Materials and methods for producing blood products

Abstract

Provided herein are materials and methods for preparing blood products. In one aspect, provided herein is a composition comprising platelets or platelet derivatives and an aqueous medium, wherein the protein concentration of the aqueous medium is less than 50% of the protein concentration of the donor apheresis plasma.

Description

[0001] Cross-references to related applications

[0002] This application claims priority to U.S. Provisional Application Serial No. 62 / 843,061, filed May 3, 2019, and U.S. Provisional Application Serial No. 62 / 936,122, filed November 15, 2019, each of which is incorporated herein by reference in its entirety.

[0003] Statement of Government Interests

[0004] This invention was completed with government support under contract number HHSO100201300021 granted by the Biomedical Advanced Research and Development Authority (BARDA) of the U.S. Department of Health and Human Services. The government holds certain rights to this invention. Technical Field

[0005] This disclosure generally relates to blood products (such as blood products with reduced HLA antibody content) and methods for producing such blood products. Background Technology

[0006] Blood is a complex mixture of many components. Generally, blood can be described as containing four main parts: red blood cells, white blood cells, platelets, and plasma. The first three are cellular or cell-like components, while the fourth (plasma) is a liquid component, which is a broad and variable mixture of salts, proteins, and many other factors required for bodily functions. The components of blood can be separated from each other using various methods. Generally, differential centrifugation is currently the most commonly used method for separating different blood components based on size and (in some applications) density.

[0007] Inactivated platelets (also commonly known as thrombocytes) are small, often irregularly shaped (e.g., disc-shaped or oval) megakaryocyte-derived components of the blood that participate in the clotting process. They help protect the body from excessive blood loss caused by trauma or injury, as well as normal physiological activities. Platelets are considered crucial for normal hemostasis, providing the first line of defense against blood leakage from injured blood vessels. Platelets typically adhere to the inner lining of a ruptured blood vessel, where they are activated, become amorphous, and interact with components of the clotting system present in the plasma or released by the platelets themselves or other components of the blood. Purified platelets have been found to treat subjects with low platelet counts (thrombocytopenia) and platelet dysfunction (thromboembolism). Concentrated platelets are commonly used to control bleeding following injury or during acquired platelet dysfunction or insufficiency, such as bleeding occurring during surgery and bleeding due to the presence of platelet inhibitors. SUMMARY

[0008] The document is based at least in part on the production of blood products (e.g., compositions comprising platelets or platelet derivatives (e.g., thrombosomes)) having reduced levels of free proteins (e.g., antibodies (e.g., human leukocyte antigen (HLA) antibodies or human neutrophil antigen (HNA) antibodies)).

[0009] Provided herein is a composition comprising platelets and an aqueous medium, wherein the protein concentration of the aqueous medium is less than 50% of the protein concentration of the donor apheresis.

[0010] Implementations can have one or more of the following features. The aqueous medium can have a protein concentration that is less than 30% of a protein concentration of the donor apheresis. The aqueous medium can have a concentration of human leukocyte antigen (HLA) class I antibodies that is less than 30% of a concentration of HLA class I antibodies in the donor apheresis. The aqueous medium can have a concentration of human leukocyte antigen (HLA) class II antibodies that is less than 30% of a concentration of HLA class II antibodies in the donor apheresis. The aqueous medium can have a concentration of human neutrophil antigen (HNA) antibodies that is less than 30% of a concentration of HNA antibodies in the donor apheresis. The protein concentration can be less than 10% of a protein concentration of the donor apheresis. The aqueous medium can have a concentration of human HLA class I antibodies that is less than 10% of a concentration of HLA class I antibodies in the donor apheresis. The aqueous medium can have a concentration of human HLA class II antibodies that is less than 10% of a concentration of HLA class II antibodies in the donor apheresis. The aqueous medium can have a concentration of human HNA antibodies that is less than 10% of a concentration of HNA antibodies in the donor apheresis. The protein concentration can be less than 5% of a protein concentration of the donor apheresis. The aqueous medium can have a concentration of human HLA class I antibodies that is less than 5% of a concentration of HLA class I antibodies in the donor apheresis. The aqueous medium can have a concentration of human HLA class II antibodies that is less than 5% of a concentration of HLA class II antibodies in the donor apheresis. The aqueous medium can have a concentration of human HNA antibodies that is less than 5% of a concentration of HNA antibodies in the donor apheresis. The protein concentration can be less than 3% of a protein concentration of the donor apheresis. The aqueous medium can have a concentration of human HLA class I antibodies that is less than 3% of a concentration of HLA class I antibodies in the donor apheresis. The aqueous medium can have a concentration of human HLA class II antibodies that is less than 3% of a concentration of HLA class II antibodies in the donor apheresis. The aqueous medium can have a concentration of human HNA antibodies that is less than 3% of a concentration of HNA antibodies in the donor apheresis. The protein concentration can be less than 1% of a protein concentration of the donor apheresis. The aqueous medium can have a concentration of human HLA class I antibodies that is less than 1% of a concentration of HLA class I antibodies in the donor apheresis. The aqueous medium can have a concentration of human HLA class II antibodies that is less than 1% of a concentration of HLA class II antibodies in the donor apheresis. The aqueous medium can have a concentration of human HNA antibodies that is less than 1% of a concentration of HNA antibodies in the donor apheresis. The protein concentration can be determined by absorbance at 280 nanometers (nm) with 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 x 10 3platelets per pL. In some embodiments, the platelet count can be at least 2250 x 10 3 platelets per pL. In some embodiments, the platelet count can be at least 2250 x 10 6 erythrocytes per pL. In some embodiments, the composition can further include erythrocytes. In some embodiments, the erythrocyte count can be less than 0.2 x 10 6red blood cells per pL. The composition can be negative for HLA class I antibodies according to regulatory agency approved testing. The composition can be negative for HLA class II antibodies according to regulatory agency approved testing. The composition can be negative for HNA antibodies according to regulatory agency approved testing. 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 as determined by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively, for the composition can be less than 5%. 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 as determined by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively, for the composition can be less than 3%. 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 as determined by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, respectively, for the composition can be less than 1%. The percentage of beads positive for HLA class I antibodies as determined by flow cytometry using beads coated with class I HLA for the composition can be less than 5%. The percentage of beads positive for HLA class I antibodies as determined by flow cytometry using beads coated with class I HLA for the composition can be less than 3%. The percentage of beads positive for HLA class I antibodies as determined by flow cytometry using beads coated with class I HLA for the composition can be less than 1%. The percentage of beads positive for HLA class II antibodies as determined by flow cytometry using beads coated with class II HLA for the composition can be less than 5%. The percentage of beads positive for HLA class II antibodies as determined by flow cytometry using beads coated with class II HLA for the composition can be less than 3%. The percentage of beads positive for HLA class II antibodies as determined by flow cytometry using beads coated with class II HLA for the composition can be less than 1%. The percentage of beads positive for HNA antibodies as determined by flow cytometry using beads coated with HNA for the composition can be less than 5%. The percentage of beads positive for HNA antibodies as determined by flow cytometry using beads coated with HNA for the composition can be less than 3%. The percentage of beads positive for HNA antibodies as determined by flow cytometry using beads coated with HNA for the composition can be less than 1%. The aqueous medium can further comprise a buffering agent, a base, a loading agent, optionally a salt, and optionally at least one organic solvent. The buffering agent can be 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid (HEPES). 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, dimethylsulfoxide, dioxane, methanol, n-propanol, isopropanol, tetrahydrofuran (THF), N-methylpyrrolidone, dimethylacetamide (DMAC), and combinations thereof. The composition can be prepared by a method comprising tangential flow filtration (TFF) of a starting material comprising platelets, centrifugation of the starting material comprising platelets, or a combination thereof. 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 as determined by flow cytometry using beads coated with HLA Class I, HLA Class II, or HNA, respectively, on the composition can be reduced by at least 50% compared to a similar composition not prepared by a method comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof. 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 as determined by flow cytometry using beads coated with HLA Class I, HLA Class II, or HNA, respectively, on the composition can be reduced by at least 75% compared to a similar composition not prepared by a method comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof. 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 as determined by flow cytometry using beads coated with HLA Class I, HLA Class II, or HNA, respectively, on the composition can be reduced by at least 90% compared to a similar composition not prepared by a method comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof. 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 as determined by flow cytometry using beads coated with HLA Class I, HLA Class II, or HNA, respectively, on the composition can be reduced by at least 95% compared to a similar composition not prepared by a method comprising tangential flow filtration of a blood product composition, centrifugation of a blood product composition, or a combination thereof. The starting material can be (a) positive for HLA Class I antibodies based on a regulatory agency approved test, (b) positive for HLA Class II antibodies based on a regulatory agency approved test, (c) positive for HNA antibodies based on a regulatory agency approved test, or (d) one or more of (a), (b), and (c). The starting material can have a protein concentration of about 60 to about 80 mg / ml. The starting material can comprise a donor blood product. The donor blood product can be a pooled donor blood product. The starting material can comprise a donor apheresis material. The TFF can comprise concentration. The TFF can comprise diafiltration. The diafiltration can comprise diafiltration with at least two diafiltration volumes. The TFF can comprise buffer exchange.TFF can be performed using membranes with pore sizes from about 0.2 μm to about 1 μm. TFF can also be performed using membranes with pore sizes from about 0.2 μm to about 0.45 μm. TFF can be performed at temperatures from 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 also 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 also 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. Finally, 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 performed until the platelet concentration is at least approximately 2000 × 10⁻⁶. 3 Platelets / μL. TFF can be performed until the platelet concentration reaches at least approximately 2250 × 10⁻⁶. 3Platelets / μL. TFF may include exchanging the buffer for a buffer comprising a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent. The buffer may be 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). The base may be sodium bicarbonate. The loading agent may be a monosaccharide, a polysaccharide, or a combination thereof. The monosaccharide may be selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose. The monosaccharide may be trehalose. The polysaccharide may be polysucrose. The salt may be sodium chloride, potassium chloride, or a combination thereof. The organic solvent may 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 may include centrifugation at 1400×g to about 1550×g. This process may lack centrifugation of the platelet-containing composition. The composition may include less than 5.0% (by scattering intensity). The composition may include less than 4.5% (by scattering intensity). The composition may include less than 4.0% (by scattering intensity). The composition may include less than 3.5% (by scattering intensity). Platelets or platelet derivatives may have at least 55% CD41 positivity. Platelets or platelet derivatives may have at least 60% CD41 positivity. Platelets or platelet derivatives may have at least 65% CD41 positivity. Platelets or platelet derivatives may have at least 80% CD42 positivity. Platelets or platelet derivatives may have at least 85% CD42 positivity. Platelets or platelet derivatives may have at least 90% CD42 positivity. Platelets or platelet derivatives may retain at least about 10% of the lactate dehydrogenase activity of the donor apheresis platelets. Platelets or platelet derivatives can retain at least approximately 15% of the lactate dehydrogenase activity of donor apheresis platelets. Platelets or platelet derivatives can retain at least approximately 20% of the lactate dehydrogenase activity of donor apheresis platelets. Platelets or platelet derivatives can have at least 70% annexin V positivity. Platelets or platelet derivatives can have at least 75% annexin V positivity. Platelets or platelet derivatives can have at least 80% annexin V positivity. Platelets or platelet derivatives can have at least 8% CD47 positivity. Platelets or platelet derivatives can have at least 10% CD47 positivity. Platelets or platelet derivatives can have at least 15% CD47 positivity. Platelets or platelet derivatives can have at least 20% CD47 positivity. Platelets or platelet derivatives can have at least 80% CD62 positivity.Platelets or platelet derivatives may have at least 82% CD62 positivity. Platelets or platelet derivatives may have at least 85% CD62 positivity. Platelets or platelet derivatives may have at least 90% CD62 positivity. Platelets or platelet derivatives may contain cell membrane-associated fibrinogen. Aqueous media may have a lactate concentration of less than 2.0 mmol / L. Aqueous media may have a lactate concentration of less than 1.5 mmol / L. Aqueous media may have a lactate concentration of about 0.4 to about 1.3 mmol / L. Aqueous media may have a lactate concentration of about 0.5 to about 1.0 mmol / L. Platelet derivatives may include thrombus bodies.

[0011] This article also provides a method for preparing a composition comprising platelets and an aqueous medium, the method comprising tangential flow filtration (TFF) of a platelet-containing starting material, centrifugation of the platelet-containing starting material, or a combination thereof, wherein the protein concentration of the aqueous medium is less than 50% of the protein concentration of donor plasma.

[0012] The implementation may have one or more of the following characteristics. The starting material may be (a) positive for HLA class I antibodies based on a regulatory-approved test, (b) positive for HLA class II antibodies based on a regulatory-approved test, (c) positive for HNA antibodies based on a regulatory-approved test, or (d) one or more of (a), (b), and (c). The starting material may have a protein concentration of about 60 to about 80 mg / ml. The starting material may include donor blood products. The donor blood products may be pooled donor blood products. The starting material may include donor apheresis material. TFF may include concentration. TFF may include perfiltration. Perfiltration may include perfiltration having at least two perfiltration volumes. 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 may be performed 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 performed until the platelet concentration is at least approximately 2000 × 10⁻⁶. 3 Platelets / μL. TFF can be performed until the platelet concentration reaches at least approximately 2250 × 10⁻⁶. 3Platelets / μL. TFF may include exchanging the buffer for a buffer comprising a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent. The buffer may be 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). The base may be sodium bicarbonate. The loading agent may be a monosaccharide, a polysaccharide, or a combination thereof. The monosaccharide may be selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose. The monosaccharide may be trehalose. The polysaccharide may be polysucrose. The salt may be sodium chloride, potassium chloride, or a combination thereof. The organic solvent may 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 may include centrifugation at 1400×g to about 1550×g. This process may lack centrifugation of the platelet-containing composition. Compared to similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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% when measured by flow cytometry using bead pairs coated with class I, class II, or HNA antibodies, respectively. Compared to similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, or a combination thereof, the percentage of beads positive for antibodies selected from the group consisting of HLA class I antibodies, class II antibodies, and HNA antibodies can be reduced by at least 75% when measured by flow cytometry using bead pairs coated with class I, class II, or HNA antibodies, respectively. Compared to similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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 90% when measured by flow cytometry using bead pairs coated with class I, class II, or HNA antibodies, respectively. The protein concentration can be less than 30% of the protein concentration of donor plasma.The concentration of human leukocyte antigen (HLA) class I antibodies in the aqueous medium can be less than 30% of the concentration of HLA class I antibodies in the donor's single-donor plasma. The concentration of human leukocyte antigen (HLA) class II antibodies in the aqueous medium can be less than 30% of the concentration of HLA class II antibodies in the donor's single-donor plasma. The concentration of human neutrophil antigen (HNA) antibodies in the aqueous medium can be less than 30% of the concentration of HNA antibodies in the donor's single-donor plasma. The protein concentration can be less than 10% of the protein concentration in the donor's single-donor plasma. The concentration of human HLA class I antibodies in the aqueous medium can be less than 10% of the concentration of HLA class I antibodies in the donor's single-donor plasma. The concentration of human HLA class II antibodies in the aqueous medium can be less than 10% of the concentration of HLA class II antibodies in the donor's single-donor plasma. The concentration of human HNA antibodies in the aqueous medium can be less than 10% of the concentration of HNA antibodies in the donor's single-donor plasma. The protein concentration can be less than 5% of the protein concentration in the donor's single-donor plasma. The concentration of human HLA class I antibodies in the aqueous medium can be less than 5% of the concentration of HLA class I antibodies in the donor's single-donor plasma. The concentration of human HLA class II antibodies in the aqueous medium can be less than 5% of the concentration of HLA class II antibodies in the donor's single-donor plasma. The concentration of human HLA antibodies in the aqueous medium can be less than 5% of the concentration of HLA antibodies in the donor's single-donor plasma. The protein concentration can be less than 3% of the protein concentration in the donor's single-donor plasma. The concentration of human HLA class I antibodies in the aqueous medium can be less than 1% of the concentration of HLA class I antibodies in the aqueous medium. The aqueous medium may contain a concentration of human HLA class II antibodies less than 1% of the concentration of HLA class II antibodies in donor apheresis plasma. The aqueous medium may contain a concentration of human HNA antibodies less than 1% of the concentration of HNA antibodies in donor apheresis plasma. The composition may be negative for HLA antibodies according to regulatory-approved tests. The composition may be negative for HLA class II antibodies according to regulatory-approved tests. The composition may be negative for HNA antibodies according to regulatory-approved tests. When the composition is measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA antibodies 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 may be less than 5%.When measured by flow cytometry using bead pairs coated with HLA class I, HLA class II, or HNA antibodies respectively, the percentage of beads positive for antibodies selected from the group consisting of HLA class I, HLA class II, and HNA antibodies can be less than 3%. When measured by flow cytometry using bead pairs coated with HLA class I, HLA class II, or HNA antibodies respectively, the percentage of beads positive for antibodies selected from the group consisting of HLA class I, HLA class II, and HNA antibodies can be less than 1%. When measured by flow cytometry using bead pairs coated with HLA class I, the percentage of beads positive for HLA class I antibodies can be less than 5%. When measured by flow cytometry using bead pairs coated with HLA class I, the percentage of beads positive for HLA class I antibodies can be less than 3%. When measured by flow cytometry using bead pairs coated with HLA class I, the percentage of beads positive for HLA class I antibodies can be less than 1%. When measured by flow cytometry using a bead-pair composition coated with class II HLA, the percentage of beads positive for HLA class II antibodies may be less than 5%. When measured by flow cytometry using a bead-pair composition coated with class II HLA, the percentage of beads positive for HLA class II antibodies may be less than 3%. When measured by flow cytometry using a bead-pair composition coated with class II HLA, the percentage of beads positive for HLA class II antibodies may be less than 1%. When measured by flow cytometry using a bead-pair composition coated with HNA, the percentage of beads positive for HNA antibodies may be less than 5%. When measured by flow cytometry using a bead-pair composition coated with HNA, the percentage of beads positive for HNA antibodies may be less than 3%. When measured by flow cytometry using a bead-pair composition coated with HNA, the percentage of beads positive for HNA antibodies may be less than 1%. According to the method of any one of claims 125-199, the composition comprises less than 5.0% (by scattering intensity) of particles. The composition may include less than 4.5% (by scattering intensity) of particles. The composition may include less than 4.0% (by scattering intensity) of particles. The composition may include less than 3.5% (by scattering intensity) of particles. Platelets or platelet derivatives may have at least 55% CD41 positivity. Platelets or platelet derivatives may have at least 60% CD41 positivity. Platelets or platelet derivatives may have at least 65% CD41 positivity. Platelets or platelet derivatives may have at least 80% CD42 positivity. Platelets or platelet derivatives may have at least 85% CD42 positivity. Platelets or platelet derivatives may have at least 90% CD42 positivity. Platelets or platelet derivatives may retain at least about 10% of the lactate dehydrogenase activity of donor apheresis platelets.Platelets or platelet derivatives can retain at least approximately 15% of the lactate dehydrogenase activity of donor apheresis platelets. Platelets or platelet derivatives can retain at least approximately 20% of the lactate dehydrogenase activity of donor apheresis platelets. Platelets or platelet derivatives can have at least 70% annexin V positivity. Platelets or platelet derivatives can have at least 75% annexin V positivity. Platelets or platelet derivatives can have at least 80% annexin V positivity. Platelets or platelet derivatives can have at least 8% CD47 positivity. Platelets or platelet derivatives can have at least 10% CD47 positivity. Platelets or platelet derivatives can have at least 15% CD47 positivity. Platelets or platelet derivatives can have at least 20% CD47 positivity. Platelets or platelet derivatives can have at least 80% CD62 positivity. Platelets or platelet derivatives may have at least 82% CD62 positivity. Platelets or platelet derivatives may have at least 85% CD62 positivity. Platelets or platelet derivatives may have at least 90% CD62 positivity. Platelets or platelet derivatives may contain cell membrane-associated fibrinogen. The aqueous medium may have a lactate concentration of less than 2.0 mmol / L. The aqueous medium may have a lactate concentration of less than 1.5 mmol / L. The aqueous medium may have a lactate concentration of about 0.4 to about 1.3 mmol / L. The aqueous medium may have a lactate concentration of about 0.5 to about 1.0 mmol / L. Platelet derivatives may include thrombus bodies. The method may further include a pathogen reduction step. The pathogen reduction step may be prior to TFF. The method may further include lyophilizing the composition containing platelets or platelet derivatives. The method may further include heat-treating the composition containing platelets or platelet derivatives.

[0013] This article also provides a composition comprising platelets and an aqueous medium prepared by any of the methods described herein.

[0014] This document also provides a method for preparing lyophilized platelets, comprising (a) preparing a composition comprising platelets and an aqueous medium using any of the methods described herein, and (b) lyophilizing the composition comprising platelets and an aqueous medium.

[0015] This article also provides a composition comprising lyophilized platelets prepared by any of the methods described herein.

[0016] This article also provides a method for preparing a composition comprising platelets or platelet derivatives and an aqueous medium, the method comprising diluting a starting material comprising platelets to form a diluted starting material, and concentrating the platelets to approximately 2250 × 10⁻⁶. 3Cells / μL (±250×10⁻⁶) 3 To form a concentrated platelet composition, and to wash the concentrated platelet composition with a preparation agent of at least 2 times the filtration volume (DV) to form a TFF-treated composition.

[0017] The implementation may include one or more of the following features. Dilution may include dilution with approximately equal weight (±10%) of the formulation. 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% of the relative plasma (as determined by plasma protein content). After washing, if the cell concentration in the TFF-treated composition is not about 2000 × 10⁻⁶ 3 Cells / μL (±300×10⁻⁶) 3 If the method further includes diluting the formulation or concentrating it to fall within that 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.

[0018] This article also provides a composition comprising platelets or platelet derivatives prepared by any of the methods described herein.

[0019] The materials and methods described in this paper offer several advantages. First, they allow for the acquisition of other donors that are otherwise delayed, and reduce competition for single-collection materials. Attached Figure Description

[0020] Figure 1A This demonstrates the use of lateral scattering relative to forward scattering to identify class I and class II HLA-FLOPRA in PBS. TM The initial gating position of the bead.

[0021] Figure 1B This demonstrates the use of phycoerythrin fluorescence relative to forward scattering to identify class I and class II HLA-FLOPRA in PBS. TM The initial gating position of the bead.

[0022] Figure 2A FLOWPRA is shown in PBS, supplier anemic platelet plasma (PPP) (triples), and HLA-gated donor anemic platelet plasma (triples). TM An exemplary FITC-H histogram of a bead.

[0023] Figure 2BFLOWPRA is shown in PBS, supplier anemic platelet plasma (PPP) (triples), and HLA-gated donor anemic platelet plasma (triples). TM An exemplary FITC-H histogram of a bead.

[0024] Figure 2C FLOWPRA is shown in PBS, supplier anemic platelet plasma (PPP) (single dataset), and HLA-gated donor anemic platelet plasma (single dataset). TM An exemplary FITC-H histogram of a bead.

[0025] Figure 2D FLOWPRA is shown in PBS, supplier anemic platelet plasma (PPP) (single dataset), and HLA-gated donor anemic platelet plasma (single dataset). TM An exemplary FITC-H histogram of a bead.

[0026] Figure 3A FLOWPRA in George King PPP gated on Class I HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0027] Figure 3B FLOWPRA in George King PPP gated on Class II HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0028] Figure 4A FLOWPRA in a donor 1PPP gated on a class I HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0029] Figure 4B FLOWPRA in a donor 1PPP gated on a type II HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0030] Figure 5A FLOWPRA in a donor 2PPP gated on a class I HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0031] Figure 5B FLOWPRA in a donor 2PPP gated on a type II HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0032] Figure 6AFLOWPRA in a donor 3PPP gated on a class I HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0033] Figure 6B FLOWPRA in a donor 3PPP gated on a type II HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0034] Figure 7A FLOWPRA in a donor 4PPP gated on a class I HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0035] Figure 7B FLOWPRA in a donor 4PPP gated on a type II HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0036] Figure 8A FLOWPRA in a 5PPP donor gated on a class I HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0037] Figure 8B FLOWPRA in a type II HLA-gated donor 5PPP is shown. TM An exemplary FITC-H histogram of a bead.

[0038] Figure 9A FLOWPRA in a donor 6PPP gated on a class I HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0039] Figure 9B FLOWPRA in a type II HLA-gated donor 6PPP is shown. TM An exemplary FITC-H histogram of a bead.

[0040] Figure 10A FLOWPRA in a donor 7PPP gated on a class I HLA is shown. TM An exemplary FITC-H histogram of a bead.

[0041] Figure 10B FLOWPRA in a type II HLA-gated donor 7PPP is shown. TM An exemplary FITC-H histogram of a bead.

[0042] Figure 11A FLOWPRA gated on class I HLA is shown. TMAn exemplary FITC-H histogram of the donor PPP of the bead collection.

