Methods for bulk isolation of extracellular vesicles

Abstract

A method of bulk lyophilizing a biological product generally includes providing the biological product, freezing the biological product under controlled conditions, and applying a vacuum to the biological product. In one or more embodiments, the biological product includes a body fluid, a cell-derived vesicle, or a target molecule of interest. In one or more embodiments, the biological product includes a blood product. In one or more embodiments, the biological product includes extracellular vesicles. In one or more embodiments, the method further includes a second step of applying a vacuum to the biological product.

Description

CROSS-REFERENCE TO RELATED APPLICATION This application claims the benefit of U.S. Provisional Patent Application No. 63 / 619,172, filed January 9, 2024, which is incorporated herein by reference in its entirety. SUMMARY This disclosure describes, in one aspect, a method of bulk lyophilizing a biological product. Generally, the method includes providing the biological product, freezing the biological product under controlled conditions, and applying a vacuum to the biological product. In one or more embodiments, the biological product includes a body fluid, a cell-derived vesicle, or a target molecule of interest. In one or more embodiments, the biological product includes a blood product. In one or more embodiments, the biological product includes extracellular vesicles. In one or more embodiments, the further includes warming the frozen biological product to a temperature from -10 °C to -85 °C prior to applying the vacuum to the biological product. In one or more embodiments, the method further includes a second step of applying a vacuum to the biological product. In one or more embodiments, the second step of applying a vacuum to the biological product is performed at a temperature above 0 °C. The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS This disclosure describes methods that allow one to lyophilize extracellular vesicles in bulk and the resulting compositions that include lyophilized extracellular vesicles. Extracellular vesicle research and clinical applications have been limited by an inability to isolate appreciable quantities of extracellular vesicles from complex solutions. Current extracellular vesicle isolation strategies (e. g., ultracentrifugation, tangential flow fdtration, size exclusion chromatography, etc.) remain difficult to scale and are often not cGMP compliant, which limits their implementation as commercial grade or commercial scale manufacturing process for human therapeutics. Lyophilizing extracellular vesicles in bulk may be advantageous because it may streamline quality control and improve consistency in treatment by reducing batch variation. In addition, bulk lyophilization may be financially advantageous. However, procedures optimized for bench scale often do not translate reliably to larger formats. This disclosure describes methods for isolating extracellular vesicles from complex biological solutions in large quantities using high throughput, scalable, cGMP compliant unit operations to overcome these barriers. The use of clarifying unit operations and terminal lyophilization of extracellular vesicles is well-established in the field. See, e.g.,U.S. Patent No. 10,596,123, U.S. Patent No. 12,036,325; U.S. Patent Application Publication No. US2021 / 0259969; and U.S. Patent Application Publication No. US2022 / 0241325 AL This disclosure describes the isolation of extracellular vesicles from a biological solution at a large scale that far exceeds what has been done previously and in a cGMP manner appropriate for clinical application. Further, this disclosure provides data establishing the stability of the bulk-lyophilized extracellular vesicles in both an accelerated stability model and in an ambient stability model. Generally, the method includes obtaining a source of extracellular vesicles. The extracellular vesicles may be obtained from any suitable source. One exemplary source of extracellular vesicles is platelets. Platelets may be obtained from any suitable blood product including, without limitation, whole blood or any suitable apheretic blood