Cryopreserved platelets, their collections, and the processes for preparing them.

Abstract

This disclosure provides processes for preparing cryopreserved platelets, compositions comprising frozen platelets in cryopreservation media, and collections of cryocontainers comprising cryopreserved platelets. In some embodiments, for example at DMSO concentrations, processes for preparing cryopreserved platelets from a platelet pool of two or more donors are provided herein to provide a collection of cryopreserved platelets with improved vial-to-vial and lot-to-lot consistency. Collections of cryocontainers comprising cryopreserved platelets, each having a biomolecular profile representing two or more platelet donors, are also provided herein. Furthermore, compositions comprising frozen platelets are provided herein, which, when stored at a temperature in the range of -10°C to -30°C for at least one month, are capable of yielding a platelet count of at least 1.0 × 10¹¹ / 35 ml of composition, and other enumerated properties.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to U.S. Provisional Application No. 63 / 505,219, filed on 31 May 2023, which is incorporated herein by reference in its entirety.

[0002] Statements regarding government interests This invention was made with government support under contract number W81XWH20C0030, granted by the Defense Health Agency (DHA) of the U.S. Department of Defense. The Government has certain rights in this invention.

[0003] Technical field This disclosure generally relates to blood products, and more specifically to cryopreserved platelets and cryopreserved platelet compositions, and includes processes for preparing them. [Background technology]

[0004] background Platelets in liquid-stored apheresis form are used to treat blood-related problems such as bleeding and thrombocytopenia. Typically, platelets used in blood-related treatments have a shelf life of 5 days. This limits the effectiveness of platelets for blood-related treatments. Furthermore, the short shelf life means that 10-20% of available platelet collections become unusable, potentially resulting in a loss of approximately $200 million per year. Currently, there are no commercially available cryopreserved platelets available for human transfusion.

[0005] The cryopreserved platelets described in this art primarily relate to a single-donor process, meaning that one apheresis unit is processed into one cryopreserved platelet unit. Both apheresis platelet units and single-donor cryopreserved platelet units are single-donor products and therefore possess inherent inter-donor variability. This variability includes several parameters such as dimethyl sulfoxide (DMSO), total platelet count, and platelet concentration in the single-donor product, as well as variations in the percentage of cryoprotective agent. Furthermore, even CPPs made from pooled platelets exhibit excessive lot-to-lot or batch-to-batch variability in these parameters. Thus, there has been a long-standing need in this art to overcome this inter-donor, lot-to-lot, or batch-to-batch variability in cryopreserved platelets or cryopreserved platelet compositions.

[0006] It is known that strict freezing temperature conditions are required for storing cryopreserved platelets, and ultrafreezers are needed to maintain a temperature of ≤-65°C to have functional platelet products for use in, for example, battlefields, hospitals, or other patient treatment centers. However, depending on the geographical location, such battlefields or hospitals may not be equipped with ultrafreezers due to the size of the freezer unit, the cost of one or especially many such freezer units, and / or the lack of adequate power supply, thereby making it difficult to store cryopreserved platelets at such temperatures. Thus, there has been a long-standing need in the art for a cryopreserved platelet composition that is effective in controlling bleeding, can be easily manufactured without highly specialized equipment, and can be stored for months or even years in a standard -20°C freezer. [Overview of the Initiative]

[0007] overview To overcome the above and additional problems in the art, the Disclosure provides embodiments and models comprising frozen platelets, frozen platelet derivatives, cryopreserved platelets, and / or cryopreserved platelet derivatives. In some embodiments and models, pooled cryopreserved platelets (CPP) are provided, which can be aliquoted to multiple doses of the final product from starting materials typically comprising multiple platelet units. The improved processes of the Disclosure produce a final product exhibiting reduced lot-to-lot variability compared to the donor-to-donor variability of current standard therapeutic products (single-donor CPP or apheresis platelet units). The novel process embodiments and models of the Specified enable increased in-process control (IPC) and reduce variability in processing compared to single-donor CPP processes. The processes provided herein facilitate increased control of excipients and frozen volume compared to single-donor processes and previous CPP processes. This increased control reduces variability in DMSO content, dosage volume, and total number of cells administered to the patient, thereby improving the safety profile of the CPP products provided herein. The ability to produce multiple doses, such as multiple cryo-containers of CPP batches from a pool of platelet units, enables quality control testing of the final product for lot releases that cannot be achieved with single-donor CPP.

[0008] Furthermore, in some embodiments, a process for preparing a cryopreserved platelet composition is provided herein, which can be stored at a temperature above -65°C, for example, in the range of -10°C to -30°C, for a period of at least one month, three months, or twelve months, or until the cryopreserved platelets are needed to treat a subject in need. Also provided is a frozen platelet composition which can be stored at a temperature above -65°C, for example, in the range of -10°C to -30°C, for a period of at least one month, three months, or twelve months, or until the cryopreserved platelets are needed to treat a subject in need.

[0009] Therefore, in one embodiment, a process for preparing a cryopreserved platelet composition comprising cryopreserved platelets, wherein the process is i) Freezing a group of platelets in a cryopreservation medium at a temperature of -50°C or lower to form an initial frozen platelet composition, ii) Exposing the initial frozen platelet composition to a temperature of -30°C or higher but below 0°C, or below -1°C, iii) A process is provided herein that includes storing an initial frozen platelet composition at a temperature of -30°C or higher but below 0°C, or below -1°C, to form a cryopreserved platelet composition containing cryopreserved platelets.

[0010] Therefore, in one embodiment, a process for preparing a cryopreserved platelet composition comprising cryopreserved platelets, wherein the process is i) Freezing a group of platelets in a cryopreservation medium at a temperature of -50°C or lower to form an initial frozen platelet composition, ii) A process is provided herein that includes storing an initial frozen platelet composition at a temperature of -30°C or higher but below 0°C, or below -1°C, for at least one month to form a cryopreserved platelet composition.

[0011] Therefore, in one embodiment, a process for preparing a batch of cryopreserved platelets, wherein the process is a) Pooling at least two platelet units in one container and at least one other platelet unit in another container, wherein the platelet units come from two or more donors. b) Centrifugation of each container to obtain a supernatant containing plasma and a pellet containing platelets, c) Resuspending the pellets in each container to form a resuspension, wherein the resuspension has a target weight determined by the number of units pooled or provided in the container. d) Pooling the resuspension from each container and forming the pooled resuspension in the pooled resuspension container, e) Adding a cryoprotectant to a pooled resuspension container having a pooled resuspension to obtain a pooled resuspension having a cryoprotectant, f) Distributing a pooled resuspension containing a cryoprotectant from a pooled resuspension container into several cryocontainers, g) a process comprising freezing a pooled resuspension having a cryoprotectant in a cryocontainer to form a batch of cryopreserved platelets is provided herein. In non-limiting examples, the target weight can be in the range of 15.0–30.0 g, 15.0–29.0 g, 15.0–28.5 g, or, in exemplary embodiments, 15.9 g to 27.9 g times the number of units pooled or provided in the container. In exemplary embodiments, the cryoprotectant is or comprises DMSO.

[0012] Accordingly, in one embodiment, a collection of cryocontainers containing cryopreserved platelets is provided herein, wherein the cryopreserved platelets in each cryocontainer have a biomolecular profile indicating two or more platelet donors, and the concentration of the cryoprotectant in the cryopreserved platelets in the first cryocontainer, DMSO in an exemplary embodiment, is within 15%, 12%, 10%, 9%, 7%, 5%, 3%, 2%, 1%, or 0.5% of the concentration of the cryoprotectant in the cryopreserved platelets in the second cryocontainer, DMSO in an exemplary embodiment. In some embodiments, the collection comprises at least 2, 5, 10, 15, 20, or more cryocontainers from one, or in exemplary embodiments, 2, 3, 4, 5, or more batches, wherein the cryocontainers in one batch have the same set of biomolecular profiles, and each batch in the collection has a different set of biomolecular profiles from any other batch in the collection. In some embodiments, each batch contains exactly 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more cryocontainers.

[0013] Accordingly, in one embodiment, a cryopreserved platelet composition comprising cryopreserved platelets, wherein the cryopreserved platelets are stored at approximately -20°C to -60°C for a period of at least one month, is provided herein.

[0014] Therefore, in one embodiment, a composition comprising platelets frozen in a cryopreservation medium in a frozen state, wherein the composition, after storage for at least 1 month, 2, 3, 4, 5, or 6 months, exhibits the following properties upon thawing: a) Being in a liquid state, and not requiring the addition of any liquid to achieve such a liquid state. b) at least 1.0 × 10 11 To exhibit the platelet count per 35 ml of composition, c) To produce a single peak corresponding to a damaged membrane peak in a membrane integrity assay, d) The composition contains less than 50% CD61-positive particles, and e) A composition is provided herein that is capable of producing one or more of the following in an in vitro thrombin generation assay:

[0015] Further details relating to aspects and embodiments of this disclosure are provided throughout this patent application. Sections and section headings are for readability purposes only and are not intended to limit any combination of disclosures, such as methods, compositions, and kits, or functional elements contained therein, across sections. Further details relating to aspects and embodiments of this disclosure are provided throughout this patent application. Sections and section headings are for readability purposes only and are not intended to limit any combination of disclosures, such as methods, compositions, or other functional elements contained therein, across sections. [Brief explanation of the drawing]

[0016] [Figure 1A]This is a non-limiting flowchart of exemplary process steps for preparing a batch or lot of cryocontainers containing cryopreserved platelets. [Figure 1B] This is a non-limiting flowchart of exemplary process steps for preparing a batch or lot of cryocontainers containing cryopreserved platelets. [Figure 1C] This is a schematic of a tube tree that can be used by one of the embodiments of the process described herein. [Figure 2A] A non-limiting flowchart of an exemplary process for preparing cryopreserved platelet compositions that can be stored at temperatures in the range of -10°C to -30°C is shown. [Figure 2B] A non-limiting flowchart of an exemplary process for preparing a cryopreserved platelet composition is shown, integrating the exemplary processes of Figures 1A and 2A. [Figure 3] This shows the target %DMSO that can be achieved using the calculations provided in Vitalant's single-donor process for preparing cryopreserved platelets. [Figure 4A] This shows the frozen volume distribution of 255 units prepared by a single-donor process using Vitalant for cryopreserved platelets. [Figure 4B] This shows the frozen volume distribution of units with aggregates observed within 6 hours after thawing and resuspension for 32 units. [Figure 5] This specification discloses a single-donor method for preparing cryopreserved platelets of Vitalant, and a comparison of the correlation between APC volume and %DMSO in the CPP pooling process. [Figure 6] This specification shows the correlation between the post-expression volume (resuscitation volume) and %DMSO of the CPP pooling process disclosed herein. [Figure 7] This shows the percentage recovery rate of platelets for batches stored at -80°C (single-temperature cryopreserved product) and -20°C (transition temperature cryopreserved product). [Figure 8]This paper shows the percentage aggregation of platelets for batches stored at -80°C (single-temperature cryopreserved products) and -20°C (transition-temperature cryopreserved products), as well as a comparison with apheresis platelets containing arachidonic acid (AA), collagen, and thrombin receptor-activating peptide 6 (TRAP-6). [Figure 9] The peak distributions for platelets stored at -80°C (single-temperature cryopreserved product), indicated by "#", and at -20°C (transition-temperature cryopreserved product), indicated by "*", as well as for apheresis platelets, indicated by "+". [Modes for carrying out the invention]

[0017] definition As used herein, “cryopreserved platelets” refers to frozen platelets that are in a liquid state upon thawing, regardless of whether any liquid is added to the frozen platelets after thawing. Therefore, cryopreserved platelets are not fresh platelets and are not freeze-dried platelet derivatives. During processing, cryopreserved platelets are not dried. The term “cryopreserved platelets” does not implicitly mean any shortest time for which such platelets exist in a frozen state. However, cryopreserved platelets are typically stable for at least 1, 2, 3, 4, 5, 6, 9, or 12 months, and in exemplary embodiments, stable for at least 18, 24, 36, or 48 hours. Cryopreserved platelets are typically suspended in a cryoprotectant in a frozen state until thawed before use. In some embodiments herein, cryopreserved platelets are stored at a temperature of -20°C for at least 1, 2, 3, 4, 5, 6, 9, or 12 months.

[0018] When used herein in the context of cryopreserved platelets, or compositions comprising frozen platelets and / or platelet derivatives, “platelet derivative” implies particles that, unlike fresh platelets, do not possess an intact cell membrane, as demonstrated, for example, by a calcein AM membrane integrity assay (see, for example, Example 8). Therefore, in some embodiments, the compositions provided herein contain platelet derivatives, and such platelet derivatives are not freeze-dried platelet derivatives.

[0019] Unless otherwise noted, technical terms are used according to their conventional usage. Definitions of common terms in molecular biology can be found in Benjamin Lewin, Genes V, published by Oxford University Press, 1994 (ISBN 0-19-854287-9), Kendrew et al. (eds), The Encyclopedia of Molecular Biology, published by Blackwell Science Ltd., 1994 (ISBN 0-632-02182-9), and Robert A. Meyers (ed.), Molecular Biology and Biotechnology: a Comprehensive Desk Reference, published by VCH Publishers, Inc., 1995 (ISBN 1-56081-569-8). Unless otherwise noted, all technical and scientific terms used herein have the same meanings as those generally understood by those skilled in the art in which this disclosure pertains. The singular terms “a,” “an,” and “the” refer to multiple objects unless the context clearly indicates otherwise. “A or B” means A or B, or A and B. Furthermore, it should be understood that all base sizes or amino acid sizes, and all molecular weight or molecular mass values ​​given for nucleic acids or polypeptides are approximate and provided for illustrative purposes only.

[0020] Furthermore, the ranges provided herein are understood to be abbreviations for all values ​​within that range. For example, the range 1–50 is understood to include any number, combination of numbers, or subrange from the group consisting of 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 1–49, 1–25, 1.7–31.9, etc. (including fractions unless the context explicitly indicates otherwise). Any concentration range, percentage range, ratio range, or integer range should be understood to include any integer values ​​within the listed range, and, where appropriate, fractions thereof (such as one-tenth and one-hundredth of an integer), unless otherwise indicated. Similarly, any numerical ranges listed herein with respect to any physical characteristics of a polymer, such as subunits, size, or thickness, should be understood to include any integers within the listed range, unless otherwise indicated. Given a range where several low values ​​and several high values ​​overlap, a person skilled in the art will recognize that the selected range includes lower values ​​less than those higher values.

[0021] As used herein, the symbol "<" may mean less than, or in the context of temperature, less than the listed temperature. As used herein, the symbol " / " may mean including a range of temperatures in the context of temperature; for example, -20°C+ / -2°C means temperatures between -18°C and -22°C. As used herein, "about" or "essentially consisting of" means ±10% of the range, value, or structure indicated, unless otherwise indicated. As used herein, the terms "include" and "comprise" are used synonymously. As used herein, "comprising" is synonymous with "including," "containing," or "characterizing," and is comprehensive or open-ended and does not exclude additional unlisted elements or method steps. As used herein, "consists of" excludes any elements, steps, or components not specified in the elements of the claims. As used herein, “essentially consisting of” does not exclude materials or steps that do not substantially affect the basic and novel characteristics of the claims. In each case herein, any of the terms “comprising,” “essentially consisting of,” and “consisting of” may be replaced with any of the other two terms. Inventions described herein exemplary may be preferably put into practice even without any elements or limitations not specifically disclosed herein.

[0022] Similar or equivalent methods and materials may be used in the practice or testing of this disclosure, but preferred methods and materials are described below. All published publications, patent applications, patents, and other references referenced herein are incorporated in their entirety by reference. In the event of any conflict with other statements, including definitions of terms, the statements herein shall prevail. In addition, the materials, methods, and examples are illustrative and not intended to limit the scope of this disclosure.

[0023] For clarity, certain features of aspects and embodiments described herein that are considered in the context of separate embodiments may also be provided in combination in a single embodiment. Conversely, various aspects and embodiments considered in the context of a single aspect or embodiment for brevity may also be provided separately or in any preferred subcombination. All combinations of aspects and embodiments are specifically encompassed and disclosed herein as if every possible combination were individually expressly disclosed. In addition, all subcombinations of various aspects and embodiments and their elements are also specifically disclosed herein, even if every possible subcombination is not individually expressly disclosed herein.

[0024] While embodiments of this disclosure are adaptable to various modifications and alternative forms, specific embodiments are shown in the drawings as examples and are described in detail below. However, the intent is not to limit this disclosure to the specific embodiments described. In contrast, this disclosure is intended to cover all modifications, equivalents, and alternatives that fall within the scope of this disclosure as defined by the appended claims.

[0025] Any invention disclosed or claimed herein is to be understood to encompass all variations, combinations, and rearrangements of any one or more features described herein. One or more features may be expressly excluded from the claims unless such specific exclusions are expressly stated herein. Furthermore, disclosure of reagents for use in a particular method is intended, unless otherwise understood by those skilled in the art, to be synonymous with (and support) the method involving the use of such reagents, either according to the specific method disclosed herein or any other method known in the art. In addition, where herein and / or claims disclose a particular method, one or more of the reagents disclosed herein may be used in that method, unless otherwise understood by those skilled in the art.

[0026] Detailed explanation This disclosure addresses many long-standing needs and problems in the art, including, but not limited to, those mentioned in the “Background Art” section of this specification. To overcome the above and additional problems in the art, this disclosure provides embodiments and models comprising frozen platelets, frozen platelet derivatives, cryopreserved platelets, and / or cryopreserved platelet derivatives. In some embodiments and models, pooled cryopreserved platelets (CPP) are provided, which can be aliquoted from a starting material typically comprising multiple platelet units into multiple doses of the final product. The processes according to this disclosure produce a final product exhibiting reduced lot-to-lot or batch-to-batch variability compared to the donor-to-donor variability of current standard therapeutic products (single-donor CPP or apheresis platelet units). The embodiments and models of this specification enable increased in-process control (IPC) and reduce variability in processing compared to single-donor CPP processes. The processes provided herein facilitate increased control of excipients and frozen volume compared to single-donor processes and previous CPP processes. This increased control reduces variability in DMSO content, dosage volume, and total number of cells administered to the patient, thereby enhancing the safety profile of the CPP product provided herein. The ability to produce multiple doses, such as multiple cryo-containers of batches of CPP from a pool of platelet units, enables quality control testing of the final product for lot releases that cannot be achieved with single-donor CPP. Additionally, because multiple doses are produced from a common pool, the process allows for direct comparisons for stability testing and the production of archival samples for subsequent analysis. Furthermore, the exemplary process herein uses approximately 90% less cryoprotective agents, such as DMSO, compared to cryoprotected platelets in current standard treatment, to achieve batches or lots of cryopreserved platelets. The exemplary process herein also provides improved batch-to-batch consistency in cryoprotective agent concentrations, such as dimethyl sulfoxide (DMSO) concentration, total platelet count, and platelet concentration, compared to current standard treatment CPP products.

[0027] For example, the process for preparing batches of cryopreserved platelets disclosed herein, and the collection of cryo-containers containing cryopreserved platelets, provide cryopreserved platelets that are uniform within a batch and across multiple batches. The observed uniformity may include, but is not limited to, the concentration of DMSO, the platelet concentration, and the total number of platelets in the cryopreserved platelets. Such uniformity is generally not observable in cryopreserved platelets produced from a single unit of platelets due to inter-donor variability or previous processes for producing CPP using a pool of donors.

[0028] Accordingly, as illustrated in Figure 1A, in some embodiments, a process is provided for preparing a batch or multiple batches of cryopreserved platelets. Steps in such embodiments are shown in boxes in Figure 1A, and optional steps in such embodiments are shown in dashed boxes. Such methods may include, in non-limiting examples, the following steps: a) Step (110) Obtaining or providing platelet units from two or more donors, b) A step (120) in which at least two platelet units are pooled in one container and at least another platelet unit in another container, such that at the end of the step there are two or more containers. c) Centrifugation of each container to obtain a supernatant containing plasma and a pellet containing platelets (130) d) step (140), a step of resuspending pellets in each container to form a resuspension, wherein the resuspension has a target weight determined by the number of units pooled in the container or provided. e) A step of pooling the resuspension from each container and forming the pooled resuspension in a pooled resuspension container (150), f) Adding a cryoprotectant to a pooled resuspension container having a pooled resuspension to obtain a pooled resuspension having a cryoprotectant (160) g) The step of distributing a pooled resuspension containing a cryoprotectant from a pooled resuspension container among several cryo containers (170), and h) Step (180) Freeze the pooled resuspension with cryoprotectant in a cryocontainer to form a batch of cryopreserved platelets.

[0029] Typically, at least 3, 4, or 5 platelet units are provided (110) and available for pooling (120). The platelet units may be from strictly or more than 2, 3, 4, or 5 donors. In non-limiting examples, the target weight may be in the range of 15.0–30.0 g, 15.0–29.0 g, 15.0–28.5 g, or, in exemplary embodiments, 15.9 g to 27.9 g times the number of units pooled or provided in the container. Steps preceding the resuspension step (140) may include removing a portion of the supernatant to achieve the target weight of the resuspension. Alternatively, steps preceding the resuspension step may include removing a portion of the supernatant and adding a buffer composition before resuspension within the target weight. In exemplary embodiments, the steps of pooling the resuspension (150) and adding a cryoprotectant (160) may be carried out using a tube tree system. In exemplary embodiments, adding a cryoprotectant (160) may include adding dimethyl sulfoxide (DMSO) as a cryoprotectant, and the addition is carried out until the target weight of DMSO to be added is achieved. Typically, the addition of DMSO (160) is carried out using the same tube tree system used to pool the resuspension (150). In exemplary embodiments, dispensing the pooled resuspension (170) may be carried out using a dosing tree system. In some embodiments, the dosing tree system may have a different design from the tube tree system, or may be different units or the same units after washing the units.

[0030] In addition to the above, to address the long-standing need to store cryopreserved platelets at temperatures above -65°C, for example, in a -20°C freezer, the Disclosure provides a process for forming cryopreserved platelets, which in some exemplary embodiments are cryopreserved platelet derivatives, the process including a transition of freezing temperature from an initial freezing temperature to a storage freezing temperature. Accordingly, in some embodiments, the Disclosure provides a process comprising the steps of forming an initial frozen platelet composition by initial freezing at a temperature of -50°C, -60°C, -65°C, -70°C, -80°C, -85°C, or below -90°C, or in the range of -50°C to -85°C or -60°C to -85°C (i.e., initial temperature), and then storing the initial frozen platelet composition in a frozen state at a temperature of -30°C or above but below 0°C (i.e., storage temperature) to form a cryopreserved platelet composition. Surprisingly, the cryopreserved platelets formed by the processes disclosed herein have been found to be stable when stored at higher temperatures compared to the temperatures required to store conventional cryopreserved platelets, and to retain their hemostatic ability. For example, cryopreserved platelets prepared by the processes disclosed herein can be stored at temperatures of approximately -30°C, -25°C, -20°C, -15°C, -10°C, or -5°C, or in the range of -30°C to -5°C or -30°C to -5°C, for at least 1 month to a maximum of 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or at least or a maximum of 1 year, 2, 3, 4, 5, or 6 years, or 6 months to 1 year, 2, 3, 4, 5, or 6 years. Thus, as illustrated in Figure 2A, in some embodiments, a process for preparing cryopreserved platelet compositions is provided. Exemplary steps of such embodiments are shown in the boxes in Figure 2A. Such methods may include, in non-limiting examples, the following steps: a) Step (280) to freeze platelets in cryopreservation medium at a temperature of -50°C or below (or, in some embodiments, -55°C or below, or -60°C or below) to form an initial frozen platelet composition, and b) The initial frozen platelet composition is stored for at least one month at a temperature in the range of -30°C to -10°C (in some embodiments, in a freezer set to about -20°C) to form a cryopreserved platelet composition (290). In some embodiments, the cryopreserved platelets formed in this manner are cryopreserved platelet derivatives. Furthermore, in exemplary embodiments, the cryopreserved platelet composition or cryopreserved platelet derivative composition has one or more of the enumerated properties provided herein for frozen platelets and / or frozen platelet derivative compositions.

[0031] In other embodiments, a process for preparing cryopreserved platelet compositions is provided herein, as illustrated in Figure 2B. Steps in such embodiments are shown in the boxes in Figure 2B. Such methods may, in non-limiting examples, include the following steps: a) Step (210) Obtaining or providing platelet units from two or more donors, b) A step (220) in which at least two platelet units are pooled in one container and at least another platelet unit in another container, such that at the end of the step there are two or more containers. c) Centrifugation of each container to obtain a supernatant containing plasma and a pellet containing platelets (230) d) step (240) a step of resuspending pellets in each container to form a resuspension, wherein the resuspension has a target weight determined by the number of units pooled in the container or provided, e) A step of pooling the resuspension from each container and forming the pooled resuspension in a pooled resuspension container (250), f) Adding a cryoprotectant to a pooled resuspension container having a pooled resuspension to obtain a pooled resuspension having a cryoprotectant (260), and g) The step of distributing the pooled resuspension having a cryoprotectant from the pooled resuspension container among several cryo containers (270). In some embodiments, the pooled resuspension in the cryo containers is frozen and stored at a temperature of -50°C or below.

[0032] Optionally, in some embodiments, the transition temperature freezing protocol is performed on pooled resuspensions in cryocontainers. Such embodiments include: h) freezing the pooled resuspensions in cryocontainers at a temperature below -50°C to form cryocontainers, each containing an initial frozen platelet composition (285); and i) storing the cryocontainers containing the initial frozen platelet compositions at a temperature in the range of -30°C to -10°C for at least one month to form cryocontainers containing cryopreserved platelet compositions (295).

[0033] Preparation of cryopreserved platelets using transitions at freezing temperatures In one embodiment, a process for preparing cryopreserved platelets is provided herein, comprising: an initial freezing step of freezing platelets in a cryopreservation medium, or freezing a pooled resuspension having a cryoprotectant disclosed herein at a temperature of -50°C, -55°C, -60°C or lower, in exemplary embodiments, at a temperature of -65°C, -70°C, -75°C, or -80°C or lower, to form an initial frozen platelet composition; and a second step of storing the initial frozen platelet composition in a frozen state at a temperature of -40°C, -35°C, -30°C, -25°C or higher, in exemplary embodiments, at a temperature of -20°C, -15°C, or -10°C or higher but below 0°C, to form a batch of cryopreserved platelets, a cryopreserved platelet composition, or a batch of cryopreserved platelets. In some embodiments, compositions containing cryopreserved platelets obtained from a process that uses a transition in freezing temperature from an initial temperature below a certain initial target temperature set to a target initial temperature or temperature range of at most -50°C, and then, after a period of time, is stored at a target storage temperature set to a target storage temperature of about -10°C to about -30°C, may be referred to as transition-temperature cryopreserved products or transition-temperature cryopreserved compositions, and such a process may be referred to as a transition-temperature cryopreservation process. Also, for convenience of distinguishing transition-temperature cryopreserved products from cryopreserved products that do not involve such temperature transitions in their preparation, in some embodiments, cryopreserved products obtained only by freezing and storing at a target temperature of -60°C or below, for example, about -80°C, are referred to as single-temperature cryopreserved products. Those skilled in the art will understand that such single-temperature cryopreserved products can actually be subjected to temperature fluctuations, but such fluctuations do not involve a transition from an initial freezing temperature below -50°C to a target storage temperature of -10°C to -30°C.Remarkably, cryopreserved platelets obtained by processes involving temperature transitions as disclosed herein, when stored at temperatures above -30°C but below 0°C, or at -5°C, can exhibit hemostatic properties upon thawing, and in exemplary embodiments, can reduce bleeding and increase platelet counts in subjects where this is necessary, for example, by increasing platelet counts in thrombocytopenic patients or by generating thrombin in in vitro thrombin generation assays, thereby addressing long-term needs in storage conditions for cryopreserved platelets.

[0034] In some embodiments, the initial frozen platelet composition can be stored at freezing temperatures above -40°C, -35°C, -30°C, -25°C, and -20°C. In some embodiments, the initial frozen platelet composition can be stored for at least 30 minutes, 1 hour, 2, 3, 6, 8, 10, 12, 18, 24 hours, 2 days, 3, 5, 7, 15, 20, 25 days, 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, or 1 year, 2, 3, 4, 5, or 6 years. In some embodiments, the initial frozen platelet composition can be stored over periods ranging from 1 month to 10 years, 1 month to 8 years, 1 month to 6 years, 1 month to 5 years, 1 month to 3 years, 1 month to 2 years, 1 month to 1 year, 6 months to 10 years, 1 year to 10 years, 2 years to 10 years, or 3 years to 10 years. In some cases, the initial frozen platelet composition can be stored at temperatures ranging from -40°C to -10°C until the cryopreserved platelets are used to treat a subject in need, in exemplary embodiments, to administer to the subject to reduce bleeding in the subject.

[0035] Typically, the initial freezing step involves freezing a cryopreservation medium containing platelets, or a pooled resuspension containing a cryoprotectant disclosed herein, to achieve a temperature disclosed herein, thereby forming an initial frozen platelet composition. For example, freezing a cryopreservation medium containing platelets, or a pooled resuspension containing a cryoprotectant disclosed herein, at a temperature in the range of -50°C to -85°C involves subjecting the cryopreservation medium or pooled suspension to that temperature range so that an initial frozen platelet composition is formed at the end of the step, and the temperature of the initial frozen platelet composition is in the range of -50°C to -85°C. The initial freezing step can be carried out for a period of time until the cryopreservation medium containing platelets or the pooled resuspension containing a cryoprotectant reaches a temperature of -50°C, -55°C, -60°C, -65°C, -70°C, -75°C, or -80°C or lower, and the time it takes for the cryopreservation medium or the pooled resuspension to reach the temperature can depend on various factors, but are not limited to, the volume of the cryocontainer, the dimensions of the cryocontainer, the volume of the cryopreservation medium containing platelets, the concentration of platelets in the cryocontainer, and the composition of the cryopreservation medium in the cryocontainer. The cryopreservation medium that can be used in the process disclosed herein may be a cryopreservation medium containing a cryoprotectant. In exemplary embodiments, the cryoprotectant comprises dimethyl sulfoxide (DMSO). In other embodiments, the cryoprotectant may be any other cryoprotectant other than DMSO. Other non-limiting examples of suitable cryoprotectants may include sugars such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, xylose, and combinations thereof. In some embodiments, the cryopreservation medium containing DMSO as a cryoprotectant can have concentrations ranging from 0.001 to 10%, 0.5 to 7%, 1 to 8%, 2 to 8%, 3 to 8%, 4 to 8%, or 5 to 8%. In some embodiments, the initial freezing step can be performed for at least 30 minutes, 1 hour, 2 hours, or 3 hours. For example, the initial freezing step can be performed over a period ranging from 30 minutes to 12 hours, 30 minutes to 10 hours, 30 minutes to 8 hours, 30 minutes to 6 hours, or 30 minutes to 4 hours.In some embodiments, the initial freezing step can be performed for 12 hours, 2 days, 3 days, 1 week, 1 month, or longer than 6 months. In some embodiments, the temperature during the initial freezing step can be in the range of -50°C to -90°C, -50°C to -85°C, -50°C to -80°C, -50°C to -75°C, -50°C to -70°C, -55°C to -90°C, -60°C to -90°C, -60°C to -85°C, -60°C to -80°C, -60°C to -75°C, or -65°C to -75°C. In exemplary embodiments, the temperature during the initial freezing step can be -65°C+ / -5°C, -65°C+ / -4°C, -65°C+ / -3°C, -65°C+ / -2°C, or -65°C+ / -1°C. In other exemplary embodiments, the temperature during the initial freezing step may be -80°C ± 5°C, -80°C ± 4°C, -80°C ± 3°C, -80°C ± 2°C, or -80°C ± 1°C. In some embodiments, the duration during the initial freezing step may depend on the temperature to be achieved. For example, the initial freezing step may include a duration ranging from 1 to 7 hours, 1 to 6 hours, 1 to 5 hours, 1 to 4 hours, or 1 to 2 hours, and a temperature ranging from -60°C to -80°C. In some embodiments, the initial freezing step includes placing a cryopreservation medium containing platelets, or a pooled resuspension containing a cryoprotectant, into a freezer set to a temperature ranging from -50°C to -90°C to form an initial frozen platelet composition.

