Purified mesenchymal stem cell compositions and methods of purifying mesenchymal stem cell compositions

Abstract

To provide pharmaceutical compositions including mesenchymal stem cells that have immunogenicity reduced relative to mesenchymal stem cell compositions purified by centrifugation.SOLUTION: A method for producing a purified mesenchymal stem cell composition, comprising the steps of: (i) obtaining a preparation containing mesenchymal stem cells cultured ex vivo; (ii) contacting the preparation with a wash solution to create a mixture; (iii) agitating the mixture with a centrifugal filtration device; and (iv) recovering the purified mesenchymal stem cell composition.SELECTED DRAWING: Figure 1

Description

[Technical Field]

[0001] Related Applications This application claims priority to U.S. Provisional Patent Application No. 61 / 088,898, filed August 14, 2008, the contents of which are incorporated herein by reference.

[0002] U.S. Federally Sponsored Research or Development [Not applicable] [Microfiche / Copyright Citation] [Not applicable] The present invention relates to purified mesenchymal stem cell compositions and methods for purifying mesenchymal stem cell compositions. [Background technology]

[0003] Mesenchymal stem cells (MSCs) can be found in bone marrow, blood, dermis, periosteum, and other body tissues and can be generated in a variety of in vivo or in vitro ways. Depending on factors and influences, they can differentiate into a variety of cell types, including adipose tissue, loose connective tissue, bone tissue, cartilage tissue, elastic tissue, bone marrow stromal tissue, muscle tissue, fibrous connective tissue, and cardiac tissue. Such cells are disclosed, for example, in U.S. Patent Nos. 5,197,985; 5,226,914; 5,486,359; 5,837,539, and 6,087,113, each of which is independently incorporated by reference in its entirety.

[0004]

[0002] MSCs have been found to engraft and selectively differentiate into lineages such as muscle, bone, cartilage, bone marrow stroma, tendon, and fat, depending on the tissue environment. Because of their function and phenotype, these cells do not provoke adverse immune responses, allowing for the development of products derived from unrelated human donors.

[0005]

[0003] Generally, MSCs are isolated from the tissue from which they are obtained, purified, and then expanded in an appropriate culture medium. The medium contains various components that support MSC expansion, such as serum, including serum proteins (e.g., serum albumin, such as bovine serum albumin); growth factors; and cytokines. After isolation, purification, and culture expansion, the MSCs undergo a series of washes and, optionally, centrifugation. The MSCs can then be frozen and stored in an appropriate cryopreservation medium, such as a cryopreservation medium containing dimethyl sulfoxide ("DMSO"). The MSCs are then thawed immediately prior to administration to the patient.

[0006]

[0004] The manufacturing process for expanding MSCs requires cell culture in the presence of non-autologous serum and cell harvest with non-autologous trypsin; in some methods, the non-autologous serum is fetal bovine serum ("FBS") and the non-autologous trypsin is porcine trypsin. Ex vivo expansion of human MSCs ("hMSCs") using animal reagents results in residual cytotoxicity in the final product. This results in the presence of macromolecules of non-human origin (e.g., porcine and bovine origin) that are associated with the proliferation of hMSCs in a matrix containing a non-human product, and increased amounts of xenogeneic material may be observed after expansion of hMSCs in a matrix containing a human product, relative to hMSCs expanded in a matrix containing a human product.

