in U.S. Patent No. 8,486,617 (e.g., Examples 1-5) and the procedures described in U.S. Patent No. 8,097,403 (e.g., Examples 1-3, etc.) (which are hereby incorporated by reference in their entirety). In some cases, the starting material (e.g., one or more donor platelet units) is first pooled in a common container. In some embodiments, the starting material may include one or more donor platelet units. In some embodiments, the starting material may include donor plasma. The starting material may or may not be acidified with an anticoagulant buffer (i.e., ACD-A) prior to centrifugation. Plasma can be aspirated from the platelet pellet after centrifugation. A cryoprotectant-containing cell-compatible buffer (e.g., a loading buffer that may be the same as or similar to the preparation) may be added to the platelet pellet before resuspending the cells in a suspension. If desired, the platelets may or may not be diluted to a predetermined concentration (e.g., 2200k / ul - 2800k / ul) with a buffer. The platelets in the buffer can be incubated at an incubation temperature of 18°C to 37°C for 0 minutes to 240 minutes. A cryoprotective extender (e.g., polysucrose) can be added to the platelets in the buffer such that the final extender concentration is 1% - 10% w / v (preferably 6% w / v). The centrifuged and processed platelets can be filled into vials, lyophilized, and heat-treated.

[0083] In some embodiments, the particle size (e.g., diameter, maximum dimension) of platelets or platelet derivatives (e.g., thrombosomes) is at least about 0.5 μm (e.g., at least at least about 0.6 μm, at least about 0.7 μm, at least about 0.8 μm, at least about 0.9 μm, at least about 1.0 μm, at least about 1.2 μm, at least about 1.5 μm, at least about 2.0 μm, at least about 2.5 μm, or at least about 5.0 μm). In some embodiments, the particle size is less than about 5.0 μm (e.g., less than about 2.5 μm, less than about 2.0 μm, less than about 1.5 μm, less than about 1.0 μm, less than about 0.9 μm, less than about 0.8 μm, less than about 0.7 μm, less than about 0.6 μm, less than about 0.5 μm, less than about 0.4 μm, or less than about 0.3 μm). In some embodiments, the particle size is from about 0.5 μm to about 5.0 μm (e.g., from about 0.5 μm to about 4.0 μm, from about 0.5 μm to about 2.5 μm, from about 0.6 μm to about 2.0 μm, from about 0.7 μm to about 1.0 μm, from about 0.5 μm to about 0.9 μm, or from about 0.6 μm to about 0.8 μm).

[0084] In some embodiments, 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 the platelet or platelet derivative (e.g., thrombosome) particles have a size in the range of about 0.5 μm to about 5.0 μm (e.g., about 0.5 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm). In some embodiments, up to 99% (e.g., up to about 95%, up to about 80%, up to about 75%, up to about 70%, up to about 65%, up to about 60%, up to about 55%, or up to about 50%) of the platelet or platelet derivative (e.g., thrombosome) is in the range of about 0.5 μm to about 5.0 μm (e.g., about 0.5 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm). In some embodiments, 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 the platelet or platelet derivative (e.g., thrombosome) is in the range of about 0.5 μm to about 5.0 μm (e.g., about 0.5 μm to about 4.0 μm, about 0.5 μm to about 2.5 μm, about 0.6 μm to about 2.0 μm, about 0.7 μm to about 1.0 μm, about 0.5 μm to about 0.9 μm, or about 0.6 μm to about 0.8 μm).

[0085] In some cases, the microparticles can be particles having a particle size (e.g., diameter, maximum dimension) of less than about 0.5 μm (less than about 0.45 μm or 0.4 μm). In some cases, the microparticles can be particles having a particle size in the range of about 0.01 μm to about 0.5 μm (e.g., about 0.02 μm to about 0.5 μm).

[0086] The particulate content of a composition (e.g., a composition prepared according to the methods described herein) comprising platelets or platelet derivatives (e.g., thrombosomes) can contribute less than about 5.0% (e.g., less than about 4.5%, less than 4.0%, less than 3.5%, less than 3.0%, less than 2.5%, less than 2.0%, less than 1.5%, less than 1.0%, or less than 0.5%) of the total scattering intensity of all particles in the composition having a radius of from about 1 nm to about 60,000 nm. As used herein, the “content of particulates by scattering intensity” refers to the content of particulates based on the scattering intensity of all particles in the composition having a radius of from about 1 nm to about 60,000 nm. The particulate content can be measured by any suitable method, e.g., dynamic light scattering (DLS). In some cases, the viscosity of the sample used for DLS can be set to (or adjusted to be) about 1.060 cP since it is approximately the viscosity of plasma.

[0087] The platelets or platelet derivatives (e.g., thrombosomes) described herein can have cell surface markers. The presence of cell surface markers can be quantified using any suitable method. In some embodiments, the presence of cell surface markers is determined using a binding protein (e.g., an antibody) specific for one or more cell surface markers and flow cytometry (e.g., as a percent positive, e.g., about 2.7×10 5It can be quantified using thrombosomes / μL and approximately 4.8 μL of anti-CD41 antibody, approximately 3.3 μL of anti-CD42 antibody, approximately 1.3 μL of annexin V, or approximately 2.4 μL of anti-CD62 antibody. Non-limiting examples of cell surface markers include CD41 (also called glycoprotein IIb or GPIIb and can be assayed using, for example, anti-CD41 antibody), CD42 (can be assayed using, for example, anti-CD42 antibody), CD62 (also called CD62P or P-selectin and can be assayed using, for example, anti-CD62 antibody), phosphatidylserine (can be assayed using, for example, annexin V (AV)), and CD47 (used for self-recognition and the absence of this marker can, in some cases, lead to phagocytosis). The positive percentage of any cell surface marker can be any appropriate positive percentage. For example, the average CD41 positive percentage of platelets or platelet derivatives (e.g., thrombosomes) (e.g., prepared by the methods described herein) can be at least 55% (e.g., at least 60%, at least 65%, at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). As another example, the average CD42 positive percentage of platelets or platelet derivatives (e.g., thrombosomes) (e.g., those described herein) can be at least 65% (e.g., at least 67%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, or at least 95%). As another example, the average CD62 positive percentage of platelets or platelet derivatives (e.g., thrombosomes) (e.g., prepared by the methods described herein) can be at least 10% (e.g., at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 82%, at least 83%, at least 84%, at least 85%, at least 90%, or at least 95%).As another example, the average annexin V positive rate of platelets or platelet derivatives (e.g., thrombosomes) (e.g., those prepared by the methods described herein) can be at least 25% (e.g., at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 97%, or at least 99%). As another example, the average CD47 positive percent of platelets or platelet derivatives (e.g., thrombosomes) (e.g., those prepared by the methods described herein) can be at least about 8% (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%).

[0088] Glycoprotein VI (GPVI) is a platelet receptor for collagen, and platelet activation occurs when collagen binds to GVPI. In thrombosomes, receptor binding can be significantly reduced compared to fresh platelets. Without being bound by any particular theory, it is thought that the manufacturing process blocks or destroys some copies of this receptor in thrombosomes, which likely results in the reduced collagen binding in thrombosomes compared to fresh platelets.

[0089] The platelets or platelet derivatives (e.g., thrombosomes) described herein may have fibrinogen associated with their cell membranes. Aggregation of activated platelets is mediated by the formation of the GPIIb / IIIa complex, which can bind fibrinogen (also called factor I) and form a blood clot. GPIIb / IIIa is a platelet fibrinogen receptor also known as the CD41 / CD61 complex. PAC-1, a GPIIb / IIIa clone, binds to the active form of GPIIb / IIIa. Without being bound to any particular theory, the presence of fibrinogen on the cell membrane is thought to indicate platelets or platelet derivatives (e.g., thrombosomes) capable of forming a blood clot. Similarly, without being bound to any particular theory, the failure of an anti-PAC1 antibody to bind to platelets or platelet derivatives (e.g., thrombosomes) (e.g., those prepared by the methods described herein) may indicate fibrinogen bound to the active form of GPIIb / GPIIIa. This is because PAC-1 binds to the same complex. In some cases, platelets or platelet derivatives (e.g., thrombosomes) (e.g., those prepared by the methods described herein) may have more bound fibrinogen if they retain a greater amount of residual plasma.

[0090] The platelets or platelet derivatives (e.g., thrombosomes) described herein are capable of generating thrombin, for example, when in the presence of reagents including tissue factor and phospholipids. For example, in some cases, the platelets or platelet derivatives (e.g., thrombosomes) described herein (e.g., about 4.8×10 3The concentration (in particles / μL) can generate a thrombin peak height (TPH) of at least 25 nM (e.g., at least 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 52 nM, 54 nM, 55 nM, 56 nM, 58 nM, 60 nM, 65 nM, 70 nM, 75 nM, or 80 nM) when in the presence of a reagent containing tissue factor (e.g., 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, or 10 pM) and optionally phospholipid. For example, in some cases, the platelets or platelet derivatives (e.g., thrombosomes) described herein (e.g., about 4.8×10 3 The concentration (in particles / μL) can generate a TPH of about 25 nM to about 100 nM (e.g., about 25 nM to about 50 nM, about 25 to about 75 nM, about 50 to about 100 nM, about 75 to about 100 nM, about 35 nM to about 95 nM, about 45 to about 85 nM, about 55 to about 75 nM, or about 60 to about 70 nM) when in the presence of a reagent containing tissue factor (e.g., 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM or 10 pM) and optionally phospholipid. In some cases, the platelets or platelet derivatives (e.g., thrombosomes) described herein (e.g., about 4.8×10 3 The concentration (in particles / μL) can generate a TPH of at least 25 nM (e.g., at least 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 52 nM, 54 nM, 55 nM, 56 nM, 58 nM, 60 nM, 65 nM, 70 nM, 75 nM, or 80 nM) using conditions including, for example, 20 μL of a PRP reagent (Thrombinoscope catalog number TS30.00) and an 80 μL composition containing about 4.8×10 3 particles / μL of platelets or platelet derivatives (e.g., thrombosomes) when in the presence of a PRP reagent (Thrombinoscope catalog number TS30.00). In some cases, the platelets or platelet derivatives (e.g., thrombosomes) described herein (e.g., about 4.8×10 3 The concentration (in particles / μL) can generate a TPH of at least 25 nM (e.g., at least 30 nM, 35 nM, 40 nM, 45 nM, 50 nM, 52 nM, 54 nM, 55 nM, 56 nM, 58 nM, 60 nM, 65 nM, 70 nM, 75 nM, or 80 nM) using conditions including, for example, 20 μL of a PRP reagent (Thrombinoscope catalog number TS30.00) and an 80 μL composition containing about 4.8×10 3Using conditions including an 80 μL composition containing platelets or platelet derivatives (e.g., thrombosomes) at a particle / μL level, TPH can be generated at about 25 nM to about 100 nM (e.g., about 25 nM to about 50 nM, about 25 to about 75 nM, about 50 to about 100 nM, about 75 to about 100 nM, about 35 nM to about 95 nM, about 45 to about 85 nM, about 55 to about 75 nM, or about 60 to about 70 nM).

[0091] The platelets or platelet derivatives (e.g., thrombosomes) described herein can generate thrombin, for example, when in the presence of reagents including tissue factor and phospholipids. For example, in some cases, the titer of platelets or platelet derivatives (e.g., thrombosomes) is 10 6 At least 1.2 (e.g., at least 1.3, 1.4, 1.5, 1.6, 1.7, 1.8, 1.9, 2.0, 2.1, 2.2, 2.3, 2.4, or 2.5) thrombin generation potency units (TGPU) per particle can be obtained. For example, in some cases, the titer of platelets or platelet derivatives (e.g., thrombosomes) is 10 6 1.2 to 2.5 TGPU per particle (e.g., 10 6 1.2 to 2.0, 1.3 to 1.5, 1.5 to 2.25, 1.5 to 2.0, 1.5 to 1.75, 1.75 to 2.5, 2.0 to 2.5, or 2.25 to 2.5 TGPU per particle) can be obtained. TGPU can be calculated as follows: TGPU / 1 million particles = [TPH (nM)] * [titer coefficient (IU) / (nM)] / [576,000 particles in the well]. Similarly, the titer coefficient of a thrombin sample can be calculated as follows: titer coefficient = calibrated activity calculated value (IU) / effective calibrated activity (nM). In some cases, the calibrated activity can be based on the WHO International Thrombin Standard.

[0092] The platelets or platelet derivatives (e.g., thrombosomes) described herein can be aggregable, for example, in quantification by using a total thrombosis analysis system (T-TAS (registered trademark)). In some cases, the platelets or platelet derivatives described herein are at least 70×10 3Particles / μL (e.g., at least 73×10 3 , 100×10 3 , 150×10 3 , 173×10 3 , 200×10 3 , 250×10 3 , or 255×10 3 Particles / μL), for example, in platelet-reduced citrate whole blood, a T-TAS occlusion time of less than 14 minutes (e.g., less than 13.5, 13, 12.5, 12, 11.5, or 11 minutes) (e.g., the time until kPa reaches 80) can be provided. In some cases, the platelets or platelet derivatives described herein, at a concentration of at least 70×10 3 Particles / μL (e.g., at least 73×10 3 , 100×10 3 , 150×10 3 , 173×10 3 , 200×10 3 , 250×10 3 , or 255×10 3 Particles / μL), in platelet-reduced citrate whole blood, can provide an area under the curve (AUC) of at least 1300 (e.g., at least 1380, 1400, 1500, 1600, or 1700).

[0093] The platelets or platelet derivatives (e.g., thrombosomes) described herein are aggregable, for example, in the presence of an aggregation agonist. Non-limiting examples of aggregation agonists include thrombin and collagen. In some cases, the percentage of aggregation of the platelets or platelet derivatives (e.g., thrombosomes) described herein is at least 5% (e.g., at least 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 67%, 70%, 75%, 85%, 90%, or 99%) in the presence of an aggregation agonist. In some cases, the percentage of aggregation of the platelets or platelet derivatives (e.g., thrombosomes) described herein is from about 25% to about 100% (e.g., from about 25% to about 50%, from about 25% to about 75%, from about 50% to about 100%, from about 75% to about 100%, from about 40% to about 95%, from about 55% to about 80%, or from about 65% to about 75%) in the presence of an aggregation agonist. The percentage of aggregation can be quantified by any suitable method, such as light transmission aggregometry.

[0094] Compositions containing the platelets or platelet derivatives (e.g., thrombosomes) described herein can have appropriate conditions and amounts of cell substrates and / or metabolites (e.g., pH, pCO 2 , pO 2 , HCO 3 concentration, total carbon dioxide (TCO 2 ), sO 2 , and lactate concentration). Lactate may be a product of glycolysis. Without being bound by any particular theory, the starting material may have a high lactate concentration because it is stored ex vivo while respiring and undergoing glycolysis for a certain period (e.g., about 3 days) until manufacture. For example, in some cases, the pH can be from about 6.0 to about 7.5 (e.g., from about 6.0 to about 7.4, from about 6.9 to about 7.5, or from about 7.0 to about 7.3). As another example, pCO 2 can be from about 10 to about 20 mmHg (e.g., from about 10 to about 15 mmHg, from about 15 to about 20 mmHg, or from about 17 to about 19 mmHg). pO 2can be about 140 to about 165 mmHg (e.g., about 140 to about 150 mmHg, about 150 to about 160 mmHg, or about 160 to about 165 mmHg). HCO 3 concentration can be about 4.5 to about 6.5 mmol / L (e.g., about 5.0 to about 6.0 mmol / L). Total carbon dioxide can be about 4 to about 8 mmol / L (e.g., about 5 to about 7 mmol / L). sO 2 can be at least about 98% (e.g., at least about 99%). Lactate concentration can be less than about 2.0 mmol / L (e.g., less than 1.5 mmol / L or less than 1.0 mmol / L). Lactate concentration can be about 0.4 to about 1.3 mmol / L (e.g., about 0.5 to about 0.6 mmol / L, about 0.5 to about 1.0 mmol / L, or about 0.8 to about 1.3 mmol / L).

[0095] The platelets or platelet derivatives (e.g., thrombosomes) described herein can retain some metabolic activity, as evidenced by, for example, lactate dehydrogenase (LDH) activity. In some cases, the platelets or platelet derivatives (e.g., thrombosomes) described herein can retain at least about 10% (e.g., at least about 12%, 15%, 20%, 25%, 30%, 35%, 40%, or 45%) of the LDH activity of donor apheresis platelets. Without being bound by any particular theory, it is thought that as the amount of polysucrose added increases, the amount of residual LDH activity increases (e.g., a product of a formulation containing 8% polysucrose retains more LDH activity than a product of a formulation containing 4% polysucrose). Similarly, without being bound by any particular theory, it is thought that heat treating a lyophilized composition containing platelets or platelet derivatives (e.g., thrombosomes) increases the amount of LDH activity retained. As another example, metabolic activity can be evidenced by retention of esterase activity (e.g., the ability of a cell to cleave the acetate group of carboxyfluorescein diacetate succinimidyl ester (CFDASE) to unmask a fluorophore).

[0096] In blood products, generally, pathogen reduction is desirable. Without being bound by any particular theory, some methods of pathogen reduction are thought to be able to cause the formation of microparticles in the treated blood product. One method of pathogen reduction involves the use of a photosensitive nucleic acid intercalating compound to alter the nucleic acid of the pathogen with illumination of an appropriate wavelength. The INTERCEPT® System (manufactured by Cerus Corporation) uses amotosalen (a nucleic acid intercalating compound that forms crosslinks with nucleic acids upon UVA illumination).

[0097] The final blood products described herein (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be prepared by any suitable method. The final blood products described herein (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be prepared by the methods disclosed herein. In some embodiments described herein, the final blood product can be a composition comprising platelets and an aqueous medium. In some embodiments, the final blood product can be the result of freeze-drying a composition comprising platelets and an aqueous medium as described herein. In some embodiments, the final blood product can be prepared using tangential flow filtration (TFF) of a starting material (e.g., an untreated blood product (e.g., donor apheresis material (e.g., pooled donor apheresis material)), or a partially treated blood product (e.g., a filtered blood product)). In some embodiments, the final blood product can be prepared using centrifugation of a starting material (e.g., an untreated blood product (e.g., donor apheresis material (e.g., pooled donor apheresis material)), or a partially treated blood product (e.g., a blood product that has undergone filtration)). The methods described herein are generally described in the context of using apheresis material as the starting material, but it will be understood that other materials, such as platelets cultured in vitro, or whole blood, may be used. In some cases, platelets can be separated from whole blood (e.g., pooled whole blood).

[0098] The starting material can be any suitable starting material. In some embodiments, the protein concentration of the starting material can be from about 60 to about 80 mg / mL. In some embodiments, the protein concentration can be based on the protein concentration in the plasma of whole blood. In some embodiments, the protein concentration can be based on the protein concentration of donor apheresis plasma. In some embodiments, the starting material can be a donor blood product (e.g., whole blood or fractionated blood). In some embodiments, the starting material can be a pooled donor blood product (e.g., pooled whole blood or pooled and fractionated blood). In some embodiments, the starting material can include donor apheresis plasma. In some embodiments, the starting material can be derived from donor apheresis plasma. As used herein, "donor apheresis plasma" can refer to the plasma component of the apheresis material, whether or not platelets or other blood cells are included.