[0036] In some embodiments, storing the initial frozen platelet composition includes exposing the initial frozen platelet composition to temperatures of -30°C, -25°C, -20°C, -15°C, or -10°C or higher but below -5°C. In exemplary embodiments, the initial frozen platelet composition is exposed to temperatures of -20°C+ / -5°C, -20°C+ / -4°C, -20°C+ / -3°C, -20°C+ / -2°C, -20°C+ / -1°C, or -20°C+ / -0.5°C. In some embodiments, storing the initial frozen platelet composition includes storing it in a freezer set to a temperature in the range of -10°C to -40°C, -10°C to -30°C, or -15°C to -25°C. Typically, an initial frozen platelet composition, when stored at or exposed to the temperatures disclosed herein, reaches the intended temperature at the end of the step for forming cryopreserved platelets, thereafter the cryopreserved platelets are stored at the temperatures disclosed herein for at least 7, 10, 15, 20, 25 days, 1 month, 2, 3, 4, 6, 8, 10, 12 months, 1 year, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, or until the cryopreserved platelets are used for administration to or treatment of a subject in need. In some embodiments, storing an initial frozen platelet composition may include storing it at a freezing temperature of -30°C or higher for at least 30 minutes, 45 minutes, 50 minutes, 60 minutes, 70 minutes, 80 minutes, 90 minutes, 2 hours, or 3 hours to form cryopreserved platelets. In some embodiments, the processes disclosed herein may include exposing an initial frozen platelet composition to a temperature of -30°C or higher, or in exemplary embodiments, in the range of -10°C to -30°C, for a period of time until the temperature of the initial frozen platelet composition reaches -30°C or higher, or in exemplary embodiments, in the range of -10°C to -30°C, to form cryopreserved platelets. Typically, once the temperature of the initial frozen platelet composition reaches the range of -10°C to -30°C to form a cryopreserved composition, the cryopreserved composition is stored at a temperature of -10°C to -30°C until the cryopreserved platelets are used to treat or to administer to a subject that needs them.

[0037] Composition containing frozen platelets In some aspects and embodiments, the compositions provided herein have one or more enumerated properties (which may also be referred to as enumerated attributes or enumerated features). It will be understood that a composition falling under such an aspect or embodiment that includes one or more enumerated properties exhibits such one or more enumerated properties, but that it is not necessary for the steps to actually be performed to demonstrate one or more enumerated properties to fall under such an aspect or embodiment that includes one or more enumerated properties. However, those skilled in the art will understand that such one or more enumerated properties of a composition can be identified by using the method described by the enumerated property or by performing known methods to determine whether a test composition has such one or more enumerated properties. Frozen compositions herein that include platelets and / or platelet derivatives exhibit the following non-limiting enumerated properties upon thawing: a) at least 1.0 × 10 in 35 ml 11 The composition exhibits one or more of the following characteristics: a) it is possible to exhibit a platelet count of 1, b) it contains approximately 50% to 99% platelets and / or platelet-derived particles in the range of approximately 1 μm to approximately 2.5 μm or 5 μm, c) it is in a liquid state and does not require the addition of liquid to achieve such a liquid state, d) it yields a single peak corresponding to a damaged membrane peak in a membrane integrity assay, e) it has a CD61-positive microparticle content of less than 50% of the CD61-positive particles in the composition, f) it has the ability to generate thrombin in an in vitro thrombin generation assay, g) it is possible to induce aggregation under in vitro aggregation conditions including an agonist, h) it exhibits swirl when the composition is visually observed, i) it exhibits a lack of aggregation when the composition is visually observed, and / or j) it exhibits lactoadherin positivity in the range of 80 to 99.5%.

[0038] In one embodiment, a composition comprising frozen platelets, or in exemplary embodiments, frozen platelet derivatives, in a cryopreservation medium in a frozen state is provided herein. In some embodiments, a composition comprising frozen platelets in a cryopreservation medium is a composition comprising cryopreserved platelets and / or cryopreserved platelet derivatives. Typically, a composition comprising frozen platelets is in a liquid state upon thawing, without the addition of any liquid such as water or buffer. Although not bound by any theory, since the cryopreservation process does not involve a drying step, platelets in the cryopreservation medium are frozen so that the cryopreservation medium is subjected to freezing temperatures, and because there is no drying step, the cryopreservation medium containing platelets is in a liquid state upon thawing. In some embodiments, a composition of the Specified, when stored at a temperature in the range of -10°C to -40°C for at least 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1 year, 2, 4, 5, 8, or 10 years, contains at least 1.0 × 10⁻¹⁰ of the composition. 11 pieces, 1.2×10 11 pieces, 1.4×10 11 pieces, 1.6×10 11 pieces, or 1.7 × 10 11 It is possible to exhibit a platelet count of 20-35 ml. For example, when the compositions herein are stored at a temperature in the range of -10°C to -40°C for at least 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1 year, 2, 4, 5, 8, or 10 years, at least 1.0 × 10¹³ plates can be found in a cryo-container, cryovial, or cryo-bag with a volume of 35, 30, 25, or 20 ml, or a volume of approximately 20-35 ml. 11It is possible to obtain a platelet count of a certain number. The platelet count can be performed using an automated blood analyzer or manually using a hemocytometer. For example, the platelet count of a sample, such as a thawed platelet sample, can be determined using a blood analyzer, such as a Beckman Coulter AcT Diff2 blood particle analyzer or a Beckman Coulter DxH blood analyzer (Beckman Coulter, beckmancoulter.com). Blood analyzers are known to be based on the Coulter principle, an electronic method for counting and classifying particles by size. While many types of particles can be counted and classified by size using the Coulter principle, a specific application of this principle in hematology is for counting and classifying leukocytes (WBCs), red blood cells (RBCs), and platelets (PLTs). In a non-limiting manner, the platelet count in the compositions herein may be derived from an internal continuous PLT / RBC histogram. Particles from 0 to 70 fL are counted and classified by size as they pass through the RBC aperture. Raw data is evaluated using proprietary platelet algorithms such as DxH (available with Beckman Coulter DxH blood analyzers) to identify platelet populations. The system also performs feature analysis to identify interference patterns at the lower and upper limits of the PLT histogram. The algorithm uses both raw PLT data and fitted histograms for this process to determine PLT interference patterns depending on the severity of the interference, and corrects or flags the results. Evaluation of the platelet histogram is improved in accuracy by excluding interference from debris, microbubbles, red blood cell fragments, or very small red blood cells. As a non-limiting example of platelet counting technology, Example 5 and Table 6 demonstrate data on platelet count per bag by using either a Beckman Coulter AcT Diff2 blood particle analyzer or a Beckman Coulter DxH blood analyzer.In some embodiments, platelets can be counted by considering platelets having diameters in the range of 0.5–5 μm, 1–4 μm, 1–3 μm, 1–2.5 μm, 1.5–3 μm, or, in exemplary embodiments, 0.5–2.5 μm or 2.5–5.0 μm, as typically measured by flow cytometry or light scattering. In some embodiments, platelets can be counted by considering platelets having a diameter of at least 0.5 μm or at least 1 μm, as typically measured by flow cytometry or light scattering. In some embodiments, particles in a composition with a diameter of less than 1 μm are typically fine particles, as typically measured by flow cytometry or light scattering. In some embodiments, particles in a composition with a diameter of less than 0.5 μm are typically fine particles, as typically measured by flow cytometry or light scattering. As a non-limiting example of platelet counting techniques, flow cytometry can be used to sort and count platelets or platelet derivatives in the compositions herein. As is known in the art, different techniques are available for measuring the particle size of platelets, platelet derivatives, and microparticles, such as platelet-derived microparticles. One such technique that can be used to measure particle size in a non-limiting manner is flow cytometry. Flow cytometry is a technique for quantifying cellular characteristics such as cell number, size and complexity, fluorescence, phenotype, and viability. Generally, forward scattering in flow cytometry is positioned along the laser intercept and is typically considered a measure of relative cell size. Lateral scattering is typically positioned perpendicular to the laser beam intercept and is used to measure the relative complexity of cells. Commercially available sizing beads can be used to obtain forward scattering values ​​for calibrating the instrument to measure particle size. The gate used to measure the particle size distribution in the compositions disclosed herein is described using a forward scattering height (FSC-H) signal produced by a latex bead of known diameter.For example, using commercially available sizing beads of 0.5 μm and 2.5 μm, the size gate range in a flow cytometry instrument for counting particles smaller than 0.5 μm, such as microparticles or platelet-derived microparticles, can be set to count platelets or platelet derivatives in the 0.5 μm and 2.5 μm range. In some embodiments, a composition comprising frozen platelets or frozen platelet derivatives, or cryopreserved platelets or cryopreserved platelet derivatives in a frozen state, exhibits a platelet count recovery rate of at least 65%, 70%, or 75% upon thawing when stored at temperatures in the range of -10°C to -40°C for at least 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12 months, 1 year, 2, 4, 5, 8, or 10 years, in exemplary embodiments. For example, the platelet recovery rate can be in the range of 60%–95%, 65%–95%, 70%–95%, or 75%–95%, 70%–99%, 72%–99%, or 75%–99%. Those skilled in the art will understand that the platelet recovery rate can be evaluated by comparing the number of platelets in the composition before freezing and after thawing to determine the number after storage. In a non-limiting example, the percentage platelet recovery rate can be evaluated using a Beckman Coulter AcT Diff 2 blood particle analyzer or a Beckman Coulter DxH blood analyzer (see Example 7).

[0039] In some embodiments, compositions comprising frozen platelets and / or platelet derivatives as herein, when stored for at least 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months at temperatures ranging from -10°C to -40°C, may have a CD61-positive particulate matter content of less than 80%, 75%, 70%, 65%, 60%, and in exemplary embodiments, less than 50%, 40%, 30%, or 25% upon thawing. In some embodiments, the CD61-positive particulate matter content from all particles, including platelets, platelet derivatives, and particulate matter, is in the range of 1-30%, 1-25%, 1-20%, 1-15%, 5-30%, 5-25%, or 5-20%. In some embodiments, particulate matter, or CD61-positive particulate matter, is typically a particle with a diameter of less than 0.5 μm, as measured by flow cytometry or light scattering. In some embodiments, microparticles, or CD61-positive microparticles, are typically particles with a diameter of less than 0.25 μm when measured by flow cytometry or light scattering. In some embodiments, microparticles, or CD61-positive microparticles, are typically particles with a diameter of less than 1 μm when measured by flow cytometry or light scattering. In some embodiments, at least 70%, 75%, or 80% of the particles in the composition, typically containing platelets, platelet derivatives, and microparticles, are positive for lactoadherin. For example, lactoadherin-positive particles in the composition may be in the range of 70% to 99%, 75% to 99%, or 80% to 99% of the particles in the composition. Analysis using flow cytometry-based sorting and counting is a non-limiting technique for calculating the percentage positivity of CD-61-positive particulate matter and lactoadherin-positive particles. Example 9 demonstrates a technique for calculating the percentage positivity of CD-61-positive particulate matter and lactoadherin-positive particles in the compositions disclosed herein, and the data are presented in Table 8. Various known techniques can be used to determine the particle sizes of various populations in the compositions disclosed herein.For example, in some embodiments, flow cytometry forward scattering is used to determine particle size. In other embodiments, light scattering methods such as Thrombolux dynamic light scattering are used to determine particle size.

[0040] In some embodiments, compositions comprising frozen platelets and / or platelet derivatives provided herein, in exemplary embodiments, include platelets and / or platelet-derived particles such as platelet derivatives having a particle size (e.g., diameter or maximum dimension) of at least about 0.4 μm, at least about 0.5 μm, at least about 0.6 μm, at least about 0.7 μm, at least about 0.8 μm, at least about 0.9 μm, or at least about 1.0 μm, when thawed. In some embodiments, the cryopreserved platelets consist of approximately 50% to approximately 99% platelets (e.g., approximately 55% to approximately 95%, approximately 60% to approximately 90%, approximately 65% ​​to approximately 85%, approximately 70% to approximately 80%) and / or platelet-derived particles in the range of approximately 0.3 μm to approximately 5.0 μm in diameter, approximately 0.5 μm to approximately 5.0 μm (e.g., approximately 0.4 μm to approximately 4.0 μm in diameter, approximately 0.5 μm to approximately 2.5 μm in diameter, approximately 0.6 μm to approximately 2.0 μm in diameter, approximately 1 μm to approximately 5.0 μm in diameter, approximately 1 μm to approximately 4.0 μm in diameter, approximately 1.5 μm to approximately 4.5 μm in diameter, or approximately 1 μm to approximately 3.0 μm in diameter).

[0041] In some embodiments, a composition comprising cryopreserved platelets and / or platelet derivatives, or frozen and stored platelets and / or platelet derivatives, can exhibit the ability to generate thrombin in an in vitro thrombin generation assay upon thawing when stored at a temperature in the range of -10°C to -40°C for at least 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months. One of ordinary skill in the art can evaluate thrombin generation using any known test(s). For example, thrombin generation can be evaluated by a thrombin generation assay, which can be performed by a semi - automated method using, for example, a calibrated automated thrombogram or a fully automated system. A thrombin generation assay is a type of coagulation test and is based on the potential of plasma to generate thrombin over time after the addition of activating factors such as phospholipids, tissue factor, and calcium. The results of the assay can typically be calculated as a thrombogram, or a thrombin generation curve, using computer software after calculation of thrombogram parameters. Non - limiting examples of assay conditions for a thrombin generation assay include incubating platelets in the presence of tissue factor and phospholipids. In some embodiments, the in vitro assay comprises incubating platelets and / or platelet derivatives in the presence of tissue factor and phospholipids but in the absence of fresh platelets. Thus, in some embodiments, the cryopreserved platelets and / or platelet derivatives disclosed herein are capable of generating thrombin, for example, in vitro in the presence of reagents containing tissue factor and phospholipids. For example, in some cases, the cryopreserved platelets and / or platelet derivatives described herein, or frozen and stored platelets and / or platelet derivatives (e.g., at least about 10×10 3 particles / μL, 20×10 3 particles / μL, 30×10 3 particles / μL, or 44×10 3In similar embodiments, a reagent containing tissue factor (e.g., at concentrations of 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, or 10 pM) and optionally in the presence of phospholipids can produce thrombin peak heights (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), 50 nM, 75 nM, 100 nM, 150 nM, 175 nM, 200 nM, 250 nM, 275 nM, or 300 nM). For example, in some cases, frozen platelets and / or platelet derivatives as described herein, or cryopreserved platelets and / or platelet derivatives (e.g., at least about 10 × 10 3 particles / μL, 20 × 10 3 particles / μL, 30 × 10 3 particles / μL, or 44 × 10⁻⁶ 3 In exemplary embodiments, TPH of about 100 nM to about 350 nM (e.g., about 125 nM to about 350 nM, or about 150 to about 350 nM) can be produced in the presence of a reagent containing tissue factor and (e.g., 0.25 pM, 0.5 pM, 1 pM, 2 pM, 5 pM, or 10 pM) and optionally phospholipids. In some cases, frozen platelets and / or platelet derivatives as described herein, or cryopreserved platelets and / or platelet derivatives (e.g., about 4.8 × 10⁻¹⁶) can be produced. 3 (At a concentration of particles / μL) In the presence of PRP reagent (catalog number TS30.00 from Thrombinoscope), for example, 20 μL of PRP reagent yields approximately 4.8 × 10⁶ particles. 3Using conditions that include 80 μL of a composition containing platelets or platelet derivatives in quantities of particles / μL, or cryopreserved platelets and / or platelet derivatives, 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) can be produced. In some cases, the frozen platelets and / or platelet derivatives described herein (e.g., about 4.8 × 10⁻¹⁶ particles / μL) can be used to produce TPH. 3 (At a concentration of particles / μL) In the presence of PRP reagent (catalog number TS30.00 from Thrombinoscope), for example, 20 μL of PRP reagent yields approximately 4.8 × 10⁶ particles. 3 Using conditions that include an 80 μL composition containing 1 / μL of frozen platelets and / or platelet derivatives, 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) can be produced. In some embodiments, the compositions herein contain at least 0.4, 0.5, 0.7 / 10 6 The particles may have an IU of 100. As a non-limiting demonstration of thrombin-generating ability, Example 9 and Table 8 demonstrate the thrombin-generating ability of platelets or platelet derivatives during storage. Those skilled in the art can evaluate the thrombin-generating potential of platelets or platelet derivatives disclosed herein using other known techniques. Accordingly, in some embodiments, the compositions herein include platelets or platelet derivatives that retain hemostatic ability even when stored for at least 12 months at temperatures in the range of -10°C to -30°C.

[0042] In some embodiments, compositions comprising frozen platelets and / or platelet derivatives, or cryopreserved platelets and / or platelet derivatives, when stored at temperatures in the range of -10°C to -40°C, in exemplary embodiments, when stored for at least 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, can occlude collagen-coated microchannels, tissue factor-coated microchannels, or collagen and tissue factor-coated microchannels in vitro upon thawing. For example, such occlusion can be determined by using, for example, a Total Thrombosis Analysis System (T-TAS®). In some embodiments, the microchannels are collagen-coated microchannels. In some embodiments, the microchannels are tissue factor-coated microchannels, e.g., thromboplastin-coated microchannels. In some embodiments, the microchannels are collagen and tissue factor-coated microchannels. In some cases, the frozen or cryopreserved platelets or platelet derivatives described herein can occlude at least 50 × 10⁶ upon thawing. 3 particles / μL, 60 × 10 3 particles / μL, or 70 × 10 3 particles / μL (e.g., at least 73 × 10⁻¹⁶ particles) 3 pieces, 100×10 3 pieces, 150×10 3 pieces, 173×10 3 pieces, 200×10 3 pieces, 250×10 3 10 pieces, or 255 x 10 3When the concentration is (100 particles / μL), for example, platelets in citrate-treated whole blood can result in T-TAS occlusion times (e.g., time to reach 60 kPa) of less than 30, 25, 20, 15, or 14 minutes, or 5 at the lower end of the range to 15, 20, or 25 at the upper end, or 10 at the lower end of the range to 15, 20, or 25 at the upper end, or 15 at the lower end of the range to 20 or 25 at the upper end. In some cases, frozen or cryopreserved platelets or platelet derivatives described herein may, upon thawing, be at least 50 × 10⁻⁶ 3 particles / μL, 60 × 10 3 particles / μL, or 70 × 10 3 particles / μL (e.g., at least 73 × 10⁻¹⁶ particles) 3 pieces, 100×10 3 pieces, 150×10 3 pieces, 173×10 3 pieces, 200×10 3 pieces, 250×10 3 10 pieces, or 255 x 10 3At a concentration of (100 particles / μL), for example, in citrate-treated whole blood with reduced platelet content, an area under the curve (AUC) of at least 1300 (e.g., at least 1380, 1400, 1500, 1600, or 1700) can be obtained. Occlusion time indicates the time it takes for a sample to form a thrombus. A shorter time indicates faster thrombus formation. Analysis can capture occlusion time (OT) and area under the curve (AUC). OT represents the delay time it takes for the flow pressure to reach a target pressure, such as 60 kPa, 70 kPa, or 80 kPa, from the baseline pressure. AUC is the area under the flow-pressure-time curve related to overall thrombus formation. Microchannels or capillaries of different dimensions can be used in the T-TAS system to determine the occlusion time of cryopreserved platelets or cryopreserved platelet derivatives, or frozen platelets or frozen platelet derivatives, under different experimental conditions, as they are offered by numerous commercial suppliers (see, for example, Zacros, Tokyo, JP). For example, T-TAS PL tips, AR tips, or HD tips can be used in occlusion (e.g., T-TAS) assays as they are commercially available. Typically, AR tips for the purpose of T-TAS assays are coated with either collagen, tissue factor such as thromboplastin, or both. Typically, HD tips for the purpose of T-TAS assays are coated with either collagen, tissue factor such as thromboplastin, or both. For example, a PL tip may have a capillary dimension of 40 μm × 40 μm, or an AR tip may have a capillary dimension of 0.3 mm × 80 μm, or an HD tip may have a capillary dimension of 0.3 mm × 50 μm. Therefore, it is conceivable that a T-TAS assay can be performed to test the ability of collagen-coated microchannels to occlude microchannels using microchannels or capillaries having dimensions in the range of 0.02–0.5, 0.1–0.5, 0.2–0.4, 0.1–0.3, or 0.2–0.3 mm × 25–200, 25–100, 50–100, 40–90, 40–80, or 50–80 μm.

[0043] In some embodiments, compositions comprising frozen platelets and / or platelet derivatives as herein, when stored at temperatures in the range of -10°C to -40°C, in exemplary embodiments, when stored for at least 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, upon thawing, typically exhibit a single peak in a membrane integrity assay based on the retention of fluorophores in platelets and / or platelet derivatives. Those skilled in the art can devise different techniques for testing the retention of fluorophores in particles such as platelets and / or platelet derivatives. One such technique is the calcein acetoxymethyl (AM) membrane integrity assay. Calcein AM is a substance that can pass through the cell membrane and reach the cytoplasm where it can be hydrolyzed by the enzyme esterase to produce fluorescence. Intact platelets and / or platelet derivatives can retain this fluorescence, but intangible platelets do not. Thus, particles can be evaluated for the integrity of their membranes based on the fluorescence they emit. Therefore, based on the calcein AM assay, in some embodiments, the platelets and / or platelet derivatives in the compositions provided herein do not have intact cell membranes, i.e., they have damaged membranes. Example 8 is a non-limiting example demonstrating damaged cell membranes in platelets and / or platelet derivatives in the compositions (see Figure 9). Not limited to theory, observations from Figure 9 probably suggest that there are two populations in the single-temperature cryopreserved product (stored at -80°C) (a second population that probably retains higher fluorescence retention due to having intact membranes (see the second peak from the left of "#" in Figure 9) compared to a first population that probably retains less calcein AM fluorescence due to damaged membranes (see the first peak from the left of "#" in Figure 9)). Furthermore, it was observed that the single peak (see "*" in Figure 9) of the cryopreserved product at all three transition temperatures (stored at -20°C) corresponds to the first population of cryopreserved products at a single temperature, showing less retention of calcein AM.Therefore, a single population of cryopreserved products (stored at -20°C) observed in the calcein AM assay can be inferred to contain platelets and / or platelet derivatives with damaged membranes. Surprisingly, however, despite the composition containing frozen platelets and / or platelet derivatives, the composition, when stored for at least 2, 4, 6, or 12 months at temperatures ranging from -10°C to -40°C, or -20°C+ / -2°C, typically yields a platelet count (at least 1 × 10¹⁶ per 20–35 ml) when counted using a blood analyzer such as those disclosed herein. 11 The criteria for parameters, including visual observations such as the number of platelets, pH (above 6.5), absence of aggregation, and presence of swirl, must be met.

[0044] Another non-limiting technique for assessing membrane integrity involves detecting lactate dehydrogenase enzyme (LDH) released by cells with damaged membranes. LDH is a stable cytoplasmic enzyme found in all cells. When the cell membrane is damaged, LDH is rapidly released into the cell culture supernatant. According to one protocol, LDH activity can be readily quantified by using nicotinamide adenine dinucleotide (NAD) + hydrogen (NADH), produced during the conversion of lactate to pyruvate, to reduce the second compound in the binding reaction to a product with readily quantifiable properties. This protocol measures the reduction of the yellow tetrazolium salt, iodonitrotetrazolium (INT), to a red water-soluble formazan class dye with NADH at an absorbance of 492 nm. The amount of formazan is directly proportional to the amount of LDH in the supernatant, which is then directly proportional to the number of cells with damaged membranes. Therefore, in some embodiments, the frozen platelets or platelet derivatives in the compositions herein, or the cryopreserved platelets or platelet derivatives, have damaged membranes as determined by an LDH assay for evaluating membrane integrity.

[0045] In some embodiments, compositions comprising frozen platelets and / or platelet derivatives, or cryopreserved platelets and / or platelet derivatives, as herein, when stored at temperatures in the range of -10°C to -40°C, in exemplary embodiments, when stored for at least 1 month, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, or 12 months, may exhibit aggregation upon thawing under aggregation conditions comprising an agonist, but not limited to arachidonic acid, collagen, and TRAP-6. In some embodiments, the aggregation conditions include an agonist but do not include fresh platelets or apheresis platelets. In some embodiments, the aggregation conditions include an agonist but do not include fresh platelets or apheresis platelets and do not include divalent cations. Non-exclusive examples of aggregation agonists include collagen, epinephrine, ristocetin, arachidonic acid, adenosine diphosphate, and thrombin receptor-associated protein (TRAP). In some embodiments, the frozen platelets and / or platelet derivatives herein, or cryopreserved platelets and / or platelet derivatives, upon thawing in the presence of arachidonic acid, exhibit aggregation in the range of 20–60%, 20–50%, or 30–50%, or at least 20%, 30%, 40%, or 50%. In some embodiments, the frozen platelets and / or platelet derivatives herein, or cryopreserved platelets and / or platelet derivatives, upon thawing in the presence of collagen, exhibit aggregation in the range of 2–50%, 2–40%, or 2–30%. In some embodiments, the frozen platelets and / or platelet derivatives herein, or cryopreserved platelets and / or platelet derivatives, upon thawing in the presence of TRAP-6, exhibit aggregation in the range of 2–50%, 2–40%, or 2–30%. A non-limiting method for determining aggregation is by using a PAP8 aggregator (Bio Data Corporation, biodatacorp.com). Examples 7 and Figure 8 demonstrate the aggregation of frozen platelets and / or platelet derivatives, or cryopreserved platelets and / or platelet derivatives, according to this specification upon thawing in the presence of collagen, TRAP-6, and arachidonic acid.

[0046] Platelet units and pooling of units The processes provided herein may include platelet units as starting material or a source of platelets. Typically, the platelet units may be apheresis platelet units (APUs), however, other sources of platelets may also be included in the processes disclosed herein. Alternative sources of platelets may include whole blood-derived platelets. Platelets can be obtained from whole blood using either the known platelet-rich plasma (PRP) method or the buffy coat method. Platelets obtained from the buffy coat method are known as buffy coat-derived platelet concentrates (BC-PCs). Those skilled in the art may use platelets from any of the available sources, from an accessibility and commercial standpoint. Platelet units, such as APUs, may be accessed from any approved blood bank or center that processes blood units. The process may be carried out in a tertiary care facility with access to a blood bank. The process may be carried out in any facility or processing center with access to a blood bank, or the platelet units may be supplied to a facility or processing center. Typically, platelets are available in two forms: a pool of whole blood-derived platelet concentrates, and platelets collected via apheresis. Platelet concentrates are prepared from donated whole blood, separated within 8 hours of collection, and contain at least 5.5 × 10⁶ plates per unit. 10 Each platelet concentrate unit contains 100 platelets, with each unit containing approximately 40-50 ml of plasma. Apheresis platelets are collected from a single donor and suspended in 200-300 ml of plasma, with at least 3 x 10⁶ platelets per unit. 11 It contains [number] platelets.

[0047] Typically, the platelet units provided herein come from two or more donors. For example, platelet units may come from 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, or more donors. The number of donors may depend on the number of platelet units required for the preparation of cryopreserved platelets. To prepare a batch of cryopreserved platelets containing more than 10 cryo-containers, platelet units may be obtained from 8, 9, or more than 10 donors. For example, 12 platelet units may be provided to prepare a batch of cryopreserved platelets containing 12 cryo-containers. In such a case, each unit may come from a different donor, so that the platelet units come from 12 donors. In other cases, two units may come from one donor, so that the platelet units come from 6 donors, or three units may come from one donor, so that the platelet units come from 4 donors. Those skilled in the art will understand that the number of donors may depend on the number of platelet units required and the availability of such units within the blood bank. In some cases, at least 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, or more units can be processed in a single batch. The processes disclosed herein may include performing the processes disclosed herein two or more times to form two or more batches of cryopreserved platelets. For example, the processes disclosed herein may be performed multiple times to form multiple batch numbers of cryopreserved platelets. For example, five batches of cryopreserved platelets can be formed by performing the process five times. Similarly, the process can be performed any number of times depending on the required number of batches of cryopreserved platelets. For example, batches of 2-500, 2-450, 2-400, 2-300, 2-250, 2-200, 2-150, or 2-100 can be formed by performing the processes disclosed herein multiple times. In such embodiments, each batch may have 3 to 50, 3 to 40, 3 to 30, 3 to 25, 3 to 20, 3 to 25, 3 to 12, 4 to 12, or 5 to 12 cryo-containers for cryopreserved platelets.

