Method for reducing the potency of biological contaminants in complex biological preparations

Abstract

A method for reducing the potency of biological contaminants in a biological product generally comprises applying electromagnetic energy with a dose of ultraviolet (UV) wavelength or lower that is effective in reducing biological contaminants in a sample of the biological product while maintaining the integrity and function of the biological product. In one or more embodiments, the electromagnetic energy includes UV-B, UV-C, X-rays, or γ-rays. In one or more embodiments, the method further comprises applying electron beam radiation to the biological product.

Description

[0001] Cross-reference of related applications

[0002] This application claims priority to U.S. provisional patent application No. 63 / 619,143 filed on January 9, 2024, the full text of which is incorporated herein by reference.

[0003] outline

[0004] In one aspect, the present application describes a method for reducing the potency of biological contaminants within a biological product. Generally, the method comprises applying electromagnetic energy with a dose of ultraviolet (UV) wavelength or lower that is effective in reducing the potency of biological contaminants within a sample of the biological product while maintaining the integrity and function of the biological product.

[0005] In one or more embodiments, the method further comprises freeze-drying the biological product.

[0006] In one or more embodiments, the biological contaminant comprises an enveloped virus or a non-enveloped DNA virus.

[0007] In one or more embodiments, the biological contaminant includes a DNA virus or an RNA virus.

[0008] In one or more embodiments, the biological contaminant comprises a single-stranded DNA (ssDNA) virus or a double-stranded DNA (dsDNA) virus.

[0009] In one or more embodiments, the biological contaminant comprises one or more of human immunodeficiency virus (HIV), rabies pseudovirus (PRV), bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), and hepatitis A virus (HAV). In one or more of these embodiments, the HIV titer is reduced by at least 10 times and / or, the PRV titer is reduced by at least 10 times and / or, the BVDV titer is reduced by at least 10 times and / or, the PPV titer is reduced by at least 6 times and / or, and the HAV titer is reduced by at least 6 times.

[0010] In one or more embodiments, the biological contaminant includes bacteria.

[0011] In one or more embodiments, the biological contaminant includes fungi.

[0012] In one or more embodiments, the electromagnetic energy includes UV-B, UV-C, X-rays, or γ-rays. In one or more of these embodiments, the biological product is subjected to UV-C at a dose of 5 mL / min to 500 mL / min.

[0013] In one or more embodiments, the ionizing radiation comprises X-rays, γ-rays, or electron beam radiation. In one or more of these embodiments, the biological product is treated with electron beam radiation of 0 kilogray (kGy) to 55 kGy, for example, electron beam radiation of 25 kGy.

[0014] In one or more embodiments, the method further comprises applying a biological product to a second form of electromagnetic energy or ionizing radiation.

[0015] In one or more embodiments, the method further comprises freeze-drying, freezing, centrifuging, or filtering the biological product.

[0016] In one or more embodiments, the biological product comprises exosomes.

[0017] In another aspect, the present disclosure describes a biological product treated to reduce at least one potency of a biological contaminant compared to an untreated sample of the biological product.

[0018] In one or more embodiments, the titer of the biological contaminant is reduced by at least 1 log compared to the untreated biological product.

[0019] In one or more embodiments, the biological product comprises exosomes.

[0020] In one or more embodiments, the biological product further comprises freeze-dried powder.

[0021] In one or more embodiments, the biological product further comprises a carrier.

[0022] In one or more embodiments, the biological contaminant comprises an enveloped virus or a non-enveloped DNA virus.

[0023] In one or more embodiments, the biological contaminant is a DNA virus or an RNA virus.

[0024] In one or more embodiments, the DNA virus is a single-stranded DNA (ssDNA) virus or a double-stranded DNA (dsDNA) virus.

[0025] In one or more embodiments, the biological contaminant includes bacteria.

[0026] In one or more embodiments, the biological contaminant includes fungi.

[0027] In one or more embodiments, the biological product has significantly the same turbidity, particle size, particle concentration, angiogenic cytokine concentration, effect on cell proliferation, antioxidant capacity, or enzyme activity as the untreated biological product.

[0028] In one or more embodiments, the biological product is derived from a mammalian blood product. In one or more of these embodiments, the mammalian blood product comprises human whole blood.

[0029] In one or more embodiments, the biological product exhibits significantly the same stability as the untreated biological product.

[0030] In another aspect, the present disclosure describes a composition comprising a biological product treated to reduce at least one potency of a biological contaminant compared to an untreated sample of the biological product, and a pharmaceutically acceptable carrier.

[0031] In another aspect, the present disclosure describes a method for treating a subject comprising administering to the subject a composition comprising a biological product treated to reduce at least one potency of a biological contaminant compared to an untreated sample of the biological product.

[0032] The foregoing overview is not intended to describe each disclosed embodiment or all implementations of the present invention. The following description further illustrates exemplary embodiments. Throughout the application, guidance is provided through a list of examples, which may be used in various combinations. In each case, the listed list serves only as a representative group and should not be interpreted as an exclusive list. Brief explanation of the drawing

[0033] Fig. 1. Morphological characterization of PEP. (A) PEP possesses a spherical vesicle structure with an intact lipid bilayer. (B) Particle size distribution analysis via NanoSight (Malvern Panalytical Ltd., Malvern, UK) showed that the average vesicle diameter of PEP ranged from 56.4 nm to 151 nm, with an average diameter of 96.9 nm ± 2.8 nm, which represents the standard size range for exosomes. By applying a dilution factor correction of 1000 to the reported particle concentrations, 1.9 × 10⁶ in 100% PEP solution 11 A final concentration of particles / mL was produced. Fig. 2. Turbidity of PEP when treated with UV-C. The absorbance of 50 μL of 100% PEP solution at the time of initial treatment (T0, left) or 100 μL after 3 months (T3, right) was measured at 600 nm using a plate reader (VARIOSKANLUX, Thermo Fisher Scientific, Inc., Waltham, Massachusetts). Passing the PEP through UV-C radiation treatment multiple times did not show a change in turbidity compared to 100% PEP (untreated) that was not exposed to UV-C. Fig. 3. Angiogenic cytokines in PEP treated with UV-C at initial treatment (T0). The concentrations of angiogenic-promoting cytokines ANG-1, EGF, PDGF-BB, and VEGF-A detected by automated ELISA (ELLA, Bio-Techne, Minneapolis, Minnesota) were slightly reduced in PEP after UV-C treatment, but not to a significant degree. Fig. 4. Angiogenic cytokines in PEP treated with UV-C 1 month after treatment (T1). 1 month after UV-C treatment, the concentrations of angiogenic-promoting cytokines ANG-1, EGF, PDGF-BB, and VEGF-A detected by ELISA (ELLA, Bio-Techne, Minneapolis, Minnesota) did not change significantly from T0. Fig. 5. Angiogenic cytokines in PEP treated with UV-C after 3 months (T3). After 3 months of UV-C treatment, the concentrations of angiogenic-promoting cytokines ANG-1, EGF, PDGF-BB, and VEGF-A detected by ELISA (ELLA, Bio-Techne, Minneapolis, Minnesota) did not change significantly from the initial (T0) or 1 month (T1) time point. Figure 6. UV-C-treated PEP supports cell proliferation. When added to cell cultures of normal human dermal fibroblasts (NHDF), PEP supported cell proliferation more than the FBS-positive control even when treated with UV-C. This was observed immediately after treatment (T0) and one month after treatment (T1 month), as measured by percentage full growth in cell culture plates. Fig. 7. UV-C-treated PEP supports cell proliferation at the 3-month stability time point (T3). When added to a cell culture of normal human dermal fibroblasts (NHDF) 3 months after UV-C treatment, PEP supported cell proliferation more than the FBS-positive control when treated with UV-C, as measured by cell counts via full growth and nuclear staining on the cell culture plate. Fig. 8. Antioxidant capacity of PEP treated with UV-C. UV-C treatment of PEP did not have a negative effect on the antioxidant capacity of PEP immediately after treatment (T0), 1 month after treatment (T1 month), or 3 months after treatment (T3 month). Fig. 9. Enzymatic activity of PEP treated with UV-C. Effect of UV-C treatment on the enzymatic activity (alkaline phosphatase and / or catalase) of PEP when passed through UV-C radiation multiple times. The effect was limited with 1-3 passes of UV-C. Fig. 10. The reconstitution time for representative PEP repeats was evaluated by reconstituting each PEP sample in 1 mL of sterile water. L-1: 2× UV-C + E-beam, repeat #1; L-2: 2× UV-C + E-beam, repeat #2; A-1: ​​2× UV-C, repeat #1; A-2: 2× UV-C, repeat #2. Fig. 11. Turbidity measurement. Pattern bar: PEP applied to 2× UV-C ± E-beam before freeze-drying; Monochromatic bar: PEP applied to 2× UV-C ± E-beam, freeze-dried, and reconstituted; L-1: 2× UV-C + E-beam, repeat #1; L-2: 2× UV-C + E-beam, repeat #2; A-1: ​​2× UV-C, repeat #1; A-2: 2× UV-C, repeat #2. Fig. 12. Determination of average particle size. Pattern bar: PEP applied to 2× UV-C ± E-beam before freeze-drying; Monochromatic bar: PEP applied to 2× UV-C ± E-beam, freeze-dried, and reconstituted; L-1: 2× UV-C + E-beam, repeat #1; L-2: 2× UV-C + E-beam, repeat #2; A-1: ​​2× UV-C, repeat #1; A-2: 2× UV-C, repeat #2. Fig. 13. Determination of particle concentration. Pattern bar: PEP applied to 2× UV-C ± E-beam before freeze-drying; Monochromatic bar: PEP applied to 2× UV-C ± E-beam, freeze-dried, and reconstituted; L-1: 2× UV-C + E-beam, repeat #1; L-2: 2× UV-C + E-beam, repeat #2; A-1: ​​2× UV-C, repeat #1; A-2: 2× UV-C, repeat #2. Fig. 14. Angiogenic cytokines. PEP reconstituted and lyophilized after application to 2× UV-C ± E-beam; L-1: 2× UV-C + E-beam, repeat #1; L-2: 2× UV-C + E-beam, repeat #2; A-1: ​​2× UV-C, repeat #1; A-2: 2× UV-C, repeat #2. Fig. 15. Inflammatory cytokines. PEP lyophilized and reconstituted after application to 2× UV-C ± E-beam; L-1: 2× UV-C + E-beam, repeat #1; L-2: 2× UV-C + E-beam, repeat #2; A-1: ​​2× UV-C, repeat #1; A-2: 2× UV-C, repeat #2. All IFN-γ and IL-1β measurements were below the lower limit of quantification (LOQ). TNF-α was detected in the samples at acceptable levels. Fig. 16. Cell proliferation. The time to induce ≥70% full-face growth of normal human dermal fibroblasts (NHDF) was measured. L-1: 73% full-face growth after 72 hours; L-2: 73% full-face growth after 69 hours; A-1: ​​76% full-face growth after 72 hours; and A-2: 74% full-face growth after 69 hours. Fig. 17. Residual moisture content of PEP that has been freeze-dried and reconstituted after application to a 2× UV-C ± E-beam. Fig. 18. Reconstruction time of PEP samples applied to E-beam (E-beam), one pass of E-beam & UV-C treatment (E-beam + 1× UV-C), two passes of E-beam & UV-C treatment (E-beam + 2× UV-C), or three passes of E-beam & UV-C treatment (E-beam + 3× UV-C) in the absence of UV-C. Fig. 19. Alkaline phosphatase activity of PEP samples applied to an E-beam (E-beam), one pass of E-beam & UV-C treatment (E-beam + 1× UV-C), two passes of E-beam & UV-C treatment (E-beam + 2× UV-C), or three passes of E-beam & UV-C treatment (E-beam + 3× UV-C) in the absence of UV-C. Fig. 20. Antioxidant capacity of PEP samples by TEAC applied to an E-beam in the absence of UV-C (E-beam), one pass of E-beam & UV-C treatment (E-beam + 1× UV-C), two passes of E-beam & UV-C treatment (E-beam + 2× UV-C), or three passes of E-beam & UV-C treatment (E-beam + 3× UV-C). Fig. 21. Cell proliferation by frontal growth of cells treated with PEP applied to an E-beam in the absence of UV-C (E-beam), one pass of E-beam & UV-C treatment (E-beam + 1× UV-C), two passes of E-beam & UV-C treatment (E-beam + 2× UV-C), or three passes of E-beam & UV-C treatment (E-beam + 3× UV-C). Percentage of frontal growth over 96 hours of imaging after treatment with PEP samples. Fig. 22. Reconstruction time of a PEP sample applied to γ ​​irradiation of an indicated dose without UV-C treatment or with one pass of UV-C treatment (1× UV-C), two passes of UV-C treatment (2× UV-C), or three passes of UV-C treatment (3× UV-C). Fig. 23. Reconstruction time of PEP samples applied to γ ​​irradiation of indicated doses without UV-C treatment or with one pass of UV-C treatment (1× UV-C), two passes of UV-C treatment (2× UV-C), or three passes of UV-C treatment (3× UV-C) after stability testing. (A) Room temperature stability condition. (B) Accelerated stability condition. Fig. 24. Average particle size of PEP samples applied to γ ​​irradiation of indicated doses without UV-C treatment or with one pass of UV-C treatment (1× UV-C), two passes of UV-C treatment (2× UV-C), or three passes of UV-C treatment (3× UV-C). Fig. 25. Average particle size of PEP samples applied to γ ​​irradiation at indicated doses without UV-C treatment or with one pass of UV-C (1× UV-C), two passes of UV-C (2× UV-C), or three passes of UV-C (3× UV-C) after stability testing. (A) Room temperature stability condition. (B) Accelerated stability condition. Fig. 26. Catalase concentration of a PEP sample applied to γ ​​irradiation of the indicated dose without UV-C treatment or with one pass of UV-C treatment (1× UV-C), two passes of UV-C treatment (2× UV-C), or three passes of UV-C treatment (3× UV-C). Fig. 27. Catalase concentration of PEP samples applied to γ ​​irradiation at indicated doses without UV-C treatment or with one pass of UV-C (1× UV-C), two passes of UV-C (2× UV-C), or three passes of UV-C (3× UV-C) after stability testing. (A) Room temperature stability conditions. (B) Accelerated stability conditions. Fig. 28. Antioxidant capacity of PEP samples applied to indicated doses of γ irradiation without UV-C treatment or with one pass of UV-C treatment (1× UV-C), two passes of UV-C treatment (2× UV-C), or three passes of UV-C treatment (3× UV-C). Fig. 29. Antioxidant capacity of PEP samples applied to indicated doses of γ irradiation without UV-C treatment or with one pass of UV-C (1× UV-C), two passes of UV-C (2× UV-C), or three passes of UV-C (3× UV-C) after stability testing. (A) Room temperature stability conditions. (B) Accelerated stability conditions. Fig. 30. Angiogenic cytokine concentrations of PEP samples applied to γ ​​irradiation of indicated doses without UV-C treatment or with 1 pass of UV-C treatment (1× UV-C), 2 passes of UV-C treatment (2× UV-C), or 3 passes of UV-C treatment (3× UV-C). (A) ANG-1; (B) EGF. Fig. 31. Angiogenic cytokine concentrations of PEP samples applied to indicated doses of γ irradiation without UV-C treatment or with one pass of UV-C treatment (1× UV-C), two passes of UV-C treatment (2× UV-C), or three passes of UV-C treatment (3× UV-C). (A) PDGF-BB; (B) VEGF-A. Fig. 32. Proliferative capacity of cells treated with PEP samples applied to indicated doses of γ irradiation without UV-C treatment or with 1 pass of UV-C (1× UV-C), 2 passes of UV-C (2× UV-C), or 3 passes of UV-C (3× UV-C). SFM: Negative control; FBS: Positive control. Specific details for implementing the invention