[0007] Bovine serum albumin ("BSA") is a significant component of FBS. Both BSA and porcine trypsin are known allergens. As such, they can trigger adverse reactions in patients sensitive to bovine and porcine macromolecules and, upon multiple exposures, can sensitize non-allergic patients, resulting in allergic reactions. (See, e.g., Colten HR et al., N Engl J Med, 1975, 292:1050; Moneret-Vautrin A. et al., Allergy, 1991, 46:228; Orta M et al., Ann Allergy Asthma Immunol 2003, 90:446; de Benito V. et al., Allergologia et Immunopathologia, 2001, 29:272.) Increased amounts of FBS present in culture-expanded MSCs may also cause undesirable side effects in patients, such as undesirable immune responses, pulmonary embolism, vasoconstriction, cardiac shock, or death. The presence of residual BSA or porcine trypsin may increase immunogenicity and promote clearance or excretion of MSCs from the recipient. Increased amounts of FBS present in pharmaceutical compositions containing culture-expanded MSCs may increase the risk of transmitting viruses, prion diseases, and xenogeneic proteins to patients receiving such MSC-based therapies. Increased amounts of FBS, specifically BSA, present in pharmaceutical compositions containing culture-expanded hMSCs may initiate an immune response against these xenogeneic substances. For example, if an MSC preparation administered to a patient contains BSA or other xenogeneic proteins, such xenogeneic proteins may trigger an undesirable immune response. The xenogeneic proteins may induce cellular or humoral immune responses (e.g., the development of anti-bovine serum protein antibodies), which may result in less effective MSC proliferation, especially if such xenogeneic proteins become associated with the MSC cell surface membrane. As such, new approaches are needed to reduce the amount of xenogeneic substances, including FBS and specifically BSA, present in pharmaceutical compositions containing culture-expanded MSCs. New approaches should reduce the amount of xenogeneic materials, including sugars, proteins, and other macromolecules, present in culture-expanded MSCs, which may increase the safety profile of the resulting MSC compositions.

[0008]

[0006] Substrates containing alternative serum, such as autologous human serum, have been considered; however, the use of autologous serum is not possible if the amount of cells required in the final MSC product exceeds the amount that can be expanded in a given amount of autologous serum. Furthermore, the use of autologous human serum presupposes that the patient has sufficient time and is sufficiently healthy to donate serum prior to the initiation of MSC therapy. Current conventional MSC culture methods typically require 2 to 10 weeks to isolate, expand, harvest, and purify the appropriate number of cells that constitute a pharmaceutical treatment. In some cases, a pharmaceutical treatment consists of a single dose. In other cases, a pharmaceutical treatment consists of two or more doses. Unfortunately, in some cases, MSC therapy is required less than about 2 weeks from the diagnosis or onset of a clinical condition, or less than about 1 week from the diagnosis or onset of a clinical condition, or less than about 48 hours from the diagnosis or onset of a clinical condition. If MSC therapy is required within a short time of diagnosis or symptom onset of a clinical condition, hMSCs that have already been produced, purified, and cryopreserved offer the significant advantage of being available at the time of diagnosis or symptom onset of acute disease.

[0009]

[0007] Furthermore, human serum, including autologous human serum, poses a statistically significant increased risk of transmitting disease, eg, viral disease, to the recipient of the MSC pharmaceutical composition. Spees et al. reported a series of experiments in fetal calf serum ("FCS") and autologous human serum. (Spees et al., Mol Therapy, 2004, 9: 747 The final composition produced by sequential combination of bases showed a >15-fold reduction in residual FCS per sample by SDS-Page electrophoresis of labeled FCS after 50 wash cycles. Protocols requiring autologous human serum and extensive washing that do not result in a more reproducible final composition, while theoretically attractive, do not provide the quality or consistency necessary to produce a pharmaceutical composition suitable for administration to humans.

[0010]

[0009] Risk doses and thresholds for clinical reactivity among allergic patients have been determined for a number of antigens. (Moneret-Vautrin A. & Kanny G., Curr Opin Allergy Clin Immunol, 2004, 4:215; Bindslev-Jensen C et al., Allergy, 2002, 57:741). These limits have been determined for oral administration of antigens, but not for intravenous ( Threshold values for intravenous (IV) exposure are unknown. (Wensing M. et al., J Allergy Clin Immunol, 2002, 110:915; Taylor SL et al., Clin Exp Allergy, 2004, 34:689) Therapeutic decisions regarding IV administration of compositions containing MSCs are complicated by the absence of threshold data and literature reports indicating that cellular and animal-derived products may cause severe adverse reactions (e.g., anaphylaxis and serum sickness-like illness). (Moneret-Vautrin A et al., Allergy, 1991, 46:228; Orta M et al., Ann Allergy Asthma Immunol 2003, 90:446; de Benito V. et al., Allergologia et Immunopathologia, 2001, 29:272).