[0043] Figure 11B FLOWPRA gated on Class II HLA is shown. TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0044] Figure 12A The image shows HLA-gated FLOWPRA after tangential flow filtration (TFF) to 50% retention of plasma proteins (by absorbance at 280 nm). TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0045] Figure 12B The image shows HLA-gated FLOWPRA after tangential flow filtration (TFF) to 50% retention of plasma proteins (by absorbance at 280 nm). TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0046] Figure 13A The FLOWPRA gated on class I HLA is shown after tangential flow filtration (TFF) to approximately 8% (by absorbance at 280 nm) of plasma proteins retained. TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0047] Figure 13B The FLOWPRA gated on class II HLA is shown after tangential flow filtration (TFF) to approximately 8% (by absorbance at 280 nm) of plasma proteins retained. TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0048] Figure 14A The FLOWPRA gated on class I HLA is shown after tangential flow filtration (TFF) to approximately 6% (by absorbance at 280 nm) of plasma proteins retained. TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0049] Figure 14B The image shows HLA-gated FLOWPRA after tangential flow filtration (TFF) with approximately 6% (by absorbance at 280 nm) of plasma proteins retained. TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0050] Figure 15AThe FLOWPRA gated on class I HLA is shown after tangential flow filtration (TFF) to approximately 1% (by absorbance at 280 nm) of plasma proteins. TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0051] Figure 15B The image shows HLA-gated FLOWPRA after tangential flow filtration (TFF) of plasma proteins retained to approximately 1% (by absorbance at 280 nm). TM An exemplary FITC-H histogram of the donor PPP of the bead collection.

[0052] Figure 16 Exemplary flow cytometry data of thrombus bodies are shown, either unstained (dark data points) or stained with anti-CD41 antibody (light data points).

[0053] Figure 17 Exemplary histograms of thrombus bodies with (light data points) and without (dark data points) calcium, incubated with annexin V, are shown.

[0054] Figure 18 Exemplary histograms of thrombus bodies incubated with anti-CD62 antibody (light data points) or with isotype control (dark data points) are shown.

[0055] Figure 19 The graph shows the thrombin peak height of thrombus bodies in the presence of PRP reagent containing tissue factor and phospholipids (solid and dashed lines) and control phospholipids (dots).

[0056] Figure 20 Examples of histogram comparisons of low plasma thrombus bodies are shown, with those unstained (black) or stained with isotype control antibody (dark gray) or FITC-labeled 9F9 antibody (light gray), and a table showing the average fluorescence intensity of two replicates.

[0057] Figure 21 Examples of histogram comparisons of low plasma thrombus bodies are shown, with one unstained (black) and the other stained with anti-PAC-1 antibody (light gray), along with a table showing the average fluorescence intensity of two replicates.

[0058] Figure 22A Thrombus bodies (1×10⁻⁶) treated with various concentrations of labeled anti-CD47 antibody or isotype control were used. 6 The average fluorescence intensity of (number of cells) is plotted.

[0059] Figure 22B These are exemplary histograms of thrombus bodies: unstained (black), stained with an isotype control antibody (dark gray), or stained with an anti-CD47 antibody (light gray).

[0060] Figure 22C This is a graph showing the average fluorescence intensity of thrombus bodies (250,000 cells) treated with various concentrations of labeled anti-CD47 antibody or isotype control.

[0061] Figure 22D These are exemplary histograms of thrombus bodies: unstained (black), stained with an isotype control antibody (dark gray), or stained with an anti-CD47 antibody (light gray).

[0062] Figure 23A This is a graph showing the percentage of particles of different radii, as determined by dynamic light scattering (DLS), in existing human platelets (batch J) and platelet derivatives derived from them (before lyophilization).

[0063] Figure 23B This is a graph showing the percentage of particles of different radii, as determined by DLS, in existing human platelet stores (batch K) and platelet derivatives derived from them (before lyophilization).

[0064] Figure 23C This is a graph showing the percentage of particles of different radii, as determined by DLS, in existing human platelet stores (batch L) and platelet derivatives derived from them (before lyophilization).

[0065] Figure 24A This is a graph showing the percentage of particles of different radii, as determined by DLS, in existing human platelet stores (batch D) and platelet derivatives derived from them (before lyophilization).

[0066] Figure 24B This is a graph showing the percentage of particles of different radii, as determined by DLS, in existing human platelet stores (batch E) and platelet derivatives derived from them (before lyophilization).

[0067] Figure 24C This is a graph showing the percentage of particles of different radii, as determined by DLS, in existing human platelet stores (batch F) and platelet derivatives derived from them (before lyophilization).

[0068] Figure 25A This is an exemplary schematic diagram of a pathogen reduction system.

[0069] Figure 25B This is a graph showing how the weight of the reaction vessel changes over time.

[0070] Figure 25C It is a graph showing the change in pressure in the reaction vessel over time.

[0071] Figure 26AThis is a graph showing the percentage of particles of different radii in rehydrated thrombus bodies with pathogens removed, as determined by DLS (batch N) or untreated (batch M).

[0072] Figure 26B This is a graph showing the percentage of particles of different radii in rehydrated thrombus bodies with pathogens removed, as determined by DLS (batch K) or untreated (batch J).

[0073] Figure 27A This is a graph showing the percentage of particles of different radii in hIDSPs with pathogen removal, as determined by DLS (batch N) or untreated (batch M).

[0074] Figure 27B This is a graph showing the percentage of particles of different radii in untreated hIDSP (batch M) and thrombus bodies derived from it, as determined by DLS.

[0075] Figure 27C This is a graph showing the percentage of particles of different radii in hIDSP processed to remove pathogens and the thrombus bodies derived from it (batch N), as determined by the DLS method.

[0076] Figure 28A This is a graph showing the percentage of particles of different radii in hIDSPs with pathogen removal, as determined by DLS (batch K) or untreated (batch J).

[0077] Figure 28B This is a graph showing the percentage of particles of different radii in untreated hIDSP (batch J) and thrombus bodies derived from it, as determined by DLS.

[0078] Figure 28C This is a graph showing the percentage of particles of different radii in hIDSP processed to remove pathogens and the thrombus bodies derived from it (batch K), as determined by DLS.

[0079] Figure 29A It is a bar graph showing the transmittance of platelets, thrombus bodies, and their combinations using optical transmission aggregation.

[0080] Figure 29B It is a bar graph showing the count of platelets, thrombus bodies, and their combinations in aggregated platelets (and / or thrombus bodies).

[0081] Figure 29C This is a bar graph showing the transmittance of platelets, thrombus bodies, and their combinations in optical transmission focusing.

[0082] Figure 30This is a bar graph showing the transmittance of thrombin-activated platelets, thrombus bodies, and their combinations in the presence and absence of GPRP.

[0083] Figure 31 This is a bar graph showing the percentage of platelets, thrombus bodies, and their combinations that are activated by PMA in the presence and absence of RGDS.

[0084] Figure 32A SEM images of activated platelets are shown (scale bar = 2 μm).

[0085] Figure 32B SEM images of activated platelets are shown (scale bar = 1 μm).

[0086] Figure 32C SEM images of rehydrated human thrombus bodies are shown (scale bar = 2 μm).

[0087] Figure 32D SEM images of rehydrated human thrombus bodies are shown (scale bar = 1 μm).

[0088] Figure 33A This is a diagram showing the adhesion of thrombus bodies cut off during thrombus removal in whole blood.

[0089] Figure 33B This is a diagram showing the adhesion of thrombus bodies under shearing in blood plasma.

[0090] Figure 33C The formation of fibrin in microcapillary channels is shown in the absence of GPRP.

[0091] Figure 33D This demonstrates the lack of fibrin formation in microcapillary channels in the presence of GPRP.

[0092] Figure 33E This is a graph showing the effect of GPRP on thrombus body adhesion under shear in plasma.

[0093] As used herein and in the appended claims, the term "platelet" may include whole platelets, fragmented platelets, and platelet derivatives.

[0094] As used herein and in the appended claims, the singular forms “a,” “an,” and “the” include the plural objects unless the context clearly specifies otherwise. Thus, for example, reference to “platelets” includes a plurality of such platelets. Furthermore, the use of terms that may be described using equivalent terms includes the use of those equivalent terms. Thus, for example, the use of the term “subject” should be understood to include the terms “patient,” “individual,” and other terms used in the art to indicate a person undergoing treatment.

[0095] Unless otherwise defined, 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 invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are described hereafter. All publications referenced herein are incorporated by way of citation to combine the methods and / or materials disclosed and described in the cited publications. This disclosure controls, to a certain extent, any conflict with any incorporated publications. Detailed Implementation

[0096] It should be understood that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting. Furthermore, where a range of values ​​is disclosed, those skilled in the art will understand that all other particular values ​​within the disclosed range are inherently disclosed by those values ​​and the ranges they represent, without the need to disclose every particular value or range herein. For example, the disclosed range of 1-10 includes 1-9, 1-5, 2-10, 3.1-6, 1, 2, 3, 4, 5, etc. Additionally, each disclosed range includes a lower value at most 5% lower and a higher value at most 5% higher. For example, the disclosed range of 4-10 includes 3.8-10.5. This concept is captured in this document by the term “about”.

[0097] Unless otherwise defined, 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 invention pertains. While any methods and materials similar to or equivalent to those described herein may be used in the practice or testing of this disclosure, preferred methods and materials are described hereafter. All publications referenced herein are incorporated by way of citation to combine the methods and / or materials disclosed and described in the cited publications. This disclosure controls, to a certain extent, any conflict with any incorporated publications.

[0098] As used herein and in the appended claims, the term "platelet" can include whole platelets, fragmented platelets, platelet derivatives, or thrombus bodies. "Platelet" as defined above can include, for example, platelets in whole blood, platelets in plasma, platelets in a buffer solution optionally supplemented with selected plasma proteins, cryopreserved platelets, dried platelets, cryopreserved platelets, thawed cryopreserved platelets, rehydrated dried platelets, rehydrated cryopreserved platelets, frozen platelets, thawed cryopreserved platelets, or rehydrated cryopreserved platelets. "Platelet" can refer to mammalian platelets, such as human platelets, or platelets from non-human mammals.

[0099] As used herein, “thrombus bodies” (sometimes also referred to as Tsomes) are platelet derivatives that have been treated with a formulation (e.g., any formulation described herein) and cryopreserved (e.g., freeze-dried). In some cases, thrombus bodies can be prepared from aggregated platelets. Thrombus bodies in dried form at ambient temperature can have a shelf life of 2–3 years and can be rehydrated with sterile water within minutes for immediate infusion. An example of thrombus bodies is… In clinical trials for the treatment of acute bleeding in patients with thrombocytopenia.

[0100] Transfusion-associated acute lung injury (TRALI) is a condition believed to be caused by antibodies present in transfusion products (such as human leukocyte antigen (HLA), human neutrophil antigen (HNA), or granulocyte antibodies) that can react with antigens in the transfusion recipient.

[0101] Plasma-based blood products from donors considered high-risk or whose tests show positive results for human leukocyte antigen (HLA) class I, II and neutrophil-specific antibodies are prohibited from use for transfusion or production of human platelet products (e.g., compositions containing platelets and / or platelet derivatives (e.g., thrombus bodies)) and are therefore omitted from the donor bank.

[0102] Using tangential flow filtration (TFF) or multichannel centrifugation can reduce the amount of antibodies in blood products to limits undetectable, for example, by currently FDA-approved testing methods. In some cases, reduction of certain plasma components (e.g., HLA antibodies) may allow this donor population to be accepted for the production of blood products (e.g., compositions containing platelets and / or platelet derivatives (e.g., thrombus bodies)). In some embodiments described herein, the blood product may be a composition containing platelets and an aqueous medium.

[0103] The production of thrombus bodies or cryopreserved platelets may be limited by the availability of licensed apheresis blood collection at blood donation centers across the United States. Competition for these products can be fierce, and the allocation of blood product manufacturing needs is often less prioritized than patient care needs. The manufacturing of blood products (e.g., scaling up) can be aided by apheresis blood collection from donors who have otherwise been postponed. One way to achieve this is by reducing the level of free antibodies in donor plasma to meet currently FDA-approved testing thresholds using tangential flow filtration (TFF) or centrifugation and plasma removal. While generally more time-consuming than TFF, centrifugation of the raw material (e.g., donor plasma) has a similar effect on the raw material. In some cases, removing donor plasma and replacing it with a buffer can allow inventors to manufacture and characterize a final product (e.g., a composition containing platelets and / or platelet derivatives (e.g., thrombus bodies)) with reduced protein (e.g., antibody (e.g., HLA or HNA antibodies)) content (e.g., as measured by absorbance at 280 nm). This product can improve the safety of the product for recipients by reducing transfusion-related causes of TRALI.

[0104] In some embodiments, the materials and methods provided herein may allow previously deferred donors (such as those screened as having a positive HLA antibody test or a donor history of HLA risk) to enter a donor pool of raw materials for manufacturing blood products (e.g., compositions containing platelets and / or platelet derivatives (e.g., thrombus bodies)). In some embodiments described herein, the blood product may be a composition containing platelets and an aqueous medium. Furthermore, reduction of HLA antibodies from raw materials (e.g., apheresis materials (e.g., platelets or pooled platelets)) may allow the final product (e.g., a composition containing platelets and / or platelet derivatives (e.g., thrombus bodies)) to be HLA-reduced, which improves the safety of the product for the recipient.

[0105] In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may not have detectable levels of HLA antibodies. In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may not have detectable levels of antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may not have detectable levels of HLA class I antibodies. In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may not have detectable levels of HLA class II antibodies. In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may not have detectable levels of HNA antibodies. In some embodiments, antibody detection may be performed using a regulatory-approved (e.g., FDA-approved) assay. The regulatory-approved assay can be any assay approved by a suitable regulatory authority. In some embodiments, the regulatory-approved test can be One Lambda's LABSCREEN. TM Mixed. In some implementations, it can be used. 100 / 200 or XY and HLA FUSION TM The software undergoes regulatory approval testing. In some embodiments described herein, the blood product may be a composition comprising platelets and an aqueous medium.

[0106] In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may have antibody levels below a reference level, said antibodies being selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may have HLA class I antibody levels below a reference level. In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may have HLA class II antibody levels below a reference level. In some embodiments, blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) may have HNA antibody levels below a reference level. The reference level can be any suitable reference level. In some embodiments described herein, the blood product may be a composition comprising platelets and an aqueous medium.

[0107] In some embodiments, in a regulatory-approved assay (e.g., an FDA-approved assay), blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) are tested negative for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, in a regulatory-approved assay (e.g., an FDA-approved assay), blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) can be tested negative for HLA class antibodies. In some embodiments, in a regulatory-approved assay (e.g., an FDA-approved assay), blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) can be tested negative for HLA class II antibodies. In some embodiments, in a regulatory-approved assay (e.g., an FDA-approved assay), blood products as described herein (e.g., compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies)) can be tested negative for HNA antibodies. In some embodiments described herein, the blood product may be a composition comprising platelets and an aqueous medium. The regulatory-approved assay may be any appropriate regulatory-approved assay. In some embodiments, the regulatory-approved test may be One Lambda's LABSCREEN. TM Mixed. In some implementations, it can be used. 100 / 200 or XY and HLA FUSION TM The software undergoes regulatory approval testing.

[0108] This document provides compositions comprising platelets and / or platelet derivatives (e.g., thrombus bodies) and an aqueous medium. In some embodiments, the aqueous medium may include a formulation (e.g., any formulation described herein). In some embodiments, the aqueous medium provided herein may have a lower-than-reference level of antibody, said antibody being selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, the aqueous medium provided herein may have a lower-than-reference level of HLA class I antibody. In some embodiments, the aqueous medium provided herein may have a lower-than-reference level of HLA class II antibody. In some embodiments, the aqueous medium provided herein may have a lower-than-reference level of HNA antibody. The reference level may be any suitable reference level. In some embodiments, in a regulatory-approved assay (e.g., an FDA-approved assay), the aqueous medium provided herein may test negative for antibodies selected from the group consisting of HLA class I antibodies, HLA class II antibodies, and HNA antibodies. In some embodiments, in a regulatory-approved assay (e.g., an FDA-approved assay), the aqueous medium provided herein may test negative for HLA class I antibodies. In some embodiments, in regulatory-approved assays (e.g., FDA-approved assays), aqueous media as provided herein can test negative for HLA class II antibodies. Regulatory-approved assays can be any suitable regulatory-approved assay. In some embodiments, a regulatory-approved assay can be OneLambda's LABSCREEN. TM Mixed. In some implementations, it can be used. 100 / 200 or XY and HLA FUSION TM The software undergoes regulatory approval testing.

[0109] In some embodiments, the aqueous medium may have a reduced amount of residual plasma compared to donor apheresis plasma (e.g., single donor apheresis plasma or pooled donor apheresis plasma), which may be a percentage of the residual plasma (e.g., less than or equal to about 50%, 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 residual plasma). In some embodiments, the aqueous medium may have a reduced amount of residual plasma compared to donor-collected plasma (e.g., single-donor-collected plasma or pooled donor-collected plasma), which may be a 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% of residual plasma). In some embodiments, the aqueous medium may have a protein concentration of less than or equal to about 50% of the protein concentration of donor plasma (e.g., single donor plasma or pooled donor plasma) (e.g., less than or equal to 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%). In some embodiments, the aqueous medium may have a protein concentration of about 5% to about 50% of the protein concentration of donor plasma (e.g., single donor plasma or pooled donor plasma) (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%). In some embodiments, the aqueous medium may have a protein concentration of about 0.1% to about 5% of the protein concentration of donor plasma (e.g., single donor plasma or pooled donor plasma) (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%). The protein concentration can be measured by any suitable method. In some embodiments, protein concentration can be measured by absorbance at 280 nm (A280).In some embodiments, the aqueous medium may have an A280 of less than 1.70 AU (e.g., less than 1.66, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 AU), wherein the path length is 0.5 cm.

[0110] In some embodiments, the aqueous medium may have an HLA class I antibody concentration less than about 70% of the concentration in donor plasma (e.g., single donor plasma or pooled donor plasma) (e.g., less than about 60%, 50%, 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%). The HLA class I antibody concentration can be measured by any suitable method.

[0111] In some embodiments, the aqueous medium may have an HLA class II antibody concentration less than about 50% of the concentration in donor plasma (e.g., single donor plasma or pooled donor plasma) (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%). The HLA class II antibody concentration can be measured by any suitable method.

[0112] In some embodiments, the aqueous medium may have an HNA antibody concentration less than about 50% of the HNA antibody concentration in donor plasma (e.g., single donor plasma or pooled donor plasma) (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%). The HNA antibody concentration can be measured by any suitable method.

[0113] In some embodiments, the composition as described herein may have at least 10 6 (For example, at least 5×10) 6 10 7 5×10 7 10 8 5×10 8 10 9 5×10 9 Or 10 10The platelet count is [missing information]. In some embodiments, the composition described herein may have a platelet count of at least about 200 × 10⁻⁶. 3 Platelets / μL (e.g., at least about 300 × 10⁶) 3 400×10 3 500×10 3 750×10 3 1000×10 3 1500×10 3 2000×10 3 Or 2500×10 3 Platelet count (platelets / μL). In some embodiments, the composition as described herein may have a platelet count of at least about 2000 × 10⁻⁶. 3 Platelets / μL (e.g., at least approximately 2050 × 10⁶) 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 Platelet count (platelets / μL). In some embodiments, the composition as described herein may have a platelet count of less than or equal to 1000 × 10⁻⁶. 4 Platelet count per μL.

[0114] In some embodiments, the compositions provided herein may include red blood cells. In some embodiments, the compositions provided herein may have a content of less than about 10 10 (For example, less than 5×10) 9 10 9 5×10 8 10 8 5×10 7 10 7 5×10 6 Or 10 6 The red blood cell count. In some embodiments, the red blood cell count may be less than 0.2 × 10⁻⁶. 6 / μL (e.g., less than 0.1×10⁻⁶) 6 / μL, 0.5×10 5 / μL or 0.1×10 5 / μL).

[0115] In some cases, flow cytometry can be used to evaluate compositions as provided herein. In some embodiments, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA respectively, the antibody concentration 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%, 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%). In some embodiments, such as when measured by flow cytometry using beads coated with class I HLA to a composition containing platelets and an aqueous medium, the percentage of beads positive for HLA class I antibodies is less than 10% (e.g., less than 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%). In some embodiments, such as when a composition comprising platelets and an aqueous medium is measured by flow cytometry using beads coated with class II HLA, the percentage of beads positive for HLA class II antibodies is less than 10% (e.g., less than 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%). In some embodiments, such as when a composition comprising platelets and an aqueous medium is measured by flow cytometry using beads coated with HNA, the percentage of beads positive for HNA antibodies is less than 10% (e.g., less than 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%).

[0116] In some embodiments, such as those measured by flow cytometry using a bead-pair aqueous medium coated with class I HLA, class II HLA, or HNA respectively, the antibody group 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%, 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%). In some embodiments, such as when measured by flow cytometry using beads coated with class I HLA to an aqueous medium, the percentage of beads positive for HLA class I antibodies is less than 10% (e.g., less than 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%). In some embodiments, such as when measured by flow cytometry using beads coated with class II HLA to an aqueous medium, the percentage of beads positive for HLA class II antibodies is less than 10% (e.g., less than 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%). In some embodiments, such as when HNA-coated beads are measured in an aqueous medium by flow cytometry, the percentage of beads that are positive for HNA antibodies is less than 10% (e.g., less than 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%).

[0117] In some embodiments, the compositions provided herein may include one or more additional components. In some embodiments, the compositions provided herein may include formulations (e.g., any formulation described herein). In some embodiments, the compositions may include a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent. The buffer may be any suitable buffer. In some embodiments, the buffer may be 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). The base may be any suitable base. In some embodiments, the base may be sodium bicarbonate. The loading agent may be any suitable loading agent. In some embodiments, the loading agent may be a monosaccharide, a polysaccharide, or a combination thereof. In some embodiments, the loading agent may be selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, and xylose. In some embodiments, the loading agent may be trehalose. In some embodiments, the polysaccharide may be polysucrose. The salt may be any suitable salt. In some embodiments, the salt may be sodium chloride, potassium chloride, or a combination thereof. The organic solvent may be any suitable organic solvent. In some embodiments, the organic solvent may 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.

[0118] The formulation may include any suitable component. In some embodiments, the formulation may comprise a liquid medium. In some embodiments, the formulation may comprise one or more salts selected from phosphates, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts that may be found in blood or blood products or known to be used for drying platelets, or any combination of two or more of these.

[0119] In some embodiments, the formulation comprises one or more salts, such as phosphates, sodium salts, potassium salts, calcium salts, magnesium salts, and any other salts that may be found in blood or blood products. Exemplary salts include sodium chloride (NaCl), potassium chloride (KCl), and combinations thereof. In some embodiments, the formulation comprises one or more salts at a concentration of about 0.5 mM to about 100 mM. In some embodiments, the formulation comprises one or more salts at a concentration of about 0.5 mM to about 100 mM (e.g., about 0.5 mM 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 mM to about 85 mM). In some embodiments, the formulation comprises one or more salts at a concentration of about 5 mM, about 75 mM, or about 80 mM. In some embodiments, the formulation comprises one or more salts at a concentration of about 0.5 mM to about 2 mM, wherein the salt is selected from calcium salts, magnesium salts, and combinations thereof.

[0120] Preferably, these salts are present in compositions containing platelets or platelet derivatives (e.g., lyophilized platelets) in amounts substantially the same as those found in whole blood.

[0121] In some embodiments, the formulation further comprises a carrier protein. In some embodiments, the carrier protein comprises 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).

[0122] The formulation can be any buffer that is non-toxic to platelets and provides sufficient buffering capacity for the solution at the temperature at which it is exposed during the methods provided herein. Therefore, the buffer can include any commercially available, known biocompatible buffers, such as phosphate-buffered saline (PBS), bicarbonate / carbonate buffers, such as sodium bicarbonate buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and tris-based buffers, such as tris-buffered saline (TBS). Similarly, it can include one or more of the following buffer solutions: propane-1,2,3-tricarboxylic acid (propanetricarboxylic acid); phenylpentacarboxylic acid; maleic acid; 2,2-dimethylsuccinic acid; EDTA; 3,3-dimethylglutaric acid; bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS); phenylhexacarboxylic acid (phenylhexacarboxylic acid); N-(2-acetamido)imino-diacetic acid (ADA); butane-1,2,3,4-tetracarboxylic acid; pyrophosphate; 1,1-cyclopentanediacetic acid (3,3-tetramethylene-glutaric acid); piperazine-1,4-bis-(2-ethanesulfonic acid) (PIP) The formulation comprises: N-(2-acetamido)-2-aminoethanesulfonic acid (ACES); 1,1-cyclohexanediacetic acid; 3,6-methylene-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-methylpropanetricarboxylic acid); 2-(N-morpholino)propanesulfonic acid (MOPS); phosphoric acid; and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES). In some embodiments, the formulation comprises one or more buffers, such as N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) or sodium bicarbonate (NaHCO3). 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 of 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 of about 10 mM, about 20 mM, about 25 mM, or about 30 mM.

[0123] 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 a polymer of polysucrose, sucrose, and epichlorohydrin. In some embodiments, the formulation comprises one or more sugars in amounts from about 10 mM to about 1,000 mM. In some embodiments, the formulation comprises one or more sugars in amounts from about 50 to about 500 mM. In some embodiments, one or more sugars are present in amounts from 10 mM to 500 mM. In some embodiments, one or more sugars are present in amounts from 50 mM to 200 mM. In some embodiments, one or more sugars are present in amounts from 100 mM to 150 mM. In some embodiments, one or more sugars are lyophilizing agents; for example, in some embodiments, the lyophilizing agent comprises trehalose, polysaccharides, or combinations thereof.

[0124] In some embodiments, the composition comprising platelets or platelet derivatives (e.g., thrombus bodies) may comprise one or more of water or saline solution. In some embodiments, the composition comprising platelets or platelet derivatives (e.g., lyophilized platelets) may comprise DMSO.