[0048] The process described herein involves pooling platelet units in containers such that a minimum number of units are processed in one container until the step of combining the contents of all such containers for the addition of a cryoprotectant. For example, at least two or three units are pooled in a container to create multiple containers having pooled platelet units. The pooling of platelet units may be based on the number of platelet units provided and / or the number of platelet units that can be pooled in one container. For example, if five platelet units are provided, two units can be pooled in a container and the remaining unit can be processed in a separate container so that two containers can each have a set of two pooled units and one container can have the remaining unit. If six platelet units are provided, two units can be pooled in a container so that three containers can each have a set of two pooled units. The pooling of platelet units may be carried out in such a way that three or more units can be pooled in one container. For example, if five platelet units are provided, three units can be pooled in a first container and two units can be pooled in a second such container. Alternatively, three units can be pooled in a first container, and one unit can be processed in each separate container. The number of units that can be pooled in a container can depend on the type of container and the volume that can be processed within it. Typically, the container can be an apheresis platelet unit (APU) bag. For example, if an APU bag can hold a volume of approximately 800–900 ml for processing, then two to three units can be pooled in one container such as an APU bag. For example, APUs can hold volumes in the range of 800–1600 mL, 800–1000 mL, or 1000–1500 mL. For example, considering an APU bag as a container according to the process disclosed herein, pooling can be done by welding a plasma transport set to the APU bag using an SCD, and then welding a second APU bag to the other end of the plasma transport set. Plasma transport sets are added to extend the working length of the tubing.Subsequently, 2 APUs can be pooled together in a single APU bag. This can be done multiple times to create multiple APU bags with pools of APUs (2 platelet units) from the initial platelet units. The sterile connection device used can be Terumo, TSCD II sterile tubing welding machine, model number 3me-SC203a (or equivalent). The plasma transport set used can be Charter Medical, 24-inch tubing, roller clamp and two puncture pins, product number 03-220-00 (or equivalent). If there is an odd number of initial platelet units such as APUs, the plasma transport set can be welded to the odd number of APUs, and a 600 mL transport bag (Terumo, TeruFlex transport bag, catalog number: 1BB*T060CB71 or equivalent) can be welded to the other end of the plasma transport set. The APCs of the odd number of APUs remain in the APU bag.

[0049] After pooling platelet units or separating each platelet unit into a separate container, the weight of apheresis platelet concentrate (APC) can be determined for each container, such as each APU bag. For example, a non-restrictive equation for calculating the APC weight for each container is as follows: Pooled or single APC weight = Pooled or single APU weight - Empty container / bag weight

[0050] The pooled APU weight can be determined using a scale (Ohaus Adventurer Precision Balance, product number AX8201 / E, or equivalent). The empty bag weight is a known value corresponding to the type of bag used for apheresis platelet collection.

[0051] The process described herein includes a step of centrifugation of a container containing pooled platelet units, or a container containing one platelet unit. The centrifugation step is for separating platelets from plasma and can therefore be achieved by centrifugation of the container at 1000g–2000g, 1000g–1500g, or 1100g–1400g for a period ranging from 5–30 minutes, 5–25 minutes, or 5–20 minutes. Typically, the container may be an APU bag, which can be held in a centrifuge cup and centrifuged at 1250g at maximum acceleration for 10 minutes, followed by 10 minutes of deceleration.

[0052] Plasma expression and resuspension The process described herein may include a step of resuspending the pellet obtained after the centrifugation step to achieve a target weight of resuspension within a specific range, for example, 12g to 32g, 13g to 30g, 14g to 29g, or 15.9g to 27.9g times the number of platelet units pooled or provided in the processed or centrifuged container. For example, if 2 units are pooled in the container, the target weight of resuspension can be 12g to 32g, 13g to 30g, 14g to 29g, or 15.9g to 27.9g, so that the target weight of resuspension is 24g to 64g, 26g to 60g, 28g to 58g, or 31.8g to 55.7g. The target weight of resuspension can be achieved by removing a portion of the supernatant containing plasma, a step also known as plasma expression. Plasma expression or removal of supernatant can be performed to achieve the target weight of the remaining supernatant and pellet. Alternatively, removing a portion of the supernatant can be carried out until the target weight of the supernatant to be removed is achieved. The target weight of the supernatant to be removed can be determined based on the weight of the remaining supernatant and pellet required. For example, if two units are pooled in a container, the target weight of the resuspension can be twice 12g-32g, 13g-30g, 14g-29g, or 15.9g-27.9g, so that the target weight of the resuspension is 24g-64g, 26g-60g, 28g-58g, or 31.8g-55.7g. Typically, if two units are provided in a container, the target weight of plasma to be removed can be determined by the weight of the resuspension, which is the platelet pellet, and the weight of the remaining plasma, in the range of 31.8g-55.7g, and in one non-limiting example, the target weight of plasma to be removed is determined by the weight of the resuspension, which is approximately 45.6g.

[0053] Typically, the target plasma removal weight for expression can be determined by subtracting 46.5 g from the pooled APC weight using the following equation. This is done to leave approximately 46.5 g of platelet pellet and plasma after the pooled APU has been expressed.

[0054] Determination of expression endpoints Plasma removal target = pooled APC weight (2 platelet units) - 46.5g

[0055] Each container, after pooling platelet units, can be removed from the centrifuge and expressed one by one (with some of the supernatant removed). The containers can be carefully removed from the centrifuge cup so as not to disturb the platelet pellet and then placed in a plasma expresser (Fenwal Inc., manual plasma extractor, product code 4R4414, or equivalent). The empty APU bag is placed on a scale and tare to weigh the expressed plasma. The pooled APU is then expressed. Expression is stopped when the plasma removal target is reached (±1.0 g). The post-expression pellet weight is determined to ensure that the weight of the platelet pellet and the remaining supernatant is within the range for further processing (31.8 g to 55.7 g). If the post-expression weight is outside the range, the supernatant can be added or removed as appropriate until the post-expression weight is within the range. Once the post-expression weight is within the range, the pellet is resuspended by gently shaking and massaging the APU bag until the pellet is no longer visible. After the pellet is no longer visible, the resuspended platelet pellet is allowed to rest at ambient temperature for 5 minutes. The resuspended pellet is then visually inspected for aggregates. If aggregates are observed at this point in the process and do not disappear after further rest and stirring, production control is notified, and the process continues. A 30-minute ambient temperature rest with gentle stirring is added to the process after the addition of 27% DMSO. To fit into an APU bag containing 1 platelet unit, the plasma removal target can be determined by the following equation:

[0056] Plasma removal target (per single platelet unit) = Odd APC weight (per platelet unit) - 23.3g

[0057] The pellet weight range after expression (weight of the remaining supernatant and pellet) is changed to 15.9g to 27.9g, taking into account that odd-numbered APUs are single APUs and not pooled APUs.

[0058] In some embodiments, the processes described herein may include resuspending pellets with a buffer composition such that a target weight range of 15.9 g to 27.9 g times the number of platelet units is the target weight range of the resuspension with the buffer composition. Typically, such a resuspension with a buffer composition can remove 90% to 99.9% of the supernatant containing plasma, and then the pellets can be resuspended with the buffer composition. The buffer composition may include a buffer, a base, one or more sugars, optionally a salt, and optionally an organic solvent. The buffer may be any buffer that is nontoxic to platelets and provides adequate buffering capacity to the solution at the temperature to which the resuspension is exposed during the processes provided herein. Thus, the buffer composition may include any of the commercially available known biocompatible buffers, such as phosphate buffers, e.g., phosphate-buffered saline (PBS), bicarbonate / carbonate, e.g., sodium bicarbonate buffer, N-2-hydroxyethylpiperazine-N'-2-ethanesulfonic acid (HEPES), and Tris-based buffers, e.g., Tris-buffered saline (TBS).Similarly, this is the buffer for the following: propane-1,2,3-tricarboxylic acid (tricarvalic acid), benzenepentacarboxylic acid, maleic acid, 2,2-dimethylsuccinic acid, EDTA, 3,3-dimethylglutaric acid, bis(2-hydroxyethyl)imino-tris(hydroxymethyl)-methane (BIS-TRIS), benzenehexacarboxylic acid (mellitic acid), N-(2-acetamide)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) (PIPES), N-(2-acetamide) It may contain one or more of the following: -2-aminoethanesulfonic acid (ACES), 1,1-cyclohexanediacetic acid, 3,6-endomethylene-1,2,3,6-tetrahydrophthalic acid (EMTA, ENDCA), imidazole, 2-(aminoethyl)trimethylammonium chloride (CHOLAMINE), N,N-bis(2-hydroxyethyl)-2-aminoethanesulfonic acid (BES), 2-methylpropane-1,2,3-triscarboxylic acid (beta-methyltricarbaryl acid), 2-(N-morpholino)propane-sulfonic acid (MOPS), phosphoric acid, and N-tris(hydroxymethyl)methyl-2-aminoethanesulfonic acid (TES).

[0059] In some embodiments, one or more buffers may be present in the resuspension in any preferred amount. In some embodiments, the buffer may be present in an amount of 1 mM to 1 M. In some embodiments, one or more buffers may be present in the resuspension in an amount of about 0.2 to about 20 mg / ml, or about 0.2 to about 2 mg / ml, or about 2 mg / ml to about 20 mg / ml. In some embodiments, the buffer composition may contain one or more salts in the resuspension in an amount of about 0.08 to about 8 mg / ml, for example, about 0.08 to about 0.8 mg / ml, or about 0.8 mg / ml to about 8 mg / ml.

[0060] The process for preparing cryopreserved platelets provided herein may also include adding to the resuspension one or more salts, such as phosphates, sodium salts (e.g., NaCl), potassium salts (e.g., KCl), calcium salts, magnesium salts, and any other salts that can be found in blood or blood products or are known to be useful for cryopreserving platelets, or any combination of two or more of these.

[0061] In some embodiments, the salt is present in the resuspension at a concentration of about 1 mM to about 1000 mM, for example, about 0.01 M to about 0.2 M. In some embodiments, one or more salts are present in the resuspension at a concentration of about 0.4 to about 40 mg / ml, or about 0.4 to about 4 mg / ml, or about 4 mg / ml to about 40 mg / ml. In some embodiments, one or more salts are present in the resuspension at a concentration of about 0.03 to about 3 mg / ml, or about 0.03 to about 0.3 mg / ml, or about 0.3 mg / ml to about 3 mg / ml.

[0062] In some embodiments, these salts are present in the resuspension in amounts nearly identical to those found in whole blood.

[0063] In some embodiments, the process for preparing a cryopreserved platelet composition includes adding an organic solvent, such as alcohol, to a suspension medium, e.g., ethanol. The organic solvent may include one or more alcohols, e.g., short-chain alcohols such as ethanol. Short-chain alcohols are alcohols having 1 to 6 carbon atoms, particularly 2, 3, or 4 carbon atoms, e.g., methanol, ethanol, and propanol, including 1-propanol and 2-propanol, preferably ethanol. The organic solvent may also be a mixture of different organic solvents. In such a resuspension medium, the solvent may be in the range of 0.1% to 5.0% (v / v). In some embodiments, one or more organic solvents are present in the resuspension at about 0.08% (v / v) to about 8% (v / v), or about 0.08% (v / v) to about 0.8% (v / v), or about 0.8% (v / v) to about 8% (v / v).

[0064] In some embodiments, the process described herein involves resuspending the pellet in a buffer composition after the removal or expression of plasma. Such a buffer composition may contain one or more sugars. The sugars may include monosaccharides, disaccharides, or polysaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, xylose, and combinations thereof. In some embodiments, the sugar used in the process of preparing cryopreserved platelets provided herein is trehalose. In some embodiments, the polysaccharide is polysucrose, or a combination of any of the above sugars, in exemplary embodiments, trehalose and polysucrose. Thus, in one embodiment, the first mixture comprises platelets, a cryoprotectant, such as a cryoprotectant comprising trehalose, polysucrose, or a combination thereof, and a solvent such as ethanol.

[0065] In some embodiments, the preferred sugar may include one or more sugar alcohols. Non-limiting examples of sugar alcohols include mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, and combinations thereof.

[0066] In various embodiments, one or more sugars may be present in the resuspension in any preferred amount. Such sugars may be, for example, cryoprotective agents or one of several cryoprotective agents when one or more sugars are present in the resuspension together with DMSO. In some embodiments, the sugars may be present at about 1 mM to about 1 M. In some embodiments, the sugars may be present at about 10 mM to about 500 mM. In some embodiments, the sugars may be present at about 20 mM to about 200 mM. In some embodiments, the sugars may be present at about 40 mM to about 100 mM. In some embodiments, one or more sugars may be present in the resuspension at about 0.04 mg / ml to about 4 mg / ml, about 0.04 mg / ml to about 0.4 mg / ml, or about 0.4 mg / ml to about 4 mg / ml. In some embodiments, one or more sugars are present in the resuspension at concentrations of approximately 3 mg / ml to approximately 300 mg / ml, approximately 3 mg / ml to approximately 30 mg / ml, or approximately 30 mg / ml to approximately 300 mg / ml.

[0067] In various embodiments, the sugars are present at different specific concentrations within the ranges listed above, and those skilled in the art will readily understand these various concentrations without the need to specifically enumerate each one herein. If two or more sugars are present in the resuspension, each sugar may be present in an amount corresponding to the range and specific concentrations disclosed herein.

[0068] Pooling of resuspension The process described herein may include pooling of resuspensions from each container, such as an APU bag, after the step of resuspending the pellets to form a resuspension. Pooling can be performed by pooling the resuspensions from each container, such as an APU bag, one by one into a pooled resuspension container to form a pooled resuspension within the pooled resuspension container. Typically, pooling of resuspensions from each container can be achieved using a pool tree system. Such a pooled resuspension container may be an APU bag capable of holding the volume of resuspensions from all containers. Typically, the pooled resuspension container is a single container to which cryoprotectant is added in a series of steps, which may contain the pooled resuspension. However, to accommodate a large number of platelet units, such as 15, 20, 30, 40, 50, or more, in a single batch, the pooled resuspensions from all containers may be pooled and divided into two, three, or more pooled resuspension containers.

[0069] A pool tree system can be designed based on the requirements and the platelet units / containers being processed. An exemplary, non-limiting example of a pool tree system is shown in Figure 1C. Referring to Figure 1C, one non-limiting example of a pool tree system or tube tree system includes a first four-port cross-style manifold (5) connecting a first 14-inch tube (1), a second 14-inch tube (1), a third 14-inch tube (1), and a fourth 4-inch tube (2); a second four-port cross-style manifold (5) connecting the fourth 4-inch tube (2) to a fifth 20-inch tube (3), a sixth 14-inch tube (1), and a seventh 4-inch tube (2); a third four-port cross-style manifold (5) connecting the seventh 4-inch tube (2) to an eighth 14-inch tube (1), a ninth 14-inch tube (1), and a tenth 14-inch tube (1); and one on / off ratchet clamp (4) for each of the tubes. A non-limiting example of the tubing is the TYGON ND100-65 tubing, which varies in size depending on the requirements, with dimensions of 3 / 32 inch ID x 5 / 32 inch OD. Further non-limiting descriptions are provided in the table below.

[0070] (Table) - Tube Tree TIFF2026521991000002.tif55148* RF seal on the end of a Tygon tube. **Zip ties (x12) on each barb and tube connection.**

[0071] For example, in a process that provides 12 platelet units, six containers, e.g., six APU bags, can be processed, and after the step of removing plasma (plasma expression), the resuspensions from each bag can be pooled into a single bag. Once the six pellets are resuspended, the six APU bags can be welded to a sterile tube tree (Optimum Processing, Inc., part number 02817, or equivalent) to create a “pool tree system” and the resuspensions from each bag can be pooled into a single APU bag. Those skilled in the art will understand that any such pool tree system can be used or custom designed for pooling resuspensions as provided herein.

[0072] Addition of freeze-protecting agent The processes described herein may include adding a cryoprotectant to a pooled resuspension, as disclosed herein. While not bound by any theory or mechanism, the cryoprotectant stabilizes proteins and other biological substances within the platelets. The identity of the cryoprotectant is not limited as long as it can enter the platelets and provide cryoprotective properties. In some embodiments, the cryoprotectant comprises dimethyl sulfoxide (DMSO). In other embodiments, the cryoprotectant is any other cryoprotectant other than DMSO. Other non-limiting examples of suitable freeze-drying agents include sugars such as monosaccharides and disaccharides, including sucrose, maltose, trehalose, glucose, mannose, dextrose, xylose, and combinations thereof. In some embodiments, the sugar used for the preparation of cryopreserved platelet compositions provided herein is trehalose.

[0073] In some embodiments, the cryoprotectant may contain one or more sugar alcohols. Non-limiting examples of sugar alcohols include mannitol, sorbitol, xylitol, maltitol, maltitol syrup, lactitol, erythritol, and combinations thereof.

[0074] In various embodiments, one or more sugars may be present in the pooled resuspension or cryopreserved platelets in any preferred amount. For example, the sugars may be present at about 1 mM to about 1 M, about 10 mM to about 500 mM, about 20 mM to about 200 mM, or about 40 mM to about 100 mM. In further non-limiting examples, one or more sugars may be present in the pooled resuspension or cryopreserved platelets at about 0.04 mg / ml to about 4 mg / ml, about 0.04 mg / ml to about 0.4 mg / ml, or about 0.4 mg / ml to about 4 mg / ml. In some embodiments, one or more sugars may be present in the pooled resuspension or cryopreserved platelets at about 3 mg / ml to about 300 mg / ml, about 3 mg / ml to about 30 mg / ml, or about 30 mg / ml to about 300 mg / ml.

[0075] In some embodiments, the cryoprotectant may include one or more polyols. For example, suitable cryoprotectants include glycerol (glycerin), ethylene glycol, polyethylene glycol, sorbitol, propylene glycol, pentaerythritol, sugars, hydroxypropyl-β-cyclodextrin, glycerol oligomers, or combinations thereof. In some embodiments, the cryoprotectant is present in amounts of about 10% (v / v) or less (e.g., about 9% (v / v), 8% (v / v), 7% (v / v), 6% (v / v), 5% (v / v), 4% (v / v), 3% (v / v), 2% (v / v), 1% (v / v), 0.5% (v / v), or 0.1% (v / v) or less). In some embodiments, the cryoprotectant is present in amounts of at least about 1% (w / v) (e.g., at least about 2% (v / v), 3% (v / v) These are amounts of 1%, 4%(v / v), 5%(v / v), 6%(v / v), 7%(v / v), 8%(v / v), 9%(v / v), or 10%(v / v). For example, cryoprotectants are present in amounts of approximately 0.1%(v / v) to 10%(v / v), approximately 0.5%(v / v) to 7%(v / v), approximately 1%(v / v) to 5%(v / v), or approximately 0.1%(v / v) to 1%(v / v). Glycerol can also be used as a cryoprotectant.

[0076] Typically, the processes of this specification involve using DMSO as a cryoprotectant. For example, the addition of DMSO can be carried out until a target weight of DMSO is added to the pooled resuspension, as disclosed herein. DMSO can be added to the pooled resuspension until the concentration of DMSO in the pooled resuspension is in the range of 0.001–10%. For example, the DMSO concentration can be in the range of 0.005–10%, 0.1–10%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, 5.5–10%, 6–10%, 6–9%, 6–8%, 0.001–9%, 0.001–8%, or 0.001–7%. In some embodiments of the processes of this specification, the DMSO concentration in the pooled resuspension can be at least 0.001% or 0.005%. 0.05%, 0.1%, 0.2%, 0.5%, 1%, 2%, 3%, 4%, 5%, 6%, 7%, 8%, 9%, or 10%. If, during the execution of the process herein, the suspensions from each container are pooled to form a pooled resuspension in a pooled resuspension container, the amount of cryoprotectant or DMSO to be added can be calculated based on the weight of the pooled resuspension obtained after pooling the resuspensions from each container, e.g., an APU bag. Typically, the weight of the pooled resuspension can be determined by summing the weights of all resuspensions from each container or APU bag. The weight of the pooled APC after removing plasma from all containers that initially had 2 platelet units can be added, if applicable, along with the weight of the APC from the containers that initially had 1 platelet unit after plasma removal to reach the total APC weight, or total APC weight. The total APC weight in this step can be the post-expression weight and can be reached by summing the weights of the resuspensions from all containers / APU bags after plasma removal.

[0077] Total weight after expression (after plasma removal) = sum of resuspension weights = total APC weight

[0078] The amount of DMSO to be added in this step can vary based on the total APC weight, the stock DMSO concentration, and the target DMSO concentration to be achieved in the cryopreserved platelets. An exemplary, non-limiting equation for calculating the weight of DMSO to be added to the pooled resuspension is given below. A stock DMSO concentration of 27% is considered, and the target DMSO concentration of 6.085% in the cryopreserved platelets in the cryo-container is considered as follows: Target weight of 27% DMSO = Total APC weight * 0.2946

[0079] After determining the target weight of DMSO to be added, the DMSO can be added to the pooled resuspension by any means that facilitates the addition of DMSO with minimal waste and leads to uniform mixing of the DMSO. The addition of DMSO can be carried out directly or indirectly in the pooled resuspension container. Typically, bags or containers (Bio Life Solutions, BloodStor® 27 NaCl Biopreservation Media, part number 327207, or equivalent) containing sterile, injectable-grade 27% DMSO and 0.66% sodium chloride in water can be welded to the pool tree system initially used to pool the resuspension from each container / APU bag. The 27% DMSO bags can be placed on a scale to weigh how much 27% DMSO flows out of the 27% DMSO bags into the pool tree system. The 27% DMSO bags can be placed higher than the pool tree system to allow the 27% DMSO to flow gravimetrically. Adding the target weight of 27% DMSO to the pool tree system can be carried out until ±3.0g, ±2.0g, or ±1.0g has been added to the pool tree system. The tubes to the 27% DMSO bag can be clamped to prevent additional 27% DMSO from entering the system. Steps involving the addition of cryoprotective agents such as DMSO can be carried out using the pool tree system that was initially used to pool the resuspension from each container / bag. In such steps, DMSO can be used to rinse the pool tree system and the container / APU bag containing the resuspension to effectively wash away any platelets that may be adhering to the pool tree system. Typically, the 27% DMSO that entered the pool tree system is used to rinse the system and recover any residual platelet material remaining in the APU bag and in the tubes of the pool tree system. The 27% DMSO can then be added to the pooled resuspension, e.g., the APU bag containing the pooled resuspension.A tube stripper can be used to remove any remaining 27% DMSO solution from the pool tree system, ensuring that all 27% DMSO is added to the platelets.

[0080] Those skilled in the art will understand that the DMSO constant of 0.2946 disclosed in the above equation can be manipulated based on the stock solution of DMSO and the target concentration of DMSO that needs to be achieved in cryopreserved platelets. For example, if a stock solution of 10% DMSO is considered instead of 27% DMSO, the DMSO constant will change.

[0081] Distribution and freezing The process described herein may include distributing a pooled resuspension with a cryoprotective agent such as DMSO into several cryocontainers for a further freezing step. The cryocontainers can be any suitable sealable container (e.g., a container closure system) in which platelets can be frozen. The cryocontainers can be suitable vials (cryovials), ampoules, or bags (e.g., cryobags). For example, a cryocontainer can be a cryobag such as a fluorinated ethylene propylene (FEP) bag or a polyvinyl chloride (PVC) bag. The cryocontainer can be a borosilicate serum vial. A non-limiting example of a cryocontainer or cryobag is a 250 mL ethylene vinyl acetate (EVA) thermoplastic container (CryoStore CS250 series, Origen, Austin, TX). The CS250 has a recommended freezing volume of 30–70 mL. EVA containers are also suitable for delivery of intravenous dosage forms, as are any other blood containers, as they are suitable for flexibility and transparency. The EVA containers are placed in polyethylene overlap bags (Helmer, Noblesville, IN) as secondary containers, and then placed in cardboard boxes (Mission City Container, San Antonio, TX), with inner dimensions of 7 inches x 5 1 / 4 inches x 1 5 / 8 inches and a test burst strength of 200 pounds, to protect the products from both light and damage.

[0082] In some embodiments of the processes described herein, the distribution of a pooled resuspension with a cryoprotective agent, such as DMSO, can be carried out by determining the packing weight or packing volume to be distributed into several cryocontainers. For example, the packing weight can be determined by dividing the pooled resuspension by the number of platelet units provided or processed, so that the pooled resuspension can be distributed into a number of cryocontainers equal to the number of platelet units provided or processed. In some embodiments, the packing weight can be determined by dividing the weight of the pooled resuspension by 1 / 3, 1 / 2, 2 / 3, or 2 times the number of platelet units provided or processed. As a non-limiting example, if 12 platelet units are processed, the weight of the pooled resuspension can be divided by 12 to form a batch containing 12 cryocontainers containing cryopreserved platelets. Alternatively, if 12 platelet units are processed, the pooled resuspension with a cryoprotective agent can be distributed into 3, 6, 8, 15, 18, or 24 cryocontainers by dividing the total weight of the pooled resuspension with a cryoprotective agent by an appropriate number.

[0083] The dispensing described herein can be carried out using any means that allows for the maintenance of a sterile environment and clear passage of the pooled resuspension with cryoprotectant. In exemplary embodiments, dispensing can be carried out by a dosing tree system. The dosing tree system may be the same as or different in design from the tube tree system. Typically, if a number of cryo containers equal to the number of platelet units is desired and 12 platelet units are provided for processing, then 12 cryo bags can be filled by welding pooled resuspension containers, such as APU bags containing the pooled resuspension (final product), to a new tube tree. This creates a “filling tree system”. The weight of the pooled resuspension with cryoprotectant (final product weight) can then be determined by weighing the pooled resuspension containers / APU bags and subtracting the weight of the empty bags. The maximum filling weight of the cryo bags can then be determined by dividing the weight of the pooled resuspension (final product) by 12. The minimum filling weight can be determined by subtracting 2.0 g from the maximum filling weight. This determines the filling weight range of the cryo bags. Furthermore, twelve cryobags (250mL EVA CryoStore freezing bags, Origen Reference CS250, or equivalent) can then be welded to the dispensing tree system. The cryobag filling procedure is performed by placing the cryobags on a scale, priming the cryobag line until just before the pooled resuspension with cryoprotectant enters the cryobags, and then tare the cryobags. The cryobags can then be filled with the pooled resuspension with cryoprotectant until it reaches the appropriate level. The filled weight of each cryobag can then be recorded, and their volume can be determined by dividing the weight by 1.03 g / mL.

[0084] The process of this specification following the step of distributing the pooled resuspension having the cryoprotectant includes freezing the pooled resuspension having the cryoprotectant. Freezing can be carried out by subjecting the pooled resuspension having the cryoprotectant to temperatures below about -1°C (e.g., about -5°C, about -10°C, about -20°C, about -30°C, about -40°C, about -50°C, about -60°C, about -70°C, about -80°C, or below about -90°C). For example, a pooled resuspension having the cryoprotectant in a cryo-container can be subject to temperatures of about -70°C to about -90°C, -50°C to about -70°C, -30°C to about -50°C, -10°C to about -30°C, or about -10°C to about -20°C). Typically, the cryo-container can be subject to temperatures of about -80°C. Typically, each cryobag can be placed in a thawing bag and freezer carton for storage in a -80°C freezer. The units can then be placed in the freezer and the start time of freezing is recorded. In exemplary embodiments, the elapsed time from the end of 27% DMSO addition to the start time of freezing may be 3 hours or less. Cryopreserved platelets can be thawed for further use by known methods, such as exposing the cryopreserved platelets to a non-freezing temperature. For example, thawing may involve immersing the cryopreserved platelets in a 37°C water bath for a suitable time, e.g., about 8 to 10 minutes.

[0085] In some embodiments, the process herein includes, after the step of distributing a pooled resuspension having a cryoprotectant, freezing the pooled resuspension having a cryoprotectant with a transition of freezing temperature from an initial freezing temperature to a storage freezing temperature. In some embodiments, the process herein includes the step of initial freezing at a temperature of -50°C, -60°C, -65°C, -70°C, -80°C, -85°C, or below -90°C, or in the range of -50°C to -85°C, or -60°C to -85°C (i.e., an initial temperature) to form an initial frozen platelet composition, and then storing the initial frozen platelet composition in a frozen state at a temperature of -30°C or above but below 0°C (i.e., a storage temperature) to form cryopreserved platelets or cryopreserved platelet compositions, thereby forming a batch of cryopreserved platelets.

[0086] In some embodiments, the processes described herein can be completed up to the freezing step within 4, 3, or 3 hours after the addition of the cryoprotectant. For example, the process can be completed up to the freezing step within a range of 1 to 3 hours, 1.5 to 3 hours, 2 to 3 hours, or 1.5 to 2.5 hours after the addition of the cryoprotectant.

[0087] A batch of cryocontainers containing cryopreserved platelets obtained from the process described herein can be evaluated for several post-manufacturing standards. A non-limiting set of such standards may include: Freezing volume: 20 mL to 35 mL Time from adding 27% DMSO to the freezer: 3 hours or less Platelet age freezing until the end of day 2 Visible aggregates in the cryobag: None %DMSO: 5.65%~6.52% Maximum DMSO per CPP unit: 2.53g (mass corresponding to the maximum freezing volume at maximum %DMSO).

[0088] Variations in cryopreserved platelets within a batch and across multiple batches Processes of the Specified Spectrum, including a process that includes a transition of freezing temperature from an initial freezing temperature to a storage freezing temperature, can provide a batch of cryopreserved platelets having a plurality of cryo-containers having cryopreserved platelet compositions, cryopreserved platelets, or cryopreserved platelets that can be more uniform than those of conventional methods, and can have reduced inter-batch variability. Such improved uniformity and reduced inter-batch variability can be expressed in some embodiments as low coefficients of variation within 15%, 14%, 13%, 12%, 11%, or 10% of a parameter, not limited to the resuspension weight or volume (post-expression weight or volume) in a container such as an APU bag, either within one batch or across multiple batches. For example, the resuspension volume in a container can have a coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.1–10%, 0.1–9%, 0.1–8%, or 0.1–7% within one batch or across multiple batches. For example, the resuspension volume in the container can have a coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.1–10%, 0.1–9%, 0.1–8%, or 0.1–7% across at least two batches. For example, the resuspension volume in the container can have a coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.1–10%, 0.1–9%, 0.1–8%, or 0.1–7% across at least five batches. For example, the resuspension volume in the container can have a coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.1–10%, 0.1–9%, 0.1–8%, or 0.1–7% over at least 15 batches.For example, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspensions across at least 2, 3, 4, 5, or 10 lots, or from 2, 3, 4, or 5 lots at the lower end to 100 lots at the upper end, has an average intra-batch coefficient of variation (average intra-batch CV) of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, or 7%. In some embodiments, the average intra-batch coefficient of variation of the resuspension in each container across at least 10 batches can be in the range of 1–20%, 1–15%, 1–10%, 1–8%, 2–8%, 3–8%, or 4–8%. For example, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspensions across 2 to 12 batches has an average intra-batch coefficient of variation (average intra-batch CV) of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, or 7%. In some embodiments, the average intra-batch coefficient of variation of resuspensions in each container across 2 to 12 batches can be in the range of 1 to 20%, 1 to 15%, 1 to 10%, or 1 to 8%, 2 to 8%, 3 to 8%, or 4 to 8%. In some embodiments, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspensions in containers across at least 5 batches or within 1 batch varies within the range of 20%, 15%, 12%, 10%, 9%, or 8%. For example, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension in each container varies over at least 5 batches or within one batch within the ranges of 3–25%, 7–20%, 7–15%, 7–14%, 7–12%, 7–10%, 3–20%, 3–15%, 3–12%, or 3–10%. In some embodiments, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension in each container varies over at least 10 batches or within one batch within the ranges of 20%, 15%, 12%, 10%, 9%, or 8%.For example, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension in each container varies over at least 10 batches or within one batch within a range of 3–25%, 7–20%, 7–15%, 7–14%, 7–12%, 7–10%, 3–20%, 3–15%, 3–12%, or 3–10%. In some embodiments, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension in each container varies over at least 20 batches or within one batch within a range of 20%, 15%, 12%, 10%, 9%, or 8%. For example, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension in each container varies over at least 5 batches or within one batch, ranging from 3–25%, 7–20%, 7–15%, 7–14%, 7–12%, 7–10%, 3–20%, 3–15%, 3–12%, or 3–10%. In some embodiments, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension in each container varies over 5–100 batches or within one batch, ranging from 20%, 15%, 12%, 10%, 9%, or 8%. For example, resuspending pellets in each container would result in resuspending in each container such that the volume of resuspension in each container varies across 5 to 100 batches or within a single batch, ranging from 3 to 25%, 7 to 20%, 7 to 15%, 7 to 14%, 7 to 12%, 7 to 10%, 3 to 20%, 3 to 15%, 3 to 12%, or 3 to 10%.