[0099] In some embodiments, the starting material can be a donor apheresis material (e.g., donor platelets or a pool of donor platelets). In some embodiments, the starting material is positive for one or more of HLA class I antibodies, HLA class II antibodies, and HNA antibodies based on an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the starting material can show a positive reaction to HLA class I antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the starting material can show a positive reaction to HLA class II antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). In some embodiments, the starting material can show a positive reaction to HNA antibodies in an assay approved by a regulatory agency (e.g., an FDA-approved assay). The assay approved by a regulatory agency can be an assay approved by any suitable regulatory agency. In some embodiments, the assay approved by a regulatory agency can be LABSCREEN™ Mixed from One Lambda. In some embodiments, the assay approved by a regulatory agency can be performed using LUMINEX® 100 / 200 or LUMINEX® XY and HLA FUSION™ software.

[0100] In some embodiments, the starting material can undergo a pathogen reduction step, e.g., through a nucleic acid intercalating compound that forms cross-links to nucleic acids upon UVA illumination.

[0101] In some embodiments, starting materials (e.g., one or more units of donor platelets) can first be pooled in a common container. The starting materials may or may not first be diluted with an acidified wash buffer (e.g., a control buffer). Without being bound by any particular theory, it is believed that washing with an acidified wash buffer can reduce platelet activation during processing. In some cases, the starting materials can undergo two general processing routes, i.e., washing with a control buffer (e.g., using TFF) until a desired residual component (e.g., percentage of residual donor plasma) is reached before concentrating to the final concentration, or concentrating the starting materials to the final concentration before washing with a control buffer (e.g., using TFF) until a desired residual component (e.g., percentage of residual donor plasma) is reached. The TFF-treated material is filled into vials, lyophilized, and heat-treated.

[0102] In some embodiments, the method can include an initial dilution step, e.g., diluting a starting material (e.g., an untreated blood product (e.g., a donor apheresis material (e.g., pooled donor apheresis material))) with a formulation (e.g., any of the formulations described herein) to form a diluted starting material. In some cases, the initial dilution step can include dilution with a formulation having a mass equal to at least about 10% (e.g., at least about 15%, 25%, 50%, 75%, 100%, 150%, or 200%) of the mass of the starting material. In some embodiments, the initial dilution step can be performed using a TFF device.

[0103] In some embodiments, the method can include concentrating (e.g., concentrating platelets) (e.g., concentrating the starting material or the diluted starting material) to form a concentrated platelet composition. For example, the concentration can be from about 1000×10 3 to about 4000×10 3 platelets / μL (e.g., from about 1000×10 3 to about 2000×10 3 , from about 2000×10 3 to about 3000×103 or about 4000×10 3 It may include concentrating to platelets / μL). In some embodiments, the concentrating step may be performed using a TFF device.

[0104] The concentration of platelets or platelet derivatives (e.g., thrombosomes) can be quantified by any suitable method. For example, a counter can be used to quantify the concentration of blood cells in a suspension using impedance (e.g., Beckman Coulter AcT 10 or AcT diff 2).

[0105] In some embodiments, TFF may include diafiltration (sometimes referred to as "washing") of a starting material, a diluted starting material, a concentrated platelet composition, or combinations thereof. In some embodiments, diafiltration may include washing at at least 2 diavolumes (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 diavolumes, or more). In some embodiments, TFF may include buffer exchange. In some embodiments, a buffer may be used with TFF. The buffer can be any suitable buffer. In some embodiments, the buffer may be a formulation (e.g., any of the formulations described herein). In some embodiments, the buffer may be the same formulation as that used for dilution. In some embodiments, the buffer may be a different formulation than that used for dilution. In some embodiments, the buffer may include a lyoprotectant including a buffering agent, a base, a bulking agent, optionally a salt, and optionally at least one organic solvent (e.g., an organic solvent selected from the group consisting of acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), or combinations thereof). The buffering agent can be any suitable buffering agent. In some embodiments, the buffering agent can be HEPES ((4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid)). The base can be any suitable base. In some embodiments, the base can be sodium bicarbonate. In some embodiments, the sugar can be a monosaccharide. In some embodiments, the bulking agent can be a sugar. In some embodiments, the sugar may include sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, the monosaccharide can be trehalose. In some embodiments, the bulking agent may include polysucrose. The salt can be any suitable salt.In some embodiments, the salt can be selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), or combinations thereof.

[0106] In some embodiments, a membrane with a pore size of about 0.1 μm to about 1 μm (e.g., about 0.1 μm to about 1 μm, about 0.1 μm to about 0.5 μm, about 0.2 to about 0.45 μm, about 0.45 to about 1 μm, about 0.1 μm, about 0.2 μm, about 0.45 μm, about 0.65 μm, or about 1 μm) can be used in TFF. The membrane can be made from any suitable material. In some cases, the membrane can be a hydrophilic membrane. In some embodiments, the membrane can be a hydrophobic membrane. In some embodiments, a membrane with a nominal molecular weight cut-off (NMWCO) of at least about 100 kDa (e.g., at least about 200, 300 kDa, 500 kDa, or 1000 kDa) can be used in TFF. TFF can be carried out at any suitable temperature. In some embodiments, TFF can be carried out at a temperature of about 20°C to about 37°C (e.g., about 20°C to about 25°C, about 20°C to about 30°C, about 25°C to about 30°C, about 30°C to about 35°C, about 30°C to about 37°C, about 25°C to about 35°C, or about 25°C to about 37°C). In some embodiments, TFF can be carried out at a flow rate of about 100 ml / min to about 800 ml / min (e.g., the circulation flow rate) (e.g., about 100 to about 200 ml / min, about 100 to about 400 ml / min, about 100 to about 600 ml / min, about 200 to about 400 ml / min, about 200 to about 600 ml / min, about 200 to about 800 ml / min, about 400 to about 600 ml / min, about 400 to about 800 ml / min, about 600 to about 800 ml / min, about 100 ml / min, about 200 ml / min, about 300 ml / min, about 400 ml / min, about 500 ml / min, about 600 ml / min, about 700 ml / min, or about 800 ml / min).

[0107] In some embodiments, TFF can be performed until a specific endpoint is reached to form a TFF-treated composition. The endpoint can be any suitable endpoint. In some embodiments, the endpoint can be the percentage of residual plasma (e.g., about 50% or less, 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less of the residual plasma). In some embodiments, the endpoint can be the relative absorbance (A280) at 280 nm. For example, the endpoint can be an A280 (e.g., using a path length of 0.5 cm) that is about 50% or less (e.g., about 40% or less, 30% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2% or less, or 0.1% or less) of the A280 (e.g., using a path length of 0.5 cm) before TFF (e.g., the starting material or the diluted starting material). In some embodiments, A280 can correspond to a system that measures 7.5% plasma = 1.66 AU. In some embodiments, the device for measuring A280 can be configured as follows, i.e., a flow cell with a 0.5 cm gap can be attached to the filtrate line of the TFF system. The flow cell can be connected to a flow cell photometer by attaching optical fiber cables (a light source cable and a photodetector cable) to each side of the flow cell. The flow cell can be fabricated using silica glass lenses on each side of the optical fiber cables. In some embodiments, the endpoint can be the absolute A280 (e.g., using a path length of 0.5 cm).For example, the endpoint can be an A280 of 1.70 AU or less (e.g., 1.66 AU or less, 1.6 AU or less, 1.5 AU or less, 1.4 AU or less, 1.3 AU or less, 1.2 AU or less, 1.1 AU or less, 1.0 AU or less, 0.9 AU or less, 0.8 AU or less, 0.7 AU or less, 0.6 AU or less, 0.5 AU or less, 0.4 AU or less, 0.3 AU or less, 0.2 AU or less, or 0.1 AU or less) using, for example, a path length of 0.5 cm. In some embodiments, the percentage of residual plasma, relative A280, or A280 can be quantified based on the aqueous medium of the composition comprising platelets and an aqueous medium. In some embodiments, the percentage of residual plasma can be quantified based on a known correlation with A280. In some embodiments, since TFF can include concentration or dilution of the sample (e.g., using a formulation), the endpoint can be the platelet concentration. For example, the endpoint is at least about 2000×10. 3 platelets / μL (e.g., at least about 2050×10 3 , 2100×10 3 , 2150×10 3 , 2200×10 3 , 2250×10 3 , 2300×10 3 , 2350×10 3 , 2400×10 3 , 2450×10 3 , or 2500×10 3 platelets / μL). As another example, the endpoint can be a platelet concentration of about 1000×10 3 to about 2500 platelets / μL (e.g., about 1000×10 3 to about 2000×10 3 , about 1500×10 3 to about 2300×10 3 , or about 1700×10 3 to about 2300×10 3 platelets / μL). In some embodiments, the endpoint can include multiple criteria (e.g., percentage of residual plasma and platelet concentration, relative A280 and platelet concentration, or absolute A280 and platelet concentration).

[0108] Typically, the TFF-treated composition is subsequently lyophilized, optionally with a heat treatment step, to form the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)). However, in some cases, the TFF-treated composition may be considered the final blood product.

[0109] In some embodiments, the blood product can be prepared using centrifugation of a blood product (e.g., an untreated blood product (e.g., donor apheresis material (e.g., pooled donor apheresis material), or a partially treated blood product (e.g., a blood product that has undergone TFF)). In some embodiments, the blood product can be prepared without centrifugation of a blood product (e.g., an untreated blood product (e.g., donor apheresis material), or a partially treated blood product (e.g., a blood product that has undergone TFF)). Centrifugation can include any suitable steps. In some embodiments, centrifugation can include a slow acceleration, a slow deceleration, or a combination thereof. In some embodiments, centrifugation can include centrifugation at about 1400×g to about 1550×g (e.g., about 1400 to about 1450×g, about 1450 to about 1500×g, or 1500 to about 1550×g, about 1400×g, about 1410×g, about 1430×g, about 1450×g, about 1470×g, about 1490×g, about 1500×g, about 1510×g, about 1530×g, or about 1550×g). In some embodiments, the duration of centrifugation can be about 10 minutes to about 30 minutes (e.g., about 10 minutes to about 20 minutes, about 20 minutes to about 30 minutes, about 10 minutes, about 20 minutes, or about 30 minutes).

[0110] In some embodiments, the final blood product can be prepared using both TFF and centrifugation (e.g., centrifugation after TFF, or TFF after centrifugation).

[0111] Also provided herein are compositions prepared by any of the methods described herein.

[0112] In some embodiments, the compositions described herein may be analyzed at multiple points during processing. In some embodiments, the starting material (e.g., a donor apheresis material (e.g., a pooled donor apheresis material)) may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments, the starting material (e.g., a donor apheresis material (e.g., a pooled donor apheresis material)) may be analyzed for protein concentration (e.g., by absorbance at 280 nm (e.g., using a 0.5 cm path length)). In some embodiments, the composition at an intermediate step of the process (e.g., when the protein concentration is reduced to 75% or less (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less) of the protein concentration of the untreated blood product) may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the blood product at an intermediate step of the process may be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the antibody content of the starting material. In some embodiments, the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments described herein, the final blood product may be a composition comprising platelets and an aqueous medium.In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the antibody content of the starting material. In some embodiments, the final blood product can have an antibody selected from the group consisting of undetectable levels of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, the aqueous medium of the compositions described herein may be analyzed as described herein.

[0113] In some embodiments, the compositions described herein may be analyzed at multiple points during processing. In some embodiments, donor apheresis plasma may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments, donor apheresis plasma may be analyzed for protein concentration (e.g., by absorbance at 280 nm). In some embodiments, the composition at an intermediate step of the process (e.g., when the protein concentration is reduced to 75% or less (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less) of the protein concentration of the untreated blood product) may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the blood product at an intermediate step of the process may be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the antibody content of the donor apheresis plasma. In some embodiments, the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) may be analyzed for antibody content (e.g., HLA or HNA antibody content). In some embodiments described herein, the final blood product may be a composition comprising platelets and an aqueous medium. In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) may be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the antibody content of the donor apheresis plasma.In some embodiments, the final blood product may have antibodies selected from the group consisting of undetectable levels of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, the aqueous medium of the compositions described herein may be analyzed as described herein.

[0114] The protein concentration of the blood product can be measured by any suitable method. In some embodiments, the protein concentration of the blood product can be measured using absorbance at 280 nm.

[0115] The antibody content of the blood product (e.g., the antibody content of HLA or HNA) can be measured by any suitable method.

[0116] In some embodiments, as the HLA detection method, One Lambda, FLOWPRA™ Screening or LABScreen Multi test kits from Thermo Fisher Scientific may be used. The raw materials can be tested before TFF or centrifugation to quantify the baseline levels of class I and II antibodies against human leukocyte antigen (HLA) and human neutrophil antigen (HNA). The test can be repeated after treatment by centrifugation or TFF to measure the removal of HLA and HNA. Additional test points can be implemented throughout the TFF procedure to maintain in-process control. To ensure reliable reduction and compliance with current FDA testing and acceptance requirements, samples can be randomly selected from the batch and qualitative testing for HLA / HNA antibodies can be performed after lyophilization and annealing.

[0117] In some embodiments, the antibody content of two blood products (e.g., HLA or HNA antibody content) can be compared by quantifying the percentage of beads that are positive for a marker (e.g., beads coated with HLA or HNA that bind to HLA or HNA antibodies respectively). Any suitable comparison method can be used. In some embodiments, the antibody content of two blood products can be compared using the methods described herein. In some embodiments, such a method can be carried out as follows. An aliquot of plasma (e.g., about 1 mL) of platelet-poor plasma can be obtained. In some embodiments, an aliquot of filtered platelet-poor plasma (PPP) (e.g., about 1 mL) (e.g., using a 0.2 μm filter) can be obtained. Beads coated with class I HLA and / or beads coated with class II HLA are added to the plasma (e.g., about 5 μL of each type of bead to about 20 μL of PPP) to form a mixture of PPP and beads. The mixture of PPP and beads can be vortexed. The mixture of PPP and beads can be incubated to form an incubated mixture. Any suitable incubation conditions can be used. For example, in some embodiments, the incubation can be carried out at a certain temperature (e.g., room temperature) for a certain period of time (e.g., about 30 minutes) using other conditions (e.g., in the dark) to form the incubated mixture. In some embodiments, the incubation can include agitation (e.g., gentle rocking). The beads in the incubated mixture can be washed using any suitable conditions. In some embodiments, the beads in the incubated mixture can be washed with a wash buffer. The washed beads can be separated from the incubated mixture by any suitable method. In some embodiments, the washed beads can be separated by centrifugation (e.g., at 9,000×g for 2 minutes) to obtain pelleted beads. In some embodiments, the washing step can be repeated. The beads can be resuspended to form a bead solution.An antibody conjugated to a detectable moiety (e.g., an antibody that binds to an assayed antibody content (e.g., HLA or HNA antibody content)) can be added to a bead solution (e.g., αIgG conjugated to a fluorescent reporter (e.g., FITC)). The antibody can be incubated with the bead solution under any suitable conditions. In some embodiments, the antibody can be incubated for a period of time (e.g., about 30 minutes) at a certain temperature (e.g., room temperature) using other conditions (e.g., in the dark) to form labeled beads. The labeled beads can be washed to remove unbound antibody conjugated to the detectable moiety. The labeled beads can be washed using any suitable conditions. In some embodiments, the labeled beads can be washed with a wash buffer. The washed labeled beads can be separated by any suitable method. In some embodiments, the washed labeled beads can be separated by centrifugation (e.g., at 9,000 g for 2 minutes) to obtain pelleted labeled beads. In some embodiments, the wash step can be repeated. The labeled beads can be detected by any suitable method. In some embodiments, the labeled beads can be detected by flow cytometry. In some embodiments, the detection can include measuring the percentage of beads that are positive for the detectable moiety compared to a negative control. In some embodiments, the negative control can be prepared as described above using a PPP sample known to be negative for an antibody (e.g., HLA class I, HLA class II, or HNA antibody).

[0118] In some embodiments, blood products (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be analyzed at multiple points during processing. In some embodiments, the starting material (e.g., donor apheresis material) can be analyzed to quantify the percentage of positive beads (e.g., beads coated with HLA or HNA). In some embodiments, the starting material (e.g., the donor's apheresis material) may be analyzed for protein concentration (e.g., by absorbance at 280 nm). In some embodiments, the blood product at an intermediate step of the process (e.g., when the protein concentration of the starting material is reduced to 75% or less (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less)) can be analyzed to quantify the percentage of positive beads (e.g., beads coated with HLA or HNA). In some embodiments, the percentage of positive beads (e.g., beads coated with HLA or HNA) from the blood product at an intermediate step of the process can be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the percentage of positive beads from the starting material.In some embodiments, the percentage of positive beads (e.g., beads coated with HLA or HNA) from the blood product at an intermediate step of the process can be 75% or less (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less) of the total amount of beads. In some embodiments, the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be analyzed to quantify the percentage of positive beads (e.g., beads coated with HLA or HNA). In some embodiments, the positive beads (e.g., beads coated with HLA or HNA) from the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more reduction) compared to the percentage of positive beads from the starting material. In some embodiments, the percentage of positive beads (e.g., beads coated with HLA or HNA) from the final blood product can be 75% or less (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less). In some embodiments, the aqueous medium of the compositions described herein may be analyzed as described herein.

[0119] In some embodiments, blood products (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be analyzed at multiple points during processing. In some embodiments, donor apheresis plasma can be analyzed to quantify the percentage of positive beads (e.g., beads coated with HLA or HNA). In some embodiments, donor apheresis plasma may be analyzed for protein concentration (e.g., by absorbance at 280 nm). In some embodiments, the blood product at an intermediate step of processing (e.g., when the protein concentration of the starting material is reduced to 75% or less (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less)) can be analyzed to quantify the percentage of positive beads (e.g., beads coated with HLA or HNA). In some embodiments, the percentage of positive beads (e.g., beads coated with HLA or HNA) from the blood product at an intermediate step of processing can be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the percentage of positive beads from the donor apheresis plasma.In some embodiments, the percent of positive beads (e.g., beads coated with HLA or HNA) from the blood product at an intermediate step of the process can be 75% or less of the total amount of beads (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less). In some embodiments, the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be analyzed to quantify the percent of positive beads (e.g., beads coated with HLA or HNA). In some embodiments, the percent of positive beads (e.g., beads coated with HLA or HNA) from the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) can be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the percent of positive beads from the donor apheresis material. In some embodiments, the percent of positive beads (e.g., beads coated with HLA or HNA) from the final blood product can be 75% or less of the total amount of beads (e.g., 70% or less, 65% or less, 60% or less, 55% or less, 50% or less, 45% or less, 40% or less, 35% or less, 30% or less, 25% or less, 20% or less, 15% or less, 10% or less, 9% or less, 8% or less, 7% or less, 6% or less, 5% or less, 4% or less, 3% or less, 2% or less, 1% or less, or less). In some embodiments, the aqueous medium of the compositions described herein may be analyzed as described herein.

[0120] The percentage of positive beads can be quantified using any suitable method. In some embodiments, the positive beads can be quantified by comparison with a negative control sample. The negative control sample can be any suitable negative control sample. In some embodiments, a negative control sample is used to determine the positive gating such that less than a particular percentage (e.g., from about 0.01% to about 1% (e.g., from about 0.01% to about 0.05%, from about 0.05% to about 0.1%, from about 0.1% to about 0.5%, from about 0.5% to about 1%, about 0.01%, about 0.05%, about 0.1%, about 0.5%, or about 1%)) of the negative control sample is present within the positive gate. In some embodiments, the negative control sample can be a buffer (e.g., PBS). In some embodiments, the negative control sample can be a synthetic plasma composition. In some embodiments, the negative control sample can be a blood product known to be negative for the antibody being assayed (e.g., an HLA antibody or an HNA antibody).