product (including leukapheresis products, plasmapheresis product, cryo poor plasma, fresh frozen plasma, pheresis platelet products, platelet rich plasma, platelet poor plasma, or any erythrocyte depleted and leukocyte depleted product). The blood or blood product may be obtained from any suitable source, including but not limited to the general population, general population age 30 or below, general population age 40 or below, post-surgical population, a pre-menopausal woman, a peripartum woman, a placenta, or umbilical cord blood. Alternative illustrative sources of extracellular vesicles include, but are not limited to, a non-blood source such as, for example, umbilical cord Wharton’s jelly, stromal vascular fraction of fat, apheresis bone marrow products, synovial fluid, cerebrospinal fluid, mesenchymal stem cells, endothelial cells, neural stem cells, embryonic stem cells, induced pluripotent stem cells, or the conditioned medium of these or any other cell sources. In one or more embodiments, extracellular vesicles derived from a blood product. In one or more embodiments, the blood product from which the extracellular vesicles are derived may be pooled from two or more separate collections. In one or more embodiments, the volume of blood product subject to the methods described herein can be at least 2.5 liters (L), at least 3 L, at least 4 L, at least 5 L, at least 6 L, at least 7 L, at least 8 L, at least 9 L, at least 9.5 L, at least 10 L, at least 12 L, at least 18 L, at least 25 L, at least 50 L, at least 100 L, or at least 500 L. In one or more embodiments, the volume of blood product can be within a range having endpoints defined by any minimum volume listed immediately above any second volume listed above that is greater than the selected minimum volume. Thus, for example, the volume of blood product can be from 2.5 L to 25 L, from 6 L to 18 L, from 9.5 L to 20 L, from 10 L to 12 L, etc. In one or more embodiments, there can be approximately 20% loss of volume throughout the lyophilization process. Thus, in one or more embodiments, extracellular vesicles derived from a blood product subjected to lyophilization can have a volume of, for example, 2 L to 20 L, such as 5 L to 15 L, such as 8 L to 10 L are lyophilized. In an exemplary embodiment, extracellular vesicles are lyophilized in bulk using a lyophilization protocol optimized to freeze dry the extracellular vesicles and minimize the amount of moisture remaining in the lyophilized extracellular vesicle product. In one or more illustrative embodiments, the method involves freezing the extracellular vesicles, then drying the extracellular vesicles as the extracellular vesicles are re-warmed in a stepwise manner. In one or more embodiments, the extracellular vesicles may be cooled to non-freezing temperature prior to being frozen, as described in Example 2. The extracellular vesicles are frozen to a temperature of at least -10 °C. In this context, the term “at least” refers to the extent to which a temperature is below 0° C. Thus, “at least -10 °C” refers to temperatures, as described in more detail below, that include -10 °C, -15 °C, -50 °C, and so on. The extracellular vesicles are frozen to a temperature of from -30 °C to -95 °C. Thus, in one or more embodiments, the extracellular vesicles may be frozen to a temperature of, for example, -30 °C, -35 °C, -40 °C, -45 °C, -50 °C, -55 °C, -60 °C, -65 °C, -70 °C, -75 °C, -80 °C, -85 °C, -90 °C, or -95 °C. In one or more embodiments, the extracellular vesicles may be frozen to a temperature that falls within a range having endpoints defined by any two of the temperatures listed immediately above. Thus, in one or more embodiments, the extracellular vesicles may be frozen to a temperature of from -30 °C to -50 °C, from -30 °C to -75 °C, from -50 °C to -85°C, and so on. In one exemplary embodiment, the extracellular vesicles are frozen to a temperature of -40°C to -60 °C, such as, for example, -50 °C. In one or more embodiments, the extracellular vesicles are frozen at a rate of cooling of from 5° C / min to 0.1° C / min. However, the methods described herein can involve cooling the extracellular vesicles at a rate that falls outside of this range. In one or more embodiments, the