[0125] In some embodiments, the formulation includes an organic solvent, such as an alcohol (e.g., ethanol). In such formulations, the amount of 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).

[0126] 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, dichloromethane, 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 includes ethanol, DMSO, or combinations thereof. The presence of an organic solvent (such as ethanol) can be beneficial for the processing of platelets, platelet derivatives, or thrombus bodies (e.g., lyophilized platelet derivatives).

[0127] In some embodiments, the formulation does not include an organic solvent. In some embodiments, the formulation includes an organic solvent. In some embodiments, the formulation includes DMSO.

[0128] The formulation can have any suitable pH. For example, in some embodiments, the formulation can have a pH of about 6.0 to about 7.4 (e.g., about 6.5 to about 6.9 or about 6.6 to about 6.8).

[0129] In some embodiments, one or more other components may bind to platelets (e.g., as part of a formulation). Exemplary components may include prostaglandin E1 or prostacyclin and / or EDTA / EGTA to prevent platelet aggregation and activation.

[0130] In some embodiments, the formulation may be buffer A, as shown in Example 1. In some embodiments, the formulation may contain buffer A, as shown in Example 1, wherein one or more components (e.g., ethanol) are present in an amount up to three times that shown in Example 1. Non-limiting examples of formulation compositions that may be used are shown in Tables P1-P6.

[0131] Table P1

[0132]

[0133] Table P1. Applicable formulations

[0134] Table P2.

[0135]

[0136] Table P2. Applicable formulations

[0137] Table P3

[0138]

[0139]

[0140] Table P3. Available Preparations

[0141] Table P4

[0142]

[0143] Table P4. Buffer B can be used when incubating platelets, for example, for flow cytometry. This incubation can be performed in the dark at room temperature. Albumin is an optional component of buffer B.

[0144] Table P5

[0145]

[0146]

[0147] Table P5 shows the concentrations of HEPES and salts in buffer B. The pH can be adjusted to 7.4 using NaOH. Albumin is an optional component of buffer B.

[0148] Table P6.

[0149]

[0150] Table P6 is another exemplary formulation.

[0151] In some embodiments, rehydrating a composition comprising platelets or platelet derivatives includes 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 solution). In some embodiments, the aqueous liquid is a saline solution. In some embodiments, the aqueous liquid is a suspension.

[0152] In some embodiments, platelets or platelet derivatives (e.g., thrombus bodies) have less than about 10%, such as less than about 8%, such as less than about 6%, such as less than about 4%, such as less than about 2%, such as less than about 0.5% of the platelet membrane through cross-linking of proteins and / or lipids present on the membrane. In some embodiments, rehydrated platelets or platelet derivatives (e.g., thrombus bodies) have less than about 10%, such as less than about 8%, such as less than about 6%, such as less than about 4%, such as less than about 2%, such as less than about 0.5% of the platelet membrane through cross-linking of proteins and / or lipids present on the membrane.

[0153] In some embodiments, platelets or aggregated platelets may be acidified to a pH of about 6.0 to about 7.4 prior to TFF, or diluted with a formulation. In some embodiments, the method includes acidifying platelets to a pH of about 6.5 to about 6.9. In some embodiments, the method includes acidifying platelets to a pH of about 6.6 to about 6.8. In some embodiments, acidification includes adding a solution containing acid-citrate-glucose (ACD) to the aggregated platelets.

[0154] In some embodiments, platelets are separated or diluted with a formulation prior to TFF. In some embodiments, the method further includes separating platelets by using centrifugation. In some embodiments, centrifugation is performed at a relative centrifugal force (RCF) of about 1000 × g to about 2000 × g. In some embodiments, centrifugation is performed at a relative centrifugal force (RCF) of about 1300 × g to about 1800 × g. In some embodiments, centrifugation is performed at a relative centrifugal force (RCF) of about 1500 × g. In some embodiments, centrifugation is performed for about 1 minute to about 60 minutes. In some embodiments, centrifugation is performed for about 10 minutes to about 30 minutes. In some embodiments, centrifugation is performed for about 30 minutes.

[0155] In some embodiments, platelets are separated, for example, in a liquid medium, before treating the subject.

[0156] In some embodiments, platelets are donor-derived platelets. In some embodiments, platelets are obtained by a method including an apheresis step. In some embodiments, platelets are aggregated platelets.

[0157] In some embodiments, platelets are collected from multiple donors. Such platelets collected from multiple donors may also be referred to herein as collected platelets. In some embodiments, there are more than 5 donors, such as more than 10, such as more than 20, such as more than 50, such as up to about 100 donors. In some embodiments, there are about 5 to about 100 donors, such as about 10 to about 50, such as about 20 to about 40, such as about 25 to about 35. The collected platelets can be used to prepare any of the compositions described herein.

[0158] In some embodiments, platelets are derived in vitro. In some embodiments, platelets are derived or prepared in a culture. In some embodiments, preparing platelets includes deriving or growing platelets from a culture of megakaryocytes. In some embodiments, preparing platelets includes deriving 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).

[0159] Therefore, in some embodiments, platelets or platelet derivatives (e.g., thrombus bodies) are prepared prior to treating the subject as described herein. In some embodiments, platelets or platelet derivatives (e.g., thrombus bodies) are lyophilized. In some embodiments, platelets or platelet derivatives (e.g., thrombus bodies) are cryopreserved. For example, in some embodiments, platelets or platelet derivatives may 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 incorporated herein by reference in its entirety).

[0160] In some embodiments, platelets (e.g., apheresis platelets, platelets separated from whole blood, aggregated platelets, or combinations thereof) form a suspension in a formulation comprising 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.

[0161] In some embodiments, the method further includes drying platelets or platelet derivatives (e.g., thrombus bodies). In some embodiments, the drying step includes lyophilizing platelets or platelet derivatives (e.g., thrombus bodies). In some embodiments, the drying step includes freeze-drying platelets or platelet derivatives (e.g., thrombus bodies). In some embodiments, the method further includes rehydrating the platelets or platelet derivatives (e.g., thrombus bodies) obtained from the drying step.

[0162] In some embodiments, platelets or platelet derivatives (e.g., thrombus bodies) are refrigerated, cryopreserved, or lyophilized (e.g., to produce thrombus bodies) prior to use in therapy or functional assays.

[0163] According to this disclosure, any known technique for drying platelets can be used, provided that the technique can achieve a final residual moisture content of less than 5%. Preferably, the technique achieves a final residual moisture content of less than 2% (e.g., 1%, 0.5%, or 0.1%). Non-limiting examples of suitable techniques are freeze-drying and spray drying. Suitable freeze-drying methods are presented in Table LA. Further exemplary freeze-drying methods can be found in U.S. Patent Nos. 7,811,558, 8,486,617, and 8,097,403. An exemplary spray drying method includes: combining nitrogen, as a drying gas, with a formulation according to the present disclosure, and then introducing the mixture into a GEA mobile miniature spray dryer from GEA Processing Engineering, Inc. (Columbia, Maryland, USA), having a dual-fluid nozzle configuration, 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 atomic rate in the range of 0.5 to 2.0 bar, an atomic rate in the range of 5 to 13 kg / h, a nitrogen dosage in the range of 60 to 100 kg / h, and an operating time of 10 to 35 minutes. The final step in the spray drying process is the preferential collection of the dried mixture. In some embodiments, the dried composition remains stable at temperatures from -20°C or lower to 90°C or higher for at least six months.

[0164] Table LA: Exemplary freeze-drying scheme

[0165]

[0166]

[0167] In some embodiments, the step of drying platelets or platelet derivatives (e.g., thrombus bodies) as disclosed herein, such as the step of freeze-drying platelets and / or platelet derivatives as disclosed herein, includes incubating the platelets and / or platelet derivatives with a lyophilizing agent (e.g., a non-reducing disaccharide). Therefore, in some embodiments, the method for preparing platelets and / or platelet derivatives further includes incubating the platelets with a lyophilizing agent. In some embodiments, the lyophilizing agent is a sugar. In some embodiments, the sugar is a disaccharide, such as a non-reducing disaccharide.

[0168] In some embodiments, platelets and / or platelet derivatives are incubated with the lyophilizing agent at a suitable temperature for a sufficient duration to allow the platelets to incubate with the lyophilizing agent. Non-limiting examples of suitable lyophilizing agents are sugars, such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, and xylose. In some embodiments, non-limiting examples of lyophilizing agents include serum albumin, dextran, polyvinylpyrrolidone (PVP), starch, and hydroxyethyl starch (HES). In some embodiments, exemplary lyophilizing agents may include high molecular weight polymers. “High molecular weight” means a polymer with an average molecular weight of about or greater than 70 kDa and up to 1,000,000 kDa. Non-limiting examples are polymers of sucrose and epichlorohydrin (e.g., polysucrose). In some embodiments, the lyophilizing agent is polysucrose. Although any amount of high molecular weight polymer can be used as a lyophilizing agent, it is preferred to use an amount reaching a final concentration of about 3% to 10% (w / v) (e.g., 3% to 7%, such as 6%).

[0169] An exemplary sugar used in the compositions disclosed herein is trehalose. Regardless of the characteristics of the sugar, it can be present in the composition in any suitable amount. For example, it can be present in amounts from 1 mM to 1 mM. In embodiments, it is present in amounts from 10 mM to 500 mM. In some embodiments, it is present in amounts from 20 mM to 200 mM. In embodiments, it is present in amounts from 40 mM to 100 mM. In various embodiments, the sugar is present at different specific concentrations within the above-described ranges, and those skilled in the art will readily understand the various concentrations without the need to specifically list each one herein. In the case where more than one sugar is present in the composition, each sugar can be present in amounts according to the above-described ranges and specific concentrations.

[0170] In some cases, the preparation of thrombus bodies further includes one or more of the procedures described in U.S. Patent Nos. 8,486,617 (e.g., Examples 1-5) and 8,097,403 (e.g., Examples 1-3) (which are incorporated herein by reference in their entirety). In some cases, starting material (e.g., one or more donor platelet units) is initially pooled into a common vessel. In some embodiments, the starting material may comprise one or more donor platelet units. In some embodiments, the starting material may comprise donor plasma. Prior to centrifugation, the starting material may or may not be acidified with an anticoagulant buffer (i.e., ACD-A). After centrifugation, plasma may be aspirated from the platelet pellet. Before resuspending the cells in the suspension, a cell compatibility buffer containing a cryoprotectant (e.g., a loading buffer that may be similar to or identical to the formulation) may be added to the platelet pellet. If desired, the platelets may or may not be diluted with a buffer to a predetermined concentration (e.g., 2200 kJ / µl to 2800 kJ / µl). Platelets in buffer solution can be incubated at temperatures ranging from 18°C ​​to 37°C for 0 to 240 minutes. A lyophilization protectant (e.g., sucrose) can be added to the platelets in buffer solution to achieve a final swelling agent concentration of 1% w / v to 10% w / v (preferably 6% w / v). The centrifuged, treated platelets can then be packaged into vials, lyophilized, and heat-treated.

[0171] In some embodiments, platelets or platelet derivatives (e.g., thrombus bodies) have a particle size (e.g., diameter, maximum size) of at least about 0.5 μm (e.g., 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 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).

[0172] 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 platelets or platelet derivatives (e.g., thrombus bodies) have a particle 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 platelets or platelet derivatives (e.g., thrombus bodies) are 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 platelets or platelet derivatives (e.g., thrombus bodies) are 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).

[0173] In some cases, the particles can be particles with a particle size (e.g., diameter, maximum size) of less than about 0.5 μm (less than about 0.45 μm or 0.4 μm). In other cases, the particles can be particles with a particle size of about 0.01 μm to about 0.5 μm (e.g., about 0.02 μm to about 0.5 μm).

[0174] In compositions containing platelets or platelet derivatives (e.g., thrombus bodies), such as those prepared according to the methods described herein, the contribution of the particle content to the total scattering intensity of all particles in the composition with radii from about 1 nm to about 60,000 nm may be less than about 5.0% (e.g., less than about 4.5%, 4.0%, 3.5%, 3.0%, 2.5%, 2.0%, 1.5%, 1.0%, or 0.5%). As used herein, the particle content “by scattering intensity” refers to the particle content based on the scattering intensity of all particles in the composition with radii from about 1 nm to about 60,000 nm. The particle content can be measured by any suitable method, such as by dynamic light scattering (DLS). In some cases, the viscosity of the sample used for DLS may be about 1.060 cP (or may be adjusted to 1.060 cP), as this is an approximate viscosity of plasma.

[0175] Platelets or platelet derivatives (e.g., thrombus bodies) as described herein may have cell surface markers. The presence of cell surface markers can be determined using any suitable method. In some embodiments, the presence of cell surface markers can be determined using a binding protein (e.g., an antibody) specifically targeting one or more cell surface markers and flow cytometry (e.g., as a positive percentage, for example, using about 2.7 × 10⁻⁶ cells / mL). 5Thrombus bodies / 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) to determine. Non-limiting examples of cell surface markers include CD41 (also known as glycoprotein IIb or GPIIb, which can be measured using, for example, anti-CD41 antibody), CD42 (which can be measured using, for example, anti-CD42 antibody), CD62 (also known as CD62P or P-selectin, which can be measured using, for example, anti-CD62 antibody), phosphatidylserine (which can be measured using, for example, annexin V (AV)), and CD47 (which is used for self-recognition; the absence of this marker may lead to phagocytosis in some cases). The positive percentage of any cell surface marker can be any appropriate positive percentage. For example, platelets or platelet derivatives (e.g., thrombus bodies), such as those prepared by the methods described herein, may have an average CD41 positivity percentage of 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, platelets or platelet derivatives (e.g., thrombus bodies), such as those described herein, may have an average CD42 positivity percentage of 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, platelets or platelet derivatives (e.g., thrombus bodies), such as those prepared by the methods described herein, may have an average CD62 positivity percentage of 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 yet another example, platelets or platelet derivatives (e.g., thrombus bodies), such as those prepared by the methods described herein, may have an average annexin V positivity of 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 yet another example, platelets or platelet derivatives (e.g., thrombus bodies), such as those prepared by the methods described herein, may have an average CD47 positivity percentage of at least about 8% (e.g., at least about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45%, 50%, or 55%).

[0176] Glycoprotein VI (GPVI) is the platelet receptor for collagen, and the binding of collagen to GVPI activates platelets. Receptor binding is significantly reduced in thrombus bodies compared to fresh platelets. Without being bound by any particular theory, it is believed that manufacturing methods that block or destroy some copies of this receptor in thrombus bodies may lead to the reduced collagen binding in thrombus bodies relative to fresh platelets.

[0177] Platelets or platelet derivatives (e.g., thrombus bodies) as described herein can possess cell membrane-associated fibrinogen. Aggregation of activated platelets is mediated by the formation of the GPIIb / IIIa complex, which can bind to fibrinogen (also known as factor 1) and form clots. GPIIb / IIIa is the platelet fibrinogen receptor, also known as the CD41 / CD61 complex. The GPIIb / IIIa clone PAC-1 binds to the active form of GPIIb / IIIa. Without being bound by any particular theory, it is believed that the presence of fibrinogen on the cell membrane may indicate the ability of platelets or platelet derivatives (e.g., thrombus bodies) to form clots. Similarly, without being bound by any particular theory, it is believed that the lack of binding of anti-PAC1 antibodies to platelets or platelet derivatives (e.g., thrombus bodies) (such as platelets or platelet derivatives prepared by the methods described herein) may indicate fibrinogen binding to the active form of GPIIb / GPIIIa, since PAC-1 binds to the same complex. In some cases, platelets or platelet derivatives (e.g., thrombus bodies) (such as platelets or platelet derivatives prepared by the methods described herein) may have a large amount of bound fibrinogen when they retain a high amount of residual plasma.

[0178] Platelets or platelet derivatives (e.g., thrombus bodies) as described herein can generate thrombin, for example, in the presence of a reagent containing tissue factor and phospholipids. For example, in some cases, when a reagent containing tissue factor (e.g., at 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, or 10 pM) and optionally phospholipids is present, platelets or platelet derivatives (e.g., thrombus bodies) as described herein (e.g., at a concentration of about 4.8 × 10⁻⁶) can generate thrombin. 3A particle / μL platelet can produce 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). For example, in some cases, when a reagent containing tissue factor (e.g., at 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, or 10 pM) and optionally phospholipids is present, platelets or platelet derivatives (e.g., thrombus bodies) as described herein (e.g., at a concentration of about 4.8 × 10⁻⁶) can be produced. 3 (particles / μL) can generate 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). In some cases, when PRP reagent (from Thrombinoscope cat#TS30.00) is present, for example, using 20 μL of PRP reagent and 80 μL of a solution containing about 4.8 × 10⁻⁶ particles / μL, TPH can be generated. 3 The conditions of the composition of platelets or platelet derivatives (e.g., thrombus bodies) per μL of platelet or platelet derivatives (e.g., thrombus bodies) as described herein (e.g., a concentration of about 4.8 × 10⁻⁶) 3 (particles / μL) can generate 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). In some cases, when a PRP reagent (from Thrombinoscope cat#TS30.00) is present, for example, using 20 μL of PRP reagent and 80 μL of [amount missing] containing approximately 4.8 × 10 [units missing] 3 The conditions of the composition of 1 particle / μL platelet or platelet derivative (e.g., thrombus body) as described herein for platelets or platelet derivative (e.g., thrombus body) (e.g., a concentration of about 4.8 × 10⁻⁶). 3 Particles per μL can produce 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).

[0179] Platelets or platelet derivatives (e.g., thrombus bodies) as described herein can produce thrombin, for example, in the presence of reagents containing tissue factors and phospholipids. For example, in some cases, platelets or platelet derivatives (e.g., thrombus bodies) may have a concentration of 10... 6 Each particle has a potency of 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 generating potency units (TGPUs). For example, in some cases, platelets or platelet derivatives (e.g., thrombus bodies) may have a potency of at least 10 TGPUs per particle. 6 The potency of 1.2 to 2.5 TPGU per particle (e.g., per 10 6 The particle count is 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 TPGUs. TPGU can be calculated as follows: TGPU / million particles = [TPH in nM] * [potency coefficient in IU / (nM)] / [576,000 particles in the well]. Similarly, the potency coefficient of a thrombin sample can be calculated as follows: Potency coefficient = calculated calibrator activity (IU) / effective calibrator activity (nM). In some cases, the calibrator activity can be based on the WHO International Thrombin Standard.

[0180] Platelets or platelet derivatives (e.g., thrombus bodies) as described herein can aggregate, for example, through the use of a total thrombosis analysis system. As determined. In some cases, as described herein, platelets or platelet derivatives, when the concentration is at least 70 × 10⁻⁶. 3 Particles / μL (e.g., at least 73 × 10⁻⁶) 3 100×10 3 150×10 3 173×10 3 200×10 3 250×10 3 Or 255×10 3 At concentrations of 1 particle / μL, T-TAS occlusion times of less than 14 minutes (e.g., less than 13.5, 13, 12.5, 12, 11.5, or 11 minutes) can result in a time to 80 kPa, for example, in citric acidified whole blood with thrombocytopenia. In some cases, platelets or platelet derivatives as described herein, when at concentrations of at least 70 × 10⁻⁶, can cause T-TAS occlusion times of less than 14 minutes (e.g., less than 13.5, 13, 12.5, 12, 11.5, or 11 minutes), for example, in citric acidified whole blood with thrombocytopenia. 3 Particles / μL (e.g., at least 73 × 10⁻⁶) 3 100×10 3 150×10 3 173×103 200×10 3 250×10 3 Or 255×10 3 When the number of particles per μL is 1, it can result in an area under the curve (AUC) of at least 1300 (e.g., at least 1380, 1400, 1500, 1600 or 1700), for example in citric acidified whole blood with thrombocytopenia.

[0181] Platelets or platelet derivatives (e.g., thrombus bodies) as described herein can aggregate, for example, in the presence of an aggregation agonist. Non-limiting examples of aggregation agonists include thrombin and collagen. In some cases, in the presence of an aggregation agonist, platelets or platelet derivatives (e.g., thrombus bodies) as described herein can have an aggregation percentage of 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 some cases, in the presence of an aggregation agonist, platelets or platelet derivatives (e.g., thrombus bodies) as described herein may have an aggregation percentage of about 25% to about 100% (e.g., about 25% to about 50%, about 25% to about 75%, about 50% to about 100%, about 75% to about 100%, about 40% to about 95%, about 55% to about 80%, or about 65% to about 75%). The aggregation percentage can be determined by any suitable method (e.g., phototransmission aggregation).

[0182] Compositions comprising platelets or platelet derivatives (e.g., thrombus bodies) as described herein may have suitable conditions and appropriate amounts of cell substrates and / or metabolites, such as pH, pCO2, pO2, HCO3 concentration, total carbon dioxide (TCO2), SO2, 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 has been stored in vitro, respired, and subjected to glycolysis for a period of time (e.g., about 3 days) until the time of manufacture. For example, in some cases, the pH may be about 6.0 to about 7.5 (e.g., about 6.0 to about 7.4, about 6.9 to about 7.5, or about 7.0 to about 7.3). As another example, pCO2 may be about 10 to about 20 mmHg (e.g., about 10 to about 15 mmHg, about 15 to about 20 mmHg, or about 17 to about 19 mmHg). pO2 can be from 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). HCO3 concentration can be from 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 from about 4 to about 8 mmol / L (e.g., about 5 to about 7 mmol / L). sO2 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 1.0 mmol / L). Lactate concentration can be from 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).

[0183] Platelets or platelet derivatives (e.g., thrombus bodies) as described herein can retain some metabolic activity, for example, as demonstrated by lactate dehydrogenase (LDH) activity. In some cases, platelets or platelet derivatives (e.g., thrombus bodies) as 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 believed that the addition of an increased amount of sucrose increases the amount of residual LDH activity (e.g., a product with a formulation containing 8% sucrose has more retained LDH activity than a product with a formulation containing 4% sucrose). Similarly, without being bound by any particular theory, it is believed that heat treatment of lyophilized compositions containing platelets or platelet derivatives (e.g., thrombus bodies) increases the amount of retained LDH activity. As another example, metabolic activity can be demonstrated by the presence of retained esterase activity, such as the ability of cells to cleave the acetate groups on carboxyfluorescein diacetate succinimide ester (CFDASE) to expose the fluorophore.

[0184] Reduction of pathogens in blood products is generally desirable. Without being bound by any particular theory, it is believed that some methods of pathogen reduction can lead to the formation of microparticles in processed blood products. One method of pathogen reduction involves using photosensitive nucleic acid intercalation compounds to alter the nucleic acids of pathogens upon irradiation with an appropriate wavelength. The system (manufactured by Cerus) uses amotosalen (a nucleic acid intercalation compound that forms cross-links in nucleic acids when irradiated with UVA).

[0185] The final blood products described herein (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)) can be prepared by any suitable method. The final blood products described herein (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)) can be prepared by methods disclosed herein. In some embodiments described herein, the final blood product may be a composition comprising platelets and an aqueous medium. In some embodiments, the final blood product may be the result of freeze-drying a composition comprising platelets and an aqueous medium, as described herein. In some embodiments, the final blood product may be prepared using tangential flow filtration (TFF) of starting materials (e.g., unprocessed blood products (e.g., apheresis materials (e.g., pooled apheresis materials)) or partially processed blood products (e.g., blood products that have already undergone filtration)). In some embodiments, the final blood product can be prepared using centrifugation of the starting material (e.g., unprocessed blood product (e.g., apheresis material (e.g., pooled apheresis material)) or partially processed blood product (e.g., filtered blood product)). It should be understood that while the methods described herein are generally based on the use of apheresis material as the starting material, other materials, such as cultured platelets or whole blood, can also be used. In some cases, platelets can be separated from whole blood (e.g., pooled whole blood).

[0186] The starting material can be any suitable starting material. In some embodiments, the starting material can have a protein concentration of about 60 to about 80 mg / mL. In some embodiments, the protein concentration can be based on the protein concentration in whole blood plasma. In some embodiments, the protein concentration can be based on the protein concentration in donor apheresis plasma. In some embodiments, the starting material can be a donor blood product (e.g., whole blood or graded blood). In some embodiments, the starting material can be a pooled donor blood product (e.g., pooled whole blood or pooled graded 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 apheresis material, regardless of whether the material contains platelets or other blood cells.

[0187] In some embodiments, the starting material may be donor apheresis material (e.g., donor platelets or a donor platelet pool). 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 a regulatory-approved assay (e.g., an FDA-approved assay). In some embodiments, the starting material may be tested for HLA class I antibodies in a regulatory-approved assay (e.g., an FDA-approved assay). In some embodiments, the starting material may be tested for HLA class II antibodies in a regulatory-approved assay (e.g., an FDA-approved assay). In some embodiments, the starting material may be tested for HNA antibodies in a regulatory-approved assay (e.g., an FDA-approved assay). The regulatory-approved assay may be any appropriate regulatory-approved assay. In some embodiments, the regulatory-approved test may be One Lambda's LABSCREEN. TM Mixed. In some implementations, it can be used. 100 / 200 or XY and HLA FUSION TM The software undergoes regulatory approval testing.

[0188] In some embodiments, the starting material may undergo a pathogen reduction step, such as a nucleic acid intercalation compound that forms cross-links in the nucleic acid upon UVA irradiation.

[0189] In some embodiments, starting material (e.g., one or more units of donor platelets) may initially be pooled into a common vessel. The starting material may initially be diluted with or may initially not 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 material may undergo two general processing pathways; either washing with a control buffer (e.g., using TFF) until the desired residual component (e.g., percentage of residual donor plasma) is reached, followed by concentration to the final concentration; or the starting material may be concentrated to the final concentration before washing with a control buffer (e.g., using TFF) until the desired residual component (e.g., percentage of residual donor plasma) is reached. The TFF-processed material can then be bottled, lyophilized, and heat-treated.