[0089] Another parameter for evaluating uniformity can be the volume or weight of the pooled resuspension with cryoprotective agent, such as DMSO, or the cryopreservation medium containing platelets, in a cryocontainer, also known as frozen volume or weight, which has a low coefficient of variation within 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 3%, 2%, or 1% within one batch or across multiple batches. For example, the volume or weight of the pooled resuspension with cryoprotective agent, or the cryopreservation medium containing platelets, in a cryocontainer can have a coefficient of variation in the range of 0.1–5%, 0.1–4%, 0.1–3%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1%–0.4%, 0.1%–0.3%, or 0.1%–0.2% within one batch or across multiple batches. For example, the volume or weight of a pooled resuspension with cryoprotectant in a cryocontainer, or a cryopreservation medium with platelets, can have a coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.10%, 0.1–9%, 0.1–8%, 0.1–7%, 0.1–6%, 0.1–5%, 0.1–4%, 0.1–3%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1–0.4%, 0.1–0.3%, or 0.1%–0.2% across at least two batches. For example, the volume or weight of a pooled resuspension with cryoprotectant in a cryocontainer, or a cryopreservation medium with platelets, can have a coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.10%, 0.1–9%, 0.1–8%, 0.1–7%, 0.1–6%, 0.1–5%, 0.1–4%, 0.1–3%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1–0.4%, 0.1–0.3%, or 0.1%–0.2% over at least 5 batches.For example, the volume or weight of a pooled resuspension with cryoprotectant in a cryocontainer, or a cryopreservation medium with platelets, can have a coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.10%, 0.1–9%, 0.1–8%, 0.1–7%, 0.1–6%, 0.1–5%, 0.1–4%, 0.1–3%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1–0.4%, 0.1–0.3%, or 0.1%–0.2% over at least 15 batches. For example, the volume or weight of a pooled resuspension with cryoprotectant in a cryocontainer, or a cryopreservation medium with platelets, can have a coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.10%, 0.1–9%, 0.1–8%, 0.1–7%, 0.1–6%, 0.1–5%, 0.1–4%, 0.1–3%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1–0.4%, 0.1–0.3%, or 0.1%–0.2% over 5–100 batches. For example, the volume or weight of pooled resuspensions in cryocontainers or cryopreservation media containing platelets across at least 10 batches have an average intra-batch coefficient of variation of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%. For example, the volume or weight of pooled resuspensions with cryoprotectant in cryocontainers, or cryopreservation media with platelets, across at least 10 batches, have an average intra-batch coefficient of variation in the range of 0.1–15%, 0.1–14%, 0.1–13%, 0.1–12%, 0.1–11%, 0.1–10%, 0.1–9%, 0.1–8%, 0.1–7%, 0.1–6%, 0.1–5%, 0.1–4%, 0.1–3%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1–0.4%, 0.1–0.3%, or 0.1–0.2%. For example, the volume or weight of pooled resuspensions with cryoprotectant in cryocontainers, or cryopreservation media with platelets, across 2 to 12 batches, have mean intra-batch coefficients of variation (mean intra-batch CV) of less than 15%, 14%, 13%, 12%, 11%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%.In some embodiments, the volume or weight of a pooled resuspension with cryoprotectant in a cryocontainer, or a cryopreservation medium containing platelets, across 2 to 12 batches, can be in the range of 0.05–20%, 0.1–20%, 0.5–20%, 1–20%, 1–15%, 1–10%, or 1–8%, 1–7%, 1–6%, 1–5%, 1–4%, 1–3%, 1–2%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1%–0.4%, 0.01%–0.3%, or 0.1%–0.2%. In some embodiments, the volume or weight of a pooled resuspension with cryoprotectant in a cryocontainer, across at least 5 batches or within 1 batch, can vary within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%. For example, a pooled resuspension with cryoprotectant across a cryocontainer, or a cryopreservation medium with platelets across at least five batches or within one batch, may vary in the range of 0.05–20%, 0.1–20%, 0.5–20%, 1–20%, 1–15%, 1–10%, or 1–8%, 1–7%, 1–6%, 1–5%, 1–4%, 1–3%, 1–2%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1%–0.4%, 0.01%–0.3%, or 0.1%–0.2%. In some embodiments, the volume or weight of pooled resuspensions with cryoprotectant in cryocontainers, or cryopreservation media with platelets, across at least 10 batches or within one batch, varies within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. For example, the volume or weight of pooled resuspensions with cryoprotectant in cryocontainers, or cryopreservation media with platelets, across at least 10 batches or within one batch, varies within the ranges of 0.05–20%, 0.1–20%, 0.5–20%, 1–20%, 1–15%, 1–10%, or 1–8%, 1–7%, 1–6%, 1–5%, 1–4%, 1–3%, 1–2%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1%–0.4%, 0.01%–0.3%, or 0.1%–0.2%.In some embodiments, the volume or weight of pooled resuspensions with cryoprotectant in cryocontainers, or cryopreservation media with platelets, across at least 20 batches or within one batch, varies within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. For example, the volume or weight of pooled resuspensions with cryoprotectant in cryocontainers, or cryopreservation media with platelets, across at least 20 batches or within one batch, varies in the range of 0.05–20%, 0.1–20%, 0.5–20%, 1–20%, 1–15%, 1–10%, or 1–8%, 1–7%, 1–6%, 1–5%, 1–4%, 1–3%, 1–2%, 0.1–2%, 0.1–1%, 0.1–0.5%, 0.1%–0.4%, 0.01%–0.3%, or 0.1%–0.2%. In some embodiments, the volume or weight of pooled resuspensions with cryoprotectant in cryocontainers, or cryopreservation media with platelets, over at least 5 to 100 batches or within one batch, varies within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. For example, the volume or weight of pooled resuspensions with cryoprotectant in cryocontainers, or cryopreservation media with platelets, over at least 5 to 100 batches or within one batch, varies within the ranges of 0.05 to 20%, 0.1 to 20%, 0.5 to 20%, 1 to 20%, 1 to 15%, 1 to 10%, or 1 to 8%, 1 to 7%, 1 to 6%, 1 to 5%, 1 to 4%, 1 to 3%, 1 to 2%, 0.1 to 2%, 0.1 to 1%, 0.1 to 0.5%, 0.1% to 0.4%, 0.01% to 0.3%, or 0.1% to 0.2%.

[0090] In some embodiments, including a process for preparing a batch of cryopreserved platelets, a process for preparing a cryopreserved platelet composition including a transition of freezing temperature from an initial freezing temperature to a storage freezing temperature, and a collection or batch containing cryopreserved platelets, the process herein provides improved uniformity with respect to the concentration of cryoprotective agents present in batches and in multiple batches of cryopreserved platelets such that the mean cryoprotective agent concentration, such as DMSO concentration in cryopreserved platelets or in cryopreservation media containing platelets, has a coefficient of variation of less than 10% over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches. For example, the DMSO concentration in cryopreserved platelets over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 5%. For example, the DMSO concentration in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 3%. For example, the DMSO concentration in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 2%. For example, the DMSO concentration in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 1%. For example, the DMSO concentration in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 0.5%. For example, the DMSO concentration in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 0.4%.Therefore, in some embodiments of the process, or embodiments of the collection of cryopreserved platelets, the DMSO concentration in cryopreserved platelets across multiple batches or within a single batch can be in the range of 0.01–2%, 0.01–1.8%, 0.01–1.6%, 0.01–1.5%, 0.01–1.3%, 0.01–1.1%, 0.01–1%, 0.01–0.8%, 0.01–0.7%, 0.01–0.5%, 0.01–0.4%, 0.02–0.4%, or 0.04–0.4%. For example, the average cryoprotectant concentration (e.g., DMSO concentration) in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 5%. In some embodiments, the average cryoprotective agent concentration (e.g., DMSO concentration) in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 1%. For example, the average cryoprotective agent concentration (e.g., DMSO concentration) in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 0.8%, 0.5%, or 0.4%. Typically, the average DMSO concentration in cryopreserved platelets across at least 10 batches can be less than 0.5%. In some embodiments, the cryoprotective agent is DMSO, and the average concentration of DMSO in cryopreserved platelets in a cryo-container across at least 10 batches has a coefficient of variation of less than 5%, 4%, 3%, 2%, 1%, or 0.5%. In some embodiments, the DMSO concentration in cryopreserved platelets across at least 10 batches or within one batch can be in the range of 0.01–2%, 0.01–1.8%, 0.01–1.6%, 0.01–1.5%, 0.01–1.3%, 0.01–1.1%, 0.01–1%, 0.01–0.8%, 0.01–0.7%, 0.01–0.5%, 0.01–0.4%, 0.02–0.4%, or 0.04–0.4%.For example, the average concentration of DMSO in cryopreserved platelets in a cryo-container across at least 20 batches has a coefficient of variation of less than 5%, 4%, 3%, 2%, 1%, or 0.5%. In some embodiments, the DMSO concentration in cryopreserved platelets across at least 20 batches or within one batch can be in the range of 0.01–2%, 0.01–1.8%, 0.01–1.6%, 0.01–1.5%, 0.01–1.3%, 0.01–1.1%, 0.01–1%, 0.01–0.8%, 0.01–0.7%, 0.01–0.5%, 0.01–0.4%, 0.02–0.4%, or 0.04–0.4%. For example, the average concentration of DMSO in cryopreserved platelets in a cryo-container across at least 50 batches has a coefficient of variation of less than 5%, 4%, 3%, 2%, 1%, or 0.5%. In some embodiments, the DMSO concentration in cryopreserved platelets across 50 batches or within one batch can be in the range of 0.01–2%, 0.01–1.8%, 0.01–1.6%, 0.01–1.5%, 0.01–1.3%, 0.01–1.1%, 0.01–1%, 0.01–0.8%, 0.01–0.7%, 0.01–0.5%, 0.01–0.4%, 0.02–0.4%, or 0.04–0.4%. For example, the average concentration of DMSO in cryopreserved platelets in a cryo-container across at least 100 batches has a coefficient of variation of 5%, 4%, 3%, 2%, 1%, or less than 0.5%. In some embodiments, the DMSO concentration in cryopreserved platelets across at least 100 batches or within one batch can be in the range of 0.01–2%, 0.01–1.8%, 0.01–1.6%, 0.01–1.5%, 0.01–1.3%, 0.01–1.1%, 0.01–1%, 0.01–0.8%, 0.01–0.7%, 0.01–0.5%, 0.01–0.4%, 0.02–0.4%, or 0.04–0.4%. For example, the average concentration of DMSO in cryopreserved platelets in a cryo-container across 5–100 batches has a coefficient of variation of less than 1% or 0.5%.In some embodiments, the DMSO concentration in cryopreserved platelets across 5 to 100 batches or within a single batch can be in the range of 0.01 to 2%, 0.01 to 1.8%, 0.01 to 1.6%, 0.01 to 1.5%, 0.01 to 1.3%, 0.01 to 1.1%, 0.01 to 1%, 0.01 to 0.8%, 0.01 to 0.7%, 0.01 to 0.5%, 0.01 to 0.4%, 0.02 to 0.4%, or 0.04 to 0.4%. For example, the average concentration of DMSO in cryopreserved platelets in a cryo-container across 5 to 500 batches has a coefficient of variation of less than 1% or 0.5%. In some embodiments, the DMSO concentration in cryopreserved platelets across 5 to 500 batches can be in the range of 0.01 to 2%, 0.01 to 1.8%, 0.01 to 1.6%, 0.01 to 1.5%, 0.01 to 1.3%, 0.01 to 1.1%, 0.01 to 1%, 0.01 to 0.8%, 0.01 to 0.7%, 0.01 to 0.5%, 0.01 to 0.4%, 0.02 to 0.4%, or 0.04 to 0.4%. In some embodiments, the DMSO concentration in cryopreserved platelets in a cryo-container within a batch has a coefficient of variation of less than 10%, 9%, 8%, 7%, 5%, 3%, 1%, 0.8%, 0.6%, or 0.5%. In some embodiments, the concentration of DMSO in cryopreserved platelets in a cryocontainer within one batch or across at least five batches varies within 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.4%. In some embodiments, the concentration of DMSO in cryopreserved platelets in a cryocontainer within one batch or across at least ten batches varies within 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.4%. In some embodiments, the concentration of DMSO in cryopreserved platelets in a cryocontainer within one batch or across 5 to 100 batches varies within 5%, 4%, 3%, 2%, 1%, 0.5%, or 0.4%.

[0091] In some embodiments of a process for preparing a batch of cryopreserved platelets, a process for preparing a cryopreserved platelet composition including a transition of freezing temperature from an initial freezing temperature to a storage freezing temperature, and a collection or batch containing cryopreserved platelets, the platelet concentration in the cryopreserved platelets in a cryo-container within one batch or across multiple batches varies within the range of 20%, 15%, 12%, 10%, or 8%. For example, the platelet concentration in the cryopreserved platelets in a cryo-container within one batch or across multiple batches varies within the range of 2-20%, 2-15%, 2-12%, 2-10%, 5-20%, 7-20%, or 10-20%. For example, the platelet concentration in the cryopreserved platelets in a cryo-container within one batch or across 5-100 batches varies within the range of 20%, 15%, 12%, 10%, or 8%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer within one batch or across 5 to 100 batches varies in the range of 2-20%, 2-15%, 2-12%, 2-10%, 5-20%, 7-20%, or 10-20%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across at least 5 batches has an average intra-batch coefficient of variation of 20%, 18%, 15%, 12%, 10%, 8%, 5%, or less than 4%. In some embodiments, the platelet concentration in cryopreserved platelets in a cryocontainer across at least 5 batches has an average intra-batch coefficient of variation in the range of 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-6%, or 2-5%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer within a batch has a coefficient of variation of less than 20%, 18%, 15%, 12%, 10%, 8%, 5%, or 4%. In some embodiments, the platelet concentration in cryopreserved platelets in a cryocontainer within a batch has a coefficient of variation in the range of 2–20%, 2–15%, 2–12%, 2–10%, 2–8%, 2–6%, 3–6%, 3–20%, 3–15%, 3–12%, 3–10%, or 3–6%.

[0092] In some embodiments of a process for preparing a batch of cryopreserved platelets, a process for preparing a cryopreserved platelet composition including a transition of freezing temperature from an initial freezing temperature to a storage freezing temperature, and a collection or batch containing cryopreserved platelets, the total number of platelets in cryopreserved platelets in a cryo-container within one batch or across multiple batches varies within 20%, 15%, 12%, 10%, or 8%. For example, the total number of platelets in cryopreserved platelets in a cryo-container within one batch or across multiple batches varies within the range of 2-20%, 2-15%, 2-12%, 2-10%, 5-20%, 7-20%, or 10-20%. For example, the total number of platelets in cryopreserved platelets in a cryo-container within one batch or across 5-100 batches varies within 20%, 15%, 12%, 10%, or 8%. For example, the total number of platelets in cryopreserved platelets in a cryo-container within one batch or across 5 to 100 batches varies in the range of 2-20%, 2-15%, 2-12%, 2-10%, 5-20%, 7-20%, or 10-20%. For example, the total number of platelets in cryopreserved platelets in a cryo-container across at least 5 batches has an average intra-batch coefficient of variation of 20%, 18%, 15%, 12%, 10%, 8%, 5%, or less than 4%. In some embodiments, the total number of platelets in cryopreserved platelets in a cryo-container across at least 5 batches has an average intra-batch coefficient of variation in the range of 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-6%, or 2-5%. For example, the total number of platelets in cryopreserved platelets in a cryo-container within one batch has an average coefficient of variation of 20%, 18%, 15%, 12%, 10%, 8%, or less than 7%. In some embodiments, the total number of platelets in cryopreserved platelets in a cryocontainer within a batch has a coefficient of variation in the range of 2-20%, 2-15%, 2-12%, 2-10%, 2-8%, 2-7%, 3-7%, 3-20%, 3-15%, 3-12%, or 3-10%.

[0093] A collection of cryogenic containers containing cryopreserved platelets. In some embodiments, a collection of cryocontainers containing cryopreserved platelets is provided herein, wherein the cryopreserved platelets in each cryocontainer have a biomolecular profile indicating two or more platelet donors. The concentration of a cryoprotective agent, such as DMSO, in the cryopreserved platelets of a first cryocontainer may be within 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% of the concentration of a cryoprotective agent, such as DMSO, in the cryopreserved platelets of a second cryocontainer. For example, the concentration of DMSO in the cryopreserved platelets of a first cryocontainer may be in the range of 0.001 to 10%, 0.001 to 8%, 0.001 to 6%, 0.001 to 4%, 0.001 to 2%, or 0.001 to 1% of the concentration of DMSO in the cryopreserved platelets of a second cryocontainer. The collection provided herein may include multiple cryocontainers containing cryopreserved platelets. A collection can be a collection of cryocontainers in a batch, for example, each batch may have at least 2, 3, 4, 5, 6, 7, 8, 9, 10, or more cryocontainers, and a collection may have at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 15, 20, 50, or more batches. Multiple cryocontainers or cryo-containers having cryopreserved platelets as herein may be referred to as a “batch” or “lot”. Typically, cryopreserved platelets in each cryocontainer have a set of biomolecular profiles indicating two or more platelet donors, and cryocontainers in one batch have the same set of biomolecular profiles. The set of biomolecular profiles indicating two or more platelet donors in one batch may differ from the set of biomolecular profiles in another batch. Therefore, the CPP from the first lot typically has a first set of donor biomolecular profiles, and the CPP from the second lot typically has a second set of donor biomolecular profiles, the second set of donors being not identical to the first set of donors.Those skilled in the art will understand that there are numerous features that can be used to distinguish CPP from a set of first donors from CPP from a set of second donors. Thus, a collection of cryocontainers containing CPP may have multiple batches such that, within one batch, a set of biomolecular profiles representing two or more platelet donors is identical across the cryocontainers in that batch and different from the cryocontainers in other batches within the collection. Typically, a collection of cryocontainers provided herein is uniform across multiple batches and within batches, with significantly less inter-donor variability. Several non-limiting parameters for assessing uniformity may include cryoprotectant concentration, platelet concentration, total platelet count, pH, thrombin production capacity (IU), and CD61-positive microparticle concentration. In some embodiments, the collection comprises frozen or cryopreserved platelets and / or platelet derivatives having one or more of the enumerated characteristics provided herein.

[0094] The collection in a cryo-container containing cryopreserved platelets according to this specification may, in some embodiments, include platelet concentrations such that the platelet concentration in the cryopreserved platelets in the cryo-container varies within a range of 30%, 25%, 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4% within a single batch or across multiple batches. For example, the platelet concentration in the cryopreserved platelets in the cryo-container varies within a range of 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across at least five batches varies within the ranges of 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across at least five batches varies within the ranges of 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across at least ten batches varies within the ranges of 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across at least 10 batches varies within the ranges of 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%. In some embodiments, the platelet concentration in cryopreserved platelets in a cryocontainer across at least 5 batches has an average intra-batch coefficient of variation within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the platelet concentrations in cryopreserved platelets in cryocontainers across at least five batches have an average intra-batch coefficient of variation within the following ranges: 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%.In some embodiments, the platelet concentration in cryopreserved platelets in a cryocontainer within a batch has a coefficient of variation of 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across a batch has a coefficient of variation of 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%.

[0095] The collection in a cryo-container containing cryopreserved platelets described herein may, in some embodiments, include a total number of platelets such that the total number of platelets in cryopreserved platelets in a cryo-container within one batch or across multiple batches varies within 30%, 25%, 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the total number of platelets in cryopreserved platelets in a cryo-container within one batch or across multiple batches varies within the range of 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%. For example, the total number of platelets in cryopreserved platelets in a cryocontainer across at least five batches varies within the ranges of 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the total number of platelets in cryopreserved platelets in a cryocontainer across at least five batches varies within the ranges of 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across at least ten batches varies within the ranges of 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across at least 10 batches varies within the ranges of 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%. In some embodiments, the platelet concentration in cryopreserved platelets in a cryocontainer across at least 5 batches has an average intra-batch coefficient of variation within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the platelet concentrations in cryopreserved platelets in cryocontainers across at least five batches have an average intra-batch coefficient of variation within the following ranges: 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%.In some embodiments, the platelet concentration in cryopreserved platelets in a cryocontainer within a batch has a coefficient of variation within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, or 4%. For example, the platelet concentration in cryopreserved platelets in a cryocontainer across a batch has a coefficient of variation within 0.5–30%, 1–30%, 2–30%, 3–30%, 0.5–25%, 0.5–20%, 0.5–15%, 0.5–10%, 0.5–7%, or 0.5–5%.

[0096] A collection of cryopreserved platelets in a cryocontainer according to this specification may, in some embodiments, include uniformity of the pH of the cryocontainer such that the pH of the cryopreserved platelets in the cryocontainer within one batch or across multiple batches varies within 5%, 4%, 3%, 2%, 1%, 0.9%, or 0.75%. For example, the pH of the cryopreserved platelets in the cryocontainer within one batch or across multiple batches varies within the range of 0.001–5%, 0.001–4%, 0.001–3%, 0.001–2%, 0.001–1%, 0.01–1%, 0.05–1%, or 0.5–1%. In some embodiments, the pH of the cryopreserved platelets in the cryocontainer across at least five batches has an average intra-batch coefficient of variation within 10%, 7%, 5%, 4%, 3%, 2%, or 1%. For example, the pH of cryopreserved platelets in a cryocontainer across at least five batches has an average intra-batch coefficient of variation in the range of 0.001–5%, 0.001–4%, 0.001–3%, 0.001–2%, 0.001–1%, 0.01–1%, 0.05–1%, or 0.5–1%. In some embodiments, the pH of cryopreserved platelets in a cryocontainer across at least five batches has a coefficient of variation in the range of 10%, 7%, 5%, 4%, 3%, 2%, or 1%. For example, the pH of cryopreserved platelets in a cryocontainer across at least five batches has a coefficient of variation in the range of 0.001–5%, 0.001–4%, 0.001–3%, 0.001–2%, 0.001–1%, 0.01–1%, 0.05–1%, or 0.5–1%.

[0097] The collection in a cryocontainer containing cryopreserved platelets described herein may, in some embodiments, include uniformity of CD61-positive particle concentration such that the concentration of CD61-positive particles in the cryopreserved platelets in the cryocontainer varies within 20%, 15%, 12%, 10%, 9%, 8%, or 7% within one batch or across multiple batches. For example, the concentration of CD61-positive particles in the cryopreserved platelets in the cryocontainer varies within the range of 1–20%, 1–15%, 1–12%, 1–10%, 3–20%, 3–15%, 3–12%, 3–10%, or 5–10%. In some embodiments, the concentration of CD61-positive microparticles in cryopreserved platelets in cryocontainers across at least five batches has an average intra-batch coefficient of variation in the range of 1–20%, 1–15%, 1–12%, 1–10%, 3–20%, 3–15%, 3–12%, 3–10%, or 5–10%. For example, the concentration of CD61-positive microparticles in cryopreserved platelets in cryocontainers across at least five batches has a coefficient of variation in the range of 1–20%, 1–15%, 1–12%, 1–10%, 3–20%, 3–15%, 3–12%, 3–10%, or 5–10%. For example, the concentration of CD61-positive microparticles in cryopreserved platelets in cryocontainers across at least five batches has a coefficient of variation in the range of 25%, 20%, 15%, 10%, or 8%.

[0098] The collection of cryopreserved platelets in a cryo-container described herein may, in some embodiments, span multiple batches or contain 10 platelets in a single batch. 6 This may include the uniformity of thrombinogenesis capacity of cryopreserved platelets such that the measure of thrombinogenesis per platelet varies within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%. Those skilled in the art will know that the measure of thrombinogenesis can be any appropriate unit based on the assay used, for example, by performing a thrombinogenesis assay. 6You will understand that thrombin production capacity can be determined in terms of IU per platelet. An exemplary and non-limiting example of a method for evaluating thrombin production is shown in Example 4. For example, across multiple batches or within a single batch of 10 6 The measure of thrombin production per platelet varies within the ranges of 0.5–20%, 0.5–15%, 0.5–12%, 0.5–10%, 0.5–8%, or 0.5–5%. For example, across multiple batches... 6 The measure of thrombin generation per platelet has an average intra-batch coefficient of variation within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, or 2%. In some embodiments, 10 across multiple batches 6 The measure of thrombin production per platelet has mean intra-batch coefficients in the range of 0.5–20%, 0.5–15%, 0.5–12%, 0.5–10%, 0.5–8%, or 0.5–5%. For example, 10 across multiple batches. 6 The measure of thrombin production per platelet has a coefficient of variation within 20%, 15%, 12%, 10%, 9%, 8%, 7%, 6%, 5%, 4%, or 3%. For example, 10 across multiple batches. 6 The thrombin generation per platelet has a coefficient of variation in the range of 0.5–20%, 0.5–15%, 0.5–12%, 0.5–10%, 0.5–8%, or 0.5–5%.

[0099] The cryogenic containers for CPPs described herein are typically prepared using platelets pooled from multiple donors (e.g., pooled platelets). Therefore, the cryogenic containers or cryovials containing CPPs described herein, as well as the processes for preparing and using them, typically contain a population of CPPs having biomolecular profiles indicating two or more platelet donors. Those skilled in the art will understand that various molecular tests exist that can be used to verify that the CPPs were prepared from multiple donors.

[0100] In some embodiments, the biomolecular profile indicating two or more platelet donors is a protein profile. For example, the biomolecular profile may be the amino acid sequences of one or more proteins, e.g., one or more proteins present in or associated with the CPP in a lot of cryovials produced from a pool of donors. Such a profile may include, for example, three or more amino acid sequences of a target protein from a single gene. These amino acid sequences may be polymorphisms of the protein. If a single donor has one or two amino acid sequences of this protein, e.g., depending on whether they are homozygous or heterozygous, then it will be understood that the presence of three or more amino acid sequences of this protein in the CPP may indicate two or more donors. Furthermore, if the set of donors whose platelets were pooled to produce a first lot of CPP is not the same as the donors used to produce a second lot of CPP, then the set of amino acid sequence variants / allels / versions of the target protein(s) in the first lot may be different from the set of amino acid sequence variants / allels / versions of the target protein(s) in the second lot. For example, if five donors are used to create a first pool of platelets for the first lot, and five different donors are used to create a second pool of platelets for the second lot, then for the target protein, there can be up to 10 different alleles / mutants / versions in each pool for the protein initially expressed from a single gene, and at least one allele / mutant / version of the target protein can be unique to each lot compared to the other lots.

[0101] In some embodiments that rely on quantitative information, the biomolecular profile indicating two or more platelet donors is the presence of two or more alleles / mutants / versions / amino acid sequences of at least one protein from at least one gene that differ significantly more than 50% in frequency within the composition. For example, a 50% frequency would be expected if such a composition came from a single donor that was heterozygous for the alleles in the first gene.

[0102] In a manner similar to the above considerations regarding target proteins (multiple), biomolecular profiles indicating two or more platelet donors can be detected and / or quantified by detecting and / or quantifying nucleic acids present in or associated with CPPs. Such detection can utilize techniques such as PCR, typically but not limited to quantitative reverse transcription polymerase chain reaction (qRT-PCR). qRT-PCR is considered one of the techniques available for quantifying RNA, such as mRNA, in a sample. The RNA identified may be specific to the platelet-donating individual or can be used to identify platelets donated by a single donor. In exemplary embodiments, such RNA molecules can be detected and / or quantified from a platelet sample to establish that the platelets were donated by two or more individuals.

[0103] In some embodiments, the cryo-container collection according to this specification may contain frozen platelets in cryopreservation medium in a frozen state, and the composition may, upon thawing, yield one or more of the properties listed herein, for example, after being stored for at least one month, two, three, four, five, six, eight, ten, or twelve months, in exemplary embodiments, at temperatures ranging from -10°C to -30°C, or -20°C+ / -5°C. a) Being in a liquid state, without the addition of any liquid. b) at least 1.0 × 10 11 To exhibit the platelet count per 35 ml of composition, c) To produce a single peak corresponding to a damaged membrane peak in a membrane integrity assay, d) The composition contains less than 50% CD61-positive particles, and e) To generate thrombin in an in vitro thrombin generation assay. In some embodiments, the composition can yield two or more, three or more, four or all of the properties. In exemplary embodiments, the composition can yield all of the properties. In some embodiments, the composition can yield properties a), b), and d). In some embodiments, the composition can yield properties a), b), d), and e).

[0104] Stability of cryopreserved platelets In some embodiments of the model, which includes a process for preparing a batch of cryopreserved platelets, a cryopreserved platelet composition and cryopreserved platelets, a process for preparing a collection or batch containing cryopreserved platelets as described herein, including a transition of freezing temperature from an initial freezing temperature to a storage freezing temperature, can be stable when stored frozen at -80°C, -70°C, -60°C, -50°C, -40°C, -30°C, -20°C, or higher. In some embodiments, cryopreserved platelets as disclosed herein, or cryopreserved platelets formed by the processes disclosed herein, in exemplary embodiments, are stable when stored at temperatures of -40°C, -35°C, -30°C, -25°C, -15°C, or -10°C but below 0°C. The stability of cryopreserved platelets present in cryocontainers provided in the collection of cryocontainers herein can be evaluated by a non-limiting list of parameters including cracks, ruptures, and breakage of the cryocontainer, such as a cryobag; visual inspection of aggregate-free swirling of cryopreserved platelets in the cryocontainer, such as a cryobag; the number of platelets per cryocontainer; and the pH of cryopreserved platelets in the cryocontainer. For example, in some non-limiting embodiments, the cryopreserved platelets herein are stable when stored at a specific temperature, e.g., about -20°C or higher, if the cryopreserved platelets swirl without any aggregates present upon visual inspection. For example, when stored at -20°C+ / -10°C, -20°C+ / -8°C, -20°C+ / -5°C, or -20°C+ / -2°C, the cryopreserved platelets swirl without any aggregates present upon visual inspection.For example, in some non-limiting embodiments, the cryopreserved platelets of this specification are stable when stored at a specific temperature, e.g., about -20°C or higher, e.g., -20°C ± 5°C, and the pH of the cryopreserved platelets is typically 6.0 or higher, typically 6.2 or higher, upon thawing. For example, when the cryopreserved platelets of this specification are stored at about -20°C, e.g., -20°C ± 5°C for periods ranging from 1 to 36 months, 1 to 30 months, 1 to 24 months, 1 to 18 months, or 1 to 12 months, they typically exhibit a pH greater than 7.0 upon thawing. In some embodiments, when the cryopreserved platelets of this specification are stored at about -20°C, e.g., -20°C ± 5°C for periods ranging from 1 to 12 months, they typically exhibit a pH greater than 6.2, 6.4, 6.6, 6.8, 7.0, or 7.2 upon thawing. For example, when cryopreserved platelets according to this specification are stored at -20°C+ / -5°C for a period ranging from 1 to 12 months, they typically exhibit a pH in the range of 6.2–7.8, 6.4–7.8, 6.6–7.8, or 7–7.8 upon thawing.

