Methods for reducing titer of biological contaminants in complex biologics

Abstract

A method of reducing the titer of a biological contaminant in a biological product generally includes subjecting the biological product to electromagnetic energy having an ultraviolet (UV) wavelength or shorter in a dose effective to reduce biological contaminants in a sample of the biological product while maintaining integrity and function of the biological product. In one or more embodiments, the electromagnetic energy comprises UV-B, UV-C, X-rays, or γ-rays. In one or more embodiments, the method further includes subjecting the biological product to electron beam radiation.

Description

[0001] METHODS FOR REDUCING TITER OF BIOLOGICAL CONTAMINANTS

[0002] IN COMPLEX BIOLOGICS

[0003] CROSS-REFERENCE TO RELATED APPLICATION

[0004] This application claims the benefit of U.S. Provisional Patent Application No. 63 / 619,143, filed January 9, 2024, which is incorporated herein by reference in its entirety.

[0005] SUMMARY

[0006] This disclosure describes, in one aspect, a method of reducing the titer of a biological contaminant in a biological product. Generally, the method includes subjecting the biological product to electromagnetic energy having an ultraviolet (UV) wavelength or shorter in a dose effective to reduce the titer of biological contaminants in a sample of the biological product while maintaining integrity and function of the biological product.

[0007] In one or more embodiments, the method further includes lyophilizing the biological product.

[0008] In one or more embodiments, the biological contaminant includes an enveloped virus or a non-enveloped DNA virus.

[0009] In one or more embodiments, the biological contaminant includes a DNA virus or an RNA virus.

[0010] In one or more embodiments, the biological contaminant includes a single-stranded DNA (ssDNA) virus or a double-stranded DNA (dsDNA) virus.

[0011] In one or more embodiments, the biological contaminant includes one or more of human immunodeficiency virus (HIV), pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), Porcine Parvovirus (PPV), hepatitis A virus (HAV). In one or more of these embodiments, embodiments, the HIV titer is reduced by at least 10-fold, the PRV titer is reduced by at least 10- fold, the BVDV titer is reduced by at least 10-fold, the PPV titer is reduced by at least 6-fold, and / or the HAV titer is reduced by at least 6-fold.

[0012] In one or more embodiments, the biological contaminant includes a bacterium.

[0013] In one or more embodiments, the biological contaminant includes a fungus. In one or more embodiments, the electromagnetic energy includes UV-B, UV-C, X-rays, or y-rays. In one or more of these embodiments, the biological product is subjected to UV-C at a dose of from 5 mL / min to 500 mL / min.

[0014] In one or more embodiments, the ionizing radiation includes X-rays, y-rays, or electron beam radiation. In one or more of these embodiments, the biological product is treated with 0 kiloGray (kGy) to 55 kGy of electron beam radiation such as, for example, 25 kGy of electron beam radiation.

[0015] In one or more embodiments, the method further includes subjecting the biological product to a second form of electromagnetic energy or ionizing radiation.

[0016] In one or more embodiments, the method further includes lyophilizing, freezing, centrifuging, or fdtering the biological product.

[0017] In one or more embodiments, the biological product includes exosomes.

[0018] In another aspect, this disclosure describes a biological product treated to reduce at least one titer of a biological contaminant compared to an untreated sample of the biological product.

[0019] In one or more embodiments, the titer of the biological contaminant is reduced by at least one log compared to an untreated biological product.

[0020] In one or more embodiments, the biological product includes exosomes.

[0021] In one or more embodiments, the biological product further includes a lyophilized powder.

[0022] In one or more embodiments, the biological product further includes a carrier.

[0023] In one or more embodiments, the biological contaminant includes an enveloped virus or a non-enveloped DNA virus.

[0024] In one or more embodiments, the biological contaminant is a DNA virus or an RNA virus.

[0025] In one or more embodiments, the DNA virus is a single- stranded DNA (ssDNA) virus or a double-stranded DNA (dsDNA) virus.

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

[0027] In one or more embodiments, the biological contaminant includes a fungus.

[0028] 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 enzymatic activity as an untreated biological product. 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 includes human full blood.

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

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

[0031] In another aspect, this disclosure describes a method of treating a subject includes administering to the subject a composition that includes that includes a biological product treated to reduce at least one titer of a biological contaminant compared to an untreated sample of the biological product.

[0032] The above summary is not intended to describe each disclosed embodiment or every implementation of the present invention. The description that follows more particularly exemplifies illustrative embodiments. In several places throughout the application, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0033] BRIEF DESCRIPTION OF THE FIGURES

[0034] FIG. 1 Morphologic characterization of PEP. (A) PEP has a spherical vesicle structure with intact lipid-bilayer. (B) Particle size distribution analysis via NanoSight (Malvern Panalytical Ltd., Malvern, United Kingdom) showing that average vesicle diameter of PEP ranged from 56.4 nm to 151 nm and the mean diameter was 96.9 nm ± 2.8 nm, representing the standard size range of exosomes. A dilution factor correction of 1000 was applied to the reported particle concentration resulting in a final concentration of 1.9* 1011particles / mL in a 100% PEP solution.

[0035] FIG. 2. Turbidity of PEP when treated with UV-C. Absorbance of 50 pL at initial treatment (TO, left) or 100 pL three months post manufacture (T3, right) of 100% PEP solution was measured at 600 nm using a plate reader (VARIOSKANLUX, Thermo Fisher Scientific, Inc., Waltham, MA). Multiple passes of UV-C radiation treatment showed no change in the turbidity of PEP compared to 100% PEP that was not passed through UV-C (untreated). FIG. 3. Angiogenic cytokines in PEP when treated with UV-C at initial treatment (TO). The concentrations of pro-angiogenic cytokines ANG-1, EGF, PDGF-BB, and VEGF-A detected via automated ELISA (ELLA, Bio-Techne, Minneapolis, MN) slightly decreased in PEP with UV-C treatment, however not to a significant extent.

[0036] FIG. 4. Angiogenic cytokines in PEP when treated with UV-C one month after treatment (Tl). One month post UV-C treatment, the concentrations of pro-angiogenic cytokines ANG-1, EGF, PDGF-BB, and VEGF-A detected via automated ELISA (ELLA, Bio-Techne, Minneapolis, MN) were not significantly changed from TO.

[0037] FIG. 5. Angiogenic cytokines in PEP when treated with UV-C at three months post treatment (T3). Three months post UV-C treatment, the concentrations of pro-angiogenic cytokines ANG-1, EGF, PDGF-BB, and VEGF-A detected via automated ELISA (ELLA, Bio- Techne, Minneapolis, MN) were not significantly changed from initial (TO) or one-month (Tl) time points.

[0038] FIG. 6. UV-C treated PEP supports cell proliferation. When added to cell culture of normal human dermal fibroblasts (NHDFs), PEP supported cell proliferation greater than the FBS positive control, even when treated with UV-C. This was observed immediately after treatment (TO) and at one month post-treatment (Tl month), as measured by percent confluence in the cell culture plate.

[0039] FIG. 7. UV-C treated PEP supports cell proliferation at three-month stability time point (T3). When added to cell culture of normal human dermal fibroblasts (NHDFs) three months post UV-C treatment, PEP supported cell proliferation greater than the FBS positive control, even when treated with UV-C, as measured by confluence in the cell culture plate and by cell count via nuclear stain.

[0040] FIG. 8. Antioxidant capacity of UV-C treated PEP. UV-C treatment of PEP had no negative impact on the antioxidant capacity of PEP immediately after treatment (TO), one month (Tl month), or three months (T3 month) after treatment.

[0041] FIG. 9. Enzymatic activity of UV-C treated PEP. UV-C treatment effect on the enzymatic activity (alkaline phosphatase and / or catalase) of PEP with multiple passes of UV-C radiation treatment. There was limited effect with 1-3 passes of UV-C.

[0042] FIG. 10. Reconstitution time for representative PEP replicates was evaluated by reconstituting each PEP sample in 1 mL of sterile water. L-l: 2* UV-C + E-beam, replicate #1; L-2: 2* UV-C + E-beam, replicate #2; A-l : 2* UV-C, replicate #1; A-2: 2* UV-C, replicate #2. FIG. 11 . Turbidity measurements. Patterned bars: PEP subjected to 2* UV-C ± E-beam, prior to lyophilization; Solid bars: PEP subjected to 2* UV-C ± E-beam, then lyophilized and reconstituted; L-l : 2x UV-C + E-beam, replicate #1; L-2: 2* UV-C + E-beam, replicate #2; A-l : 2x UV-C, replicate #1; A-2: 2x UV-C, replicate #2.

[0043] FIG. 12. Determination of mean particle size. Patterned bars: PEP subjected to 2x UV-C ± E-beam, prior to lyophilization; Solid bars: PEP subjected to 2x UV-C ± E-beam, then lyophilized and reconstituted; L-l : 2x UV-C + E-beam, replicate #1; L-2: 2x UV-C + E-beam, replicate #2; A-l: 2x UV-C, replicate #1; A-2: 2x UV-C, replicate #2.

[0044] FIG. 13. Determination of particle concentration. Patterned bars: PEP subjected to 2x UV-C ± E-beam, prior to lyophilization; Solid bars: PEP subjected to 2x UV-C ± E-beam, then lyophilized and reconstituted; L-l : 2x UV-C + E-beam, replicate #1; L-2: 2x UV-C + E-beam, replicate #2; A-l : 2x UV-C, replicate #1; A-2: 2x UV-C, replicate #2.

[0045] FIG. 14. Angiogenic cytokines. PEP subjected to 2x UV-C ± E-beam, then lyophilized and reconstituted; L-l : 2x UV-C + E-beam, replicate #1; L-2: 2x UV-C + E-beam, replicate #2; A-l: 2x UV-C, replicate #1; A-2: 2x UV-C, replicate #2.

[0046] FIG. 15. Proinflammatory cytokines. PEP subjected to 2x UV-C ± E-beam, then lyophilized and reconstituted; L-l : 2x UV-C + E-beam, replicate #1; L-2: 2x UV-C + E-beam, replicate #2; A-l : 2x UV-C, replicate #1; A-2: 2x UV-C, replicate #2. All measurements of IFN- y and IL- 1 were below the lower limit of quantification (LOQ). TNF-a was detected in the samples at an acceptable level.

[0047] FIG. 16. Cell proliferation. The time to induce >70% confluency of normal human dermal fibroblasts (NHDFs) was measured. L-l: 73% confluency in 72 hours; L-2: 73% confluency in 69 hours; A-l : 76% confluency in 72 hours; and A-2: 74% confluency in 69 hours.

[0048] FIG. 17. Residual moisture content of PEP subjected to 2x UV-C ± E-beam, then lyophilized and reconstituted.

[0049] FIG. 18. Reconstitution time of PEP samples subjected to E-beam with no UV-C (E- beam), one pass of UV-C treatment (E-beam + U UV-C), two passes of UV-C treatment (E- beam + 2x UV-C), or three passes of UV-C treatment (E-beam + 3x UV-C).

[0050] FIG. 19. Alkaline phosphatase activity of PEP samples subjected to E-beam with no UV- C (E-beam), one pass of UV-C treatment (E-beam + U UV-C), two passes of UV-C treatment (E-beam + 2x UV-C), or three passes of UV-C treatment (E-beam + 3x UV-C). FIG. 20. Antioxidative capacity by TEAC of PEP samples subjected to E-beam with no UV-C (E-beam), one pass of UV-C treatment (E-beam + lx UV-C), two passes of UV-C treatment (E-beam + 2x UV-C), or three passes of UV-C treatment (E-beam + 3x UV-C).