[0190] In some embodiments, the method may include an initial dilution step, whereby starting material (e.g., unprocessed blood products (e.g., apheresis material (e.g., pooled apheresis material)) may be diluted with a formulation (e.g., any formulation described herein) to form a diluted starting material. In some cases, the initial dilution step may include dilution with a formulation, wherein the mass of the formulation is equal to at least about 10% of the mass of the starting material (e.g., at least about 15%, 25%, 50%, 75%, 100%, 150%, or 200% of the mass of the starting material). In some embodiments, a TFF device may be used for the initial dilution step.

[0191] In some embodiments, the method may include concentration (e.g., concentrated platelets) (e.g., concentrated starting material or diluted starting material) to form a concentrated platelet composition. For example, concentration may include concentrating to about 1000 × 10⁻⁶. 3 Approximately 4000×10 3 Platelets / μL (e.g., approximately 1000 × 10⁻⁶) 3 Approximately 2000×10 3 Approximately 2000 × 10 3 Approximately 3000×10 3 Or approximately 4000×10 3 (platelets / μL). In some embodiments, a TFF device may be used for the concentration step.

[0192] The concentration of platelets or platelet derivatives (e.g., thrombus bodies) can be determined 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).

[0193] In some embodiments, TFF may include perfiltration (sometimes referred to as “washing”) of starting material, diluted starting material, concentrated platelet composition, or combinations thereof. In some embodiments, perfiltration may include washing with at least 2 (e.g., at least 3, 4, 5, 6, 7, 8, 9, 10 or more) perfiltration volumes. In some embodiments, TFF may include buffer exchange. In some embodiments, buffers may be used in TFF. The buffer may be any suitable buffer. In some embodiments, the buffer may be a formulation (e.g., any formulation described herein). In some embodiments, the buffer may be the same formulation used for dilution. In some embodiments, the buffer may be a different formulation from the formulation used for dilution. In some embodiments, the buffer may include a lyophilizing agent, including a buffer, base, loading agent, optionally a salt, and optionally at least one organic solvent, such as 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. The buffer may be any suitable buffer. In some embodiments, the buffer may be 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES). The base may be any suitable base. In some embodiments, the base may be sodium bicarbonate. In some embodiments, the sugar may be a monosaccharide. In some embodiments, the loading agent may 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 may be trehalose. In some embodiments, the loading agent may include polysucrose. The salt may be any suitable salt. In some embodiments, the salt may be selected from the group consisting of sodium chloride (NaCl), potassium chloride (KCl), or combinations thereof.

[0194] In some embodiments, membranes with pore sizes 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 μm to about 0.45 μm, about 0.45 μm 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 for TFF. The membrane can be made of 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, membranes with a nominal molecular weight limit (NMWCO) of at least about 100 kDa (e.g., at least about 200 kDa, 300 kDa, 500 kDa, or 1000 kDa) can be used for TFF. TFF can be performed at any suitable temperature. In some embodiments, TFF can be performed at temperatures 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 performed at a flow rate (e.g., circulation flow rate) of about 100 ml / min to about 800 ml / min (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).

[0195] 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., less than or equal to about 50%, 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 residual plasma). In some embodiments, the endpoint can be the relative absorbance (A280) at 280 nm. For example, the endpoint can be A280 (e.g., using a path length of 0.5 cm), which is less than or equal to approximately 50% of the A280 (e.g., using a path length of 0.5 cm) prior to TFF (e.g., starting material or diluted starting material) (e.g., less than or equal to approximately 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%). In some embodiments, A280 can be relative to a system measuring 7.5% plasma = 1.66 AU. In some embodiments, the instrument for measuring A280 can be configured such that a 0.5 cm gap flow cell can be attached to the filtrate line of the TFF system. The flow cell can be connected to a photometer, wherein fiber optic cables are connected to each side of the flow cell (light source cable and photodetector cable). The flow cell can be made with quartz glass lenses on each side of the fiber optic cable. In some embodiments, the endpoint can be an absolute A280 (e.g., using a path length of 0.5 cm). For example, the endpoint can be an A280 less than or equal to 1.70 AU (e.g., less than or equal to 1.66, 1.6, 1.5, 1.4, 1.3, 1.2, 1.1, 1.0, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 AU) (e.g., using a path length of 0.5 cm). In some embodiments, the percentage of residual plasma, relative A280, or A280 can be determined based on an aqueous medium comprising a composition of platelets and an aqueous medium. In some embodiments, the percentage of residual plasma can be determined based on a known correlation with A280. In some embodiments, the endpoint can be a platelet concentration, since TFF can include the concentration or dilution of the sample (e.g., using a formulation). For example, the endpoint can be at least about 2000 × 10⁻⁶. 3 Platelets / μL (e.g., at least approximately 2050 × 10⁶) 3 2100×10 3 2150×10 3 2200×10 3 2250×103 2300×10 3 2350×10 3 2400×10 3 2450×10 3 Or 2500×10 3 Platelet concentration (platelets / μL). As another example, the endpoint could be approximately 1000 × 10⁻⁶. 3 To approximately 2500 platelets / μL (e.g., approximately 1000 × 10⁻⁶). 3 Approximately 2000×10 3 Approximately 1500×10 3 Approximately 2300×10 3 Or approximately 1700×10 3 Approximately 2300×10 3 Platelet concentration (platelets / μL). In some embodiments, the endpoint may include more than one criterion (e.g., percentage of residual plasma and platelet concentration, relative A280 and platelet concentration, or absolute A280 and platelet concentration).

[0196] Typically, TFF-treated compositions are subsequently lyophilized, optionally with a heat treatment step, to form the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)). However, in some cases, TFF-treated compositions may be considered as the final blood product.

[0197] In some embodiments, blood products can be prepared using centrifugation of blood products (e.g., untreated blood products (e.g., apheresis material (e.g., pooled apheresis material)) or partially treated blood products (e.g., blood products that have undergone TFF)). In some embodiments, blood products can be prepared without centrifuging blood products (e.g., untreated blood products (e.g., apheresis material) or partially treated blood products (e.g., blood products that have undergone TFF)). Centrifugation can include any suitable steps. In some embodiments, centrifugation can include slow acceleration, slow deceleration, or a combination thereof. In some embodiments, centrifugation may include centrifugation at a rate of 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 may 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).

[0198] In some embodiments, both TFF and centrifugation (e.g., TFF followed by centrifugation or centrifugation followed by TFF) can be used to prepare the final blood product.

[0199] This document also provides compositions prepared by any of the methods described herein.

[0200] In some embodiments, the compositions as described herein can be analyzed at multiple points during processing. In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the starting material (e.g., donor apheresis material (e.g., pooled donor apheresis material)) can be analyzed. In some embodiments, the protein concentration (e.g., by absorbance at 280 nm (e.g., using a path length of 0.5 cm)) of the starting material (e.g., donor apheresis material (e.g., pooled donor apheresis material)) can be analyzed. In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the compositions in intermediate steps of processing (e.g., when the protein concentration decreases to less than or equal to 75% of the protein concentration of the untreated blood product (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower)) can be analyzed. In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the blood product in the intermediate steps of processing 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 antibody content (e.g., HLA or HNA antibody content) of the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)) can be analyzed. 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., thrombus bodies)) 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 may not have detectable levels of HLA class I antibodies, HLA... Antibodies comprising a group consisting of class II antibodies and HNA antibodies. In some embodiments, the aqueous medium of the compositions described herein can be used for analysis as described herein.

[0201] In some embodiments, the compositions as described herein can be analyzed at multiple points during processing. In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the donor apheresis plasma can be analyzed. In some embodiments, the protein concentration of the donor apheresis plasma can be analyzed (e.g., by absorbance at 280 nm). In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the compositions in intermediate steps of processing can be analyzed (e.g., when the protein concentration decreases to less than or equal to 75% of the protein concentration of the unprocessed blood product (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower)). In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of the blood product in the intermediate steps of processing 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 donor plasma. 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., thrombus bodies)) can be analyzed. 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., thrombus bodies)) 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 donor apheresis plasma. In some embodiments, the final blood product may not have detectable levels of HLA-selected antibodies. Antibodies comprising a group consisting of class I antibodies, class II HLA antibodies, and HNA antibodies. In some embodiments, the aqueous medium of the compositions described herein can be used for analysis as described herein.

[0202] The protein concentration of blood products can be measured by any suitable method. In some embodiments, the absorbance at 280 nm can be used to measure the protein concentration of blood products.

[0203] The antibody content (e.g., HLA or HNA antibody content) of blood products can be measured by any suitable method.

[0204] In some embodiments, FLOWPRA from One Lambda and Thermo Fisher Scientific can be used. TM Screening or LABScreen Multi assay kits can be used as a method for HLA detection. Raw materials can be tested prior to TFF or centrifugation to determine baseline levels of class I and II antibodies against human leukocyte antigen (HLA) and human neutrophil antigen (HNA). After processing, the tests can be repeated by centrifugation or TFF to measure HLA and HNA removal rates. Additional test points can be performed throughout the TFF procedure to maintain process control. After lyophilization and annealing, random samples can be selected from the batch for qualitative HLA / HNA antibody testing to ensure reduction and compliance with current FDA testing and acceptance requirements.

[0205] In some embodiments, the antibody content (e.g., HLA or HNA antibody content) of two blood products can be compared by determining the percentage of beads that are positive for a marker (e.g., HLA or HNA-coated beads 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, this method can be performed as follows: An aliquot of anemic platelet plasma (e.g., about 1 mL) can be obtained. In some embodiments, an aliquot of filtered (e.g., using a 0.2 μm filter) anemic platelet plasma (PPP) (e.g., about 1 mL) can be obtained. Beads coated with type I HLA and / or beads coated with type II HLA can be added to the plasma (e.g., about 5 μL of each type of bead can be added 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, incubation may be performed under other conditions (e.g., in the dark) at a certain temperature (e.g., at room temperature) for a period of time (e.g., about 30 minutes) to form an incubated mixture. In some embodiments, incubation may include agitation (e.g., gentle shaking). The beads in the incubated mixture may be washed using any suitable conditions. In some embodiments, the beads in the incubated mixture may be washed with a washing buffer. The washed beads may be separated from the incubated mixture by any suitable method. In some embodiments, the washed beads may be separated by centrifugation (e.g., at 9,000 × g for 2 minutes) to obtain precipitated beads. In some embodiments, the washing step may be repeated. The beads may be resuspended to form a bead solution. Antibodies conjugated to the detectable portion (e.g., antibodies binding to the measured antibody content (e.g., HLA or HNA antibody content)) may be added to the bead solution (e.g., αIgG conjugated to a fluorescent reporter, such as FITC). The antibodies may be incubated with the bead solution under any suitable conditions. In some embodiments, antibodies may be incubated under other conditions (e.g., in the dark) at a temperature (e.g., room temperature) for a period of time (e.g., about 30 minutes) to form labeled beads. The labeled beads may be washed to remove unbound antibodies conjugated to the detectable portion. The labeled beads may be washed using any suitable conditions. In some embodiments, the labeled beads may be washed with a washing buffer. The washed labeled beads may be separated by any suitable method. In some embodiments, the washed labeled beads may be separated by centrifugation (e.g., at 9,000 g for 2 minutes) to obtain precipitated labeled beads. In some embodiments, the washing step may be repeated. The labeled beads may be detected by any suitable method.In some embodiments, the labeled beads can be detected by flow cytometry. In some embodiments, the detection may include measuring the percentage of beads that are positive for the detectable portion compared to a negative control. In some embodiments, a negative control can be prepared as described above using a PPP sample known to be negative for antibodies (e.g., HLA class I, HLA class II, or HNA antibodies).

[0206] In some embodiments, blood products (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)) can be analyzed at multiple points during processing. In some embodiments, the starting material (e.g., donor apheresis material) can be analyzed to determine the percentage of positive beads (e.g., HNA or HNA-coated beads). In some embodiments, the protein concentration of the starting material (e.g., donor apheresis material) can be analyzed (e.g., by absorbance at 280 nm). In some embodiments, the percentage of positive beads (e.g., HLA or HNA-coated beads) can be determined by analyzing the blood product in intermediate steps of processing (e.g., when the protein concentration decreases to less than or equal to 75% of the protein concentration of the starting material (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower)). In some embodiments, the percentage of positive beads (e.g., HLA or HNA-coated beads) can be determined by analyzing the blood product in intermediate steps of processing (e.g., when the protein concentration decreases to less than or equal to 75% of the protein concentration of the starting material (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower)). In some embodiments, the percentage of positive beads from blood products (e.g., HLA or HNA-coated beads) in intermediate processing steps 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 percentage of positive beads from the starting material. In some embodiments, the percentage of positive beads from blood products (e.g., HLA or HNA-coated beads) in intermediate processing steps may be less than or equal to 75% of the total number of beads (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less). In some embodiments, the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)) can be analyzed to determine the percentage of positive beads (e.g., HLA or HNA-coated beads).In some embodiments, the percentage of positive beads (e.g., HLA or HNA-coated beads) from the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)) 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., HLA or HNA-coated beads) from the final blood product may be less than or equal to 75% of the total number of beads (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, or less). In some embodiments, the aqueous medium of the composition as described herein may be analyzed as described herein.

[0207] In some embodiments, blood products (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)) may be analyzed at multiple points during processing. In some embodiments, donor apheresis plasma may be analyzed to determine the percentage of positive beads (e.g., HLA or HNA-coated beads). In some embodiments, the protein concentration of donor apheresis plasma may be analyzed (e.g., by absorbance at 280 nm). In some embodiments, blood products in intermediate steps of processing may be analyzed (e.g., when the protein concentration decreases to less than or equal to 75% of the protein concentration of the starting material (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower)) to determine the percentage of positive beads (e.g., HLA or HNA-coated beads).

[0208] In some embodiments, the percentage of positive beads (e.g., HLA or HNA-coated beads) can be determined by analyzing the blood product in intermediate steps of processing (e.g., when the protein concentration decreases to less than or equal to 75% of the protein concentration of the starting material (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or lower)).

[0209] In some embodiments, the percentage of positive beads (e.g., HLA or HNA-coated beads) from the blood product in intermediate processing steps 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 percentage of positive beads from donor apheresis plasma. In some embodiments, the percentage of positive beads (e.g., HLA or HNA-coated beads) from the blood product in intermediate processing steps may be less than or equal to 75% of the total number of beads (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less). In some embodiments, the percentage of positive beads (e.g., HLA- or HNA-coated beads) from the final blood product (e.g., platelets, cryopreserved platelets, freeze-dried platelets, etc.) can be determined by analyzing the final blood product. In some embodiments, the percentage of positive beads (e.g., HLA- or HNA-coated beads) from the final blood product can be reduced by at least 5% (e.g., 10%, 20%, 30%, 40%, 50%, 60%) compared to the percentage of positive beads from the donor apheresis material. 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or more). In some embodiments, the percentage of positive beads (e.g., HLA or HNA-coated beads) from the final blood product may be less than or equal to 75% of the total number of beads (e.g., less than or equal to 70%, 65%, 60%, 55%, 50%, 45%, 40%, 35%, 30%, 25%, 20%, 15%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1% or less). In some embodiments, the aqueous medium of the composition as described herein may be analyzed as described herein.

[0210] The percentage of positive beads can be determined using any suitable method. In some embodiments, positive beads can be identified by comparison with a negative control sample. The negative control sample can be any suitable negative control sample. In some embodiments, the negative control sample can be used to determine a positive gate such that less than a certain percentage (e.g., about 0.01% to about 1% (e.g., about 0.01% to about 0.05%, about 0.05% to about 0.1%, about 0.1% to about 0.5%, 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 solution (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 measured (e.g., HLA or HNA antibody).

[0211] This document also provides a method for reducing the percentage of antibodies (e.g., HLA antibodies (e.g., HLA class I or HLA class II antibodies) or HNA antibodies) in a platelet-containing composition (e.g., a blood product), the method comprising filtering the composition by tangential flow filtration. This document also provides a method for reducing the amount of antibodies (e.g., HLA antibodies (e.g., HLA class I or HLA class II antibodies) or HNA antibodies) in a platelet-containing composition (e.g., a blood product), the method comprising filtering the composition by tangential flow filtration. This document also provides a method for reducing the percentage of beads positive for antibodies (e.g., HLA antibodies (e.g., HLA class I or HLA class II antibodies) or HNA antibodies) in a platelet-containing composition (e.g., a blood product), the method comprising filtering the composition by tangential flow filtration.

[0212] This document also provides a method for reducing the percentage of antibodies (e.g., HLA antibodies (e.g., HLA class I or HLA class II antibodies) or HNA antibodies) in a platelet-containing composition (e.g., a blood product), the method comprising filtering the composition by centrifugation. This document also provides a method for reducing the amount of antibodies (e.g., HLA antibodies (e.g., HLA class I or HLA class II antibodies) or HNA antibodies) in a platelet-containing composition (e.g., a blood product), the method comprising filtering the composition by centrifugation. This document also provides a method for reducing the percentage of beads positive for antibodies (e.g., HLA antibodies (e.g., HLA class I or HLA class II antibodies) or HNA antibodies) in a platelet-containing composition (e.g., a blood product), the method comprising filtering the composition by centrifugation.

[0213] In some embodiments of any of the methods described herein, the amount of antibodies (e.g., HLA antibodies (e.g., HLA class I or HLA class II antibodies) or HNA antibodies) in the composition (e.g., a blood product) may be reduced below a reference level. The reference level may be any suitable reference level. In some embodiments of any of the methods described herein, the percentage of beads positive for antibodies (e.g., HLA antibodies (e.g., HLA class I or HLA class II antibodies) or HNA antibodies) in the composition (e.g., a blood product) may be reduced compared to a blood product prior to undergoing any of the methods described herein. The percentage of antibody-positive beads may be reduced by any appropriate amount. In some embodiments, the percentage of antibody-positive beads may 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 a blood product prior to undergoing any of the methods described herein.

[0214] In some embodiments, the compositions described herein may undergo any suitable additional process steps. In some embodiments, the compositions described herein may be lyophilized. In some embodiments, lyophilized platelets may be heat-treated (e.g., at about 80°C for about 24 hours).

[0215] For example, in some embodiments, the composition may be cryopreserved or freeze-dried. In some embodiments, the first composition (e.g., a composition comprising platelets and an aqueous medium as described herein) may be treated with a mixture. In some embodiments, the mixture may include a lyophilizing agent, comprising a base, a loading agent, and optionally at least one organic solvent, such as 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 comprising platelets. In some embodiments, the loading agent may be a sugar. In some embodiments, the sugar may be a monosaccharide. In some embodiments, the sugar may be sucrose, maltose, trehalose, glucose (e.g., dextrose), mannose, or xylose. In some embodiments, the loading agent may be a polysucrose.

[0216] In some embodiments, the first or second composition may be dried. In some embodiments, the first or second composition may be dried using a cryoprotectant. In some embodiments, the cryoprotectant may include sugar, optionally an alkali, and optionally at least one organic solvent, such as 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 may be polysucrose.

[0217] In some embodiments, the first or second composition may be freeze-dried. In some embodiments, the first or second composition may be freeze-dried using a cryoprotectant. In some embodiments, the cryoprotectant may include sugar, optionally an alkali, and optionally at least one organic solvent, such as 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, freeze-drying may be performed at a temperature of about -40°C to about 5°C. In some embodiments, freeze-drying may be performed at a gradient (e.g., about -40°C to about 5°C). In some embodiments, a secondary drying step may be performed (e.g., at about 20°C to about 40°C).

[0218] This document also provides blood products prepared by any of the methods described herein (e.g., platelets, cryopreserved platelets, freeze-dried platelets (e.g., thrombus bodies)).

[0219] In some embodiments, compared to similar compositions not prepared by methods including tangential flow filtration of the platelet-containing composition, centrifugation of the platelet-containing composition, 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, as determined by flow cytometry using beads coated with class I HLA, class II HLA, or HNAs respectively, is reduced by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%).

[0220] In some embodiments, compared to similar compositions not prepared by methods including tangential flow filtration of the platelet-containing composition, centrifugation of the platelet-containing composition, or a combination thereof, the percentage of beads positive for HLA class I antibodies, as determined by flow cytometry using beads coated with class I HLA, is reduced by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%).

[0221] In some embodiments, compared to similar compositions not prepared by methods including tangential flow filtration of the platelet-containing composition, centrifugation of the platelet-containing composition, or a combination thereof, the percentage of beads positive for HLA class II antibodies, as determined by flow cytometry using beads coated with class II HLA, is reduced by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%).

[0222] In some embodiments, compared to similar compositions not prepared by methods including tangential flow filtration of the platelet-containing composition, centrifugation of the platelet-containing composition, or a combination thereof, the percentage of beads positive for HNA antibodies, as determined by flow cytometry using HNA-coated beads, is reduced by at least 10% (e.g., at least 20%, 30%, 40%, 50%, 60%, 70%, 75%, 80%, 85%, 90%, 95%, or 99%).

[0223] In the methods provided herein for preparing the compositions provided herein, the optional addition of a lyophilizing agent may be the last step before drying. However, in some embodiments, the lyophilizing agent may be added simultaneously with or before other components of the composition, such as salts, buffers, optionally cryoprotectants, or other components. In some embodiments, the lyophilizing agent is added to the formulation, thoroughly mixed to form a drying solution, dispensed into a drying container (e.g., a glass or plastic serum vial, lyophilized bag), and subjected to conditions permissible for drying the TFF-treated composition to form a dried composition.

[0224] In various embodiments, the freeze-drying bag is a gas-permeable bag configured to allow gas to pass through at least a portion or all of the bag during processing. The gas-permeable bag allows for the exchange of gases inside the bag with atmospheric gases present in the surrounding environment. The gas-permeable bag can be permeable to gases such as oxygen, nitrogen, water, air, hydrogen, and carbon dioxide, thereby allowing gas exchange to occur in the compositions provided herein. In some embodiments, the gas-permeable bag allows for the removal of some carbon dioxide present inside the bag by allowing carbon dioxide to permeate through its walls. In some embodiments, releasing carbon dioxide from the bag can be beneficial in maintaining the desired pH level of the compositions contained in the bag.