[0105] For example, in some non-limiting embodiments, when the cryopreserved platelets described herein are stored at a specific temperature, e.g., about -20°C or higher, e.g., -20°C+ / -5°C, the total number of platelets in the cryo-container is typically 1.5 × 10⁶ upon thawing. 11 pieces, 1.6×10 11 pieces, or 1.7 × 10 11 It is stable when there are more than 1.5 × 10¹⁶ units. In exemplary embodiments, the cryopreserved platelets of this specification, when stored at -20°C+ / -5°C for at least 1 month, 2, 3, 4, 6, 8, 10, 12 months, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years, are stable upon thawing, with a capacity of 1.5 × 10¹⁶ units. 11 pieces, 1.6×10 11 pieces, or 1.7 × 10 11The cryo-container contains a total number of platelets of a certain magnitude or greater. In some embodiments, the cryopreserved platelets according to this specification typically have a particle size, e.g., 0.5 μm to 2.5 μm, 1 μm to 5 μm, 1 μm to 4 μm, or 0.5 μm to 5.0 μm, such that at least 50% of the platelets have a diameter in the range of 0.5 μm to 2.5 μm, 1 μm to 5 μm, 1 μm to 4 μm, or 0.5 μm to 5.0 μm after thawing. In some embodiments, the cryopreserved platelets described herein, when stored for at least one month, two, three, four, six, eight, ten, twelve months, two, three, four, five, six, seven, eight, nine, or ten years at temperatures ranging from -40°C to -5°C, -30°C to -5°C, or -20°C to -5°C, typically exhibit a particle size upon thawing, with diameters ranging from 0.5 μm to 2.5 μm, 1 μm to 5 μm, 1 μm to 4 μm, or 0.5 μm to 5.0 μm, and a particle size of 1.5 × 10⁻¹⁶. 11 pieces, 1.6×10 11 pieces, or 1.7 × 10 11 The total number of platelets in the cryo-vessel is one or more. In some embodiments, at least 50%, 60%, 70%, or 75% of the cryopreserved platelets have a diameter in the range of 0.5 μm to 2.5 μm, 1 μm to 5 μm, 1 μm to 4 μm, or 0.5 μm to 5.0 μm upon thawing. In an exemplary embodiment, the cryopreserved platelets of this specification have a diameter of 1.5 × 10⁻⁶ upon thawing. 11 pieces, 1.6×10 11 pieces, or 1.7 × 10 11 Having a total number of platelets in one or more cryo-vessels, typically, cryopreserved platelets retain, upon thawing, hemostatic properties such as generating thrombin under in vitro conditions, the ability to reduce bleeding in a subject, or the ability to increase platelets in a subject where an increase in platelet count is necessary. In exemplary embodiments, the ability to reduce bleeding in a subject is based, for example, on the administration of 0.5 to 3 units of frozen platelets, frozen platelet derivatives, cryopreserved platelets, or cryopreserved platelet derivatives. In exemplary embodiments, 1 unit is 2.5 × 10⁶ 11 pieces + / -4.2×10 11This corresponds to individual frozen or cryopreserved platelets and / or platelet derivatives. In some embodiments, the methods herein include administering to a subject a liquid composition of frozen platelets, frozen platelet derivatives, cryopreserved platelets, or thawed compositions of cryopreserved platelet derivatives, prepared according to and / or any method provided herein, to restore hemostasis, reduce bleeding, or stop bleeding in the subject.

[0106] Stability can also be determined by evaluating certain parameters after thawing cryopreserved platelets stored at temperatures of -40°C, -35°C, -30°C, -25°C, -15°C, or above -10°C but below 0°C, in exemplary embodiments, at temperatures in the range of -40°C to -10°C. In some embodiments, thawing cryopreserved platelets disclosed herein, or cryopreserved platelets obtained by processes disclosed herein, can be done by exposing the cryopreserved platelets to a temperature above the freezing temperature. For example, exposing cryopreserved platelets to a temperature above 0°C, e.g., at least 5°C, 10°C, 15°C, 20°C, 25°C, 30°C, or 35°C. In exemplary embodiments, thawing includes exposing cryopreserved platelets to temperatures in the range of 20°C to 40°C, 22°C to 40°C, 25°C to 40°C, 30°C to 40°C, or 32°C to 40°C. In some embodiments, thawing involves subjecting cryopreserved platelets to temperatures of 37°C+ / -5°C, 37°C+ / -4°C, 37°C+ / -3°C, 37°C+ / -2°C, 37°C+ / -1°C, or 37°C+ / -0.5°C. Thawing as herein typically involves subjecting a cryocontainer or cryovial containing cryopreserved platelets as disclosed herein to a water bath set to 37°C+ / -2°C for a period of time until the contents of the cryocontainer are completely thawed. Those skilled in the art can assume that the time required for the contents to be completely thawed may vary depending on the volume of cryopreserved platelets, the dimensions of the cryocontainer, and the temperature at which the cryocontainer was stored before thawing. Therefore, thawing can be performed by placing the cryocontainer in a water bath set to a temperature of 37°C+ / -2°C for at least 1 minute, 2, 3, 4, 5, 6, 7, 8, 9, or 10 minutes, for example, in the range of 2-10, 2-9, 2-8, 2-7, or 2-6 minutes.

[0107] Exemplary Embodiments In this section of exemplary embodiments, non-limiting exemplary embodiments are provided herein and will be further discussed throughout this specification. For brevity and convenience, not all embodiments and embodiments disclosed herein, nor all possible combinations thereof, are listed in this section. Additional embodiments and embodiments are provided in other sections of this specification. Furthermore, it will be understood that, for example, as will be discussed throughout this disclosure, specific embodiments for many embodiments are provided that can be combined with any other embodiments. Given the complete disclosure described herein, any individual embodiments listed below or in this complete disclosure are intended to be combined with any embodiments listed below or in this complete disclosure. This applies to cases where there are additional elements that can be added to a certain embodiment, or narrower elements of elements already present in a certain embodiment. Such combinations may be provided as non-limiting representative combinations and / or may be discussed more specifically in other sections of this detailed description.

[0108] In one embodiment, a process for preparing a cryopreserved platelet composition containing cryopreserved platelets, or a cryopreserved platelet derivative composition containing a cryopreserved platelet derivative, the process being: i) A population of platelets in a cryopreservation medium is frozen at a temperature of -50°C or lower, in an exemplary embodiment, for the time until the cryopreservation medium freezes, to form an initial frozen platelet composition. ii) Transferring the initial frozen platelet composition into a freezer set to a temperature of -30°C or higher but below 0°C, iii) A process is provided herein that includes storing an initial frozen platelet composition in a freezer for at least 90 minutes to form a cryopreserved platelet composition containing cryopreserved platelets, or a cryopreserved platelet derivative composition containing a cryopreserved platelet derivative.

[0109] In one embodiment, a process for preparing a cryopreserved platelet composition comprising cryopreserved platelets, the process is: i) Freezing a group of platelets in a cryopreservation medium at a temperature of -50°C or lower to form an initial frozen platelet composition, ii) A process is provided herein that includes storing an initial frozen platelet composition at a temperature in the range of -10°C to -30°C for at least one month to form a cryopreserved platelet composition.

[0110] In one embodiment, a process for preparing a cryopreserved platelet composition comprising cryopreserved platelets, the process is: i) Freezing a population of platelets in a cryopreservation medium at a temperature below -50°C to form an initial frozen platelet composition, ii) A process is provided herein that includes storing an initial frozen platelet composition in a freezer set to a temperature of -20°C+ / -2°C for at least one month to form a cryopreserved platelet composition.

[0111] In one embodiment, a process for preparing a batch of cryopreserved platelets, wherein the process comprises: a) Pooling at least two platelet units in one container and at least one other platelet unit in another container, wherein in exemplary embodiments, there are at least three, four, or five platelet units, and the platelet units come from two or more donors, for example, two, three, four, or more donors. b) Centrifugation of each container to obtain a supernatant containing plasma and a pellet containing platelets, c) Resuspend the pellets in each container to form a resuspension, d) Pooling the resuspension from each container and forming the pooled resuspension in the pooled resuspension container, e) Adding a cryoprotectant to a pooled resuspension container having a pooled resuspension to obtain a pooled resuspension having a cryoprotectant, f) Distributing a pooled resuspension containing a cryoprotectant from a pooled resuspension container into several cryocontainers, g) a process comprising freezing a pooled resuspension having a cryoprotectant in a cryocontainer to form a batch of cryopreserved platelets is provided herein. In exemplary embodiments, the cryoprotectant is dimethyl sulfoxide (DMSO), and the pooled resuspension, together with DMSO, forms a pooled resuspension having DMSO. In some embodiments, the resuspension has a target weight that is X times the number of units pooled or provided in the container. In some embodiments, X is in the range of 10 g to 40 g times the number of units pooled or provided in the container. Thus, the resuspension has a target weight in the range of 10 g to 40 g times the number of units pooled or provided in the container. In exemplary embodiments, the resuspension has a target weight in the range of 15.9 g to 27.9 g times the number of units pooled or provided in the container.

[0112] In one embodiment, a process for preparing a batch of cryopreserved platelets, wherein the process comprises: a) Pooling at least two platelet units in one container and at least one other platelet unit in another container, wherein in exemplary embodiments, there are at least three, four, or five platelet units, and the platelet units come from two or more donors, for example, two, three, four, or more donors. b) Centrifugation of each container to obtain a supernatant containing plasma and a pellet containing platelets, c) Resuspend the pellets in each container to form a resuspension, d) Pooling the resuspension from each container and forming the pooled resuspension in the pooled resuspension container, e) Adding a cryoprotectant to a pooled resuspension container having a pooled resuspension to obtain a pooled resuspension having a cryoprotectant, f) Distributing a pooled resuspension containing a cryoprotectant from a pooled resuspension container into several cryocontainers, g) Freezing the pooled resuspension containing the cryoprotectant in the cryocontainer at a temperature of -50°C or lower to form an initial frozen platelet composition in the cryocontainer, h) A process is provided herein that includes storing an initial frozen platelet composition in a cryo-container at a temperature of -30°C or higher but below 0°C, for example in a freezer at -20°C, to form a batch of cryopreserved platelets. In exemplary embodiments, storage is performed for at least 7, 10, 15, 20, 25 days, 1 month, 2, 4, 6, 8, 10, 12 months, 2, 4, 6, 8, or 10 years.

[0113] In one embodiment, a collection of cryocontainers containing cryopreserved platelets, wherein the cryopreserved platelets in each cryocontainer have a set of biomolecular profiles indicating two or more platelet donors, and in an exemplary embodiment, one batch of cryocontainers has the same set of biomolecular profiles, and each batch in the collection has a different set of biomolecular profiles from any other batch in the collection. The collection includes multiple cryocontainers of at least two, three, four, or more batches. Each batch of cryocontainers includes at least 2, 3, 4, or 5 cryocontainers. A collection of cryo-containers is provided herein, wherein the coefficient of variation of the mean DMSO concentration in cryopreserved platelets across multiple batches is less than 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1%. In exemplary embodiments, the coefficient of variation of the mean DMSO concentration in cryopreserved platelets across multiple batches is in the range of 0.001–10%, 0.001–8%, 0.001–6%, 0.001–5%, 0.001–3%, 0.001–2%, 0.001–1%, 0.001–0.5%, or 0.001–0.1%.

[0114] In one embodiment, a collection of cryocontainers containing cryopreserved platelets, wherein the cryopreserved platelets in each cryocontainer have biomolecular profiles indicating two or more platelet donors, A collection of cryocontainers is provided herein, wherein the concentration of DMSO in the cryopreserved platelets in the first cryocontainer is within 25%, 20%, 15%, 10%, 5%, or 1% of the concentration of DMSO in the cryopreserved platelets in the second cryocontainer.

[0115] In one embodiment, the following composition is provided herein, comprising platelets frozen in a cryopreservation medium in a frozen state, wherein, upon thawing, the composition is capable of yielding one or more of the following properties after being stored for at least one month, two, three, four, five, six, eight, ten, or twelve months, in exemplary embodiments, at temperatures ranging from -10°C to -30°C, or -20°C+ / -5°C. a) Being in a liquid state, without the addition of any liquid, b) at least 1.0 × 10 11 To exhibit the platelet count per 35 ml of composition, c) To produce a single peak corresponding to a damaged membrane peak in a membrane integrity assay, d) The composition contains less than 50% CD61-positive particles, and e) To generate thrombin in an in vitro thrombin generation assay. In some embodiments, the composition can yield two or more, three or more, four or all of the properties. In exemplary embodiments, the composition can yield all of the properties. In some embodiments, the composition can yield properties a), b), and d). In some embodiments, the composition can yield properties a), b), d), and e).

[0116] In one embodiment, the following composition is provided herein, comprising platelets frozen in a cryopreservation medium in a frozen state, wherein, upon thawing, the composition is capable of yielding one or more of the following properties after being stored for at least one month, two, three, four, five, six, eight, ten, or twelve months, in exemplary embodiments, at temperatures ranging from -10°C to -30°C, or -20°C+ / -5°C. a) at least 1.0 × 10 11 To exhibit the platelet count per 35 ml of composition, b) To produce a single peak corresponding to a damaged membrane peak in a membrane integrity assay, c) The composition contains less than 50% CD61-positive particles, and d) In an in vitro thrombin generation assay, the composition generates thrombin, wherein the composition does not contain freeze-dried platelet derivatives or lyophilized platelet derivatives. In some embodiments, the composition may yield one or more, two or more, three or all of the properties. In some embodiments, the composition may yield properties a), c), and d).

[0117] In one embodiment, the Specified Publicly Provided Cryopreserved Platelet Composition comprises cryopreserved platelets, wherein the cryopreserved platelets are stored at a temperature of about -20°C for a period of at least 12, 18, or 24 months.

[0118] In one embodiment, the Specified Publicly Provided Cryopreserved Platelet Composition comprises cryopreserved platelets, wherein the cryopreserved platelets are stored at approximately -20°C for a period ranging from 1 month to 12, 18, 24, or 36 months.

[0119] In one embodiment, a method for administering a cryopreserved platelet composition to a subject is provided herein, the method comprising: thawing a cryovial of cryopreserved platelets (CPP) from a collection from any one of the embodiments or models herein, or thawing a cryovial containing a cryopreserved platelet composition from any one of the embodiments or models herein, to prepare a liquid containing CPP; and administering the liquid containing CPP to a subject. In exemplary embodiments, bleeding in the subject is reduced after administration.

[0120] In some embodiments of any aspect or embodiment of this specification, which includes a process for preparing a batch of cryopreserved platelets or a cryopreserved platelet composition, dispensing is performed, and in exemplary embodiments, dispensing in step f) is performed in several cryo-containers equal to the total number of units provided in the first step, exemplary embodiment, step a). In some embodiments, dispensing is performed, and in exemplary embodiments, dispensing in step f) is performed in each cryo-container until a target filling weight is achieved, the target filling weight being determined by the weight of the pooled resuspension with DMSO, and in exemplary embodiments, the target filling weight being determined by dividing the weight of the pooled resuspension with DMSO by the number of platelet units provided in the first step, exemplary embodiment, step a). In some embodiments, removal of a portion of the supernatant containing plasma is performed before resuspension, exemplary embodiment, step c) until a target weight of pellet and remaining plasma is achieved, the target weight being in the range of 15.9 g to 27.9 g times the number of units pooled or provided in the container. In some embodiments, the removal of a portion of the supernatant is carried out until a weight of + / - 10g, + / - 9g, + / - 8g, + / - 7g, + / - 6g, + / - 5g, + / - 4g, + / - 3g, + / - 2g, or + / - 1g of the target weight of the pellet and the remaining supernatant is achieved. In some embodiments, in step d), pooling the resuspension from each container is carried out using a tube tree system, and in exemplary embodiments, in step e), adding DMSO to the pooled resuspension container is carried out using a tube tree system. In some embodiments, in step f), distributing the pooled resuspension with DMSO from the pooled resuspension container to several cryo containers is carried out using a dosing tree system. In some embodiments, the process is carried out two or more times to form two or more batches of cryopreserved platelets.In some embodiments, the process is carried out multiple times to form multiple batches of cryopreserved platelets.

[0121] In some embodiments of any aspect or embodiment of this specification, which include a process for preparing a batch of cryopreserved platelets or a cryopreserved platelet composition, in step a), an odd number of platelet units are provided, and one platelet unit is processed in a separate container. In some embodiments, in step a), two platelet units are pooled in one container to form a plurality of containers. In some embodiments, in step c), the target weight is in the range of 31.8 g to 55.7 g for a container with two platelet units and in the range of 15.9 g to 27.9 g for a container with one platelet unit. In some embodiments, in step c), the target weight is in the range of 43.5 g to 49.5 g for a container with two units and in the range of 20.3 g to 26.3 g for a container with one unit. In some embodiments, in step c), the target weight is in the range of 45.5 g to 47.5 g for a container with two units and in the range of 22.3 g to 24.3 g for a container with one unit. In some embodiments, in step c), the target weight is approximately 46.5 g for a container with 2 platelet units and approximately 23.3 g for a container with 1 platelet unit. In some embodiments, in step a), 5, 7, 9, or 11 units are provided, each unit from a different donor. In some embodiments, in step a), 5 units are provided from 5 donors. In some embodiments, an even number of units are provided in step a). In some embodiments, an even number of units are provided, and 2 platelet units are pooled in one container to form a plurality of containers, each having 2 platelet units. In some embodiments, an even number of units are provided, and the target weight is in the range of 31.8 g to 55.7 g for each container. In some embodiments, an even number of units are provided, and the target weight is in the range of 43.5 g to 49.5 g for each container. In some embodiments, an even number of units are provided, and the target weight is in the range of 45.5 g to 47.5 g for each container.In some embodiments, an even number of units are provided in step a), and in step a), 6, 8, 10, 12, 14, 16, 18, 20, or more units are provided, each unit from a different donor. In some embodiments where an even number of units are provided, pooling is performed for 12 units from 12 donors. In some embodiments, at least 2, 3, 4, 5, 6, 7, or 8 units are pooled in the container.

[0122] In some embodiments of any aspect or embodiment of this specification, which include a process for preparing a batch of cryopreserved platelets or a cryopreserved platelet composition, in step e), the addition of DMSO is in the following amounts: 0.001-10%, 0.001-9%, 0.001-8%, 0.001-7%, 0.001-6%, 0.001-5%, 0.001-4%, 0.001-3%, 0.001-2%, 0.001-1%, 0.001-0.9%, 0.001-0.8%, 0.001-0.7%, 0.001-0.6%, 0. The process is carried out to achieve DMSO concentrations in the following ranges: 0.001-0.5%, 0.001-0.4%, 0.001-0.3%, 0.001-0.2%, 0.001-0.1%, 0.001-0.05%, 0.001-0.01%, 0.01-10%, 0.01-9%, 0.01-5.5%, 0.05-10%, 0.1-10%, 0.5-10%, 1-10%, 1-8%, 1-7%, 1-6%, 1-4%, 2-10%, 2-8%, 2-7%, 3-10%, 3-8%, 3-7%, 4-10%, 4-8%, 4-7%, 5-10%, 5-9%, 5-8%, 5-7%, or 6-10%. In some embodiments, the addition of DMSO is carried out using a stock solution of 27% DMSO. In some embodiments, the time from the addition of DMSO to cryopreservation is 1 hour, 2, 3, 4, 5, or 6 hours or less. In some embodiments, over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches, a) the coefficient of variation of the average resuspension volume over multiple batches is less than 10%, and / or b) the coefficient of variation of the average pooled resuspension volume with DMSO in the cryo-container over multiple batches is less than 5%.In some embodiments, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension across at least 10 batches has an average intra-batch coefficient of variation (average intra-batch CV) of 30%, 25%, 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1%, or can be in the range of 0.1–30%, 0.1–20%, 0.1–15%, 0.1–12%, 0.1–10%, 0.1–8%, 0.1–6%, 0.1–4%, 0.1–2%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, or 6–10%. In some embodiments, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension over at least 20 batches has an average intra-batch coefficient of variation (average intra-batch CV) of 30%, 25%, 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1%, or can be in the range of 0.1–30%, 0.1–20%, 0.1–15%, 0.1–12%, 0.1–10%, 0.1–8%, 0.1–6%, 0.1–4%, 0.1–2%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, or 6–10%. In some embodiments, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension across containers in one batch has a coefficient of variation of less than 30%, 25%, 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1%, or can be in the range of 0.1–30%, 0.1–20%, 0.1–15%, 0.1–12%, 0.1–10%, 0.1–8%, 0.1–6%, 0.1–4%, 0.1–2%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, or 6–10%.In some embodiments, resuspending pellets in each container leads to a resuspension in each container such that the volume of resuspension across at least 10 batches or within one batch varies by 30%, 25%, 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% or less, or can be in the range of 0.1–30%, 0.1–20%, 0.1–15%, 0.1–12%, 0.1–10%, 0.1–8%, 0.1–6%, 0.1–4%, 0.1–2%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, or 6–10%. In some embodiments, the volume of pooled resuspensions having DMSO in cryocontainers over at least 10 batches may have an average intra-batch coefficient of variation of 30%, 25%, 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1%, or may be in the range of 0.1–30%, 0.1–20%, 0.1–15%, 0.1–12%, 0.1–10%, 0.1–8%, 0.1–6%, 0.1–4%, 0.1–2%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, or 6–10%. In some embodiments, the volume of pooled resuspensions having DMSO in cryocontainers over at least 20 batches may have an average intra-batch coefficient of variation of 30%, 25%, 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1%, or may be in the range of 0.1–30%, 0.1–20%, 0.1–15%, 0.1–12%, 0.1–10%, 0.1–8%, 0.1–6%, 0.1–4%, 0.1–2%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, or 6–10%. In some embodiments, the volume of pooled resuspension in a cryo-container having DMSO in one batch may have a coefficient of variation of 30%, 25%, 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1%, or may be in the range of 0.1–30%, 0.1–20%, 0.1–15%, 0.1–12%, 0.1–10%, 0.1–8%, 0.1–6%, 0.1–4%, 0.1–2%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, or 6–10%.In some embodiments, the volume of the pooled resuspension having DMSO in a cryo-container across at least 10 batches or within one batch may vary to 30%, 25%, 20%, 15%, 12%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or 1% or less, or may be in the range of 0.1–30%, 0.1–20%, 0.1–15%, 0.1–12%, 0.1–10%, 0.1–8%, 0.1–6%, 0.1–4%, 0.1–2%, 1–10%, 2–10%, 3–10%, 4–10%, 5–10%, or 6–10%. In some embodiments, in step c), the resuspension is carried out using a buffer composition comprising a buffer, a base, and one or more sugars. In some embodiments, the sugars include one or more monosaccharides, disaccharides, polysaccharides, or combinations thereof. In some embodiments, the sugars include trehalose and polysucrose. In some embodiments, the buffer further includes salts and organic solvents. In some embodiments, the container in step a) is an apheresis platelet unit (APU) bag. In some embodiments, the APU bag has a volume of at least 600, 700, or 800 mL. In some embodiments, the APU bag has a maximum volume of at least 1000 or at least 1500 mL. In some embodiments, the APU bag has a maximum volume of 1600 mL. In some embodiments, the pooled resuspension container is one or more APU bags.

[0123] In some embodiments of any aspect or embodiment, including a collection of cryocontainers containing cryopreserved platelets, a process for preparing batches of cryopreserved platelets, a process for preparing a cryopreserved platelet composition, or a composition containing frozen platelets, the collection includes a plurality of cryocontainers of at least 3, 4, 5, 10, 15, 20, 50, 75, or 100 batches. In some embodiments, the biomolecular profile indicating two or more platelet donors is the presence of two amino acid sequences of a first protein from a first gene that differ significantly by more than 50% in frequency within the cryopreserved platelets, or three or more amino acid sequences of a first protein. In some embodiments, the set of biomolecular profiles in one batch is different from the set of biomolecular profiles in another batch. In some embodiments, the coefficient of variation of the mean DMSO concentration in cryopreserved platelets across multiple batches is less than 15%, 10%, 8%, 6%, 5%, and 4%, and in exemplary embodiments, less than 3%, 2.5%, 2%, 1.5%, 1%, 0.75%, 0.5%, 0.25%, or 0.1%. For example, the coefficient of variation of the mean DMSO concentration across multiple batches can be in the range of 15–0.01%, 10–0.01%, 8–0.01%, 6–0.01%, 5–0.01%, 4–0.01%, 3–0.01%, 2–0.01%, or 1–0.01%. In some embodiments, the DMSO concentration in the cryopreserved platelets in the first cryo-vessel is within 20%, 15%, 12%, 10%, 8%, and 6%, while in exemplary embodiments, the DMSO concentration in the cryopreserved platelets in the second cryo-vessel is within 5%, 3%, and 1%. In some embodiments, the biomolecular profile indicating two or more platelet donors is the presence of two amino acid sequences of a first protein from a first gene that differ significantly in frequency of more than 50% within the cryopreserved platelets, or the presence of three or more amino acid sequences of a first protein.In some embodiments, the collection comprises several batches of cryocontainers, each batch of cryocontainers having the same biomolecular profile, and each batch of cryocontainers having a different biomolecular profile from any other batch of cryocontainers in the collection. In some embodiments, when thawed, the cryopreserved platelets in the first cryocontainer have a volume that varies by 30%, 25%, 20%, 15%, in exemplary embodiments, 10%, 8%, 6%, 5%, 3%, or 1% or less of the volume in the second cryocontainer. In some embodiments, the first and second cryocontainers are from different batches of the collection of cryocontainers. In some embodiments, the mean DMSO concentrations in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches have coefficients of variation less than 10%, 8%, 5%, 3%, or 2%, and in exemplary embodiments, less than 1%, 0.9%, 0.8%, 0.75%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%. In some embodiments, the average DMSO concentration in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation in the range of 0.01–5%, 0.01–4%, 0.01–3%, 0.01–2%, 0.01–1%, 0.05–1%, 0.05–0.75%, 0.05–0.50%, or 0.05–0.4%. In some embodiments, the DMSO concentration in cryopreserved platelets in a cryo-container within one batch or across at least five batches varies by 5%, 4%, 3%, 2%, 1% or less, and in exemplary embodiments, by 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.05% or less. In some embodiments, the cryocontainer in a batch or collection contains cryopreserved platelets having DMSO in the range of 0.001–10%, 0.01–5.5%, 1–6%, 5–7%, 0.001–8%, 1–10%, 1–8%, 1–7%, 2–10%, 2–8%, 2–7%, 3–10%, 3–8%, 3–7%, 4–10%, 4–9%, 4–8%, or 4–7%.

[0124] In some embodiments of any aspect or embodiment of this specification, which includes a process for preparing a batch of cryopreserved platelets, a collection of cryocontainers containing cryopreserved platelets, or a composition containing cryopreserved platelets, the cryopreserved platelets are stable at a temperature of -20°C or -20°C+ / -5°C for at least 1 month, 2, 3, 4, 6, 8, 10 months, 1 year, 2, 3, 4, 5, 6, 7, 8, 9, or 10 years. For example, cryopreserved platelets can be stable for 1 month to 5 years, 1 month to 12 months, 3 months to 5 years, 6 months to 5 years, 1 to 5 years, or 2 to 5 years. In some embodiments, the platelet concentration in cryopreserved platelets in a cryocontainer within one batch or across at least five batches varies by 20%, 15% or less, and in exemplary embodiments by 10%, 8%, 5%, 3%, 2%, or 1% or less. In some embodiments, the platelet concentration in cryopreserved platelets in cryocontainers across at least five batches has an average intra-batch coefficient of variation of 20%, less than 15%, and in exemplary embodiments, 10%, 8%, 6%, 5%, 3%, or less than 1%. In some embodiments, the platelet concentration in cryopreserved platelets in cryocontainers within one batch has an average coefficient of variation of 20%, less than 15%, and in exemplary embodiments, 10%, 8%, 6%, 5%, 3%, or less than 1%. In some embodiments, the total number of platelets in cryopreserved platelets in cryocontainers within one batch or across at least five batches varies by 20%, 15%, 10% or less, and in exemplary embodiments, 8%, 6%, 5%, 3%, or less than 1%. In some embodiments, the total number of platelets in cryopreserved platelets in cryocontainers across at least five batches has an average intra-batch coefficient of variation of 20%, 15%, less than 10%, and in exemplary embodiments, 8%, 6%, 5%, 3%, or less than 1%. In some embodiments, the total number of platelets in cryopreserved platelets in a cryocontainer within a batch has a coefficient of variation of 25%, less than 20%, and in exemplary embodiments, 15%, 10%, 8%, 6%, 5%, 4%, 3%, 2%, or less than 1%.In some embodiments, the total number of platelets in cryopreserved platelets in a cryo-container within a batch has a coefficient of variation in the range of 0.01-25%, 0.01-20%, 0.01-10%, 0.01-8%, 0.01-6%, 0.01-5%, 0.01-3%, 0.01-2%, 0.5-10%, 0.5-8%, 0.5-5%, 0.5-3%, 1-10%, 1-8%, 1-6%, or 1-5%. In some embodiments, the cryopreserved platelets contain plasma in the range of 50-95%, 50-90%, 50-85%, 50-80%, 50-75%, 60-95%, 65-95%, 70-95%, or 70-85% (v / v). In some embodiments, the cryopreserved platelets contain DMSO in the range of 1-10%, 1-8%, 2-10%, 2-8%, 3-10%, 3-9%, 3-8%, 4-10%, or 4-8% (v / v). In some embodiments, the cryopreserved platelets contain sodium chloride in the range of 5-30%, 5-25%, 5-20%, 5-15%, or 7-15% (w / v). In some embodiments, the cryopreserved platelets contain a buffer, salt, one or more sugars, and at least one organic solvent. In some embodiments, the sugars include trehalose and polysucrose. In some embodiments, the cryo-container is a cryo-bag.