[0051] FIG. 21. Cellular proliferation by confluence of cells treated with PEP subjected to E- beam with no UV-C (E-beam), one pass of UV-C treatment (E-beam + l x UV-C), two passes of UV-C treatment (E-beam + 2x UV-C), or three passes of UV-C treatment (E-beam + 3x UV-C). Confluence percentage over 96 hours of imagine following treatment with PEP samples.

[0052] FIG. 22. Reconstitution time of PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1 x UV-C), two passes of UV-C treatment (2x UV-C), or three passes of UV-C treatment (3* UV-C).

[0053] FIG. 23. Reconstitution time of PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1 x UV-C), two passes of UV-C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C) after stability testing. (A) Ambient stability conditions. (B) Accelerated stability conditions.

[0054] FIG. 24. Average particle size in PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1xUV-C), two passes of UV-C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C).

[0055] FIG. 25. Average particle size in PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1xUV-C), two passes of UV-C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C) after stability testing. (A) Ambient stability conditions. (B) Accelerated stability conditions.

[0056] FIG. 26. Catalase concentration in PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1 x UV-C), two passes of UV-C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C).

[0057] FIG. 27. Catalase concentration in PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1xUV-C), two passes of UV-C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C) after stability testing. (A) Ambient stability conditions. (B) Accelerated stability conditions.

[0058] FIG. 28. Antioxidant capacity of PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (l x UV-C), two passes of UV-C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C). FIG. 29. Antioxidant capacity of PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1 x UV-C), two passes of UV-C treatment (2 UV-C), or three passes of UV-C treatment (3x UV-C) after stability testing. (A) Ambient stability conditions. (B) Accelerated stability conditions.

[0059] FIG. 30. Concentration of angiogenic cytokines in PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1 x UV-C), two passes of UV- C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C). (A) ANG-1; (B) EGF.

[0060] FIG. 31. Concentration of angiogenic cytokines in PEP samples subjected to the indicated dose of y irradiation with or without one pass of UV-C treatment (1 x UV-C), two passes of UV- C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C). (A) PDGF-BB; (B) VEGF-A.

[0061] FIG. 32. Proliferative capacity of cells treated with PEP subjected to the indicated dose of Y irradiation with or without one pass of UV-C treatment (1xUV-C), two passes of UV-C treatment (2x UV-C), or three passes of UV-C treatment (3x UV-C). SFM: negative control; FBS: positive control.

[0062] DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

[0063] This disclosure describes methods for reducing the titer of one or more biological contaminants in a biological product such as, but not limited to, a purified exosome product. In one or more embodiments, the biological product may be a therapeutic product that, after being subjected to one or more titer-reducing methods described herein, is suitable for administering to a subject. Thus, while described below in the context of an illustrative embodiment in which the biological product is a purified exosome product, the methods and compositions described herein can include the use of any suitable biologic. Illustrative alternative biologies include, but are not limited to, antibodies, cellular therapies, or liposomal therapies.

[0064] Purified exosome product (PEP)

[0065] PEP is a purified exosome product prepared using a cryodesiccation step that produces a product having a structure that is distinct from exosomes prepared using conventional methods. For example, PEP typically has a spherical or spheroidal structure and an intact lipid bilayer rather than a crystalline structure that results from the reaggregation of lipids of the exosome lipid bilayer after exosomes are disrupted during convention exosome preparation methods. As used herein, a “spheroid” structure is shaped like a three-dimensional sphere with flattened poles. The spherical or spheroid exosome structures generally have a diameter of no more than 300 nanometers (nm). Typically, a PEP preparation contains spherical or spheroid exosome structures that have a relatively narrow size distribution. An example size distribution of a PEP preparation is shown in FIG. IB. Here, the mean particle size diameter was 96.9 nm + 52.2 nm. In some preparations, PEP includes spherical or spheroidal exosome structures with a mean diameter of 110 nm + 90 nm, with most of the exosome structures having a mean diameter of 110 nm + 50 nm such as, for example, 110 nm + 30 nm.

[0066] Production of purified exosome product (PEP) involves separating plasma from blood and isolating a solution of exosomes from separated plasma with filtration and centrifugation. PEP is fully characterized and methods for preparing PEP are described in International Patent Application No. PCT / US2018 / 065627 (published as International Publication No. WO 2019 / 118817), U.S. Patent Publication No. 2021 / 0169812 Al, and U.S. Patent No. 10,596,123, each of which is incorporated by reference herein in its entirety.

[0067] Morphologic Characterization of PEP

[0068] In one or more embodiments, the vesicle size of PEP exosomes may be measured to characterize the preparation. Vesicle size may be measured, for example, by electron microscopy, such as transmission electron microscopy or scanning electron microscopy. Transmission electron microscopy images indicate that exosomes of PEP exhibit the typical spherical vesicles with an intact lipid-bilayer structure (FIG. 1A). Vesicle sizes of PEP ranged from 56.4 nm to 151 nm with a mean size of 96.9 nm ± 2.8 nm, representing the standard size range of exosomes. PEP solution at 100% was calculated to contain 1.9* 1011PEP particles / mL (FIG. IB).

[0069] Titer-reducing methods

[0070] Biological products, such as PEP, are typically 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 held to a rigorous standard of sterilization to ensure that the biological product does not spread disease. For example, blood donations are often sterilized before they can be administered to recipients. One common method of sterilizing donated blood is ionizing radiation. Additional methods of sterilizing biological products, such as blood, include heat denaturation, acid denaturation, the use of detergents, and ultrafiltration. While many of these methods are effective at neutralizing viruses, the conditions are typically harsh and result in degradation of the biological product.

[0071] In addition, sterilizing complex biological products (e.g., PEP) presents unique challenges of removing biological contaminants without disrupting the active biological components. As used herein, the term “biological contaminants” refers to any biological material or entity that is not desired to be 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.

[0072] In one or more embodiments in which the biological contaminant includes one or more viruses, the biological contaminant can include one or more enveloped viruses, one or more nonenveloped DNA viruses, one or more DNA viruses and / or one or more RNA viruses. In one or more embodiments in which the biological contaminant includes one or more DNA viruses, the biological contaminant can include one or more single-stranded DNA (ssDNA) viruses and / or one or more double-stranded DNA (dsDNA) viruses. Thus, for example, the biological contaminant can include 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.

[0073] In one or more embodiments, the biological contaminant includes one or more bacterial species.

[0074] In one or more embodiments, the biological contaminant includes one or more fungal species.

[0075] Methods of sterilization typically do not predictably distinguish between different types of biological components. For example, heat denaturing a sample may inactivate a contaminating virus, but it may also inactivate a therapeutic enzyme.

[0076] Accordingly, developing a method that selectively inactivates contaminants without disrupting the activity of desired biological components is often unpredictable and presents a significant technical challenge. Multiple methods of reducing the titers of biological contaminants and their compatibility with PEP are described herein.

[0077] In one aspect, the present disclosure relates to methods of treating a biological product. In one or more embodiments, treating the biological product includes reducing the titer of one or more biological contaminants in a sample of the biological product. Typically, methods of reducing the titer of biological contaminants in a biological product yields a biological product that is at least partially sterilized. In one or more embodiments, the treated biological product includes 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, at least in that cells treated with the treated biological product are less likely to become modified (e.g., infected) by a contaminant (e g., a virus).

[0078] In one or more embodiments, the treated biological product exhibits similar stability to an untreated sample of the biological product. For example, a treated biological product may have a shelf life similar to that of an untreated biological product under comparable storage conditions.

[0079] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product does not include adding an excipient to the biological product. For example, a method of treating a biological product may not require or otherwise include adding riboflavin, psoralen, and / or amotosalen to the biological product. Additionally, the method of reducing the titer of one or more biological contaminants in a biological product may not include the use of detergents, solvents, or pH modifying reagents.

[0080] Process-based methods of reducing the titer of biological contaminants

[0081] As is described in greater detail herein, PEP is typically prepared from a blood product. Preparing PEP typically includes centrifugation, fdtration, freezing the sample, and optional lyophilization. Each of these steps may reduce the viral titer of the sample, thereby contributing to reducing the titer of any biological contaminants in the sample.

[0082] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product, whether an exosome product such as PEP or another biological product, includes centrifuging a biological product. In one or more of these embodiments, the method can include centrifuging a blood product so that plasma is separated from the blood. As is described herein, centrifuging a blood product to separate plasma may additionally separate out one or more contaminants. Further centrifugation may further remove contaminants. In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes centrifuging the biological product. In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes removing white blood cells from a blood product, also referred to as leukor eduction. White blood cells may be removed, for example, by fdtration.

[0083] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes filtering the biological product. In one or more of these embodiments, the method includes filtering a blood product, such as plasma. The filter size may be selected to retain exosomes while allowing smaller material to pass through the filter. Alternatively, the filter size may be selected to retain larger contaminants while allowing exosomes to pass through the filter. In one or more embodiments, the method includes using a filter having a molecular weight cut-off (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 includes using a filter having a MWCO of at most 1000 kDa, at most 800 kDa, at most 500 kDa, at most 300 kDa, at most 150 kDa, at most 100 kDa, or at most 80 kDa.

[0084] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes freezing and thawing the biological product. In one or more embodiments, the method includes freezing and thawing a blood product. Freezing and thawing a biological product or a blood product may include freezing the product at least once and, therefore, 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 the product will be frozen once more than it is thawed to yield a frozen final product.

[0085] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes freezing the biological product. In one or more embodiments, freezing a biological product includes 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 a biological product includes incubating the biological product at a temperature of at most 0°C, at most -20 °C, at most -50 °C, or at most -80 °C.

[0086] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes lyophilizing the biological product. Lyophilizing, sometimes referred to as freeze-drying, includes bringing a product to a low temperature in a vacuum and removing frozen water via sublimation. In one or more embodiments, lyophilizing a biological product includes using a commercial freeze dryer. In one or more embodiments, lyophilizing a biological product includes lowering the temperature of the biological product to at most -50 °C, or at most -60 °C. In one or more embodiments, lyophilizing a biological product includes lowering the temperature of the biological product to at least -80 °C, at least -70 °C, or at least -60 °C.

[0087] In one or more embodiments, a method of reducing the titer of a biological contaminant in a biological product can include subjecting the biological product to electromagnetic energy and / or ionizing radiation. As used herein, electromagnetic energy refers to radiant energy having a wavelength and a frequency. Exemplary forms of electromagnetic energy include, but are not limited to, UV-B, UV-C, X-rays, and y-rays. As used herein, ionizing radiation refers to subatomic particles or electromagnetic waves that have sufficient energy to ionize atoms or molecules. Exemplary forms of ionizing radiation include, but are not limited to, X-rays, y-rays, or electron beam radiation. Because some electromagnetic waves have sufficient energy to ionize atoms or molecules, electromagnetic energy and ionizing radiation are not mutually exclusive.

[0088] For the case of viral inactivation, concentration of virus is typically defined as Plaque Forming Units per mL (PFU / mL). In the case of bacterial inactivation, concentration of bacteria is typically defined as Colony Forming Units per mL (CFU / mL).