extracellular vesicles may be cooled at a rate of, for example, 0.1 °C / min, 0.2 °C / min, 0.3 °C / min, 0.4 °C / min, 0.5 °C / min, 0.6 °C / min, 0.7 °C / min, 0.8 °C / min, 0.9 °C / min, 1.0 °C / min, 2.0 °C / min, 3.0 °C / min, 4.0 °C / min, or 5.0 °C / min. The cooling rate may or may not be constant throughout the freezing process. Thus, in one or more embodiments, the extracellular vesicles may be cooled at one or more rates that fall within a range having endpoints defined by any two of the cooling rates listed above. Thus, the extracellular vesicles may be cooled at a rate of from 0.1 °C / min to 1.0 °C / min, from 0.5 °C / min to 5 °C / min, 0.8 °C / min to 3.0 °C / min, and so on. In one exemplary embodiment, the extracellular vesicles are cooled at a rate of 0.5 °C / min. After freezing, the extracellular vesicles are warmed and dried in a stepwise manner. That is, the extracellular vesicles are warmed and then held at a first warmed temperature (or within a first range of warmed temperatures, as described in more detail below) while subjected to a vacuum. Then the extracellular vesicles are warmed a second time, then once again held at a second warmed temperature (or within a second range of warmed temperatures, as described in more detail below) while a vacuum is applied. In one or more embodiments, at each warming step, the extracellular vesicles are warmed at a rate of from 5° C / min to 0.1° C / min. However, the methods described herein can involve warming the extracellular vesicles at a rate that falls outside of this range. In one or more embodiments, the extracellular vesicles may be warmed at a rate of, for example, 0.1 °C / min, 0.2 °C / min, 0.3 °C / min, 0.4 °C / min, 0.5 °C / min, 0.6 °C / min, 0.7 °C / min, 0.8 °C / min, 0.9 °C / min, 1.0 °C / min, 2.0 °C / min, 3.0 °C / min, 4.0 °C / min, or 5.0 °C / min. The warming rate may or may not be constant throughout the warming process. Thus, in one or more embodiments, the extracellular vesicles may be warmed at one or more rates that fall within a range having endpoints defined by any two of the warming rates listed above. Thus, the extracellular vesicles may be warmed at a rate of from 0.1 °C / min to 1.0 °C / min, from 0.5 °C / min to 5 °C / min, 0.8 °C / min to 3.0 °C / min, and so on. In one exemplary embodiment, the extracellular vesicles are warmed at a rate of 0.5 °C / min. The first warmed temperature may be any temperature from -35 °C to -10 °C that is warmer than the temperature at which the extracellular vesicles were frozen. Thus, in one or more embodiments, the first warmed temperature may be -35 °C, -34 °C, -33 °C, -32 °C, -31 °C, -30 °C, -29 °C, -28 °C, -27 °C, -26 °C, -25 °C, -24 °C, -23 °C, -22 °C, -21 °C, -20 °C, -19 °C, -18 °C, -17 °C, -16 °C, -15 °C, -14 °C, -13 °C, -12 °C, -11 °C, or -10 °C. While a vacuum is applied, the first warming temperature may or may not be held constant. Thus, in one or more embodiments, the first warming temperature may be, or may fluctuate, within a range having endpoints defined by any two of the first warming temperatures listed above. For example, the first warming temperature may be from -32 °C to -12 °C, from -27 °C to -17 °C, -25 °C to -20 °C, and so on. In one exemplary embodiment, the first warming temperature is -22 °C ± 10 °C. At the first warming temperature, the extracellular vesicles may be dried by subjecting the extracellular vesicles to a vacuum pressure of from 10 mTorr to 300 mTorr. Thus, in one or more embodiments, the extracellular vesicles may be subjected to a vacuum pressure of 10 mTorr, 20 mTorr, 30 mTorr, 40 mTorr, 50 mTorr, 60 mTorr, 70 mTorr, 80 mTorr, 90 mTorr, 100 mTorr, 110 mTorr, 120 mTorr, 130 mTorr, 140 mTorr, 150 mTorr, 175 mTorr, 200 mTorr, 225 mTorr, 250 mTorr, 275 mTorr, or 300 mTorr. During the first drying step, the vacuum pressure may or may not be held constant. Thus, in one or more embodiments, the vacuum pressure of the first drying step may be, or may fluctuate, within a range having endpoints defined by any two of the first vacuum pressures listed above. For example, the first drying step may be performed at a vacuum pressure of from 100 mTorr to 120 mTorr, from 90 mTorr to 130 mTorr, etc. The second warmed temperature may