[0125] In any of the embodiments or models of this specification, including a process for preparing cryopreserved platelets or cryopreserved platelet compositions, a process for preparing a batch of cryopreserved platelets, or a process for preparing cryopreserved platelets or cryopreserved platelet compositions, freezing may include freezing a population of platelets in a cryopreservation medium at a temperature of -50°C, -55°C, -60°C, or -65°C or lower, or freezing a pooled resuspension having the cryoprotectant of this specification to form an initial frozen platelet composition; and storage may include storing the initial frozen platelet composition at a temperature of -45°C, -40°C, -35°C, -30°C, -25°C, or -20°C or higher for at least 2, 4, 6, 7, 10, 15, 20, or 25 days, 1 month, 2, 4, 6, 8, 10, or 12 months, 1 year, 2, 4, 6, 8, or 10 years to form a cryopreserved platelet composition. In exemplary embodiments, storage includes storing the initial frozen platelet composition in a freezer set to a temperature of -20°C + / -2°C. In some embodiments, freezing includes subjecting a population of platelets in a cryopreservation medium or pooled resuspension having a cryoprotectant disclosed herein to that temperature for at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 7 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, or 6 months to form an initial frozen platelet composition. For example, freezing can be performed for periods of 24 hours to 6 months, 24 hours to 5 months, 24 hours to 4 months, 1 day to 6 months, 1 day to 5 months, or 1 day to 4 months. In some embodiments, this process further includes thawing the cryopreserved platelets to form a liquid platelet composition and administering an effective amount of the liquid platelet composition to a subject in need. In some embodiments, the storage of the initial frozen platelet composition can be carried out for a period ranging from 1 month to 10 years, 1 month to 8 years, 1 month to 7 years, 1 month to 5 years, 1 month to 3 years, or 1 month to 1 year.In some embodiments, the cryopreservation medium contains dimethyl sulfoxide (DMSO) in concentrations ranging from 5% to 8%, for example, DMSO can be 6%+ / -1%, + / 0.8%, + / -0.6%, + / -0.5%, + / -0.4%, + / -0.3%, + / -0.2%, + / -0.1%. In some embodiments, the cryopreservation medium contains DMSO in concentrations ranging from 0.5% to 8%, 2% to 8%, 3% to 8%, or 5% to 8%. In some embodiments, the initial frozen platelet composition is stored at a temperature in the range of -10°C to -30°C for at least 1 month, 2, 3, 4, 5, or 6 months to form cryopreserved platelets, which, upon thawing, possess the following: i) the ability to reduce bleeding in subjects where it is needed; ii) a recovery rate of at least 65%, 70%, or 75% of the platelet count; iii) a pH of 6.2 or higher; iv) aggregate-free swirling upon visual inspection; and v) 1.7 × 10⁻¹. 11The product exhibits one or more of the following characteristics: a platelet count of 10% / bag or platelet / cryocontainer or a platelet count of 20–35 ml. In some embodiments, the mean DMSO concentration in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation of less than 10%, 8%, 5%, 3%, or 2%, and in exemplary embodiments, less than 1%, 0.9%, 0.8%, 0.75%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, or 0.1%. In some embodiments, the average DMSO concentration in cryopreserved platelets across at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation in the range of 0.01–5%, 0.01–4%, 0.01–3%, 0.01–2%, 0.01–1%, 0.05–1%, 0.05–0.75%, 0.05–0.50%, or 0.05–0.4%. In some embodiments, the DMSO concentration in cryopreserved platelets in a cryo-container within one batch or across at least five batches varies by 5%, 4%, 3%, 2%, 1% or less, and in exemplary embodiments, by 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.05% or less. In some embodiments, the cryocontainer in a batch or collection contains cryopreserved platelets having DMSO in the range of 0.001–10%, 0.01–5.5%, 1–6%, 5–7%, 0.001–8%, 1–10%, 1–8%, 1–7%, 2–10%, 2–8%, 2–7%, 3–10%, 3–8%, 3–7%, 4–10%, 4–9%, 4–8%, or 4–7%. In some embodiments, the cryopreserved platelets, upon thawing, have diameters in the range of 0.5–5 μm, 1–4 μm, 1–3 μm, or 0.5–2.5 μm, and the composition, upon thawing, has a CD61-positive microparticle content in the range of 10–30%, 10–35%, 10–40%, 10–45%, or 10–50%.In some embodiments, a therapeutically effective amount of cryopreserved platelets or cryopreserved platelet derivatives, frozen platelets or frozen platelet derivatives, when stored at temperatures of -45°C, -40°C, -35°C, -30°C, -25°C, or above -20°C for at least 2, 4, 6, 7, 10, 15, 20, or 25 days, 1 month, 2, 4, 6, 8, 10, or 12 months, 1 year, 2, 4, 6, 8, or 10 years, retains the ability to reduce bleeding in a subject in need upon thawing. In some embodiments, the therapeutically effective amount is the amount capable of reducing bleeding in a subject compared to bleeding before administration of the thawed, cryopreserved platelets or cryopreserved platelet derivatives, frozen platelets or frozen platelet derivatives. Thus, in some embodiments, the therapeutically effective amount can be at least 0.25 units, 0.5 units, 0.75 units, 1 unit, 2, or 3 units of thawed, cryopreserved platelets or frozen platelets. For example, the therapeutically effective dose can range from 0.25 units to 5 units, and in exemplary embodiments, it can be in the range of 0.5 units at the lower end and 3, 2, or 1 unit at the upper end, or 1 unit at the lower end and 4, 3, or 2 units at the upper end. In some embodiments, 1 unit is at least 1 × 10 in a volume of at least about 40 ml, 45 ml, 50 ml, 55 ml, or 60 ml. 11 pieces, 1.5×10 11 pieces, 2×10 11 1, or 2.5 × 10 11 It corresponds to 1 x 10¹⁶ platelets. In some embodiments, one unit is 1 x 10¹⁶ of at least 40 or 50 ml. 11 ~5×10 11 pieces, 1×10 11 ~4×10 11 pieces, 1×10 11 ~4×10 11 pieces, or 2 × 10 11 ~3×10 11 It corresponds to a range of platelets of a certain number. In an exemplary embodiment, one unit is 2.5 × 10¹⁶ in 50 + / - 4 ml 11 + / - 4.2 × 10 11corresponds to individual platelets. This unit can refer to a unit of frozen platelets, frozen platelet derivatives, cryopreserved platelets, or cryopreserved platelet derivatives, as understood in the context.

[0126] In some embodiments of any of the aspects or embodiments herein, including a composition comprising frozen platelets or cryopreserved platelets in a cryopreservation medium in a frozen state disclosed herein, a collection disclosed herein, or a process for preparing a cryopreserved platelet composition or a process for preparing a batch of cryopreserved platelets, the composition exhibits a platelet count of at least 1.0×10 11 per composition of 20 - 35 ml upon thawing. For example, at least 1.0×10 11 per 20 ml of composition, 1.0×10 11 per 25 ml of composition, 1.0×10 11 per 30 ml of composition, 1.0×10 11 per 35 ml of composition. In some embodiments, the compositions herein exhibit a platelet count of at least 1.1×10 11 per 20 - 35 ml of composition, 1.2×10 11 per 20 - 35 ml of composition, 1.3×10 11 per 20 - 35 ml of composition, 1.4×10 11 per 20 - 35 ml of composition, 1.5×10 11 per 20 - 35 ml of composition, 1.6×10 11 per 20 - 35 ml of composition, or 1.7×10 11The composition exhibits a platelet count of 20-35 ml per particle. In some embodiments, the cryopreserved platelets herein are cryopreserved platelet derivatives. In some embodiments, the frozen platelets herein are frozen platelet derivatives. Typically, the compositions disclosed herein are in a liquid state upon thawing and do not require the addition of any liquid to achieve such a liquid state. In some embodiments, the cryopreserved platelets, or frozen platelets herein, do not contain freeze-dried platelet derivatives or lyophilized platelet derivatives. In some embodiments, the composition yields a single peak upon thawing that corresponds to a damaged membrane peak in a membrane integrity assay. In some embodiments, the membrane integrity assay comprises incubating the composition with calcein acetoxymethyl (AM) for 20 minutes before performing flow cytometry, and the flow cytometry comprises detecting fluorescence produced by metabolic calcein AM and retained by particles in the treated composition. In some embodiments, the in vitro thrombin generation assay comprises generating thrombin in the presence of tissue factor and phospholipids. In some embodiments, the in vitro thrombin generation assay comprises generating thrombin in the presence of tissue factor and phospholipids. In some embodiments, the frozen platelets or cryopreserved platelets herein have a diameter in the range of 0.5–5 μm, 1–4 μm, 1–3 μm, or 0.5–2.5 μm upon thawing. In some embodiments, the compositions comprising the frozen platelets herein have a CD61-positive microparticle content in the range of 5–50%, 5–45%, 5–40%, 5–35%, 5–30%, 10–30%, 10–40%, 10–50%, or 10–60% upon thawing. In some embodiments, the compositions disclosed herein contain CD61-positive microparticles with a diameter of less than 1 μm, 0.8 μm, or 0.5 μm.In some embodiments, the compositions herein have a pH of 6.0, 6.2, or 6.5 or higher upon thawing. In some embodiments, the compositions herein have a pH in the range of 6.2 to 8.0 upon thawing. In some embodiments, the compositions herein have dispersion properties such that the frozen platelets within the composition do not form visible aggregates upon visual inspection after thawing. In some embodiments, the compositions have dispersion suspension properties such that no aggregation of platelets in the composition is observed by visual inspection of the composition after thawing. In some embodiments, the compositions have swirling properties such that swirling of the composition can be observed by visual inspection of the composition after thawing. In some embodiments, the compositions comprise a cryopreservation medium containing dimethyl sulfoxide (DMSO) at concentrations of 0.5% to 10%, 1% to 8%, or 3% to 8%. In some embodiments, in the compositions herein, less than 50% of the frozen platelet derivatives are positive for CD62 or annexin V upon thawing. For example, in the compositions of this specification, particles positive for CD62 account for 90%, 85%, 80%, 70%, 65%, 60%, or less than 50%. For example, in the compositions of this specification, particles positive for annexin V account for 90%, 85%, 80%, 70%, 65%, 60%, or less than 50%. For example, in the compositions of this specification, particles positive for annexin V are in the range of 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 5-50%, 5-40%, 5-30%, or 5-20%. For example, in the compositions of this specification, particles positive for CD62 are in the range of 1-50%, 1-40%, 1-30%, 1-20%, 1-10%, 5-50%, 5-40%, 5-30%, or 5-20%. In some embodiments, the frozen or cryopreserved platelets in the compositions herein are less active compared to freeze-dried or lyophilized platelet derivatives. In some embodiments, the frozen or cryopreserved platelets in the compositions are not lyophilized or freeze-dried platelet derivatives. In some embodiments, the compositions herein contain at least 1.0 × 10. 11Exhibits the platelet count of 20 ml per composition. In some embodiments, the composition, when stored at a temperature in the range of -5°C to -30°C for at least 1 day, 2, 4, 6, 7, 10, 15, 20, 25 days, 1 month, 2, 4, 6, 8, 10, or 12 months, 1 year, 2, 4, 5, 6, 8, or 10 years, in exemplary embodiments, exhibits a CD61-positive microparticle content of less than 50% of the CD61-positive particles in the composition upon thawing. In some embodiments, the composition, when stored at a temperature in the range of -5°C to -30°C for at least 1 day, 2, 4, 6, 7, 10, 15, 20, 25 days, 1 month, 2, 4, 6, 8, 10, or 12 months, 1 year, 2, 4, 5, 6, 8, or 10 years, in exemplary embodiments, results in a single peak corresponding to the membrane peak impaired in the membrane integrity assay upon thawing. In some embodiments, the composition, when stored at a temperature in the range of -5°C to -30°C for at least 1 day, 2, 4, 6, 7, 10, 15, 20, 25 days, 1 month, 2, 4, 6, 8, 10, or 12 months, 1 year, 2, 4, 5, 6, 8, or 10 years, in exemplary embodiments, generates thrombin in the in vitro thrombin generation assay upon thawing. In exemplary embodiments, the compositions herein are capable of providing one or more, two or more, three or more, or all of the properties after storage for 12 months. In some embodiments, a composition comprising frozen platelets in a cryopreservation medium in a frozen state, when stored at a temperature in the range of -10°C to -30°C, or -20°C + / - 5°C for at least 1 month, 2, 3, 4, 5, 6, 8, 10, or 12 months, in exemplary embodiments, exhibits a liquid state without adding a liquid upon thawing and has at least 1.0×10 11 Exhibits the platelet count of 35 ml per composition, exhibits a CD61-positive microparticle content of less than 50% of the CD61-positive particles in the composition, and generates thrombin in the in vitro thrombin generation assay.

[0127] Compositions produced by any of the methods described herein are also provided herein. In some embodiments, any of the compositions provided herein can be prepared by any of the methods described herein. Specific embodiments disclosed herein may be further limited in the claims using the language “~consisting of” or “essentially consisting of.”

[0128] The following non-limiting examples are provided purely for illustrative purposes of the embodiments and are not intended to limit the scope or spirit of this disclosure. [Examples]

[0129] Example 1. Single donor method A single-donor CPP unit consists of a single transfusion-ready dose of apheresis platelets concentrated by centrifugation and expression, and is cryopreserved in approximately 6% DMSO at -65°C or below (in a -80°C freezer). The following is a description of the single-donor process, also known as the Vitalant method, as described in Valeri, C. Robert et al. Transfusion 45.12(2005):1890-1898.

[0130] Step 1 - Initial inspection of the APU upon receipt After receiving the APU, visual inspection is performed for swirl, aggregates, RBC contamination, and intact ports. Units are checked against indicators where the units were irradiated. If no irradiation indicators are present, Vitalant irradiates the units later in the process (step 4). It is confirmed that the elapsed time from the end of collection to the receipt of the APU is 48 hours or less.

[0131] Step 2 - Determining the initial APC volume The initial APC volume is determined. Through the single donor process, the volume of the component is determined using the weight / volume conversion method, which divides the total weight of the component (after subtracting the tare weight of the empty bag) by the specific gravity of the component. For the purposes of this process, the units for various measurements are as follows: weight is in grams (g), volume is in milliliters (mL), and specific gravity is in grams / milliliter (g / mL). Equation 1 is used to determine the APC weight and volume. (Equation 1): Determination of APC weight and volume APC weight = APU weight - empty bag weight TIFF2026521991000003.tif8128

[0132] The APU weight includes the APC weight and the weight of the empty apheresis collection bag. The terms APC weight and APC volume describe the weight and volume of apheresis platelet concentrate (APC) contained within an APU (apheresis platelet unit). Since the APU contains the APC volume within it, the APU volume and APC volume can be used interchangeably. The following steps are used in the manufacturing process to determine the APC volume. The APU weight is determined using a scale. The APC weight is found by subtracting the weight of the empty collection bag from the APU weight. Vitalant used the empty collection bag weight listed in TMRL.801, Common Tare Weights Job Aid. The APC weight is converted to volume using a specific gravity of 1.027 g / mL. This specific gravity is from TMRL.301 V.3 “Cryopreservation (Closed System) of Apheresis Platelet Units Procedure” and is considered the known specific gravity of APC according to the literature and standard blood bank practices. This APC specific gravity is used throughout this specification.

[0133] Step 3 - Reduce APC volume (if necessary) If the APC volume exceeds 375 mL, the sample is typically removed to reduce the APC volume until it is within the processing range. A 5 mL transport set (Charter Medical, 6-inch tube with puncture pin, removable 5 mL BD® syringe, product number 03-220-XL, or equivalent) is connected to the APU using a sterile connection device (SCD, Terumo TSCD® Sterile Tubing Welder, SC-201A). This 5 mL transport set is then used to remove the sample. This sample removal method is used throughout the single donor process to remove the sample. After sample removal, the APC volume is re-determined.

[0134] Step 4 - APU irradiation (if necessary) If the APU has not been irradiated beforehand, it will be irradiated at this point in the process.

[0135] Step 5 - APU sampling and pre-manufacturing specification verification Next, the APU is placed on a platelet incubator / stirrer for at least 30 minutes. The APU is again examined for swirl and aggregates. Then, the APU is sampled for pH / blood gas and platelet concentration analysis. A copy of the pH / blood gas printout is placed in the batch record. After sampling, the APC volume is determined again. Equation 2 is used to determine the total platelet count in the APU using the platelet concentration and APC volume. (Equation 2): Determination of total platelet count at APU Total platelet count = APC volume * platelet concentration

[0136] Once it is confirmed that the APU meets all pre-manufacturing standards, the cryopreservation process can proceed. The following is an excerpt from TMRL.301V.3: "If the APU does not meet the standards (negative swirl present, aggregates present, <3.0 × 10⁻¹⁰)" 11If the platelet yield is less than 100%, or if the volume is <165 or >375 mL, return the APU to the platelet incubator / stirrer and discuss the course of action with the TMRL Director (or designated person). Occurrence / deviation reporting procedure: Complete deviation reports as necessary, in accordance with TMRL113. This describes how to handle APUs that do not meet pre-manufacturing specifications.

[0137] Step 6 - Addition of 27% DMSO Formulate APC to a final DMSO percentage (%DMSO) of 5.65%–6.52% using a sterile, injectable-grade bag containing 27% DMSO and 0.66% sodium chloride in water (Bio Life Solutions, BloodStor® 27 NaCl Biopreservation Media, part number 327207, or equivalent). Throughout this report, APC and DMSO solutions will be referred to as APC / DMSO. Formulate APU with 27% DMSO using Table 1.

[0138] (Table 1) TMRL.806V.1, “Weight of 27% DMSO to Add for Freezing Platelet Job Aid” TIFF2026521991000004.tif18160

[0139] A 27% DMSO bag is welded to the APU using an SCD. The APU is placed on a scale to measure the weight of the 27% DMSO to be added to the APU. The 27% DMSO bag is suspended on the APU to allow the 27% DMSO to flow gravimetrically for the 27% DMSO addition step. The 27% DMSO is then slowly added to the APU, and the APU is gently massaged to facilitate mixing. The 27% DMSO is added at a rate of 10-15 g / min until the target weight of 27% DMSO is reached. The start and end times of the 27% DMSO addition are recorded. If the 27% DMSO addition rate falls outside the desired range of 10-15 g / min, a deviation report is written and production continues.

[0140] Step 7 - Calculation of %DMSO Next, using equations 3 and 4, weigh the APU to find the APC / DMSO volume and %DMSO. (Equation 3): Determination of APC / DMSO volume TIFF2026521991000005.tif10128

[0141] Vitalant is listed in TMRL.301V.3, Apheresis Platelet Unit Cryopreservation (Closed System) Procedure, with an APC / DMSO specific gravity of 1.03 g / mL.

[0142] (Equation 4): Determination of %DMSO Dispensed 27% DMSO (mL) = APC / DMSO volume - APC volume TIFF2026521991000006.tif7128

[0143] The %DMSO content is recorded, and it is determined whether the %DMSO content falls within the product specification range (5.65% to 6.52%).

[0144] Step 8 - Sampling for APU culture after DMSO addition Remove APC / DMSO from the sample for aerobic and anaerobic culture purposes using BacT / ALERT.

[0145] Step 9 - Platelet Material Transport A tube extension set (such as a plasma transport set, Charter Medical, 24-inch tubing, roller clamp, and two puncture pins, product number 03-220-00, or equivalent) is welded between the cryobag (500mL EVA CryoStore freezer bag, Origen Reference CS500, or equivalent) and the APU to connect the two bags and extend the working length of the tubing for centrifugation and expression. The APC / DMSO solution is then transferred from the APU bag to the cryobag. Excess air in the cryobag is expressed into the empty APU bag, and the tubing is clamped.

[0146] Step 10 - Centrifugation Next, the cryobag and empty APU bag are subjected to centrifugation at 1250G for 10 minutes with the maximum acceleration set to 3, no brakes, and slow-stop setting (Thermo Scientific, Sorvall RC3BP+ centrifuge).

[0147] Step 11 - Expression The cryobag is carefully removed from the centrifuge cup so as not to interfere with the platelet pellet and placed in a plasma expresser (Fenwal Inc., manual plasma extractor, product code 4R4414, or equivalent). If any leakage is noticed, the unit is usually not used for patient infusion. The plasma expresser is used to remove the supernatant without losing platelets. After expression is complete, the cryobag is weighed. The cryobag should weigh 50g to 55g to meet the required volume specifications and account for the additional volume removed for pre-freeze sampling. If the weight is less than 50g, the tube clamp is released and the material is slowly returned from the plasma / DMSO drainage bag to the cryobag to achieve a weight of 50g to 55g. This range corresponds to 25.5mL to 30.4mL. The pre-sampling volume is then determined to ensure that the post-sampling volume is 20 to 35mL.

[0148] Step 12 - Rest and resuspend platelet pellet A resting period of 30 minutes (±5 minutes) at 20-24°C is required before resuspending the platelet pellet. Resuspend the pellet for several minutes by gentle manual agitation. The cryobag is inspected for visible aggregates, and any aggregates present are recorded.

[0149] Step 13 - Sampling after resuspension If the volume obtained after sampling is less than 20 mL, sampling is skipped. If the volume is sufficient to continue, approximately 2 mL of the pre-freezing sample is removed. The sample is removed by slowly filling a 5 mL syringe (part of the 5 mL transport set) and discharging it several times to mix with the platelet suspension and obtain a well-mixed sample.

[0150] Step 14 - Determining the volume before freezing The pre-freezing volume is determined using a weight / volume conversion with a standard empty cryobag tare weight of 22.8 g and an APC / DMSO specific gravity of 1.03 g / mL. This determination of the post-freezing volume is to ensure that the volume is within the freezing range. The pre-freezing volume ranges from 20 mL to 35 mL.

[0151] Step 15 - Freeze Label the cryobags and perform a final visual inspection to ensure the bags are intact and leak-free. The excess tubing on the cryobags is sealed using a tube sealer (SEBRA® Handheld RF Tube Sealing System, Model No. 2380, or equivalent), and a safety seal is added to the remaining tubing. Place the cryobags in thawing bags and freezer cartons for storage in a -80°C freezer. Place the units in the freezer and record the start time of freezing.

[0152] Step 16 - Timing Standard The elapsed time from the end of 27% DMSO addition to the start of freezing is typically maintained at 2 hours and 45 minutes or less. The elapsed time from the end of APU to placement in the freezer is typically 57 hours or less. If these time standards are not met, a deviation report will be filed.

[0153] Post-manufacturing specifications: TMRL.805V.2 lists the following post-manufacturing specifications for CPP units: ●Freezing volume: 20mL~35mL ●Time from adding 27% DMSO to the freezer: 2 hours 45 minutes or less ●Elapsed time at the end of processing: 57 hours or less. ● Visible aggregates: None ●Sterility test: No growth observed. ●%DMSO: 5.65%~6.52% ●Maximum DMSO per CPP unit: 2.53g (mass corresponding to the maximum freezing volume at maximum %DMSO) Products that do not meet the final acceptance criteria (non-conforming products) may be designated as other products or disposed of at the discretion of the TMRL Director.

[0154] Analysis of specific key steps in the single donor process 27% DMSO added DMSO at concentrations of 5% to 10% is commonly used as a cryoprotectant in biological applications and is added to APCs due to its cryoprotective properties.

[0155] Disadvantages of the previous 27% DMSO addition method The Vitalant method, which involves adding 27% DMSO based on Table 1, is a very simplified method for formulating the product, but it has several drawbacks. Dosing 27% DMSO in this manner creates a process with a variable %DMSO target. This leads to a product with an inherently variable %DMSO. Additionally, Table 1 can lead to %DMSO units outside the specification. Figure 3 illustrates these problems. Figure 3 shows the target %DMSO achieved using Table 1. The target %DMSO was determined using equations 1, 3, and 4. The variability of %DMSO shown in Figure 3 is equal to a mean percent difference of 10.6% from the mean of the 27% DMSO dosing range. Analysis A describes the mathematics used in determining all the data points in Figure 3.

[0156] Analysis A: Analysis of Table 1 A 189.0 mL APU falls within the 27% DMSO dosage range of 50 g according to Table 1. Using a volume / weight conversion like Equation 1, the APC weight of this APU is 194.1 g. After adding the 27% DMSO of 50 g required by Table 1, the APC / DMSO weight is 244.1 g, or 237.0 mL. Determining the percentage DMSO using Equation 4 shows that a 189 mL APU administered with 27% DMSO of 50 g is formulated to 5.47% DMSO, which is below the stated specification.

[0157] Freezing volume The primary objective of the centrifugation and expression steps is to concentrate platelets to a lower volume, so that less DMSO is injected when CPP transfusion is performed. Centrifugation achieves this overall concentration and volume reduction by pelleting platelets from plasma / DMSO during spinning, and the excess plasma / DMSO can then be aspirated using a plasma expressor. The expression step (step 11) is a determinant of the frozen volume. The overall variability present in the Vitalant expression step and frozen volume is demonstrated in Figure 4A (Figure 2A from the long-term stability properties of Vitalant in cryopreserved platelets at -80°C, protocol 11-5-J). A wide range of volumes is observed, suggesting slight control over this processing step. In the long-term stability properties of Vitalant in cryopreserved platelets at -80°C, protocol 11-5-J, aggregates were observed in CPP units with low frozen volumes. This demonstrates the importance of controlling the expression and packing volume in the process. Figure 4B (Figure 2B from the stability test) shows the distribution of frozen volumes of units in which agglutination was observed after thawing, and agglutination was observed only at low frozen volumes.

[0158] The previous expression step instructed the manufacturing staff to "remove as much supernatant as possible without losing platelets." This method, using visual inspection, is uncontrolled and variable, as shown in Figure 4A.

[0159] Freezing protocol The freezing protocol ensures uniform freezing across units. All CPP units are placed in thawing bags and then laid flat in cardboard boxes. The flat orientation of the CPP units ensures uniform heat transfer from the cryobags to the freezer shelves. The same product enclosure system is used for all units, and all units are placed on shelves in a freezer at -80°C to minimize variability between units.

[0160] Example 2. An exemplary improved process for preparing cryopreserved platelets. As disclosed in the Examples, a non-limiting, exemplary, improved process for preparing cryopreserved platelets using a pool of platelet units as a starting material is referred to in these Examples as the pooled CPP process or the exemplary pooled CPP process. The exemplary pooled CPP process is adapted to the inclusion and pooling of platelets from 12 apheresis platelet units (APUs) with the aim of improving product control, reducing variability of the final product, and increasing scalability. The exemplary pooled CPP process incorporates all the major process steps present in the single-donor CPP process of Example 1, however, the process is improved to streamline the process and produce a more uniform batch of final product while limiting batch-to-batch variability of the final product. Figure 1B is a non-limiting flowchart of the exemplary pooled CPP process. A summary of the major steps between the exemplary pooled CPP process and the single-donor process is given below.

[0161] (Table 2) TIFF2026521991000007.tif83132

[0162] The exemplary pooled CPP process produces multiple doses of a homogeneous final product. This allows for representative product characterization testing of all pooled CPP units sent for injection and enables product archiving of all batches produced. A single-donor process does not allow for quality assurance of the same type of product. Multiple doses from the same final product also allow for initiating stability testing that directly compares the same product at different points in time, enabling a true representation of product stability. This protocol maintains the same product formulation (ratio of platelets, plasma, saline, and DMSO) and a similar overall process for cryopreserved platelets.

[0163] The following are exemplary, non-limiting processes for preparing cryopreserved platelets as disclosed herein.

[0164] The exemplary pooled CPP process is designed to combine up to 12 apheresis platelet units (APUs) from 5-10 donors into a single pool to produce up to 12 pooled CPP units. While the process can be completed with fewer than 12 APUs, a minimum of 5 APUs from 5 donors is required. The initial number of APUs is equal to the number of pooled CPP units produced. The pooled CPP product has the same composition as the single-donor product (77.5% residual plasma from the APUs, 16.5% 0.66% NaCl saline, and approximately 6% DMSO). Similar to single-donor CPP units, pooled CPP units consist of a single transfusion-ready dose of apheresis platelets concentrated by centrifugation and plasma expression, and are cryopreserved in approximately 6% DMSO at -65°C or below (in a -80°C freezer).

[0165] Step 1 - Initial Quality Control (QC) of the APU upon receipt Before APUs are released for manufacturing, the QC department will verify, in accordance with QCT-001, that all APUs meet the following pre-manufacturing standards. Leukocyte removal: 5×10 6 Responsibility for facilities that collect fewer than one WBC. Platelet age: Day 2 or earlier. Responsibility for gamma ray irradiation at 25 Gy and the collection facility. The APU ports are undamaged. No signs of red blood cell (RBC) contamination. APC swirl without aggregates. pH: pH6.2 or higher. Average APU total platelet count: 2.5 × 10⁻⁶ 11 One or more platelets.

[0166] Step 2 - Quality Assurance (QA) Line Clearance The QA department completes the line clearance for the process before manufacturing begins.

[0167] Step 3 - APU manufacturing and transport of the APU to manufacturing inspection. Following QA and QC obligations, APUs are released for manufacturing. The start time of manufacturing is recorded when the APUs are removed from the QC platelet incubator and physically handed over to the manufacturing staff. Upon receiving the released APUs, the manufacturing staff completes several quality checks to verify that the APUs are acceptable. The APUs are visually inspected for swirl, aggregates, and red blood cell contamination to confirm that the APUs are irradiated. The donor ID and expiration date of the APUs are recorded in the batch record for traceability and tracking purposes.

[0168] Step 4 - Creation of a 2-unit pool at APU After all units are determined to be acceptable, the plasma transport set is welded to the APU using an SCD, and then the second APU is welded to the other end of the plasma transport set. The plasma transport set is added to extend the working length of the tubing. The two APUs are then pooled together into a single APU bag. This is done six times to create six pools of two APUs from the initial 12 APUs. The sterile connection device used is a Terumo, TSCD II sterile tubing welder, model number 3me-SC203a (or equivalent). The plasma transport set used is a Charter Medical, 24-inch tubing, roller clamp, two puncture pins, product number 03-220-00 (or equivalent).

[0169] Step 5 - Determining the pooled APC weight Determine the pooled APC weights and record them for use in later expression steps. For each of the six pooled APUs, the pooled APC weight is determined using the following equation: Pooled APC weight = Pooled APU weight - Empty bag weight

[0170] The pooled APU weight is determined using a scale (Ohaus Adventurer Precision Balance, product number AX8201 / E, or equivalent). The empty bag weight is a known value corresponding to the type of bag used for apheresis platelet collection.

[0171] Step 6 - Centrifugation Each pooled APU is placed in a centrifuge cup. The cup is weight-balanced (if necessary) and mounted in the centrifuge. The pooled APUs undergo centrifugation at 1250G for 10 minutes at maximum acceleration, followed by a 10-minute deceleration period (Beckman-Coulter Avanti J-HC centrifuge).