[0089] UV-C radiation

[0090] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes treating the biological product with ultraviolet (UV)-C radiation. UV-C radiation is typically defined as electromagnetic radiation having a wavelength of 100 nm to 280 nm. UV-C radiation typically has photon energy of 4.43 electronvolt (eV) to 12.4 eV, or 0.710 attojoule (aJ) to 1.987 al.

[0091] The efficacy of inactivation is measured in log kill and is defined as follows:

[0092] Log kill where: No = the initial concentration of biological contaminant (PFU / mL, CFU / mL, etc.), and f = the initial concentration of biological contaminant (PFU / mL, CFU / mL, etc.).

[0093] In one or more embodiments, the relationship between log kill and UV-C fluence is linear, with a small deviation at low fluence values, where no inactivation is detected. This can be described by the following model: where: k = viral inactivation coefficient (cm2 / mJ)

[0094] F = UV-C fluence (mJ / cm2) b = y-intercept offset

[0095] The y-intercept offset models the low fluence values where no inactivation is detected. Some studies have shown that very high fluence values can see a diminishing returns effect, where increases in fluence do not yield an increase in inactivation. These values are typically excluded from calculations to keep the linear model accurate.

[0096] In one or more embodiments, treating a sample with UV-C radiation includes treating the sample with electromagnetic radiation having a wavelength of at least 100 nm to at most 280 nm. However, it should be understood that some electromagnetic radiation may include wavelengths of less than 100 nm or more than 280 nm. In one or more embodiments, treating a sample with UV-C radiation includes treating the sample with electromagnetic radiation having a range of wavelengths, wherein the electromagnetic radiation includes some radiation having a wavelength of at least 100 nm to at most 280 nm. In one or more embodiments, the sample may be treated with electromagnetic radiation having a wavelength of at least 200 nm to at most 280 nm. In one or more embodiments, the sample may be treated with electromagnetic radiation having a range of wavelengths, wherein the electromagnetic radiation includes some radiation having a wavelength of at least 200 nm to at most 280 nm.

[0097] In one or more embodiments, a source of UV-C radiation includes a light electrode diode (LED). One example of a suitable UV-C radiation source is the PEARLAQUA line of water disinfection products (AquiSense Technologies, Erlanger, KY), such as the PEARLAQUA Micro. Such machines expose a sample to UV-C radiation as it flows through the machine. In this way, the dose of UV-C radiation administered to the sample is inversely proportionate 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.

[0098] A sample may be treated with any suitable dose of UV-C radiation. In one or more embodiments, treating a sample with UV-C radiation includes 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 a sample with UV-C radiation includes treating the sample at a maximum flow rate of at most 700 mL / min, at most 650 mL / min, at most 600 mL / min, at most 575 mL / min, at most 550 mL / min, at most 525 mL / min, at most 500 mL / min, at most 475 mL / min, at most 450 mL / min, at most 425 mL / min, at most 400 mL / min, at most 375 mL / min, at most 350 mL / min, at most 325 mL / min, or at most 300 mL / min. In one or more embodiments, treating a sample with UV-C radiation includes treating the sample with UV-C radiation at a flow rate that falls within a range having endpoints defined by any minimum flow rate listed above and any maximum flow rate that is greater than the selected minimum flow rate. Thus, in some embodiments, treating a sample with UV-C radiation includes treating the sample with UV-C radiation at a flow rate of from 150 mL / min to 300 mL / min, from 275 mL / min to 500 mL / min, from 200 mL / min to 275 mL / min, and so on. In one or more embodiments, treating a sample with UV-C radiation includes treating the sample at a flow rate of approximately 250 mL / min. In one or more embodiments, treating a sample with UV-C radiation includes treating the sample at a flow rate of approximately 500 mL / min.

[0099] In one or more embodiments, treating a sample with UV-C radiation includes 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 second, 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 a sample with UV-C radiation includes treating the sample for at most 10 minutes, at most 9 minutes, at most 8 minutes, at most 7 minutes, at most 6 minutes, at most 5 minutes, at most 4 minutes, at most 3 minutes, at most 2 minutes, or at most 1 minute.

[0100] In one or more embodiments, treating the sample with UV-C radiation can include delivering UV-C having an intensity of from 1 mJ / cm2to 250 mJ / cm2. Thus, in one or more embodiments, the delivered UV-C intensity can be a minimum of least 1 mJ / cm2, 2 ml / cm2, 3 mJ / cm2, 4 mJ / cm2, 5 mJ / cm2, 6 mJ / cm2, 7 mJ / cm2, 8 ml / cm2, 9 mJ / cm2, 10 mJ / cm2, 12 mJ / cm2, 15 mJ / cm2, or 20 mJ / cm2. In one or more embodiments, the delivered UV-C intensity can be a maximum of no more than 250 mJ / cm2, no more than 200 ml / cm2, no more than 150 mJ / cm2, no more than 100 mJ / cm2, no more than 90 mJ / cm2, no more than 85 mJ / cm2, no more than 80 ml / cm2, no more than 75 ml / cm2, no more than 70 mJ / cm2, no more than 65 mJ / cm2, no more than 60 mJ / cm2, no more than 5 mJ / cm2, no more than 50 mJ / cm2, no more than 45 mJ / cm2, no more than 40 mJ / cm2, no more than 35 mJ / cm2, or no more than 30 mJ / cm2. In one or more embodiments, the UV-C intensity can be fall within a range having endpoints defined by any minimum intensity listed above and any maximum intensity listed above that is greater than the selected minimum intensity. Thus, for example, in one or more embodiments, the method can include delivering UV-C having an intensity of from 1 mJ / cm2to 150 mJ / cm2, from 10 mJ / cm2to 250 mJ / cm2, from 5 mJ / cm2to 150 mJ / cm2, from 5 mJ / cm2to 100 mJ / cm2, from 5 mJ / cm2to 300 mJ / cm2, from 10 mJ / cm2to 30 mJ / cm2, from 10 mJ / cm2to 80 mJ / cm2, from 10 mJ / cm2to 60 mJ / cm2, from 10 mJ / cm2to 50 mJ / cm2, from 20 mJ / cm2to 30 mJ / cm2, from 20 mJ / cm2to 60 mJ / cm2, from 20 mJ / cm2to 75 mJ / cm2, and so on.

[0101] In one or more embodiments, treating a sample with UV-C ration includes treating the sample with radiation having a predetermined fluence. As used herein, fluence refers to the time- integrated flux of a radiation stream.

[0102] UV-B radiation

[0103] Another method of reducing the titer of one or more biological contaminants in a biological sample includes treating with UV-B radiation. UV-B radiation typically includes electromagnetic radiation having a wavelength of 280 nm to 315 nm. Notably, UV-B radiation is not ionizing, unlike UV-C radiation.

[0104] Typically, reducing the titer of a biological contaminant in a biological sample using UV- B radiation 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 a sample prior to treatment with UV-B radiation. However, in some cases, adding excipients may be undesirable. Thus, when an excipient is used, the excipient concentration may be reduced to render the process more amenable for use in treating biological products. For example, in one or more embodiments, treating the sample with UV-B radiation can include reducing the riboflavin concentration to a final concentration of less than 5% volume / volume in the total solution.

[0105] Electron beam (E-beam) radiation

[0106] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes treating the biological product with electron beam (E-beam) radiation. E-beam radiation is a beam of concentrated, highly charged electrons generated by accelerators capable of producing continuous or pulsed beams. This form of ionizing energy has low penetration and high-dosage rates. As material is passed through the E- beam, energy from the electron is absorbed, disrupting chemical bonds leading to damaged DNA and proteins, rendering microorganisms incapable of replication.

[0107] In one or more embodiments, the method includes treating the biological product with at least 10 kiloGray (kGy), at least 15 kGy, at least 20 kGy, or at least 25 kGy of electron beam radiation. In one or more embodiments, the method includes treating the biological product with at most 60 kGy, at most 55 kGy, at most 50 kGy, at most 45kGy, at most 40 kGy, at most 35 kGy, at most 30 kGy, or at most 25 kGy of electron beam radiation.

[0108] Electron beam radiation can be applied to either frozen or liquid preparations. Thus, in one or more embodiments, the sample may be frozen during exposure to the electron beam radiation.

[0109] As is described herein, treating a biological product with electron beam radiation alone may not be sufficient to reduce the titer of biological contaminants to desired degree. Thus, in one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes treating the biological product with electron beam radiation in addition to a secondary method — e.g., such as UV-C radiation — of reducing the titer of biological contaminants in the biological product.

[0110] Gamma irradiation

[0111] Gamma irradiation utilizes the radioactive beta decay of Cobalt-60 to create a gamma ray field of ionizing radiation. Gamma radiation has a high level of penetrance and can treat material inside of shipping containers, minimizing material handling. Because it relies on the rate of decay of Cobalt-60, gamma dosing is time dependent and can be long and potentially generate heat. Therefore, when gamma radiation is used to reduce the titer of biological contaminants in complex biologies (e.g., exosomes), considerations include exposure time, dose, and temperature of the material. In one or more embodiments, gamma irradiation doses < 50 kGy are sufficient to remove virus from biological products while preserving product potency. Specifically, in an illustrative embodiment, a total final dose of 25 kGy is sued to reduce the titer of biological contaminants while retaining potency of the exosome preparation.

[0112] Combinations of methods In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes subjecting the biological product to more than one round of methods that reduce the titer of biological contaminants. In one or more of these embodiments, the biological product may be subjected to two or more rounds of treatment of the same method to reduce the titer of the biological contaminant. In such cases, the specific parameters used in one round of treatment using a given method may be the same or may be different than the parameters used in another round of treatment using the same method.

[0113] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product includes subjecting the biological product to more than one method that reduce the titer 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 concurrently. However, a sample may be treated with more than one treatment method concurrently as feasible.

[0114] Further, when a combination of treatment methods is used, one may achieve the desired degree of titer reduction using a lower intensity of radiation for each method, which may minimize, or even eliminate, damage to the biological product. Illustrative combinations of treatment methods include, but are not limited to, UV-B + UV-C, UV-B + E-beam, UV-B + y- ray, UV-B + X-ray, UV-C + E-beam, UV-C + y-ray, UV-C + X-ray, y-ray + E-beam, y-ray + X- ray, E-beam + X-ray, etc.

[0115] Viral titer calculation

[0116] Viral titer can be calculated using the Spearman-Karber method, which is known to the art. Briefly, the Spearman-Karber method calculates the negative common logarithm for each tested volume that causes an infection in 50% of the analyzed cultures (TCID50) according to the following formula: wherein m is equal to the common logarithm of the viral titer (logTCIDso / mL), wherein xo is equal to the common logarithm of the delusion factor of the lowest dose that induces viral CPEs in all parallel wells, wherein d is equal to the common logarithm of the serial dilution factor (logio3), wherein p, is equal to the proportion of CPE positive wells at dose z (summed from xo, and wherein vwis equal to the common logarithm of volume conversion factor (mL, logio0.2 for endpoint titration, logio0.3 for LVP). This formula can be used to calculate titer using methods A, B, and C below.

[0117] Method A is applied to endpoint titrations when at least the highest dose (lowest dilution factor) is virus-positive in all tested parallel cultures and higher tested dilutions show a reduced proportion of infections.

[0118] Method B is applied with endpoint titrations where the highest dose is not virus-positive in all tested parallel cultures and higher tested dilutions show a reduced proportion of infections. It is assumed that at a three-fold higher dose, all parallel cultures are virus-positive. The titer is calculated per Method A, with the addition of a virtual dose at a three-fold higher concentration than the highest dose tested.