[0121] Also provided herein are methods for reducing the percentage of antibodies (e.g., HLA antibodies (e.g., HLA class I antibodies or HLA class II antibodies) or HNA antibodies) in a composition comprising platelets (e.g., a blood product), the method comprising filtering the composition by tangential flow filtration. Also provided herein are methods for reducing the amount of antibodies (e.g., HLA antibodies (e.g., HLA class I antibodies or HLA class II antibodies) or HNA antibodies) in a composition comprising platelets (e.g., a blood product), the method comprising filtering the composition by tangential flow filtration. Also provided herein are methods for reducing the percentage of beads that are positive for antibodies (e.g., HLA antibodies (e.g., HLA class I antibodies or HLA class II antibodies) or HNA antibodies) in a composition comprising platelets (e.g., a blood product), the method comprising filtering the composition by tangential flow filtration.

[0122] Also provided herein is a method for reducing the percentage of antibodies (e.g., HLA antibodies (e.g., HLA class I antibodies or HLA class II antibodies) or HNA antibodies) in a composition containing platelets (e.g., a blood product), which includes filtering the composition by centrifugation. Also provided herein is a method for reducing the amount of antibodies (e.g., HLA antibodies (e.g., HLA class I antibodies or HLA class II antibodies) or HNA antibodies) in a composition containing platelets (e.g., a blood product), which includes filtering the composition by centrifugation. Also provided herein is a method for reducing the percentage of beads that are positive for antibodies (e.g., HLA antibodies (e.g., HLA class I antibodies or HLA class II antibodies) or HNA antibodies) in a composition containing platelets (e.g., a blood product), which includes filtering the composition by centrifugation.

[0123] In some embodiments of any of the methods described herein, the amount of antibodies (e.g., HLA antibodies (e.g., HLA class I antibodies or HLA class II antibodies) or HNA antibodies) in the composition (e.g., a blood product) can be reduced below a reference level. The reference level can be any suitable reference level. In some embodiments of any of the methods described herein, the percentage of beads that are positive for antibodies (e.g., HLA antibodies (e.g., HLA class I antibodies or HLA class II antibodies) or HNA antibodies) in the composition (e.g., a blood product) can be reduced compared to the blood product before passing through the methods described herein. The percentage of beads that are positive for antibodies can be reduced by any suitable amount. In some embodiments, the percentage of beads that are positive for antibodies can be reduced by at least 5% (e.g., at least 10%, 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more) compared to the blood product before passing through any of the methods described herein.

[0124] In some embodiments, the compositions described herein can undergo any suitable additional processing steps. In some embodiments, the compositions described herein can be freeze-dried. In some embodiments, the freeze-dried platelets can be thermally treated (e.g., at about 80 °C for about 24 hours).

[0125] For example, in some embodiments, the composition can be cryopreserved or freeze-dried. In some embodiments, a first composition (e.g., a composition comprising platelets and an aqueous medium as described herein) can be treated with a mixture. In some embodiments, the mixture can include a lyoprotectant containing a base, a bulking agent, and optionally at least one organic solvent (e.g., an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), or combinations thereof) to form a second composition containing platelets. In some embodiments, the bulking agent can be a sugar. In some embodiments, the sugar can be a monosaccharide. In some embodiments, the saccharide can be sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, the bulking agent can be polysucrose.

[0126] In some embodiments, the first composition or the second composition can be dried. In some embodiments, the first composition or the second composition can be dried with a cryoprotectant. In some embodiments, the cryoprotectant can include a sugar, optionally a base, and optionally at least one organic solvent (e.g., an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), or combinations thereof) to form a third composition. In some embodiments, the cryoprotectant can be polysucrose.

[0127] In some embodiments, the first composition or the second composition can be freeze-dried. In some embodiments, the first composition or the second composition can be freeze-dried using a cryoprotectant. In some embodiments, the cryoprotectant can include a sugar, optionally a base, and optionally at least one organic solvent (e.g., an organic solvent selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), or combinations thereof) to form a fourth composition. In some embodiments, the freeze-drying can be performed at a temperature of about -40°C to about 5°C. In some embodiments, the freeze-drying can be performed with a gradient (e.g., about -40°C to about 5°C). In some embodiments, a secondary drying step can be performed (e.g., at about 20°C to about 40°C).

[0128] Also provided herein are blood products (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombosomes)) produced by any of the methods described herein.

[0129] In some embodiments, the percentage of beads that are positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) lower compared to a similar composition not prepared by a process comprising tangential flow filtration of a composition comprising platelets, centrifugation of a composition comprising platelets, or a combination thereof, in the quantification of the compositions described herein by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0130] In some embodiments, the percentage of beads that are positive for an HLA class I antibody is at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) lower compared to a similar composition not prepared by a process comprising tangential flow filtration of a composition comprising platelets, centrifugation of a composition comprising platelets, or a combination thereof, in the quantification of the compositions described herein by flow cytometry using beads coated with class I HLA.

[0131] In some embodiments, the percentage of beads that are positive for an HLA class II antibody is at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) lower compared to a similar composition not prepared by a process comprising tangential flow filtration of a composition comprising platelets, centrifugation of a composition comprising platelets, or a combination thereof, in the quantification of the compositions described herein by flow cytometry using beads coated with class II HLA.

[0132] In some embodiments, the percentage of beads positive for the HNA antibody is at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%) lower compared to a similar composition not prepared by a process comprising tangential flow filtration of a composition comprising platelets, centrifugation of a composition comprising platelets, or a combination thereof, in the quantification of the compositions described herein by flow cytometry using beads coated with HNA.

[0133] Among the processes for making the compositions provided herein, the optional addition of a lyoprotectant can be the last step before drying. However, in some embodiments, the lyoprotectant can be added simultaneously with, or prior to, other components of the composition (e.g., salts, buffers, optionally cryoprotectants, or other components). In some embodiments, the lyoprotectant is added to the formulation, mixed well to form a drying solution, dispensed into a drying container (e.g., a glass or plastic serum vial, a lyophilization bag), and subjected to conditions that allow drying of the TFF-treated composition to form a dried composition.

[0134] In various embodiments, the lyophilization bag is a gas-permeable bag configured such that gas can pass through at least a portion or all of the bag during processing. The gas-permeable bag can allow the gas inside the bag to exchange with the gas in the atmosphere present in the surrounding environment. The gas-permeable bag is permeable to gases such as oxygen, nitrogen, water, air, hydrogen, carbon dioxide, etc., and allows gas exchange in the compositions provided herein. In some embodiments, the gas-permeable bag allows removal of some of the carbon dioxide present inside the bag by permeating carbon dioxide through its walls. In some embodiments, the release of carbon dioxide from the bag can be advantageous for maintaining the desired pH level of the composition contained within the bag.

[0135] In some embodiments, the container of the process herein is a closed or sealed gas-permeable container. In some embodiments, the container is a closed or sealed container, a portion of which is gas-permeable. In some embodiments, the surface area of the gas-permeable portion of the closed or sealed container (e.g., a bag) relative to the volume of the formulation contained within the container (hereinafter referred to as the "SA / V ratio") can be adjusted to improve the pH maintenance of the compositions provided herein. For example, in some embodiments, the SA / V ratio of the container is at least about 2.0 cm 2 / mL (e.g., at least about 2.1 cm 2 / mL, at least about 2.2 cm 2 / mL, at least about 2.3 cm 2 / mL, at least about 2.4 cm 2 / mL, at least about 2.5 cm 2 / mL, at least about 2.6 cm 2 / mL, at least about 2.7 cm 2 / mL, at least about 2.8 cm 2 / mL, at least about 2.9 cm 2 / mL, at least about 3.0 cm 2 / mL, at least about 3.1 cm 2 / mL, at least about 3.2 cm 2 / mL, at least about 3.3 cm 2 / mL, at least about 3.4 cm 2 / mL, at least about 3.5 cm 2 / mL, at least about 3.6 cm 2 / mL, at least about 3.7 cm 2 / mL, at least about 3.8 cm 2 / mL, at least about 3.9 cm 2 / mL, at least about 4.0 cm 2 / mL, at least about 4.1 cm 2 / mL, at least about 4.2 cm 2 / mL, at least about 4.3 cm 2 / mL, at least about 4.4 cm 2 / mL, at least about 4.5 cm 2 / mL, at least about 4.6 cm 2 / mL and at least about 4.7 cm 2 / mL and at least about 4.8 cm 2 / mL and at least about 4.9 cm 2 / mL or at least about 5.0 cm 2 / mL. In some embodiments, the SA / V ratio of the container is at most about 10.0 cm 2 / mL (e.g., at most about 9.9 cm 2 / mL, at most about 9.8 cm 2 / mL, at most about 9.7 cm 2 / mL, at most about 9.6 cm 2 / mL, at most about 9.5 cm 2 / mL, at most about 9.4 cm 2 / mL, at most about 9.3 cm 2 / mL, at most about 9.2 cm 2 / mL, at most about 9.1 cm 2 / mL, at most about 9.0 cm 2 / mL, at most about 8.9 cm 2 / mL, at most about 8.8 cm 2 / mL, at most about 8.7 cm 2 / mL, at most about 8.6 cm 2 / mL, at most about 8.5 cm 2 / mL, at most about 8.4 cm 2 / mL, at most about 8.3 cm 2 / mL, at most about 8.2 cm 2 / mL, at most about 8.1 cm 2 / mL, at most about 8.0 cm 2 / mL, at most about 7.9 cm 2 / mL, at most about 7.8 cm 2 / mL, at most about 7.7 cm 2 / mL, at most about 7.6 cm 2 / mL, at most about 7.5 cm 2 / mL, at most about 7.4 cm 2 / mL, at most about 7.3 cm 2 / mL, at most about 7.2 cm 2 / mL, at most about 7.1 cm 2 / mL, at most about 6.9 cm 2 / mL, at most about 6.8 cm 2 / mL, at most about 6.7 cm 2 / mL, at most about 6.6 cm 2 / mL, up to approximately 6.5 cm 2 / mL, up to approximately 6.4 cm 2 / mL, up to approximately 6.3 cm 2 / mL, up to approximately 6.2 cm 2 / mL, up to approximately 6.1 cm 2 / mL, up to approximately 6.0 cm 2 / mL, up to approximately 5.9 cm 2 / mL, up to approximately 5.8 cm 2 / mL, up to approximately 5.7 cm 2 / mL, up to approximately 5.6 cm 2 / mL, up to approximately 5.5 cm 2 / mL, up to approximately 5.4 cm 2 / mL, up to approximately 5.3 cm 2 / mL, up to approximately 5.2 cm 2 / mL, up to approximately 5.1 cm 2 / mL, up to approximately 5.0 cm 2 / mL, up to approximately 4.9 cm 2 / mL, up to approximately 4.8 cm 2 / mL, up to approximately 4.7 cm 2 / mL, up to approximately 4.6 cm 2 / mL, up to approximately 4.5 cm 2 / mL, up to approximately 4.4 cm 2 / mL, up to approximately 4.3 cm 2 / mL, up to approximately 4.2 cm 2 / mL, up to approximately 4.1 cm 2 / mL, or up to approximately 4.0 cm 2 / mL). In some embodiments, the SA / V ratio of the container is from about 2.0 to about 10.0 cm 2 / mL (e.g., about 2.1 cm 2 / mL to about 9.9 cm 2 / mL, about 2.2 cm 2 / mL to about 9.8 cm 2 / mL, about 2.3 cm 2 / mL to about 9.7 cm 2 / mL, about 2.4 cm 2 / mL to about 9.6 cm 2 / mL, about 2.5 cm 2 / mL to about 9.5 cm 2 / mL, about 2.6 cm 2 / mL to about 9.4 cm 2 / mL, approximately 2.7 cm 2 / mL ~ approximately 9.3 cm 2 / mL, approximately 2.8 cm 2 / mL ~ approximately 9.2 cm 2 / mL, approximately 2.9 cm 2 / mL ~ approximately 9.1 cm 2 / mL, approximately 3.0 cm 2 / mL ~ approximately 9.0 cm 2 / mL, approximately 3.1 cm 2 / mL ~ approximately 8.9 cm 2 / mL, approximately 3.2 cm 2 / mL ~ approximately 8.8 cm 2 / mL, approximately 3.3 cm 2 / mL ~ approximately 8.7 cm 2 / mL, approximately 3.4 cm 2 / mL ~ approximately 8.6 cm 2 / mL, approximately 3.5 cm 2 / mL ~ approximately 8.5 cm 2 / mL, approximately 3.6 cm 2 / mL ~ approximately 8.4 cm 2 / mL, approximately 3.7 cm 2 / mL ~ approximately 8.3 cm 2 / mL, approximately 3.8 cm 2 / mL ~ approximately 8.2 cm 2 / mL, approximately 3.9 cm 2 / mL ~ approximately 8.1 cm 2 / mL, approximately 4.0 cm 2 / mL ~ approximately 8.0 cm 2 / mL, approximately 4.1 cm 2 / mL ~ approximately 7.9 cm 2 / mL, approximately 4.2 cm 2 / mL ~ approximately 7.8 cm 2 / mL, approximately 4.3 cm 2 / mL ~ approximately 7.7 cm 2 / mL, approximately 4.4 cm 2 / mL ~ approximately 7.6 cm 2 / mL, approximately 4.5 cm 2 / mL ~ approximately 7.5 cm 2 / mL, approximately 4.6 cm 2 / mL ~ approximately 7.4 cm 2 / mL, approximately 4.7 cm 2 / mL ~ approximately 7.3 cm 2 / mL, approximately 4.8 cm 2 / mL ~ approximately 7.2 cm2 / mL, about 4.9 cm 2 / mL to about 7.1 cm 2 / mL, about 5.0 cm 2 / mL to about 6.9 cm 2 / mL, about 5.1 cm 2 / mL to about 6.8 cm 2 / mL, about 5.2 cm 2 / mL to about 6.7 cm 2 / mL, about 5.3 cm 2 / mL to about 6.6 cm 2 / mL, about 5.4 cm 2 / mL to about 6.5 cm 2 / mL, about 5.5 cm 2 / mL to about 6.4 cm 2 / mL, about 5.6 cm 2 / mL to about 6.3 cm 2 / mL, about 5.7 cm 2 / mL to about 6.2 cm 2 / mL, or about 5.8 cm 2 / mL to about 6.1 cm 2 / mL).

[0136] The gas-permeable closed container (e.g., a bag) or a part thereof can be made from one or more various gas-permeable materials. In some embodiments, the gas-permeable bag can be made from one or more polymers including fluoropolymers (e.g., polytetrafluoroethylene (PTFE) and perfluoroalkoxy (PFA) polymers), polyolefins (e.g., low density polyethylene (LDPE), high density polyethylene (HDPE)), fluorinated ethylene propylene (FEP), polystyrene, polyvinyl chloride (PVC), silicone, and any combination thereof.

[0137] In some embodiments, the dried platelets or platelet derivatives (e.g., thrombosomes) can be subjected to heat treatment. The heating can be carried out at a temperature above about 25°C (e.g., about 40°C, 50°C, 60°C, 70°C, 80°C, or higher). In some embodiments, the heating is carried out at about 70°C to about 85°C (e.g., about 75°C to about 85°C, or about 75°C or 80°C). The temperature of the heating can be selected in combination with the length of time for which the heating is carried out. Any suitable time can be used, but typically, the lyophilized platelets are heated for at least 1 hour and up to 36 hours. Thus, in embodiments, the heating is carried out for at least 2 hours, at least 6 hours, at least 12 hours, at least 18 hours, at least 20 hours, at least 24 hours, or at least 30 hours. For example, the lyophilized platelets can be heated for 18 hours, 19 hours, 20 hours, 21 hours, 22 hours, 23 hours, 24 hours, 25 hours, 26 hours, 27 hours, 28 hours, 29 hours, or 30 hours. Non-limiting exemplary combinations include heating the dried platelets or platelet derivatives (e.g., thrombosomes) at a temperature higher than 30°C for at least 30 minutes; heating the dried platelets or platelet derivatives (e.g., thrombosomes) at a temperature higher than 50°C for at least 10 hours; heating the dried platelets or platelet derivatives (e.g., thrombosomes) at a temperature higher than 75°C for at least 18 hours; and heating the dried platelets or platelet derivatives (e.g., thrombosomes) at 80°C for 24 hours. In some embodiments, the heating can be carried out in a sealed container (e.g., a vial with a cap). In some embodiments, the sealed container is subjected to a vacuum before heating. The heat treatment step has been found to improve the stability and shelf life of the freeze-dried platelets, particularly in the presence of cryoprotectants such as albumin or polysucrose. Indeed, a specific combination of serum albumin or polysucrose and the heat treatment step after lyophilization has provided more favorable results than cryoprotectants without the heat treatment step.A cryoprotectant (e.g., sucrose) can be present in any suitable amount (e.g., about 3% to about 10% of the mass or volume of platelets or platelet derivatives (e.g., thrombosomes)).

[0138] In some cases, a composition containing platelets or platelet derivatives (e.g., thrombosomes) can be rehydrated with water (e.g., sterile water for injection) at about room temperature for about 10 minutes. Generally, the rehydration volume is approximately equal to the amount used to fill each vial of thrombosomes before drying.

[0139] In some embodiments, the storage stability of platelets or platelet derivatives (e.g., thrombosomes) prepared as disclosed herein is at least approximately equal to the storage stability of platelets before preparation.

[0140] In some embodiments, the method further includes cryopreserving the platelets or platelet derivatives (e.g., using a preparation, e.g., a preparation described herein) before administering the platelets or platelet derivatives.

[0141] In some embodiments, the method further includes drying a composition containing platelets or platelet derivatives (e.g., thrombosomes) (e.g., using a preparation described herein) before administering the platelets or platelet derivatives. In some embodiments, the method can further include heating the composition after the drying step. In some embodiments, the method can further include rehydrating the composition after the freeze-drying step or the heating step.

[0142] In some embodiments, the method further includes lyophilizing a composition comprising platelets or platelet derivatives (e.g., thrombosomes) (e.g., using a formulation described herein) prior to administering the platelets or platelet derivatives. In some embodiments, the method may further include heating the composition after the lyophilization step. In some embodiments, the method may further include rehydrating the composition after the lyophilization step or the heating step.

[0143] In some embodiments, the method further includes cryopreserving platelets, platelet derivatives, or thrombosomes (e.g., using a formulation, e.g., a formulation described herein) prior to administering the platelets, platelet derivatives, or thrombosomes.

[0144] Examples of storage conditions include standard room temperature storage (e.g., storing at a temperature in the range of about 20 to about 30 °C) or cryopreservation (e.g., storing at a temperature in the range of about 1 to about 10 °C). In some embodiments, the method further includes freezing, lyophilizing, thawing, rehydrating, and combinations thereof, a composition comprising platelets or platelet derivatives (e.g., thrombosomes) (e.g., using a formulation described herein) prior to administering the platelets or platelet derivatives. For example, in some embodiments, the method further includes drying a composition comprising platelets or platelet derivatives (e.g., for forming thrombosomes) (e.g., using a formulation described herein) prior to administering the platelets or platelet derivatives. In some embodiments, the method may further include rehydrating the composition obtained from the drying step.