[0034] The present disclosure describes a method for reducing the potency of one or more biological contaminants in a biological product, such as, but not limited to, purified exosomal products. In one or more embodiments, the biological product may be a therapeutic product suitable for administration to a subject after being applied to one or more potency-reduction methods described herein. Accordingly, although the following description is in the context of an exemplary embodiment where the biological product is a purified exosomal product, the methods and compositions described herein may involve the use of any suitable biological agent. Exemplary alternative biological agents include, but are not limited to, antibodies, cell therapies, or liposomal therapies.

[0035] Purified Exosome Products (PEP)

[0036] PEP is a purified exosome product produced using a freeze-drying step that produces a product having a structure distinct from exosomes manufactured by conventional methods. For example, PEP typically has a spherical or globular structure and an intact lipid bilayer that is not a crystalline structure, which is formed from the lipid re-aggregation of the exosome lipid bilayer after exosomes are destroyed during conventional exosome manufacturing methods. As used herein, a "spherical" structure is a shape resembling a three-dimensional sphere with flattened poles. Spherical or globular exosome structures generally have a diameter of 300 nanometers (nm) or less. Typically, PEP formulations contain spherical or globular exosome structures with a relatively narrow size distribution. An exemplary size distribution of a PEP formulation is presented in FIG. 1B. Here, the average particle size diameter was 96.9 nm ± 52.2 nm. In some formulations, PEP contains spherical or globular exosome structures with an average diameter of 110 nm ± 90 nm, and most exosome structures have an average diameter of 110 nm ± 50 nm, e.g., 110 nm ± 30 nm.

[0037] The production of purified exosome products (PEP) involves separating plasma from blood and isolating exosome solutions from the separated plasma by filtration and centrifugation. PEP has been fully characterized, and methods for manufacturing PEP have been described in International Patent Application No. PCT / US2018 / 065627 (published as International Publication No. WO 2019 / 118817), U.S. Patent Publication No. 2021 / 0169812 A1, and U.S. Patent No. 10,596,123, each of which is incorporated herein by reference in its entirety.

[0038] Morphological characteristics of PEP

[0039] In one or more embodiments, the formulation can be characterized by measuring the vesicle size of PEP exosomes. For example, the vesicle size can be measured by electron microscopy, such as transmission electron microscopy or scanning electron microscopy. Transmission electron microscopy images indicate that the PEP exosomes exhibit typical spherical vesicles with an intact lipid-bilayer structure (Fig. 1A). The vesicle size of PEP ranged from 56.4 nm to 151 nm, and the average size was 96.9 nm ± 2.8 nm, which represents the standard size range for exosomes. A 100% PEP solution is 1.9 × 10⁶ 11 It was calculated to contain 1 PEP particle / mL (Fig. 1B).

[0040] Method to reduce potency

[0041] Typically, biological products, such as PEP, are subject to strict safety requirements that must be met before they can be administered to patients. In particular, biological products prepared from cell or tissue donor samples are often subject to strict sterilization standards to ensure that the biological product does not spread disease. For example, donated blood is often sterilized before being administered to recipients. One common method for sterilizing donated blood is ionizing radiation. Additional methods for sterilizing biological products, such as blood, include thermal denaturation, acid denaturation, the use of detergents, and ultrafiltration. While many of these methods are effective for virus neutralization, the conditions are typically harsh and lead to the degradation of the biological product.

[0042] Additionally, the sterilization of complex biological products (e.g., PEP) presents a unique challenge in removing biological contaminants without destroying the active biological components. As used herein, the term “biological contaminant” refers to any biological material or entity that is undesirable to be present in the final biological product. Biological contaminants include, but are not limited to, pathogens (e.g., viruses, bacteria, fungi, etc.) and / or biological entities that may interfere with the desired biological activity of the biological product.

[0043] In one or more embodiments in which the biological contaminant comprises one or more viruses, the biological contaminant may comprise one or more enveloped viruses, one or more non-enveloped DNA viruses, one or more DNA viruses and / or one or more RNA viruses. In one or more embodiments in which the biological contaminant comprises one or more DNA viruses, the biological contaminant may comprise one or more single-stranded DNA (ssDNA) viruses and / or one or more double-stranded DNA (dsDNA) viruses. Thus, for example, the biological contaminant may comprise human immunodeficiency virus (HIV), pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), hepatitis A virus (HAV), or any combination of two or more viruses.

[0044] In one or more embodiments, the biological contaminant comprises one or more bacterial species.

[0045] In one or more embodiments, the biological contaminant includes one or more fungal species.

[0046] Sterilization methods typically do not predictably distinguish between different types of biological components. For example, heat denaturation of a sample can inactivate contaminating viruses, but it can also inactivate therapeutic enzymes.

[0047] Therefore, the development of methods to selectively inactivate contaminants without destroying the activity of desired biological components is often unpredictable and presents significant technical challenges. Various methods for reducing the potency of biological contaminants and their compatibility with PEP are described herein.

[0048] In one aspect, the present disclosure relates to a method for treating a biological product. In one or more embodiments, the treatment of the biological product comprises reducing the potency of one or more biological contaminants in a sample of the biological product.

[0049] Typically, a biological product is produced that is at least partially sterilized in a manner that reduces the potency of biological contaminants within the biological product. In one or more embodiments, the treated biological product contains a reduced level of biological contaminants. In one or more embodiments, the treated biological product exhibits the same beneficial properties as an untreated sample of the biological product. In one or more embodiments, the treated biological product exhibits improved properties in that cells treated with the treated biological product are less likely to be altered (e.g., infected) by contaminants (e.g., viruses).

[0050] In one or more embodiments, the treated biological product exhibits stability similar to that of an untreated sample of the biological product. For example, the treated biological product may have a shelf life similar to that of the untreated biological product under similar storage conditions.

[0051] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product does not include adding excipients to the biological product. For example, a method for treating a biological product may not require the addition of riboflavin, psoralen, and / or amotosalen to the biological product, or may not otherwise include them. Additionally, a method for reducing the potency of one or more biological contaminants in a biological product may not include the use of detergents, solvents, or pH-modifying reagents.

[0052] Process-based method for reducing the potency of biological contaminants

[0053] As described in more detail herein, PEP is typically prepared from blood products. The preparation of PEP typically involves centrifugation, filtration, freezing of the sample, and optional lyophilization. Each of these steps can contribute to reducing the titer of any biological contaminants in the sample by reducing the viral titer of the sample.

[0054] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises centrifuging the biological product, whether it is an exosomal product such as PEP or another biological product. In one or more of these embodiments, the method may comprise centrifuging a blood product to separate plasma from the blood. As described herein, centrifuging the blood product to separate plasma may additionally separate one or more contaminants. Additional centrifugation may further remove contaminants. In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises centrifuging the biological product.

[0055] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises removing leukocytes from a blood product, which is also referred to as leukopenia. For example, leukocytes may be removed by filtration.

[0056] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises filtering the biological product. In one or more of these embodiments, the method comprises filtering a blood product, e.g., plasma. The filter size may be selected to allow smaller substances to pass through the filter while retaining exosomes. Alternatively, the filter size may be selected to allow exosomes to pass through the filter while retaining larger contaminants. In one or more embodiments, the method comprises using a filter having a molecular weight cutoff value (MWCO) of at least 30 kilodaltons (kDa), at least 50 kDa, at least 80 kDa, or at least 100 kDa. In one or more embodiments, the method comprises using a filter having an MWCO of up to 1000 kDa, up to 800 kDa, up to 500 kDa, up to 300 kDa, up to 150 kDa, up to 100 kDa, or up to 80 kDa.

[0057] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises freezing and thawing the biological product. In one or more embodiments, the method comprises freezing and thawing a blood product. Freezing and thawing the biological product or blood product may include freezing the product at least once and thus may include multiple freeze-thaw cycles—e.g., at least two freeze-thaw cycles, at least three freeze-thaw cycles, at least four freeze-thaw cycles, etc. Typically, the biological product will be frozen and thawed the same number of times, or will be frozen one more time than the product is thawed to yield a frozen final product.

[0058] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises freezing the biological product. In one or more embodiments, freezing the biological product comprises incubating the biological product at a temperature of at least -20°C, at least -50°C, or at least -80°C. In one or more embodiments, freezing the biological product comprises incubating the biological product at a temperature of up to 0°C, up to -20°C, up to -50°C, or up to -80°C.

[0059] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises freeze-drying the biological product. Freeze-drying (sometimes referred to as freeze-drying) comprises lowering the product to a low temperature under vacuum and removing frozen water through sublimation. In one or more embodiments, freeze-drying the biological product comprises using a commercial freeze dryer. In one or more embodiments, freeze-drying the biological product comprises lowering the temperature of the biological product to a maximum of -50°C or a maximum of -60°C. In one or more embodiments, freeze-drying the biological product comprises lowering the temperature of the biological product to at least -80°C, at least -70°C, or at least -60°C.

[0060] In one or more embodiments, a method for reducing the potency of biological contaminants in a biological product may include applying electromagnetic energy and / or ionizing radiation to the biological product. As used herein, electromagnetic energy refers to radiant energy having a wavelength and frequency. Exemplary forms of electromagnetic energy include, but are not limited to, UV-B, UV-C, X-rays, and γ-rays. As used herein, ionizing radiation refers to subatomic particles or electromagnetic waves having sufficient energy to ionize atoms or molecules. Exemplary forms of ionizing radiation include, but are not limited to, X-rays, γ-rays, or electron beam radiation. Since some electromagnetic waves have sufficient energy to ionize atoms or molecules, electromagnetic energy and ionizing radiation are not mutually exclusive.