[0119] Method C is used with LVP analyses where the proportion of virus-positive wells is 15% to <50%. The procedure is the same as for mode B, but the proportion of infections is evaluated for only a single dilution.

[0120] For methods A, B, and C, the standard error (se) may be calculated using the following formula: wherein df is equal to the common logarithm of the serial dilution factor (logio3), wherein pt is equal to the proportion of CPE positive wells at dilution z, and wherein nt is equal to the number of parallel wells tested at dilution z.

[0121] Additionally or alternatively, the Taylor series may be used to calculate viral titer. The Taylor series can be used to calculate titer for LVP analyses where few (<15%) positive wells are detected. The titer is assumed to be in units of TCIDso / mL. The following formula can be used: m = log

[0122] Wherein m is equal to the common logarithm of the viral titer (logTCIDso / mL), wherein D is equal to the sample dilution factor, wherein Vw is equal to the inoculation volume per well, and wherein pwis equal to the proportion of CPE positive wells. If now CPEs are detected in any wells, the titer is reported as less than or equal to the assay limit of detection (LOD). The LOD is calculated from the Poisson distribution based on the probability of a false negative result and is reported as the upper 95% confidence limit. There are two methods of calculating the Poisson distribution. The titer is assumed to be in units of TCIDso / mL.

[0123] First, the standard method of calculating the LOD is typically used when a significant amount of the process fraction volume is analyzed. This method uses the formula:

[0124] , — lag wherein m is equal to the common logarithm of the viral titer (logTCIDso / mL), wherein p is equal to the probability of a false negative result (typically 0.05), wherein Lis equal to the process fraction volume in mL, and wherein v is equal to the volume tested in mb.

[0125] An alternative method can be used when the sampled volume is less than 1% of the total fraction volume. This method uses the formula: wherein m is equal to the common logarithm of the viral titer (logTCIDso / mL), p is equal to the probability of a false negative result (typically 0.05), and v is equal to the volume tested in mL.

[0126] For a single fraction, the total viral load can be calculated using the following formula: wherein At is equal to the common logarithm of the total viral load for fraction z, wherein mi is equal to the common logarithm of the viral titer (TCIDso / mL or VE / mL), and wherein E is equal to the total volume of fraction z in m .

[0127] For a combined pool of fractions, the total viral load can be calculated using the following formula: wherein AT is equal to the common logarithm of the total viral load for the combined fractions, and wherein^ / is equal to the common logarithm of the total viral load for fraction i. Viral titer reduction calculation

[0128] The log reduction value (R) of viral titer can be calculated using the following formula:

[0129] Wherein R is equal to the log reduction value, wherein Ao is equal to the common logarithm of the total viral load for the stating material, and wherein is equal to the common logarithm of the total viral load for the final material. Typically, the starting material is the material prior to processing. The starting material may be “spiked” with a known amount of virus. Typically, the final material is the processed material (e.g., chromatography eluate, virus filtration filtrate, etc.).

[0130] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product results in a log reduction in viral titer of at least 1-fold, at least 2-fold, at least 3-fold, at least 4-fold, at least 5-fold, at least 6-fold, at least 7-fold, at least 8- fold, at least 9-fold, or at least 10-fold in the treated product relative to the untreated product. In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product results in a log reduction of viral titer of at most 15-fold, at most 14-fold, at most 13-fold, at most 12-fold, at most 11-fold, at most 10-fold, or at most 9-fold. In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product results in a reduction in viral titer of 6-fold to 12-fold.

[0131] The desired level of reduction typically varies between viruses. Accordingly, a person having ordinary skill in the art should understand that the fold reduction in viral titer may differ between viruses.

[0132] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product results in a log reduction of human immunodeficiency virus (HIV) titer of at least 10-fold. In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product results in a log reduction of pseudorabies virus (PRV) titer of at least 10-fold. In one or more embodiments, the results in a log reduction of bovine viral diarrhea virus (BVDV) titer of at least 10-fold.

[0133] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product results in a log reduction of Porcine Parvovirus (PPV) titer of at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold. In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product results in a log reduction of hepatitis A virus (HAV) titer of at least 6-fold, at least 7-fold, at least 8-fold, at least 9-fold, or at least 10-fold.

[0134] Typically, a method of reducing the titer of one or more biological contaminants in a biological product results in a log reduction of multiple virus titers. In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product results in a reduction of HIV titer, PRY titer, BVDV titer, PPV titer, HAV titer, or a combination thereof.

[0135] Turbidity

[0136] To determine turbidity, liquid samples of exosomes treated with 1-5 passes of UV-C radiation were pipetted into a 96-well clear bottom plate in triplicate wells. For TO and Tl, 50 pL of samples were pipetted into the plate at 50 pL per well. T3 samples were pipetted at 100 pL per well. Absorbance was measured at 600 nm on the VARIOSKAN Lux plate reader. The method did not significantly alter the turbidity of the samples (FIG. 2). Similar results were obtained on samples subjected to a combination of UV-C radiation and E-beam radiation (FIG. H).

[0137] Thus, in one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product does not significantly change the turbidity of the biological product. In one or more embodiments, a method of treating a biological product may not increase turbidity beyond a predetermined threshold. In one or more embodiments, a method of treating a biological product may not decrease turbidity beyond a predetermined threshold.

[0138] Size and concentration

[0139] To measure the size of treated exosomes, 100 nm polystyrene standard beads were diluted 1 : 1000. Focus was adjusted to sharpen the edges of the beads while minimizing overexposure of the beads. This focus was maintained throughout the remainder of the sample analysis. Test samples were diluted 1 : 1000 prior to running on the NANOSIGHT in particle-free water. Three thirty-second videos of the particles within the flow cell were recorded. The syringe pump infusion rate was set to 50. Analysis parameters were auto-set within the NTA software and results were exported. The concentration of the treated biological product was determined by analyzing videos of the particles in the flow cell. The size and concentration of exosomes in the sample were not significantly changed. Similar results were obtained when samples were subjected to a combination of UV-C radiation and E-beam radiation (FIG. 12; FIG. 13). Further, similar results were obtained when samples were treated with a combination of y radiation coupled with varying degrees of UV-C radiation, prior to (FIG. 24) and following stability testing (FIG. 25).

[0140] Thus, in one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product does not significantly change the size of a biological product. In particular, a method of treating a biological product may not significantly decrease particle size.

[0141] In one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product does not significantly change the concentration of a biological product. In particular, a method of treating a biological product may not significantly decrease particle concentration.

[0142] Angiogenic cytokines

[0143] Samples of exosome product 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, MN). Treatment to reduce the titer of biological contaminants produced no significant change 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).

[0144] Similar results were obtained when the samples were treated with a combination of UV-C radiation and E-beam radiation (FIG. 14). Further, similar results were obtained when samples were treated with a combination of y radiation coupled with varying degrees of UV-C radiation (FIG. 30; FIG. 31).

[0145] Thus, in one or more embodiments, a method of reducing the titer 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 decrease 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).

[0146] Cell proliferation To determine the effect of biological-contaminant-tier-reducing treatment on exosome- induced cell proliferation, UV-C-treated exosomes were added to cultured fibroblasts. Normal human dermal fibroblasts (NHDFs) were seeded in a 96-well plate at a density of 3,200 cells / well and left to adhere overnight. Each PEP testing sample was resuspended in 1 mb H2O and passed through a 0.22 pm syringe filter, then diluted to 10% with serum-free media with heparin (1 U / mL final concentration).

[0147] For the TO and T1 month samples, 200 pL of each diluted sample was added to the 96- well plate the next day after seeding. Cells were left to incubate for at least 96 hours in the INCUCYTE with scans acquired every three hours in phase contrast at 10x magnification. Confluence percentage was normalized to percentage at point of treatment using subtraction.

[0148] For the T3 month samples, one day after plating, cells were incubated with 100 pL of NUCLIGHT (nuclear stain) for 45 minutes. Then, 100 pL of each diluted PEP sample was added to the 96-well plate to create a final 4000-fold NUCLIGHT dilution in 10% PEP. Cells were left to incubate for 96+ hours in the INCUCYTE with scans set to acquire every three hours in phase contrast at 10* magnification (for confluency analysis as in in TO and T1 month) and red fluorescence channel (excitation: 567-607 nm; emission: 622-704 nm for the NUCLIGHT red stain) at 10x magnification (to analyze cell count). Confluence percentage and cell count were normalized to the point of treatment using subtraction. FIG. 6 and FIG. 7 show that reducing the titer of biological contaminants did not significantly affect cell proliferation induced by exosomes.

[0149] Similar results were obtained when samples were subjected to a combination of UV-C radiation and E-beam radiation (FIG. 16; FIG. 21). Further, similar results were obtained when samples were treated with a combination of y radiation coupled with varying degrees of UV-C radiation (FIG. 32).

[0150] In one or more embodiments, cell proliferation may be measured using a nuclear stain, such as NUCLIGHT. The nuclear stain may be used to mark individual cells, and individual cells may be counted to determine the total number of cells at any point during growth. In one or more embodiments, cell proliferation may be measured using microscopy to measure the total area covered by the cultured cells. An increase in the area covered by the cells may be interpreted as cell growth.

[0151] Thus, in one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product does not significantly change the biological product’s effect on cell proliferation. Tn particular, the method may not significantly decrease the effect that the biological product has on cell proliferation. In other words, untreated PEP and a treated PEP biological product may have the same effect on cell proliferation.

[0152] Antioxidant capacity

[0153] The CELL BIOLABS Trolox Equivalent Antioxidant Capacity (TEAC) assay kit was used to test the antioxidant capacity of a sample of exosome product treated to reduce the titer of biological contaminants. The TEAC assay measures total antioxidant capacity in a sample by introducing an oxidized ABTS*+ radical. Hydrogen-donating antioxidants in the sample reduce the radical to neutral ABTS in a concentration-dependent manner. The blue ABTS*+ radical (read at 410 nm) becomes colorless as it is converted back to its neutral form. Briefly, PEP test samples were diluted 1 :20 in assay buffer. Trolox standards were prepared from a stock. Each standard and sample was plated in triplicate into a 96-well clear bottom plate. The ABTS*+ Reagent was added to each well, mixed thoroughly, and incubated for five minutes on an orbital shaker. The plate was read using a plate reader (VARIOSKAN LUX, Thermo Fisher Scientific, Inc., Waltham, MA) at 410 nm and pM Trolox equivalent antioxidant capacity was calculated for each PEP sample based on the Trolox standard curve. FIG. 8 shows that treatment to reduce the titer of biological contaminants did not significant affect antioxidant capacity of exosomes.

[0154] Similar results were obtained when samples were subjected to a combination of E-beam radiation and varying degrees of UV-C radiation (FIG. 20). Further, similar results were obtained when samples were treated with a combination of y radiation coupled with varying degrees of UV-C radiation, prior to (FIG. 28) and following stability testing (FIG. 29).

[0155] Thus, in one or more embodiments, a method of reducing the titer of one or more biological contaminants in a biological product does not significantly change the antioxidant capacity of the biological product. In particular, the method may not decrease the antioxidant capacity of the biological product.