[0172] Step 7 - Expression The target weight of plasma removal for expression is determined using Equation 5 by subtracting 46.5 g from the weight of the pooled APC. This is done to leave approximately 46.5 g of platelet pellet and plasma after the pooled APU has been expressed. (Equation 5): Determination of expression endpoints Plasma removal target = pooled APC weight - 46.5g

[0173] Each pooled APU is removed from the centrifuge and expressed one by one. The pooled APUs are carefully removed from the centrifuge cup so as not to interfere with the platelet pellet and then placed into a plasma expresser (Fenwal Inc., manual plasma extractor, product code 4R4414, or equivalent). The empty APU bag is placed on a scale and tare is performed to weigh the expressed plasma. The pooled APUs are then expressed. Expression is stopped when the plasma removal target is reached (±1.0 g).

[0174] Step 8 - Checking weight after expression The post-expression pellet weight is determined to ensure that the weights of the platelet pellet and supernatant are within the target range (31.8g to 55.7g) for further processing. If the post-expression weight is outside the range, supernatant is added or removed as appropriate until the post-expression weight is within the range.

[0175] Step 9 - Resuspension Once the post-expression weight is within the range, the pellets are resuspended by gently shaking and massaging the APU bag until the pellets are no longer visible. After the pellets are no longer visible, the resuspended platelet pellets are allowed to rest at ambient temperature for 5 minutes. The resuspended pellets are then visually inspected for aggregates. If aggregates are observed at this point in the process and they do not disappear after further rest and stirring, production control is notified, and the process is continued with the addition of 27% DMSO, followed by a 30-minute ambient temperature rest with gentle stirring.

[0176] Step 10 - Pooling Once the six pellets are resuspended, the six APU bags are welded to a sterile tube tree (Optimum Processing, Inc., part number 02817 or equivalent) to create a “pool tree system” and pool the resuspended platelet pellet material into a single APU bag. Figure 1C is an image of the tube tree used to prepare one batch or one lot of cryocontainers using the method described herein.

[0177] Step 11 - Calculation of DMSO target weight for 27% Using Equation 6, the total post-expression weight is determined using the sum of previously measured post-expression weights. The total post-expression weight is used in Equation 7 to calculate the 27% DMSO target weight required to formulate the pooled resuspended platelet material into approximately 6% DMSO. Equation 7 uses the "DMSO constant" of 0.2946 obtained in the section titled "Development of a method for adding 27% DMSO for pooled CPPs". (Equation 6): Determination of total post-expression weight Total post-expression weight = total platelet pellet weight (Equation 7): Determination of the 27% DMSO target weight 27% DMSO target weight = APC weight * 0.2946

[0178] Step 12 - Add 27% DMSO Next, a bag containing sterile, injectable grade 27% DMSO and 0.66% sodium chloride in water (Bio Life Solutions, BloodStor® 27 NaCl Biopreservation Media, part number 327207, or equivalent) is welded to the pool tree system. The 27% DMSO bag is placed on a scale to weigh how much 27% DMSO flows out of the bag into the pool tree system. The 27% DMSO bag is positioned higher than the pool tree system to allow the 27% DMSO to flow gravimetrically. The target weight ±1.0g of 27% DMSO is added to the pool tree system. The tube to the 27% DMSO bag is clamped to prevent any additional 27% DMSO from entering the system.

[0179] Step 13 - Rinsing Rinse the pool tree system with 27% DMSO and collect any residual platelet material remaining in the APU bags and tubing of the pool tree system. Then, add the solution to the APU containing the pooled, resuspended platelet pellets. Use a tube stripper to remove any remaining solution from the pool tree system, ensuring that all 27% DMSO is added to the platelets.

[0180] Step 14 - Determining the filling volume of the cryobag The APU bags containing the final product are welded to a new tube tree, filling it with 12 cryobags. This creates the "filling tree system". The APU bags are then weighed, and the weight of the final product is determined by subtracting the weight of the empty bags (determining the weight of the empty bags is explained in step 5, "Determining the pooled APC weight"). The maximum filling weight of the cryobags is determined by dividing the final product weight by 12. The minimum filling weight is determined by subtracting 2.0g from the maximum filling weight. This determines the filling weight range of the cryobags.

[0181] Step 15 - Cryobag Filling Procedure Next, twelve cryobags (250mL EVA CryoStore freezer bags, Origen Reference CS250, or equivalent) are welded to the dispensing tree system. The cryobag filling procedure is performed by placing the cryobags on a scale, priming the cryobag line until just before the product enters the cryobag, and then tare the cryobags. The cryobags are then filled with the final product until it reaches the desired level. The filled weight of each cryobag is recorded, and their volumes are determined by dividing the weight by 1.03 g / mL.

[0182] Step 16 - Freeze Next, each cryobag is placed in a thawing bag and freezer carton for storage in a -80°C freezer. Then, the units are placed in the freezer and the start time of freezing is recorded. If necessary, after freezing for a certain period in a -80°C freezer, a certain number of cryobags are transferred to a freezer set to -20°C and stored at that temperature to form cryopreserved products at the transition temperature. The elapsed time from the end of 27% DMSO addition to the start time of freezing is typically 3 hours or less.

[0183] Post-manufacturing specifications: All CPP units manufactured using the exemplary pooled CPP process typically meet the following specifications prior to freezing. Frozen volume: 20 mL to 35 mL Time from addition of 27% DMSO to freezer: 3 hours or less Freezing by end of day 2 of platelet age Visible aggregates in cryobag: None % DMSO: 5.65% to 6.52% Maximum DMSO in CPP unit: 2.53 g (mass corresponding to maximum frozen volume at maximum % DMSO).

[0184] Summary of batches with odd initial APUs If there are odd initial APUs, the following steps are modified to include additional steps that allow the pooled process to conform to a single APU. The single APU, and all other steps, calculations, and specifications of the pooled process are not changed.

[0185] Step 4 (Creation of pool of 2 unit APUs): Weld the plasma transfer set to the odd APU and weld a 600 mL transfer bag (Terumo, TeruFlex transfer bag, catalog number: 1BB*T060CB71, or equivalent) to the other end of the plasma transfer set. The APC of the odd APU remains in the APU bag.

[0186] Step 7 (Expression): Equation 5 for calculating the plasma removal target to determine the expression endpoint is modified to account for the odd APU being a single APU and not a pooled APU. Plasma removal target (for single APU) = odd APC weight - 23.3 g

[0187] Step 8 (Check weight after expression) The target pellet weight range after expression is changed to 15.9 g to 27.9 g to account for the odd APU being a single APU and not a pooled APU.

[0188] Example 3. Process development for pooled CPP disclosed herein Standardization of weighing practices: 1. To improve accuracy and precision, a standard orientation is implemented for weighing all bags for the purpose of weight determination and weight / volume conversion. APU bags are weighed by folding the lower third of the bag itself and centering it at the rear end of the scale, ensuring that all bags are on the scale and no unnecessary tubes are on the scale. Cryo bags are placed on the scale in a similar manner, but their smaller profile does not require folding.

[0189] Incoming APC volume exclusion standards: The single-donor process had a pre-processed APC volume specification of 165 mL to 375 mL. This volume specification was included to ensure that the APU was acceptable in that process. APU outside this specification would not comply with the 27% DMSO addition table (Table 1). Due to changes in centrifugation practices and the method of adding 27% DMSO, this volume specification is no longer relevant to the pooled process. The upper limit of this volume specification was included due to processing constraints resulting from the previously used cryobag size and the centrifugation practices of the single-donor method. The single-donor method requires formulating the APC in approximately 6% DMSO before centrifugation, transferring the APC / DMSO to a cryobag, and then centrifuging the APC / DMSO in the cryobag. An incoming APU with a volume of 375 mL becomes 473 mL after the addition of 27% DMSO. Because a 500 mL cryobag was used for centrifugation, anything exceeding approximately 473 mL would not fit in the cryobag or would result in significant "pillowing" of the cryobag. The pooled process does not add 27% DMSO before centrifugation, so there is no increase in the working volume of platelet material before centrifugation. The pooled process also uses APU bags for centrifugation rather than cryobags. Apheresis platelet bags can hold a maximum volume of approximately 1600 mL and can comfortably hold approximately 800 mL without pillowing. Therefore, volume processing constraints are not necessary in the pooled process. Eliminating this volume standard makes more APU acceptable for processing. Additionally, since the cryobags are not subjected to centrifugation, unwanted stress on the cryobags is removed from the process. This practice of centrifugation within cryobags allows for the possibility of the cryobags rupturing in the centrifuge, causing unwanted stress on the cryobags. This unwanted stress can cause loss of integrity and increase the potential for further downstream breakage in the freezing and thawing steps of product handling.

[0190] Derivation of the DMSO constant: The DMSO constant in equation 7 is derived from the C1V1=C2V2 calculation used to calculate %DMSO in equation 4. The derivation of the DMSO constant is shown below. C1*V1=C2*V2 C1 = 27% DMSO Volume of DMSO with V1 = 27% = X (target value) DMSO with C2 = 6.085% (intermediate %DMSO standard) V2 = APC / DMSO volume = APC volume + 27% DMSO volume = APC volume + X Substitute V1 and V2 into the equation C1*V1=C2*V2: C1*X = C2*(APC volume + X) Substitute the known DMSO percentages (C1 and C2): 27%*X = 6.085%*(APC volume + X) Rearranging the equation to find X, we get the following: TIFF2026521991000008.tif7128X = 0.2909 * APC volume = 27% DMSO volume

[0191] This constant of 0.2909, when multiplied by the APC volume, calculates the 27% DMSO volume needed to achieve 6.085% DMSO. Since the in-process measurement is by weight, the constant is converted to account for this, eliminating unnecessary weight / volume conversions and simplifying the calculations that the operator would need to perform. This conversion was done by using the specific gravity of 27% DMSO and APC.

[0192] This specific gravity of TIFF2026521991000009.tif81281.04 g / mL is derived from the excipient SDS.

[0193] TIFF2026521991000010.tif81281.027 g / mL is the known specific gravity of APC.

[0194] Substitute these equations for the APC volume and the 27% DMSO volume into the previous equation used to find the 27% DMSO volume. TIFF2026521991000011.tif7128

[0195] Rearrange the equations to find the weight of 27% DMSO. TIFF2026521991000012.tif7128

[0196] TIFF2026521991000013.tif7128

[0197] This DMSO constant (0.2946) is a unitless value obtained by multiplying it by the weight of APC, and it determines the 27% by weight of DMSO required to formulate APC / DMSO into a 6.085% DMSO (6.09% DMSO when rounded) mixture.

[0198] Calculation of 27% DMSO addition For an exemplary improved pooled process or exemplary pooled CPP process, a method for formulating the CPP product to a fixed %DMSO was developed to eliminate the problems identified in the analysis of the previous 27%DMSO addition method in Example 1 (Table 1 and Analysis A). This exemplary pooled CPP process is intended to reduce batch-to-batch variability of the product formulation and ensure that APC / DMSO consistently falls within the DMSO (%DMSO) specification range. By using a DMSO constant, a method is produced in which all or virtually all pooled CPP batches are formulated to achieve the same target percentage DMSO. To ensure that the percentage DMSO is always within specification, a tolerance of ±1.0 g of 27% DMSO is included. To demonstrate this theory, Analysis B applies the DMSO constant method to formulate 189 mL of APU as performed for the improved exemplary pooled CPP process. A comparison of Analysis B and Analysis A demonstrates how the DMSO constant can eliminate the problems in the conventional single-donor method of Example 1. (Analysis B): Case study of adding DMSO to 27% of pooled CPP Required 27% DMSO weight = APC weight * DMSO constant Required weight of 27% DMSO = 194.1 g * 0.2946 = 57.2 g of 27% DMSO

[0199] Using the same mathematical approach as in Analysis A, when 57.2 g of 27% DMSO is administered to 189.0 mL of APU, the APU of Analysis B is formulated to 6.09% DMSO.

[0200] Figure 5 is similar to Figure 5 and compares the correlation between the APC volume and % DMSO for two 27% DMSO addition methods. Figure 5 illustrates the increased precision and accuracy of an exemplary pooled CPP process. The old method refers to the process of single-donor CPP and the use of Table 1 used in Analysis A. The exemplary pooled CPP process refers to the process of an exemplary pooled CPP and the use of Equation 7 used in Analysis B. The plot of the exemplary pooled CPP process also shows a line with a tolerance of ±1 g from the 27% DMSO target weight.

[0201] Figure 6 illustrates the correlation between the post-expression volume and percent DMSO for an exemplary pooled CPP process.

[0202] Figure 6 encompasses the entire range of post-expression volumes possible with the pooled process. The lowest achievable post-expression volume is from a 5-unit batch where all APUs are expressed to the lower limit of the post-expression range. The highest achievable post-expression volume is from a 12-unit batch where all APUs are expressed to the upper limit of the post-expression range. A line with a tolerance of ±1 g from the 27% DMSO target weight is also included. Figure 6 demonstrates that the percent DMSO of the exemplary pooled CPP process is always within specifications for all batch sizes (including the minimum batch size of 5 APUs), thereby eliminating the need for confirmatory DMSO percent calculations. Table 3 consists of in-process percent DMSO data from all cGMP-like pooled CPP batches produced (including 12 batches and 136 pooled CPP units).

[0203] (Table 3) TIFF2026521991000014.tif19164

[0204] Table 3 demonstrates the precision (low CV) and accuracy (target %DMSO is 6.09%) obtained in the embodiment of the 27% DMSO addition method provided in Example 2 (an exemplary pooled CPP process step).

[0205] Optimization of excipient use: The single-donor process of Example 1 requires the addition of 27% DMSO before the platelets are concentrated by centrifugation and plasma expression. Adding 27% DMSO before centrifugation increases the working volume of the solution by approximately 30%, which limits the scalability of the single-donor process. A further obstacle to the scalability of the single-donor process is the fact that this method also results in the removal of most of the added 27% DMSO during plasma expression, requiring the disposal of the majority of the 27% DMSO as waste. For example, in Analysis B, 189 mL of APU has 57.2 g of 27% DMSO added to the total APU. After centrifugation, if the plasma / DMSO is expressed to retain 28.0 mL of residual plasma / DMSO and platelet pellet, 216.0 mL of plasma / DMSO is removed. This is equal to 48.7 mL of 27% DMSO removed from the CPP unit, and 6.3 mL of 27% DMSO is retained in the cryobag along with the platelets. This is the only inefficient use of excipients in the product, as approximately 90% of the excipients are removed from the final product and disposed of as waste. The pooled process optimizes the use of excipients by adding 27% DMSO after centrifugation and plasma removal, so that none are discarded as waste and only the amount of 27% DMSO necessary to formulate the product to the target % DMSO is consumed.

[0206] Addition of in-process control of plasma removal targets: Equation 5 was created to determine the expression endpoint for standardizing the expression step. This equation calculates the expression endpoint weight using the weight before centrifugation. Plasma is weighed at the time of expression to determine when the endpoint is reached. Expression is stopped when the plasma removal target weight (±1.0 g) is reached. A tolerance for the plasma removal target is included to ensure that the expression step does not lead to a post-expression weight that is outside the range necessary to continue processing. These additions to the expression step are included to increase control over the process and reduce batch-to-batch variability. The plasma removal target is calculated using the following calculation: (Equation 5): Determination of expression endpoints Plasma removal target = pooled APC weight - 46.5g

[0207] This method typically ensures that approximately 46.5 g of platelet pellet and plasma remain after expression. After expression, there is typically a weight check to ensure that the weight of the platelet pellet and supernatant is within the range for further processing (31.8 g to 55.7 g). If the post-expression weight is outside the range, supernatant is added / removed as appropriate until it is within the range.

[0208] Determination of the post-expression range: Single-donor plasma expression was performed after the addition of 27% DMSO, while the pooled process centrifugation and plasma expression steps were performed before the addition of 27% DMSO. This had to be taken into consideration when determining the post-expression weight range. The calculation for determining the post-expression weight range is as follows:

[0209] Firstly, the contribution of APC to the final APC / DMSO solution is typically determined. The volume fraction of APC in the APC / DMSO solution is typically calculated to determine this. TIFF2026521991000015.tif8128TIFF2026521991000016.tif8128APC / DMSO volume = APC volume + 27% DMSO volume

[0210] Substitute the APC / DMSO volume equation into the APC volume fraction equation. TIFF2026521991000017.tif8128

[0211] As mentioned above in the "Determination of DMSO Constant" section, when APC / DMSO is formulated into 6.09% DMSO, the formula for 27% DMSO volume is as follows: 27% DMSO volume = 0.2909 * APC volume

[0212] Substitute this equation for 27% DMSO volume into the APC volume fraction equation. TIFF2026521991000018.tif8128

[0213] Since the APC volume is in both the numerator and denominator of the equation, this value can be eliminated from the equation, and the APC volume fraction can be solved. TIFF2026521991000019.tif8128

[0214] This means that APC, after being formulated with 6.09% DMSO, constitutes 77.5% of the CPP product.

[0215] The single donor frozen volume range is used to convert the volume range of the APC / DMSO product to the equivalent pre-DMSO range, and this APU volume fraction of 0.775 is typically used: TIFF2026521991000020.tif10159

[0216] The pooled process involves pooling two APUs for centrifugation and expression, so these values ​​are doubled, and the weight is converted to facilitate use in the process using an APC specific gravity of 1.027 g / mL.

[0217] TIFF2026521991000021.tif13161

[0218] Determination of the target weight after expression of 46.5g: The single-donor process of Vitalant had a post-expression volume range of 25.5 mL to 31.3 mL. The midpoint of this range is 28 mL, or 28.8 g. Rounding to 1 g intervals, this becomes 29 g. This 29 g APC / DMSO weight was chosen as the starting point for determining the post-expression target weight of the exemplary pooled CPP process. To account for the potential dead space loss introduced by the pooled process, this value of 29 g was increased by 1 g to 30 g (or 29.1 mL). This was then converted to an equivalent amount of DMSO pre-product using the same conversion as in the previous section. 29.1 mL of APC / DMSO * APC volume fraction = 29.1 mL * 0.775 = 22.6 mL of APC

[0219] 29.1 mL of APC / DMSO consists of 22.6 mL of APC. Since the pooled process involves pooling two APUs for centrifugation and expression, this value of 22.6 mL of APC was then doubled and then converted to weight using an APC specific gravity of 1.027 g / mL to facilitate use in the process. TIFF2026521991000022.tif6128

[0220] Next, this value of 46.4g was rounded to the nearest half-gram interval to obtain a post-emergence target weight of 46.5g.

[0221] Implementation of new expression and filling methods Table 4 comprises in-process post-expression and filled / frozen volume data from all cGMP-like pooled batches produced to date using the improved exemplary pooled CPP method of Example 2. These 12 pooled CPP batches encompassed 69 expression steps and 136 manufactured pooled CPP units. Specification ranges for single-donor post-expression and frozen volumes are listed, along with the midpoints of these ranges. Pooled CPP post-expression volumes have been converted to equivalent DMSO post-values ​​using the previously discussed APC / DMSO volume to APC volume conversion to enable appropriate comparisons. Minimum and maximum batch averages from the pooled CPP batches are listed, along with the average intra-batch CV.

[0222] (Table 4): Summary of new expression and filling methods TIFF2026521991000023.tif37164

[0223] As illustrated in the analysis of the single-donor process, the expression step and freezing volume are critical to the product, and therefore, in exemplary examples, they should be controlled as much as possible. The pooled CPP post-expression mean was 0.2 mL away from the single-donor post-expression volume of 28.0 mL (a difference of 0.7%), and the CV was less than 10%, thus the addition to the expression step made for an accurate and precise method. In addition, the pooled process post-expression range is within the single-donor post-expression range of the process in Example 1. Furthermore, all pooled CPP batches produced to date using the improved exemplary pooled CPP process of Example 2 are within the pooled CPP post-expression range and do not require additional handling, manipulation, and weight readjustment. This demonstrates the reliability of the method improvement. In-process calculation and control of the expression and filling operations ensure that all cryobags are filled within the freezing volume range of 20 mL to 35 mL. The pooled process allows for greater control of the freezing volume because each bag is filled individually from a shared pool of product. The frozen volume CV demonstrates that the pooled process has a highly controlled filling procedure, which reduces variability in frozen volume. The pooled CPP frozen volume data demonstrates that improvements in the expression step, coupled with the filling procedure, lead to a more acceptable frozen volume. With the improved exemplary process provided herein in Example 2, the resulting 12 batches of CPP have an average in-batch CV of 7.7% for post-expression volume (resuscitation volume) and an average in-batch CV of 1.6% for frozen volume (pooled resuspension with cryoprotectant in each cryobag).

[0224] Example 4. Uniformity of pooled CPP within a batch Batch homogeneity was tested across 28 units or cryo-containers from six cGMP-like pooled CPP batches. The pooled CPP batches were prepared according to the method of Example 2. Platelet count per bag, platelets / μL, IU / 10 6TGA measurements, CD61 particle positivity, and pH in individual platelets were determined across 28 units from 6 batches or cryogenic vessels. Platelet counts per bag and per μL were obtained using either a Beckman Coulter AcT Diff 2 blood particle analyzer or a Beckman Coulter DxH blood analyzer (Beckman Coulter, beckmancoulter.com).

[0225] The thrombin generation assay was performed according to the following steps: The Thrombinoscope CAT software was opened and the instrument was set up according to the manufacturer's guidelines. PRP reagent containing tissue factor and phospholipids, fluorescein buffer, and fluorescein substrate were prepared according to the manufacturer's guidelines (Stago, stago.com). A buffer containing 30% Octaplus was prepared by combining thawed Octaplus® (Octaphama, octaphramausa.com) and thawed TGA dilution buffer. Cephalin was diluted 1:50 using the combined buffer and used as a positive control. CPP was measured using Octaplus based on platelet count data at 1584 × 10⁶ 3 Diluted to / μL.

[0226] 1584×10 3 Using a CPP diluent of / μL, 325 × 10 using Octaplus 3 / μL, 160×10 3 / μL, and 80×10 3 A series of dilutions of CPP at 325 × 10⁻¹ / μL was prepared. Using a multichannel pipette, 20 μL of PRP reagent and 20 μL of calibrator were added to each test well. Then, 325 × 10⁻¹⁰ 3 / μL, 160×10 3 / μL, and 80×10 315 μL of sample (IU / μL) was added to each of the test and calibration wells. 65 μL of Octaplus was added to all test and calibration wells. 80 μL of 1:50 cephalin was added to the positive control well. Each plate was placed in a tray and incubated at 41°C for 10 minutes. After incubation, fluorescein buffer and fluorescein substrate were distributed to the active wells. The plates were read at 20-second intervals for 75 minutes to capture the complete thrombin generation profile. The thrombin generation profile measurement was expressed as IU / 10 6 It was reported as an individual particle reading.

[0227] Flow cytometry results for CD61 microparticle positivity were obtained using Novocyte Flow Cytometry (Agilent, agilent.com) according to the following method. Novocyte Flow Cytometry was prepared according to the manufacturer's guidelines. CPP samples were diluted 100-fold in saline. 10 μL of anti-human CD61-APC (BD Biosciences, bdbiosciences.com) was diluted to 70 μL. A gated isotope control staining mixture was prepared by combining 20 μL of mouse IgG1-APC (BD Biosciences, bdbiosciences.com) with 60 μL of saline. A test staining mixture was prepared by combining 20 μL of diluted Cd61-APC with 60 μL of saline. Control samples were stained in triplicate by adding 5 μL of 1:100 CPP to 20 μL of the gated isotope control staining mixture. Next, the test samples were stained in triplicate by adding 5 μL of 1:100 CPP to 20 μL of the test staining mixture. All samples were incubated at room temperature for 20–30 minutes, away from light. After incubation was complete, 400 μL of saline was added to each sample, followed by 100 μL of each sample in each well, starting with the gating control sample, and then added to the test sample. The well plates were then placed in a tray and the plates were run. After sample acquisition was complete, the gates were adjusted according to the control and used to determine the number of CD61-positive particles on the test samples. The table below shows the results for 28 units across 6 batches.

[0228] (Table 5) TIFF2026521991000024.tif133166

[0229] The mean intra-batch coefficient of variation is substantially less than 10%, demonstrating that the pooled process exhibits very low variability between units having a batch. The low CV clearly indicates that the pooled process produces a batch of uniform units, which is not the case for CPP units produced from single-donor APUs, which are known to have inter-donor variability. The improved exemplary pooled CPP process of Example 2 averages out donor variability, thereby reducing inter-batch variability of the product. The mean intra-batch CV achieved using the improved exemplary pooled CPP process of Example 2 is less than 5% (4.2%), and for platelet ( / ul) concentration it is less than 4% (3.7%), and for thrombin production capacity (IU / 10 6 The concentration of platelets was less than 3% (2%), the concentration of CD61-positive particles was less than 7% (6.9%), and the concentration of pH was less than 1% (0.8%).

[0230] Example 5. One-year stability test of pooled CPP product stored at -20°C (product stored frozen at transition temperature). Pooled CPP products were prepared according to the method of Example 2. The stability of the pooled product was tested at different points in time over a one-year storage period. Seven apheresis platelet units (APUs) were pooled, yielding seven units of pooled CPP product or a cryo-container. First, the seven units were stored in a freezer at ≤-65°C for a minimum of 24 hours to form the initial frozen platelet composition. After the initial storage, the cryo-container was transferred to a freezer at -20°C for storage to form cryopreserved platelets (cryopreserved product at transition temperature). One unit of the pooled CPP product (cryopreserved product at transition temperature) was removed at different points in time and tested according to the following criteria. Visual inspection of unit bags for cracks, ruptures, and damage. Visual inspection of the absence of aggregates in the product within the bag. Platelet count per bag. pH of the product.

[0231] Before starting each test, the product was removed from the freezer, thawed in a 37°C water bath for 8 minutes, and then rehydrated by adding 25 mL of 0.9% physiological saline. Visual inspection for breakage, visual inspection for agglutination-free swirl, platelet count per bag, and pH of the product are indicators of product release criteria. Platelet count per bag was obtained using either a Beckman Coulter AcT Diff 2 blood particle analyzer or a Beckman Coulter DxH blood analyzer (Beckman Coulter, beckmancoulter.com). The following table details the results at each test point.

[0232] (Table 6) TIFF2026521991000025.tif73149

[0233] The stabilization test results demonstrated the product stability of the cryopreserved product at transition temperature after storage at -20°C for 12 months. The product passed all release criteria at all time points. There were no cracks, breakage, or leaks on visual inspection. Aggregate-free swirls were confirmed at all time points. Minimum pH and minimum platelet count / bag criteria were achieved at all time points. A gradual increase in pH was observed over 12 months but did not affect the viability of the product. Platelet count / bag was consistent at different time points. The findings herein confirm the stability of pooled CPP product (cryopreserved product at transition temperature) at a storage temperature of -20°C for one year.

[0234] Example 6: Exemplary CPP manufacturing method This embodiment illustrates one method for producing cryopreserved platelets, which was then used to compare cryopreserved platelets obtained by a process involving a transition in freezing temperature, as exemplified in this method, also referred to as a transition-temperature cryopreserved product, with cryopreserved platelets stored only at -80°C, also referred to as a single-temperature cryopreserved product.

[0235] Cryopreserved platelets were produced using a single APU per batch of mixed ABO and Rh types for all three batches prepared. The APUs were acidified to a pH range of 6.6–6.8 using an anticoagulant dextrose citrate (ACD) solution. pH levels were tested using a Fisher brand Accument XL250 pH meter (Oakton Instruments). After acidifying the APUs to pH 6.6–6.8, a final DMSO concentration of 6% was achieved using a 27% DMSO solution. This was done by first determining the platelet count in the APU and then calculating the volume required to produce the desired number of aliquots. The volume obtained from the APU was then multiplied by a constant of 0.2909 to obtain the exact volume of DMSO solution to be added. For the purposes of this study, platelet counts were determined using an AcT diff 2 whole blood cell count (CBC) analyzer (Beckman Coulter, beckmancoulter.com). Once a 6% DMSO concentration was obtained, the 6% DMSO and platelet-rich plasma (PRP) solution derived from APU was centrifuged at 1250 g for 20 minutes. After centrifugation, the supernatant plasma / 6% DMSO solution was aspirated and set aside for resuspension. The platelets were then resuspended in approximately half to 1 mL of plasma / 6% DMSO solution, and the platelet count was determined by concentration testing with a 1:20 dilution (50 μL of product + 950 μL of PBS). The platelet count was then used to determine 8,000 × 10⁶ 3 The required volume of pre-aspirated plasma / 6% DMSO supernatant was calculated to achieve a platelet concentration of 1000 platelets / μL. The number of platelets was then verified before preparing aliquots of platelets in 6% DMSO (standard preparation). The final product was aliquoted into 5 mL cryovials in a volume of 1.5 mL. The aliquots were transferred to a -80°C freezer for 48 hours. After the 48-hour period, half of the produced aliquots were transferred to a -20°C freezer to form a cryopreserved product at the transition temperature, while the other aliquots remained at -80°C to form a cryopreserved product at a single temperature.

[0236] Example 7: Comparison of cryopreserved products stored at a transition temperature of -20°C and cryopreserved products stored at a single temperature of -80°C. Batch 1, 2, and 3 of the cryopreserved products at each transition temperature and the single-temperature cryopreserved products, prepared by the method of Example 6, were thawed by transferring the cryotubes to a 37°C water bath for 8-10 minutes. After thawing, 1.5 mL of physiological saline was added to each cryotube. The products were then allowed to rest at room temperature with stirring for 30 minutes. After the resting period, stability and agglutination tests were performed.

[0237] For stability testing, the percentage platelet recovery rate was determined. Percent platelet recovery was performed using AcT diff 2, and the percentage yield was determined. Figure 7 shows the percentage platelet recovery rates for batches stored at -80°C (single-temperature cryopreserved product) and -20°C (transition-temperature cryopreserved product). The average percentage recovery rate between batches at -80°C (single-temperature cryopreserved product) was 83% compared to 77% when stored at -20°C (transition-temperature cryopreserved product). The objective of the experiment was to obtain recovery rates exceeding 70% obtained by all batches at both storage temperatures. Therefore, the experiment showed similar percentage recovery rates at both storage temperatures, thereby demonstrating that batches with transition-temperature cryopreserved product (stored at -20°C) are more stable when stored at higher temperatures than single-temperature cryopreserved product stored at -80°C.