[0061] For viral inactivation, viral concentration is typically defined in plaque-forming units per mL (PFU / mL). For bacterial inactivation, bacterial concentration is typically defined in colony-forming units per mL (CFU / mL).

[0062] UV-C radiation

[0063] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises treating the biological product with ultraviolet (UV)-C radiation. UV-C radiation is typically defined as electromagnetic radiation with a wavelength of 100 nm to 280 nm. UV-C radiation typically has a photon energy of 4.43 electron-volts (eV) to 12.4 eV, or 0.710 attojoules (aJ) to 1.987 aJ.

[0064] Inactivation efficiency is measured in log death and is defined as follows:

[0065]

[0066] [During the meal, N 0 = initial concentration of biological contaminants (PFU / mL, CFU / mL, etc.),

[0067] N f = Initial concentration of biological contaminants (PFU / mL, CFU / mL, etc.)].

[0068] In one or more embodiments, the relationship between log inactivation and UV-C fluence is linear, with small deviation at low fluence values ​​where inactivation is not detected. This can be described by the following model:

[0069]

[0070] [During the meal, k = Virus inactivation factor (cm²) 2 / mJ)

[0071] F = UV-C fluence (mJ / cm²) 2 )

[0072] b = y-intercept offset].

[0073] The y-intercept offset models low fluence values ​​where inactivation is not detected. Some studies have indicated that very high fluence values ​​can show diminishing returns, where an increase in fluence does not yield an increase in inactivation. These values ​​are typically excluded from calculations to maintain the accuracy of the linear model.

[0074] In one or more embodiments, treating the sample with UV-C radiation involves treating the sample with electromagnetic radiation having a wavelength of at least 100 nm to a maximum of 280 nm. However, it should be understood that some electromagnetic radiation may include wavelengths less than 100 nm or greater than 280 nm. In one or more embodiments, treating the sample with UV-C radiation involves treating the sample with electromagnetic radiation of a certain range of wavelengths, wherein the electromagnetic radiation includes some radiation having a wavelength of at least 100 nm to a maximum of 280 nm. In one or more embodiments, the sample may be treated with electromagnetic radiation having a wavelength of at least 200 nm to a maximum of 280 nm. In one or more embodiments, the sample may be treated with electromagnetic radiation of a certain range of wavelengths, wherein the electromagnetic radiation includes some radiation having a wavelength of at least 200 nm to a maximum of 280 nm.

[0075] In one or more embodiments, the UV-C radiation source comprises a photoelectrode diode (LED). An example of a suitable UV-C radiation source is a water disinfection product of the PEARLAQUA series (AquiSense Technologies, Erlanger, Kentucky), such as the PEARLAQUA Micro. Such a device exposes the sample to UV-C radiation as the sample flows through the device. In this manner, the dose of UV-C radiation administered to the sample is inversely proportional to the flow rate. In other words, a slower flow rate typically means that the sample is exposed to a higher dose of UV-C radiation.

[0076] The sample may be treated with any suitable dose of UV-C radiation. In one or more embodiments, treating the sample with UV-C radiation comprises treating the sample at a minimum flow rate of at least 5 mL / min, at least 10 mL / min, at least 20 mL / min, at least 30 mL / min, at least 40 mL / min, at least 50 mL / min, at least 75 mL / min, at least 100 mL / min, at least 125 mL / min, at least 150 mL / min, at least 175 mL / min, at least 200 mL / min, at least 225 mL / min, at least 250 mL / min, at least 275 mL / min, or at least 300 mL / min. In one or more embodiments, treating the sample with UV-C radiation comprises treating the sample at a maximum flow rate of up to 700 mL / min, up to 650 mL / min, up to 600 mL / min, up to 575 mL / min, up to 550 mL / min, up to 525 mL / min, up to 500 mL / min, up to 475 mL / min, up to 450 mL / min, up to 425 mL / min, up to 400 mL / min, up to 375 mL / min, up to 350 mL / min, up to 325 mL / min, or up to 300 mL / min. In one or more embodiments, treating the sample with UV-C radiation comprises treating the sample with UV-C radiation at a flow rate within a range where the endpoint is defined by any of the aforementioned minimum flow rates and any maximum flow rate greater than the selected minimum flow rate. Accordingly, in some embodiments, treating the sample with UV-C radiation comprises treating the sample with UV-C radiation at flow rates such as 150 mL / min to 300 mL / min, 275 mL / min to 500 mL / min, 200 mL / min to 275 mL / min, etc. In one or more embodiments, treating the sample with UV-C radiation comprises treating the sample at a flow rate of about 250 mL / min.In one or more embodiments, treating the sample with UV-C radiation includes treating the sample at a flow rate of about 500 mL / min.

[0077] In one or more embodiments, treating the sample with UV-C radiation comprises treating the sample for at least 0.05 seconds, at least 0.1 seconds, at least 0.5 seconds, at least 0.6 seconds, at least 0.7 seconds, at least 0.8 seconds, at least 0.9 seconds, at least 1.0 seconds, at least 2.0 seconds, at least 5.0 seconds, at least 10 seconds, at least 15 seconds, at least 20 seconds, at least 30 seconds, at least 60 seconds, at least 90 seconds, at least 2 minutes, at least 3 minutes, at least 4 minutes, or at least 5 minutes. In one or more embodiments, treating the sample with UV-C radiation comprises treating the sample for up to 10 minutes, up to 9 minutes, up to 8 minutes, up to 7 minutes, up to 6 minutes, up to 5 minutes, up to 4 minutes, up to 3 minutes, up to 2 minutes, or up to 1 minute.

[0078] In one or more embodiments, treating the sample with UV-C radiation at an intensity of 1 mJ / cm² 2 Up to 250 mJ / cm² 2 It may include delivering UV-C. Accordingly, in one or more embodiments, the delivered UV-C intensity has a minimum value of at least 1 mJ / cm² 2 , 2 mJ / cm 2 , 3 mJ / cm 2 , 4 mJ / cm 2 , 5 mJ / cm 2 , 6 mJ / cm 2 , 7 mJ / cm 2 , 8 mJ / cm 2 , 9 mJ / cm 2 , 10 mJ / cm 2 , 12 mJ / cm 2 , 15 mJ / cm 2 , or 20 mJ / cm² 2 It may be. In one or more embodiments, the delivered UV-C intensity has a maximum value of 250 mJ / cm².2 Below, 200 mJ / cm² 2 Below, 150 mJ / cm² 2 Below, 100 mJ / cm² 2 Below, 90 mJ / cm² 2 Below, 85 mJ / cm² 2 Below, 80 mJ / cm² 2 Below, 75 mJ / cm² 2 Below, 70 mJ / cm² 2 Below, 65 mJ / cm² 2 Below, 60 mJ / cm² 2 Below, 5 mJ / cm² 2 Below, 50 mJ / cm² 2 Below, 45 mJ / cm² 2 Below, 40 mJ / cm² 2 Below, 35 mJ / cm² 2 Less than, or 30 mJ / cm² 2 It may be less than or equal to. In one or more embodiments, the UV-C intensity may fall within a range where the endpoint is defined by any of the aforementioned minimum intensities and any of the aforementioned maximum intensities greater than the selected minimum intensity. Thus, for example, in one or more embodiments, the method has an intensity of 1 mJ / cm² 2 Up to 150 mJ / cm² 2 , 10 mJ / cm 2 Up to 250 mJ / cm² 2 , 5 mJ / cm 2 Up to 150 mJ / cm² 2 , 5 mJ / cm 2 Up to 100 mJ / cm² 2 , 5 mJ / cm 2 Up to 300 mJ / cm 2 , 10 mJ / cm 2 Up to 30 mJ / cm² 2 , 10 mJ / cm 2 Up to 80 mJ / cm² 2 , 10 mJ / cm 2 Up to 60 mJ / cm² 2 , 10 mJ / cm 2 Up to 50 mJ / cm² 2 , 20 mJ / cm2 Up to 30 mJ / cm² 2 , 20 mJ / cm 2 Up to 60 mJ / cm² 2 , 20 mJ / cm 2 Up to 75 mJ / cm² 2 It may include delivering UV-C.

[0079] In one or more embodiments, treating a sample with UV-C radiation involves treating the sample with radiation of a predetermined fluence. As used herein, fluence refers to the time-integrated flux of the radiation flow.

[0080] UV-B radiation

[0081] Another method to reduce the titer of one or more biological contaminants in biological samples involves treatment with UV-B radiation. UV-B radiation typically comprises electromagnetic radiation with wavelengths ranging from 280 nm to 315 nm. In particular, unlike UV-C radiation, UV-B radiation is not ionizing radiation.

[0082] Typically, using UV-B radiation to reduce the potency of biological contaminants in a biological sample requires adding one or more excipients to the sample and subsequently exposing the sample to UV-B radiation. In one or more embodiments, riboflavin is added to the sample prior to UV-B radiation treatment. However, in some cases, adding an excipient may not be desirable. Therefore, if an excipient is used, the concentration of the excipient may be reduced to make the process more suitable for use in treating biological products. For example, in one or more embodiments, treating the sample with UV-B radiation may involve reducing the riboflavin concentration to a final concentration of less than 5% (volume / volume) in the total solution.

[0083] Electron beam (E-beam) radiation

[0084] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises treating the biological product with electron beam (E-beam) radiation. E-beam radiation is a beam of concentrated and highly charged electrons generated by an accelerator capable of producing continuous or pulsed beams. This form of ionization energy has low penetrating power and a high dose rate. When a material passes through the E-beam, energy from the electrons is absorbed, breaking chemical bonds, which leads to DNA and protein damage and renders microorganisms unable to replicate.

[0085] In one or more embodiments, the method comprises treating the biological product with electron beam radiation of at least 10 kilogray (kGy), at least 15 kGy, at least 20 kGy, or at least 25 kGy. In one or more embodiments, the method comprises treating the biological product with electron beam radiation of up to 60 kGy, up to 55 kGy, up to 50 kGy, up to 45 kGy, up to 40 kGy, up to 35 kGy, up to 30 kGy, or up to 25 kGy.

[0086] Electron beam radiation can be applied to frozen or liquid formulations. Accordingly, in one or more embodiments, the sample can be frozen while exposed to electron beam radiation.

[0087] As described herein, treating a biological product with electron beam radiation alone may not be sufficient to reduce the potency of biological contaminants to a desired degree. Accordingly, in one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises, in addition to treating the biological product with electron beam radiation, a secondary method for reducing the potency of biological contaminants in a biological product, e.g., UV-C radiation.

[0088] Gamma irradiation

[0089] Gamma irradiation utilizes the radioactive beta decay of Cobalt-60 to generate a gamma field of ionizing radiation. Gamma radiation has a high level of penetrating power and can treat materials inside transport containers, thereby minimizing material handling. Because this depends on the decay rate of Cobalt-60, the gamma dose is time-dependent, can be prolonged, and can potentially generate heat. Therefore, when gamma radiation is used to reduce the potency of biological contaminants within complex bio-preparations (e.g., exosomes), considerations include exposure time, dose, and the temperature of the material. In one or more embodiments, a gamma irradiation dose of < 50 kGy is sufficient to eliminate viruses from the biological product while maintaining product efficacy. Specifically, in an exemplary embodiment, a total final dose of 25 kGy is used to reduce the potency of biological contaminants while maintaining the efficacy of the exosomal preparation.

[0090] Method combination

[0091] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises applying the biological product to the method for reducing the potency of biological contaminants more than once. In one or more of these embodiments, the biological product may be applied to be treated two or more times by the same method to reduce the potency of biological contaminants. In such cases, a specific parameter used in one treatment using a given method may be the same or different from the parameter used in another number of treatments using the same method.

[0092] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product comprises applying the biological product to more than one method for reducing the potency of biological contaminants. For example, a sample may be treated with UV-C radiation and electron beam radiation. Typically, when a sample is treated with more than one treatment method, each method is applied sequentially rather than concomitantly. However, where feasible, a sample may be treated concomitantly with more than one treatment method.