[0156] Enzymatic activity (catalase and alkaline phosphatase)

[0157] The ABNOVA fluorescent alkaline phosphatase kit was used to test the enzymatic activity of each PEP test sample, according to the manufacturer’s instructions. This assay measures the alkaline phosphatase enzymatic activity in a sample by hydrolysis of 4- methylumbelliferyl. In the presence of alkaline phosphatase enzyme, 4-methylumbelliferyl is converted to 4-methylumbelliferone (which is highly fluorescent) and phosphate. The rate at which alkaline phosphatase is hydrolyzed into 4-methylumbelliferone (the rate of fluorescence increase) is directly proportional to the enzyme’s activity. Briefly, PEP test samples were diluted 1 :2 in water, then 10 pL per well was plated in triplicate in a 96-well plate. 90 pL of he provided reagent was added to each well. The samples were incubated for one minute, then read on the plate reader once every minute for at least 16 minutes at an excitation wavelength of 360 nm and an emission wavelength of 450 nm. The average rate (slope of fluorescence over time) of each sample was used to determine sample-to- sample differences in alkaline phosphatase enzymatic activity.

[0158] A catalase enzymatic activity kit may be run using the INVITROGEN Catalase Colorimetric Activity Kit. A bovine catalase standard is provided to generate a standard curve for the assay. Samples were 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. The supplied colorimetric detection reagent was added, followed by diluted horseradish peroxidase (HRP), and incubated at room temperature for 15 minutes. The HRP reacts with the substrate in the presence of hydrogen peroxide to convert the colorless substrate into a pink-colored product. The colored product was read at 560 nm using a plate reader (VARIOSKAN LUX, Thermo Fisher Scientific, Inc., Waltham, MA). Increasing levels of catalase in the samples causes a decrease in hydrogen peroxide concentration and a reduction in pink product. Results are shown in FIG. 9.

[0159] Similar results were obtained when samples were subjected to a combination of E-beam radiation and varying degrees of UV-C radiation (FIG. 19). Further, similar results were obtained when samples were treated with a combination of y radiation coupled with varying degrees of UV-C radiation, prior to (FIG. 26) and following stability testing (FIG. 27).

[0160] Thus, in one or more embodiments, a method of reducing the titer 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 decrease the enzymatic activity of the biological product beyond a predetermined threshold value. As it is used in this context, “enzymatic activity” may refer to the individual activity of one enzyme or the combined activity of two or more enzymes in the biological product. In one or more embodiments, “enzymatic activity” refers to the activity of catalase and / or alkaline phosphatase. Although exemplified in the context of illustrative embodiments in which alkaline phosphatase and / or catalase are measured as proxies for enzymes contained within exosomes, any suitable enzyme or enzyme of particular interest may be used to assess the effects of treating a sample to reduce the titer of biological contaminants on enzymatic activity.

[0161] Reconstitution time

[0162] FIG. 10 provides data showing the time needed to reconstitute lyophilized samples in sterile water. Similar results were obtained when samples were subjected to a combination of E- beam radiation and varying degrees of UV-C radiation (FIG. 18). Further, similar results were obtained when samples were treated with a combination of y radiation coupled with varying degrees of UV-C radiation, prior to (FIG. 22) and following stability testing (FIG. 23).

[0163] Thus, various treatments intended to reduce the titer of biological contaminants did not significantly change the reconstitutability of samples subjected to the treatments.

[0164] Compositions and kits including biological products

[0165] In another aspect, the present disclosure relates to compositions including biological products treated to reduce the titer of biological contaminants. In one or more embodiments, the composition is a pharmaceutically acceptable composition, meaning that the composition is suitable for administration to a subject, such as a mammal.

[0166] The 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, diluent, antibacterial, and / or antifungal agent, isotonic agent, absorption delaying agent, buffer, hydrogel, carrier solution, suspension, colloid, water, and the like. The use of such media and / or agents for pharmaceutical active substances is well known in the art. Except insofar as any conventional media or agent is incompatible with the treated biological product, its use in the therapeutic compositions is contemplated. Supplementary active ingredients also can be incorporated into the compositions. As used herein, “pharmaceutically acceptable” refers to a material that is not biologically or otherwise undesirable, i.e., the material may be administered to an individual along with the treated biological product without causing any undesirable biological effects or interacting in a deleterious manner with any of the other components of the pharmaceutical composition in which it is contained. As noted above, in a surgical setting, exemplary suitable carriers include surgical glue, tissue adhesive, or supportive matrix (e.g., a collagen scaffold). As used herein, “collagen scaffold” refers to a three-dimensional network that includes collagen, such as a hydrogel.

[0167] In one or more embodiments, the supportive matrix includes 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 wherein the composition includes collagen, the collagen may be provided as procollagen, fibrillar collagen, such as type I collagen, type III collagen, or a combination thereof. In embodiments wherein the composition includes collagen, the collagen may be provided as a collagen scaffold. In one or more other embodiments, the extracellular matrix components may be supplied in any suitable form, such as purified recombinant protein.

[0168] A pharmaceutical composition including the treated biological product may be formulated in a variety of forms adapted to a preferred route of administration. Thus, a pharmaceutical composition can be administered via known routes including, for example, oral, parenteral (e.g., intradermal, transcutaneous, subcutaneous, intramuscular, intravenous, intraperitoneal, etc.), or topical (e.g., application to tendon tissue exposed during surgery, intranasal, intrapulmonary, intramammary, intravaginal, intrauterine, intradermal, transcutaneous, rectally, etc.). A pharmaceutical composition can be administered to a mucosal surface, such as by administration to, for example, the nasal or respiratory mucosa (e.g., by spray or aerosol). A pharmaceutical composition also can be administered via a sustained or delayed release.

[0169] Thus, a pharmaceutical composition may be provided in any suitable form including but not limited to a solution, a suspension, an emulsion, a spray, an aerosol, or any form of mixture. The pharmaceutical composition may be delivered in formulation with any pharmaceutically acceptable excipient, carrier, or vehicle. For example, the formulation may be delivered in a conventional topical dosage form such as, for example, a cream, an ointment, an aerosol formulation, a non-aerosol spray, a gel, a lotion, and the like. In embodiments wherein the formulation is a gel, the gel may have any suitable density. For example, for certain embodiments in a surgical setting, the pharmaceutical composition may be formulated as a gel having sufficient density to keep the formulation in a desired location. The formulation may further include one or more additives including such as, for example, an adjuvant, a skin penetration enhancer, a colorant, a fragrance, a flavoring, a moisturizer, a thickener, and the like. Suitable excipients may include, for example, human or bovine collagen, hyaluronic acidbased compounds, human fibrinogen, or human thrombin.

[0170] In one or more embodiments, the compositions described herein may be lyophilized. The lyophilized composition including the treated biological product may be combined with an additional excipient, which may additionally be lyophilized. Components of the lyophilized composition may be co-packaged or may be separately provided and mixed before use to create an exosome-loaded biocompatible scaffold. The lyophilized excipient may be, for example, lyophilized human or bovine collagen, hyaluronic acid-based compounds, human fibrinogen, human thrombin, or other lyophilized powders that form a biocompatible gel when put in contact with bodily fluids (ex. blood or interstitial fluid).

[0171] In one or more embodiments, the compositions described herein may be reconstituted from a lyophilized powder. Lyophilized powder may be reconstituted, for example, by adding the dry powder to a liquid, typically water or a pharmaceutically acceptable buffer. In one or more embodiments, a composition reconstituted from a lyophilized powder may be frozen. The frozen composition may be prepared by first mixing the lyophilized powder with a liquid, such as water or a pharmaceutically acceptable buffer, then bringing the mixture to a temperature at or below the freezing point of the mixture.

[0172] Methods of administering biological products

[0173] In another aspect, the present disclosure relates to methods of administering to a subject a biological product that has been treated to reduce the titer of biological contaminants. In one or more embodiments, a method includes administering a treated biological product to a subject in need thereof. A subject may have or be at risk of having a condition that may benefit from the administration of a treated biological product.

[0174] As used herein, a “subject” can be a human or any non-human animal. Exemplary nonhuman animal subjects include, but are not limited to, a livestock animal or a companion animal. Exemplary non-human animal subjects include, but are not limited to, animals that are hominid (including, for example chimpanzees, gorillas, or orangutans), bovine (including, for instance, cattle), caprine (including, for instance, goats), ovine (including, for instance, sheep), porcine (including, for instance, swine), equine (including, for instance, horses), members of the family Cervidae (including, for instance, deer, elk, moose, caribou, reindeer, etc.), members of the family Bison (including, for instance, bison), feline (including, for example, domesticated cats, tigers, lions, etc ), canine (including, for example, domesticated dogs, wolves, etc.), avian (including, for example, turkeys, chickens, ducks, geese, etc.), a rodent (including, for example, mice, rats, etc.), a member of the family Leporidae (including, for example, rabbits or hares), members of the family Mustelidae (including, for example ferrets), or member of the order Chiroptera (including, for example, bats).

[0175] In one or more embodiments, a composition described herein are administered via injection into / onto a surgical site. A composition as described herein may be administered alone or in addition to traditional surgical repair methods, such as sutures or staples. A composition as described herein also may also be used to enhance the biocompatibility and therapeutic effect of tendon sutures, anchors, patches, or other devices used to repair tendinous injures.

[0176] A formulation may be conveniently presented in unit dosage form and may be prepared by methods well known in the art of pharmacy. Methods of preparing a composition with a pharmaceutically acceptable carrier include the step of bringing the treated biological product into association with a carrier that constitutes one or more accessory ingredients. In general, a formulation may be prepared by uniformly and / or intimately bringing the treated biological product into association with a liquid carrier, a finely divided solid carrier, or both, and then, if necessary, shaping the product into the desired formulations.

[0177] The amount of treated biological product administered can vary depending on various factors including, but not limited to, the content and / or source of the treated biological product being administered, the weight, physical condition, and / or age of the subject, and / or the route of administration. Thus, the absolute weight of treated biological product included in a given unit dosage form can vary widely, and depends upon factors such as the species, age, weight, and physical condition of the subject, and / or the method of administration. Accordingly, it is not practical to set forth generally the amount that constitutes an amount of the biological product effective for all possible applications. Those of ordinary skill in the art, however, can readily determine the appropriate amount with due consideration of such factors.

[0178] In one or more embodiments, the biological product may be a purified exosome product (PEP). In one or more embodiments, a dose of PEP can be measured in terms of the number of PEP exosomes delivered in a dose. Thus, in one or more embodiments, the method can include administering sufficient PEP to provide a dose of, for example, from l * 106PEP exosomes to 1 x 101?PEP exosomes to the subject, although in one or more embodiments the methods may be performed by administering PEP in a dose outside this range. In one or more embodiments, therefore, the method can include administering sufficient PEP to provide a minimum dose of at least 1 x 106PEP exosomes, at least 1 x 107PEP exosomes, at least 1 x 108PEP exosomes, at least 1 x io9PEP exosomes, at least 1 x 1O10PEP exosomes, at least I x lO11PEP exosomes, at least 2x lOnPEP exosomes, at least 3x lOnPEP exosomes, at least 4x lOnPEP exosomes, at least 5x lOnPEP exosomes, at least 6x lOnPEP exosomes, at least 7x 1011PEP exosomes, at least 8x 1011PEP exosomes, at least 9 1011PEP exosomes, at least I x lO12PEP exosomes, 2xl012PEP exosomes, at least 3xl012PEP exosomes, at least 4x io12PEP exosomes, or at least 5x l012PEP exosomes, at least Ix lO13PEP exosomes, or at least I x lO14PEP exosomes.