[0238] Agglutination tests were performed using a PAP8 aggregator (Bio Data Corporation, biodatacorp.com). Batch 1, 2, and 3 of each product were diluted to 250 k / μl in hexamethylenetetramine (HMTA) based on the AcT count. 225 μl of the diluted sample was added to each cuvette. The cuvettes were then transferred to the incubation wells of the PAP8 aggregator. Either 25 μl of collagen (final concentration 10 μg / ml, stock solution 100 μg / ml), 25 μl of TRAP-6 (final concentration 20 μM), or 25 μl of arachidonic acid (AA) (final concentration 500 μg / ml, stock solution 5 mg / ml) was added, and measurements were taken after 2 minutes. Figure 8 shows the percentage aggregation of platelets in batches stored at -80°C (single-temperature cryopreserved product) and -20°C (transition-temperature cryopreserved product), as well as a comparison with apheresis platelets containing arachidonic acid (AA), collagen, and TRAP-6. The aggregation data collection included apheresis platelets for comparison. A two-sided t-test was performed to compare the batch of single-temperature cryopreserved product stored at -80°C with the transition-temperature cryopreserved product stored at -20°C. The AA agonist produced a p-value of 0.80, with no significant difference between the groups. The collagen agonist produced a p-value of 0.59, but with no significant difference. TRAP-6 produced a p-value of 0.45, similarly with no significant difference. Since no significant differences were observed between batches of both products, this experiment demonstrates that cryopreserved platelets of the transition temperature cryopreserved product stored at -20°C exhibit hemostatic properties such as stability and aggregation, as well as the single temperature cryopreserved product stored at -80°C, thereby addressing the long-standing need to store cryopreserved platelets at temperatures above -65°C.

[0239] Example 8: Calcein acetoxymethyl (AM) cell membrane test Batch 1, 2, and 3 of the cryopreserved products at each transition temperature and the single-temperature cryopreserved products, prepared by the method of Example 6, were thawed by transferring the cryotubes to a 37°C water bath for 8-10 minutes. After thawing, 1.5 mL of physiological saline was added to each cryotube. The products were then allowed to rest at room temperature with stirring for 30 minutes. After the products had rested, the calcein AM assay was performed. Calcein AM is a substance that can pass through the cell membrane and reach the cytoplasm where it is hydrolyzed by the enzyme esterase to produce fluorescence. Intact platelets can retain this fluorescence, but platelets that are not intact do not. Based on the AcT count, 400 μL of sample was prepared at a concentration of 1,000,000 / μL. A 1 mM calcein AM stock solution was diluted in 10 μM HMTA (1:100). Replication samples of cryopreserved products at each transition temperature and single temperature were prepared from 1 μL of 10 μM calcein and 9 μL of diluted cryopreserved platelets. All samples were incubated at room temperature for 20 minutes, away from light. Each sample was diluted with 990 μL of PBS. 100 μL of each sample was transferred to individual wells in a 96-well plate. Each sample was acquired on a flow cytometer under the following conditions and parameters: FSC, SSC, FB530 (FITC), stop condition: 30 μL or 30,000 events, FSC-H threshold > 1,000, and flow rate: moderate. Apheresis platelets were included in the assay for comparison. Figure 9 shows the findings performed for the calcein AM test. As expected, apheresis platelets, indicated by "+", showed a single peak and were able to retain more calcein AM compared to cryopreserved products at transition temperatures and single temperature. In batches 1, 2, and 3, cryopreserved products stored at transition temperatures of -20°C, indicated by "*", showed a single peak indicating a single cell population based on membrane integrity, while cryopreserved products stored at a single temperature of -80°C, indicated by "#", showed two peaks indicating two cell populations based on membrane integrity.Furthermore, all three batches of cryopreserved products at transition temperatures stored at -20°C exhibited similar moderate-sized peaks, likely demonstrating similar levels of membrane integrity across batches of cryopreserved products at transition temperatures. This observation suggests the existence of two distinct populations in single-temperature cryopreserved products: a first population capable of retaining calcein AM fluorescence, possibly due to damaged membranes (see the first peak from the left of "#" in Figure 9), and a second population showing higher fluorescence retention, possibly due to intact membranes (see the second peak from the left of "#" in Figure 9). Moreover, it was observed that the single peak in all three batches of cryopreserved products at transition temperatures (see "*" in Figure 9) corresponds to the first population of single-temperature cryopreserved products exhibiting less calcein AM retention. Therefore, though not limited to theory, it is thought that the single population of cryopreserved products at transition temperatures observed in the calcein AM assay contains platelets with damaged membranes. However, surprisingly, as shown in Example 7, all batches 1, 2, and 3 of the cryopreserved products at transition temperatures showed platelet recovery rates exceeding 70%. Similarly, as shown in Example 5, Table 6, the cryopreserved products at transition temperatures prepared by the process disclosed in Example 2 were able to pass all criteria related to aggregate-free swirling, pH, and platelet count after being stored at -20°C for even 12 months.

[0240] Example 9: Characterization of cryopreserved products stored at -20°C at various transition temperatures. Three batches of cryopreserved platelets, W4464-22-000010, W4464-22-000013, and W4464-22-000016, were prepared by the process disclosed in Example 2, which uses transition-mode freezing, by freezing the resulting cryo-containers at a temperature of ≤-65°C for 141 days, 85 days, and 43 days, respectively, to form an initial frozen platelet composition, and then storing the initial frozen platelet composition at a temperature of -20°C for 1 month, 3 months, 6 months, and 12 months to form cryopreserved platelets (cryopreserved products at transition temperatures). The data included in this example are for only one batch of batches up to 12 months, specifically batch W4464-22-000010. Each cryo-container consisted of a volume of 20–35 ml in 6% DMSO. Upon reaching the specified time, the cryopreserved platelets were thawed from the cryo-containers and tested for the following parameters. Vision test: Pass / Fail ·Agglomerate free: Pass / Fail pH (≧6.2) • Platelet count per bag (≧1.7E+11) • Thrombin generation assay (TGA) (20K / μL IU / 10 6 (Individual particles) ·CD61 positive particles x 10 6 / μL • Lactoadherin-positive particles (%)

[0241] Platelet count Platelet counts per bag were obtained using either a Beckman Coulter AcT Diff 2 blood particle analyzer or a Beckman Coulter DxH blood analyzer (Beckman Coulter, beckmancoulter.com).

[0242] TGA Protocol To evaluate thrombin generation of the products described herein, a Fluoroskan Ascent instrument was used. The concentration of cryopreserved platelets or platelet derivatives from one of the cryocontainers of the batch of this embodiment to be tested was determined using a Beckman Coulter® AcT Diff and AcT Diff 2 blood analyzer (EQU-045) or a DxH520 blood analyzer. After determining the concentration, the platelets were diluted with Octaplus (solvent-detergent treated pooled plasma manufactured by Octapharma) to 352 × 10⁻⁶. 3 pieces, 160×10 3 pieces, 80×10 3 A serial dilution tube with a platelet concentration of individual platelets / μl was prepared. To obtain the output of the sample, 44 × 10 3 Platelet platelet in-platelet diluent at a concentration of 1 / μl, appropriate volume of PRP reagent (tissue factor and phospholipids), and Octaplus were added to the wells of a 96-well plate along with platelets and the fluorescent thrombin substrate FluCa (Stago). The raw fluorescence intensity of FluCa was measured over time using a Fluoroskan Ascent instrument. TGA count checks and TGA particle parameters were generated from the Fluoroskan Ascent instrument. A detailed protocol is provided below.

[0243] Frozen platelets were thawed for 8 minutes in a water bath or plasma thawer set to 37°C.

[0244] After thawing, the platelets were diluted by adding 25 mL of 0.9% physiological saline.

[0245] The manufacturer's guidelines were followed in the CAT software, and the equipment was set up according to the manufacturer's guidelines.

[0246] PRP reagents containing tissue factor and phospholipids, calibrators, and fluorine buffer and fluorine substrate were prepared according to the manufacturer's guidelines.

[0247] Octaplus and TGA diluted buffer were thawed in a 37°C water bath for 10 minutes.

[0248] Thawed Octaplus was added to TGA dilution buffer to prepare a buffer containing 30% Octaplus.

[0249] Reconstituted cephalin was diluted 1:50 using 30% Octaplus solution and used as a positive control.

[0250] Using OctaPlus, based on platelet count data from a DxH blood analyzer, thawed platelets are processed into 1584 × 10⁴ 3 Diluted to / μL.

[0251] Thawed platelets 1584 × 10 3 Using a dilution of / μL, use OctaPlus to measure 325 × 10 3 / μL, 160×10 3 / μL, and 80×10 3 A serial dilution of platelets was prepared in μL volumes.

[0252] 20 μL of PRP reagent was added to each test well, and 20 μL of calibrator was added to each calibration well.

[0253] 325×10 3 / μL, 160×10 3 / μL, and 80×10 3 15 μL of sample ( / μL) was added to each of the test and calibration wells.

[0254] 65 μL of Octaplus was added to all test and calibration wells.

[0255] 80 μL of 1:50 cephalin was added to the positive control well.

[0256] The plate was placed in a tray and incubated at 41°C for 10 minutes. After incubation, fluorescein buffer was dispensed, and a fluorescein substrate mixture (containing a fluorescently labeled peptide that generates a fluorescent signal when cleaved by thrombin) was added to the active wells.

[0257] The plate was read at 20-second intervals for 75 minutes to capture the complete thrombin generation profile.

[0258] Calculation of CD61-positive particles and lactoadherin-positive particles Approximately 1 ml of frozen platelets, after thawing, were diluted with 2:1 saline solution and then further diluted 100 times by serial dilution.

[0259] CD61, also known as integrin 3 (GPlla), is a glycoprotein subunit of the fibrinogen receptor. GPllb / lla (and other integrin complexes such as the Vitronectin receptor av3) are found on the surface of platelets. These are found at high levels on normal platelets, and CD61 is used in this protocol to identify platelet-derived particles. Allophycocyanin (APC) is a fluorescent dye with a maximum excitation value at 650 nm and a maximum emission value at 660 nm. This fluorescent dye is detected on channel R660 on the NovoCyte instrument. Appropriate dilutions of CD61-APC were prepared.

[0260] Lactoadherin is a milk fat globule-epidermal growth factor-8 protein that binds to phosphatidylserine in a calcium-independent manner. Fluorescein isothiocyanate (FITC) is a fluorescent dye with maximum excitation at 495 nm and maximum emission at 519 nm. This fluorescent dye is detected on channel B530 of the NovoCyte instrument. Appropriate dilutions of lactoadherin-FITC were prepared.

[0261] For test staining, platelets were stained with CD61-APC and then stained with lactoadherin-FITC in the tube by first adding the staining mixture to the tube, followed by the addition of diluted platelets. All samples were incubated away from light for approximately 20 minutes. Each sample was acquired on a flow cytometer under the following conditions and parameters: FSC, SSC, FB530 (for FITC), R660 (for APC), stop condition: 50 μL or 30,000 events, FSC-H threshold greater than 500, and flow rate: moderate (35 μl / min).

[0262] The "CD61+" range gate was adjusted on the APC-H histogram to include 0.8%–1.0% of the most fluorescent events. The CD61+ gate was copied from the APC-H histogram of the gating control sample and pasted directly into the APC-H histogram of the corresponding test sample. Each test sample was examined and replicated to confirm that the "CD61+ microparticles" polygon gate on the APC-H vs. FSC-H bivariate plot contained microparticle populations but not larger platelet populations between CD61+ events in each replicate. The "Lact+" range gate was adjusted on the FITC-H histogram to include 0.8%–1.0% of the most fluorescent events. The Lact+ gate was copied from the FITC-H histogram of the gating control sample and pasted directly into the FITC-H histogram of the corresponding test.

[0263] Table 7 shows the results of the standards related to quality criteria.

[0264] (Table 7) TIFF2026521991000026.tif48168

[0265] The results for the remaining standards are shown in Table 8.

[0266] (Table 8) TIFF2026521991000027.tif77168

[0267] The results from Table 7 indicate that cryopreserved products at transition temperatures, i.e., cryopreserved platelets, when stored at -20°C for 1 month, 3, 6, and 12 months, do not exhibit visible aggregates upon thawing, pass visual inspection of the swirl test, and meet the criteria for platelet count and pH. Therefore, these results demonstrate that cryopreserved platelets at transition temperatures as described herein exhibit the properties shown in Table 7 when stored at -20°C for at least 12 months.

[0268] Furthermore, the results shown in Table 8 demonstrate that cryopreserved platelets retain their thrombin-producing ability upon thawing, even after being stored at -20°C for at least 12 months. It can also be observed that cryopreserved platelets exhibit a CD61-positive microparticle content ranging from 21% (1 month) to 14% (12 months) upon thawing. Therefore, it is observed that the percentage of CD61-positive microparticles gradually decreases over storage time. Additionally, the cryopreserved platelets described herein exhibit lactoadherin positivity in the range of 80–99.5%.

[0269] All references throughout this application, such as patent documents, published patent applications, and non-patent documents or other sources, including issued or granted patents or equivalents, are incorporated herein by reference in whole, as if each reference were incorporated individually, provided that it does not conflict at least partially with the disclosures of this application (for example, partially conflicting references are incorporated by reference with the partially conflicting portion of the reference removed).

[0270] The terms and expressions used herein are for illustrative purposes only, not limitation, and in using such terms and expressions, there is no intention to exclude any equivalents of the exhibited and described features or any part thereof, but it is recognized that various modifications are possible within the scope of the claimed invention. Accordingly, although the invention has been specifically disclosed by preferred embodiments, representative embodiments, and optional features, it should be understood that optional features, modifications, and variations of the concepts disclosed herein may be made by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims. The specific embodiments provided herein are representative examples of useful embodiments of the invention, and it will be apparent to those skilled in the art that the invention may be carried out using numerous variations of the devices, device components, and method steps described herein. As will be apparent to those skilled in the art, useful methods and devices for the present method may include numerous optional compositions and processing elements and steps.

[0271] All patents and publications referenced herein represent the state of the art for those skilled in the art to which the present invention relates. References cited herein are incorporated herein by whole reference to show the state of the art as of the date of their publication or filing, and it is intended that this information may be taken into account herein, where necessary, to exclude specific aspects of the prior art. For example, if a composition of a substance is claimed, it should be understood that compounds known and available in the art prior to the applicant's invention, including compounds for which a valid disclosure is provided in the references cited herein, are not intended to be included in the claims of a composition of a substance described herein.

[0272] Those skilled in the art will understand that starting materials, biological substances, reagents, synthesis methods, purification methods, analytical methods, assay methods, and biological methods other than those specifically provided as representative examples can be employed in the practice of the present invention without relying on excessive experimentation. All functional equivalents of any such materials and methods known to those skilled in the art are intended to be included in the present invention. The terms and expressions used are for illustrative purposes only, not limitation, and in the use of such terms and expressions there is no intention to exclude any equivalent of the shown and described features or any part thereof, but it should be recognized that various modifications are possible within the scope of the claimed invention. Accordingly, although the present invention has been specifically disclosed by preferred embodiments and optional features, it should be understood that optional features, modifications, and variations of the concepts disclosed herein may be reused by those skilled in the art, and such modifications and variations are considered to be within the scope of the invention as defined by the appended claims.

[0273] The disclosed embodiments, examples, and experiments are not intended to limit the scope of this disclosure or to represent that the following experiments are all or only experiments to be performed. While efforts have been made to ensure accuracy with respect to the numerical values ​​used (e.g., quantities, temperatures, etc.), some experimental errors and deviations should be taken into consideration. It should be understood that modifications in the methods described may be made without altering the fundamental aspects that the experiments attempt to illustrate.

[0274] A person skilled in the art can devise many modifications and other embodiments within the scope and spirit of this disclosure. Indeed, the materials, methods, drawings, experiments, examples, and variations in embodiments described may be made by a person skilled in the art without altering the fundamental aspects of this disclosure. Any of the disclosed embodiments can be used in combination with other disclosed embodiments.

[0275] In some cases, several concepts are described with reference to specific embodiments. However, those skilled in the art will understand that various modifications and changes can be made without departing from the scope of the invention as set forth in the following claims. Accordingly, this specification and the figures should be interpreted as illustrative rather than restrictive, and all such modifications are intended to fall within the scope of the invention.

Claims

1. A process for preparing a batch of cryopreserved platelets, wherein the process comprises: a) Pooling at least two platelet units in one container and at least one other platelet unit in another container, wherein there are at least five platelet units and the platelet units come from two or more donors, b) Centrifugation of each container to obtain a supernatant containing plasma and a pellet containing platelets, c) Resuspending the pellets in each container to form a resuspension, wherein the resuspension has a target weight based on the number of units pooled or provided in the container. d) Pooling the resuspension from each container and forming the pooled resuspension in the pooled resuspension container, e) Adding dimethyl sulfoxide (DMSO) to the pooled resuspension container having the pooled resuspension to obtain a pooled resuspension having DMSO, f) Distributing the pooled resuspension containing the DMSO from the pooled resuspension container to several cryo containers, g) Freezing the pooled resuspension containing the DMSO in the cryo-container to form a batch of cryopreserved platelets. The process including the process described above.

2. The process according to claim 1, wherein in step f), the distribution is carried out for a number of cryocontainers equal to the total number of units provided in step a).

3. The process according to claim 1, wherein in step f), the dispensing is carried out until a target filling weight is achieved in each cryo-container, the target filling weight being determined by the weight of the pooled resuspension having the DMSO.

4. The process according to claim 3, wherein the target filling weight is determined by dividing the weight of the pooled resuspension having the DMSO by the number of platelet units provided in step a).

5. The process according to claim 1, wherein, prior to step c), a portion of the supernatant containing the plasma is removed until a target weight of the pellet and the remaining plasma is achieved, the target weight being in the range of 15.9 g to 27.9 g times the number of units pooled or provided in the container.

6. The process according to claim 5, wherein the removal of the portion of the supernatant is carried out until the weight of the pellet and the remaining supernatant is within ±1 g of the target weight.

7. The process according to claim 1, wherein in step d), the resuspension is pooled from each container using a tube tree system, and in step e), DMSO is added to the pooled resuspension container using the tube tree system.

8. The process according to claim 1, wherein in step f), distributing the pooled resuspension having the DMSO from the pooled resuspension container to several cryo containers is carried out using a dosing tree system.

9. The process according to claim 1, wherein the freezing comprises freezing the pooled resuspension at a temperature of -50°C or lower to form an initial frozen platelet composition in the cryo-container, and storing the initial frozen platelet composition in the cryo-container at a temperature of -30°C or higher to 0°C or below -5°C to form the batch of cryopreserved platelets.

10. The process according to any one of claims 1 to 9, wherein the process is carried out two or more times to form two or more batches of the cryopreserved platelets.

11. A process for preparing a cryopreserved platelet composition containing cryopreserved platelets, i) Freezing The process involves freezing a group of platelets in a storage medium at a temperature of -50°C or lower to form an initial frozen platelet composition, ii) The initial frozen platelet composition is stored at a temperature in the range of -10°C to -30°C for at least one month to form the cryopreserved platelet composition. The process including the process described above.

12. The process according to claim 11, wherein the storage includes storing the initial frozen platelet composition in a freezer set to a temperature of -20°C ± 2°C.

13. The process according to claim 11, wherein the freezing comprises subjecting the population of platelets in the cryopreservation medium to the temperature for at least 30 minutes, 1 hour, 2 hours, 3 hours, 4 hours, 8 hours, 12 hours, 1 day, 2 days, 7 days, 1 week, 2 weeks, 1 month, 2 months, 3 months, or 6 months to form the initial frozen platelet composition.

14. The process according to claim 11, further comprising thawing the cryopreserved platelets to form a liquid platelet composition, and administering an effective amount of the liquid platelet composition to a subject in need.

15. The process according to claim 11, wherein the storage is carried out for a period ranging from one month to five years, one month to three years, or one month to one year.

16. The process according to claim 11, wherein the cryopreservation medium contains dimethyl sulfoxide (DMSO) at a concentration in the range of 5% to 8%.

17. A collection of cryogenic containers containing cryopreserved platelets, Each cryogenically preserved platelet in a cryogenic container has a set of biomolecular profiles indicating two or more platelet donors, one batch of cryogenic containers has the same set of biomolecular profiles, and each batch in the collection has a set of biomolecular profiles different from any other batch in the collection. The collection includes a plurality of cryo containers, at least two batches of each container. Each batch of cryocontainers includes at least five cryocontainers. The coefficient of variation of the average DMSO concentration in the cryopreserved platelets across multiple batches is less than 1%. The aforementioned collection of cryogenic containers.

18. The collection of cryocontainers according to claim 17, wherein the collection comprises a plurality of batches of at least 3, 4, 5, 10, 15, 20, 50, 75, or 100 cryocontainers.

19. The cryo-container collection according to claim 17, wherein the biomolecular profile indicating two or more platelet donors is the presence of two amino acid sequences of a first protein from a first gene that differ significantly by more than 50% in frequency within the cryopreserved platelets, or three or more amino acid sequences of the first protein.

20. The collection of cryocontainers according to claim 17, wherein the set of biomolecular profiles in one batch is different from the set of biomolecular profiles in another batch.

21. The process according to any one of claims 1 to 10, wherein the freezing comprises freezing the pooled resuspension at a temperature of -50°C or lower to form an initial frozen platelet composition in the cryo-container, and storing the initial frozen platelet composition in the cryo-container at a temperature in the range of -10°C to -30°C to form the batch of cryopreserved platelets.

22. The process according to claim 21, wherein the freezing includes subjecting the pooled resuspension to a temperature of -50°C or lower for at least 30 minutes, 45 minutes, 1 hour, 2 hours, or 3 hours.

23. The process according to any one of claim 21 or 22, wherein the storage includes storing the initial frozen platelet composition in the cryo container in a freezer set to a temperature of -20°C ± / -2°C.

24. The process according to any one of claims 21 or 22, wherein the initial frozen platelet composition in the cryo container is stored at a temperature in the range of -10°C to -30°C for at least 90 minutes, 2 hours, or 3 hours.

25. The process according to any one of claim 21 or 22, wherein the initial frozen platelet composition in the cryo container is stored at a temperature in the range of -10°C to -30°C for a period of one month to five years.

26. The process according to any one of claims 1 to 9, wherein the freezing comprises freezing the pooled resuspension at a temperature in the range of -50°C to -85°C to form the batch of cryopreserved platelets.

27. The process according to any one of claims 1 to 10 or 21 to 26, wherein in step a), an odd number of platelet units are provided, and one platelet unit is processed in a separate container.

28. The process according to claim 27, wherein in step a), two platelet units are pooled in one container to form a plurality of containers.

29. The process according to claim 28, wherein in step c), the target weight is in the range of 31.8 g to 55.7 g for the container having 2 platelet units and in the range of 15.9 g to 27.9 g for the container having 1 platelet unit.

30. The process according to claim 27, wherein in step c), the target weight is in the range of 43.5 g to 49.5 g for the container having two units and in the range of 20.3 g to 26.3 g for the container having one unit.

31. The process according to claim 27, wherein in step a), 5, 7, 9, or 11 units are provided, each unit being from a different donor.

32. The process according to any one of claims 1 to 10 or 21 to 26, wherein an even number of units are provided in step a).

33. The process according to claim 32, wherein in step a), two platelet units are pooled in one container to form a plurality of containers, each having two platelet units.

34. The process according to claim 32, wherein the target weight is in the range of 31.8 g to 55.7 g for each of the containers.

35. The process according to claim 32, wherein in step a), 6, 8, 10, or 12 units are provided, and each unit is from a different donor.

36. The process according to any one of claims 1 to 10 or 21 to 35, wherein two units are pooled in a container.

37. The process according to any one of claims 1 to 10 or 21 to 35, wherein at least three units are pooled in a container.

38. The process according to any one of claims 1 to 10 or 21 to 35, wherein in step e), the addition of the DMSO is carried out to achieve a concentration of DMSO in the pooled resuspension ranging from 3 to 8%.

39. The process according to claim 38, wherein the concentration of the DMSO in the pooled resuspension is in the range of 5 to 7%.

40. Over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches, a) The coefficient of variation of the average resuspension volume across multiple batches is less than 10%, and / or b) The coefficient of variation of the average pooled resuspension volume with DMSO in cryocontainers across multiple batches is less than 5%. The process according to claim 10.

41. The process according to claim 10, wherein resuspending the pellets in each container leads to a resuspension in each container such that the volume of the resuspension over at least 10 batches has an average intra-batch coefficient of variation (average intra-batch CV) of less than 15%.

42. The process according to any one of claims 1 to 10 or 21 to 39, wherein resuspending the pellets in each container results in a resuspension in each container such that the volume of the resuspension across containers in one batch has a coefficient of variation of less than 15%.

43. The process according to any one of claims 1 to 10 or 21 to 39, wherein the resuspension of the pellets in each container leads to a resuspension in each container such that the volume of the resuspension in the container varies by 20% or less over at least 10 batches or within one batch.

44. The process according to claim 10, wherein the volume of the pooled resuspension having the DMSO in a cryocontainer over at least 10 batches has an average intra-batch coefficient of variation of less than 5%.

45. The process according to any one of claims 1 to 10 or 21 to 39, wherein the volume of the pooled resuspension in a cryocontainer having DMSO in one batch has a coefficient of variation of less than 5%.

46. The process according to any one of claims 1 to 10 or 21 to 39, wherein the volume of the pooled resuspension having the DMSO in a cryocontainer varies by 10% or less across at least 10 batches or within one batch.

47. The process according to any one of claims 1 to 10 or 21 to 46, wherein the container in step a) is an apheresis platelet unit (APU) bag.

48. The process according to any one of claims 1 to 10 or 21 to 46, wherein the pooled resuspension container is one or more APU bags.

49. The collection or process of a cryocontainer according to any one of claims 10 or 17-20, wherein the coefficient of variation of the average DMSO concentration in the cryopreserved platelets across multiple batches is less than 0.5%.

50. A collection or process of a cryocontainer according to any one of claims 10 or 17-20, wherein the average DMSO concentration in the cryopreserved platelets over at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 50, 75, or 100 batches has a coefficient of variation in the range of 0.05 to 1%.

51. A collection or process of cryocontainers according to any one of claims 9, 10, or 17-20, wherein the concentration of DMSO in the cryopreserved platelets in the cryocontainer within one batch or over at least five batches varies between 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, or 0.05% or less.

52. A collection or process of cryocontainers according to any one of claims 9, 10, or 17-20, wherein the cryocontainers in the batch or collection contain the cryopreserved platelets having DMSO in the range of 1 to 10%.

53. The collection or process of a cryocontainer according to claim 52, wherein the DMSO is in the range of 5 to 8%.

54. A collection of cryocontainers for the process according to claim 53, wherein the DMSO is in the range of 6% ± 1%, 6% ± 0.75%, 6% ± 0.5%, 6% ± 0.25%, 6% ± 0.1%, or 6% ± 0.5%.

55. A collection or process of a cryo-container according to any one of claims 9, 10, or 11-20, wherein the cryopreserved platelets are stable for at least one year at a temperature in the range of -10°C to -30°C.

56. A collection or process of a cryocontainer according to any one of claims 9, 10, or 17-20, wherein the concentration of platelets in the cryopreserved platelets in the cryocontainer varies by 10% or less within one batch or over at least five batches.

57. A collection or process of a cryocontainer according to any one of claims 10 or 17-20, wherein the total number of platelets in the cryopreserved platelets in the cryocontainer over at least five batches has an average intra-batch coefficient of variation of less than 6%.

58. A collection or process of a cryocontainer according to any one of claims 10 or 17-20, wherein the platelet concentration (platelets / μl) in the cryopreserved platelets in the cryocontainer over at least five batches has an average intra-batch coefficient of variation of less than 5%.

59. A collection or process of cryovessels according to any one of claims 10 or 17-20, wherein the thrombin-producing ability of the cryopreserved platelets in the cryovessels over at least five batches has an average intra-batch coefficient of variation of less than 5%.

60. A collection or process of a cryocontainer according to any one of claims 10 or 17-20, wherein the percentage of CD61-positive microparticles in the cryocontainer containing the cryopreserved platelets over at least five batches has an average intra-batch coefficient of variation of less than 10%.

61. A collection or process of cryocontainers according to any one of claims 10 or 17-20, wherein the pH of the cryopreserved platelets in the cryocontainers over at least five batches has an average intra-batch coefficient of variation of less than 3%.

62. The process according to any one of claims 9, 10, or 11-16, wherein the initial frozen platelet composition is stored at a temperature in the range of -10°C to -30°C for at least six months.

63. A process or collection of a cryocontainer according to any one of claims 1 to 61, wherein a therapeutically effective amount of cryopreserved platelets retains, upon thawing, the ability to reduce bleeding in a subject in need.

64. The process or cryo-container collection according to any one of claims 1 to 61, wherein the cryopreserved platelets exhibit a recovery rate of at least 65% of the platelet count upon thawing.

65. The process or collection of a cryo-container according to any one of claims 1 to 61, wherein the frozen-stored composition exhibits a pH of 6.2 or higher upon thawing.

66. The process or collection of a cryo-container according to any one of claims 1 to 61, wherein the cryopreserved platelets exhibit aggregate-free swirling upon thawing and visual inspection.

67. The aforementioned cryopreserved platelet composition, upon thawing, contains 1.7 × 10 １１ A process or collection of cryocontainers according to any one of claims 1 to 61, exhibiting a platelet count of one or more platelets / bags or platelets / cryocontainers.

68. The process or collection of a cryo-container according to any one of the prior claims, wherein the cryopreserved platelets comprise plasma in the range of 70–85% (v / v), DMSO in the range of 4–8% (v / v), and sodium chloride in the range of 7–15% (w / v).

69. The process or collection of cryocontainers according to any one of the prior claims, wherein the cryocontainer is a cryobag.

70. A cryopreserved platelet composition or batch of cryopreserved platelets prepared by a process according to any one of claims 1 to 16, 21 to 48, or 62 to 69.

71. A composition comprising platelets frozen in a cryopreservation medium in a frozen state, wherein the composition, after storage for 6 months and upon thawing, exhibits the following properties: a) Being in a liquid state, and not requiring the addition of any liquid to achieve such a liquid state. b) at least 1.0 × 10 １１ To exhibit a platelet count of 35 ml per composition, c) To produce a single peak corresponding to a damaged membrane peak in a membrane integrity assay. d) The composition contains less than 50% CD61-positive particles, and e) In an in vitro thrombin generation assay, generate thrombin. The composition is capable of bringing about the following.

72. The process, collection, or composition according to any one of claims 1, 11, 17, or 70, wherein the cryopreserved platelets are cryopreserved platelet derivatives, or the frozen platelets are frozen platelet derivatives.

73. The composition according to claim 71, wherein the membrane integrity assay comprises incubating the composition with calcein acetoxymethyl (AM) to form a treated composition, and analyzing the treated composition by flow cytometry.

74. The composition, process, or collection according to any one of claims 71 or 72, wherein the in vitro thrombin generation assay comprises generating thrombin in the presence of tissue factor and phospholipids.

75. The composition, process, or collection according to any one of claims 71 or 72, wherein the frozen platelets, upon thawing, have a diameter in the range of 0.5 to 5 μm, 1 to 4 μm, 1 to 3 μm, or 0.5 to 2.5 μm, and the composition, upon thawing, has a CD61-positive microparticle content in the range of 10 to 30%.

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