[0011]

[0010] As an example, the risk dose and clinical reactivity among allergic patients Limits and thresholds have been determined for a large number of antigens, most of which relate to the food allergen category (Moneret-Vautrin A. & Kanny G., Curr Opin Allergy Clin Immunol, 2004, 4:21). Because these limits were determined for oral administration of the antigen, they were not significant for IV administration. The threshold values for IV exposure to allergens are likely to be different from those for IV administration. Again, the threshold values for IV exposure to allergens remain unknown (Taylor SL et al., Clin Exp Allergy, 2004, 34:689). The absence of threshold data and literature reports indicating that cellular and animal-derived products may cause serious adverse reactions (e.g., anaphylaxis and serum sickness-like illness) preclude the use of therapeutic agents manufactured in the presence of bovine or porcine products (Orta M. et al., Ann Allergy Asthma Immunol 2003, 90:446).

[0012]

[0011] Perotti et al. describe a technique useful for removing DMSO from umbilical cord blood for cryopreservation. (Perotti CG et al., Transfusion, 2004, 44(6):900-906). Calmels et al. mention centrifugal filtration as a useful technique for removing DMSO from hematopoietic stem cell grafts (Calmels B et al., Bone Marrow Transplant., 2003, 31(9):823-828). Hampson et al. mention a method for washing cultured bone marrow mononuclear cells. (US2008 / 0175825). Residual BSA levels after washing from cell culture supernatants were reported to be less than about 3 μg / ml. Using a Cytomate instrument to wash bone marrow mononuclear cells, Hampson et al. obtained a post-wash cell viability of about 70%. Hampson et al. suggested that this significant drop in cell viability may have been due to cell damage caused by mechanical forces applied during the process.

[0013] The protocol, which requires extensive washing of the cells, is suitable for administration to humans. They do not provide the quality or consistency necessary to manufacture pharmaceutical compositions. Furthermore, the effect of adequate washing protocols on cell viability and the efficacy of pharmaceutical compositions containing such cells is unknown.

[0014] Furthermore, many published purification protocols involve the transfer of MSC-containing intermediate products. The development of a closed manufacturing system for the production of an MSC pharmaceutical composition is considered a significant achievement in the art, as it involves at least one step involving the inclusion of a product in a closed system, where the product is exposed to the external environment (i.e., it is not a closed system). A closed system carefully controls the quantity and quality of materials entering and leaving the system, as well as the manner in which these materials enter and exit. [Prior art documents] [Patent documents]

[0015] [Patent Document 1] U.S. Patent No. 5,197,985 [Patent Document 2] U.S. Patent No. 5,226,914 [Patent Document 3] U.S. Patent No. 5,486,359 [Patent Document 4] U.S. Patent No. 5,837,539 [Patent Document 5] U.S. Patent No. 6,087,113 [Patent Document 6] US2008 / 0175825 [Non-patent literature]

[0016] [Non-Patent Document 1] Colten HR et al., N Engl J Med, 1975, 292:1050 [Non-patent document 2] Moneret-Vautrin A. et al., Allergy, 1991, 46:228 [Non-patent document 3] Orta M et al., Ann Allergy Asthma Immunol 2003, 90:446 [Non-patent document 4] de Benito V. et al., Allergologia et Immunopathologia, 2001, 29:272 [Non-Patent Document 5] Spees et al., Mol Therapy, 2004, 9: 747 [Non-patent document 6] Moneret-Vautrin A. & Kanny G., Curr Opin Allergy Clin Immunol, 2004, 4:215 [Non-Patent Document 7] Bindslev-Jensen C et al., Allergy, 2002, 57:741 [Non-patent document 8] Wensing M. et al., J Allergy Clin Immunol, 2002, 110:915 [Non-Patent Document 9] Taylor SL et al., Clin Exp Allergy, 2004, 34:689 [Non-Patent Document 10] Moneret-Vautrin A. & Kanny G., Curr Opin Allergy Clin Immunol, 2004, 4:21 [Non-Patent Document 11] Perotti CG et al., Transfusion, 2004, 44(6):900-906 [Non-Patent Document 12] Calmels B et al., Bone Marrow Transplant., 2003, 31(9):823-828 Summary of the Invention [Problem to be solved by the invention]

[0017] In consideration of these challenges, (1) the patient's allergies to residual ingredients in the product It is necessary to determine a threshold dose that will minimize the risk of sexual reactions; (2) provide a method for purifying hMSC compositions to reduce the amount of residual components, including allergens, to below a threshold level while minimizing cell damage and maintaining cell viability; and (3) provide hMSC compositions that contain less than a threshold amount of residual components, including allergens, limited cell damage, and a high proportion of viable cells.