[0145] In some embodiments, provided herein is a method for preparing a composition comprising platelets or platelet derivatives (e.g., thrombosomes). The method includes diluting a starting material comprising platelets with a preparation agent (e.g., buffer A shown in Example 1) to approximately equal weight (±10%), and concentrating the platelets to about 2250×10 3 cells / μL (±250×10 3 ), and then washing with 2 to 4 diavolumes (DV) (e.g., about 2 diavolumes) of the preparation agent to form a TFF-treated composition. The residual plasma percentage can be less than about 15% relative plasma (quantified by plasma protein content). After washing, if the concentration of cells in the TFF-treated composition is not about 2000×10 3 cells / μL (±300×10 3 ), the cells can be diluted or concentrated with the preparation agent to be within this range. The method can further include lyophilizing the TFF-treated composition and then treating the lyophilized composition comprising platelets or platelet derivatives (e.g., thrombosomes) at about 80°C for about 24 hours. In some embodiments, the method can further include a pathogen reduction step, e.g., before diluting the starting material.

[0146] Also provided herein are compositions produced by any of the methods described herein.

[0147] In some embodiments, any composition provided herein can be made by the methods described herein.

[0148] The specific embodiments disclosed herein may be further limited in the claims using the recitations “consisting of” or “consisting essentially of”.

[0149] Exemplary Embodiments Embodiment 1 is a composition comprising platelets or platelet derivatives and an aqueous medium, wherein the aqueous medium has a protein concentration that is 50% or less of the protein concentration of donor apheresis plasma.

[0150] Embodiment 2 is the composition according to Embodiment 1, wherein the protein concentration of the aqueous medium is 30% or less of the protein concentration of the donor apheresis plasma.

[0151] Embodiment 3 is the composition according to Embodiment 1 or 2, wherein the aqueous medium has a human leukocyte antigen (HLA) class I antibody concentration that is less than 30% of the HLA class I antibody concentration in the donor apheresis plasma.

[0152] Embodiment 4 is the composition according to any one of Embodiments 1 to 3, wherein the aqueous medium has a human leukocyte antigen (HLA) class II antibody concentration that is less than 30% of the HLA class II antibody concentration in the donor apheresis plasma.

[0153] Embodiment 5 is the composition according to any one of Embodiments 1 to 4, wherein the aqueous medium has a human neutrophil antigen (HNA) antibody concentration that is less than 30% of the HNA antibody concentration in the donor apheresis plasma.

[0154] Embodiment 6 is the composition according to any one of Embodiments 1 to 5, wherein the protein concentration is 10% or less of the protein concentration of the donor apheresis plasma.

[0155] Embodiment 7 is the composition according to any one of Embodiments 1 to 6, wherein the aqueous medium has a concentration of human HLA class I antibody that is less than 10% of the HLA class I antibody concentration in the donor apheresis plasma.

[0156] Embodiment 8 is the composition according to any one of Embodiments 1 to 7, wherein the aqueous medium has a concentration of human HLA class II antibody that is less than 10% of the HLA class II antibody concentration in the donor apheresis plasma.

[0157] Embodiment 9 is the composition according to any one of Embodiments 1 to 8, wherein the aqueous medium has a human HNA antibody concentration of less than 10% of the HNA antibody concentration in donor apheresis plasma.

[0158] Embodiment 10 is the composition according to any one of Embodiments 1 to 9, wherein the protein concentration is 5% or less of the protein concentration in donor apheresis plasma.

[0159] Embodiment 11 is the composition according to any one of Embodiments 1 to 10, wherein the aqueous medium has a concentration of human HLA class I antibody that is less than 5% of the HLA class I antibody concentration in donor apheresis plasma.

[0160] Embodiment 12 is the composition according to any one of Embodiments 1 to 11, wherein the aqueous medium has a concentration of human HLA class II antibody that is less than 5% of the HLA class II antibody concentration in donor apheresis plasma.

[0161] Embodiment 13 is the composition according to any one of Embodiments 1 to 12, wherein the aqueous medium has a human HNA antibody concentration of less than 5% of the HNA antibody concentration in donor apheresis plasma.

[0162] Embodiment 14 is the composition according to any one of Embodiments 1 to 13, wherein the protein concentration is 3% or less of the protein concentration in donor apheresis plasma.

[0163] Embodiment 15 is the composition according to any one of Embodiments 1 to 14, wherein the aqueous medium has a concentration of human HLA class I antibody that is less than 3% of the HLA class I antibody concentration in donor apheresis plasma.

[0164] Embodiment 16 is the composition according to any one of Embodiments 1 to 15, wherein the aqueous medium has a concentration of human HLA class II antibody that is less than 3% of the HLA class II antibody concentration in donor apheresis plasma.

[0165] Embodiment 17 is the composition according to any one of Embodiments 1 to 16, wherein the aqueous medium has a human HNA antibody concentration of less than 3% of the HNA antibody concentration in donor apheresis plasma.

[0166] Embodiment 18 is the composition according to any one of Embodiments 1 to 17, wherein the protein concentration is 1% or less of the protein concentration of donor apheresis plasma.

[0167] Embodiment 19 is the composition according to any one of Embodiments 1 to 18, wherein the aqueous medium has a concentration of human HLA class I antibody that is less than 1% of the HLA class I antibody concentration in donor apheresis plasma.

[0168] Embodiment 20 is the composition according to any one of Embodiments 1 to 19, wherein the aqueous medium has a concentration of human HLA class II antibody that is less than 1% of the HLA class II antibody concentration in donor apheresis plasma.

[0169] Embodiment 21 is the composition according to any one of Embodiments 1 to 20, wherein the aqueous medium has a human HNA antibody concentration of less than 1% of the HNA antibody concentration in donor apheresis plasma.

[0170] Embodiment 22 is the composition according to any one of Embodiments 1 to 21, wherein the protein concentration is quantified by the absorbance at 280 nanometers (nm) using a path length of 0.5 cm.

[0171] Embodiment 23 is the composition according to Embodiment 22, wherein the absorbance at 280 nm is 1.7 AU or less.

[0172] Embodiment 24 is the composition according to Embodiment 22, wherein the absorbance at 280 nm is 1.66 AU or less.

[0173] Embodiment 25 is the composition according to Embodiment 22, wherein the absorbance at 280 nm is 1.6 AU or less.

[0174] Embodiment 26 is the composition according to any one of Embodiments 1 to 25, wherein the platelet count is at least 200×10 3 platelets / μL.

[0175] Embodiment 27 is the composition according to Embodiment 26, wherein the platelet count is at least 2250×10 3 platelets / μL.

[0176] Embodiment 28 is the composition according to any one of Embodiments 1 to 27, wherein the composition has a red blood cell count of less than 0.2×10 6 red blood cells / μL.

[0177] Embodiment 29 is the composition according to any one of Embodiments 1 to 27, wherein the composition further contains red blood cells.

[0178] Embodiment 30 is the composition according to Embodiment 29, wherein the red blood cell count is less than 0.2×10 6 red blood cells / μL.

[0179] Embodiment 31 is the composition according to any one of Embodiments 1 to 30, wherein the composition is negative for HLA class I antibodies based on a test approved by a regulatory agency.

[0180] Embodiment 32 is the composition according to any one of Embodiments 1 to 31, wherein the composition is negative for HLA class II antibodies based on a test approved by a regulatory agency.

[0181] Embodiment 33 is the composition according to any one of Embodiments 1 to 32, wherein the composition is negative for HNA antibodies based on a test approved by a regulatory agency.

[0182] Embodiment 34 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is less than 5% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0183] Embodiment 35 is the composition according to any one of Embodiments 1 to 34, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is less than 3% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0184] Embodiment 36 is the composition according to any one of Embodiments 1 to 35, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is less than 1% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0185] Embodiment 37 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for an HLA class I antibody is less than 5% in the quantification of the composition by flow cytometry using beads coated with class I HLA.

[0186] Embodiment 38 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for an HLA class I antibody is less than 3% in the quantification of the composition by flow cytometry using beads coated with class I HLA.

[0187] Embodiment 39 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for HLA class I antibody is less than 1% in the quantification of the composition by flow cytometry using beads coated with class I HLA.

[0188] Embodiment 40 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for HLA class II antibody is less than 5% in the quantification of the composition by flow cytometry using beads coated with class II HLA.

[0189] Embodiment 41 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for HLA class II antibody is less than 3% in the quantification of the composition by flow cytometry using beads coated with class II HLA.

[0190] Embodiment 42 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for HLA class II antibody is less than 1% in the quantification of the composition by flow cytometry using beads coated with class II HLA.

[0191] Embodiment 43 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for HNA antibody is less than 5% in the quantification of the composition by flow cytometry using beads coated with HNA.

[0192] Embodiment 44 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for HNA antibody is less than 3% in the quantification of the composition by flow cytometry using beads coated with HNA.

[0193] Embodiment 45 is the composition according to any one of Embodiments 1 to 33, wherein the percentage of beads positive for HNA is less than 1% in the quantification of the composition by flow cytometry using beads coated with HNA.

[0194] Embodiment 46 is the composition according to any one of Embodiments 1 to 45, wherein the aqueous medium further contains a buffer, a base, a filler, optionally a salt, and optionally at least one organic solvent.

[0195] Embodiment 47 is the composition according to Embodiment 46, wherein the buffer is HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0196] Embodiment 48 is the composition according to any one of Embodiments 46 to 47, wherein the base is sodium bicarbonate.

[0197] Embodiment 49 is the composition according to any one of Embodiments 46 to 48, wherein the filler is a monosaccharide, a polysaccharide, or a combination thereof.

[0198] Embodiment 50 is the composition according to Embodiment 49, wherein the monosaccharide is selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose.

[0199] Embodiment 51 is the composition according to Embodiment 49, wherein the monosaccharide is trehalose.

[0200] Embodiment 52 is the composition according to any one of Embodiments 49 to 51, wherein the polysaccharide is polysucrose.

[0201] Embodiment 53 is the composition according to any one of Embodiments 46 to 52, wherein the salt is sodium chloride, potassium chloride, or a combination thereof.

[0202] Embodiment 54 is the composition according to any one of Embodiments 46 to 53, wherein the organic solvent is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), and combinations thereof.

[0203] Embodiment 55 is the composition according to any one of Embodiments 1 to 54, which is prepared by a process comprising tangential flow filtration (TFF) of a starting material containing platelets, centrifugation of a starting material containing platelets, or a combination thereof.

[0204] Embodiment 56 is the composition according to Embodiment 55, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 50% reduced compared to a similar composition not prepared by a process comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0205] Embodiment 57 is the composition according to Embodiment 55, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 75% reduced compared to a similar composition not prepared by a process comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0206] Embodiment 58 is the composition according to embodiment 55, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 90% reduced compared to a similar composition not prepared by a process comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0207] Embodiment 59 is the composition according to embodiment 55, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 95% reduced compared to a similar composition not prepared by a process comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof, in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0208] Embodiment 60 is the composition according to any one of embodiments 55-59, wherein the starting material is a) positive for HLA class I antibodies based on an assay approved by a regulatory agency, b) positive for HLA class II antibodies based on an assay approved by a regulatory agency, c) positive for HNA antibodies based on an assay approved by a regulatory agency, or d) one or more of a), b), or c). Embodiment 61 is the composition according to any one of embodiments 55-60, wherein the starting material has a protein concentration of about 60 to about 80 mg / ml.

[0209] Embodiment 61 is the composition according to any one of embodiments 55-60, wherein the starting material has a protein concentration of about 60 to about 80 mg / ml.

[0210] Embodiment 62 is the composition according to any one of Embodiments 55 to 61, wherein the starting material contains a donor blood preparation.

[0211] Embodiment 63 is the composition of Embodiment 62, wherein the donor blood preparation is a pooled donor blood preparation.

[0212] Embodiment 64 is the composition according to any one of Embodiments 62 to 63, wherein the starting material contains a donor apheresis material.

[0213] Embodiment 65 is the composition according to any one of Embodiments 55 to 64, wherein TFF includes concentration.

[0214] Embodiment 66 is the composition according to any one of Embodiments 55 to 65, wherein TFF includes diafiltration.

[0215] Embodiment 67 is the composition according to Embodiment 66, wherein the diafiltration includes diafiltration at at least 2 diavolumes.

[0216] Embodiment 68 is the composition according to any one of Embodiments 55 to 67, wherein TFF includes buffer exchange.

[0217] Embodiment 69 is the composition according to any one of Embodiments 55 to 68, wherein TFF is performed using a membrane with a pore size of about 0.2 μm to about 1 μm.

[0218] Embodiment 70 is the composition according to any one of Embodiments 55 to 68, wherein TFF is performed using a membrane having a pore size of about 0.2 μm to about 0.45 μm.

[0219] Embodiment 71 is the composition according to any one of Embodiments 55 to 70, wherein TFF is performed at a temperature of about 20°C to about 37°C.

[0220] Embodiment 72 is the composition according to any one of Embodiments 55 to 71, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 50% or less of the absorbance of the starting material at 280 nm.

[0221] Embodiment 73 is the composition according to any one of Embodiments 55 to 71, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 30% or less of the absorbance of the starting material at 280 nm.

[0222] Embodiment 74 is the composition according to any one of Embodiments 55 to 71, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 10% or less of the absorbance of the starting material at 280 nm.

[0223] Embodiment 75 is the composition according to any one of Embodiments 55 to 71, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 5% or less of the absorbance of the starting material at 280 nm.

[0224] Embodiment 76 is the composition according to any one of Embodiments 55 to 71, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 3% or less of the absorbance of the starting material at 280 nm.

[0225] Embodiment 77 is the composition according to any one of Embodiments 55 to 71, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 1% or less of the absorbance of the starting material at 280 nm.

[0226] Embodiment 78 is the composition according to any one of Embodiments 55 to 77, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 1.70 AU or less.

[0227] Embodiment 79 is the composition according to any one of Embodiments 55 to 77, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 1.66 AU or less.

[0228] Embodiment 80 is the composition according to any one of Embodiments 55 to 77, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 1.60 AU or less.

[0229] Embodiment 81 is the composition according to any one of Embodiments 55 to 80, wherein TFF is performed until the platelet concentration becomes at least about 2000×10 3 platelets / μL.

[0230] Embodiment 82 is the composition according to any one of Embodiments 55 to 80, wherein TFF is performed until the platelet concentration becomes at least about 2250×10 3 platelets / μL.

[0231] Embodiment 83 is the composition according to any one of Embodiments 55 to 82, wherein TFF includes buffer exchange with a preparation containing a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

[0232] Embodiment 84 is the composition according to Embodiment 83, wherein the buffer is HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0233] Embodiment 85 is the composition according to any one of Embodiments 83 to 84, wherein the base is sodium bicarbonate.

[0234] Embodiment 86 is the composition according to any one of Embodiments 83 to 85, wherein the loading agent is a monosaccharide, a polysaccharide, or a combination thereof.

[0235] Embodiment 87 is the composition according to Embodiment 86, wherein the monosaccharide is selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose.

[0236] Embodiment 88 is the composition according to Embodiment 86, wherein the monosaccharide is trehalose.

[0237] Embodiment 89 is the composition according to any one of Embodiments 86 to 88, wherein the polysaccharide is polysucrose.

[0238] Embodiment 90 is the composition according to any one of Embodiments 83 to 89, wherein the salt is sodium chloride, potassium chloride, or a combination thereof.

[0239] Embodiment 91 is the composition according to any one of Embodiments 83 to 90, wherein the organic solvent is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), and combinations thereof.

[0240] Embodiment 92 is the composition according to any one of Embodiments 55 to 91, wherein the centrifugation includes centrifugation at 1400×g to about 1550×g.

[0241] Embodiment 93 is the composition according to any one of Embodiments 55 to 91, wherein the centrifugation includes centrifugation at 1450×g to about 1500×g.

[0242] Embodiment 94 is the composition according to any one of Embodiments 55 to 91, wherein the process does not include centrifugation of the composition containing platelets.

[0243] Embodiment 95 is the composition according to any one of Embodiments 1 to 94, wherein the composition contains less than 5.0% (by scattering intensity) of fine particles.

[0244] Embodiment 96 is the composition according to any one of Embodiments 1 to 94, wherein the composition contains less than 4.5% of fine particles (by scattering intensity).

[0245] Embodiment 97 is the composition according to any one of Embodiments 1 to 94, wherein the composition contains less than 4.0% of fine particles (by scattering intensity).

[0246] Embodiment 98 is the composition according to any one of Embodiments 1 to 94, wherein the composition contains less than 3.5% of fine particles (by scattering intensity).

[0247] Embodiment 99 is the composition according to any one of Embodiments 1 to 98, wherein the platelet or platelet derivative has at least 55% CD41 positive percentage.

[0248] Embodiment 100 is the composition according to any one of Embodiments 1 to 98, wherein the platelet or platelet derivative has at least 60% CD41 positive percentage.

[0249] Embodiment 101 is the composition according to any one of Embodiments 1 to 98, wherein the platelet or platelet derivative has at least 65% CD41 positive percentage.

[0250] Embodiment 102 is the composition according to any one of Embodiments 1 to 101, wherein the platelet or platelet derivative has at least 65% CD42 positive percentage.

[0251] Embodiment 103 is the composition according to any one of Embodiments 1 to 101, wherein the platelet or platelet derivative has at least 80% CD42 positive percentage.

[0252] Embodiment 104 is the composition according to any one of Embodiments 1 to 101, wherein the platelet or platelet derivative has at least 90% CD42 positive percentage.

[0253] Embodiment 105 is the composition according to any one of Embodiments 1 to 104, wherein the platelet or platelet derivative retains at least about 10% of the lactate dehydrogenase activity of donor apheresis platelets.

[0254] Embodiment 106 is the composition according to any one of Embodiments 1 to 104, wherein the platelet or platelet derivative retains at least about 15% of the lactate dehydrogenase activity of donor apheresis platelets.

[0255] Embodiment 107 is the composition according to any one of Embodiments 1 to 104, wherein the platelet or platelet derivative retains at least about 20% of the lactate dehydrogenase activity of donor apheresis platelets.

[0256] Embodiment 108 is the composition according to any one of Embodiments 1 to 107, wherein the platelet or platelet derivative has at least 25% annexin V positive percentage.

[0257] Embodiment 109 is the composition according to any one of Embodiments 1 to 107, wherein the platelet or platelet derivative has at least 50% annexin V positive percentage.

[0258] Embodiment 110 is the composition according to any one of Embodiments 1 to 107, wherein the platelet or platelet derivative has at least 70% annexin V positive percentage.

[0259] Embodiment 111 is the composition according to any one of Embodiments 1 to 110, wherein the platelet or platelet derivative has at least 8% CD47 positive percentage.

[0260] Embodiment 112 is the composition according to any one of Embodiments 1 to 110, wherein the platelet or platelet derivative has at least 10% CD47 positive percentage.

[0261] Embodiment 113 is the composition according to any one of Embodiments 1 to 110, wherein the platelet or platelet derivative has at least 15% CD47 positive percentage.

[0262] Embodiment 114 is the composition according to any one of Embodiments 1 to 110, wherein the platelet or platelet derivative has at least 20% CD47 positive percentage.

[0263] Embodiment 115 is the composition according to any one of Embodiments 1 to 114, wherein the platelet or platelet derivative has at least 10% CD62 positive percentage.

[0264] Embodiment 116 is the composition according to any one of Embodiments 1 to 114, wherein the platelet or platelet derivative has at least 50% CD62 positive percentage.

[0265] Embodiment 117 is the composition according to any one of Embodiments 1 to 114, wherein the platelet or platelet derivative has at least 80% CD62 positive percentage.

[0266] Embodiment 118 is the composition according to any one of Embodiments 1 to 114, wherein the platelet or platelet derivative has at least 90% CD62 positive percentage.