[0225] In some embodiments, the container of the method described herein is a closed or sealed gas-permeable container. In some embodiments, the container is closed or sealed and a portion thereof 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 product contained in the container (hereinafter referred to as the "SA / V ratio") can be adjusted to improve pH retention of the compositions provided herein. For example, in some embodiments, the SA / V ratio of the container may be at least about 2.0 cm². 2 / mL (e.g., at least about 2.1cm) 2 / mL, at least about 2.2cm 2 / mL, at least about 2.3cm 2 / mL, at least about 2.4cm 2 / mL, at least about 2.5cm 2 / mL, at least about 2.6cm 2 / mL, at least about 2.7cm 2 / mL, at least about 2.8cm 2 / mL, at least about 2.9cm 2 / mL, at least about 3.0cm 2 / mL, at least about 3.1cm 2 / mL, at least about 3.2cm 2 / mL, at least about 3.3cm 2 / mL, at least about 3.4cm 2 / mL, at least about 3.5cm 2 / mL, at least about 3.6cm 2 / mL, at least about 3.7cm 2 / mL, at least about 3.8cm 2 / mL, at least approximately 3.9cm 2 / mL, at least about 4.0cm 2 / mL, at least about 4.1cm 2 / mL, at least about 4.2cm 2 / mL, at least about 4.3cm 2 / mL, at least approximately 4.4cm 2 / mL, at least about 4.5cm 2 / mL, at least about 4.6cm 2 / mL, at least about 4.7cm 2 / mL, at least about 4.8cm 2 / mL, at least approximately 4.9cm 2 / mL or at least about 5.0cm 2 / mL. In some embodiments, the SA / V ratio of the container can be up to about 10.0 cm⁻¹. 2 / mL (for example, up to about 9.9cm) 2 / mL, up to approximately 9.8cm 2 / mL, up to approximately 9.7cm 2 / mL, up to approximately 9.6cm 2 / mL, up to approximately 9.5cm 2 / mL, up to approximately 9.4cm 2 / mL, up to approximately 9.3cm 2 / mL, up to approximately 9.2cm 2 / mL, up to approximately 9.1cm 2 / mL, up to approximately 9.0cm 2 / mL, up to approximately 8.9cm 2 / mL, up to approximately 8.8cm 2 / mL, up to approximately 8.7cm 2 / mL, up to approximately 8.6cm 2 / mL, up to approximately 8.5cm 2 / mL, up to approximately 8.4cm 2 / mL, up to approximately 8.3cm 2 / mL, up to approximately 8.2cm 2 / mL, up to approximately 8.1cm 2 / mL, up to approximately 8.0cm 2 / mL, up to approximately 7.9cm 2 / mL, up to approximately 7.8cm 2 / mL, up to approximately 7.7cm 2 / mL, up to approximately 7.6cm 2 / mL, up to approximately 7.5cm 2 / mL, up to approximately 7.4cm 2 / mL, up to approximately 7.3cm 2 / mL, up to approximately 7.2cm 2 / mL, up to approximately 7.1cm 2 / mL, up to approximately 6.9cm 2 / mL, up to approximately 6.8cm 2 / mL, up to approximately 6.7cm 2 / mL, up to approximately 6.6cm 2 / mL, up to approximately 6.5cm 2 / mL, up to approximately 6.4cm 2 / mL, up to approximately 6.3cm 2 / mL, up to approximately 6.2cm 2 / mL, up to approximately 6.1cm 2 / mL, up to approximately 6.0cm 2 / mL, up to approximately 5.9cm 2 / mL, up to approximately 5.8cm 2 / mL, up to approximately 5.7cm 2 / mL, up to approximately 5.6cm 2 / mL, up to approximately 5.5cm 2 / mL, up to approximately 5.4cm 2 / mL, up to approximately 5.3cm 2 / mL, up to approximately 5.2cm 2 / mL, up to approximately 5.1cm 2 / mL, up to approximately 5.0cm 2 / mL, up to approximately 4.9cm 2 / mL, up to approximately 4.8cm 2 / mL, up to approximately 4.7cm 2 / mL, up to approximately 4.6cm 2 / mL, up to approximately 4.5cm 2 / mL, up to approximately 4.4cm 2 / mL, up to approximately 4.3cm 2 / mL, up to approximately 4.2cm 2 / mL, up to approximately 4.1cm 2 / mL or at most about 4.0cm 2 / mL. In some embodiments, the SA / V ratio of the container can range from about 2.0 to about 10.0 cm⁻¹. 2 / mL (e.g., approximately 2.1cm) 2 / mL to approximately 9.9cm 2 / mL, approximately 2.2cm 2 / mL to approximately 9.8cm 2 / mL, approximately 2.3cm 2 / mL to approximately 9.7cm 2 / mL, approximately 2.4cm 2 / mL to approximately 9.6cm 2 / mL, approximately 2.5cm 2 / mL to approximately 9.5cm2 / mL, approximately 2.6cm 2 / mL to approximately 9.4cm 2 / mL, approximately 2.7cm 2 / mL to approximately 9.3cm 2 / mL, approximately 2.8cm 2 / mL to approximately 9.2cm 2 / mL, approximately 2.9cm 2 / mL to approximately 9.1cm 2 / mL, approximately 3.0cm 2 / mL to approximately 9.0cm 2 / mL, approximately 3.1cm 2 / mL to approximately 8.9cm 2 / mL, approximately 3.2cm 2 / mL to approximately 8.8cm 2 / mL, approximately 3.3cm 2 / mL to approximately 8.7cm 2 / mL, approximately 3.4cm 2 / mL to approximately 8.6cm 2 / mL, approximately 3.5cm 2 / mL to approximately 8.5cm 2 / mL, approximately 3.6cm 2 / mL to approximately 8.4cm 2 / mL, approximately 3.7cm 2 / mL to approximately 8.3cm 2 / mL, approximately 3.8cm 2 / mL to approximately 8.2cm 2 / mL, approximately 3.9cm 2 / mL to approximately 8.1cm 2 / mL, approximately 4.0cm 2 / mL to approximately 8.0cm 2 / mL, approximately 4.1cm 2 / mL to approximately 7.9cm 2 / mL, approximately 4.2cm 2 / mL to approximately 7.8cm 2 / mL, approximately 4.3cm 2 / mL to approximately 7.7cm 2 / mL, approximately 4.4cm 2 / mL to approximately 7.6cm 2 / mL, approximately 4.5cm 2 / mL to approximately 7.5cm 2 / mL, approximately 4.6cm 2 / mL to approximately 7.4cm 2 / mL, approximately 4.7cm 2 / mL to approximately 7.3cm 2 / mL, approximately 4.8cm 2 / mL to approximately 7.2cm 2 / mL, approximately 4.9cm 2 / mL to approximately 7.1cm 2 / mL, approximately 5.0cm 2 / mL to approximately 6.9cm 2 / mL, approximately 5.1cm 2 / mL to approximately 6.8cm 2 / mL, approximately 5.2cm 2 / mL to approximately 6.7cm 2 / mL, approximately 5.3cm 2 / mL to approximately 6.6cm 2 / mL, approximately 5.4cm 2 / mL to approximately 6.5cm 2 / mL, approximately 5.5cm 2 / mL to approximately 6.4cm 2 / mL, approximately 5.6cm 2 / mL to approximately 6.3cm 2 / mL, approximately 5.7cm 2 / mL to approximately 6.2cm 2 / mL or approximately 5.8cm 2 / mL to approximately 6.1cm 2 / mL).

[0226] Gas-permeable sealed containers (e.g., bags) or portions thereof may be made of one or more of a variety of gas-permeable materials. In some embodiments, gas-permeable bags may be made of one or more polymers, including fluoropolymers (such as polytetrafluoroethylene (PTFE) and perfluoroalkoxy (PFA) polymers), polyolefins (such as low-density polyethylene (LDPE), high-density polyethylene (HDPE)), fluorinated ethylene propylene (FEP), polystyrene, polyvinyl chloride (PVC), silicone, and any combination thereof.

[0227] In some embodiments, dried platelets or platelet derivatives (e.g., thrombus bodies) may undergo heat treatment. Heating may be performed at temperatures above about 25°C (e.g., greater than about 40°C, 50°C, 60°C, 70°C, 80°C, or higher). In some embodiments, heating may be performed between about 70°C and about 85°C (e.g., between about 75°C and about 85°C, or at about 75°C or 80°C). The temperature used for heating may be selected in conjunction with the length of time for which heating is to be performed. While any suitable time may be used, freeze-dried platelets are typically heated for at least 1 hour but not more than 36 hours. Thus, in embodiments, heating is performed 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, freeze-dried platelets may be heated for 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, or 30 hours. Non-limiting exemplary combinations include: heating dried platelets or platelet derivatives (e.g., thrombus bodies) at temperatures above 30°C for at least 30 minutes; heating dried platelets or platelet derivatives (e.g., thrombus bodies) at temperatures above 50°C for at least 10 hours; heating dried platelets or platelet derivatives (e.g., thrombus bodies) at temperatures above 75°C for at least 18 hours; and heating dried platelets or platelet derivatives (e.g., thrombus bodies) at 80°C for 24 hours. In some embodiments, heating may be performed in a sealed container (such as a capped vial). In some embodiments, the sealed container is evacuated before heating. It has been found that heat treatment steps, particularly in the presence of cryoprotectants (such as albumin or sucrose), can improve the stability and shelf life of lyophilized platelets. In fact, certain combinations of using serum albumin or sucrose and post-lyophilization heat treatment steps have yielded favorable results compared to those cryoprotectants without a heat treatment step. Cryoprotectants (e.g., sucrose) may be present in any suitable amount (e.g., about 3% to about 10% by mass or volume of platelets or platelet derivatives (e.g., thrombus bodies)).

[0228] In some cases, compositions containing platelets or platelet derivatives (e.g., thrombus bodies) can be rehydrated with water (e.g., sterile water for injection) at about room temperature for about 10 minutes. Generally, the rehydrated volume is approximately equal to the volume used to fill each vial of thrombus bodies before drying.

[0229] In some embodiments, the storage stability of platelets or platelet derivatives (e.g., thrombus bodies) prepared as disclosed herein is at least approximately equal to the storage stability of platelets before preparation.

[0230] In some embodiments, the method further includes cryopreserving platelets or platelet derivatives (e.g., with a formulation, such as the formulation described herein) prior to administration of platelets or platelet derivatives.

[0231] In some embodiments, the method further includes drying the composition containing platelets or platelet derivatives (e.g., with a formulation, such as those described herein) prior to the administration of platelets or platelet derivatives (e.g., thrombus bodies). In some embodiments, the method may further include heating the composition after the drying step. In some embodiments, the method may further include rehydrating the composition after a freeze-drying step or a heating step.

[0232] In some embodiments, the method further includes freeze-drying a composition containing platelets or platelet derivatives (e.g., a formulation, such as those described herein) prior to administration of platelets or platelet derivatives (e.g., thrombus bodies). In some embodiments, the method may further include heating the composition after the freeze-drying step. In some embodiments, the method may further include rehydrating the composition after the freeze-drying step or the heating step.

[0233] In some embodiments, the method further includes refrigerating the platelets, platelet derivatives, or thrombus bodies (e.g., with a formulation, such as the formulation described herein) prior to administration of the platelets, platelet derivatives, or thrombus bodies.

[0234] Storage conditions include, for example, standard room temperature storage (e.g., storage at temperatures ranging from about 20 to about 30°C) or refrigeration (e.g., storage at temperatures ranging from about 1 to about 10°C). In some embodiments, the method further includes cryopreserving, freeze-drying, thawing, rehydrating, and combinations thereof (e.g., with a formulation, such as those described herein) of the composition containing platelets or platelet derivatives (e.g., thrombus bodies) prior to application of platelets or platelet derivatives (e.g., thrombus bodies). For example, in some embodiments, the method further includes drying (e.g., freeze-drying) the composition containing platelets or platelet derivatives (e.g., with a formulation, such as those described herein) (e.g., to form thrombus bodies) prior to application of platelets or platelet derivatives (e.g., thrombus bodies). In some embodiments, the method may further include rehydrating the composition obtained from the drying step.

[0235] In some embodiments, this document provides a method for preparing a composition comprising platelets or platelet derivatives (e.g., thrombus bodies). The method may include diluting a platelet-containing starting material with about an equal weight (±10%) of a formulation (e.g., buffer A as provided in Example 1) to concentrate the platelets to approximately 2250 × 10⁻⁶. 3 Cells / μL (±250×10⁻⁶) 3 Then, wash with 2-4 percolation volumes (DV) of formulation (e.g., about 2 DV) to form a TFF-treated composition. The residual plasma percentage can be less than about 15% of the relative plasma (as determined by plasma protein content). After washing, if the cell concentration in the TFF-treated composition is not about 2000 × 10⁻⁶, 3 Cells / μL (±300×10⁻⁶) 3 If the cells are diluted with a formulation or concentrated to fall within that range, the method may further include lyophilizing the TFF-treated composition and subsequently treating the lyophilized composition containing platelets or platelet derivatives (e.g., thrombus bodies) at about 80°C for about 24 hours. In some embodiments, the method may further include a pathogen reduction step, for example, prior to diluting the starting material.

[0236] This document also provides compositions produced by any of the methods described herein.

[0237] In some embodiments, any composition provided herein may be prepared by the methods described herein.

[0238] The specific embodiments disclosed herein may be further limited by the use of language such as “consisting of” or “substantially consisting of” in the claims.

[0239] Exemplary embodiments

[0240] Example 1 is a composition comprising platelets or platelet derivatives and an aqueous medium, wherein the protein concentration of the aqueous medium is less than or equal to 50% of the protein concentration of donor plasma.

[0241] Example 2 is the composition described in Example 1, wherein the protein concentration of the aqueous medium is less than or equal to 30% of the protein concentration of the donor plasma.

[0242] Example 3 is the composition described in Example 1 or 2, wherein the aqueous medium has a concentration of human leukocyte antigen (HLA) class I antibody less than 30% of the concentration of human leukocyte antigen (HLA) class I antibody in donor plasma.

[0243] Example 4 is the composition of any one of Examples 1 to 3, wherein the aqueous medium has a concentration of human leukocyte antigen (HLA) class II antibody less than 30% of the concentration of human leukocyte antigen (HLA) class II antibody in donor plasma.

[0244] Example 5 is the composition of any one of Examples 1 to 4, wherein the aqueous medium has a concentration of human neutrophil antigen (HNA) antibody less than 30% of the concentration of HNA antibody in donor plasma.

[0245] Example 6 is the composition of any one of Examples 1 to 5, wherein the protein concentration is less than or equal to 10% of the protein concentration of donor plasma.

[0246] Example 7 is the composition of any one of Examples 1 to 6, wherein the aqueous medium has a concentration of human HLA class I antibodies that is less than 10% of the concentration of HLA class I antibodies in donor plasma.

[0247] Example 8 is the composition of any one of Examples 1 to 7, wherein the aqueous medium has a concentration of human HLA class II antibodies that is less than 10% of the concentration of HLA class II antibodies in donor plasma.

[0248] Example 9 is the composition of any one of Examples 1 to 8, wherein the aqueous medium has a concentration of human HNA antibody less than 10% of the concentration of HNA antibody in donor plasma.

[0249] Example 10 is the composition of any one of Examples 1 to 9, wherein the protein concentration is less than or equal to 5% of the protein concentration of donor plasma.

[0250] Example 11 is the composition of any one of Examples 1 to 10, wherein the aqueous medium has a concentration of human HLA class I antibodies that is less than 5% of the concentration of HLA class I antibodies in donor plasma.

[0251] Example 12 is the composition of any one of Examples 1 to 11, wherein the aqueous medium has a concentration of human HLA class II antibodies that is less than 5% of the concentration of HLA class II antibodies in donor plasma.

[0252] Example 13 is the composition of any one of Examples 1 to 12, wherein the aqueous medium has a concentration of human HNA antibody less than 5% of the concentration of HNA antibody in donor plasma.

[0253] Example 14 is the composition of any one of Examples 1 to 13, wherein the protein concentration is less than or equal to 3% of the protein concentration of donor plasma.

[0254] Example 15 is the composition of any one of Examples 1 to 14, wherein the aqueous medium has a concentration of human HLA class I antibodies that is less than 3% of the concentration of HLA class I antibodies in donor plasma.

[0255] Example 16 is the composition of any one of Examples 1 to 15, wherein the aqueous medium has a concentration of human HLA class II antibodies that is less than 3% of the concentration of HLA class II antibodies in donor plasma.

[0256] Example 17 is the composition of any one of Examples 1 to 16, wherein the aqueous medium has a concentration of human HNA antibody less than 3% of the concentration of HNA antibody in donor plasma.

[0257] Example 18 is the composition of any one of Examples 1 to 17, wherein the protein concentration is less than or equal to 1% of the protein concentration of donor plasma.

[0258] Example 19 is the composition of any one of Examples 1 to 18, wherein the aqueous medium has a concentration of human HLA class I antibodies less than 1% of the concentration of HLA class I antibodies in donor plasma.

[0259] Example 20 is the composition of any one of Examples 1 to 19, wherein the aqueous medium has a concentration of human HLA class II antibodies less than 1% of the concentration of HLA class II antibodies in donor plasma.

[0260] Example 21 is the composition of any one of Examples 1 to 20, wherein the aqueous medium has a concentration of human HNA antibody less than 1% of the concentration of HNA antibody in donor plasma.

[0261] Example 22 is the composition of any one of Examples 1 to 21, wherein the protein concentration is determined by absorbance at 280 nanometers (nm) and the path length is 0.5 cm.

[0262] Example 23 is the composition described in Example 22, wherein the absorbance at 280 nm is less than or equal to 1.7 AU.

[0263] Example 24 is the composition described in Example 22, wherein the absorbance at 280 nm is less than or equal to 1.66 AU.

[0264] Example 25 is the composition described in Example 22, wherein the absorbance at 280 nm is less than or equal to 1.6 AU.

[0265] Example 26 is the composition of any one of Examples 1 to 25, wherein the platelet count is at least 200 × 10⁻⁶. 3 Platelets / μL.

[0266] Example 27 is the composition described in Example 26, wherein the platelet count is at least 2250 × 10⁻⁶. 3 Platelets / μL.

[0267] Example 28 is the composition of any one of Examples 1 to 27, wherein the composition has a content of less than 0.2 × 10⁻⁶. 6 Red blood cell count per μL.

[0268] Example 29 is the composition of any one of Examples 1 to 27, wherein the composition further comprises red blood cells.

[0269] Example 30 is the composition described in Example 29, wherein the red blood cell count is less than 0.2 × 10⁻⁶. 6 Red blood cells / μL.

[0270] Example 31 is the composition of any one of Examples 1 to 30, wherein the composition is negative for HLA antibodies based on a test approved by a regulatory agency.

[0271] Example 32 is the composition of any one of Examples 1 to 31, wherein the composition is negative for HLA class II antibodies based on a test approved by a regulatory agency.

[0272] Example 33 is the composition of any one of Examples 1 to 32, wherein the composition is negative for HNA antibodies based on a test approved by a regulatory agency.

[0273] Example 34 is the composition of any one of Examples 1 to 33, wherein, as measured 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 is less than 5%.

[0274] Example 35 is the composition of any one of Examples 1 to 34, wherein, as measured 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 is less than 3%.

[0275] Example 36 is the composition of any one of Examples 1 to 35, wherein, as measured 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 is less than 1%.

[0276] Example 37 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HLA class I antibodies, as determined by flow cytometry using beads coated with class I HLA, is less than 5%.

[0277] Example 38 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HLA class I antibodies, as determined by flow cytometry using beads coated with class I HLA, is less than 3%.

[0278] Example 39 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HLA class I antibodies, as determined by flow cytometry using beads coated with class I HLA, is less than 1%.

[0279] Example 40 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HLA class II antibodies, as determined by flow cytometry using beads coated with class II HLA, is less than 5%.

[0280] Example 41 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HLA class II antibodies, as determined by flow cytometry using beads coated with class II HLA, is less than 3%.

[0281] Example 42 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HLA class II antibodies, as determined by flow cytometry using beads coated with class II HLA, is less than 1%.

[0282] Example 43 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HNA antibodies, as determined by flow cytometry using HNA-coated beads, is less than 5%.

[0283] Example 44 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HNA antibodies, as determined by flow cytometry using HNA-coated beads, is less than 3%.

[0284] Example 45 is the composition of any one of Examples 1 to 33, wherein the percentage of beads positive for HNA antibodies, as determined by flow cytometry using HNA-coated beads, is less than 1%.

[0285] Example 46 is the composition of any one of Examples 1 to 45, wherein the aqueous medium further comprises a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

[0286] Example 47 is the composition described in Example 46, wherein the buffer is 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES).

[0287] Example 48 is the composition of any one of Examples 46 to 47, wherein the base is sodium bicarbonate.

[0288] Example 49 is the composition of any one of Examples 46 to 48, wherein the loading agent is a monosaccharide, a polysaccharide, or a combination thereof.

[0289] Example 50 is the composition described in Example 49, wherein the monosaccharide is selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose and xylose.

[0290] Example 51 is the composition described in Example 49, wherein the monosaccharide is trehalose.

[0291] Example 52 is the composition of any one of Examples 49 to 51, wherein the polysaccharide is a polysaccharide.

[0292] Example 53 is the composition of any one of Examples 46 to 52, wherein the salt is sodium chloride, potassium chloride, or a combination thereof.

[0293] Example 54 is a composition of any one of Examples 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.

[0294] Example 55 is the composition of any one of Examples 1 to 54, wherein the composition is prepared by a method comprising tangential flow filtration (TFF) of a platelet-containing starting material, centrifugation of a platelet-containing starting material, or a combination thereof.

[0295] Example 56 is the composition described in Example 55, wherein, compared to similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, is reduced by at least 50%.

[0296] Example 57 is the composition described in Example 55, wherein, compared to similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, is reduced by at least 75%.

[0297] Example 58 is the composition described in Example 55, wherein, compared to similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, is reduced by at least 90%.

[0298] Example 59 is the composition described in Example 55, wherein, compared to similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, is reduced by at least 95%.

[0299] Example 60 is the composition of any one of Examples 55 to 59, wherein the starting materials are:

[0300] a) Positive for HLA class I antibodies based on a test approved by the regulatory agency;

[0301] b) Positive for HLA class II antibodies based on a test approved by the regulatory agency;

[0302] c) A positive result for HNA antibodies based on a test approved by a regulatory agency; or

[0303] d) one or more of a), b) or c).

[0304] Example 61 is the composition of any one of Examples 55 to 60, wherein the starting material has a protein concentration of about 60 to about 80 mg / ml.

[0305] Example 62 is the composition of any one of Examples 55 to 61, wherein the starting material comprises donor blood products.

[0306] Example 63 is the composition described in Example 62, wherein the donor blood product is a collected donor blood product.

[0307] Example 64 is the composition of any one of Examples 62 to 63, wherein the starting material comprises donor-harvested material.

[0308] Example 65 is the composition of any one of Examples 55 to 64, wherein the TFF comprises a concentrate.

[0309] Example 66 is the composition of any one of Examples 55 to 65, wherein the TFF comprises percolation.

[0310] Example 67 is the composition described in Example 66, wherein percolation includes percolation with at least two percolation volumes.

[0311] Example 68 is the composition of any one of Examples 55 to 67, wherein the TFF comprises buffer exchange.

[0312] Example 69 is the composition of any one of Examples 55 to 68, wherein a membrane with a pore size of about 0.2 μm to about 1 μm is used for TFF.

[0313] Example 70 is the composition of any one of Examples 55 to 68, wherein a membrane with a pore size of about 0.2 μm to about 0.45 μm is used for TFF.

[0314] Example 71 is the composition of any one of Examples 55 to 70, wherein the TFF is performed at a temperature of about 20°C to about 37°C.

[0315] Example 72 is the composition of any one of Examples 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 is less than or equal to 50% of the absorbance of the starting material at 280 nm.

[0316] Example 73 is the composition of any one of Examples 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 is less than or equal to 30% of the absorbance of the starting material at 280 nm.

[0317] Example 74 is the composition of any one of Examples 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 is less than or equal to 10% of the absorbance of the starting material at 280 nm.

[0318] Example 75 is the composition of any one of Examples 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 is less than or equal to 5% of the absorbance of the starting material at 280 nm.

[0319] Example 76 is the composition of any one of Examples 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 is less than or equal to 3% of the absorbance of the starting material at 280 nm.

[0320] Example 77 is the composition of any one of Examples 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 is less than or equal to 1% of the absorbance of the starting material at 280 nm.

[0321] Example 78 is the composition of any one of Examples 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 is less than or equal to 1.70 AU.

[0322] Example 79 is the composition of any one of Examples 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 is less than or equal to 1.66 AU.

[0323] Example 80 is the composition of any one of Examples 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 is less than or equal to 1.60 AU.

[0324] Example 81 is the composition of any one of Examples 55 to 80, wherein TFF is performed until the platelet concentration is at least about 2000 × 10⁻⁶. 3 Platelets / μL.

[0325] Example 82 is the composition of any one of Examples 55 to 80, wherein TFF is performed until the platelet concentration is at least about 2250 × 10⁻⁶. 3 Platelets / μL.

[0326] Example 83 is the composition of any one of Examples 55 to 82, wherein the TFF comprises exchanging a buffer solution for a formulation comprising a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

[0327] Example 84 is the composition described in Example 83, wherein the buffer is 4-(2-hydroxyethyl)-1-piperazine ethane sulfonic acid (HEPES).

[0328] Example 85 is the composition of any one of Examples 83 to 84, wherein the base is sodium bicarbonate.

[0329] Example 86 is the composition of any one of Examples 83 to 85, wherein the loading agent is a monosaccharide, a polysaccharide, or a combination thereof.

[0330] Example 87 is the composition described in Example 86, wherein the monosaccharide is selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose and xylose.

[0331] Example 88 is the composition described in Example 86, wherein the monosaccharide is trehalose.

[0332] Example 89 is the composition of any one of Examples 86 to 88, wherein the polysaccharide is a polysaccharide.

[0333] Example 90 is the composition of any one of Examples 83 to 89, wherein the salt is sodium chloride, potassium chloride, or a combination thereof.

[0334] Example 91 is a composition of any one of Examples 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.

[0335] Example 92 is the composition of any one of Examples 55 to 91, wherein centrifugation includes centrifugation at 1400×g to about 1550×g.

[0336] Example 93 is the composition of any one of Examples 55 to 91, wherein centrifugation includes centrifugation at 1450×g to about 1500×g.

[0337] Example 94 is the composition of any one of Examples 55 to 91, wherein the method does not include centrifuging the composition containing platelets.

[0338] Example 95 is the composition of any one of Examples 1 to 94, wherein the composition contains less than 5.0% (in terms of scattering intensity) of particles.

[0339] Example 96 is the composition of any one of Examples 1 to 94, wherein the composition contains less than 4.5% (in terms of scattering intensity) of particles.

[0340] Example 97 is the composition of any one of Examples 1 to 94, wherein the composition contains less than 4.0% (in terms of scattering intensity) of particles.

[0341] Example 98 is a composition of any one of Examples 1 to 94, wherein the composition contains less than 3.5% (in terms of scattering intensity) of particles.

[0342] Example 99 is the composition of any one of Examples 1 to 98, wherein the platelets or platelet derivatives have a CD41 positive percentage of at least 55%.

[0343] Example 100 is the composition of any one of Examples 1 to 98, wherein the platelets or platelet derivatives have a CD41 positive percentage of at least 60%.

[0344] Example 101 is the composition of any one of Examples 1 to 98, wherein the platelets or platelet derivatives have a CD41 positive percentage of at least 65%.

[0345] Example 102 is the composition of any one of Examples 1 to 101, wherein the platelets or platelet derivatives have a CD42 positive percentage of at least 65%.

[0346] Example 103 is the composition of any one of Examples 1 to 101, wherein the platelets or platelet derivatives have a CD42 positive percentage of at least 80%.

[0347] Example 104 is the composition of any one of Examples 1 to 101, wherein the platelets or platelet derivatives have a CD42 positive percentage of at least 90%.

[0348] Example 105 is the composition of any one of Examples 1 to 104, wherein the platelet or platelet derivative retains at least about 10% of the lactate dehydrogenase activity of the donor apheresis platelet.

[0349] Example 106 is the composition of any one of Examples 1 to 104, wherein the platelet or platelet derivative retains at least about 15% of the lactate dehydrogenase activity of the donor apheresis platelet.

[0350] Example 107 is the composition of any one of Examples 1 to 104, wherein the platelet or platelet derivative retains at least about 20% of the lactate dehydrogenase activity of the donor apheresis platelet.