[0018] In summary, the state of the art regarding methods for producing pharmaceutical MSC compositions is This includes one long felt need, namely, reducing the immunogenicity of MSC compositions cultured in non-human serum. Furthermore, the present technology described and claimed herein surprisingly identifies a challenge not previously recognized as a significant drawback in the conventional art, namely, reducing MSC aggregation. [Means for solving the problem]

[0019] Some aspects of the present technology relate to MSC compositions purified by centrifugation. The present invention discloses pharmaceutical compositions comprising MSCs with reduced immunogenicity. Some aspects of the present technology include the step of administering to a subject a pharmaceutical composition, comprising administering to the subject a pharmaceutical composition, a pharmaceutical composition, or a method for ... Regarding the decreased D 90 The present invention discloses a pharmaceutical composition comprising MSCs that exhibit the following:

[0020] Some embodiments of the present technology exhibit reduced adhesion of individual MSCs to each other. A pharmaceutical composition comprising MSCs is disclosed. Some aspects of the present technology are pharmaceutical compositions comprising MSCs, wherein discloses a pharmaceutical composition in which MSCs are purified by centrifugal filtration after expansion in culture.

[0021]

[0020] Some aspects of the present technology simultaneously (i) reduce the immunogenicity of the MSC composition. and (ii) a pharmaceutical composition comprising MSCs purified by centrifugal filtration, which reduces the average size of MSC aggregates by reducing the adhesiveness of individual MSCs.

[0022] Some embodiments of the present technology have reduced immunogenicity and reduced aggregation tendency. Another aspect of the present technology discloses a pharmaceutical composition comprising purified MSCs purified by centrifugal filtration, which simultaneously (i) reduces the immunogenicity of the MSC composition; and (ii) reduces the average size of the MSC aggregates.

[0023] Some embodiments of the present technology disclose a pharmaceutical composition comprising MSCs and DMSO. do.

[0023] Furthermore, some aspects of the present technology may be used to identify cells that remain after expansion in a medium containing, for example, BSA. Disclosed are pharmaceutical compositions comprising MSCs that have been purified to reduce the amount of foreign material, such as proteins, present in the MSCs, which exhibit a superior safety profile, for example, due to reduced immunogenicity of such compositions.

[0024] Another aspect of the present technology is the detection of cell surface membrane molecules, extracellular nucleic acids (DNA / RNA) and Disclosed is a pharmaceutical composition comprising purified MSCs, which is reduced in the amount of substances including other cell debris.Such pharmaceutical composition can show a good safety profile, for example, by reducing the adhesiveness of individual MSCs, thereby reducing the average size of MSC aggregates.Such reduction in adhesiveness can achieve the reduction in the average size of MSC aggregates.

[0025] Furthermore, some embodiments of the present technology allow for the production of comparable amounts of unpurified culture-enhanced M The present invention relates to compositions comprising culture-expanded hMSCs with reduced amounts of residual FBS components, specifically BSA, relative to SCs. In some of these embodiments, the amount of BSA in the composition comprising purified, culture-expanded MSCs is about 10-1,000-fold less than the amount present in a comparable amount of unpurified, culture-expanded MSCs.

[0026]

[0026] Yet another aspect of the present technology is a purified human mesenchymal stem cell and a human mesenchymal stem cell. The present technology relates to methods for purifying cells. More specifically, certain embodiments relate to pharmaceutical compositions comprising hMSCs, wherein the amount of extracellular, cell surface, and transmembrane molecules in such compositions is reduced by one log (the term "log" as used herein means base 10 log) relative to a comparable amount of unpurified, culture-expanded hMSCs. Other embodiments relate to one or more pharmaceutical compositions containing less than about 10 μg / mL of residual BSA. Some embodiments of this technology utilize a D of about 18 μm to about 25 μm. 90 The present invention relates to a pharmaceutical composition comprising MSCs exhibiting the following:

[0027] In an additional aspect, the present technology provides a pharmaceutical composition comprising culture-expanded hMSCs. Methods for producing such compositions, wherein the amount of extracellular, cell surface, and transmembrane molecules in such compositions is reduced by one log relative to a comparable amount of unpurified, culture-expanded hMSCs. Another embodiment relates to a method for producing a pharmaceutical composition comprising culture-expanded hMSCs containing less than about 10 μg / mL residual BSA. Yet another embodiment relates to a method for producing a pharmaceutical composition comprising culture-expanded hMSCs containing less than about 10 μg / mL residual BSA. A further embodiment relates to a method for producing a pharmaceutical composition comprising culture-expanded hMSCs having a D of about 18 μm to about 25 μm. 90 Some embodiments of the present technology relate to methods for producing a pharmaceutical composition comprising culture-expanded hMSCs, the pharmaceutical composition comprising hMSCs exhibiting a D of about 18 μm to about 25 μm, the hMSCs comprising less than about 10 μg / mL of residual BSA, and 90 The present invention relates to a composition exhibiting the following formula:

[0028]

[0028] Yet another aspect of the present technology is the ability to produce comparable amounts of unpurified culture-expanded MSCs. In contrast, compositions comprising culture-expanded hMSCs have reduced amounts of xenogeneic material, including sugars, proteins, and other macromolecules. In some embodiments, the amount of xenogeneic material in a composition comprising purified culture-expanded MSCs is about 1 log less than the amount present in a comparable amount of unpurified culture-expanded MSCs.

[0029] In yet additional embodiments, the present technology provides a method for administering such products to patients, particularly to patients receiving I It concerns the determination of certain limits of residual components in hMSC products that would minimize the risk of allergic reactions in patients receiving them via the V route.

[0030] Furthermore, in some embodiments, the present technology provides a method for purifying hMSCs, The present invention relates to a method of purifying an hMSC preparation by contacting the hMSC preparation with a wash solution, agitating the preparation, and recovering the purified hMSCs. [Brief explanation of the drawings]

[0031] [Figure 1] FIG. 1 is a representative apparatus that can be used to wash and purify mesenchymal stem cells according to aspects of the present technology. [Figure 2]

[0032] FIG. 2 is a photograph (10x magnification) of the crude MSC composition. [Figure 3]

[0033] FIG. 3 is a photograph (10x magnification) of an MSC composition purified by centrifugation. [Figure 4]

[0034] FIG. 4 is a photograph (10x magnification) of an MSC composition purified by centrifugal filtration. DETAILED DESCRIPTION OF THE INVENTION

[0032]

[0035] Some embodiments of the present technology provide pharmaceutical MSC compositions with reduced amounts of xenogeneic material. Some aspects of the present technology further identify and solve the previously unrecognized challenge of providing pharmaceutical MSC compositions with reduced aggregation tendencies. Individually and collectively, these two solutions provide pharmaceutical MSC compositions, particularly pharmaceutical hMSC compositions, that exhibit enhanced therapeutic efficacy and a superior safety profile.

[0033]

[0036] In at least one aspect, the technology described herein provides purified MSCs. The present invention provides a composition comprising: a composition comprising: a) a residual BSA of less than about 55 μg / mL; b) a composition comprising: a) a residual BSA of less than about 55 μg / mL; c) a composition comprising: a) a residual BSA of less than about 55 μg / mL; d ... e) some embodiments related to this aspect of the technology, the composition comprises less than about 42 μg / mL; h) some embodiments, the composition comprises less than about 25 μg / mL; h) some embodiments, the composition comprises less than about 13 μg / mL; h) some embodiments, the composition comprises less than about 10 μg / mL; i) other embodiments, the composition comprises between about 7 μg / mL and about 15 μg / mL of residual BSA; i) still other embodiments, the composition comprises between about 8 μg / mL and about 12 μg / mL of residual BSA. In some embodiments related to this aspect of the technology, the composition comprises purified MSCs, wherein the composition comprises less than about 50 μg / mL residual BSA; alternatively, less than about 45 μg / mL residual BSA; alternatively, less than about 40 μg / mL residual BSA; alternatively, less than about 35 μg / mL residual BSA; alternatively, less than about 30 μg / mL residual BSA; alternatively, less than about 25 μg / mL residual BSA; alternatively, less than about 20 μg / mL residual BSA; or alternatively, less than about 15 μg / mL residual BSA. Residual BSA obtained by published methods is generally less than 1×10 6 The reported dose is approximately 30-700 μg of BSA per cell (Spees et al., Mol Therapy, 2004, 9: 747 As detailed in the Examples below, the over 200-fold reduction in BSA between compositions and methods published in the art represents a significant, surprisingly, and unexpected increase in the safety margin of MSC pharmaceutical compositions.