[0093] Additionally, when a combination of treatment methods is used, a desired degree of potency reduction can be achieved by using lower intensity radiation for each method, which may minimize or even eliminate damage to the biological product. Exemplary combinations of treatment methods include, but are not limited to, UV-B + UV-C, UV-B + E-beam, UV-B + γ-ray, UV-B + X-ray, UV-C + E-beam, UV-C + γ-ray, UV-C + X-ray, γ-ray + E-beam, γ-ray + X-ray, E-beam + X-ray, etc.

[0094] Virus titer calculation

[0095] Viral titers can be calculated using the Spearman-Kaerber method known in the relevant technical field. Briefly, the Spearman-Kaerber method is the negative common logarithm (TCID) for each test volume that causes infection in 50% of the analyzed cultures according to the following formula. 50 Calculate )

[0096]

[0097] [In the formula, m is the common logarithm of the viral titer (logTCID 50 Same as / mL, x 0 is equal to the common logarithm of the lowest dose of dilution factor that induces viral CPE in all parallel wells, andd is the common logarithm of the serial dilution factor (log 10 Same as 3), and p i is capacity i Equivalent to the ratio of CPE positive wells in ( x Summed from 0), v w is the common logarithm of the volume conversion factor (mL, log for endpoint titration). 10 0.2, in the case of LVP log 10 [It is the same as 0.3]. This formula can be used to calculate the titer using methods A, B, and C.

[0098] Method A is applied to the endpoint titration where all tested parallel cultures are virus-positive at least at the highest dose (lowest dilution factor), and higher tested dilutions show a decrease in the infection rate.

[0099] Method B is applied to the endpoint titration where the highest dose is not virus-positive in all tested parallel cultures, and a higher tested dilution shows a reduction in the infection rate. At a dose three times higher, it is assumed that all parallel cultures are virus-positive. The titer is calculated according to Method A, and a hypothetical dose at a concentration three times higher than the highest tested dose is added.

[0100] Method C is used for LVP analysis when the proportion of virus-positive wells is 15% to <50%. The procedure is the same as in Mode B, but the infection rate is evaluated for only a single dilution.

[0101] For methods A, B, and C, the standard error (s) is calculated using the following formula. e Can calculate )

[0102]

[0103] [During the meal, d f is the common logarithm of the serial dilution factor (log 10Same as 3), and p i is dilution i It is the same as the ratio of CPE positive wells in, n i is dilution i [It is the same as the number of parallel wells tested in].

[0104] Additionally or alternatively, viral titers can be calculated using a Taylor series. Titers can be calculated for LVP analyses where a small number (<15%) of positive wells are detected. Titers are TCID 50 It is assumed to be in units of / mL. The following formula can be used:

[0105]

[0106] [In the formula, m is the common logarithm of the viral titer (logTCID 50 Same as / mL, D is the same as the sample dilution factor, and V w is equal to the inoculation volume per well, p w is equal to the ratio of CPE positive wells].

[0107] If CPE is not detected in any well, the titer is reported to be below the Limit of Detection (LOD). The LOD is calculated from a Poisson distribution based on the probability of false negative results and is reported as the upper 95% confidence limit. There are two methods for calculating the Poisson distribution. The titer is TCID 50 It is assumed to be in units of / mL.

[0108] First, the standard method for calculating LOD is typically used when a significant amount of process fraction volume is analyzed. This method uses the following formula:

[0109]

[0110] [In the formula, m is the common logarithm of the viral titer (logTCID 50Same as / mL, p is equal to the probability of a false negative result (typically 0.05), and V is equal to the process fraction volume (mL), and v is the same as the tested volume (mL).

[0111] When the sampling volume is less than 1% of the total fraction volume, an alternative method may be used. This method uses the following formula:

[0112]

[0113] [During the meal, m is the commercial log of virus titers (logTCID 50 Same as / mL, p is equal to the probability of a false negative result (typically 0.05), and v is the same as the tested volume (mL).

[0114] For a single fraction, the total viral load can be calculated using the following formula:

[0115]

[0116] [During the meal, A i is fraction i Identical to the commercial log of the total virus load for, m i is the commercial log of virus titers (TCID 50 It is the same as / mL or VE / mL), and V i is fraction i [It is equal to the total volume (mL) of].

[0117] For combined fraction pools, the total virus load can be calculated using the following formula:

[0118]

[0119] [During the meal, A T is equal to the common log of the total viral load for the combined fraction, and A i is fraction i [Identical to the commercial log of the total virus load for].

[0120] Virus titer reduction calculation

[0121] The log decrease value (R) of the virus titer can be calculated using the following formula:

[0122]

[0123] [During the meal, R is equal to the log diminished value, and A 0 is equal to the common logarithm of the total viral load for the starting material, and A f [is equal to the common logarithm of the total viral load on the final material]. Typically, the starting material is the material before processing. A known amount of virus may be "spiked" into the starting material. Typically, the final material is the processed material (e.g., chromatographic eluent, viral filtrate, etc.).

[0124] In one or more embodiments, a method for reducing the titer of one or more biological contaminants in a biological product produces a log reduction in viral titer of at least 1, at least 2, at least 3, at least 4, at least 5, at least 6, at least 7, at least 8, at least 9, or at least 10 times in the treated product compared to the untreated product. In one or more embodiments, a method for reducing the titer of one or more biological contaminants in a biological product produces a log reduction in viral titer of up to 15, up to 14, up to 13, up to 12, up to 11, up to 10, or up to 9 times. In one or more embodiments, a method for reducing the titer of one or more biological contaminants in a biological product produces a reduction in viral titer of 6 to 12 times.

[0125] The desired level of reduction typically varies from virus to virus. Therefore, a person skilled in the art should understand that the multiple reduction of viral titers may differ depending on the virus.

[0126] In one or more embodiments, a method for reducing the titer of one or more biological contaminants in a biological product results in at least a 10-fold log reduction in human immunodeficiency virus (HIV) titer. In one or more embodiments, a method for reducing the titer of one or more biological contaminants in a biological product results in at least a 10-fold log reduction in pseudorabies virus (PRV) titer. In one or more embodiments, a 10-fold log reduction in bovine viral diarrhea virus (BVDV) titer.

[0127] In one or more embodiments, a method for reducing the titer of one or more biological contaminants in a biological product results in a log reduction of at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold porcine parvovirus (PPV) titer. In one or more embodiments, a method for reducing the titer of one or more biological contaminants in a biological product results in a log reduction of at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold hepatitis A virus (HAV) titer.

[0128] Typically, a method for reducing the titer of one or more biological contaminants in a biological product results in a log reduction of multiple viral titers. In one or more embodiments, a method for reducing the titer of one or more biological contaminants in a biological product results in a reduction of HIV titer, PRV titer, BVDV titer, PPV titer, HAV titer, or a combination thereof.

[0129] Turbidity

[0130] To determine turbidity, liquid samples of exosomes treated with 1–5 passes of UV-C radiation were pipetted into triple wells of a 96-well clear-bottom plate. For T0 and T1, 50 μL of the sample was pipetted into the plate at a rate of 50 μL per well. For T3, 100 μL of the sample was pipetted per well. Absorbance was measured at 600 nm using a Barioscan Lux plate reader. The method did not significantly alter the turbidity of the samples (Fig. 2). Similar results were obtained for samples subjected to a combination of UV-C and E-beam radiation (Fig. 11).

[0131] Accordingly, in one or more embodiments, the method of reducing the potency of one or more biological contaminants in the biological product does not significantly change the turbidity of the biological product. In one or more embodiments, the method of treating the biological product may not increase the turbidity beyond a predetermined threshold. In one or more embodiments, the method of treating the biological product may not decrease the turbidity beyond a predetermined threshold.

[0132] Size and concentration

[0133] To measure the size of the treated exosomes, 100 nm polystyrene standard beads were diluted 1:1000. The focus was adjusted to ensure sharp edges of the beads while minimizing overexposure. This focus was maintained throughout the analysis of the remaining samples. The test samples were diluted 1:1000 in particle-free water before running on NANOSIGHT. Three 30-second videos of the particles within the flow cell were recorded. The syringe pump injection rate was set to 50. The analysis parameters were automatically set in the NTA software, and the results were exported. The concentration of the treated biological product was determined by analyzing the videos of the particles within the flow cell. The size and concentration of exosomes within the samples did not change significantly.

[0134] Similar results were obtained when the sample was applied to a combination of UV-C radiation and E-beam radiation (Fig. 12; Fig. 13). Additionally, similar results were obtained when the sample was treated with a combination of UV-C radiation and γ radiation coupled with varying degrees before (Fig. 24) and after (Fig. 25) the stability test.

[0135] Accordingly, in one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product does not significantly change the size of the biological product. In particular, a method for treating the biological product may not significantly reduce the particle size.

[0136] In one or more embodiments, a method for reducing the potency of one or more biological contaminants in a biological product does not significantly change the concentration of the biological product. In particular, a method for treating the biological product may not significantly reduce the particle concentration.

[0137] angiogenic cytokines

[0138] Samples of exosome products treated to reduce the titer of biological contaminants were analyzed for changes in the presence of angiogenic cytokines. PEP samples were lysed and analyzed using an automated ELISA system (ELLA, Bio-Techne, Minneapolis, Minnesota). Treatment to reduce the titer of biological contaminants did not cause significant changes in the presence of angiopoietin-1 (ANG1), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB), or vascular epidermal growth factor-A (VEGF-A) (Figs. 3-5).

[0139] Similar results were obtained when the sample was treated with a combination of UV-C radiation and E-beam radiation (Fig. 14). Additionally, similar results were obtained when the sample was treated with a combination of UV-C radiation and γ radiation coupled with varying degrees (Fig. 30; Fig. 31).

[0140] Accordingly, in one or more embodiments, a method of reducing the potency of one or more biological contaminants in a biological product does not significantly change the presence of angiogenic cytokines in a biological product. In particular, a method of treating a biological product may not significantly reduce the presence of angiogenic cytokines. Examples of angiogenic cytokines include angiopoietin-1 (ANG1), epidermal growth factor (EGF), platelet-derived growth factor-BB (PDGF-BB), and vascular epidermal growth factor-A (VEGF-A).

[0141] Cell proliferation

[0142] To determine the effect of treatments reducing biological contaminant titers on exosome-induced cell proliferation, UV-C-treated exosomes were added to cultured fibroblasts. Normal human dermal fibroblasts (NHDF) were seeded into 96-well plates at 3,200 cells / well and allowed to attach overnight. Each PEP test sample was resuspended in 1 mL of H2O, passed through a 0.22 μm syringe filter, and then diluted to 10% with serum-free medium + heparin (final concentration of 1 U / mL).

[0143] For T0 and T1 month samples, 200 μL of each diluted sample was added to a 96-well plate the day after seeding. Cells were incubated in an incubator for at least 96 hours, and scans were acquired every 3 hours using a phase-contrast microscope at 10× magnification. The percentage of overall growth was normalized to the treatment time using subtraction.

[0144] For T3-month samples, cells were incubated with 100 μL of Nucleite (nuclear staining) for 45 minutes, 1 day after plating. Subsequently, 100 μL of each diluted PEP sample was added to a 96-well plate to ultimately produce a 4000-fold Nucleite dilution in 10% PEP. Cells were incubated in the incubator for 96+ hours, and scans were set to be acquired every 3 hours using a 10× magnification phase-contrast microscope (for full growth analysis, as in T0 and T1 months) and a 10× magnification red fluorescence channel (excitation 567–607 nm for Nucleite red staining; emission: 622–704 nm) (for cell count analysis). The full growth percentage and cell count were normalized to the percentage at the time of treatment using subtraction. Figures 6 and 7 show that reducing the potency of biological contaminants did not significantly affect exosome-induced cell proliferation.

[0145] Similar results were obtained when the samples were treated with a combination of UV-C radiation and E-beam radiation (Fig. 16; Fig. 21). Additionally, similar results were obtained when the samples were treated with a combination of UV-C radiation and γ radiation coupled with varying degrees (Fig. 32).

[0146] In one or more embodiments, cell proliferation was measured using nuclear staining, such as nucleites. Nuclear staining can be used to label individual cells, and the total number of cells at any point during growth can be determined by counting the individual cells. In one or more embodiments, cell proliferation can be measured using a microscope to measure the total area covered by the cultured cells. An increase in the area covered by the cells can be interpreted as cell growth.

[0147] Accordingly, in one or more embodiments, the method of reducing the potency of one or more biological contaminants in the biological product does not significantly alter the effect of the biological product on cell proliferation. In particular, the method may not significantly reduce the effect of the biological product on cell proliferation. In other words, the untreated PEP and the treated PEP biological products may have the same effect on cell proliferation.