[0351] Example 108 is the composition of any one of Examples 1 to 107, wherein the platelets or platelet derivatives have at least 25% annexin V positive percentage.

[0352] Example 109 is the composition of any one of Examples 1 to 107, wherein the platelets or platelet derivatives have at least 50% annexin V positive percentage.

[0353] Example 110 is the composition of any one of Examples 1 to 107, wherein the platelets or platelet derivatives have at least 70% annexin V positive percentage.

[0354] Example 111 is the composition of any one of Examples 1 to 110, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 8%.

[0355] Example 112 is the composition of any one of Examples 1 to 110, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 10%.

[0356] Example 113 is the composition of any one of Examples 1 to 110, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 15%.

[0357] Example 114 is the composition of any one of Examples 1 to 110, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 20%.

[0358] Example 115 is the composition of any one of Examples 1 to 114, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 10%.

[0359] Example 116 is the composition of any one of Examples 1 to 114, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 50%.

[0360] Example 117 is the composition of any one of Examples 1 to 114, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 80%.

[0361] Example 118 is the composition of any one of Examples 1 to 114, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 90%.

[0362] Example 119 is the composition of any one of Examples 1 to 118, wherein the platelet or platelet derivative has fibrinogen that is bound to the cell membrane.

[0363] Example 120 is the composition of any one of Examples 1 to 119, wherein the aqueous medium has a lactate concentration of less than 2.0 mmol / L.

[0364] Example 121 is the composition of any one of Examples 1 to 119, wherein the aqueous medium has a lactate concentration of less than 1.5 mmol / L.

[0365] Example 122 is the composition of any one of Examples 1 to 121, wherein the aqueous medium has a lactate concentration of about 0.4 to about 1.3 mmol / L.

[0366] Example 123 is the composition of any one of Examples 1 to 121, wherein the aqueous medium has a lactate concentration of about 0.5 to about 1.0 mmol / L.

[0367] Example 124 is the composition of any one of Examples 1 to 123, wherein the platelet derivative comprises thrombus bodies.

[0368] Example 125 is a composition of any one of Examples 1 or 22 to 124, wherein the protein concentration is about 5% to about 50% of the protein concentration of donor plasma.

[0369] Example 126 is a composition of any one of Examples 1 to 5 or 22 to 125, wherein the protein concentration is about 5% to about 30% of the protein concentration of donor plasma.

[0370] Example 127 is a composition of any one of Examples 1 to 5 or 22 to 126, wherein the protein concentration is about 5% to about 15% of the protein concentration of donor plasma.

[0371] Example 128 is a composition of any one of Examples 1 to 9 or 22 to 127, wherein the protein concentration is about 8% to about 10% of the protein concentration of donor plasma.

[0372] Example 129 is a composition of any one of Examples 1 to 9 or 22 to 128, wherein the protein concentration is about 7% to about 10% of the protein concentration of donor plasma.

[0373] Example 130 is the composition of any one of Examples 1 to 129, wherein, when in the presence of a reagent containing tissue factor and phospholipids, platelets or platelet derivatives are present at approximately 4.8 × 10⁻⁶. 3 At a concentration of 1 particle / μL, it produces a thrombin peak height (TPH) of at least 25 nM.

[0374] Example 131 is the composition of any one of Examples 1 to 129, wherein, when in the presence of a reagent containing tissue factor and phospholipids, platelets or platelet derivatives are present at approximately 4.8 × 10⁻⁶. 3 At a concentration of 1 particle / μL, it produces a thrombin peak height (TPH) of at least 50 nM.

[0375] Example 132 is the composition of any one of Examples 1 to 129, wherein the platelets or platelet derivatives have a concentration of 10 6Each particle has an efficacy of at least 1.5 thrombin generating power units (TGPUs).

[0376] Example 133 is the composition of any one of Examples 1 to 129, wherein the platelets or platelet derivatives are at least about 70 × 10⁻⁶. 3 At a concentration of particles / μL, an occlusion time of less than 14 minutes was observed in the Total Thrombosis Analysis System (T-TAS).

[0377] Example 134 is the composition of any one of Examples 1 to 129, wherein the platelets or platelet derivatives are at least about 70 × 10⁻⁶. 3 At a concentration of particles / μL, it produces an occlusion time of less than 12 minutes in Total Thrombosis Analysis System (T-TAS) measurements.

[0378] Example 135 is a method for preparing a composition comprising platelets or platelet derivatives and an aqueous medium, the method comprising:

[0379] Compositions comprising platelets or platelet derivatives and an aqueous medium are prepared by tangential flow filtration (TFF) of starting materials containing platelets, diluted starting materials containing platelets, concentrated platelet compositions, or combinations thereof.

[0380] The protein concentration of the aqueous medium is less than or equal to 50% of the protein concentration of the donor plasma.

[0381] Example 136 is the method described in Example 125, wherein the starting materials are:

[0382] a) Positive for HLA class I antibodies based on a test approved by the regulatory agency;

[0383] b) Positive for HLA class II antibodies based on a test approved by the regulatory agency;

[0384] c) A positive result for HNA antibodies based on a test approved by a regulatory agency; or

[0385] d) one or more of a), b) and c).

[0386] Example 137 is the method of any one of Examples 135 to 136, wherein the starting material has a protein concentration of about 60 to about 80 mg / mL.

[0387] Example 138 is a method described in any one of Examples 135 to 137, wherein the starting material includes donor blood products.

[0388] Example 139 is the method described in Example 138, wherein the donor blood product is a pooled donor blood product.

[0389] Example 140 is the method of any one of Examples 135 to 139, wherein the starting material includes donor-harvested material.

[0390] Example 141 is the method of any one of Examples 135 to 140, wherein the TFF includes a concentration.

[0391] Example 142 is the method of any one of Examples 135 to 141, wherein TFF includes percolation.

[0392] Example 143 is the method described in Example 142, wherein the percolation includes percolation having at least two percolation volumes.

[0393] Example 144 is the method of any one of Examples 135 to 143, wherein the TFF includes buffer exchange.

[0394] Example 145 is the method of any one of Examples 135 to 144, wherein a membrane with a pore size of about 0.2 μm to about 1 μm is used for TFF.

[0395] Example 146 is the method of any one of Examples 135 to 145, wherein a membrane with a pore size of about 0.2 μm to about 0.45 μm is used for TFF.

[0396] Example 147 is the method of any one of Examples 135 to 146, wherein TFF is performed at a temperature of about 20°C to about 37°C.

[0397] Example 148 is the method of any one of Examples 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 is less than or equal to 50% of the absorbance of the starting material at 280 nm.

[0398] Example 149 is the method of any one of Examples 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 is less than or equal to 30% of the absorbance of the starting material at 280 nm.

[0399] Example 150 is the method of any one of Examples 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 is less than or equal to 10% of the absorbance of the starting material at 280 nm.

[0400] Example 151 is the method of any one of Examples 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 is less than or equal to 5% of the absorbance of the starting material at 280 nm.

[0401] Example 152 is the method of any one of Examples 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 is less than or equal to 3% of the absorbance of the starting material at 280 nm.

[0402] Example 153 is the method of any one of Examples 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 is less than or equal to 1% of the absorbance of the starting material at 280 nm.

[0403] Example 154 is the method of any one of Examples 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 is less than or equal to 1.70 AU.

[0404] Example 155 is the method of any one of Examples 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 is less than or equal to 1.66 AU.

[0405] Example 156 is the method of any one of Examples 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 is less than or equal to 1.60 AU.

[0406] Example 157 is the method of any one of Examples 135 to 156, wherein TFF is performed until the platelet concentration is at least about 2000 × 10⁻⁶. 3 Platelets / μL.

[0407] Example 158 is the method of any one of Examples 135 to 156, wherein TFF is performed until the platelet concentration is at least about 2250 × 10⁻⁶. 3 Platelets / μL.

[0408] Example 159 is the method of any one of Examples 135 to 158, wherein TFF comprises percolation with a formulation comprising a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

[0409] Example 160 is the method of any one of Examples 135 to 159, wherein TFF comprises exchanging a buffer solution for a formulation comprising a buffer, a base, a loading agent, optionally a salt, and optionally at least one organic solvent.

[0410] Example 161 is the method of any one of Examples 149 to 160, wherein the buffer is 4-(2-hydroxyethyl)-1-piperazine ethanesulfonic acid (HEPES).

[0411] Example 162 is the method of any one of Examples 149 to 161, wherein the base is sodium bicarbonate.

[0412] Example 163 is the method of any one of Examples 149 to 162, wherein the loading agent is a monosaccharide, a polysaccharide, or a combination thereof.

[0413] Example 164 is the method described in Example 163, wherein the monosaccharide is selected from the group consisting of sucrose, maltose, trehalose, glucose, mannose, xylose, and combinations thereof.

[0414] Example 165 is the method described in Example 163, wherein the monosaccharide is trehalose.

[0415] Example 166 is the method of any one of Examples 163 to 165, wherein the polysaccharide is a sucrose.

[0416] Example 167 is the method of any one of Examples 159 to 166, wherein the salt is sodium chloride, potassium chloride, or a combination thereof.

[0417] Example 168 is the method of any one of Examples 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.

[0418] Example 169 is the method of any one of Examples 135 to 168, wherein the method does not include centrifuging starting material containing platelets, diluted starting material containing platelets, concentrated platelet composition or a combination thereof.

[0419] Example 170 is the method of any one of Examples 135 to 169, wherein the method does not include centrifuging a composition containing platelets.

[0420] Example 171 is the method of any one of Examples 135 to 170, wherein, compared with similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, is reduced by at least 50%.

[0421] Example 172 is the method of any one of Examples 135 to 170, wherein, compared with similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, is reduced by at least 75%.

[0422] Example 173 is the method of any one of Examples 135 to 170, wherein, compared with similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, is reduced by at least 90%.

[0423] Example 174 is the method of any one of Examples 135 to 170, wherein, compared with similar compositions not prepared by methods including tangential flow filtration of the blood product composition, centrifugation of the blood product composition, 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, as measured by flow cytometry using beads coated with class I HLA, class II HLA, or HNA, is reduced by at least 95%.

[0424] Example 175 is the method of any one of Examples 135 to 174, wherein the protein concentration is less than or equal to 30% of the protein concentration of the donor plasma.

[0425] Example 176 is the method of any one of Examples 135 to 175, wherein the aqueous medium has a concentration of human leukocyte antigen (HLA) class I antibody less than 30% of the concentration of human leukocyte antigen (HLA) class I antibody in donor plasma.

[0426] Example 177 is the method of any one of Examples 135 to 176, wherein the aqueous medium has a concentration of human leukocyte antigen (HLA) class II antibody less than 30% of the concentration of human leukocyte antigen (HLA) class II antibody in donor plasma.

[0427] Example 178 is the method of any one of Examples 135 to 17, wherein the aqueous medium has a concentration of human neutrophil antigen (HNA) antibody less than 30% of the concentration of HNA antibody in donor plasma.

[0428] Example 179 is the method of any one of Examples 135 to 178, wherein the protein concentration is less than or equal to 10% of the protein concentration of the donor plasma.

[0429] Example 180 is the method of any one of Examples 135 to 179, wherein the aqueous medium has a concentration of human HLA class I antibodies that is less than 10% of the concentration of HLA class I antibodies in donor plasma.

[0430] Example 181 is the method of any one of Examples 135 to 180, wherein the aqueous medium has a concentration of human HLA class II antibodies that is less than 10% of the concentration of HLA class II antibodies in donor plasma.

[0431] Example 182 is the method of any one of Examples 135 to 181, wherein the aqueous medium has a concentration of human HNA antibody less than 10% of the concentration of HNA antibody in donor plasma.

[0432] Example 183 is the method of any one of Examples 135 to 182, wherein the protein concentration is less than or equal to 5% of the protein concentration of the donor plasma.

[0433] Example 184 is the method of any one of Examples 135 to 183, wherein the aqueous medium has a concentration of human HLA class I antibodies that is less than 5% of the concentration of HLA class I antibodies in donor plasma.

[0434] Example 185 is the method of any one of Examples 135 to 184, wherein the aqueous medium has a concentration of human HLA class II antibodies that is less than 5% of the concentration of HLA class II antibodies in donor plasma.

[0435] Example 186 is the method of any one of Examples 135 to 185, wherein the aqueous medium has a concentration of human HNA antibody less than 5% of the concentration of HNA antibody in donor plasma.

[0436] Example 187 is the method of any one of Examples 135 to 186, wherein the protein concentration is less than or equal to 3% of the protein concentration of the donor plasma.

[0437] Example 188 is the method described in any one of Examples 135 to 187, wherein the aqueous medium has a concentration of human HLA class I antibodies below 3% of the concentration of HLA class I antibodies in donor plasma.

[0438] Example 189 is the method of any one of Examples 135 to 188, wherein the aqueous medium has a concentration of human HLA class II antibodies that is less than 3% of the concentration of HLA class II antibodies in donor plasma.

[0439] Example 190 is the method of any one of Examples 135 to 189, wherein the aqueous medium has a concentration of human HNA antibody less than 3% of the concentration of HNA antibody in donor plasma.

[0440] Example 191 is the method of any one of Examples 135 to 190, wherein the protein concentration is less than or equal to 1% of the protein concentration of the donor plasma.

[0441] Example 192 is the method of any one of Examples 135 to 191, wherein the aqueous medium has a concentration of human HLA class I antibodies less than 1% of the concentration of HLA class I antibodies in donor plasma.

[0442] Example 193 is the method of any one of Examples 135 to 192, wherein the aqueous medium has a concentration of human HLA class II antibodies less than 1% of the concentration of HLA class II antibodies in donor plasma.

[0443] Example 194 is the method of any one of Examples 135 to 193, wherein the aqueous medium has a concentration of human HNA antibody less than 1% of the concentration of HNA antibody in donor plasma.

[0444] Example 195 is the method of any one of Examples 135 to 194, wherein the composition is negative for HLA antibodies based on a test approved by a regulatory agency.

[0445] Example 196 is the method of any one of Examples 135 to 195, wherein the composition is negative for HLA class II antibodies based on a test approved by a regulatory agency.

[0446] Example 197 is the method of any one of Examples 135 to 196, wherein the composition is negative for HNA antibodies based on a test approved by a regulatory agency.

[0447] Example 198 is the method of any one of Examples 135 to 197, wherein, as measured 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 is less than 5%.

[0448] Example 199 is the method of any one of Examples 135 to 198, wherein, when measured 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 is less than 3%.

[0449] Example 200 is the method of any one of Examples 135 to 199, wherein, as measured 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 is less than 1%.

[0450] Example 201 is the method of any one of Examples 135 to 200, wherein the percentage of beads positive for HLA class I antibodies, as determined by flow cytometry using beads coated with class I HLA, is less than 5%.

[0451] Example 202 is the method of any one of Examples 135 to 201, wherein the percentage of beads positive for HLA class I antibodies, as determined by flow cytometry using beads coated with class I HLA, is less than 3%.

[0452] Example 203 is the method of any one of Examples 135 to 202, wherein the percentage of beads positive for HLA class I antibodies, as determined by flow cytometry using beads coated with class I HLA, is less than 1%.

[0453] Example 204 is the method of any one of Examples 135 to 203, wherein the percentage of beads positive for HLA class II antibodies, as determined by flow cytometry using beads coated with class II HLA, is less than 5%.

[0454] Example 205 is the method of any one of Examples 135 to 204, wherein the percentage of beads positive for HLA class II antibodies, as determined by flow cytometry using beads coated with class II HLA, is less than 3%.

[0455] Example 206 is the method of any one of Examples 135 to 205, wherein the percentage of beads positive for HLA class II antibodies, as determined by flow cytometry using beads coated with class II HLA, is less than 1%.

[0456] Example 207 is the method of any one of Examples 135 to 206, wherein the percentage of beads positive for HNA antibodies, as determined by flow cytometry using HNA-coated beads, is less than 5%.

[0457] Example 208 is the method of any one of Examples 135 to 207, wherein the percentage of beads positive for HNA antibodies, as determined by flow cytometry using HNA-coated beads, is less than 3%.

[0458] Example 209 is the method of any one of Examples 135 to 208, wherein the percentage of beads positive for HNA antibodies, as determined by flow cytometry using HNA-coated beads, is less than 1%.

[0459] Example 210 is the method of any one of Examples 135 to 209, wherein the composition comprises less than 5.0% (in terms of scattering intensity) of particles.

[0460] Example 211 is the method of any one of Examples 135 to 209, wherein the composition contains less than 4.5% (in terms of scattering intensity) of particles.

[0461] Example 212 is the method of any one of Examples 135 to 209, wherein the composition contains less than 4.0% (in terms of scattering intensity) of particles.

[0462] Example 213 is the method of any one of Examples 135 to 209, wherein the composition comprises less than 3.5% (in terms of scattering intensity) of particles.

[0463] Example 214 is the method of any one of Examples 135 to 213, wherein the platelets or platelet derivatives have a CD41 positive percentage of at least 55%.

[0464] Example 215 is the method of any one of Examples 135 to 213, wherein the platelets or platelet derivatives have a CD41 positive percentage of at least 60%.

[0465] Example 216 is the method of any one of Examples 135 to 213, wherein the platelets or platelet derivatives have a CD41 positive percentage of at least 65%.

[0466] Example 217 is the method of any one of Examples 135 to 216, wherein the platelets or platelet derivatives have a CD42 positive percentage of at least 65%.

[0467] Example 218 is the method of any one of Examples 135 to 216, wherein the platelets or platelet derivatives have a CD42 positive percentage of at least 80%.

[0468] Example 219 is the method of any one of Examples 135 to 216, wherein the platelets or platelet derivatives have a CD42 positive percentage of at least 90%.

[0469] Example 220 is the method of any one of Examples 135 to 219, wherein the platelet or platelet derivative retains at least about 10% of the lactate dehydrogenase activity of the donor apheresis platelet.

[0470] Example 221 is the method of any one of Examples 135 to 219, wherein the platelet or platelet derivative retains at least about 15% of the lactate dehydrogenase activity of the donor apheresis platelet.

[0471] Example 222 is the method of any one of Examples 135 to 219, wherein the platelet or platelet derivative retains at least about 20% of the lactate dehydrogenase activity of the donor apheresis platelet.

[0472] Example 223 is the method of any one of Examples 135 to 222, wherein the platelets or platelet derivatives have at least a 25% annexin V positive percentage.

[0473] Example 224 is the method of any one of Examples 135 to 222, wherein the platelets or platelet derivatives have at least a 50% annexin V positive percentage.

[0474] Example 225 is the method of any one of Examples 135 to 222, wherein the platelets or platelet derivatives have at least 75% annexin V positive percentage.

[0475] Example 226 is the method of any one of Examples 135 to 225, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 8%.

[0476] Example 227 is the method of any one of Examples 135 to 225, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 10%.

[0477] Example 228 is the method of any one of Examples 135 to 225, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 15%.

[0478] Example 229 is the method of any one of Examples 135 to 225, wherein the platelets or platelet derivatives have a CD47 positive percentage of at least 20%.

[0479] Example 230 is the method of any one of Examples 135 to 229, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 10%.

[0480] Example 231 is the method of any one of Examples 135 to 229, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 50%.

[0481] Example 232 is the method of any one of Examples 135 to 229, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 80%.

[0482] Example 233 is the method of any one of Examples 135 to 229, wherein the platelets or platelet derivatives have a CD62 positive percentage of at least 90%.

[0483] Example 234 is the method of any one of Examples 135 to 233, wherein the platelet or platelet derivative has fibrinogen that binds to the cell membrane.

[0484] Example 235 is the method of any one of Examples 135 to 234, wherein the aqueous medium has a lactate concentration of less than 2.0 mmol / L.

[0485] Example 236 is the method of any one of Examples 135 to 234, wherein the aqueous medium has a lactate concentration of less than 1.5 mmol / L.

[0486] Example 237 is the method of any one of Examples 135 to 236, wherein the aqueous medium has a lactate concentration of about 0.4 to about 1.3 mmol / L.

[0487] Example 238 is the method of any one of Examples 135 to 236, wherein the aqueous medium has a lactate concentration of about 0.5 to about 1.0 mmol / L.

[0488] Example 239 is the method of any one of Examples 135 to 238, wherein the platelet derivative comprises a thrombus body.

[0489] Example 240 is the method of any one of Examples 135 to 239, further comprising a pathogen reduction step.

[0490] Example 241 is the method described in Example 240, wherein the pathogen reduction step is performed before TFF.

[0491] Example 242 is the method of any one of Examples 135 to 241, further comprising freeze-drying a composition containing platelets or platelet derivatives.

[0492] Example 243 is the method of any one of Examples 135 to 241, further comprising cryopreservation of a composition containing platelets or platelet derivatives.

[0493] Example 244 is the method of any one of Examples 135 to 243, further comprising heat treatment of a composition containing platelets or platelet derivatives.

[0494] Example 245 is the method of any one of Examples 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 the donor plasma.

[0495] Example 246 is the method of any one of Examples 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 the donor plasma.

[0496] Example 247 is the method of any one of Examples 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 the donor plasma.

[0497] Example 248 is the method of any one of Examples 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 the donor plasma.

[0498] Example 249 is the method of any one of Examples 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 the donor plasma.

[0499] Example 250 is the method of any one of Examples 135 to 249, wherein when a reagent containing tissue factor and phospholipids is present, platelets or platelet derivatives are present at approximately 4.8 × 10⁻⁶. 3 At a concentration of 1 particle / μL, it produces a thrombin peak height (TPH) of at least 25 nM.

[0500] Example 251 is the method of any one of Examples 135 to 249, wherein when a reagent containing tissue factor and phospholipids is present, platelets or platelet derivatives are present at approximately 4.8 × 10⁻⁶.3 At a concentration of 1 particle / μL, it produces a thrombin peak height (TPH) of at least 50 nM.

[0501] Example 252 is the method of any one of Examples 135 to 249, wherein platelets or platelet derivatives have a concentration of 10 6 Each particle has an efficacy of at least 1.5 thrombin generating power units (TGPUs).

[0502] Example 253 is the method of any one of Examples 135 to 249, wherein the platelets or platelet derivatives are at least about 70 × 10⁻⁶. 3 At a concentration of particles / μL, an occlusion time of less than 14 minutes was observed in the Total Thrombosis Analysis System (T-TAS).

[0503] Example 254 is the method of any one of Examples 135 to 249, wherein the platelets or platelet derivatives are at least about 70 × 10⁻⁶. 3 At a concentration of particles / μL, it produces an occlusion time of less than 12 minutes in Total Thrombosis Analysis System (T-TAS) measurements.

[0504] Example 255 is a composition comprising platelets or platelet derivatives and an aqueous medium prepared by any one of Examples 135 to 254.

[0505] Example 256 is a method for preparing lyophilized platelets, comprising:

[0506] a) Prepare a composition comprising platelets and an aqueous medium using the method of any one of Examples 135 to 254; and

[0507] b) Freeze-drying a composition containing platelets and an aqueous medium.

[0508] Example 257 is a composition comprising lyophilized platelets prepared by the method described in Example 235.

[0509] Example 258 is a method for preparing a composition comprising platelets or platelet derivatives and an aqueous medium, the method comprising:

[0510] Dilute the starting material containing platelets to form diluted starting material;

[0511] The concentrated and diluted starting material resulted in platelets with a size of approximately 2250 × 10⁻⁶. 3 Cells / μL (±250×10⁻⁶) 3 The concentration of ) to form a concentrated platelet composition; and

[0512] The concentrated platelet composition was washed with at least two filtration volumes (DV) of the formulation to form a TFF-treated composition.

[0513] Example 259 is the method of Example 258, wherein dilution includes dilution with approximately equal weight (±10%) of the formulation.

[0514] Example 260 is the method of any one of Examples 258 to 259, further comprising a pathogen reduction step.

[0515] Example 261 is the method of Example 260, wherein the pathogen reduction step occurs before the dilution of the starting material.

[0516] Example 262 is the method of any one of Examples 258 to 261, wherein the percentage of residual plasma is less than or equal to about 15% of the relative plasma (as determined by plasma protein content).

[0517] Example 263 is the method of any one of Examples 258 to 262, wherein after washing, if the concentration of cells in the TFF-treated composition is not about 2000 × 10⁻⁶ 3 Cells / μL (±300×10⁻⁶) 3 If the preparation is diluted, it may be concentrated to fall within that range.

[0518] Example 264 is the method of any one of Examples 258 to 263, further comprising lyophilizing the TFF-treated composition to form a lyophilized composition.

[0519] Example 265 is the method of Example 264, further comprising treating the lyophilized composition at about 80°C for about 24 hours.

[0520] Example 266 is a composition containing platelets or platelet derivatives prepared by any one of Examples 258 to 265.

[0521] Example 267 is a method of treating a coagulation-related disease or condition in a subject who requires it, comprising administering to the subject a therapeutically effective amount of any one of Examples 1 to 134, 255 or 266.

[0522] Example 268 is the method of Example 267, wherein the coagulation-related disease or condition is selected from the group consisting of: Von Willebrand disease, hemophilia, thromboembolism, thrombocytopenia, thrombocytopenic purpura, trauma, or a combination thereof.

[0523] Example 269 is a method for treating coagulation-related diseases or conditions in subjects who require it, comprising administering to the subject a therapeutically effective amount of a composition prepared by any one of Examples 135 to 254.

[0524] Example 270 is the method described in Example 269, wherein the coagulation-related disease or condition is selected from the group consisting of: von Willebrand disease, hemophilia, thromboembolism, thrombocytopenia, thrombocytopenic purpura, trauma, or a combination thereof.