be any temperature from 0 °C to 30 °C. Thus, in one or more embodiments, the second warmed temperature may be 0 °C, 1 °C, 2 °C, 3 °C, 4 °C, 5 °C, 6 °C, 7 °C, 8 °C, 9 °C, 10 °C, 11 °C, 12 °C, 13 °C, 14 °C, 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C. While a vacuum is applied, the second warming temperature may or may not be held constant. Thus, in one or more embodiments, the second warming temperature may be, or may fluctuate, within a range having endpoints defined by any two of the second warming temperatures listed above. For example, the second warming temperature may be from 5 °C to 25 °C, from 10 °C to 20 °C, and so on. In one exemplary embodiment, the second warming temperature is 15 °C ± 10 °C. At the second warming temperature, the extracellular vesicles may be dried by subjecting the extracellular vesicles to a vacuum pressure of from 10 mTorr to 300 mTorr. Thus, in one or more embodiments, the extracellular vesicles may be subjected to a vacuum pressure of 10 mTorr, 20 mTorr, 30 mTorr, 40 mTorr, 50 mTorr, 60 mTorr, 70 mTorr, 80 mTorr, 90 mTorr, 100 mTorr, 110 mTorr, 120 mTorr, 130 mTorr, 140 mTorr, 150 mTorr, 175 mTorr, 200 mTorr, 225 mTorr, 250 mTorr, 275 mTorr, or 300 mTorr. During the second drying step, the vacuum pressure may or may not be held constant. Thus, in one or more embodiments, the vacuum pressure of the second drying step may be, or may fluctuate, within a range having endpoints defined by any two of the second vacuum pressures listed above. For example, the second drying step may be performed at a vacuum pressure of from 100 mTorr to 120 mTorr, from 90 mTorr to 130 mTorr, etc. In one or more illustrative embodiments, the extracellular vesicles are frozen, warmed to a first warmed temperature of -22 °C, dried at a vacuum pressure of 100 mTorr, then warmed to 15 °C where drying continues at a vacuum pressure of 100 mTorr. In one or more embodiments, one or both drying steps can be performed at a vacuum pressure of 120 mTorr. As shown in Table 1 below (Example 2), each of the cooling step, the freezing step (labeled Freezing 3 in Table 1), and the drying step at the first warmed temperature (labeled Drying 2 in Table 1) can include a hold time—i.e., the duration at which the temperature and vacuum pressure parameters are held. Generally, the hold times for the cooling step and the freezing step are sufficient for the extracellular vesicles to reach temperature equilibrium. Since the vacuum is not applied to the extracellular vesicles at these steps, the precise hold time is not critical and, therefore, can vary greatly. Thus, the hold time at the cooling step and the freezing step can be at least 20 minutes, at least 30 minutes, at least 40 minutes, at least 60 minutes, at least 90 minutes, at least 120 minutes, at least 150 minutes, at least 180 minutes, at least 210 minutes, at least 240 minutes, at least 270 minutes, at least 300 minutes, at least 330 minutes, at least 360 minutes, at least 390 minutes, at least 420 minutes, at least 450 minutes, and so on without any results-dependent maximum limit. For convenience and efficiency of production, the hold times for the cooling step and the freezing step in one or more embodiments can be no greater than 600 minutes, no greater than 570 minutes, no greater than 540 minutes, no greater than 510 minutes, no greater than 480 minutes, no greater than 420 minutes, no greater than 390 minutes, no greater than 360 minutes, no greater than 300 minutes, no greater than 240 minutes, no greater than 210 minutes, no greater than 180 minutes, no greater than 150 minutes, no greater than 120 minutes, no greater than 100 minutes, no greater than 90 minutes, no greater than 80 minutes, no greater than 70 minutes, no greater than 60 minutes, no greater than 50 minutes, no greater than 40 minutes, or no greater than 30 minutes. In one or more embodiments, the hold time in the cooling step, the freezing step, or both, may independently of one another be expressed as a range having endpoints defined by any minimum hold time listed above and any maximum hold time listed above that is greater than the selected minimum hold time. Thus, for example, the hold time at the cooling step and / or the freezing step may be 60 