[0267] Embodiment 119 is the composition according to any one of Embodiments 1 to 118, wherein the platelet or platelet derivative has fibrinogen associated with the cell membrane.

[0268] Embodiment 120 is the composition according to any one of Embodiments 1 to 119, wherein the aqueous medium has a lactate concentration of less than 2.0 mmol / L.

[0269] Embodiment 121 is the composition according to any one of Embodiments 1 to 119, wherein the aqueous medium has a lactate concentration of less than 1.5 mmol / L.

[0270] Embodiment 122 is the composition according to any one of Embodiments 1 to 121, wherein the aqueous medium has a lactic acid concentration of about 0.4 to about 1.3 mmol / L.

[0271] Embodiment 123 is the composition according to any one of Embodiments 1 to 121, wherein the aqueous medium has a lactic acid concentration of about 0.5 to about 1.0 mmol / L.

[0272] Embodiment 124 is the composition according to any one of Embodiments 1 to 123, wherein the platelet derivative contains thrombosomes.

[0273] Embodiment 125 is the composition according to any one of Embodiments 1 or 22 to 124, wherein the protein concentration is about 5% to about 50% of the protein concentration of donor apheresis plasma.

[0274] Embodiment 126 is the composition according to any one of Embodiments 1 to 5 or 22 to 125, wherein the protein concentration is about 5% to about 30% of the protein concentration of donor apheresis plasma.

[0275] Embodiment 127 is the composition according to any one of Embodiments 1 to 5 or 22 to 126, wherein the protein concentration is about 5% to about 15% of the protein concentration of donor apheresis plasma.

[0276] Embodiment 128 is the composition according to any one of Embodiments 1 to 9 or 22 to 127, wherein the protein concentration is about 8% to about 10% of the protein concentration of donor apheresis plasma.

[0277] Embodiment 129 is the composition according to any one of Embodiments 1 to 9 or 22 to 128, wherein the protein concentration is about 7% to about 10% of the protein concentration of donor apheresis plasma.

[0278] Embodiment 130 is such that the platelet or platelet derivative is about 4.8×10 3The composition according to any one of Embodiments 1 to 129, which generates a thrombin peak height (TPH) of at least 25 nM in the presence of a reagent containing tissue factor and phospholipid at a concentration of particles / μL.

[0279] Embodiment 131 is such that the platelet or platelet derivative is about 4.8×10 3 The composition according to any one of Embodiments 1 to 129, which generates a thrombin peak height (TPH) of at least 50 nM in the presence of a reagent containing tissue factor and phospholipid at a concentration of particles / μL.

[0280] Embodiment 132 is such that the platelet or platelet derivative is 10 6 The composition according to any one of Embodiments 1 to 129, which has a titer of at least 1.5 thrombin generating potency units (TGPU) per particle.

[0281] Embodiment 133 is such that the platelet or platelet derivative is at least about 70×10 3 The composition according to any one of Embodiments 1 to 129, which results in an occlusion time of less than 14 minutes in a total thrombosis analysis system (T-TAS) assay at a concentration of particles / μL.

[0282] Embodiment 134 is such that the platelet or platelet derivative is about 70×10 3 The composition according to any one of Embodiments 1 to 129, which results in an occlusion time of less than 12 minutes in a total thrombosis analysis system (T-TAS) assay at a concentration of particles / μL.

[0283] Embodiment 135 is a process for preparing a composition comprising a platelet or platelet derivative and an aqueous medium, comprising preparing a composition comprising a platelet or platelet derivative and an aqueous medium by tangential flow filtration (TFF) of a starting material containing platelets, a diluted starting material containing platelets, a concentrated platelet composition, or a combination thereof. The process is such that the aqueous medium has a protein concentration of 50% or less of the protein concentration of donor apheresis plasma.

[0284] Embodiment 136 is such that the starting material is a) positive for HLA class I antibodies based on tests approved by a regulatory agency, b) positive for HLA class II antibodies based on tests approved by a regulatory agency, c) positive for HNA antibodies based on tests approved by a regulatory agency, or d) one or more of a), b), and c), which is the process according to Embodiment 125.

[0285] Embodiment 137 is the process according to any one of Embodiments 135 to 136, wherein the starting material has a protein concentration of about 60 to about 80 mg / mL.

[0286] Embodiment 138 is the process according to any one of Embodiments 135 to 137, wherein the starting material contains a donor blood product.

[0287] Embodiment 139 is the process of Embodiment 138, wherein the donor blood product is a pooled donor blood product.

[0288] Embodiment 140 is the process according to any one of Embodiments 135 to 139, wherein the starting material contains a donor apheresis material.

[0289] Embodiment 141 is the process according to any one of Embodiments 135 to 140, wherein the TFF includes concentration.

[0290] Embodiment 142 is the process according to any one of Embodiments 135 to 141, wherein the TFF includes diafiltration.

[0291] Embodiment 143 is the process according to Embodiment 142, wherein diafiltration includes diafiltration at at least 2 diavolumes.

[0292] Embodiment 144 is the process according to any one of Embodiments 135 to 143, wherein TFF includes buffer exchange.

[0293] Embodiment 145 is the process according to any one of Embodiments 135 to 144, wherein TFF is performed using a membrane with a pore size of about 0.2 μm to about 1 μm.

[0294] Embodiment 146 is the process according to any one of Embodiments 135 to 145, wherein TFF is performed using a membrane with a pore size of about 0.2 μm to about 0.45 μm.

[0295] Embodiment 147 is the process according to any one of Embodiments 135 to 146, wherein TFF is carried out at a temperature of about 20°C to about 37°C.

[0296] Embodiment 148 is the process according to any one of Embodiments 135 to 147, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 50% or less of the absorbance of the starting material at 280 nm.

[0297] Embodiment 149 is the process according to any one of Embodiments 135 to 148, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 30% or less of the absorbance of the starting material at 280 nm.

[0298] Embodiment 150 is the process according to any one of Embodiments 135 to 149, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 10% or less of the absorbance of the starting material at 280 nm.

[0299] Embodiment 151 is the process according to any one of Embodiments 135 to 150, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 5% or less of the absorbance of the starting material at 280 nm.

[0300] Embodiment 152 is the process according to any one of Embodiments 135 to 151, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 3% or less of the absorbance of the starting material at 280 nm.

[0301] Embodiment 153 is the process according to any one of Embodiments 135 to 152, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 1% or less of the absorbance of the starting material at 280 nm.

[0302] Embodiment 154 is the process according to any one of Embodiments 135 to 153, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 1.70 AU or less.

[0303] Embodiment 155 is the process according to any one of Embodiments 135 to 154, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 1.66 AU or less.

[0304] Embodiment 156 is the process according to any one of Embodiments 135 to 155, wherein TFF is performed using a path length of 0.5 cm until the absorbance of the aqueous medium at 280 nm becomes 1.60 AU or less.

[0305] Embodiment 157 is the process according to any one of Embodiments 135 to 156, wherein TFF is performed until the platelet concentration becomes at least about 2000×10 3 platelets / μL.

[0306] Embodiment 158 is the process according to any one of Embodiments 135 to 156, wherein TFF is performed until the platelet concentration reaches at least about 2250×10 3 platelets / μL.

[0307] Embodiment 159 is the process according to any one of Embodiments 135 to 158, wherein TFF includes diafiltration using a preparation containing a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

[0308] Embodiment 160 is the process according to any one of Embodiments 135 to 159, wherein TFF includes buffer exchange with a preparation containing a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

[0309] Embodiment 161 is the process according to any one of Embodiments 149 to 160, wherein the buffer is HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid).

[0310] Embodiment 162 is the process according to any one of Embodiments 149 to 161, wherein the base is sodium bicarbonate.

[0311] Embodiment 163 is the process according to any one of Embodiments 149 to 162, wherein the loading agent is a monosaccharide, a polysaccharide, or a combination thereof.

[0312] Embodiment 164 is the process according to Embodiment 163, wherein the monosaccharide is selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, xylose, and combinations thereof.

[0313] Embodiment 165 is the process according to Embodiment 163, wherein the monosaccharide is trehalose.

[0314] Embodiment 166 is the process according to any one of Embodiments 163 to 165, wherein the polysaccharide is polydextrose.

[0315] Embodiment 167 is the process according to any one of Embodiments 159 to 166, wherein the salt is sodium chloride, potassium chloride, or a combination thereof.

[0316] Embodiment 168 is the process according to any one of Embodiments 159 to 167, wherein the organic solvent is selected from the group consisting of ethanol, acetic acid, acetone, acetonitrile, dimethylformamide, dimethyl sulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), and combinations thereof.

[0317] Embodiment 169 is the process according to any one of Embodiments 135 to 168, wherein the process does not include centrifugation of a starting material containing platelets, a diluted starting material containing platelets, a concentrated platelet composition, or a combination thereof.

[0318] Embodiment 170 is the process according to any one of Embodiments 135 to 169, wherein the process does not include centrifugation of a composition containing platelets.

[0319] Embodiment 171 is such that the percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 50% reduced compared to a similar composition not prepared by a process including tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof, in the quantification of a composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively. It is the process according to any one of Embodiments 135 to 170.

[0320] Embodiment 172 is a process according to any one of Embodiments 135 to 170, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 75% lower in the quantification of a composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively, compared to a similar composition not prepared by a process comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof.

[0321] Embodiment 173 is a process according to any one of Embodiments 135 to 170, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 90% lower in the quantification of a composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively, compared to a similar composition not prepared by a process comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof.

[0322] Embodiment 174 is a process according to any one of Embodiments 135 to 170, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is at least 95% lower in the quantification of a composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively, compared to a similar composition not prepared by a process comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof.

[0323] Embodiment 175 is a process according to any one of Embodiments 135 to 174, wherein the protein concentration is 30% or less of the protein concentration of donor apheresis plasma.

[0324] Embodiment 176 is the process according to any one of Embodiments 135 to 175, wherein the aqueous medium has a human leukocyte antigen (HLA) class I antibody concentration of less than 30% of the HLA class I antibody concentration in donor apheresis plasma.

[0325] Embodiment 177 is the process according to any one of Embodiments 135 to 176, wherein the aqueous medium has a human leukocyte antigen (HLA) class II antibody concentration of less than 30% of the HLA class II antibody concentration in donor apheresis plasma.

[0326] Embodiment 178 is the process according to any one of Embodiments 135 to 177, wherein the aqueous medium has an HNA antibody concentration of less than 30% of the HNA antibody concentration in donor apheresis plasma.

[0327] Embodiment 179 is the process according to any one of Embodiments 135 to 178, wherein the protein concentration is 10% or less of the protein concentration of donor apheresis plasma.

[0328] Embodiment 180 is the process according to any one of Embodiments 135 to 179, wherein the aqueous medium has a concentration of human HLA class I antibody that is less than 10% of the HLA class I antibody concentration in donor apheresis plasma.

[0329] Embodiment 181 is the process according to any one of Embodiments 135 to 180, wherein the aqueous medium has a concentration of human HLA class II antibody that is less than 10% of the HLA class II antibody concentration in donor apheresis plasma.

[0330] Embodiment 182 is the process according to any one of Embodiments 135 to 181, wherein the aqueous medium has a human HNA antibody concentration that is less than 10% of the HNA antibody concentration in donor apheresis plasma.

[0331] Embodiment 183 is the process according to any one of Embodiments 135 to 182, wherein the protein concentration is 5% or less of the protein concentration of the donor apheresis plasma.

[0332] Embodiment 184 is the process according to any one of Embodiments 135 to 183, wherein the aqueous medium has a concentration of human HLA class I antibody that is less than 5% of the HLA class I antibody concentration in the donor apheresis plasma.

[0333] Embodiment 185 is the process according to any one of Embodiments 135 to 184, wherein the aqueous medium has a concentration of human HLA class II antibody that is less than 5% of the HLA class II antibody concentration in the donor apheresis plasma.

[0334] Embodiment 186 is the process according to any one of Embodiments 135 to 185, wherein the aqueous medium has a concentration of human HNA antibody that is less than 5% of the HNA antibody concentration in the donor apheresis plasma.

[0335] Embodiment 187 is the process according to any one of Embodiments 135 to 186, wherein the protein concentration is 3% or less of the protein concentration of the donor apheresis plasma.

[0336] Embodiment 188 is the process according to any one of Embodiments 135 to 187, wherein the aqueous medium has a concentration of human HLA class I antibody that is less than 3% of the HLA class I antibody concentration in the donor apheresis plasma.

[0337] Embodiment 189 is the process according to any one of Embodiments 135 to 188, wherein the aqueous medium has a concentration of human HLA class II antibody that is less than 3% of the HLA class II antibody concentration in the donor apheresis plasma.

[0338] Embodiment 190 is the process according to any one of Embodiments 135 to 189, wherein the aqueous medium has a human HNA antibody concentration that is less than 3% of the HNA antibody concentration in the donor apheresis plasma.

[0339] Embodiment 191 is the process according to any one of Embodiments 135 to 190, wherein the protein concentration is 1% or less of the protein concentration of the donor apheresis plasma.

[0340] Embodiment 192 is the process according to any one of Embodiments 135 to 191, wherein the aqueous medium has a concentration of human HLA class I antibody that is less than 1% of the HLA class I antibody concentration in the donor apheresis plasma.

[0341] Embodiment 193 is the process according to any one of Embodiments 135 to 192, wherein the aqueous medium has a concentration of human HLA class II antibody that is less than 1% of the HLA class II antibody concentration in the donor apheresis plasma.

[0342] Embodiment 194 is the process according to any one of Embodiments 135 to 193, wherein the aqueous medium has a human HNA antibody concentration that is less than 1% of the HNA antibody concentration in the donor apheresis plasma.

[0343] Embodiment 195 is the process according to any one of Embodiments 135 to 194, wherein the composition is negative for HLA class I antibodies based on a test approved by a regulatory agency.

[0344] Embodiment 196 is the process according to any one of Embodiments 135 to 195, wherein the composition is negative for HLA class II antibodies based on a test approved by a regulatory agency.

[0345] Embodiment 197 is the process according to any one of Embodiments 135 to 196, wherein the composition is negative for HNA antibodies based on a test approved by a regulatory agency.

[0346] Embodiment 198 is the process according to any one of Embodiments 135 to 197, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is less than 5% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0347] Embodiment 199 is the process according to any one of Embodiments 135 to 198, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is less than 3% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0348] Embodiment 200 is the process according to any one of Embodiments 135 to 199, wherein the percentage of beads positive for an antibody selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies is less than 1% in the quantification of the composition by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively.

[0349] Embodiment 201 is the process according to any one of Embodiments 135 to 200, wherein the percentage of beads positive for HLA class I antibodies is less than 5% in the quantification of the composition by flow cytometry using beads coated with class I HLA.

[0350] Embodiment 202 is the process according to any one of Embodiments 135 to 201, wherein the percentage of beads positive for HLA class I antibodies is less than 3% in the quantification of the composition by flow cytometry using beads coated with class I HLA.

[0351] Embodiment 203 is the process according to any one of Embodiments 135 to 202, wherein the percentage of beads positive for HLA class I antibody is less than 1% in the quantification of the composition by flow cytometry using beads coated with class I HLA.

[0352] Embodiment 204 is the process according to any one of Embodiments 135 to 203, wherein the percentage of beads positive for HLA class II antibody is less than 5% in the quantification of the composition by flow cytometry using beads coated with class II HLA.

[0353] Embodiment 205 is the process according to any one of Embodiments 135 to 204, wherein the percentage of beads positive for HLA class II antibody is less than 3% in the quantification of the composition by flow cytometry using beads coated with class II HLA.

[0354] Embodiment 206 is the process according to any one of Embodiments 135 to 205, wherein the percentage of beads positive for HLA class II antibody is less than 1% in the quantification of the composition by flow cytometry using beads coated with class II HLA.

[0355] Embodiment 207 is the process according to any one of Embodiments 135 to 206, wherein the percentage of beads positive for HNA antibody is less than 5% in the quantification of the composition by flow cytometry using beads coated with HNA.

[0356] Embodiment 208 is the process according to any one of Embodiments 135 to 207, wherein the percentage of beads positive for HNA antibody is less than 3% in the quantification of the composition by flow cytometry using beads coated with HNA.

[0357] Embodiment 209 is the process according to any one of Embodiments 135 to 208, wherein the percentage of beads that are positive for HNA is less than 1% in the quantification of the composition by flow cytometry using beads coated with HNA.

[0358] Embodiment 210 is the process according to any one of Embodiments 135 to 209, wherein the composition contains less than 5.0% (by scattering intensity) of fine particles.

[0359] Embodiment 211 is the process according to any one of Embodiments 135 to 209, wherein the composition contains less than 4.5% (by scattering intensity) of fine particles.

[0360] Embodiment 212 is the process according to any one of Embodiments 135 to 209, wherein the composition contains less than 4.0% (by scattering intensity) of fine particles.

[0361] Embodiment 213 is the process according to any one of Embodiments 135 to 209, wherein the composition contains less than 3.5% (by scattering intensity) of fine particles.

[0362] Embodiment 214 is the process according to any one of Embodiments 135 to 213, wherein the platelet or platelet derivative has at least 55% CD41 positive percentage.

[0363] Embodiment 215 is the process according to any one of Embodiments 135 to 213, wherein the platelet or platelet derivative has at least 60% CD41 positive percentage.

[0364] Embodiment 216 is the process according to any one of Embodiments 135 to 213, wherein the platelet or platelet derivative has at least 65% CD41 positive percentage.

[0365] Embodiment 217 is the process according to any one of Embodiments 135 to 216, wherein the platelet or platelet derivative has at least 65% CD42 positive percentage.

[0366] Embodiment 218 is the process according to any one of Embodiments 135 to 216, wherein the platelet or platelet derivative has at least 80% CD42 positive percentage.

[0367] Embodiment 219 is the process according to any one of Embodiments 135 to 216, wherein the platelet or platelet derivative has at least 90% CD42 positive percentage.

[0368] Embodiment 220 is the process according to any one of Embodiments 135 to 219, wherein the platelet or platelet derivative retains at least about 10% of the lactate dehydrogenase activity of donor apheresis platelets.

[0369] Embodiment 221 is the process according to any one of Embodiments 135 to 219, wherein the platelet or platelet derivative retains at least about 15% of the lactate dehydrogenase activity of donor apheresis platelets.

[0370] Embodiment 222 is the process according to any one of Embodiments 135 to 219, wherein the platelet or platelet derivative retains at least about 20% of the lactate dehydrogenase activity of donor apheresis platelets.

[0371] Embodiment 223 is the process according to any one of Embodiments 135 to 222, wherein the platelet or platelet derivative has at least 25% annexin V positive percentage.

[0372] Embodiment 224 is the process according to any one of Embodiments 135 to 222, wherein the platelet or platelet derivative has at least 50% annexin V positive percentage.

[0373] Embodiment 225 is the process according to any one of Embodiments 135 to 222, wherein the platelet or platelet derivative has at least 75% annexin V positive percentage.

[0374] Embodiment 226 is the process according to any one of Embodiments 135 to 225, wherein the platelet or platelet derivative has at least 8% CD47 positive percentage.

[0375] Embodiment 227 is the process according to any one of Embodiments 135 to 225, wherein the platelet or platelet derivative has at least 10% CD47 positive percentage.

[0376] Embodiment 228 is the process according to any one of Embodiments 135 to 225, wherein the platelet or platelet derivative has at least 15% CD47 positive percentage.