[0525] Example 271 is a method for treating a coagulation-related disease or condition in a subject who requires it, the method comprising administering to the subject a therapeutically effective amount of a composition prepared by any one of Examples 258 to 266.

[0526] Example 272 is the method of Example 271, wherein the coagulation-related disease or condition is selected from the group consisting of: von Willebrand disease, hemophilia, thromboembolism, thrombocytopenia, thrombocytopenic purpura, trauma, or a combination thereof.

[0527] Example

[0528] Example 1. Tangential Flow Filtering (TFF)

[0529] Platelets collected via apheresis are subjected to tangential flow filtration according to standard operating procedures, including the following steps: platelet dilution, platelet concentration, and platelet washing.

[0530] Initially, platelet donor units are collected into a shared container. The platelets are initially diluted with or without acidified wash buffer (e.g., control buffer) to minimize platelet activation during processing. Platelets can undergo two processing pathways: first washing with control buffer until the desired residual fraction (e.g., donor plasma) is achieved, followed by concentration to the final product concentration; or first concentrating the platelets to the final product concentration, followed by washing with control buffer until the desired residual fraction (e.g., donor plasma) is achieved. The TFF-treated platelets are then packaged into vials, lyophilized, and heat-treated.

[0531] One specific solution is as follows.

[0532] For all steps of the TFF method in this example, buffer A was used. The method was performed at a temperature of 18–24 °C.

[0533] Buffer A

[0534] Component Value (± 1%) HEPES 7.6 mM NaCl 60 mM KCl 3.84 mM Dextrose 2.4 mM NaHCO3 9.6 mM Trehalose 80 mM Ethanol 0.8％ Polysucrose 6% (w / v) pH 6.6-6.8

[0535] Platelets were loaded onto a TFF (PendoTECH controller system) prepared using a Repligen TFF cartridge (XPM45L01E). The platelets were diluted with an equal weight (±10%) of buffer A. The platelets were concentrated to approximately 2250 × 10⁻⁶. 3 Cells / μL (±250×10⁻⁶) 3 Then wash with approximately 2 osmotic volumes (DV) of buffer A. The target plasma percentage is typically less than 15% of the relative plasma (as determined by plasma protein content). Removal of plasma proteins is monitored by 280 nm UV absorbance relative to known correlations. After washing, if the cell concentration is not 2000 × 10⁻⁶, [further action is taken]. 3 Cells / μL (±300×10⁻⁶) 3 If the cells are diluted with buffer A or concentrated to fall within this range, they are then typically lyophilized and subsequently heat-treated at 80°C for 24 hours to form thrombus bodies, although sometimes cells are used before lyophilization (sometimes referred to as thrombus body "pre-lyo"). Thrombus bodies are typically rehydrated with water within 10 minutes at room temperature. Generally, the rehydrated volume is equal to the volume used to fill each vial of thrombus bodies before drying.

[0536] In some cases, samples were drawn at UV readings associated with approximately 51% relative plasma volume, approximately 8.1% relative plasma volume, approximately 6.0% relative plasma volume, and approximately 1.3% relative plasma volume. At each processing step, low-volume aliquots were sampled using approximately 6.0% or less of the sample.

[0537] Example 2. Test Plan and Measurement Protocol

[0538] Test plan:

[0539] Using the TFF method described in Example 1, batch A thrombus bodies were produced using apheresis platelets collected from a high αHLA titer donor, as reported by the platelet supplier.

[0540] Individual donor units, donor pools, and time points along the TFF process were collected for αHLA assays. Plasma was added to HLA beads (One Lambda FLOWPRA). TM Screening test), αHLA-IgG binding was assessed by staining with αIgG secondary antibody and by flow cytometry (Novocyte 3005 configuration).

[0541] Two bead types were evaluated: one coated with HLA class I antigens and the other coated with HLA class II antigens. (e.g., FLOWPRA) TMBead gating was performed as described in the screening test instructions. The αHLA-positive population was gating based on a George King (GK) PPP (platelet-rich plasma anemia) negative control and a single donor fresh aspiration negative control. Additional negative controls were collected post-production to confirm the ideal location of these positive gating points.

[0542] A compensation setting was established using mouse IgG conjugated to FITC and PE on Spherotech COMPtrol compensation beads. HLA class II beads fluoresced in PE, and a secondary antibody for IgG detection was conjugated to FITC.

[0543] Measurement protocol:

[0544] 1. One Lambda FLOWPRA TM Thaw the components of the screening test kit to 4°C.

[0545] 2. Obtain approximately 1 mL aliquots of PPP filtered through a 0.2 μm filter from each desired sample point.

[0546] 3. Transfer 5 μL of type I HLA beads and 5 μL of type II HLA beads to a 1.7 mL microcentrifuge tube. Add 20 μL of filtered test plasma. Vortex to mix.

[0547] 4. Incubate the plasma with HLA-coated beads for 30 minutes at room temperature in the dark with gentle shaking / stirring.

[0548] 5. Dilute the 10x washing buffer with deionized water to an appropriate volume of 1x working stock solution.

[0549] 6. Wash the beads with 1 mL of washing buffer. Vortex at 9,000 g for 2 minutes and centrifuge to allow the beads to precipitate. Aspirate the supernatant.

[0550] 7. Repeat step 6.

[0551] 8. Dilute 100×αIgG-FITC with washing buffer to an appropriate volume of working stock.

[0552] 9. Add 100 μL of αIgG-FITC (1x concentration) to the tube containing the washed HLA beads. Vortex to mix.

[0553] 10. Gently shake / stir in the dark at room temperature while incubating the HLA beads with αIgG-FITC for 30 minutes.

[0554] 11. Repeat steps 6 and 7 to wash away unbound αIgG-FITC.

[0555] 12. Resuspend the washed HLA beads in 200 μL of PBS. Vortex to mix.

[0556] 13. Transfer approximately 100 μL of the HLA bead suspension into the appropriate well of a 96-well U-shaped bottom microplate and dock it onto a NovoSampler.

[0557] 14. Use NovoCyte to collect events via flow cytometry.

[0558] a. In the previously determined FLOWPRA TM 10,000 events are slowly collected in the bead-gated system, with an FSC-H threshold of 10,000.

[0559] b. The secondary stop condition is a 2-minute run time and a total sample volume of 40 μL.

[0560] c. Clean the SIP between each sample to minimize residue between wells. Run the PBS in the wells between each triplicate set to minimize residue between test points.

[0561] Result

[0562] Example 3. Gating location and negative control

[0563] The initial gating sites for identifying class I and class II HLA beads were determined using FLOWPRA in PBS. TM The pearl is used to determine this. Figure 1A and Figure 1B )

[0564] Background / nonspecific binding was established using GK PPP and fresh donor PPP (in triplicate). (Exemplary data are available in...) Figure 2A , Figure 2B , Figure 3A and Figure 3B (As shown in the image). It should be noted that GK PPP shows a higher offset on FITC-H compared to fresh donor plasma. This may be due to donor variability or the freeze / thaw effect on GK plasma. Additional sampling is necessary. A positive gate is set so that <1% of GK PPP returns to FITC positivity.

[0565] Example 4. Single donor results

[0566] A representative FITC-H histogram for each HLA class is provided for each sample. Positive results >1% are considered positive. Fluorescence ratios relative to GK PPP negative controls (FITC-H intensity of class I and II beads) are reported. Fluorescence ratios >1.0 are considered positive. These positive gates and fluorescence ratios will be updated as additional negative controls are collected.

[0567] If both the positive percentage and fluorescence ratio for this bead type are ≤1, then the population is susceptible to HLA class I or II antibodies. All It was negative.

[0568] Donor 1: HLA type II positive. The mean positivity rate from the three data points in type I was 0.2%, with a fluorescence ratio of 0.3; the mean positivity rate in type II was 16.5%, with a fluorescence ratio of 1.6. (Exemplary data are available in...) Figure 4A and Figure 4B (as shown in the image).

[0569] Donor 2: HLA type I and II positive. The mean positivity rate from the three data points for type I was 1.3%, with a fluorescence ratio of 1.4; the mean positivity rate for type II was 20.3%, with a fluorescence ratio of 1.9. (Exemplary data are available in...) Figure 5A and Figure 5B (as shown in the image).

[0570] Donor 3: HLA type I and II positive. The mean positivity rate from the three data points for type I was 85.2%, with a fluorescence ratio of 14.4. The mean positivity rate for type II was 12.0%, with a fluorescence ratio of 0.8. (Exemplary data are available in...) Figure 6A and Figure 6B (as shown in the image).

[0571] Donor 4: HLA type I and II positive. The mean positivity rate from the three data points for type I was 83.5%, with a fluorescence ratio of 14.5. The mean positivity rate for type II was 12.6%, with a fluorescence ratio of 0.8. (Exemplary data are in...) Figure 7A and Figure 7B (as shown in the image).

[0572] Donor 5: HLA type I and II positive. The mean positivity rate from the three data points for type I was 4.9%, with a fluorescence ratio of 1.2. The mean positivity rate for type II was 1.3%, with a fluorescence ratio of 0.8. (Exemplary data are in...) Figure 8A and Figure 8B (as shown in the image).

[0573] Donor 6: HLA type I positive. The mean positivity rate from the three data points in type I was 2.7%, with a fluorescence ratio of 0.9. The mean positivity rate in type II was 0.3%, with a fluorescence ratio of 0.7. (Exemplary data are in...) Figure 9A and Figure 9B (as shown in the image).

[0574] Donor #7: HLA type II positive. The mean positivity rate from the three data points in type I was 0.7%, with a fluorescence ratio of 0.5. The mean positivity rate in type II was 9.0%, with a fluorescence ratio of 1.3. (Exemplary data are in...) Figure 10A and Figure 10B (as shown in the image).

[0575] The results for donors 1-7 are also shown in Table 1.

[0576] Table 1.

[0577]

[0578] Since HLA-positive donors may be present in the GK PNP pool, Table 2 shows the results relative to HLA-negative donors with N=1.

[0579] Table 2.

[0580]

[0581] Example 5. Filtering Results

[0582] Pool: HLA Group I and II positive. The mean positivity rate from the three data sets for Group I was 61.1%, with a fluorescence ratio of 4.6. The mean positivity rate for Group II was 9.0%, with a fluorescence ratio of 1.1. (Exemplary data are in...) Figure 11A and Figure 11B (as shown in the image).

[0583] Initial dilution (51%): HLA class I and II positive. The mean positivity rate from the three data sets for class I was 48.2%, with a fluorescence ratio of 4.2. The mean positivity rate for class II was 5.9%, with a fluorescence ratio of 0.9. (Exemplary data are in...) Figure 12A and Figure 12B (as shown in the image).

[0584] 20% of plasma (8.1%): HLA class I positive. The mean positivity rate from the three data points for class I was 2.4%, with a fluorescence ratio of 1.0. The mean positivity rate for class II was 0.2%, with a fluorescence ratio of 0.5. (Exemplary data are in...) Figure 13A and Figure 13B (as shown in the image).

[0585] <10% plasma (6.0%): borderline HLA class positive. The mean positivity rate from the three data points in class I was 1.1%, with a fluorescence ratio of 1.0. The mean positivity rate in class II was 0.2%, with a fluorescence ratio of 0.5. (Exemplary data are in...) Figure 14A and Figure 14B (as shown in the image).

[0586] <3% plasma (1.3%): HLA negative. The mean positivity rate from the three data points in category I was 0.4%, with a fluorescence ratio of 0.2. The mean positivity rate in category II was 0.1%, with a fluorescence ratio of 0.4. (Exemplary data are in...) Figure 15A and Figure 15B (as shown in the image).

[0587] The filtering results are also shown in Tables 3A and 3B.

[0588] Table 3A.

[0589]

[0590] Table 3B. Subtract the background fluorescence measured using HLA beads in PBS from the sample fluorescence before calculating the percentage decrease in average fluorescence intensity (a measure of reduced antibody binding).

[0591]

[0592] A value ≥100% indicates a complete reduction in detectable HLA antibodies binding to the specified bead.

[0593] Since HLA-positive donors may be present in the GK PNP pool, Table 4 shows the results relative to HLA-negative donors with N=1.

[0594] Table 4.

[0595]

[0596] Example 6. Surface markers and thrombin formation.

[0597] Thrombosome batches were produced using the TFF method described in Example 1, and cell surface marker expression was determined using flow cytometry.

[0598] Flow cytometry was used to assess the expression of CD41, CD62, and phosphatidylserine (PS) in thrombus bodies. During staining, the sample contained approximately 270,000 / μL of thrombus bodies, and was diluted approximately 1:34 before analysis in a hematology counter. The thrombus body sample was rehydrated and diluted 1:2 in deionized water. Anti-CD41 stock solution was diluted by adding 47.6 μL of antibody to 52.4 μL of HMTA. Anti-CD41 stained samples were prepared by adding 10 μL of diluted thrombus bodies to 10 μL of HMTA and 10 μL of diluted CD41 antibody. Anti-CD62 master mixture was prepared by combining 12 μL of anti-CD62 with 23.8 μL of anti-CD41 and 64.2 μL of HMTA. Isotype control mixtures were prepared in the same manner. Samples stained with anti-CD62 were prepared by adding 10 μL of diluted thrombus bodies to 20 μL of anti-CD62 master mixture. An allotype master mixture was used to prepare allotype control samples in the same manner. Annexin V (AV) master mixture was prepared by combining 11.7 μL of AV with 83.3 μL of anti-CD41 and 80 μL of HMTA. Samples stained with AV were prepared by adding 20 μL of diluted thrombus bodies containing 50 mM GPRP to 20 μL of HMTA containing 15 mM CaCl2 and 20 μL of AV master mixture. Negative gated control samples were prepared in the same manner using calcium-free HMTA 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 to dilute the AV test samples contained 5 mM CaCl2. Anti-CD41 binding was used to identify the target population. CD62 and PS expression were assessed by binding of anti-CD62 and AV in CD41-positive individuals.

[0599] The expression of glycoprotein IIb (GPIIb, also known as antigen CD41) was measured using anti-CD41 antibody (4.8 μL, Beckman Coulter part #IM1416U). Thrombus bodies measured showed CD41 positivity (Table 5). Figure 16 )

[0600] Table 5.

[0601]

[0602]

[0603] Annexin V (AV) (1.3 μL, BD Biosciences catalog number 550475) was used to determine phosphatidylserine (PS) expression. AV is a calcium-dependent phospholipid-binding protein. Thrombus bodies measured showed AV positivity (Table 6;). Figure 17 ).

[0604] Table 6.

[0605] Lot AV positive (%) 1 96.7 2 89.9 3 95.3 4 95.4 5 95.9 6 96.2 7 93.5 Mean 94.7

[0606] The expression of P-selectin (also known as CD62P) was measured using an anti-CD62P antibody (2.4 μL, BD Bioscience catalog number 550888). Thrombus bodies measured showed CD62 positivity (Table 7). Figure 18 )

[0607] Table 7.

[0608] Lot CD62 positive (%) 1 94.2 2 93.1 3 89.8 4 92.4 5 92.5 6 87.3 7 90.7 Mean 91.4

[0609] Using the following protocol, in the presence of a PRP reagent containing tissue factor and phospholipids, at a concentration of 4.8 × 10⁻⁶... 3 Thrombin generation was measured using thrombus bodies per μl. The average thrombin peak height (TPH) of the thrombus body samples was 60.3 nM. Cerebroside was used as a positive control. (Table 8;) Figure 19 )

[0610] For each test vial, the rehydrated thrombus body sample was diluted to 7,200 particles / μL based on flow cytometry particle count using 30% Octaplas solution in control buffer. Sample wells were generated in 96-well plates by adding 20 μL of PRP reagent (Diagnostica Stago catalog number 86196) and 80 μL of diluted thrombus bodies. Calibration wells were generated by adding 20 μL of thrombin calibrator reagent (Diagnostica Stago catalog number 86197) to the 80 μL diluted thrombus bodies. The plates were loaded into a plate reader and incubated in the dark at 40°C for 10 minutes. During sample incubation, a FluCa solution was prepared by adding 40 μL of FluCa substrate (Diagnostica Stago catalog number 86197) to 1.6 mL of Fluo-Buffer (Diagnostica Stago catalog number 86197) heated to 37 °C and vortexed. The FluCa solution was aspirated into a dispensing syringe, and 20 μL was mechanically dispensed into each well to achieve a final thrombus body concentration of 4,800 particles / μL in each well, initiating the thrombin generation reaction. Thrombin generation was measured in each well by fluorescence over 75 minutes.

[0611] An exemplary step-by-step approach is as follows:

[0612] 1. Open the CAT software; set up the instrument; and prepare PRP reagents (including tissue factor and some phospholipids), calibrators, fluorescence buffer, and fluorescence substrate according to the manufacturer's instructions.

[0613] 2. Thaw the Octaplas and TGA dilution buffer in a 37°C water bath for 10 minutes.

[0614] 3. Add the thawed Octaplas to the TGA dilution buffer to prepare a buffer containing 30% Octaplas.

[0615] 4. Recombinant phospholipids were diluted 1:50 with 30% Octaplas mixture as a positive control.

[0616] 5. Rehydrate the thrombus bodies with cell culture grade water for 10 minutes, then dilute with 30% Octaplas to 7,200 thrombus bodies / μL.

[0617] 6. Using a multichannel pipette, add 20 μL of PRP reagent to each test well. Add 20 μL of calibrator to each calibration well.

[0618] 7. Add 80 μL of sample to each test and calibration well. Add 80 μL of 30% Octaplas to the negative control well and add 1:50 phosphatidylcholine to the positive control well.

[0619] 8. Insert the plate into the tray and incubate at 40°C for 10 minutes. After incubation, dispense the mixture of fluorescent buffer and fluorescent substrate (containing fluorescently labeled peptides that will produce a fluorescent signal when cleaved by thrombin) into the active wells.

[0620] 9. Read the plate at 20-second intervals for 75 minutes to obtain a complete thrombin generation profile.

[0621] Table 8.

[0622]

[0623]

[0624] Table 9 summarizes the data from these measurements.

[0625] Table 9.

[0626]

[0627] 1Particle diameter can be assessed using the size beat of forward scattering in flow cytometry.

[0628] Example 7.9F9 and PAC-1 combined.

[0629] Activated platelet aggregation is mediated by the formation of the GPIIb / IIIa complex, which can bind to fibrinogen (also known as factor 1) and form a clot. GPIIb / IIIa is the platelet fibrinogen receptor, also known as the CD41 / CD61 complex. ADP promotes the active form of the GPIIb / IIIa complex in this process. Antibody 9F9 binds to cell membrane-associated fibrinogen. Therefore, the presence of fibrinogen on the cell membrane indicates that thrombus bodies can form blood clots.

[0630] One vial of thrombus bodies prepared according to Example 1 was rehydrated with 10 mL of deionized water. The aliquots of thrombus bodies were diluted to 1 × 10⁻⁶ using HMTA (HEPES-modified Tyrode's protein). 5 The final concentration of particles / μL was determined. Samples were prepared as shown in Table 11. Unstained samples were prepared by adding 10 μL of diluted thrombus bodies to 20 μL of HMTA. FITC isotype control samples were prepared by adding 10 μL of diluted thrombus bodies to 10 μL of isotype control antibody (BD Biosciences catalog number 555748) and 10 μL of HMTA. Samples stained with 9F9 were prepared by adding 10 μL of diluted thrombus bodies to 10 μL of 9F9 antibody (BD Biosciences catalog number 340507) and 10 μL of HMTA. Samples stained with PAC-1 were prepared by adding 10 μL of diluted thrombus bodies to 5 μL of isotype control antibody and 15 μL of HMTA. A total of 1 × 10⁻⁶ particles were used for each reaction mixture. 6 Two copies of each particle were prepared for all samples. Samples were incubated at room temperature in the dark for 20 minutes. After incubation, all samples were diluted with 1 mL of HBS and collected using an ACEA NovoCyte flow cytometer. Fluorescence signals generated by PAC-1 were used to determine the expression of activated GPIIb / IIIa receptors in the absence of bound fibrinogen. Fluorescence signals from 9F9 were used to determine the binding of fibrinogen to surface receptors on thrombus bodies.

[0631] HTMA (HEPES-modified Tyrode's albumin).

[0632]

[0633] Table 10.

[0634] Unstained FITC Iso 9F9 PAC-1 Cells (uL) 10 10 10 10 HMTA (uL) 20 10 10 15 Antibody (uL) 0 10 10 5

[0635] The samples were analyzed by flow cytometry, and the presence of surface-bound fibrinogen was demonstrated after rehydration. Figure 20 Anti-PAC-1 antibody showed no significant binding. Figure 21 This further demonstrates that the thrombus bodies prepared by TFF include fibrinogen bound to the active forms of GPIIb / GPIIIa, since PAC-1 binds to the same complex.

[0636] Example 8. Evaluation of CD47 binding

[0637] CD47 is a cell surface marker used for self-recognition. In some cases, the absence of this marker can lead to phagocytosis.

[0638] A vial of thrombus bodies prepared as described in Example 1 was rehydrated with 10 mL of sterile water for injection and stained with a volume-enhancing anti-CD47 antibody conjugated to Pacific Blue (BD Biosciences catalog number 561564) or a corresponding isotype control (BD Biosciences catalog number 560373). All samples contained 1 million cells. The maximum fluorescence signal produced by this titration was approximately 5 times that relative to the background. Figure 22A The overall CD47 positivity rate was approximately 40% (Table 12). Figure 22B An exemplary histogram is shown in the figure.

[0639] Aliquots of the CD47 antibody conjugated to V450 were prepared at HMTA dilutions of 1:10, 1:5, and 1:2. The initial concentration of the thrombus body samples was determined using an AcT diff 2 assay, and the concentration of each 1 mL aliquot was adjusted to 100 × 10⁻⁶ using HMTA. 3 / μL. TFF thrombus bodies were stained in duplicate with each antibody dilution by adding 10μL of antibody to 10μL of diluted thrombus bodies. Samples stained with undiluted antibody were produced in the same manner. Unstained control samples were prepared by adding 10μL of HMTA to 10μL of diluted thrombus bodies. This sample preparation was repeated using an isotype control antibody instead of anti-CD47. All samples were incubated at room temperature in the dark for 20 minutes. After incubation, the samples were diluted with 500μL of HBS, and 15,000 events were acquired for each sample using an ACEA NovoCyte flow cytometer. V450 fluorescence in the test samples was used to assess antibody binding to CD47 on the thrombus body surface. Nonspecific binding was monitored using V450 fluorescence from the isotype control samples.

[0640] Table 11 shows the average fluorescence intensity of samples containing different amounts of antibody (anti-CD47 or isotype control).

[0641] Table 11.

[0642]

[0643] Table 12 shows the percentage of CD47 positivity for different concentrations of anti-CD47 antibody.

[0644] Table 12.

[0645]

[0646] Second vials of TFF thrombus bodies from different batches were rehydrated and stained with increased-volume anti-CD47 conjugated with Pacific Blue or the corresponding isotype control. All samples contained 250,000 cells. Again, the fluorescence signal was approximately 5 to 6 times higher than the background. Figure 22C The overall CD47 positivity rate was approximately 50% (Table 15). Figure 22D An exemplary histogram is shown in the figure.

[0647] A second round of testing was performed on the new TFF thrombus body samples using an increased amount of antibody and a reduced number of thrombus bodies per sample to improve the signal intensity induced by anti-CD47 binding to thrombus bodies. The initial concentration of the thrombus body samples was determined using AcT diff 2, and the concentration of 1 mL aliquots was adjusted to 25 × 10⁻⁶ using HMTA. 3 / μL. According to Table 13 below, stain the samples in duplicate with the increased amount of antibody. The final volume of each sample should remain constant at 40μL. The total number of thrombus bodies in each sample should remain constant at 250×10⁻⁶. 3 / μL. This sample preparation was repeated using an isotype control antibody instead of anti-CD47.

[0648] Table 13.

[0649] Volume of thrombosomes Volume of AB (uL) Volume of HMTA (uL) Total 10 0 30 40 10 5 25 40 10 15 15 40 10 25 5 40 10 30 0 40

[0650] All samples were incubated at room temperature in the dark for 20 minutes. After incubation, the samples were diluted with 500 μL of HBS, and 15,000 events were acquired for each sample using an ACEANovoCyte flow cytometer. V450 fluorescence in the test samples was used to assess antibody binding to CD47 on the surface of thrombus bodies. V450 fluorescence in isotype control samples was used to monitor nonspecific binding.

[0651] Table 14 shows the average fluorescence intensity of samples containing different amounts of antibody (anti-CD47 or isotype control).

[0652] Table 14.

[0653]

[0654] Table 15 shows the percentage of CD47 positivity for different concentrations of anti-CD47 antibody.

[0655] Table 15.

[0656]

[0657] Example 9. Reduced particulate matter content

[0658] Dynamic light scattering was used to compare the particle content of human shelf-life stored platelets (hIDSP) with thrombus bodies prepared according to Example 1 (but not lyophilized). Results were obtained in... Figure 23A -C and as shown in Table 16. Figure 23A -C is a histogram that is normalized to relative intensity such that the sum of the intensities of each data point equals 1.0. For example, if a particular data point has a y-axis value of 0.1, it can generally be interpreted as representing 10% of the sample's scattering intensity.