minutes to 180 minutes, 300 minutes to 420 minutes, 90 minutes to 150 minutes, 240 minutes to 42 minutes and so on. As shown in the exemplary embodiment in Example 2 (Table 1) the hold time for the cooling step may be different than the hold time for the freezing step. In one or more embodiments, however, the hold time for the cooling step may be the same as the hold time for the freezing step. The hold time for the drying step at the first warmed temperature (labeled Drying 2 in Table 1) is typically of longer duration than the hold times for the cooling step and the freezing step. Further, because the drying step is performed under vacuum pressure, the hold time at this drying step is more results-determinative than the hold times for the cooling step and the freezing step. The hold time for the drying step may be from 3000 minutes to 5000 minutes. Thus, the hold time at the drying step can be at least 3000 minutes, at least 3160 minutes, at least 3320 minutes, at least 3480 minutes, at least 3640 minutes, at least 3820 minutes, or at least 3960 minutes. Further, the hold time at the drying step can be no more than 5000 minutes, no more than 4820 minutes, no more than 4660 minutes, no more than 4500 minutes, no more than 4440 minutes, no more than 4340 minutes, no more than 4180 minutes, or no more than 4020 minutes. The hold time at the drying step can be expressed as a range having endpoints defined by any minimum hold time for the drying step listed above and any maximum hold time for the drying step listed above that is greater than the selected minimum hold time. Thus, the hold time at the drying step can be 3000 minutes to 4020 minutes, 3480 minutes to 4440 minutes, and so on. In one or more embodiments, at least 50 g, at least 100 g, at least 200 g, at least 300 g, at least 400 g, at least 500 g, at least 1 kg, at least 2 kg, at least 3 kg, at least 4 kg, at least 5 kg, at least 6 kg, at least 7 kg, at least 8 kg, at least 9 kg, at least 10 kg, at least 15 kg, at least 20 kg, at least 25 kg, at least 30 kg, at least 35 kg, at least 40 kg, at least 45 kg or at least 50 kg of lyophilized extracellular vesicles are produced from one lyophilization cycle. The stability of the lyophilized extracellular vesicles is illustrated in Example 3 and Example 4. Example 3 shows stability of the lyophilized extracellular vesicles, in both an accelerated stability model and in ambient storage conditions, when stored in glass vials. Example 4 shows stability data of the lyophilized extracellular vesicles, in both an accelerated stability model and in ambient storage conditions, when stored in a foil pouch. While described herein in the context of extended storage in a foil pouch, other methods of storage are contemplated. In one or more embodiments, the lyophilized extracellular vesicles are stored in a glass container. In one or more embodiments, the lyophilized extracellular vesicles are stored under an inert gas, such as argon. It may be advantageous to prevent the lyophilized extracellular vesicles from absorbing moisture. In one or more embodiments, the lyophilized extracellular vesicles are stored with a desiccant. Extracellular vesicles remain stable after one year under ambient storage conditions, as evidenced by the antioxidant capacity measured in the Trolox equivalent antioxidant capacity (TEAC) assay. One example of a TEAC assay that may be used is the OXISELECT Assay Kit from Cell Biolabs (Cat. No. XAN-5040). While extracellular vesicles may be stored under ambient conditions, they may alternatively be stored at a lower temperature (e.g., lower than 22 °C). Thus, with the proper processing parameters, extracellular vesicles can be isolated and stabilized in large quantities. The lyophilized extracellular vesicle may then serve as, for example, a source material as a clinical-grade therapeutic or starting material for further manipulation. Further manipulations include, but are not limited to, loading with an exogenous therapeutic (e.g., small molecule drug, an imaging agent, a therapeutic polynucleotide, etc.). The extracellular vesicle lipid bilayers remain intact and the cargo remains potent. Lyophilization can be achieved without the adding any excipients and in closed, scalable