[0179] In one or more embodiments, the method can include administering sufficient PEP to provide a maximum dose of no more than I x lO15PEP exosomes, no more than I x lO14PEP exosomes, no more than I x lO13PEP exosomes, no more than I x lO12PEP exosomes, no more than I x lO11PEP exosomes, or no more than Ix lO10PEP exosomes.

[0180] In one or more embodiments, the method can include administering sufficient PEP to provide a dose characterized by a range having endpoints defined by any a minimum dose identified above and any maximum dose that is greater than the minimum dose. For example, in one or more embodiments, the method can include administering sufficient PEP to provide a dose of from I x lO11to I xlO13PEP exosomes such as, for example, a dose of from I x lO11to 5x io12PEP exosomes, a dose of from I x lO12to I x lO13PEP exosomes, or a dose of from 5x l012to 1 x 1013PEP exosomes. In certain embodiments, the method can include administering sufficient PEP to provide a dose that is equal to any minimum dose or any maximum dose listed above. Thus, for example, the method can involve administering a dose of I x lO10PEP exosomes, I x lO11PEP exosomes, Sx lO11PEP exosomes, I xlO12PEP exosomes, 5x l012PEP exosomes, I x lO13PEP exosomes, or Ix lO14PEP exosomes.

[0181] Alternatively, a dose of exosome product can be measured in terms of the concentration of PEP upon reconstitution from a lyophilized state. Thus, in one or more embodiments, the methods can include administering PEP to a subject at a dose of, for example, from a 0.01% solution to a 100% solution to the subject, although in one or more embodiments the methods may be performed by administering PEP in a dose outside this range. As used herein, a 100% solution of PEP refers to one vial of PEP (2x lOnexosomes or 75 mg) solubilized in 1 ml of a liquid or gel carrier (e.g., water, phosphate buffered saline, serum free culture media, surgical glue, tissue adhesive, etc.). For comparison, a dose of 0.01% PEP is roughly equivalent to a standard dose of exosomes prepared using conventional methods of obtaining exosomes such as exosome isolation from cells in vitro using standard cell conditioned media.

[0182] In one or more embodiments, therefore, the method can include 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%.

[0183] In one or more embodiments, the method can include administering sufficient PEP to provide a maximum dose of no more than 100%, no more than 90%, no more than 80%, no more than 70%, no more than 60%, no more than 50%, no more than 40%, no more than 30%, no more than 20%, no more than 10%, no more than 9.0%, no more than 8.0%, no more than 7.0%, no more than 6.0%, no more than 5.0%, no more than 4.0%, no more than 3.0%, no more than 2.0%, no more than 1.0%, no more than 0.9%, no more than 0.8%, no more than 0.7%, no more than 0.6%, no more than 0.5%, no more than 0.4%, no more than 0.3%, no more than 0.2%, or no more than 0.1%.

[0184] In one or more embodiments, the method can include administering sufficient PEP to provide a dose characterized by a range having endpoints defined by any a minimum dose identified above and any maximum dose that is greater than the minimum dose. For example, in one or more embodiments, the method can include administering sufficient exosome product to provide a dose of from 1% to 50% such as, for example, a dose of from 5% to 20%. In certain embodiments, the method can include administering sufficient PEP to provide a dose that is equal to any minimum dose or any maximum dose listed above. Thus, for example, the method can involve administering a dose of 0.05%, 0.25%, 1.0%, 2.0%, 5.0%, 20%, 25%, 50%, 80%, or 100%.

[0185] A single dose may be administered all at once, continuously for a prescribed period of time, or in multiple discrete administrations. When multiple administrations are used, the amount of each administration may be the same or different. For example, a prescribed daily dose of may be administered as a single dose, continuously over 24 hours, as two administrations, which may be equal or unequal. When multiple administrations are used to deliver a single dose, the interval between administrations may be the same or different. In one or more certain embodiments, the treated biological product may be administered from a one-time administration, for example, during a surgical procedure.

[0186] In one or more certain embodiments in which multiple administrations of the treated biological product composition are administered to the subject, the treated biological product composition may be administered as needed to heal and / or repair the tendon-bone interface to the desired degree. Alternatively, the treated biological product composition may be administered twice, three times, four times, five times, six times, seven times, eight times, nine times, or at least ten times. The interval between administrations can be a minimum of at least one day such as, for example, at least three days, at least five days, at least seven days, at least ten days, at least 14 days, or at least 21 days. The interval between administrations can be a maximum of no more than six months such as, for example, no more than three months, no more than two months, no more than one month, no more than 21 days, or no more than 14 days.

[0187] In one or more embodiments, the method can include multiple administrations of treated biological product to at an interval (for two administrations) or intervals (for more than two administrations) characterized by a range having endpoints defined by any a minimum interval identified above and any maximum interval that is greater than the minimum interval. For example, in one or more embodiments, the method can include multiple administrations of treated biological product at an interval or intervals of from one day to six months such as, for example, from three days to ten days. In one or more certain embodiments, the method can include multiple administrations of treated biological product at an interval of that is equal to any minimum interval or any maximum interval listed above. Thus, for example, the method can involve multiple administrations of treated biological product at an interval of three days, five days, seven days, ten days, 14 days, 21 days, one month, two months, three months, or six months.

[0188] In the preceding description and following claims, the term “and / or” means one or all of the listed elements or a combination of any two or more of the listed elements; the terms “comprises,” “comprising,” and variations thereof are to be construed as open ended — i.e., additional elements or steps are optional and may or may not be present; unless otherwise specified, “a,” “an,” “the,” and “at least one” are used interchangeably and mean one or more than one; and the recitations of numerical ranges by endpoints include all numbers subsumed within that range (e.g., 1 to 5 includes 1, 1.5, 2, 2.75, 3, 3.80, 4, 5, etc.). In the preceding description, particular embodiments may be described in isolation for clarity. Reference throughout this specification to “one embodiment,” “an embodiment,” “certain embodiments,” “one or more embodiments,” or “some embodiments,” etc., means that a particular feature, configuration, composition, or characteristic described in connection with the embodiment is included in at least one embodiment of the disclosure. Thus, the appearances of such phrases in various places throughout this specification are not necessarily referring to the same embodiment of the disclosure. Furthermore, the particular features, configurations, compositions, or characteristics may be combined in any suitable manner in one or more embodiments. Thus, features described in the context of one embodiment may be combined with features described in the context of a different embodiment except where the features are necessarily mutually exclusive.

[0189] In several places throughout the above description, guidance is provided through lists of examples, which examples can be used in various combinations. In each instance, the recited list serves only as a representative group and should not be interpreted as an exclusive list.

[0190] For any method disclosed herein that includes discrete steps, the steps may be performed in any feasible order. And, as appropriate, any combination of two or more steps may be performed simultaneously.

[0191] As used herein, the terms “preferred” and “preferably” refer to embodiments of the invention that may afford certain benefits under certain circumstances. However, other embodiments may also be preferred under the same or other circumstances. Furthermore, the recitation of one or more preferred embodiments does not imply that other embodiments are not useful and is not intended to exclude other embodiments from the scope of the invention.

[0192] EXAMPLES

[0193] The present invention is illustrated by the following examples. It is to be understood that the particular examples, materials, amounts, and procedures are to be interpreted broadly in accordance with the scope and spirit of the invention as set forth herein.

[0194] Example 1 - PEP generation PEP was generated from at 53 donor units, generating a total of 13 L of material aliquoted across six 2 L bioprocessing bags at varied volumes. The pooled material was then frozen at -80 °C for more than 12 hours.

[0195] The frozen material was thawed in a 37 °C water bath the next day and pooled into bioprocessing bags optimized for centrifugation. The centrifugation bioprocessing bags were processed in two runs of centrifugation, both runs were run at identical parameters of Relative Centrifugal Force (RCF) sufficient to pellet cellular debris. The supernatant from both centrifuge runs were pooled into one 20 L bag. The supernatant was then run through a filter train with pore sizes sufficient to remove macroscale (e.g., 0.2 pm and larger) cellular debris. The yield after filtration was 6.8 L. Approximately 700 m of the clarified material was aliquoted into a smaller bag for easier handling. All material was frozen at -80 °C for more than 12 hours.

[0196] Some aliquots were set aside for lyophilization. 1 mL of the liquid PEP preparation was added per vial. The vials had stoppers inserted at the first ridge and placed into the BT-9EG Benchtop Lyophilizer. The lyophilizer was pulled to <200 mTorr and -80 °C and run for a time sufficient to dry the material. Methods of lyophilizing complex biological products include, but are not limited to, methods described in International Patent Application No. PCT / US2025 / 010770. All vials were taken from the lyophilizer, fully stoppered, and capped.

[0197] Example 2 - Treating PEP 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 the DS. MIRASOL treatment was performed according to the manufacturer’s instructions using a UV-B dose of 5%.

[0199] Viral titer was calculated as described above. Porcine Parvovirus (PPV) titer was measured as a proxy for total viral titer. PPV viral titer was calculated for the sample pretreatment and post-treatment. It was determined that the average log reduction in viral titer across multiple replicates was 3.76.

[0200] Turbidity, particle size, particle concentration, angiogenic cytokine presence, effect on cell proliferation, antioxidant capacity, and enzymatic 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 treated samples. It was determined that MIRASOL treatment of PEP samples adequately reduced the titer of biological contaminants but requires adding excipients to the sample, whereas other techniques for reducing the titer of biological contaminants did not require the use of excipients.

[0201] Example 3 - Treating PEP with electron beam radiation

[0202] The liquid PEP preparation of Example 1 was thawed (Liquid PEP preparation), thawed and lyophilized (Lyophilized powder), or remained frozen (Frozen PEP preparation), then treated with either 50 kiloGray (kGy) or 25 kGy of electron beam radiation according to manufacturer’s instructions.

[0203] Table 1. Viral titer reduction in exosome product treated with electron beam radiation

[0204] Electron beam radiation reduced viral titer less when treating lyophilized powder than when treating either the liquid PEP preparation or the frozen PEP preparation.

[0205] Turbidity, particle size, particle concentration, angiogenic cytokine presence, effect on cell proliferation, antioxidant capacity, and enzymatic activity were measured as described above.

[0206] The level of PDGF-BB in the samples was reduced by approximately 4.5%. Alkaline phosphatase activity was reduced by 5% to 18%. Catalase activity was reduced by 58% The effect of the treated PEP on cell proliferation was slightly reduced compared to untreated PEP.

[0207] Example 4 - Treating PEP with UV-C radiation

[0208] The smaller aliquot (-700 mL) of material generated in Example 1 was thawed in a 37 °C water bath the next day for UV-C treatment. The UV-C chamber (PEARLAQUA, Aqui Sense Technologies, Erlanger, KY) was assembled and prepared for use. An aliquot of untreated material was taken as the control sublot for the batch. This control material was held within the lab unagitated at room temperature throughout the duration of the other treatments. The UV-C chamber was activated and feed material was then passed through the chamber at 250 mL / min, once all material had passed through, the chamber was deactivated, the treated material bag was sealed, and air was pumped through the tubing to clear any residual material, an aliquot was taken of the treated material. This remaining treated material was then used as feed material for another treatment step, repeated as the same above. This was done five times to achieve five sublots of material at varying levels of treatment. Each pass through the UV-C system took approximately 1-3 minutes. In total, the 5-pass group took approximately 30 minutes to complete all treatments.