[0148] Antioxidant capacity

[0149] The antioxidant capacity of samples of exosome products treated to reduce the titer of biological contaminants was tested using the CELL BIOLABS Trolox Equivalent Antioxidant Capacity (TEAC) assay kit. The TEAC assay measures the total antioxidant capacity of a sample by introducing oxidized ABTS*+ radicals. Hydrogen-donating antioxidants in the sample reduce radicals to neutral ABTS in a concentration-dependent manner. Blue ABTS*+ radicals (read at 410 nm) become colorless when converted to their neutral form. Briefly, the PEP test samples were diluted 1:20 in assay buffer. Trolox standards were prepared from the stock solution. Each standard and sample were triple-plated in 96-well clear-bottom plates. ABTS*+ reagents were added to each well, thoroughly mixed, and incubated in an orbital shaker for 5 minutes. The plates were read at 410 nm using a plate reader (Varioscan Lux, Thermo Fisher Scientific Inc., Waltham, Massachusetts), and the μM Trolox equivalent antioxidant capacity for each PEP sample was calculated based on the Trolox standard curve. Figure 8 indicates that treatment to reduce the titer of biological contaminants did not significantly affect the antioxidant capacity of exosomes.

[0150] Similar results were obtained when the sample was applied to a combination of E-beam radiation and various degrees of UV-C radiation (Fig. 20). Additionally, similar results were obtained when the sample was treated with a combination of various degrees of UV-C radiation and coupled γ radiation before (Fig. 28) and after (Fig. 29) the stability test.

[0151] Accordingly, in one or more embodiments, the method of reducing the potency of one or more biological contaminants in the biological product does not significantly change the antioxidant capacity of the biological product. In particular, the method may not reduce the antioxidant capacity of the biological product.

[0152] Enzyme activity (catalase and alkaline phosphatase)

[0153] In accordance with the manufacturer's instructions, the enzymatic activity of each PEP test sample was tested using the ABNOVA Fluorescent Alkaline Phosphatase Kit. This assay measures the alkaline phosphatase enzymatic activity of the sample by the hydrolysis of 4-methylumbelliferone. In the presence of alkaline phosphatase enzymes, 4-methylumbelliferone is converted to 4-methylumbelliferone (which is highly fluorescent) and phosphate. The rate at which alkaline phosphatase hydrolyzes 4-methylumbelliferone (the rate of fluorescence increase) is directly proportional to the enzymatic activity. Briefly, after diluting the PEP test samples 1:2 in water, 10 μL per well was plated in triples on a 96-well plate. 90 μL of the provided reagent was added to each well. After incubating the samples for 1 minute, they were read on a plate reader at a rate of once per minute for at least 16 minutes with an excitation wavelength of 360 nm and an emission wavelength of 450 nm. Using the average rate (time-dependent fluorescence gradient) of each sample, the sample-to-sample difference in alkaline phosphatase enzyme activity was determined.

[0154] The catalase enzyme activation kit can be run using the Invitrogen Catalase Colorimetric Activation Kit. Bovine catalase standards are provided to generate a standard curve for the assay. The sample was diluted 1:10 in the provided assay buffer and added to the wells of a half-area clear plate. Hydrogen peroxide was added to each well, and the plate was incubated at room temperature for 30 minutes. After adding the supplied colorimetric detection reagent, diluted horseradish peroxidase (HRP) was added, and the plate was incubated at room temperature for 15 minutes. HRP reacts with the substrate in the presence of hydrogen peroxide, converting the colorless substrate into a pink product. The colored product was read at 560 nm using a plate reader (Varioscan Lux, Thermo Fisher Scientific, Inc., Waltham, Massachusetts). An increase in catalase levels in the sample leads to a decrease in hydrogen peroxide concentration and a decrease in the pink product. The results are presented in Figure 9.

[0155] Similar results were obtained when the sample was applied to a combination of E-beam radiation and various degrees of UV-C radiation (Fig. 19). Additionally, similar results were obtained when the sample was treated with a combination of various degrees of UV-C radiation and coupled γ radiation before (Fig. 26) and after (Fig. 27) the stability test.

[0156] Accordingly, in one or more embodiments, the method for reducing the potency of one or more biological contaminants in a biological product does not change the enzymatic activity of the biological product beyond a predetermined level. In particular, the method does not reduce the enzymatic activity of the biological product beyond a predetermined threshold value. When used in this context, "enzymatic activity" may refer to the individual activity of a single enzyme in the biological product or the combined activity of two or more enzymes. In one or more embodiments, "enzymatic activity" refers to the activity of catalase and / or alkaline phosphatase. Although exemplified in the context of exemplary embodiments in which alkaline phosphatase and / or catalase were measured as surrogates for enzymes contained within exosomes, any suitable enzyme or enzyme of particular interest may be used to evaluate the effect on enzymatic activity of treating a sample to reduce the potency of biological contaminants.

[0157] Reconstruction time

[0158] Figure 10 provides data indicating the time required to reconstitute a freeze-dried sample in sterile water. Similar results were obtained when the sample was subjected to a combination of E-beam radiation and various degrees of UV-C radiation (Figure 18). Additionally, similar results were obtained when the sample was treated with a combination of various degrees of UV-C radiation and coupled γ radiation before (Figure 22) and after (Figure 23) the stability test.

[0159] Therefore, various treatments intended to reduce the potency of biological contaminants did not significantly alter the reconstructibility of the samples to which the treatment was applied.

[0160] Composition and kit containing biological products

[0161] In another aspect, the present disclosure relates to a composition comprising a biological product treated to reduce the potency of a biological contaminant. In one or more embodiments, the composition is a pharmaceutically acceptable composition, which means that the composition is suitable for administration to a subject, e.g., a mammal.

[0162] A treated biological product may be formulated with a pharmaceutically acceptable carrier to form a pharmaceutical composition. As used herein, "carrier" includes any solvent, dispersion medium, vehicle, coating agent, diluent, antimicrobial and / or antifungal agent, isotonic agent, absorption retardant, buffer, hydrogel, carrier solution, suspension, colloid, water, etc. The use of such media and / or agents with pharmaceutical active substances is widely known in the art. It is conceived to use any conventional media or agent in a therapeutic composition unless it is incompatible with the treated biological product. A co-active ingredient may also be incorporated into the composition. As used herein, "pharmaceutically acceptable" refers to a substance that is biologically or otherwise undesirable, that is, the substance may be administered to an individual with the treated biological product without causing any undesirable biological effects or without interacting in a harmful manner with any other component of the pharmaceutical composition containing it. As mentioned above, in a surgical setting, exemplary suitable carriers include surgical adhesives, tissue adhesives, or support matrices (e.g., collagen scaffolds). As used herein, "collagen scaffold" refers to a three-dimensional network comprising collagen, such as a hydrogel.

[0163] In one or more embodiments, the support matrix comprises at least one extracellular matrix component. Suitable extracellular matrix components include, but are not limited to, proteins such as collagen, elastin, fibronectin, or laminin, proteoglycans, and hyaluronic acid. In embodiments where the composition comprises collagen, the collagen may be provided as procollagen, fibrilated collagen, such as type I collagen, type III collagen, or a combination thereof. In embodiments where the composition comprises collagen, the collagen may be provided as a collagen scaffold. In one or more other embodiments, the extracellular matrix component may be supplied in any suitable form, such as purified recombinant protein.

[0164] Pharmaceutical compositions comprising the treated biological product may be formulated in various forms suitable for the preferred route of administration. Accordingly, the pharmaceutical composition may be administered via known routes, including, for example, oral, parenteral (e.g., intradermal, transdermal, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., application to exposed tendon tissue during surgery, intranasal, intrapulmonary, intramammary, vaginal, intrauterine, intradermal, transdermal, rectal, etc.). The pharmaceutical composition may be administered to mucosal surfaces, such as by administration to the nasal or respiratory mucosa (e.g., by spray or aerosol). The pharmaceutical composition may also be administered via sustained or delayed release.

[0165] Accordingly, the pharmaceutical composition may be provided in any suitable form, including but not limited to a solution, suspension, emulsion, spray, aerosol, or any form of mixture. The pharmaceutical composition may be delivered in a formulation containing any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form, e.g., a cream, ointment, aerosol formulation, non-aerosol spray, gel, lotion, etc. In an embodiment where the formulation is a gel, the gel may have any suitable density. For example, in a specific embodiment for a surgical setting, the pharmaceutical composition may be formulated as a gel having a density sufficient to hold the formulation in a desired location. The formulation may further include one or more additives, which include, for example, an ajuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring agent, a humectant, a thickener, etc.

[0166] Suitable excipients may include, for example, human or bovine collagen, hyaluronic acid-based compounds, human fibrinogen, or human thrombin.

[0167] In one or more embodiments, the composition described herein may be freeze-dried. A freeze-dried composition comprising a treated biological product may be combined with additional excipients, which may be further freeze-dried. The components of the freeze-dried composition may be co-packaged or provided separately and mixed before use to produce a biocompatible scaffold loaded with exosomes. The freeze-dried excipient may be, for example, freeze-dried human or bovine collagen, a hyaluronic acid-based compound, human fibrinogen, human thrombin, or other freeze-dried powders that form a biocompatible gel upon contact with body fluids (e.g., blood or interstitial fluid).

[0168] In one or more embodiments, the composition described herein may be reconstituted from a freeze-dried powder. For example, the freeze-dried powder may be reconstituted by adding the dry powder to a liquid, typically water or a pharmaceutically acceptable buffer. In one or more embodiments, the composition reconstituted from the freeze-dried powder may be frozen. The frozen composition may be prepared by first mixing the freeze-dried powder with a liquid, such as water or a pharmaceutically acceptable buffer, and then raising the mixture to a temperature below the freezing point of the mixture.

[0169] Method of administering biological products

[0170] In another aspect, the present disclosure relates to a method of administering a treated biological product to a subject to reduce the potency of a biological contaminant. In one or more embodiments, the method comprises administering the treated biological product to a subject who requires it. The subject may have a condition for which administration of the treated biological product may be beneficial, or may be at risk of having such a condition.

[0171] As used herein, "object" may be a human or any non-human animal. Examples of non-human animal objects include, but are not limited to, domestic animals or companion animals. Exemplary non-human animal subjects include, but are not limited to, animals of the Hominidae family (e.g., chimpanzees, gorillas, or orangutans), Bovidae (e.g., cattle), Capridae (e.g., goats), Shepherdae (e.g., sheep), Pswigae (e.g., pigs), Equidae (e.g., horses), members of the Cervidae family (e.g., deer, elk, moose, caribou, reindeer, etc.), members of the Bisonae family (e.g., bison), Felidae (e.g., domestic cats, tigers, lions, etc.), Canidae (e.g., domestic dogs, wolves, etc.), birds (e.g., turkeys, chickens, ducks, geese, etc.), rodents (e.g., mice, rats, etc.), members of the Leporidae family (e.g., rabbits or hares), members of the Mustelidae family (e.g., ferrets), or members of the Chiroptera order (e.g., bats).

[0172] In one or more embodiments, the composition described herein is administered by injection into or over the surgical site. The composition as described herein may be administered alone or in addition to traditional surgical repair methods, such as sutures or staples. The composition as described herein may also be used to enhance the biocompatibility and therapeutic effects of tendon sutures, anchors, patches, or other devices used to repair tendon damage.

[0173] The formulation can be conveniently presented in a unit dosage form and can be manufactured by methods widely known in the pharmaceutical industry. A method for manufacturing a composition having a pharmaceutically acceptable carrier comprises the step of associating a treated biological product with a carrier constituting one or more auxiliary components. Generally, the formulation can be manufactured by uniformly and / or closely associating the treated biological product with a liquid carrier, a finely ground solid carrier, or both, and then, if necessary, molding the product into a desired formulation.

[0174] The dosage of a treated biological product may vary depending on various factors, including but not limited to the content and / or source of the administered treated biological product, the subject's body weight, physical condition and / or age, and / or route of administration. Accordingly, the absolute weight of the treated biological product contained in a given unit dosage form may vary widely and depends on factors such as the subject's species, age, body weight and physical condition, and / or method of administration. Therefore, it is not practical to generally state the amount constituting the quantity of the biological product effective for all possible uses. However, a person skilled in the art can easily determine an appropriate amount by taking these factors into full consideration.

[0175] In one or more embodiments, the biological product may be a purified exosome product (PEP). In one or more embodiments, the dose of PEP may be measured in terms of the number of PEP exosomes delivered in a dose. Thus, in one or more embodiments, the method provides a subject, for example, 1 × 10⁶ 6 1 to 1×10 PEP exosomes 15 The method may involve administering sufficient PEP to provide a dose of PEP exosomes, but in one or more embodiments, the method may be performed by administering PEP outside this range.