configurations. In the preceding description and following claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended—i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). As used herein, “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” or the like are used in their open-ended inclusive sense, and generally mean “include, but not limited to,” “includes, but not limited to,” or “including, but not limited to.” Further, wherever embodiments are described herein with the language “have,” “has,” “having,” “include,” “includes,” “including,” “comprise,” “comprises,” “comprising” and the like, otherwise analogous embodiments described in terms of “consisting of’ and / or “consisting essentially of’ are also provided. The term “consisting of’ means including, and limited to, that which follows the phrase “consisting of.” That is, “consisting of’ indicates that the listed elements are required or mandatory, and that no other elements may be present. The term “consisting essentially of’ indicates that any elements listed after the phrase are included, and that other elements than those listed may be included provided that those elements do not interfere with or contribute to the activity or action specified in the disclosure for the listed elements. As used herein, the word “exemplary” means to serve as an illustrative example and should not be construed as preferred or advantageous over other embodiments. As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention. In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive. In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list. For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously. EXAMPLES The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein. Example 1 - Bulk Pooled Platelet (BPP) preparation Platelets stored in platelet collection bags at -80 °C were thawed in a 36.5 °C water bath until all material was thawed or a maximum of 40 minutes. 9.5 L of thawed platelets were transferred from the platelet bags to a 10 L pooling bag using gravity and pressure applied to the platelet bag. The pooling bag was inverted five times to mix the contents. The pooled platelets were filtered using a 40-pm filter and a peristaltic pump to remove large debris, leaving a clarified flow through containing exosomes. Approximately 7.5 L of filtered exosome preparation was collected stored a minimum of 12 hours at -80 °C. Example 2 - Lyophilization The filtered exosome preparation from Example 1 was thawed in a water bath set to 36.5 °C until no frozen material was observed. The thawed exosomes were aliquoted into LYOGUARD (W. L. Gore & Associates, Inc., Newark, DE) trays by gravity, with up to 1.8 kg of thawed exosome preparation being aliquoted per tray. 5          The lyophilizer (VIRTIS, SP Industries Inc., Warminster, PA) was programmed so that the lyophilization cycles were set as indicated in Table 1. Table 1. Step Temperature (°C) Ramp Time (min) Hold Time (min) Vacuum Pressure (mTorr) Cooling 1 5.0 0 120 NA Freezing 2 -50.0 108 0 NA Freezing 3 -50.0 0 360 NA Drying 1 -22.0 48 0 100 Drying 2 -22.0 0 3960 100 Drying 3 15.0 80 0 100 Drying 4 15.0 0 720 100 10 After the lyophilization cycle was initiated, trays were loaded into the lyophilizer once the shelf temperature reached 5 °C ± 2 °C. After the lyophilization cycle was completed, trays were removed from the lyophilizer and placed in a foil pouch (LYOGUARD, W. L. Gore & Associates, Inc., Newark, DE). Approximately 550 grams of lyophilized exosome material was produced. 15 Example 3 - Stability following vial storage Lyophilized samples were labeled, numbered, and weighed prior to analysis. Vials were initially filled to maximum capacity based on volume. Multiple lots were mixed to provide a homogeneous product prior to packaging. Analyses were performed at packaging, after two 20 weeks, one month, two months, three months, 12 months of storage at either 25 °C / 60% relative humidity (RH) or 40 °C / 75% RH. Analytical results are summarized in Table 2. Table 2 Analytical Test Storage Condition Time Points Month 0 Week 2 Month 1 Month 2 Month 3 Year 1 Appearance 25°C / 60% RH Off white to light yellow powder NA Off white to light yellow powder light yellow powder Weight