[0377] Embodiment 229 is the process according to any one of Embodiments 135 to 225, wherein the platelet or platelet derivative has at least 20% CD47 positive percentage.

[0378] Embodiment 230 is the process according to any one of Embodiments 135 to 229, wherein the platelet or platelet derivative has at least 10% CD62 positive percentage.

[0379] Embodiment 231 is the process according to any one of Embodiments 135 to 229, wherein the platelet or platelet derivative has at least 50% CD62 positive percentage.

[0380] Embodiment 232 is the process according to any one of Embodiments 135 to 229, wherein the platelet or platelet derivative has at least 80% CD62 positive percentage.

[0381] Embodiment 233 is the process according to any one of Embodiments 135 to 229, wherein the platelet or platelet derivative has at least 90% CD62 positive percentage.

[0382] Embodiment 234 is the process according to any one of Embodiments 135 to 233, wherein the platelet or platelet derivative has fibrinogen associated with the cell membrane.

[0383] Embodiment 235 is the process according to any one of Embodiments 135 to 234, wherein the aqueous medium has a lactate concentration of less than 2.0 mmol / L.

[0384] Embodiment 236 is the process according to any one of Embodiments 135 to 234, wherein the aqueous medium has a lactate concentration of less than 1.5 mmol / L.

[0385] Embodiment 237 is the process according to any one of Embodiments 135 to 236, wherein the aqueous medium has a lactate concentration of about 0.4 to about 1.3 mmol / L.

[0386] Embodiment 238 is the process according to any one of Embodiments 135 to 236, wherein the aqueous medium has a lactate concentration of about 0.5 to about 1.0 mmol / L.

[0387] Embodiment 239 is the process according to any one of Embodiments 135 to 238, wherein the platelet derivative contains thrombosomes.

[0388] Embodiment 240 is the process according to any one of Embodiments 135 to 239, further comprising a pathogen reduction step.

[0389] Embodiment 241 is the process according to Embodiment 240, wherein the pathogen reduction step precedes TFF.

[0390] Embodiment 242 is the process according to any one of Embodiments 135 to 241, further comprising lyophilizing the composition containing platelets or platelet derivatives.

[0391] Embodiment 243 is the process according to any one of Embodiments 135 to 241, further comprising cryopreserving the composition containing platelets or platelet derivatives.

[0392] Embodiment 244 is the process according to any one of Embodiments 135 to 243, further comprising thermally treating the composition containing platelets or platelet derivatives.

[0393] Embodiment 245 is the process according to any one of Embodiments 135 to 148, 154 to 174, or 195 to 244, wherein the protein concentration is about 5% to about 50% of the protein concentration of donor apheresis plasma.

[0394] Embodiment 246 is the process according to any one of Embodiments 135 to 149, 154 to 178, or 195 to 245, wherein the protein concentration is about 5% to about 30% of the protein concentration of donor apheresis plasma.

[0395] Embodiment 247 is the process according to any one of Embodiments 135 to 148, 154 to 178, or 195 to 246, wherein the protein concentration is about 5% to about 15% of the protein concentration of donor apheresis plasma.

[0396] Embodiment 248 is the process according to any one of Embodiments 135 to 148, 154 to 182, or 195 to 247, wherein the protein concentration is about 8% to about 10% of the protein concentration of donor apheresis plasma.

[0397] Embodiment 249 is the process according to any one of Embodiments 135 to 148, 154 to 182, or 195 to 248, wherein the protein concentration is about 7% to about 10% of the protein concentration of donor apheresis plasma.

[0398] Embodiment 250 is the process according to any one of Embodiments 135 to 249, wherein the platelet or platelet derivative produces a thrombin peak height (TPH) of at least 25 nM when in the presence of a reagent containing tissue factor and phospholipid at a concentration of about 4.8×10 3 particles / μL.

[0399] Embodiment 251 is the process according to any one of Embodiments 135 to 249, wherein the platelet or platelet derivative produces a thrombin peak height (TPH) of at least 50 nM when in the presence of a reagent containing tissue factor and phospholipid at a concentration of about 4.8×10 3 particles / μL.

[0400] Embodiment 252 is the process according to any one of Embodiments 135 to 249, wherein the platelet or platelet derivative has a titer of at least 1.5 thrombin generating potency units (TGPU) per particle. 6

[0401] Embodiment 253 is the process according to any one of Embodiments 135 to 249, wherein the platelet or platelet derivative provides an occlusion time of less than 14 minutes in a total thrombosis analysis system (T-TAS) assay at a concentration of at least about 70×10 3 particles / μL.

[0402] Embodiment 254 is the process according to any one of Embodiments 135 to 249, wherein the platelet or platelet derivative provides an occlusion time of less than 12 minutes in a total thrombosis analysis system (T-TAS) assay at a concentration of at least about 70×10 3 particles / μL.

[0403] Embodiment 255 is a composition comprising a platelet or platelet derivative and an aqueous medium, prepared by the process according to any one of Embodiments 135 to 254.

[0404] Embodiment 256 is a process for preparing freeze-dried platelets, comprising: a) preparing a composition comprising platelets and an aqueous medium using the process according to any one of Embodiments 135 to 254; b) freeze-drying the composition comprising platelets and the aqueous medium. This is the process.

[0405] Embodiment 257 is a composition comprising freeze-dried platelets prepared by the process according to Embodiment 235.

[0406] Embodiment 258 is a method for preparing a composition comprising platelets or platelet derivatives and an aqueous medium, comprising: diluting a starting material comprising platelets to form a diluted starting material; concentrating the diluted starting material such that the platelets have a concentration of about 2250×10 3 cells / μL (±250×10 3 ) to form a concentrated platelet composition; washing the concentrated platelet composition with at least 2 diavolumes (DV) of a preparation to form a TFF-treated composition. This is the method.

[0407] Embodiment 259 is the method according to Embodiment 258, wherein the diluting comprises diluting with a preparation of approximately the same weight (±10%).

[0408] Embodiment 260 is the method according to any one of Embodiments 258 to 259, further comprising a pathogen reduction step.

[0409] Embodiment 261 is the method according to Embodiment 260, wherein the pathogen reduction step is performed before diluting the starting material.

[0410] Embodiment 262 is the method according to any one of Embodiments 258 to 261, wherein the residual plasma percentage is about 15% relative plasma or less (quantification by plasma protein content).

[0411] Embodiment 263 is that after washing, if the concentration of cells in the TFF-treated composition is not about 2000×10 3 cells / μL (±300×10 3 ), the preparation may be diluted or concentrated to fall within this range, which is the method according to any one of Embodiments 258 to 262.

[0412] Embodiment 264 is the method according to any one of Embodiments 258 to 263, further comprising lyophilizing the TFF-treated composition to form a lyophilized composition.

[0413] Embodiment 265 is the method according to Embodiment 264, further comprising treating the lyophilized composition at about 80°C for about 24 hours.

[0414] Embodiment 266 is a composition comprising platelets or platelet derivatives prepared by the method according to any one of Embodiments 258 to 265.

[0415] Embodiment 267 is a method for treating a blood coagulation-related disease or condition in a subject in need thereof, comprising administering a therapeutically effective amount of the composition according to any one of Embodiments 1 to 134, 255, or 266 to the subject.

[0416] Embodiment 268 is the method according to Embodiment 267, wherein the blood coagulation-related disease or condition is selected from the group consisting of von Willebrand disease, hemophilia, thrombasthenia, thrombocytopenia, thrombocytopenic purpura, trauma, or combinations thereof.

[0417] Embodiment 269 is a method for treating a blood coagulation-related disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition prepared by the process according to any one of Embodiments 135 to 254.

[0418] Embodiment 270 is the method according to Embodiment 269, wherein the blood coagulation-related disease or condition is selected from the group consisting of von Willebrand disease, hemophilia, thrombasthenia, thrombocytopenia, thrombocytopenic purpura, trauma, or a combination thereof.

[0419] Embodiment 271 is a method for treating a blood coagulation-related disease or condition in a subject in need thereof, the method comprising administering to the subject a therapeutically effective amount of a composition prepared by the process according to any one of Embodiments 258 to 266.

[0420] Embodiment 272 is the method according to Embodiment 271, wherein the blood coagulation-related disease or condition is selected from the group consisting of von Willebrand disease, hemophilia, thrombasthenia, thrombocytopenia, thrombocytopenic purpura, trauma, or a combination thereof.

Examples

[0421] Example 1. Tangential Flow Filtration (TFF) Method Apheresis platelets were subjected to tangential flow filtration according to standard operating procedures (including the following process steps: platelet dilution, platelet concentration, platelet washing).

[0422] Platelet donor units are first pooled in a common container. Platelets may or may not be diluted initially with an acidified wash buffer (e.g., control buffer) to reduce platelet activation during processing. Platelets can pass through two processing pathways, namely, a pathway of washing with control buffer until reaching the desired residual components (e.g., donor plasma) before concentrating to the final product concentration, or a pathway of concentrating the platelets to the final product concentration before washing with control buffer until reaching the desired residual components (e.g., donor plasma). The TFF-treated platelets are then filled into vials, lyophilized, and heat-treated.

[0423] One specific protocol is as follows.

[0424] Buffer A was used in all steps of the TFF process in this example. This process was carried out at a temperature of 18 - 24 °C.

[0425] Buffer A TIFF2025081548000008.tif63128

[0426] Load the platelets into the TFF (PendoTECH controller system), which was prepared with a Repligen TFF Cassette (XPM45L01E). Dilute the platelets with buffer A of equal weight (±10%) and concentrate to approximately 2250×10 3 cells / μL (±250×10 3 ), and then wash with approximately 2 diavolumes (DV) of buffer A. The target plasma percentage was typically less than 15% relative plasma (quantified by plasma protein content). Removal of plasma proteins was monitored by UV absorbance at 280 nm based on known correlations. After washing, the cell concentration was 2000×10 3 cells / μL (±300×10 3) If not, the cells were diluted or concentrated with buffer A to fit within this range. The cells were typically lyophilized and then heat-treated at 80 °C for 24 hours, thereby forming thrombosomes, although the cells were sometimes used before lyophilization (sometimes referred to as "pre-lyo" thrombosomes). Typically, the thrombosomes were rehydrated at room temperature for 10 minutes. Generally, the rehydration volume was equal to the amount used to fill each vial of thrombosomes before drying.

[0427] In some cases, samples were taken at UV readings corresponding to relative plasma volumes of approximately 51%, approximately 8.1%, approximately 6.0%, and approximately 1.3%. Low-volume aliquots were sampled over each processing step using samples of approximately 6.0% or less.

[0428] Example 2. Test Plan and Assay Protocol Test Plan: Thrombosome batch A was produced by the TFF method described in Example 1, using apheresis platelets collected from a donor with a high αHLA titer as reported by the platelet supplier.

[0429] For αHLA testing, individual donor units, donor pools, and time points along the TFF process were collected. Plasma was added to HLA beads (One Lambda FLOWPRA™ Screen Test), stained with an αIgG secondary antibody, and αHLA-IgG binding was evaluated by flow cytometry (Novocyte 3005 configuration).

[0430] Two types of beads, namely beads coated with HLA class I antigen and beads coated with HLA class II antigen, were evaluated. Bead gating was performed as described in the instructions for the FLOWPRA™ Screen Test. The "αHLA positive" population was gated based on the George King (GK) PPP (platelet-poor plasma) negative control and the single donor freshly collected negative control. Additional negative controls were collected after production to confirm the ideal placement of these positive gates.

[0431] Compensation settings were established using FITC- and PE-conjugated mouse IgG on Spherotech COMPtrol compensation beads. HLA class II beads fluoresce within PE, and the secondary antibody used for IgG detection is FITC-conjugated.

[0432] Assay protocol: 1. Thaw the components of the One Lambda FLOWPRA™ Screen Test kit and bring to 4°C. 2. From each desired collection point, obtain a 1 mL aliquot of PPP filtered through 0.2 μm. 3. Pipette 5 μL of class I HLA beads and 5 μL of class II HLA beads into a 1.7 mL microcentrifuge tube. Add 20 μL of filtered test plasma. Vortex to mix. 4. Incubate the plasma with the HLA-coated beads for 30 minutes at room temperature and in the dark with gentle rocking / stirring. 5. Dilute the 10× wash buffer with deionized water to an appropriate volume of 1× working stock. 6. Wash the beads with 1 mL of wash buffer. Vortex and centrifuge at 9,000 g for 2 minutes to pellet the beads. Aspirate the supernatant. 7. Repeat step 6. 8. Dilute the 100× αIgG-FITC with wash buffer to an appropriate volume of 1× working stock. 9. Add 100 μL of 1×αIgG-FITC to the tube containing the washed HLA beads. Vortex to mix. 10. Incubate the HLA beads with αIgG-FITC for 30 minutes at room temperature and in the dark with gentle rocking. 11. Repeat Steps 6 and 7 to wash away unbound αIgG-FITC. 12. Resuspend the washed HLA beads in 200 μL of PBS. Vortex to mix. 13. Pipette 100 μL of the HLA bead suspension into the appropriate well(s) of a 96-well U-bottom microplate and dock to the NovoSampler. 14. Use NovoCyte to collect events by flow cytometry. a. In the FLOWPRA™ Beads gate previously quantified, slowly collect 10,000 events at an FSC-H threshold of 10,000. b. Set the secondary stop conditions to a run time of 2 minutes and a total sample volume of 40 μL. c. To minimize carryover between wells, wash the SIP between each sample. To minimize carryover between test points, run PBS wells between each triplicate set.

[0433] Results Example 3. Gate Setup and Negative Controls The initial gate setup for discriminating Class I and Class II HLA beads is determined using FLOWPRA™ beads in PBS. (Figures 1A and 1B)

[0434] Background / non-specific binding is established in triplicate using GK PPP and fresh donor PPP. (Exemplary data are shown in Figures 2A, 2B, 3A, and 3B.) Note that the PPP of GK is shifted higher with FITC-H than fresh donor plasma. This may be due to donor variability and the freezing / thawing effect of GK plasma. Additional sample collection is required. Place the positive gate so that less than 1% of GK PPP becomes FITC positive.

[0435] Example 4. Results of single donors Representative FITC-H histograms for each HLA class are shown for each sample. A positive rate > 1% is considered positive. The fluorescence ratio is reported relative to the GK PPP negative control (FITC-H intensity of class I and class II beads). A fluorescence ratio > 1.0 is considered positive. These positive gates and fluorescence ratios are updated as additional negative controls are collected.

[0436] A population of HLA class I or class II antibodies is considered negative if both the positive rate and the fluorescence ratio are 1 or less in that bead type.

[0437] Donor 1: HLA class II positive. The average positive rate for class I from triplicate data is 0.2% and the fluorescence ratio is 0.3, and the average positive rate for class II is 16.5% and the fluorescence ratio is 1.6. (Exemplary data are shown in Figures 4A and 4B.)

[0438] Donor 2: HLA class I and class II positive. The average positive rate for class I from triplicate data is 1.3% and the fluorescence ratio is 1.4, and the average positive rate for class II is 20.3% and the fluorescence ratio is 1.9. (Exemplary data are shown in Figures 5A and 5B.)

[0439] Donor 3: HLA class I and class II positive. The average positive rate of class I data from triplicate data is 85.2% and the fluorescence ratio is 14.4. The average positive rate for class II is 12.0% and the fluorescence ratio is 0.8. (Exemplary data are shown in Figures 6A and 6B.)

[0440] Donor 4: HLA class I and class II positive. The average positive rate of class I data from the triplicate data is 83.5%, and the fluorescence ratio is 14.5. The average positive rate of class II is 12.6%, and the fluorescence ratio is 0.8. (Exemplary data are shown in FIGS. 7A and 7B)

[0441] Donor 5: HLA class I and class II positive. The average positive rate of class I data from the triplicate data is 4.9%, and the fluorescence ratio is 1.2. The average positive rate of class II is 1.3%, and the fluorescence ratio is 0.8. (Exemplary data are shown in FIGS. 8A and 8B)

[0442] Donor 6: HLA class I positive. The average positive rate of class I data from the triplicate data is 2.7%, and the fluorescence ratio is 0.9. The average positive rate of class II is 0.3%, and the fluorescence ratio is 0.7. (Exemplary data are shown in FIGS. 9A and 9B)

[0443] Donor 7: HLA class II positive. The average positive rate of class I from the triplicate data is 0.7%, and the fluorescence ratio is 0.5. The average positive rate of class II is 9.0%, and the fluorescence ratio is 1.3. (Exemplary data are shown in FIGS. 10A and 10B)

[0444] The results of Donors 1 - 7 are also shown in Table 1.

[0445] (Table 1) TIFF2025081548000009.tif53162

[0446] Since there may be HLA - positive donors in the GK PNP pool, Table 2 shows the results for N = 1 HLA - negative donors.

[0447] (Table 2) TIFF2025081548000010.tif52161

[0448] Example 5. Results of Filtration Pool: HLA class I and class II positive. The average positive rate of class I data from triplicate data is 61.1%, and the fluorescence ratio is 4.6. The average positive rate of class II is 9.0%, and the fluorescence ratio is 1.1. (Exemplary data are shown in FIGS. 11A and 11B)

[0449] Initial dilution (51%): HLA class I and class II positive. The average positive rate of class I data from triplicate data is 48.2%, and the fluorescence ratio is 4.2. The average positive rate of class II is 5.9%, and the fluorescence ratio is 0.9. (Exemplary data are shown in FIGS. 12A and 12B)

[0450] "20%" plasma (8.1%): HLA class I positive. The average positive rate of class I data from triplicate data is 2.4%, and the fluorescence ratio is 1.0. The average positive rate of class II is 0.2%, and the fluorescence ratio is 0.5. (Exemplary data are shown in FIGS. 13A and 13B)

[0451] "<10%" plasma (6.0%): Borderline HLA class I positive. The average positive rate of class I data from triplicate data is 1.1%, and the fluorescence ratio is 1.0. The average positive rate of class II is 0.2%, and the fluorescence ratio is 0.5. (Exemplary data are shown in FIGS. 14A and 14B)

[0452] "<3%" plasma (1.3%): HLA negative. The average positive rate of class I data from triplicate data is 0.4%, and the fluorescence ratio is 0.2. The average positive rate of class II is 0.1%, and the fluorescence ratio is 0.4. (Exemplary data are shown in FIGS. 15A and 15B)

[0453] The results of filtration are also shown in Tables 3A and 3B.

[0454] (Table 3A) TIFF2025081548000011.tif52165

[0455] (Table 3B) The background fluorescence measured using HLA beads in PBS was subtracted from the sample fluorescence, and then the percent reduction in average fluorescence intensity (a measure of antibody binding reduction) was calculated. TIFF2025081548000012.tifValues above 138145100% indicate that the binding of detectable HLA antibodies to the indicated beads was completely reduced.

[0456] Since there may be HLA-positive donors in the GK PNP pool, Table 4 shows the results for N = 1 HLA-negative donors.

[0457] (Table 4) TIFF2025081548000013.tif43161

[0458] Example 6. Surface markers and thrombin generation. Thrombosome batches were produced by the TFF method described in Example 1, and the expression of cell surface markers was assayed using flow cytometry.