[0176] Accordingly, in one or more embodiments, the method is at least 1×10 6 10 PEP exosomes, at least 1×10 7 10 PEP exosomes, at least 1×10 8 10 PEP exosomes, at least 1×10 9 10 PEP exosomes, at least 1×10 10 10 PEP exosomes, at least 1×10 11 10 PEP exosomes, at least 2×10 11 10 PEP exosomes, at least 3×10 11 10 PEP exosomes, at least 4×10 11 10 PEP exosomes, at least 5×10 11 10 PEP exosomes, at least 6×10 11 10 PEP exosomes, at least 7×10 11 10 PEP exosomes, at least 8×10 11 10 PEP exosomes, at least 9×10 11 10 PEP exosomes, at least 1×10 12 1 PEP exosome, 2×10 12 10 PEP exosomes, at least 3×10 12 10 PEP exosomes, at least 4×10 12 10 PEP exosomes, or at least 5×10 12 10 PEP exosomes, at least 1×10 13 10 PEP exosomes, or at least 1×10 14 It may include administering sufficient PEP to provide a minimum dose of PEP exosomes.

[0177] In one or more embodiments, the method is 1×10 15 Number of PEP exosomes or fewer, 1×10 14 Number of PEP exosomes or fewer, 1×10 13 Number of PEP exosomes or fewer, 1×10 12 Number of PEP exosomes or fewer, 1×10 11 Number of PEP exosomes or fewer, or 1×10 10It may include administering sufficient PEP to provide a maximum dose of PEP exosomes of 1 or fewer.

[0178] In one or more embodiments, the method may comprise administering sufficient PEP to provide a dose characterized by a range in which the endpoint is defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in one or more embodiments, the method is 1×10 11 Up to 1×10 13 The dose of PEP exosomes, e.g., 1 × 10⁶ 11 Up to 5×10 12 Dosage of PEP exosomes, 1×10 12 Up to 1×10 13 A dose of PEP exosomes, or 5×10 12 Up to 1×10 13 The method may include administering sufficient PEP to provide a dose of PEP exosomes. In certain embodiments, the method may include administering sufficient PEP to provide a dose equal to any minimum dose or any maximum dose listed above. Thus, for example, the method may include 1 × 10⁶ 10 1 x 10 PEP exosomes, 1 × 10 11 1 PEP exosomes, 5×10 11 1 x 10 PEP exosomes, 1 × 10 12 1 PEP exosomes, 5×10 12 1 x 10 PEP exosomes, 1 × 10 13 1 x PEP exosomes, or 1 × 10⁶ 14 It may involve administering a dose of PEP exosomes.

[0179] Alternatively, the volume of the exosome product may be measured in terms of the PEP concentration when reconstituted from the lyophilized state. Accordingly, in one or more embodiments, the method may comprise administering PEP to a subject in a volume, for example, of a 0.01% solution to a 100% solution, but in one or more embodiments, the method may be performed by administering PEP in a volume outside these ranges. As used herein, a 100% solution of PEP is one vial of PEP (2×10⁻⁶) dissolved in 1 ml of a liquid or gel carrier (e.g., water, phosphate-buffered chloride, serum-free culture medium, surgical adhesive, tissue adhesive, etc.). 11 It refers to 75 mg of exosomes. For comparison, the dose of 0.01% PEP is approximately equivalent to the standard dose of exosomes prepared using conventional methods of obtaining exosomes, such as isolating exosomes from cells in vitro using standard cell conditioning medium.

[0180] Accordingly, in one or more embodiments, the method may comprise administering sufficient PEP to provide a minimum dose of at least 0.01%, at least 0.05%, at least 0.1%, at least 0.25%, at least 0.5%, at least 1.0%, at least 2.0%, at least 3.0%, at least 4.0%, at least 5.0%, at least 6.0%, at least 7.0%, at least 8.0%, at least 9.0%, at least 10%, at least 15%, at least 20%, at least 25%, at least 30%, at least 35%, at least 40%, at least 45%, at least 50%, at least 60%, or at least 70%.

[0181] In one or more embodiments, the method may comprise administering sufficient PEP to provide a maximum dose of 100% or less, 90% or less, 80% or less, 70% or less, 60% or less, 50% or less, 40% or less, 30% or less, 20% or less, 10% or less, 9.0% or less, 8.0% or less, 7.0% or less, 6.0% or less, 5.0% or less, 4.0% or less, 3.0% or less, 2.0% or less, 1.0% or less, 0.9% or less, 0.8% or less, 0.7% or less, 0.6% or less, 0.5% or less, 0.4% or less, 0.3% or less, 0.2%, or 0.1% or less.

[0182] In one or more embodiments, the method may include administering sufficient PEP to provide a dose characterized by a range in which the endpoint is defined by any minimum dose identified above and any maximum dose greater than the minimum dose. For example, in one or more embodiments, the method may include administering sufficient exosome product to provide a dose of 1% to 50%, e.g., 5% to 20%. In certain embodiments, the method may include administering sufficient PEP to provide a dose equal to any minimum dose or any maximum dose listed above. Thus, for example, the method may involve administering a dose of 0.05%, 0.25%, 1.0%, 2.0%, 5.0%, 20%, 25%, 50%, 80%, or 100%.

[0183] A single dose may be administered at once, continuously over a prescribed period, or as multiple individual doses. If multiple doses are used, each dose may be the same or different. For example, a prescribed daily dose may be administered as a single dose, continuously over 24 hours, or as two doses that may or may not be the same. If multiple doses are used to deliver a single dose, the intervals between doses may be the same or different. In one or more specific embodiments, the treated biological product may be administered in a single dose, for example, during a surgical procedure.

[0184] In one or more specific embodiments in which multiple doses of the treated biological product composition are administered to a subject, the treated biological product composition may be administered as needed to heal and / or repair the tendon-bone junction to a desired degree. Alternatively, the treated biological product composition may be administered 2, 3, 4, 5, 6, 7, 8, 9, or at least 10 times. The interval between administrations may be at least 1 day, e.g., at least 3 days, at least 5 days, at least 7 days, at least 10 days, at least 14 days, or at least 21 days. The interval between administrations may be up to 6 months, e.g., 3 months or less, 2 months or less, 1 month or less, 21 days or less, or 14 days or less.

[0185] In one or more embodiments, the method may comprise multiple administrations of the treated biological product in intervals (in the case of two administrations) or intervals (in the case of more than two administrations) characterized by a range in which the endpoint is defined by any minimum interval identified above and any maximum interval greater than the minimum interval. For example, in one or more embodiments, the method may comprise multiple administrations of the treated biological product in intervals or intervals of 1 day to 6 months, for example, 3 days to 10 days. In one or more specific embodiments, the method may comprise multiple administrations of the treated biological product in intervals equal to any minimum interval or any maximum interval listed above. Thus, for example, the method may involve multiple administrations of the treated biological product in intervals of 3 days, 5 days, 7 days, 10 days, 14 days, 21 days, 1 month, 2 months, 3 months, or 6 months.

[0186] In the foregoing description and the following claims, the term “and / or” means one or all of the enumerated elements or any combination of two or more of the enumerated elements; the terms “comprising,” “comprising,” and variations thereof shall be interpreted as open-ended, i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “one” and “at least one” are used interchangeably and mean one or more than one; and the description of a numerical range by an endpoint includes all numbers encompassed within such range (e.g., 1 to 5 include 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.).

[0187] In the foregoing description, specific embodiments may be described separately for clarity. Throughout this specification, the references to “one embodiment,” “an embodiment,” “a specific embodiment,” “one or more embodiments,” or “some embodiments” mean that a specific feature, configuration, composition, or characteristic described in relation to an embodiment is included in at least one embodiment of this disclosure. Accordingly, the appearance of such phrases in various places throughout this specification does not necessarily refer to the same embodiment of this disclosure. Furthermore, a specific feature, configuration, composition, or characteristic may be combined in any appropriate manner in one or more embodiments. Thus, unless the features are necessarily mutually exclusive, a feature described in the context of one embodiment may be combined with a feature described in the context of a different embodiment.

[0188] Throughout the foregoing description, guidelines are provided through lists of examples, and these examples may be used in various combinations. In each case, the enumerated list serves only as a representative group and should not be interpreted as an exclusive list.

[0189] For any method disclosed herein comprising separate steps, the steps may be performed in any feasible order. And, where appropriate, any combination of two or more steps may be performed simultaneously.

[0190] As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may provide specific advantages under specific circumstances. However, other embodiments may also be preferred under the same or different conditions. Furthermore, the enumeration of one or more preferred embodiments does not imply that other embodiments are not useful, nor is it intended to exclude other embodiments from the scope of the invention.

[0191] Examples

[0192] The present invention is illustrated by the following examples. It should be understood that specific examples, materials, quantities, and procedures should be interpreted broadly in accordance with the scope and spirit of the invention as described herein.

[0193] Example 1 - PEP generation

[0194] PEP was generated from 53 donor units, producing a total of 13 L of material, which was divided into six 2 L bioprocessing bags in varying volumes. Subsequently, the pooled material was frozen at -80°C for more than 12 hours.

[0195] The following day, the frozen material was thawed in a 37°C water bath and pooled into a bioprocessing bag optimized for centrifugation. The centrifugation bioprocessing bag was processed in two centrifugation runs, both executed at the same relative centrifugal force (RCF) parameter sufficient to pellet the cell debris. The supernatant from both centrifugation runs was pooled into a single 20 L bag. The supernatant was then run through a filter train with a pore size sufficient to remove macroscopic cell debris (e.g., 0.2 μm or larger). The yield after filtration was 6.8 L. Approximately 700 mL of the purified material was aliquoted into a smaller bag for easier handling. All material was frozen at -80°C for more than 12 hours.

[0196] A portion of the aliquot was set aside for freeze-drying. 1 mL of liquid PEP formulation was added to each vial. A stopper was inserted into the first ridge of the vial, and it was placed in the BT-9EG benchtop freeze-dryer. The freeze-dryer was pooled to <200 mTorr and -80°C and run for a sufficient amount of time to dry the material. The method for freeze-drying the complex biological product includes, but is not limited to, the method described in International Patent Application No. PCT / US2025 / 010770. All vials were removed from the freeze-dryer, the stoppers were completely closed, and the caps were placed.

[0197] Example 2 - PEP Treatment with UV-B Radiation

[0198] Approximately 300 grams of liquid PEP preparation was transferred into a MIRASOL treatment bag. 15 mL of riboflavin was added to the treatment bag and gently mixed with DS. MIRASOL treatment was performed according to the manufacturer's instructions using a UV-B dose of 5%.

[0199] Viral titers were calculated as described above. Porcine parvovirus (PPV) titers were measured as a proxy for total viral titers. PPV viral titers were calculated for pre-treatment and post-treatment samples. The mean log decrease in viral titers across multiple replicates was determined to be 3.76.

[0200] Turbidity, particle size, particle concentration, presence of angiogenic cytokines, effects on cell proliferation, antioxidant capacity, and enzyme activity were measured as described above. MIRASOL treatment with UV-B radiation did not significantly affect the turbidity, particle size, particle concentration, or antioxidant capacity of the treated samples.

[0201] Although MIRASOL treatment of PEP samples adequately reduced the titer of biological contaminants, it required the addition of excipients to the samples, whereas other techniques for reducing the titer of biological contaminants were determined not to require the use of excipients.

[0202] Example 3 - PEP Treatment with Electron Beam Radiation

[0203] The liquid PEP preparation of Example 1 was thawed (liquid PEP preparation), thawed and freeze-dried (freeze-dried powder), or kept frozen (frozen PEP preparation), and then treated with electron beam radiation of 50 kilogray (kGy) or 25 kGy according to the manufacturer's instructions.

[0204] Reduction in viral titers in exosome products treated with electron beam radiation

[0205]

[0206] Electron beam radiation reduced viral titers less when treating lyophilized powder than when treating liquid or frozen PEP preparations.

[0207] Turbidity, particle size, particle concentration, presence of angiogenic cytokines, effect on cell proliferation, antioxidant capacity, and enzyme activity were measured as described above.

[0208] The PDGF-BB levels of the sample decreased by approximately 4.5%. Alkaline phosphatase activity decreased by 5% to 18%. Catalase activity decreased by 58%. The effect of treated PEP on cell proliferation was slightly reduced compared to untreated PEP.