[0209] Table 2. Viral titer reduction in exosome product treated with UV-C radiation

[0210] Example 5 - Treating PEP with UV-C radiation and electron beam radiation

[0211] Material from Example 1 was thawed and treated with UV-C as indicated in Table 1. After UV-C treatment, the material was either frozen, lyophilized, or remained liquid, and material in each state was subjected to electron beam radiation as indicated in Table 3.

[0212] Table 3. Viral titer reduction in exosome product treated with electron beam radiation and UV-C radiation This Example demonstrates that treating the liquid PEP preparation or the frozen PEP preparation with the combination of UV-C radiation and electron beam radiation resulted in at least a 6.88-fold reduction in PPV titer.

[0213] Example 6 - Pharmacological characterization of PEP treated with UV-C radiation over time.

[0214] Samples generated are listed in Table 4. Samples were tested immediately (TO) and put on stability for analysis at one month (Tl) and three months (T3) at room temperature.

[0215] Table 4. Overview of samples treated with UV-C radiation

[0216] Each sample was resuspended in 1 mb water and placed on an orbital shaker. For TO and Tl, samples were shaken at 60 rpm. The method was updated between Tl and T3 stability pulls, so the T3 samples were shaken at 200 rpm. The vials were observed and timed until the PEP powder cake was fully resuspended. Reconstitution time for each sample was recorded. Each sample was characterized for relevant characteristics, including turbidity, exosome size, exosome concentration, presence of angiogenic cytokines, effect on cell proliferation, antioxidant capacity, and enzymatic activity. Methods of measuring each of these characteristics are described in greater detail herein. The results of each assay are discussed below.

[0217] Turbidity

[0218] There was no trend in turbidity from 1 to 5 UV-C passes in either the room temperature or accelerated one-month stability samples. There was no trend in turbidity from 1 to 5 UV-C passes in the room temperature stability samples at three months. However, all samples had slightly higher absorbance at three months compared to their counterpart at Tl month when comparing the 100 pL samples (FIG. 2).

[0219] Size and concentration

[0220] There was no significant difference in the average particle size between the samples, all falling around 150 nm. There was an insignificant decreasing trend in sample concentration with an increased number of passes through UV-C for samples measured immediately after treatment (TO). There was no significant difference in the concentration of particles in any sample at TO, Tl, or T3.

[0221] Angiogenic cytokines

[0222] Immediately following treatment, levels of ANG-1, EGF, and PDGF-BB were reduced in all samples treated with UV-C radiation. Multiple passes showed differences in the levels of VEGF-A, but these differences are within known product variability (FIG. 3D). While these angiogenic cytokines were slightly reduced with UV-C treatment, they were not thought to be biologically significant. Slight or no further reduction in ANG-1, EGF, PDGF-BB, and VEGF-A was observed in treated samples after one month of storage (FIG. 4) and three months of storage (FIG. 5).

[0223] Cell proliferation

[0224] There were no significant differences observed in the effect of any sample on cell proliferation at TO, Tl, or T3. UV-C treated PEP supports cell proliferation. When added to cell culture of normal human dermal fibroblasts (NHDFs), PEP supported cell proliferation greater than the FBS positive control, even when treated with UV-C. This was observed immediately after treatment (TO) and at one month post-treatment (T l month), as measured by percent confluence in the cell culture plate (FIG. 6).

[0225] UV-C treated PEP supports cell proliferation at three-month stability time point (T3). When added to cell culture of normal human dermal fibroblasts (NHDFs) three months post UV- C treatment, PEP supported cell proliferation greater than the FBS positive control, even when treated with UV-C, as measured by confluence in the cell culture plate and by cell count via nuclear stain (FIG. 7). Antioxidant capacity

[0226] There were no significant differences observed in the antioxidant capacity, as measured by Trolox equivalent antioxidant capacity (TEAC) assay, of any sample at TO, Tl, or T3 (FIG. 8).

[0227] Enzymatic activity

[0228] Catalase activity was significantly reduced after treatment with UV-C radiation (FIG. 9). Alkaline phosphatase activity was insignificantly reduced after treatment with UV-C radiation.

[0229] From this example, it was determined that treating an exosome product with UV-C radiation resulted in generally pharmaceutically acceptable properties and acceptably decreased viral titer. In addition, it was determined that treating an exosome product with UV-C radiation did not significantly affect the stability of the exosome product over time.

[0230] Example 7 - Treating PEP with UV-C radiation and quantifying multiple viral titers

[0231] Samples were treated with UV-C radiation at a high or low dose as describe in Example 4. The titer of multiple viruses, including HIV, PRV, BVDV, PPV, and HAV was measured in each sample. The justification for selecting these viruses is shown in Table 5. The initial titer of each virus and the titer after each treatment is shown below in Table 6.

[0232] Table 6.

[0233] 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 six-fold. The high dose of UVC radiation used was a flow rate of 250 mL / min. The low dose of UVC radiation used was a flow rate of 500 mL / min. Cumulative reduction represents the log viral titer reduction following treatment with high dose UV-C radiation, lyophilization, and inherent reduction.

[0234] For PPV and HAV, sufficient reduction in viral titer was achieved following cumulative treatment. For PRV and BVDV, cumulative treatment was not sufficient to reduce viral titer by the desired amount. For HIV, it was unclear whether the viral titer reduction was sufficient due to the maximum reduction achieved at each step, which is limited by original virus titer.

[0235] Example 8 - Treating PEP with UV-C, E-beam irradiation, and lyophilization

[0236] 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 with material being spiked with virus directly preceding the dosing / material generation step. For the UV-C evaluation, samples were spiked with HIV, PRV, BVDV, PPV, or HAV and subjected to two passes through a UV-C irradiation system at a dose of 250 mL / min. For the E-beam and lyophilization evaluation, samples were subjected to two passes of UV-C at 250 mL / min with no virus spiked into the solution. Then samples were split and half were spiked with HIV, PRV, BVDV, PPV, or HAV before all samples were subjected to E-beam irradiation at a dose of 25 kGy. The separate un-spiked, E-beam irradiated aliquots were then spiked with HIV, PRV, BVDV, PPV, or HAV, and then lyophilized.

[0237] Table 7 is a summary of the viral reduction results generated from combining UV-C irradiation with E-beam irradiation as sequential steps, evaluating the viral reduction at each step. By combining two passes of UV-C irradiation at 250 mL / minute through a turbulent flow cell, a cumulative E-Beam irradiation dose at 25 kGy achieved by dosing each face, and lyophilization in 2 mL aliquots, a minimum total log reduction of at least 10.13 is achieved for all five model viruses. Table 7. Logio viral reduction

[0238] CHARACTERISTICS OF TREATED SAMPLES

[0239] Appearance

[0240] Lyophilized powder cakes were reported as light yellow powder cakes and reconstituted vials were reported as opaque, yellow solutions free of particles.

[0241] Reconstitution time

[0242] Each sample was resuspended in 1 mL of sterile water within the glass vial and placed on an orbital shaker at 60 rpm. The vials were observed and timed until the PEP powder cake was fully resuspended. Reconstitution time for each sample was recorded. Reconstitution times for four illustrative aliquots are down in FIG. 10.

[0243] Turbidity

[0244] Turbidity of PEP subjected to two passes of UV-C, either with or without subsequent E- beam treatment, was measured before and after lyophilization and reconstitution. Results are shown in FIG. 11.

[0245] Particle Size and Concentration

[0246] The mean particle size of PEP subjected to two passes of UV-C, either with or without subsequent E-beam treatment, was determined before and after lyophilization and reconstitution. Results are shown in FIG. 12. Particle concentration was determined for PEP subjected to two passes of UV-C, either with or without subsequent E-beam treatment, was determined before and after lyophilization and reconstitution. Results are shown in FIG. 13.

[0247] Angiogenic cytokines

[0248] The presence of angiogenic cytokines in PEP subjected to two passes of UV-C, either with or without subsequent E-beam treatment, was determined using an automated ELISA system (ELLA, Bio-Techne, Minneapolis, MN) and a multiplex cartridge kit (SIMPLE PLEX, Bio-Techne, Minneapolis, MN) for detecting ANG-1, EGF, PDGF-BB, and VEGF-A. PEP samples were lysed by combining 200 pL resuspended PEP + 50 pL 5X RIPA / protease inhibitor. Samples were vortexed for 15 seconds then incubated at room temperature for five minutes. Samples were centrifuged at 20,000* for 10 minutes at room temperature. Before plating in the automated ELISA cartridge, samples were diluted to a final 10-fold dilution with SD13 (87.5 mb SD13 + 12.5 mL lysed PEP sample). Wash buffer, samples, and high and low controls were plated on the automated ELISA cartridge and run on the automated LISA system instrument according to the manufacturer’s instructions. Results are shown in FIG. 14.

[0249] Proinflammatory Cytokines

[0250] The presence of proinflammatory cytokines IFN-y and IL-ip in PEP subjected to two passes of UV-C, either with or without subsequent E-beam treatment, was determined after lyophilization and reconstitution. Results are shown in FIG. 15.

[0251] Cellular Proliferation

[0252] The ability of PEP, subjected to two passes of UV-C, either with or without subsequent E-beam treatment, to induce cell proliferation of normal human dermal fibroblasts (NHDFs) cultured in standard fibroblast basal media at 37 °C and 5% CO2 after being lyophilized and reconstituted was measured. Results are shown in FIG. 16. The times to >70% confluency are: L- 1 : 73% confluency in 72 hours, L-2: 73% confluency in 69 hours, A-l: 76% confluency in 72 hours, and A-2: 74% confluency in 69 hours.

[0253] Residual Moisture The residual moisture in PEP, subjected to two passes of UV-C, either with or without subsequent E-beam treatment, then lyophilized and reconstituted was measured. Results are shown in FIG. 17.

[0254] Example 9 - Treating PEP with UV-C, y irradiation, and lyophilization

[0255] PEP Preparation

[0256] Platelets were thawed and pooled, then centrifuged at 7186 Relative Centrifugal Force (RCF) for 24 minutes. The supernatant was collected and sent through a fdter train consisting of a 0.45 pm and a 0.22 pm BPS filters (Critical Process Filtration, Inc., Nashua, NH). Approximately IL of the filtrate was partitioned. The remaining filtrate was split into five groups per sublot for different levels of UV-treatment: Untreated, Untreated w / UV handling, U 250 mL / min, 2* 250 mL / min, 3x 250 mL / min. Each group was aliquoted into 50 mb bags (Entegris, Inc., Billerica, MA) and frozen.

[0257] UV-C Treatment

[0258] The UV-C treatment was carried out in a thin film unit (PEARLLAB, AquiSense Technologies, Erlanger, KY). The dose of UV-C radiation received during each pass-through was controlled by the material flowrate and power delivered to the UV-C LEDs. All passes of material were done at 250 mL / min and 100% Intensity, done in one batch per sublot, where material was aliquoted after each pass for storage or for further treatment. The Handling control (MC-#01) was done separate to the other three treated options. Table 8 below outlines the total quantity processed at each step and final yields. Material from these conditions was further partitioned according to the sampling plan.

[0259] Table 8. UV-C treatment yields

[0260] Electron beam Treatment The samples (100 mL) were submitted to E-BEAM Services, Inc. (Lebanon, OH) for treatment. All sample groups, except for the shipping control (ES-#01), received the same dose of 25 kGy. Sample treatments are summarized in Table 9. Table 9

[0261] Gamma Treatment

[0262] The samples (100 mL) were submitted to Steris Life Sciences (Mentor, OH) for treatment. Sample groups received a dose of either 12 kGy or a 25 kGy dose. Sample treatments are summarized in Table 10.