Loss NA 0.0% 0.1% Catalase Activity 35.101 mU / mL 600,800 mU / mL NA TEAC NA NA 492 pM Residual Moisture 3.12% 1.20% 1.61% Aerobic Plate Count 10 CFU / g <10 CFU / g <10 CFU / g Yeast Count <10 CFU / g <10 CFU / g <10 CFU / g Mold Count <10 CFU / g <10 CFU / g <10 CFU / g Appearance 40°C / 75% RH Light yellow powder Light yellow powder Light yellow powder Light yellow powder Light yellow powder NA Weight Loss NA 0.0% 0.0% 0.0% 0.1% Catalase Activity' 635.600 mU / mL 2,286.000 mU / mL NA NA NA TEAC NA NA 864 pM 1038 pM 1358 pM Residual Moisture 1.38% 1.78% 1.78% 2.21% 1.18% Aerobic Plate Count <10 CFU / g <10 CFU / g <10 CFU / g 80 CFU / g 5 CFU / g Yeast Count <10 CFU / g <10 CFU / g <10 CFU / g <10 CFU / g <10 CFU / g Mold Count <10 CFU / g <10 CFU / g <10 CFU / g <10 CFU / g <10 CFU / g Example 4 - Stability following storage in foil pouches Lyophilized samples were sealed in foil pouches (LYOGUARD, W. L. Gore & 5 Associates, Inc., Newark, DE), either with or without a desiccant. Samples were subjected to accelerated stability conditions (40°C / 75% RH) for three months or ambient stability conditions (25°C / 60% RH) for one year. Table 3 provides summary data for the accelerated stability testing. Analyses were performed at packaging and after two weeks, one month, six weeks, and three months of storage. Table 3 - Accelerated testing results Analytical Test Sample Month 0 Week! Month 1 Week 6 Month 3 Appearance With Desiccant Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Without Desiccant Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Residual Moisture With Desiccant 4.21% 3.09% 3.40% 3.21% 2.49% Without Desiccant 3.55% 3.80% 2.28% 1.68% TEAC Assay With Desiccant 855 pM 705 pM 1015 pM 810 pM 850 pM Without Desiccant 690 pM 830 pM 895 pM 830 pM Table 4 provides summary data for ambient stability testing. Analyses were performed at packaging and after three months, six months, and one year of storage. 10 Table 4 - Ambient testing results Analytical Test Sample Month 0 Month 3 Month 6 Year 1 Appearance With Desiccant Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Light yellow powder Without Desiccant Off white to light yellow powder / powder cake Off white to light yellow powder / powder cake Light yellow powder Residual Moisture With Desiccant 4.21% 2.39% 3.79% 4.78% Without Desiccant 2.56% 4.95% 5.41% TEAC Assay With Desiccant 855uM 970 pM 960 pM 713 pM Without Desiccant 975 pM 950 pM 760 pM The complete disclosure of all patents, patent applications, and publications, and 5 electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein 10 by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims. 15           Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of 5 equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific 10 examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements. All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified. 15

Claims

1. A method of bulk lyophilizing a biological product, the method comprising: providing the biological product;freezing the biological product at a temperature no greater than -15 °C; and applying a vacuum to the biological product.

2. The method of claim 1, wherein the biological product comprises a body fluid, a cell-derived vesicle, or a target molecule of interest.

3. The method of claim 2, wherein the biological product comprises a blood product.

4. The method of claim 2, wherein the biological product comprises extracellular vesicles.

5. The method of any preceding claim, further comprising warming the frozen biologicalproduct to a temperature from -10 °C to -85 °C prior to applying the vacuum to the biological product.

6. The method of any preceding claim, further comprising a second step of applying a vacuum to the biological product.

7. The method of claim 6, wherein the second step of applying a vacuum to the biological product is performed at a temperature above 0 °C.

8. The method of claim 1, wherein the biological product has a volume of at least 5 L.

9. The method of claim 5, wherein providing the biological product comprises providing atleast 8 L of a blood product comprising platelets and filtering the blood product to remove the platelets, thereby producing the biological product.