[0209] Example 4 - PEP Treatment with UV-C Radiation

[0210] The material produced in Example 1 from a smaller fraction (~700 mL) was thawed in a 37°C water bath for UV-C treatment the following day. A UV-C chamber (PearlAqua, Aquisense Technologies, Erlanger, Kentucky) was assembled and prepared for use. The fraction of untreated material was taken as a control sublot for the batch. This control material was maintained at room temperature without shaking in the laboratory during the different treatment periods. After activating the UV-C chamber, the feed material was passed through the chamber at 250 mL / min; once all the material had passed through the chamber, the chamber was deactivated, the treated material bag was sealed, air was pumped through the piping to purify any residual material, and the treated material was aliquoted. Subsequently, this remaining treated material was used as the feed material for another treatment step, and the process was repeated as described above. This was performed five times to obtain five sublots of material at various treatment levels. Each pass through the UV-C system took approximately 1–3 minutes. In total, the group that passed five times took about 30 minutes to complete all processing.

[0211] Reduction in viral titers in exosome products treated with UV-C radiation

[0212]

[0213] Example 5 - PEP Treatment with UV-C Radiation and Electron Beam Radiation

[0214] The material from Example 1 was thawed and treated with UV-C as indicated in Table 1. After UV-C treatment, the material was frozen, freeze-dried, or kept in liquid form, and the material in each state was subjected to electron beam radiation as indicated in Table 3.

[0215] Reduction in viral titers in exosome products treated with electron beam radiation and UV-C radiation

[0216]

[0217] These examples demonstrate that treating liquid PEP formulations or frozen PEP formulations with a combination of UV-C radiation and electron beam radiation resulted in at least a 6.88-fold reduction in PPV potency.

[0218] Example 6 - Pharmacological Characterization of PEP Treated with Time-Related UV-C Radiation

[0219] The generated samples are listed in Table 4. The samples were tested immediately (T0) and stored for stability testing for analysis at 1 month (T1) and 3 months (T3) at room temperature.

[0220] Overview of UV-C Radiation-Treated Samples

[0221]

[0222] Each sample was resuspended in 1 mL of water and placed on an orbital shaker. For T0 and T1, the samples were shaken at 60 rpm. The method was updated between the T1 and T3 stability pooling, and the T3 samples were shaken at 200 rpm. The vials were observed and timed until the PEP powder cake was completely resuspended. The reconstitution time for each sample was recorded. Each sample was characterized for relevant properties including turbidity, exosome size, exosome concentration, presence of angiogenic cytokines, effects on cell proliferation, antioxidant capacity, and enzymatic activity. The methods for measuring each of these properties are described in more detail herein. The results of each assay are discussed below.

[0223] Turbidity

[0224] There was no turbidity trend for one to five UV-C passes in the room temperature or accelerated 1-month stability samples. There was no turbidity trend for one to five UV-C passes in the 3-month room temperature stability samples. However, when comparing 100 μL samples, all samples had slightly higher absorbance at 3 months compared to their counterparts at T1 month (Fig. 2).

[0225] Size and concentration

[0226] There was no significant difference in average particle size among the samples, and all were approximately 150 nm. For the sample measured immediately after treatment (T0), there was a tendency for the sample concentration to decrease slightly as the number of UV-C passes increased. There was no significant difference in particle concentration in any T0, T1, or T3 sample.

[0227] angiogenic cytokines

[0228] Immediately after treatment, levels of ANG-1, EGF, and PDGF-BB were reduced in all samples treated with UV-C radiation. Multiple passes showed differences in VEGF-A levels, but these differences were within known product variability (Fig. 3D). Although these angiogenic cytokines were slightly reduced by UV-C treatment, they were not considered biologically significant. In the treated samples, slight additional reductions in ANG-1, EGF, PDGF-BB, and VEGF-A were observed after 1 month of storage (Fig. 4) and 3 months of storage (Fig. 5), or no additional reductions were observed.

[0229] Cell proliferation

[0230] No significant difference in the effect on cell proliferation was observed in any sample at T0, T1, or T3. UV-C-treated PEP supports cell proliferation. When added to cell cultures of normal human dermal fibroblasts (NHDF), PEP supported cell proliferation more than the FBS-positive control, even when treated with UV-C. When measured by percentage full growth in cell culture plates, this was observed immediately after treatment (T0) and one month after treatment (T1 month) (Fig. 6).

[0231] UV-C-treated PEP supports cell proliferation at the 3-month stability time point (T3). When added to a cell culture of normal human dermal fibroblasts (NHDF) 3 months after UV-C treatment, PEP supported more cell proliferation than the FBS-positive control when treated with UV-C, as measured by cell counts via nuclear staining and full growth in the cell culture plate (Fig. 7).

[0232] Antioxidant capacity

[0233] When measured by the Trolox equivalent antioxidant capacity (TEAC) assay, no significant difference in antioxidant capacity was observed in any sample at T0, T1, or T3 (Fig. 8).

[0234] Enzyme activity

[0235] Catalase activity was significantly reduced after UV-C radiation treatment (Fig. 9). Alkaline phosphatase activity was slightly reduced after UV-C radiation treatment.

[0236] From these examples, it was determined that treating exosome products with UV-C radiation produced generally pharmaceutically acceptable properties and acceptablely reduced viral titers. Additionally, it was determined that treating exosome products with UV-C radiation did not significantly affect the stability of exosome products over time.

[0237] Example 7 - PEP Treatment with UV-C Radiation and Quantification of Multiple Viral Titers

[0238] Samples were treated with high or low dose UV-C radiation as described in Example 4. The titers of multiple viruses, including HIV, PRV, BVDV, PPV, and HAV, were measured in each sample. The justification for selecting these viruses is presented in Table 5. The initial titers of each virus and the titers after each treatment are presented in Table 6 below.

[0239] Justification for Virus Selection

[0240]

[0241] Table 6

[0242]

[0243] For HIV, PRV, and BVDV, the desired log reduction in viral titer was 10-fold. For PPV and HAV, the desired log reduction in viral titer was 6-fold. The high dose of UVC radiation used was a flow rate of 250 mL / min. The low dose of UVC radiation used was 500 mL / min. Cumulative reduction represents the log reduction in viral titer after treatment with high-dose UV-C radiation, lyophilization, and intrinsic reduction.

[0244] For PPV and HAV, a sufficient reduction in viral titer was achieved after cumulative treatment. For PRV and BVDV, cumulative treatment was insufficient to reduce viral titer to the desired amount. For HIV, it was unclear whether the reduction in viral titer was sufficient, as the maximum reduction achieved at each stage was limited by the original viral titer.

[0245] Example 8 - PEP treatment by UV-C, E-beam irradiation, and freeze-drying

[0246] PEP was prepared as described in Example 7. UV-C irradiation, E-beam irradiation, and lyophilization were evaluated. To facilitate maximum viral reduction results, each step was evaluated individually, and the virus was spiked into the material immediately prior to the dose setting / material generation step. For UV-C evaluation, HIV, PRV, BVDV, PPV, or HAV was spiked into the sample and passed twice through a UV-C irradiation system at a dose of 250 mL / min. For E-beam and lyophilization evaluation, the sample was passed twice through UV-C at 250 mL / min without spiked into the solution. Subsequently, the sample was divided, and after spiked with HIV, PRV, BVDV, PPV, or HAV into half, all samples were subjected to E-beam irradiation at a dose of 25 kGy. Afterward, HIV, PRV, BVDV, PPV, or HAV was spiked into a separate E-beam irradiated portion without spikes, and then lyophilized.

[0247] Table 7 is a summary of the virus reduction results generated from combining UV-C irradiation and E-beam irradiation in sequential steps and evaluating the virus reduction at each step. By combining two passes of UV-C irradiation at 250 mL / min through a turbulent flow cell, a cumulative E-beam irradiation dose of 25 kGy achieved by irradiating from each side, and lyophilization of 2 mL aliquots, a minimum total log reduction of at least 10.13 is achieved for all five model viruses.

[0248] log 10 Virus reduction

[0249]

[0250] Characteristics of the processed sample

[0251] appearance

[0252] The freeze-dried powder cake was reported as a pale yellow powder cake, and the reconstituted vial was reported as a particle-free opaque yellow solution.

[0253] Reconstruction time

[0254] Each sample was reconstituted in 1 mL of sterile water in a glass vial and placed on an orbital shaker at 60 rpm. The vial was observed and timed until the PEP powder cake was completely resuspended. The reconstitution time for each sample was recorded. The reconstitution times for four exemplary fractions are presented in Fig. 10.

[0255] Turbidity

[0256] The turbidity of PEP applied to two UV-C passes was measured before and after freeze-drying and reconstruction, with or without subsequent E-beam treatment. The results are presented in Fig. 11.

[0257] Particle size and concentration

[0258] The average particle size of PEP applied to two UV-C passes was determined before and after freeze-drying and reconstruction, with or without subsequent E-beam treatment. The results are presented in Fig. 12.

[0259] Particle concentrations for PEP applied to two UV-C passes were determined before and after freeze-drying and reconstitution with or without subsequent E-beam treatment. The results are presented in Fig. 13.

[0260] angiogenic cytokines

[0261] The presence of angiogenic cytokines in PEP applied by two UV-C passes, with or without subsequent E-beam treatment, was determined using an automated ELISA system (ELLA, Bio-Techne, Minneapolis, Minnesota) and a multi-cartridge kit for detecting ANG-1, EGF, PDGF-BB, and VEGF-A (SIMPLE PLEX, Bio-Techne, Minneapolis, Minnesota). PEP samples were lysed by combining 200 μL of resuspended PEP with 50 μL of 5X RIPA / protease inhibitor. The samples were vortexed for 15 seconds and then incubated at room temperature for 5 minutes. The samples were centrifuged at 20,000×g for 10 minutes at room temperature. Before plating on the automated ELISA cartridge, the samples were diluted with SD13 to a final 10-fold dilution (87.5 mL of SD13 + 12.5 mL of lysed PEP sample). The washing buffer, samples, and high-dose and low-dose controls were plated onto an automated ELISA cartridge and run on an automated ELISA system device according to the manufacturer's instructions. The results are presented in Fig. 14.

[0262] Inflammatory cytokines

[0263] The presence of the pro-inflammatory cytokines IFN-γ and IL-1β in PEPs applied by two UV-C passes, with or without subsequent E-beam treatment, was determined after freeze-drying and reconstitution. The results are presented in Figure 15.

[0264] Cell proliferation

[0265] The ability of PEP applied in two UV-C passes, with or without subsequent E-beam treatment, to induce cell proliferation in normal human dermal fibroblasts (NHDFs) grown in standard fibroblast basal medium at 37°C and 5% CO2 after freeze-drying and reconstitution was measured. The results are presented in Figure 16. The times to ≥70% full growth are as follows: L-1: 73% full growth after 72 hours; L-2: 73% full growth after 69 hours; A-1: ​​76% full growth after 72 hours; and A-2: 74% full growth after 69 hours.

[0266] Residual moisture

[0267] The residual moisture of the reconstituted PEP was measured after being freeze-dried and reconstituted following two passes of UV-C, with or without subsequent E-beam treatment. The results are presented in Fig. 17.

[0268] Example 9 - PEP treatment by UV-C, γ irradiation and freeze-drying

[0269] PEP manufacturing

[0270] Platelets were thawed and pooled, then centrifuged at 7186 Relative Fusion (RCF) for 24 minutes. The supernatant was collected and passed through a filter train consisting of 0.45 μm and 0.22 μm BPS filters (Critical Process Filtration, Inc., Nashua, New Hampshire). Approximately 1 L of filtrate was dispensed. The remaining filtrate was divided into five groups per sublot for different UV-treatment levels: untreated, untreated + UV treated, 1 × 250 mL / min, 2 × 250 mL / min, and 3 × 250 mL / min. Each group was dispensed into a 50 mL bag (Entegris, Inc., Billerica, Massachusetts) and frozen.

[0271] UV-C treatment

[0272] UV-C treatment was performed at the Thin Film Unit (PEARLLAB, Aquisense Technologies, Erlanger, Kentucky). The dose of UV-C radiation delivered during each pass was controlled by the material flow rate and the power delivered to the UV-C LED. All material passes were performed at 250 mL / min and 100% intensity, in one batch per sublot, where the material was aliquoted for storage or further processing after each pass. A handling control (MC-#01) was performed separately from the other three treatment options. Table 8 below outlines the total amount processed and the final yield at each stage. The material from these conditions was further distributed according to a sampling plan.

[0273] UV-C Treatment Yield

[0274]

[0275] Electron beam processing

[0276] Samples (100 mL) were submitted to E-BEAM Services, Inc. (Lebanon, Ohio) for treatment. An identical dose of 25 kGy was administered to all sample groups, except for the transport control (ES-#01). Sample treatment is summarized in Table 9.