[0459] Flow cytometry was used to evaluate the expression of CD41, CD62, and phosphatidylserine (PS) on thrombosomes. The sample contained approximately 270,000 / μL thrombosomes at the time of staining and was diluted approximately 1:34 before analyzing the sample with a cytometer. The thrombosome sample was rehydrated and diluted 1:2 with deionized water. The stock of anti-CD41 was diluted by adding 47.6 μL of the antibody to 52.4 μL of HMTA. A sample stained with anti-CD41 was prepared by adding 10 μL of the diluted thrombosomes to 10 μL of HMTA and 10 μL of the diluted CD41 antibody. An anti-CD62 master mix was prepared by combining 12 μL of anti-CD62 with 23.8 μL of anti-CD41 and 64.2 μL of HMTA. An isotype control mix was prepared in the same manner. A sample stained with anti-CD62 was prepared by adding 10 μL of the diluted thrombosomes to 20 μL of the anti-CD62 master. An isotype control sample was prepared in the same manner using the isotype master mix. An annexin V (AV) master mix was prepared by combining 11.7 μL of AV with 83.3 μL of anti-CD41 and 80 μL of HMTA. A sample stained with AV was prepared by adding 20 μL of the diluted thrombosomes containing 50 mM GPRP to 20 μL of HMTA containing 15 mM CaCl 2 and 20 μL of the AV master mix. A negative gating control sample was prepared in the same manner using HMTA without calcium to prevent AV from binding to PS. All samples were incubated at room temperature for 20 minutes. After incubation, 1 mL of HBS was added to all samples. The HBS used for dilution of the AV test samples contained 5 mM CaCl 2 . Anti-CD41 binding was used to identify the target population. CD62 expression and PS expression were evaluated by the binding of anti-CD62 and AV within the CD41-positive population.

[0460] The expression of glycoprotein IIb (GPIIb, also known as antigen CD41) was assayed using an anti-CD41 antibody (4.8 μL, Beckman Coulter, part number IM1416U). The assayed thrombosomes were CD41 positive (Table 5; Figure 16).

[0461] (Table 5) TIFF2025081548000014.tif53128

[0462] The expression of phosphatidylserine (PS) was assayed using annexin V (AV) (1.3 μL, BD Biosciences, catalog number 550475). AV is a calcium-dependent phospholipid-binding protein. The assayed thrombosomes were AV positive (Table 6; Figure 17).

[0463] (Table 6) TIFF2025081548000015.tif53128

[0464] The expression of P-selectin (also called CD62P) was assayed using an anti-CD62P antibody (2.4 μL, BD Biosciences, catalog number 550888). The assayed thrombosomes were CD62 positive (Table 7, Figure 18).

[0465] (Table 7) TIFF2025081548000016.tif53128

[0466] Thrombin generation was measured at 4.8 × 10 3 thrombosomes / μL in the presence of a PRP reagent containing tissue factor and phospholipids using the following protocol. On average, the thrombin peak height (TPH) of the thrombosome sample was 60.3 nM. Cephalin was used as a positive control. (Table 8; Figure 19)

[0467] For each tested vial, the rehydrated sample of thrombosomes was diluted to 7,200 particles / μL based on the particle count of flow cytometry using a 30% solution of Octaplas in the control buffer. In a 96-well plate, sample wells were generated by adding 20 μL of PRP reagent (Diagnostica Stago, catalog number 86196) and 80 μL of the diluted thrombosomes. Calibrator wells were generated by adding 20 μL of thrombin calibrator reagent (Diagnostica Stago, catalog number 86197) to 80 μL of the diluted thrombosomes. This plate was loaded into a plate reader and incubated at 40 °C in the dark for 10 minutes. During the incubation of the samples, a FluCa solution was prepared by adding 40 μL of the FluCa substrate (Diagnostica Stago catalog number 86197) to 1.6 mL of Fluo-Buffer (Diagnostica Stago catalog number 86197) warmed to 37 °C and vortexing to mix. The FluCa solution was aspirated into a dispensing syringe and mechanically dispensed 20 μL each into each reaction well to bring the final concentration of thrombosomes in each well to 4,800 particles / μL and initiate the thrombin generation reaction. Thrombin generation was measured by the fluorescence of each well over 75 minutes.

[0468] An exemplary step-by-step protocol is as follows. 1. Open the CAT software, set up the device, and prepare the PRP reagent (containing tissue factor and some phospholipids), calibrator, and fluo-buffer and fluo-substrate according to the manufacturer's guidelines. 2. Thaw Octaplas and TGA dilution buffer in a 37 °C water bath for 10 minutes. 3. Add the thawed Octaplas to the TGA dilution buffer to create a buffer containing 30% Octaplas. 4. Dilute the reconstituted cephalin 1:50 using the 30% Octaplas mix and use as a positive control. 5. Rehydrate the thrombosomes with cell culture grade water for 10 minutes and then dilute with 30% Octaplas to 7,200 thrombosomes / μL. 6. Using a multi-channel pipette, add 20 μL of PRP reagent to each test well. Add 20 μL of calibrator to each calibration well. 7. Add 80 μL of sample to each test and calibration well. Add 80 μL of 30% Octaplas to the negative control well and 1:50 cephalin to the positive control well. 8. Insert the plate into the tray and incubate at 40 °C for 10 minutes. After incubation, dispense the mixture of fluo-buffer and fluo-substrate (containing a fluorescently labeled peptide that generates a fluorescent signal when cleaved by thrombin) into the active wells. 9. Read the plate every 20 seconds for 75 minutes to fully capture the thrombin generation profile.

[0469] (Table 8) TIFF2025081548000017.tif53128

[0470] Summarize the data of these assays in Table 9.

[0471] (Table 9) TIFF2025081548000018.tif68160 1 Particle size evaluated using the sizing bead of flow cytometry forward scatter.

[0472] Example 7. Binding of 9F9 and PAC-1. The aggregation of activated platelets is mediated by the formation of the GPIIb / IIIa complex, which can bind to fibrinogen (also called factor I) and form a blood clot. GPIIb / IIIa is a platelet fibrinogen receptor also known as the CD41 / CD61 complex. In this process, ADP promotes the active form of the GPIIb / IIIa complex. Antibody 9F9 binds to fibrinogen associated with the cell membrane. Thus, the presence of fibrinogen on the cell membrane indicates the presence of thrombosomes capable of forming a blood clot.

[0473] Vials of thrombosomes prepared according to Example 1 were rehydrated with 10 mL of deionized water. An aliquot of the thrombosomes was diluted with HMTA (HEPES-modified Tyrode's albumin) to a final concentration of 1×10 5 particles / μL. Samples were prepared as shown in Table 11. An unstained sample was prepared by adding 10 μL of the diluted thrombosomes to 20 μL of HMTA. The FITC isotype control sample was prepared by adding 10 μL of the diluted thrombosomes to 10 μL of an isotype control antibody (BD Biosciences, catalog number 555748) and 10 μL of HMTA. The sample stained with 9F9 was prepared by adding 10 μL of the diluted thrombosomes to 10 μL of the 9F9 antibody (BD Biosciences, catalog number 340507) and 10 μL of HMTA. The sample stained with PAC-1 was prepared by adding 10 μL of the diluted thrombosomes to 5 μL of the isotype control antibody and 15 μL of HMTA. All samples were prepared in duplicate using a total of 1×10 6 particles per reaction mixture. The samples were incubated at room temperature for 20 minutes in the dark. After incubation, all samples were diluted with 1 mL of HBS and acquired using an ACEA NovoCyte flow cytometer. The fluorescence signal generated by PAC-1 was used to quantify the expression of the activated GPIIb / IIIa receptor without bound fibrinogen. The fluorescence signal from 9F9 was used to quantify the binding of fibrinogen to the surface receptor of the thrombosomes.

[0474] HTMA (HEPES-modified tyrode albumin). TIFF2025081548000019.tif47128

[0475] (Table 10) TIFF2025081548000020.tif38160

[0476] When the sample was assayed by flow cytometry, it was shown that fibrinogen bound to the surface was present after rehydration (Figure 20), while the anti-PAC-1 antibody did not show significant binding (Figure 21). This is further proof that the thrombosomes prepared by TFF contain fibrinogen bound to the active form of GPIIb / GPIIIa (PAC-1 also binds to the same complex).

[0477] Example 8. Evaluation of CD47 Binding CD47 is a cell surface marker used for self-recognition. Without this marker, phagocytosis may occur in some cases.

[0478] One vial of the thrombosomes prepared as described in Example 1 was rehydrated with 10 mL of sterile water for injection and stained with an anti-CD47 antibody conjugated with a gradually increasing volume of Pacific Blue (BD Biosciences catalog number 561564) or the corresponding isotype control (BD Biosciences catalog number 560373). All samples contained 1 million cells. As a result of this titration, the maximum fluorescence signal was about 5 times the background (Figure 22A), and the overall CD47 positive rate was about 40% (Table 12). An exemplary histogram is shown in Figure 22B.

[0479] An aliquot of the CD47 antibody conjugated with V450 was prepared at dilutions of 1:10, 1:5, and 1:2 using HMTA. The initial concentration of the thrombosome sample was quantified using AcT diff 2, and the concentration of a 1 mL aliquot was 100×103 Adjusted to / μL. TFF thrombosomes were stained in duplicate at each antibody dilution by adding 10 μL of antibody to 10 μL of diluted thrombosomes. Samples stained with undiluted antibody were generated in the same manner. Unstained control samples were prepared by adding 10 μL of HMTA to 10 μL of diluted thrombosomes. This sample preparation was repeated using an isotype control antibody instead of anti-CD47. All samples were incubated for 20 minutes at room temperature in the dark. After incubation, the samples were diluted with 500 μL of HBS, and 15,000 events were acquired for each sample using an ACEA NovoCyte flow cytometer. Antibody binding to CD47 on the surface of thrombosomes was evaluated using V450 fluorescence in the test samples. Nonspecific binding was monitored using V450 fluorescence in the isotype control samples.

[0480] Table 11 shows the mean fluorescence intensity of samples with various amounts of antibody (anti-CD47 or isotype control).

[0481] (Table 11) TIFF2025081548000021.tif30131

[0482] Table 12 shows the percentage of CD47 positive in various concentrations of anti-CD47 antibody.

[0483] (Table 12) TIFF2025081548000022.tif34131

[0484] A second vial of TFF thrombosomes from different lots was rehydrated and stained while gradually increasing the volume of anti-CD47 conjugated to Pacific Blue or the corresponding isotype control. All samples contained 250,000 cells. Again, a fluorescence signal about 5 to 6 times the background was observed (Figure 22C), and the overall CD47 positive rate was about 50% (Table 15). An exemplary histogram is shown in Figure 22D.

[0485] The second test was performed on a new TFF thrombosome sample, increasing the amount of antibody incrementally and decreasing the number of thrombosomes per sample while using it to improve the intensity of the signal generated by the binding of anti-CD47 to the thrombosome. The initial concentration of the thrombosome sample was quantified using AcT diff 2, and the concentration of a 1 mL aliquot was adjusted to 25×10 3 / μL using HMTA. The samples were stained in duplicate while incrementally increasing the amount of antibody according to Table 13 below. The final volume of each sample was kept constant at 40 μL. The total number of thrombosomes in each sample was kept constant at 250×10 3 / μL. This sample preparation was repeated using an isotype control antibody instead of anti-CD47.

[0486] (Table 13) TIFF2025081548000023.tif37160

[0487] All samples were incubated for 20 minutes at room temperature in the dark. After incubation, the samples were diluted with 500 μL of HBS, and 15,000 events were acquired for each sample using an ACEA NovoCyte flow cytometer. Antibody binding to CD47 on the surface of the thrombosome was evaluated using V450 fluorescence in the test samples. Non-specific binding was monitored using V450 fluorescence in the isotype control samples.

[0488] Table 14 shows the mean fluorescence intensity of the samples with various amounts of antibody (anti-CD47 or isotype control).

[0489] (Table 14) TIFF2025081548000024.tif33131

[0490] Table 15 shows the percentage of CD47 positivity at various concentrations of anti-CD47 antibody.

[0491] (Table 15) TIFF2025081548000025.tif29131

[0492] Example 9. Reduction of microparticle content The microparticle content of human In-Date Stored Platelets (hIDSP) was compared using dynamic light scattering with thrombosomes prepared according to Example 1 (without lyophilization). The results are shown in FIGS. 23A to 23C and Table 16. FIGS. 23A to 23C are histograms normalized based on relative intensity such that the sum of the intensities of each data point equals 1.0. For example, if the y-axis value of a particular data point is 0.1, it can typically be interpreted that the data point accounts for 10% of the scattering intensity of the sample.

[0493] A pool of apheresis units used in the production of a batch of thrombosomes was prepared and analyzed. This sample type is designated as "hIDSP". A 1 mL aliquot was taken from this hIDSP (human In-Date Stored Platelet) pool and analyzed by dynamic light scattering (DLS; Thrombolux (Light Integra)). A sample from this aliquot was taken into a capillary and inserted into the DLS apparatus. The capillary was placed in the apparatus for 1 minute to equilibrate the temperature and movement. The internal temperature of the machine was 37°C. After 1 minute of equilibration, the viscosity setting of the sample was selected. The DLS apparatus has a viscosity setting incorporated for samples in plasma (e.g., apheresis units). This viscosity setting was used for the hIDSP sample. The viscosity of this setting was 1.060 cP (centipoise). After selecting the plasma viscosity setting, the sample was analyzed. A second and third sample were taken into capillaries from the same hIDSP aliquot and analyzed in triplicate using this hIDSP protocol. The percentage of microparticles was then quantified from this data.

[0494] The "before lyophilization" samples are intermediate samples from the thrombosome manufacturing process. This sample type is the material taken immediately before lyophilization. To analyze these samples by DLS, the viscosity of the samples was measured. The viscometer (Rheosense μVISC) is equipped with an oven and is used to bring the samples to the temperature of the DLS instrument (37 °C). The oven must be heated to 37 °C before the viscosity analysis of the samples. To quantify the viscosity of the before lyophilization samples, 400 - 350 μL of the sample was taken into a syringe and inserted into the viscometer. After inserting the sample into the viscometer, the temperature of the instrument needs to reach 37 °C again. After the oven reached 37 °C, the sample was analyzed. All settings were set to AUTO except for setting "Measurement Volume" to 400 μL. This viscosity was used for the DLS measurement of the same sample. A 1 mL aliquot was taken from this before lyophilization sample and analyzed by dynamic light scattering (DLS; Thrombolux - LightIntegra). The sample from this aliquot was taken into a capillary and inserted into the DLS instrument. The capillary was placed in the instrument for 1 minute to equilibrate the temperature and movement. The internal temperature of the instrument is 37 °C. After 1 minute of equilibration, the previously measured viscosity was entered into the viscosity setting of the DLS instrument. After entering the viscosity, the sample was analyzed. From the same before lyophilization aliquot, second and third samples were taken into capillaries and analyzed in triplicate using this before lyophilization protocol. Then, the percentage of microparticles was quantified from this data.

[0495] Thrombosomes were rehydrated according to the standard protocol and diluted 1:5 in a mixture of SeraSub (CST Technologies, Inc.) and ACD. The SeraSub / ACD diluent is ACD diluted 1:9 in SeraSub. A 1:5 dilution of 1 mL of thrombosomes was prepared and analyzed by DLS. A sample of the thrombosome diluent was taken up in a capillary and inserted into the DLS instrument. The capillary was placed in the instrument for 1 minute to equilibrate the temperature and movement. The internal temperature of the machine was 37 °C. After 1 minute of equilibration, the viscosity setting for the sample was selected. The viscosity used for the sample was 1.200 cP. After entering the viscosity, the sample was analyzed. Second, third, and fourth samples were taken up in the capillary and analyzed in quadruplicate using this thrombosome protocol. The percentage of microparticles was then quantified from the data (and platelet radius where applicable).

[0496] (Table 16) TIFF2025081548000026.tif53128

[0497] In additional experiments, the microparticle content of human in-date stored platelets (hIDSP) relative to rehydrated thrombosomes prepared according to Example 1 was compared using dynamic light scattering (DLS). The results are shown in FIGS. 24A - C and Table 17.

[0498] (Table 17) TIFF2025081548000027.tif53128

[0499] Example 12. Analysis of Metabolites Table 18 shows the analysis of the pH and metabolites present in the preparation of thrombosomes described in Example 1, which includes analysis of the platelet raw material after initial dilution, after platelet derivative concentration, and at the end of the diafiltration process, which was quantified using an i-STAT handheld blood analyzer and a CG4+ cartridge.

[0500] Platelet samples for iStat analysis were collected in small volumes (1 ml) at different processing steps. The first sample for iStat analysis, referred to as the "raw material", was collected after pooling platelet donor units and before any processing was performed. The pooled platelet units were named "initial diluent" and were diluted 1:1 with a control buffer before being subjected to platelets for TFF treatment. At the end of the concentration step of TFF, an "end of concentration" sample was collected from the platelet preparation. After washing the cells, a "DV end (before lyophilization)" sample was collected as representative of the preparation when entering the lyophilizer.

[0501] (Table 18) TIFF2025081548000028.tif78160

[0502] Example 11. Pathogen reduction In blood products, pathogen reduction is generally desirable. One method of pathogen reduction involves the use of photosensitive nucleic acid intercalating compounds to alter the nucleic acids of pathogens with illumination of an appropriate wavelength.

[0503] The INTERCEPT® system (manufactured by Cerus Corporation) uses amotosalen (a nucleic acid intercalating compound that forms crosslinks with nucleic acids upon UVA illumination). Exemplary parameters for use in this system are shown in Table 19, a schematic diagram of the system is shown in Figure 25A, and exemplary process data for the processing of 2.6 L of material at 198 minutes (approximately 14 / minute average) are shown in Figures 25B - C.

[0504] DLS was performed as described in Example 9.

[0505] (Table 19) TIFF2025081548000029.tif64128

[0506] Exemplary comparative data of the pH and metabolites of the thrombosomes prepared as in Example 1 with or without treatment by the INTERCEPT® system are shown in Table 20.

[0507] (Table 20) TIFF2025081548000030.tif93166

[0508] Exemplary comparative data of functional property evaluation (AcT number and aggregation parameter) and cell surface markers are shown in Table 21 (hIDSP), Table 22 (before lyophilization), and Table 23 (lyophilized and rehydrated to a concentration of approximately 1.8×10 6 / μL (the number of individual samples is shown in Table 23)).

[0509] (Table 21) TIFF2025081548000031.tif73161

[0510] (Table 22) TIFF2025081548000032.tif73161

[0511] (Table 23) TIFF2025081548000033.tif68161

[0512] Also, the particulate content at various stages of the thrombosome preparation was quantified as described in Example 9. Figures 26A - 26B show the similarity of rehydrated thrombosomes prepared with or without pathogen reduction treatment. An overview of these data is shown in Table 24. Figure 27A shows the particulate content of hiDSP with or without pathogen reduction treatment. Figures 27B - 27C compare the particulate content of hiDSP shown in Figure 29A and the rehydrated thrombosomes prepared therefrom. An overview of these data is shown in Table 25. Figure 28A shows the particulate content of hiDSP with or without pathogen reduction treatment. Figures 28B - 28C compare the particulate content of hiDSP shown in Figure 28A and the rehydrated thrombosomes prepared therefrom. An overview of these data is shown in Table 26.

[0513] (Table 24) TIFF2025081548000034.tif61128

[0514] (Table 25) TIFF2025081548000035.tif83128

[0515] (Table 26) TIFF2025081548000036.tif83128

[0516] Example 12. Interaction between platelets and thrombosomes. In this example, the "platelets" are platelets isolated from whole blood treated with citrate approximately 3 hours after collection. The thrombosomes are Batch D prepared by the method described in Example 1. Table 27 shows the sample layout of the experiment in this example.