[0263] Table 10

[0264] Stability models

[0265] Vials with PEP lyophilized powder cake were placed in a stability chamber at either 25 °C / 65% Relative Humidity (RH) for ambient / long term storage evaluation or 40 °C / 75% RH for Accelerated stability evaluation. Aliquots of the vials were then removed from the chambers at intervals of either one month or three months and subjected to analytical testing.

[0266] E-BEAM WITH AND WITHOUT UV-C

[0267] Reconstitution time

[0268] Reconstitution time of PEP samples was measured as described in Example 8.

[0269] Reconstitution time of PEP samples did not differ across treatment types, despite minor variability from each lot (FIG. 18). In an ambient stability test, reconstitution time was similar for all samples after one month (approximately four minutes) and after three months (approximately 2-2.5 minutes). Ina an accelerated stability test, reconstitution time for all samples were similar at both the one-month and three-month time points, approximately 2-3 minutes.

[0270] Alkaline phosphatase activity

[0271] Alkaline phosphatase activity, an indicator of enzymatic activity, was measured as described in Example 6. Enzymatic activity was slightly reduced across all samples (FIG. 19), indicating that the extent of UV-C treatment did not affect enzymatic activity.

[0272] Over the course of the stability study, all lots of PEP had increased alkaline phosphatase activity when compared to the initial timepoint (FIG. 19), increasing to approximately 25-30 average rate per minute after one month and approximately 40 average rate per minute (ambient) or approximately 30-35 average rate per minute (accelerated) after three months.

[0273] Antioxidant capacity Antioxidative capacity was measured by TEAC assay. At the initial timepoint (FIG. 20), antioxidative capacity was similar across all PEP samples and slightly elevated compared to the control sample. Over the three-month stability study, antioxidative capacity decreased in all PEP samples at one month but were greater at three months than the initial values at To in both the ambient and accelerated conditions.

[0274] Angiogenic cytokine activity

[0275] Angiogenic cytokines ANG-1, EGF, PDGF-BB, and VEGF-A were analyzed by automated ELISA (ELLA, Bio-Techne, Minneapolis, MN). Concentrations of angiogenic cytokines were variable across PEP samples at baseline, with the most common reduction in concentration observed in the E-Beam + 3 / UV-C sample. Variation between other lots is minor and unlikely to be of biological relevance.

[0276] All four angiogenic cytokines exhibited similar patterns across three-month stability under ambient and accelerated conditions. Specifically, when held under ambient (RT) conditions for three months, the concentrations of angiogenic cytokines decreased slightly or did not change significantly at one month but increased at three months compared to baseline. However, under accelerated (Acc) conditions, the concentrations of angiogenic cytokines increased slightly or did not change significantly at one month but decreased at three months compared to baseline, which is consistent with known product sensitivities.

[0277] Cellular proliferation

[0278] Cellular proliferation upon treatment with PEP samples was measured by confluency as described in Example 8. The results from the initial timepoint (To) are shown in FIG. 21.

[0279] GAMMA IRRADIATION WITH AND WITHOUT UV-C

[0280] Reconstitution time

[0281] Reconstitution time of PEP samples was measured as described in Example 8. Reconstitution time varied somewhat across PEP samples, where treated samples took slightly longer to become homogenous upon reconstitution when compared to the control at To (FIG. 22). Following stability holds under ambient conditions, reconstitution time increased at three months and returned to baseline at six months (FIG. 23 A). For all PEP samples under accelerated conditions, reconstitution time increased across all samples over three months (FIG. 23B).

[0282] Extracellular vesicle size

[0283] Average extracellular vesicle size of PEP samples was measured by NANOSIGHT particle tracking analysis (Malvern Panalytical Ltd., Malvern, United Kingdom) Extracellular vesicle size was consistent across PEP treated with multiple passes of UV-C and irradiation at the initial timepoint (To, FIG. 24), indicating the EV size is not affected by UVC and y exposure.

[0284] In the stability study, average EV size initially decreased but was restored at longer time points. (FIG. 25).

[0285] Alkaline phosphatase activity

[0286] Catalase concentration, as an indicator of enzymatic activity, was measured as described in Example 6.

[0287] Enzymatic activity by catalase concentration was reduced by increasing viral inactivation techniques at the initial timepoint (To, FIG. 26). During the stability study, all PEP samples had reduced catalase concentration when compared to the initial timepoint. This trend was true for both ambient (FIG. 27A) and accelerated (FIG. 27B) conditions.

[0288] Antioxidant capacity

[0289] Antioxidative capacity was measured by TEAC assay. At the initial timepoint (To, FIG. 28), antioxidative capacity was similar across all lots of PEP Drug Product. A minor increase in antioxidative capacity was observed with increased viral inactivation treatment. This increase is unlikely to be biologically detrimental to PEP efficacy. Over the three-month stability study, antioxidative capacity decreased in all PEP samples at one month but increased at three months for PEP stored in ambient stability conditions (FIG. 29A). However, antioxidative capacity of PEP samples stored in accelerated conditions decreased at one month and again at three months for all samples (FIG. 29B). Angiogenic cytokine activity

[0290] Angiogenic cytokines ANG-1, EGF, PDGF-BB, and VEGF-A were analyzed by automated ELISA (ELLA, Bio-Techne, Minneapolis, MN). Concentrations of angiogenic cytokines were consistent across PEP samples for each cytokine at baseline.

[0291] All four angiogenic cytokines were reduced at three-months under both ambient and accelerated conditions (data not shown).

[0292] Cellular proliferation

[0293] Cellular proliferation upon treatment with PEP samples was measured by confluency as described in Example 8. All PEP samples dramatically reduced doubling time.

[0294] The complete disclosure of all patents, patent applications, and publications, and electronically available material (including, for instance, nucleotide sequence submissions in, e.g., GenBank and RefSeq, and amino acid sequence submissions in, e.g., SwissProt, PIR, PRF, PDB, and translations from annotated coding regions in GenBank and RefSeq) cited herein are incorporated by reference in their entirety. In the event that any inconsistency exists between the disclosure of the present application and the disclosure(s) of any document incorporated herein by reference, the disclosure of the present application shall govern. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exact details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

[0295] Unless otherwise indicated, all numbers expressing quantities of components, molecular weights, and so forth used in the specification and claims are to be understood as being modified in all instances by the term “about.” Accordingly, unless otherwise indicated to the contrary, the numerical parameters set forth in the specification and claims are approximations that may vary depending upon the desired properties sought to be obtained by the present invention. At the very least, and not as an attempt to limit the doctrine of equivalents to the scope of the claims, each numerical parameter should at least be construed in light of the number of reported significant digits and by applying ordinary rounding techniques. Notwithstanding that the numerical ranges and parameters setting forth the broad scope of the invention are approximations, the numerical values set forth in the specific examples are reported as precisely as possible. All numerical values, however, inherently contain a range necessarily resulting from the standard deviation found in their respective testing measurements.

[0296] All headings are for the convenience of the reader and should not be used to limit the meaning of the text that follows the heading, unless so specified.

Claims

What is claimed is:

1. A method of treating a biological product to reduce titer of a biological contaminant in the biological product, the method including subjecting the biological product to electromagnetic energy having an ultraviolet (UV) wavelength or shorter or ionizing radiation.

2. The method of claim 1, further comprising lyophilizing the biological product.

3. The method of claim 1 or claim 2, wherein the biological contaminant comprises an enveloped virus or a non-enveloped DNA virus.

4. The method of any previous claim, wherein the biological contaminant is a DNA virus or an RNA virus.

5. The method of any previous claim, wherein the DNA virus is a single-stranded DNA (ssDNA) virus or a double-stranded DNA (dsDNA) virus.

6. The method of any preceding claim, 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).

7. The method of claim 6, wherein the HIV titer is reduced by at least 10-fold, the PRV titer is reduced by at least 10-fold, the BVDV titer is reduced by at least 10-fold, the PPV titer is reduced by at least 6-fold, and / or the HAV titer is reduced by at least 6-fold.

8. The method of claim 1 or claim 2, wherein the biological contaminant comprises a bacterium.

9. The method of claim 1 or claim 2, wherein the biological contaminant comprises a fungus.

10. The method of any preceding claim, wherein the electromagnetic energy comprises UV- B, UV-C, X-rays, or y-rays.

11. The method of claim 10, wherein the biological product is subjected to UV-C at a dose of from 5 mL / min to 500 mL / min.

12. The method of any preceding claim, wherein the ionizing radiation comprises X-rays, y- rays, or electron beam radiation.

13. The method of claim 12, wherein the biological product is treated with 0 kiloGray (kGy) to 55 kGy of electron beam radiation.

14. The method of claim 13, wherein the biological product is treated with 25 kGy of electron beam radiation.

15. The method of any preceding claim, further comprising subjecting the biological product to a second form of electromagnetic energy or ionizing radiation.

16. The method of any preceding claim, further comprising lyophilizing, freezing, centrifuging, or filtering the biological product.

17. The method of any preceding claim, wherein the biological product comprises exosomes.

18. A biological product treated to reduce at least one titer of a biological contaminant compared to an untreated sample of the biological product.

19. The biological product of claim 18, wherein the titer of the biological contaminant is reduced by at least one log compared to an untreated biological product.

20. The biological product of claim 18 or claim 19, wherein the biological product comprises exosomes.

21. The biological product of any one of claims 18-20, wherein the biological product further comprises a lyophilized powder.

22. The biological product of any one of claims 18-21, wherein the biological product further comprises a carrier.

23. The biological product of claim 22, wherein the carrier comprises a solvent.

24. The biological product of any one of claims 18-23, wherein the biological contaminant comprises an enveloped virus or a non-enveloped DNA virus.

25. The biological product of any one of claims 18-24, wherein the biological contaminant is a DNA virus or an RNA virus.

26. The biological product of any one of claims 18-25, wherein the DNA virus is a singlestranded DNA (ssDNA) virus or a double-stranded DNA (dsDNA) virus.

27. The biological product of any one of claims 18-26, wherein the one or more reduced viral titers includes at least one of human immunodeficiency virus (HIV), pseudorabies virus (PRV), bovine viral diarrhea virus (BVDV), Porcine Parvovirus (PPV), or hepatitis A virus (HAV).

28. The biological product of claim 27, wherein the HIV titer is reduced by at least 10-fold, the PRV titer is reduced by at least 10-fold, the BVDV titer is reduced by at least 10-fold, the PPV titer is reduced by at least 6-fold, and / or the HAV titer is reduced by at least 6-fold.

29. The biological product of any one of claims 18-23, wherein the biological contaminant comprises a bacterium.

30. The biological product of any one of claims 18-23, wherein the biological contaminant comprises a fungus.

31. The biological product of any one of claims 18-30, wherein the biological product comprises significantly the same turbidity, particle size, particle concentration, angiogenic cytokine concentration, effect on cell proliferation, antioxidant capacity, or enzymatic activity as an untreated biological product.

32. The biological product of any one of claims 18-31, wherein the biological product is derived from a mammalian blood product.

33. The biological product of claim 32, wherein the mammalian blood product comprises human full blood.

34. The biological product of any one of claims 18-33, wherein the biological product exhibits significantly the same stability as an untreated biological product.

35. A composition comprising: the biological product of any one of claims 18-34; and a pharmaceutically acceptable carrier.

36. A method of treating a subject comprising administering the composition of claim 35 to the subject.

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