[0659] An apheresis cell used to produce a batch of thrombus bodies was analyzed. This sample type is designated "hIDSP". A 1 mL aliquot of this hIDSP (human shelf-life stored platelets) cell was used for dynamic light scattering (DLS; Thrombolux-Light Integra) analysis. The sample from this aliquot was then drawn into a capillary and inserted into the DLS instrument. The capillary was left in the instrument for 1 minute to allow temperature and movement to reach equilibrium. The internal temperature of the instrument was 37°C. After 1 minute of equilibrium, the viscosity setting for the sample was selected. The DLS instrument has a built-in viscosity setting for samples in plasma, such as apheresis cells. This viscosity setting was used for hIDSP samples. The viscosity of this setting was 1.060 cP (centipoise). After selecting the plasma viscosity setting, the sample was analyzed. From the same hIDSP aliquot, a second and third aliquot were drawn into capillaries and analyzed using the hIDSP protocol for triplicate analysis. The particle percentage was then determined based on the data.

[0660] “Pre-Lyo” samples are samples taken during the manufacturing process of thrombus bodies. This sample type is material collected just before lyophilization. Viscosity measurements were performed on the samples for DLS analysis. The viscometer (Rheosense μVISC) has a built-in oven to heat the sample to the DLS instrument temperature (37°C). The oven must be heated to 37°C before performing viscosity analysis on the sample. To determine the viscosity of the pre-lyo sample, 400–350 μL of sample is drawn into a syringe and inserted into the viscometer. After inserting the sample into the viscometer, the instrument temperature needs to be raised back to 37°C. After the oven reaches 37°C, the sample is analyzed using all settings on AUTO (except for the “Measurement Volume” set to 400 μL). This viscosity is used for DLS measurements of the same sample. A 1 mL aliquot of this pre-lyo sample is taken for dynamic light scattering (DLS; Thrombolux-LightIntegra) analysis. The sample from this aliquot is then drawn into a capillary and inserted into the DLS instrument. Place the capillary tube in the instrument for 1 minute to allow temperature and movement to reach equilibrium. The internal temperature of the instrument is 37°C. After 1 minute of equilibrium, place the previously measured viscosity into the viscosity setting of the DLS instrument. After entering the viscosity, analyze the sample. From the same pre-lyo aliquot, draw the second and third portions of the sample into the capillary tube and analyze them using this Pre-Lyo protocol for triplicate analysis. Then determine the particle percentage based on the data.

[0661] Thrombosomes were rehydrated according to a standard protocol and diluted 1:5 in a mixture of SeraSub (CST Technologies, Inc.) and ACD. The SeraSub / ACD diluent consisted of a 1:9 ratio of ACD diluent in SeraSub. One mL of the 1:5 diluted thrombosomes was prepared for analysis by DLS. The diluted thrombosome sample was drawn into a capillary and inserted into the DLS instrument. The capillary was placed in the instrument for 1 minute to allow temperature and movement to reach equilibrium. The internal temperature of the instrument was 37°C. After 1 minute of equilibrium, the viscosity setting for the sample was selected. A viscosity of 1.200 cP was used for the sample. After entering the viscosity, the sample was analyzed. Second, third, and fourth aliquots of the sample were drawn into capillary tubes and analyzed using the thrombosome protocol for quadruplicate analysis. The percentage of particles was then determined based on the data (and platelet radius, if applicable).

[0662] Table 16.

[0663] Lot number hIDSP % MP Pre-Lyo % MP Lot J 9.47％ 0.49％ Lot K 7.55％ 0.65％ Lot L 7.73％ 0.59％ Mean 8.25％ 0.58％

[0664] In another experiment, dynamic light scattering (DLS) was used to compare the particle content of human shelf-life stored platelets (hIDSP) with rehydrated thrombus bodies prepared according to Example 1. Figure 24A The results are shown in -C and Table 17.

[0665] Table 17.

[0666]

[0667] Example 12. Metabolite Analysis

[0668] Table 18 shows the analysis of pH and metabolites present during the thrombus body preparation process as described in Example 1, including the analysis of platelet raw materials after initial dilution, after concentration of platelet derivatives, and after the end of the percolation process, as determined using an i-STAT handheld hematology analyzer and a CG4+ filter.

[0669] Platelet samples for iStat analysis were collected in small volumes (1 ml) at different processing steps. An initial sample, designated "raw material," was collected for iStat analysis after the platelet donor units were pooled but before any processing. The pooled platelet units were diluted 1:1 with control buffer before TFF treatment, designated "initial dilution." At the end of the TFF concentration phase, a "concentration end" sample was drawn from the platelet product. After washing the cells, a "DV end (Pre-Lyo)" sample was drawn as a representative of the product entering the lyophilizer.

[0670] Table 18.

[0671]

[0672] Example 11. Pathogen reduction

[0673] Reduction of pathogens in blood products is generally desirable. One method for reducing pathogens involves using photosensitive nucleic acid intercalation compounds to alter the nucleic acids of pathogens upon irradiation with an appropriate wavelength.

[0674] The system (manufactured by Cerus) uses amotosalen (a nucleic acid intercalation compound that forms cross-links in nucleic acids upon UVA irradiation). Exemplary parameters for this system are shown in Table 19, and... Figure 25A A schematic diagram of the system is shown in the figure, while Figure 25B -C shows exemplary processing data for 2.6L of processed material over 198 minutes (approximately 14 / minute average).

[0675] Perform DLS as described in Example 9.

[0676] Table 19.

[0677]

[0678]

[0679] Table 20 shows thrombus bodies prepared as in Example 1 with or without the use of... Exemplary comparative data on pH and metabolites under system processing conditions.

[0680] Table 20.

[0681]

[0682] Exemplary comparative data of functional characterization (AcT count and aggregation parameters) and cell surface markers are shown in Tables 21 (hIDSP), 22 (before lyophilization), and 23 (lyophilized and rehydrated to approximately 1.8 × 10⁻⁶ in 10 mL sterile water for injection). 6 After the concentration was calculated in μL (individual sample counts are shown in Table 23).

[0683] Table 21.

[0684]

[0685]

[0686] Table 22.

[0687]

[0688] Table 23.

[0689]

[0690] As described in Example 9, the microparticle content at each stage of thrombus body preparation was also determined. Figure 26A -B shows the similarity of rehydrated thrombus bodies prepared with and without pathogen reduction treatment. A summary of these data is shown in Table 24. Figure 27A The particulate content of hiDSP is shown with or without pathogen reduction treatment. Figure 27B -C compares Figure 29A The microparticle content of hiDSP and the rehydrated thrombus bodies prepared therefrom is shown. A summary of these data is presented in Table 25. Figure 28A The particulate content of hiDSP is shown with or without pathogen reduction treatment. Figure 28B -C compares Figure 28AThe microparticle content of hiDSP and the rehydrated thrombus bodies prepared therefrom is shown. A summary of these data is presented in Table 26.

[0691] Table 24.

[0692]

[0693] Table 25.

[0694]

[0695] Table 26.

[0696]

[0697]

[0698] Example 12. Interaction between platelets and thrombus bodies.

[0699] In this example, "platelets" are platelets separated from citric acidified whole blood approximately 3 hours after collection. Thrombus bodies are batch D, prepared using the method described in Example 1. Table 27 shows the sample layout for the experiments in this example.

[0700] Table 27.

[0701]

[0702] Platelet and thrombus body co-aggregation was assessed using a light transmission aggregation assay. Platelets and thrombus bodies were co-incubated and evaluated using 4β-phorbol-12-myristate-13-acetate (PMA) via an aggregation assay to positively / negatively activate platelets. For fresh platelets isolated from whole blood, 100 ng / mL of PMA was used. For stored platelets (i.e., apheresis platelets), 1000 ng / mL of PMA was used.

[0703] Fresh platelets were isolated from ACD-anticoagulated whole blood, washed, and diluted in HMTA to 250,000 cells / μL. Thrombus bodies were rehydrated according to standard protocol and diluted in HMTA to 250,000 cells / μL. Equal aliquots of platelets and thrombus bodies from HMTA were mixed proportionally. Platelets, thrombus bodies, and the mixed sample were assessed by Helena AggRAM in response to phorbol-myristate-acetate (PMA; 100 ng / mL) activation. The mixed sample was evaluated with and without a stir bar to assess the effect of stirring-induced shear on observed platelet-thrombus body co-aggregation.

[0704] Figure 29AThe transmittance of the samples in Table 30 is shown in the presence and absence of an agonist. A mixture of positive and negative agonists (black), thrombus bodies, and freshly drawn platelets induces platelet activation and aggregation. The amplitude of PMA (grey) activated platelets and Δtransmittance indicates aggregation mixed with thrombus bodies. In the absence of shear, there is no activation or co-aggregation less than [value missing]. Figure 29A The amplitude observed in [the study].

[0705] Platelet and thrombus body AcT counts were also assessed before and after aggregation assays. Figure 29B The counts after aggregation are shown. White bars larger than other bars indicate that thrombus bodies have merged into platelet aggregates. In the absence of an agonist (black), the absolute decrease in particle count is particularly significant and unexpected.

[0706] The effect of shearing on aggregation was also evaluated. The mixed aggregation assay (1:1 platelet:thrombus body (by count)) was repeated with and without a stir bar. Results showed... Figure 29C These results indicate that shearing is necessary for observable coagulation in the absence of platelet agonists. The magnitudes of coagulation with + / - agonists measured in plasma were slightly reduced compared to buffer.

[0707] Example 13. Inhibition of fibrin capture using GPRP.

[0708] In this example, platelets were separated from whole blood approximately 1 hour after collection. Thrombus bodies were batch H, prepared using the method described in Example 1.

[0709] Fresh platelets were isolated from ACD-anticoagulated whole blood, washed, and diluted in HMTA to 250,000 cells / μL. Thrombus bodies were rehydrated according to standard protocol and diluted in HMTA to 250,000 cells / μL. Equal aliquots of platelets and thrombus bodies from HMTA were mixed proportionally. Each aliquot of platelets, thrombus bodies, or mixed suspension was partitioned equally; one aliquot was treated with 1 mM GPRP to inhibit fibrin polymerization, while the other remained untreated. Gly-Pro-Arg-Pro (GPRP; Sigma-Aldrich catalog number G1895) is a peptide that prevents fibrin polymerization. Platelets, thrombus bodies, and mixed samples were evaluated by Helena AggRAM assay in response to thrombin (2.5 U / mL) activation.

[0710] Figure 30Results of coagulation experiments using platelets, thrombus bodies, and 2:1 and 1:1 mixtures of platelets and thrombus bodies (both activated by thrombin) in the presence or absence of GPRP (1 mM) are shown. In the mixed case, the total measured aggregation decreased with increasing thrombus body population, suggesting that the platelet-thrombus body interaction is partly caused by fibrin capture. However, most of the coagulation interaction is platelet-mediated and independent of fibrin capture, as demonstrated even with GPRP through high measured aggregation.

[0711] Examples 12-13 demonstrate that platelets and thrombus bodies co-aggregate under shear in the presence (and to a lesser extent, without) of platelet activation. The fibrin polymerization inhibitor GPRP only slightly inhibits platelet-thrombus body co-aggregation after thrombin activation.

[0712] Example 14. Co-aggregation of RGDS suppression.

[0713] In this example, platelets were separated from whole blood approximately 1 hour after collection. Thrombus bodies were batch H, prepared using the method described in Example 1.

[0714] Fresh platelets were isolated from ACD-anticoagulated whole blood, washed, and diluted in HMTA to 250,000 cells / μL. Thrombus bodies were rehydrated according to standard protocol and diluted in HMTA to 250,000 cells / μL. Equal aliquots of platelets and thrombus bodies from HMTA were mixed in equal proportions. Each aliquot of platelets, thrombus bodies, or mixed suspension was partitioned equally; one aliquot was treated with 100 μM RGDS to inhibit fibrinogen binding to platelets, while the other aliquot remained untreated. RGDS (Arg-Gly-Asp-Ser; Cayman Chemical, catalog number 15359) is a peptide sequence that binds to platelet surface integrins, particularly GPIIb / IIIa. It inhibits platelet binding to fibrinogen and other adhesion proteins. Platelets, thrombus bodies, and mixed samples were evaluated by light transmission aggregation assay in response to phorbol myristate acetate (PMA; 100 ng / mL) activation.

[0715] Co-aggregation experiments were performed using 100 μM RGDS and activated with PMA to investigate whether the interaction was caused by fibrinogen bridging between platelets and thrombus bodies. Results were... Figure 31 As shown in the figure, RGDS blocked >50% of the measured coagulation, suggesting that the interaction between platelets and thrombus bodies is likely largely caused by fibrinogen binding.

[0716] Examples 12-14 demonstrate the ease with which thrombus bodies co-aggregate with activated platelets (e.g., as demonstrated by light transmission aggregation assays). Spontaneous co-aggregation is induced by shearing. Platelet-thrombus body interactions are evident in both buffer and plasma. While co-aggregation is substantially not inhibited by GPRP, it is substantially inhibited by RGDS. This indicates a crucial role for active platelet-fibrinogen binding in the co-aggregation mechanism, and that co-aggregation is not solely caused by passive fibrin capture.

[0717] Example 15. Scanning electron microscope (SEM).

[0718] Ten-mL aliquots of the rehydrated thrombus bodies were centrifuged at 2000 RPM for 30 minutes. The supernatant from the centrifuged sample was removed to 1 mL and discarded. The sample was gently agitated to resuspend the thrombus bodies. The concentrated thrombus bodies were treated with 3% glutaraldehyde in 0.1 M dimethylarsinate buffer at pH 7.4 for 2 hours, with agitation every 15 minutes. The thrombus bodies were rinsed three times with sterile water and transferred to 1% osmium tetroxide solution for 1 hour, with agitation every 15 minutes. The sample was then rinsed more than three times with sterile water, and a 0.5 mL droplet was transferred onto a polysulfone filter membrane. The mounted sample was frozen in liquid nitrogen and dried under vacuum, and then gold sputtered and imaged using scanning electron microscopy.

[0719] Figure 32A -D displays SEM images of platelets and human thrombus bodies. Freshly activated platelets are shown... Figure 32A (Scale = 2μm) and Figure 32B (Scale bar = 1 μm). The rehydrated human thrombus bodies prepared in Example 1 are shown... Figure 32C (Scale = 2μm) and Figure 32D (Scale bar = 1 μm)

[0720] Example 16. Thrombus body data.

[0721] In total thrombosis analysis system ( In FUJIMORI KOGYO CO.,LTD), mineral oil is used to force the sample through collagen-coated microchannels. Pressure changes are used to assess thrombosis. Occlusion initiation time is the time required to reach Δ10 kPa, while occlusion time is the time required to reach each Δ80 kPa using an AR chip (Zacros catalog number TC0101).

[0722] According to FUJIMORI KOGYO CO.,LTD, the AR chip can be used to analyze the formation of mixed white thrombi primarily composed of fibrin and activated platelets. It features flow paths (300 μm wide × 50 μm high) coated with collagen and tissue factor, and can be used to analyze coagulation and platelet function. In contrast, the PL chip can be used to analyze the formation of platelet thrombi primarily composed of activated platelets. The PL chip features flow paths coated only with collagen and can be used to analyze platelet function.

[0723] The reagent (CaCTI, AR chip) was heated to 37°C and the thrombus bodies were rehydrated according to a standard protocol. Aliquots of the rehydrated thrombus bodies were washed by centrifugation at 3900 g for 10 minutes and resuspended in sodium citrate-anticoagulated anemic platelet-poor plasma (PPP) to approximately 300,000 cells / μL. CaCTI (20 μL) was mixed with the thrombus bodies in PPP (480 μL) and passed through the T-TAS AR chip under high shear. The pressure in the system was monitored over 30 minutes, or until the maximum back pressure in the channel was reached.

[0724] Prepare for use according to the manufacturer's instructions. Instrumentation. Heat the AR chip (Diapharma catalog number TC0101) and AR Chip calcium corn trypsin inhibitor (CaCTI; Diapharma catalog number TR0101) to room temperature. Transfer 300 μL of rehydrated thrombus bodies to a 1.7 mL microcentrifuge tube and centrifuge at 3900 g × 10 min to precipitate. Resuspend the thrombus body precipitate in normal human plasma collected by George King (GK) or autologous plasma with or without autologous platelets to a concentration of approximately 100,000–450,000 / μL, as determined by an Act count (Beckman Coulter AcT Diff2Cell Counter). Gently pipette 20 μL of CaCTI into 480 μL of the thrombus body sample from GK plasma. Follow the manufacturer's instructions. Load and run the sample.

[0725] Table 28 shows the platelet-reducing whole blood, citric acid-enriched whole blood supplemented with different concentrations of thrombus bodies prepared as in Example 1, and George King anemic platelet-poor plasma (GKPPP) supplemented with different concentrations of thrombus bodies prepared as in Example 1, in experiments run according to the manufacturer's instructions using an AR chip and high-shear instrument settings. result.

[0726] Table 28.

[0727]

[0728]

[0729] *The test reached a peak of approximately 75 kPa before a rapid decline. Possible erroneous results.

[0730] Test timed out.

[0731] exist Figure 33A -B shows the results over time. As measured by shortened occlusion time, increased concentrations of thrombus bodies in whole blood with thrombocytopenia promote more robust thrombus formation. Figure 33A As measured by shortened occlusion time, increased concentrations of thrombus bodies in anemic platelet-rich plasma (PPP) promote more robust thrombus formation. Figure 33B ).

[0732] The effect of GPRP (1 mM) on occlusion activity was also determined. Table 29 shows the platelet-poor plasma levels in the presence and absence of GPRP, with and without thrombus bodies. Results. Adding GPRP to prevent fibrinogen formation did not prevent samples containing thrombus bodies from reaching the occlusive pressure. Although adding GPRP to thrombus body samples in plasma prevented fibrinogen formation in microcapillary channels ( Figure 33C (Without GPRP) and 33D (GPRP), both in GK PPP), but adding GPRP to thrombus bodies (PPP) did not prevent thrombus formation. Figure 33E ).

[0733] Table 29.

[0734]

[0735] Test timeout

[0736] While embodiments of the invention may have various modifications and alternatives, specific embodiments have been illustrated by way of example in the accompanying drawings and are described in detail below. However, the purpose is not to limit the invention to the specific embodiments described. Rather, the invention is intended to cover all modifications, equivalents, and alternatives falling within the scope of the invention as defined by the appended claims.

Claims

1. A method for preparing a heat-treated platelet derivative composition, the method comprising: A platelet composition is subjected to tangential flow filtration (TFF), the platelet composition comprising whole platelets in a formulation containing a buffer, 10 mM to 500 mM of trehalose, and 3% to 10% (w / v) of sucrose, thereby preparing platelets containing at least 1000 × 10⁻⁶ particles in an aqueous medium having a particle size of less than 5.0% by scattering intensity. 3 The TFF-treated composition with platelet count / μl Freeze-drying a TFF-treated composition containing platelets in the aqueous medium to form a freeze-dried platelet derivative composition containing freeze-dried platelet derivatives; as well as The freeze-dried platelet derivative composition is heated in a temperature range of 60°C to 85°C for at least 1 hour to no more than 36 hours to heat-treat the freeze-dried platelet derivatives in the freeze-dried platelet derivative composition, thereby forming a heat-treated platelet derivative composition comprising freeze-dried platelet derivatives. The heat-treated platelet derivative in the heat-treated platelet derivative composition described herein has in vitro occlusive activity, such that when at a temperature of at least 255 × 10⁻⁶, the platelet derivative exhibits occlusive activity. 3 When forced through collagen-coated microchannels at a concentration of particles / μL, pressures of 80 kPa were achieved in less than 14 minutes in thrombocytopenic citric acid whole blood in a Total Thrombosis Analysis System (T-TAS) assay. In the heat-treated platelet derivative composition, at least 75% of the heat-treated platelet derivatives are CD62-positive, and In the heat-treated platelet derivative composition, at least 70% of the heat-treated platelet derivatives are CD42 positive.

2. The method according to claim 1, wherein the amount of trehalose in the formulation is in the range of 50 mM to 500 mM and the amount of sucrose is in the range of 3% to 7% (w / v).

3. The method according to any one of claims 1 or 2, wherein the TFF-treated composition comprises less than 3.0% microparticles in terms of scattering intensity.

4. The method according to any one of claims 1 to 3, wherein at least 70% of the heat-treated platelet derivatives in the heat-treated platelet derivative composition are CD41 positive, at least 75% of the heat-treated platelet derivatives in the heat-treated platelet derivative composition are annexin V positive, and at least 80% of the heat-treated platelet derivatives in the heat-treated platelet derivative composition are CD62 positive.

5. The method according to any one of claims 1 to 4, wherein the heat-treated platelet derivative in the heat-treated platelet derivative composition has in vitro occlusive activity, such that when at least 255 × 10⁻⁶, the platelet derivative exhibits occlusive activity. 3 When forced through collagen-coated microchannels at a concentration of particles / μL, pressures of 80 kPa are achieved in less than 12 minutes in thrombocytopenic citric whole blood during Total Thrombosis Analysis System (T-TAS) assays.

6. The method according to any one of claims 1 to 5, wherein when a reagent containing tissue factor and phospholipids is present, the heat-treated platelet derivative in the heat-treated platelet derivative composition is at 4.8 × 10⁻⁶. 3 At a concentration of 1 particle / μL, it produces a thrombin peak height (TPH) of at least 25 nM.

7. The method according to any one of claims 1 to 6, wherein: In the heat-treated platelet derivative composition, at least 75% of the heat-treated platelet derivatives are CD41 positive; and At least 75% of the heat-treated platelet derivatives in the heat-treated platelet derivative composition are CD42 positive.

8. The method according to any one of claims 1 to 7, wherein prior to performing TFF, the method comprises diluting the platelet composition with a formulation equal to at least 25% of the mass of the platelet composition.

9. The method according to any one of claims 1 to 8, wherein the TFF is performed until an endpoint is reached, wherein the endpoint is a target absorbance of the protein concentration of the TFF-treated composition, and wherein the target absorbance of the TFF-treated composition is set to an absorbance unit value less than or equal to 10% of residual plasma.

10. The method according to any one of claims 1 to 8, wherein the TFF is performed until an endpoint is reached, wherein the endpoint is a target absorbance for measuring the protein concentration of the TFF-treated composition, wherein the target absorbance is measured at 280 nm, and wherein the target absorbance of the TFF-treated composition is set to a value less than or equal to 1.70 AU using a path length of 0.5 cm, wherein the 280 nm absorbance is equal to 1.66 AU relative to a system measuring 7.5% plasma.

11. The method according to any one of claims 1 to 10, wherein the step of performing TFF comprises diluting the platelet composition in a formulation to form a diluted platelet composition; The diluted platelet composition is concentrated to form a concentrated platelet composition, such that the platelet concentration in the concentrated platelet composition is 2000 × 10⁻⁶. 3 cells / μl up to 2500×10 3 cells / μl; and The concentrated platelet composition was prepared by subjecting the concentrated platelet composition to TFF with a formulation of at least 2 times the filtration volume (DV).

12. The method according to any one of claims 1 to 11, wherein a membrane with a pore size in the range of 0.45 to 0.65 μm is used for TFF.

13. The method according to any one of claims 1 to 12, wherein at least some of the heat-treated platelet derivatives in the heat-treated platelet derivative composition have fibrinogen bound to their cell membranes.

14. The method according to any one of claims 1 to 12, wherein the heat-treated platelet derivative composition: a) Negative for HLAI antibodies based on a regulatory-approved HLAI antibody test; b) Negative for HLA class II antibodies based on a regulatory-approved HLA class II antibody test; c) A negative result for HNA antibodies based on a regulatory-approved HNA antibody test; or d) one or more of a), b) and c).

15. The method according to any one of claims 1 to 14, wherein the formulation further comprises one or more salts selected from the group consisting of phosphates, sodium salts, potassium salts, calcium salts and magnesium salts, and having a concentration in the range of 0.5 mM to 100 mM.

16. The method according to any one of claims 1 to 15, wherein the buffer comprises N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES) and sodium bicarbonate (NaHCO3), and its concentration is in the range of 5 mM to 50 mM.

17. The method according to any one of claims 1 to 16, wherein the method does not involve centrifugation of the composition containing platelets.

18. The method according to any one of claims 1 to 17, wherein the heat-treated platelet derivative in the heat-treated platelet derivative composition has a shelf life of 2-3 years.

19. The method according to any one of claims 1 to 18, wherein the formulation has a pH of 6.0 to 7.

4.

20. The method according to any one of claims 1 to 19, further comprising a pathogen reduction step prior to the TFF step.

21. The method according to any one of claims 1 to 20, wherein the TFF-treated composition comprises 1000 × 10⁻⁶ ppm in an aqueous medium. 3 Up to 4000×10 3 Platelets per μl.

22. The method according to any one of claims 1 to 21, wherein the heat-treated platelet derivative composition: a) Negative for HLAI antibodies based on a regulatory-approved HLAI antibody test; b) A negative result for HLA-II antibodies based on a regulatory-approved HLA-II antibody test; and c) Negative for HNA antibodies based on a regulatory-approved HNA antibody test.

23. The method according to any one of claims 1 to 22, wherein the amount of sucrose in the formulation is in the range of 3% to 6% (w / v).

24. The method according to any one of claims 1 to 23, wherein the heating of the freeze-dried platelet derivative composition is carried out in a temperature range of 70°C to 85°C.

25. The method according to any one of claims 1 to 24, wherein a membrane with a pore size of 0.45 μm is used for TFF.

26. The method according to any one of claims 1 to 25, wherein the freeze-dried platelet derivative composition is heated at a temperature range of 75°C to 85°C for 6 to 36 hours to heat-treat the freeze-dried platelet derivative, thereby forming a heat-treated platelet derivative composition.

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