[0517] (Table 27) TIFF2025081548000037.tif69160

[0518] The co-aggregation of platelets and thrombosomes was evaluated by light transmission aggregometry. Platelets and thrombosomes were co-incubated and evaluated by aggregometry + / - platelet activation with 4β-phorbol-12-myristate-13-acetate (PMA). For freshly isolated platelets from whole blood, 100 ng / mL of PMA was used. For stored platelets (i.e., apheresis platelets), 1000 ng / mL of PMA was used.

[0519] Fresh platelets were isolated from ACD anticoagulated whole blood, washed, and diluted to 250,000 cells / μL with HMTA. Thrombosomes were rehydrated according to the standard protocol and diluted to 250,000 cells / μL with HMTA. Aliquots of platelets in HMTA and thrombosomes in HMTA were mixed at the same ratio. Platelets, thrombosomes, and the mixed samples were evaluated by light transmission aggregometry (Helena AggRAM) in response to activation with phorbol-myristate-acetate (PMA; 100 ng / mL). The mixed samples were evaluated with or without a stir bar, and the effect of the shear force induced by stirring on platelet-thrombosome coaggregation was evaluated.

[0520] Figure 29A shows the transmittance of the samples in Table 30 with and without agonist. Mixing without agonist with shear (black) induced activation and aggregation of platelets by thrombosomes and freshly collected platelets. PMA (gray) activated the platelets. Also, the magnitude of Δ transmittance suggests mixed aggregation with thrombosomes. In the absence of shear, either no activation or coaggregation on a smaller scale than that observed in Figure 29A was observed.

[0521] Also, the AcT numbers of platelets and thrombosomes were evaluated before and after aggregation measurement. Figure 29B shows the numbers after aggregation. When the white bar is larger than the other bar(s), it is suggested that thrombosomes are incorporated into platelet aggregates. The absolute decrease in the number of particles in the absence of agonist (black) is particularly dramatic and unexpected.

[0522] Also, the effect of shear on aggregation was evaluated. The mixed aggregation assay (platelet:thrombosome number 1:1) was repeated with or without a stir bar. The results are shown in Figure 29C. These results show that shear is required for observable coaggregation in the absence of a platelet agonist. The measured numbers and the scale of coaggregation were slightly decreased in plasma compared to the buffer.

[0523] Example 13. Inhibition of fibrin trap by GPRP. In this example, "platelets" are isolated from whole blood approximately 1 hour after blood collection. The thrombosomes are batch H prepared by the method described in Example 1.

[0524] Fresh platelets were isolated from ACD-anticoagulated whole blood, washed, and diluted to 250,000 cells / μL with HMTA. Thrombosomes were rehydrated according to the standard protocol and diluted to 250,000 cells / μL with HMTA. Aliquots of platelets in HMTA and thrombosomes in HMTA were mixed at the same ratio. Each group of platelets, thrombosomes, or the mixed suspension was divided equally, one group was treated with 1 mM GPRP to inhibit fibrin polymerization, and one group was left untreated. The peptide Gly-Pro-Arg-Pro (GPRP; Sigma-Aldrich, item G1895) is a peptide that prevents fibrin polymerization. Platelets, thrombosomes, and the mixed samples were evaluated by light transmission aggregometry (Helena AggRAM) in response to the activation of thrombin (2.5 U / mL).

[0525] Figure 30 shows the results of a co-aggregation experiment in which platelets, thrombosomes, and 2:1 and 1:1 mixtures of platelets and thrombosomes were all activated with thrombin and used in the presence or absence of GPRP (1 mM). In the case of the mixtures, as the thrombosome population increased, the total aggregation measurement decreased. This suggests that the interaction between platelets and thrombosomes is partially caused by fibrin capture. However, most of the co-aggregation interaction is mediated by platelets and is not dependent on fibrin capture, as evidenced by the high aggregation measurements even in the presence of GPRP.

[0526] Examples 12 and 13 show that platelets and thrombosomes co-aggregated under shear conditions with (and to a lesser extent without) platelet activation. The fibrin polymerization inhibitor GPRP only slightly inhibited platelet-thrombosome co-aggregation after thrombin activation.

[0527] Example 14. Coaggregation inhibition of RGDS. In this example, "platelets" are isolated from whole blood approximately 1 hour after blood collection. The thrombosomes are batch H prepared by the method described in Example 1.

[0528] Fresh platelets were isolated from ACD-anticoagulated whole blood, washed, and diluted to 250,000 cells / μL with HMTA. Thrombosomes were rehydrated according to the standard protocol and diluted to 250,000 cells / μL with HMTA. Aliquots of platelets in HMTA and thrombosomes in HMTA were mixed at the same ratio. Each group of platelets, thrombosomes, or the mixed suspension was evenly divided, and one group was treated with 100 μM RGDS to inhibit fibrinogen binding to platelets, while the other group was left untreated. RGDS (Arg-Gly-Asp-Ser; Cayman Chemical, item 15359) is a peptide sequence that binds to platelet surface integrin, especially GPIIb / IIIa. This inhibits platelets from binding to fibrinogen and other adhesion proteins. Platelets, thrombosomes, and the mixed samples were evaluated by light transmission aggregometry in response to activation with phorbol myristate acetate (PMA; 100 ng / mL).

[0529] A coaggregation experiment was performed using 100 μM RGDS, activated with PMA, and examined whether an interaction occurred through fibrinogen bridging between platelets and thrombosomes. The results are shown in Figure 31. RGDS blocked more than 50% of the coaggregation measurement values. This suggests that the interaction between platelets and thrombosomes can be largely caused by fibrinogen binding.

[0530] Examples 12 to 14 show that thrombosomes readily coaggregate with activated platelets (e.g., as demonstrated by light transmission aggregation measurement). Spontaneous coaggregation is induced by shear. Platelet-thrombosome interactions are evident in both buffer and plasma. GPRP does not substantially inhibit coaggregation, while RGDS substantially inhibits coaggregation. This suggests that active platelet-fibrinogen binding plays an important role in the mechanism of coaggregation and that coaggregation is not caused by passive fibrin trapping alone.

[0531] Example 15. Scanning electron microscope (SEM). A 10 mL aliquot of rehydrated thrombosomes was centrifuged at 2000 RPM for 30 minutes. The supernatant of the centrifuged sample was removed, made up to 1 mL, and discarded. The sample was gently stirred to resuspend the thrombosomes. The concentrated thrombosomes were treated with 3% glutaraldehyde in 0.1 M cacodylate buffer at pH 7.4 for 2 hours with stirring every 15 minutes. The thrombosomes were rinsed three times with sterile water and transferred to a 1% osmium tetroxide solution for 1 hour with stirring every 15 minutes. The sample was then washed three more times with sterile water, and 0.5 mL droplets were transferred to a polysulfone filter membrane. The mounted samples were frozen in liquid nitrogen, dried under vacuum, sputter-coated with gold, and imaged using a scanning electron microscope.

[0532] Figures 32A to 32D show SEMs of platelets and human thrombosomes. Platelets immediately after activation are shown in Figures 32A (scale bar = 2 μm) and 32B (scale bar = 1 μm). Rehydrated human thrombosomes prepared as in Example 1 are shown in Figures 32C (scale bar = 2 μm) and 32D (scale bar = 1 μm).

[0533] Example 16. T-TAS (registered trademark) thrombosome data. In the Total Thrombus-formation Analysis System (T-TAS (registered trademark), FUJIMORI KOGYO CO., LTD), a sample is pushed into a collagen-coated microchannel using mineral oil. The change in pressure is used to evaluate thrombus formation. The occlusion start time is the time until Δ10 kPa is reached, and the occlusion time is the time until Δ80 kPa is reached using an AR chip (Zacros, item number TC0101).

[0534] According to FUJIMORI KOGYO CO., LTD, the AR chip can be used mainly to analyze the formation of a mixed white thrombus composed mainly of fibrin and activated platelets. The chip has a flow channel (width 300 μm × height 50 μm) coated with collagen and tissue factor and can be used to analyze coagulation function and platelet function. In contrast, the PL chip can be used mainly to analyze the formation of a platelet thrombus composed mainly of activated platelets. The PL chip has a flow channel coated only with collagen and can be used to analyze platelet function.

[0535] The T-TAS (registered trademark) reagent (CaCTI, AR chip) was warmed to 37°C, and thrombosomes were rehydrated according to the standard protocol. An aliquot of the rehydrated thrombosomes was washed by centrifugation at 3900 g for 10 minutes and resuspended in platelet-poor plasma (PPP) anticoagulated with sodium citrate to approximately 300,000 cells / μL. CaCTI (20 μL) was mixed with thrombosomes (480 μL) in PPP and passed through the T-TAS AR chip under high shear. The pressure inside the system was monitored for 30 minutes or until the maximum backpressure in the channel was achieved.

[0536] Preparation was made to use the T-TAS (registered trademark) device according to the manufacturer's instructions. The AR chip (Diapharma, catalog number TC0101) and the AR chip Calcium Corn Trypsin Inhibitor (CaCTI; Diapharma, catalog number TR0101) were warmed to room temperature. 300 uL of rehydrated thrombosomes were transferred to a 1.7 mL microcentrifuge tube and pelleted by centrifugation at 3900 g for 10 minutes. The thrombosome pellet was resuspended in George King (GK) pooled normal human plasma or autologous plasma with or without autologous platelets and adjusted to a concentration of approximately 100,000 - 450,000 / uL in quantification by the AcT number (Beckman Coulter AcT Diff 2 Cell Counter). 20 uL of CaCTI and 480 uL of the thrombosome sample in GK plasma were mixed by gentle pipetting. The sample was loaded into the T-TAS (registered trademark) and run according to the manufacturer's instructions.

[0537] Table 28 shows the results of T-TAS (registered trademark) in citrate whole blood, platelet-reduced citrate whole blood with thrombosomes prepared in Example 1 added at various concentrations, and George King platelet-poor plasma (GK PPP) with thrombosomes prepared in Example 1 added at various concentrations, in experiments performed according to the manufacturer's instructions using the AR chip and high shear device settings.

[0538] (Table 28) TIFF2025081548000038.tif100161

[0539] The results over time are shown in FIGS. 33A - 33B. Increasing the concentration of thrombosomes in platelet-reduced whole blood promoted more robust thrombus formation by shortening the occlusion time (FIG. 33A). Increasing the concentration of thrombosomes in platelet-poor plasma (PPP) promoted more robust thrombus formation by shortening the occlusion time (FIG. 33B).

[0540] The effect of GPRP (1 mM) on occlusion activity was also assayed. Table 29 shows the results of T-TAS (registered trademark) of platelet-poor plasma with or without thrombosomes in the presence and absence of GPRP. Even when GPRP was added to prevent fibrinogen formation, samples containing thrombosomes were not prevented from reaching the occlusion pressure. When GPRP was added to the thrombosome sample in plasma, fibrin formation in the microcapillary channel was prevented (FIG. 33C (without GPRP), FIG. 33D (with GPRP), both in GK PPP), but when GPRP was added to thrombosomes (PPP), thrombus formation was not prevented (FIG. 33E).

[0541] (Table 29) TIFF2025081548000039.tif91160

[0542] Embodiments of the present invention are applicable to various changes and alternative forms. Specific embodiments are illustrated by way of example in the drawings and are described in detail below. However, it is not intended to limit the present invention to the specific embodiments described. On the contrary, the present invention is intended to cover all changes, equivalents, and alternative forms that fall within the scope of the present invention as defined by the appended claims.

Claims

1. 1. A process for preparing a composition comprising platelets or a platelet derivative and an aqueous medium, comprising preparing the composition comprising platelets or a platelet derivative and an aqueous medium by tangential flow filtration (TFF) of a starting material comprising platelets, a diluted starting material comprising platelets, a concentrated platelet composition, or a combination thereof, comprising: The aqueous medium has a protein concentration that is 50% or less than the protein concentration of donor apheresis plasma. process.

2. 10. The process of claim 1 further comprising a pathogen reduction step.

3. 3. The process of claim 2, wherein the pathogen reduction step precedes TFF.

4. 4. The process of any one of claims 1 to 3, wherein the starting material has a protein concentration of about 60 to about 80 mg / mL.

5. The process of any one of claims 1 to 4, wherein the TFF comprises diafiltering in at least two diavolumes.

6. 6. The process of any one of claims 1 to 5, wherein the TFF comprises diafiltration with a formulation comprising a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

7. 7. The process of any one of claims 1 to 6, wherein TFF comprises buffer exchange into a formulation comprising a buffering agent, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

8. 8. The process of any one of claims 6 to 7, wherein the formulation comprises a buffer comprising HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), a base comprising sodium bicarbonate, and a loading agent comprising trehalose, polysucrose, or a combination thereof.

9. The process of any one of claims 6 to 8, wherein the formulation comprises an organic solvent comprising ethanol, DMSO, or a combination thereof.

10. 10. The process of any one of claims 1 to 9, wherein the protein concentration of the aqueous medium is no more than 30% of the protein concentration of donor apheresis plasma.

11. 11. The process of any one of claims 1 to 10, wherein the protein concentration of the aqueous medium is no more than 10% of the protein concentration of donor apheresis plasma.

12. 11. The process of any one of claims 1 to 10, wherein the protein concentration of the aqueous medium is about 5% to about 15% of the protein concentration of donor apheresis plasma.

13. 13. The process of any one of claims 1 to 12, wherein the protein concentration of the aqueous medium is about 7% to about 10% of the protein concentration of donor apheresis plasma.

14. The process of any one of claims 1 to 13, wherein the composition contains less than 5.0% fine particles (by scattering intensity).

15. The process of any one of claims 1 to 13, wherein the composition contains less than 4.0% fine particles (by scattering intensity).

16. The process of any one of claims 1 to 15, further comprising lyophilizing and / or cryopreserving the composition comprising platelets or platelet derivatives.

17. The process of any one of claims 1 to 16, further comprising thermally treating the composition comprising platelets or platelet derivatives.

18. The process according to any one of claims 1 to 17, wherein the platelets or platelet derivatives have a CD41 positive percentage of at least 55%.

19. The process according to any one of claims 1 to 18, wherein the platelets or platelet derivatives have a CD42 positive percentage of at least 80%.

20. The process according to any one of claims 1 to 19, wherein the platelets or platelet derivatives have an Annexin V positive percentage of at least 70%.

21. The process according to any one of claims 1 to 20, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 8%.

22. The process according to any one of claims 1 to 21, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 80%.

23. The process of any one of claims 1 to 22, wherein the platelets or platelet derivatives have fibrinogen associated with their cell membrane.

24. The platelets or platelet derivatives are about 4.8×10 ３ 24. The process of any one of claims 1 to 23, which produces a thrombin peak height (TPH) of at least 25 nM in the presence of a reagent comprising tissue factor and phospholipids at a concentration of particles / μL.

25. The platelets or platelet derivatives are ６ 25. The process of any one of claims 1 to 24, having a titer of at least 1.5 Thrombin Generating Titer Units (TGPU) per particle.

26. The platelets or platelet derivatives are at least about 70×10 ３ 26. The process of any one of claims 1 to 25, which at a concentration of particles / μL results in an occlusion time of less than 14 minutes in a Total Thrombosis Analysis System (T-TAS) assay.

27. The process of any one of claims 1 to 26, wherein the platelet derivative comprises a thrombosome.

28. The starting material is (a) positive for HLA class I antibodies based on a test approved by a regulatory agency; (b) positive for HLA class II antibodies based on a test approved by a regulatory agency; (c) is positive for HNA antibodies based on a test approved by a regulatory agency; or (d) one or more of (a), (b), and (c); The process according to any one of claims 1 to 27.

29. The composition, (a) is negative for HLA class I antibodies based on a test approved by a regulatory agency; (b) is negative for HLA class II antibodies based on a test approved by a regulatory agency; (c) is negative for HNA antibodies based on a test approved by a regulatory agency; or (d) one or more of (a), (b), and (c); The process according to any one of claims 1 to 28.

30. A composition comprising platelets or a platelet derivative and an aqueous medium, prepared by the process of any one of claims 1 to 29.

31. 1. A composition comprising platelets or platelet derivatives and an aqueous medium, the aqueous medium having a protein concentration that is 50% or less than the protein concentration of donor apheresis plasma.

32. 32. The composition of claim 31, wherein the protein concentration of the aqueous medium is no more than 30% of the protein concentration of donor apheresis plasma.

33. 33. The composition of claim 31 or 32, wherein the protein concentration of the aqueous medium is no more than 10% of the protein concentration of donor apheresis plasma.

34. 33. The composition of claim 31 or 32, wherein the protein concentration of the aqueous medium is about 5% to about 15% of the protein concentration of donor apheresis plasma.

35. 35. The composition of any one of claims 31-34, wherein the protein concentration of the aqueous medium is about 8% to about 10% of the protein concentration of donor apheresis plasma.

36. The composition of any one of claims 31 to 35, wherein the aqueous medium further comprises a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

37. 37. The process of any one of claims 31 to 36, wherein the aqueous medium comprises a buffer comprising HEPES (4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid), a base comprising sodium bicarbonate, and a loading agent comprising trehalose, polysucrose, or a combination thereof.

38. 38. The process of any one of claims 31 to 37, wherein the aqueous medium comprises an organic solvent comprising ethanol, DMSO, or a combination thereof.

39. The composition of any one of claims 31 to 38, comprising less than 5.0% fine particles (by scattering intensity).

40. 39. The composition of any one of claims 31 to 38, comprising less than 4.0% fine particles (by scattering intensity).

41. The composition of any one of claims 31 to 40, wherein the platelets or platelet derivatives have a CD41 positive percentage of at least 55%.

42. The composition of any one of claims 31 to 41, wherein the platelets or platelet derivatives have a CD42 positive percentage of at least 80%.

43. The composition of any one of claims 31 to 42, wherein the platelets or platelet derivatives have an Annexin V positive percentage of at least 70%.

44. The composition of any one of claims 31 to 43, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 8%.

45. The composition of any one of claims 31 to 44, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 80%.

46. The composition of any one of claims 31 to 45, wherein the platelets or platelet derivatives have fibrinogen associated with their cell membrane.

47. The platelets or platelet derivatives are about 4.8×10 ３ 47. The composition of any one of claims 31 to 46, which, at a concentration of particles / μL, produces a thrombin peak height (TPH) of at least 50 nM when in the presence of a reagent comprising tissue factor and phospholipids.

48. The platelets or platelet derivatives are ６ 48. The composition of any one of claims 31 to 47, having a titer of at least 1.5 thrombin generating titer units (TPGU) per particle.

49. The platelets or platelet derivatives are at least about 70×10 ３ 49. The composition of any one of claims 31 to 48, which, at a concentration of particles / μL, provides an occlusion time of less than 14 minutes in a Total Thrombosis Analysis System (T-TAS) assay.

50. The composition of any one of claims 31 to 49, wherein the platelet derivative comprises a thrombosome.

51. (a) is negative for HLA class I antibodies based on a test approved by a regulatory agency; (b) is negative for HLA class II antibodies based on a test approved by a regulatory agency; (c) is negative for HNA antibodies based on a test approved by a regulatory agency; or (d) one or more of (a), (b), and (c); The composition according to any one of claims 31 to 50.

52. 52. A method of treating a blood clotting related disease or condition in a subject in need of such treatment, comprising administering to the subject a therapeutically effective amount of a composition according to any one of claims 30 to 51.

53. 53. The method of claim 52, wherein the blood clotting-related disease or condition is selected from the group consisting of von Willebrand's disease, hemophilia, thrombasthenia, thrombocytopenia, thrombocytopenic purpura, trauma, or a combination thereof.

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