[0277] Table 9

[0278]

[0279] Gamma treatment

[0280] Samples (100 mL) were submitted to Steris Life Sciences (Mentor, Ohio) for treatment. A dose of 12 kGy or 25 kGy was provided to the sample group. Sample treatment is summarized in Table 10.

[0281]

[0282]

[0283] Stability model

[0284] Vials containing PEP freeze-dried powder cakes were placed in a stability chamber at 25°C / 65% relative humidity (RH) for room temperature / long-term storage evaluation or at 40°C / 75% RH for accelerated stability evaluation. Subsequently, aliquots from the vials were removed from the chamber at intervals of one or three months and applied to analytical tests.

[0285] E-beam in the presence or absence of UV-C

[0286] Reconstruction time

[0287] The reconstitution time of the PEP samples was measured as described in Example 8. The reconstitution time of the PEP samples did not differ across treatment types, despite slight variability from each lot (Fig. 18). In the room temperature stability test, the reconstitution time was similar for all samples after 1 month (approx. 4 min) and after 3 months (approx. 2-2.5 min). In the accelerated stability test, the reconstitution time for all samples was similar at both the 1-month and 3-month time points, at approximately 2-3 min.

[0288] Alkaline phosphatase activity

[0289] Alkaline phosphatase activity, an indicator of enzyme activity, was measured as described in Example 6. Enzyme activity decreased slightly across all samples (Fig. 19), indicating that the degree of UV-C treatment did not affect enzyme activity.

[0290] Throughout the stability study process, alkaline phosphatase activity increased in all PEP lots compared to the initial time point (Fig. 19), and after 1 month, it increased to an average rate of about 25-30 per minute, and after 3 months, to an average rate of about 40 per minute (at room temperature) or an average rate of about 30-35 per minute (accelerated).

[0291] Antioxidant capacity

[0292] Antioxidant capacity was measured using the TEAC assay. At the initial time point (Fig. 20), antioxidant capacity was similar across all PEP samples and slightly increased compared to the control sample. Over the 3-month stability study, antioxidant capacity decreased in all PEP samples at 1 month, but at 3 months, it was greater than the initial value of T0 under both room temperature and accelerated conditions.

[0293] angiogenic cytokine activity

[0294] Angiogenic cytokines ANG-1, EGF, PDGF-BB, and VEGF-A were analyzed by automated ELISA (ELLA, Bio-Techne, Minneapolis, Minnesota). The concentrations of angiogenic cytokines varied across PEP samples from baseline, with the most typical decrease in concentration observed in E-beam + 3× UV-C samples. Variation between different lots was minimal and unlikely to be biologically relevant.

[0295] The angiogenic cytokines of all four showed similar patterns over 3 months of stability under room temperature and accelerated conditions. Specifically, when maintained for 3 months under room temperature (RT) conditions, the concentration of angiogenic cytokines decreased slightly or did not change significantly at 1 month, but increased compared to baseline at 3 months. However, under accelerated (Acc) conditions, the concentration of angiogenic cytokines increased slightly or did not change significantly at 1 month, but decreased compared to baseline at 3 months, which is consistent with known product sensitivity.

[0296] Cell proliferation

[0297] Cell proliferation upon PEP sample treatment was measured by full growth as described in Example 8. Results from the initial time point (T0) are presented in FIG. 21.

[0298] Gamma irradiation in the presence and absence of UV-C

[0299] Reconstruction time

[0300] The reconstitution time of the PEP samples was measured as described in Example 8. The reconstitution time varied somewhat across the PEP samples, and the treated samples took slightly longer to become homogeneous upon reconstitution compared to the control group of T0 (Fig. 22).

[0301] After maintaining stability under room temperature conditions, the reconstitution time increased at 3 months and returned to baseline at 6 months (Fig. 23A). For all PEP samples under accelerated conditions, the reconstitution time increased over 3 months across all samples (Fig. 23B).

[0302] Extracellular vesicle size

[0303] The average extracellular vesicle size of PEP samples was measured by nano-site particle tracking analysis (Molburn Panological Limited, Molburn, UK).

[0304] The extracellular vesicle size was consistent across PEPs treated with multiple UV-C passes and γ irradiation at the initial time point (T0, Fig. 24), which indicates that EV size is not affected by UVC and γ exposure.

[0305] In the stability study, the average EV size decreased initially but recovered over a longer period (Fig. 25).

[0306] Alkaline phosphatase activity

[0307] The catalase concentration as an indicator of enzyme activity was measured as described in Example 6.

[0308] Enzyme activity by catalase concentration was reduced by increasing the virus inactivation technique at the initial time point (T0, Fig. 26). During the stability study, catalase concentration decreased in all PEP samples compared to the initial time point. This trend was true for both room temperature (Fig. 27A) and accelerated (Fig. 27B) conditions.

[0309] Antioxidant capacity

[0310] Antioxidant capacity was measured using the TEAC assay. At the initial time point (T0, Fig. 28), antioxidant capacity was similar across all lots of the PEP drug products. A slight increase in antioxidant capacity was observed as the viral inactivation treatment increased. This increase is unlikely to be biologically detrimental to PEP efficacy. Over the 3-month stability study, antioxidant capacity for PEP stored under room temperature stability conditions decreased in all samples at 1 month but increased at 3 months (Fig. 29A). However, for PEP samples stored under accelerated conditions, antioxidant capacity decreased for all samples at 1 month and decreased again at 3 months (Fig. 29B).

[0311] angiogenic cytokine activity

[0312] Angiogenic cytokines ANG-1, EGF, PDGF-BB, and VEGF-A were analyzed using an automated ELISA (ELLA, Bio-Techne, Minneapolis, Minnesota). The concentrations of angiogenic cytokines were consistent across PEP samples for each cytokine from baseline.

[0313] The angiogenic cytokines of all four were reduced at 3 months under both room temperature and accelerated conditions (data not presented).

[0314] Cell proliferation

[0315] Cell proliferation upon PEP sample treatment was measured by full growth as described in Example 8. All PEP samples dramatically reduced doubling time.

[0316] The full disclosures of all patents, patent applications, and publications cited herein, and electronically available materials (e.g., nucleotide sequence submissions from GenBank and RefSeq, and amino acid sequence submissions from SwissProt, PIR, PRF, and PDB, and translations from the annotation coding regions of GenBank and RefSeq, etc.) are incorporated by reference in their entirety. In the event of any discrepancy between the disclosure(s) of this application and the disclosure(s) of any document incorporated by reference herein, the disclosure(s) of this application shall prevail. The above detailed description and examples are provided solely for the purpose of clarifying understanding. No unnecessary limitations should be interpreted from them. Because variations obvious to a person skilled in the art will be included in the invention defined by the claims, the invention is not limited to the exact details presented and described.

[0317] Unless otherwise specified, all numbers expressing quantities such as ingredients, molecular weights, etc. used in the specification and claims should be understood as being modified by the term "approximately" in all cases. Accordingly, unless otherwise indicated, numerical parameters described in the specification and claims are approximations that may vary depending on the desired properties to be obtained by the present invention. At least, and without attempting to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should be interpreted in terms of the number of reported significant figures and by applying conventional rounding techniques.

[0318] Although the numerical ranges and parameters describing the broad scope of the invention are approximations, the numerical values ​​described in the specific embodiments are reported as accurately as possible. However, all numerical values ​​inherently contain a range inevitably generated from the standard deviation identified in each test measurement.

[0319] All headings are for the convenience of the reader and, unless otherwise specified, should not be used to limit the meaning of the body text following the heading.

Claims

Claim 1 A method for treating a biological product to reduce the potency of biological contaminants within the biological product, comprising applying the biological product to electromagnetic energy or ionizing radiation with a wavelength of ultraviolet (UV) or lower. Claim 2 A method according to claim 1, further comprising freeze-drying a biological product. Claim 3 A method according to claim 1 or 2, wherein the biological contaminant comprises an enveloped virus or a non-enveloped DNA virus. Claim 4 A method in which, in any one of paragraphs 1 to 3, the biological contaminant is a DNA virus or an RNA virus. Claim 5 A method according to any one of claims 1 to 4, wherein the DNA virus is a single-stranded DNA (ssDNA) virus or a double-stranded DNA (dsDNA) virus. Claim 6 A method according to any one of claims 1 to 5, wherein the biological contaminant comprises one or more of human immunodeficiency virus (HIV), pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), or hepatitis A virus (HAV). Claim 7 A method according to claim 6 in which the HIV titer is reduced by at least 10 times and / or the PRV titer is reduced by at least 10 times and / or the BVDV titer is reduced by at least 10 times and / or the PPV titer is reduced by at least 6 times and / or the HAV titer is reduced by at least 6 times. Claim 8 A method according to claim 1 or 2, wherein the biological contaminant comprises bacteria. Claim 9 A method according to paragraph 1 or 2 in which the biological contaminant includes fungi. Claim 10 A method according to any one of claims 1 to 9, wherein the electromagnetic energy comprises UV-B, UV-C, X-rays, or γ-rays. Claim 11 In paragraph 10, a method in which a biological product is applied to UV-C at a dose of 5 mL / min to 500 mL / min. Claim 12 A method according to any one of claims 1 to 11, wherein the ionizing radiation comprises X-rays, γ-rays, or electron beam radiation. Claim 13 A method according to paragraph 12 in which a biological product is treated with electron beam radiation of 0 kilogray (kGy) to 55 kGy. Claim 14 In paragraph 13, a method in which a biological product is treated with electron beam radiation of 25 kGy. Claim 15 A method according to any one of claims 1 to 14, further comprising applying a biological product to a second form of electromagnetic energy or ionizing radiation. Claim 16 A method according to any one of claims 1 to 15, further comprising freeze-drying, freezing, centrifuging, or filtering the biological product. Claim 17 A method according to any one of claims 1 to 16, wherein the biological product comprises exosomes. Claim 18 A biological product treated to reduce at least one titer of biological contaminant compared to an untreated sample of the biological product. Claim 19 In paragraph 18, a biological product in which the potency of the biological contaminant is reduced by at least 1 log compared to an untreated biological product. Claim 20 A biological product according to paragraph 18 or 19, wherein the biological product comprises exosomes. Claim 21 A biological product according to any one of paragraphs 18 to 20, wherein the biological product further comprises freeze-dried powder. Claim 22 A biological product according to any one of paragraphs 18 through 21, wherein the biological product further comprises a carrier. Claim 23 A biological product in which the carrier comprises a solvent, as described in paragraph 22. Claim 24 A biological product according to any one of paragraphs 18 through 23, wherein the biological contaminant comprises an enveloped virus or a non-enveloped DNA virus. Claim 25 A biological product in which the biological contaminant is a DNA virus or an RNA virus in any one of paragraphs 18 through 24. Claim 26 A biological product in which, in any one of paragraphs 18 to 25, the DNA virus is a single-stranded DNA (ssDNA) virus or a double-stranded DNA (dsDNA) virus. Claim 27 A biological product according to any one of claims 18 to 26, wherein one or more reduced viral titers comprise at least one of human immunodeficiency virus (HIV), rabies pseudovirus (PRV), bovine viral diarrhea virus (BVDV), porcine parvovirus (PPV), or hepatitis A virus (HAV). Claim 28 A biological product in which, in paragraph 27, the HIV titer is reduced by at least 10 times and / or the PRV titer is reduced by at least 10 times and / or the BVDV titer is reduced by at least 10 times and / or the PPV titer is reduced by at least 6 times and / or the HAV titer is reduced by at least 6 times. Claim 29 A biological product in which the biological contaminant in any one of paragraphs 18 through 23 comprises bacteria. Claim 30 A biological product in which the biological contaminant in any one of paragraphs 18 through 23 comprises fungi. Claim 31 A biological product according to any one of claims 18 to 30, wherein the biological product has significantly the same turbidity, particle size, particle concentration, angiogenic cytokine concentration, effect on cell proliferation, antioxidant capacity, or enzyme activity as an untreated biological product. Claim 32 A biological product that is derived from a mammalian blood product in any one of paragraphs 18 through 31. Claim 33 In paragraph 32, a biological product in which a mammalian blood product contains human whole blood. Claim 34 A biological product that, in any one of paragraphs 18 through 33, exhibits significantly the same stability as an untreated biological product. Claim 35 A composition comprising: a biological product of any one of claims 18 to 34; and a pharmaceutically acceptable carrier. Claim 36 A method of treating a subject comprising administering the composition of claim 35 to the subject.

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