[0034]

[0037] During incubation of MSCs in BSA-containing media, BSA acts as a barrier to MSC cell proliferation. To accurately assess total BSA levels after incubation of MSCs in BSA-supplemented cell culture medium, measurements must be obtained that account for BSA in the supernatant as well as BSA that has become bound to the MSCs. For example, cells in an aliquot of the suspension can be lysed and then assayed for BSA levels. The method provides total BSA levels, including both free and cell-bound BSA.

[0035]

[0038] Furthermore, some aspects of the present technology provide compositions comprising purified MSCs, MSCs with a D of approximately 18 μm to approximately 30 μm 90 In some embodiments, the MSCs have a D of about 18 μm to about 25 μm. 90 In some embodiments, MSCs have a D of about 20 μm to about 25 μm. 90 In some embodiments, the MSCs have a D of less than about 30 μm; alternatively, less than about 25 μm; or alternatively, less than about 20 μm. 90 Shows.

[0036]

[0039] Some embodiments of the present technology include pharmaceutical MSC compositions having less than about 55 μg / mL containing less than 100% residual BSA, and the MSCs have a D of about 18 μm to about 30 μm. 90 In other embodiments of this aspect of the technology, the composition comprises less than about 42 μg / mL of residual BSA, and the MSCs have a D of about 18 μm to about 30 μm. 90 In yet other embodiments, the composition comprises less than about 25 μg / mL of residual BSA, and the MSCs have a D of about 18 μm to about 30 μm. 90 In some embodiments, the composition comprises less than about 13 μg / mL of residual BSA, and the MSCs have a D of about 18 μm to about 30 μm. 90In some embodiments, the composition comprises less than about 10 μg / mL of residual BSA, and the MSCs have a D of about 18 μm to about 30 μm. 90 In other embodiments, the composition comprises about 7 μg / mL to about 15 μg / mL of residual BSA, and the MSCs have a D of about 18 μm to about 30 μm. 90 In yet other embodiments, the composition comprises between about 8 μg / mL and about 12 μg / mL of residual BSA, and the MSCs have a D of between about 18 μm and about 30 μm. 90 In some embodiments, MSCs have a D of about 18 μm to about 25 μm. 90 and the composition contains less than about 55 μg / mL of residual BSA. In some embodiments, the MSCs have a D of about 20 μm to about 25 μm. 90 and the composition contains less than about 55 μg / mL of residual BSA. Some embodiments of the present technology are compositions comprising purified MSCs, wherein the MSCs have a D of about 18 μm to about 25 μm. 90 and the composition comprises about 8 μg / mL to about 12 μg / mL of residual BSA. In some embodiments, the MSCs have a D of about 20 μm to about 25 μm. 90 and the composition comprises about 8 μg / mL to about 12 μg / mL of residual BSA.

[0037]

[0040] As used herein, "aggregate" refers to a single or multiple particles, including aggregates, agglomerates, and aggregations. "Aggregation" refers to the totality of a plurality of individual cells that have gathered together into clusters through adhesion or greater. As used herein, "aggregation" refers to the tendency of cells to aggregate. It was initially hypothesized, but later demonstrated through experiments detailed in the Examples section of this patent application, that purified MSC preparations exhibit a reduced tendency to form aggregates. Without wishing to be bound by theory, it is believed that these MSC aggregates do not disperse efficiently after administration and are of sufficient size to potentially cause fatal pulmonary embolism.

[0038]

[0041] This technology has been shown to be effective in preventing the formation of aggregates containing MSCs, which may lead to pulmonary embolism. We were the first to recognize that increased amounts of xenogenous material can specifically cause increased cell adhesion, possibly due to certain xenogenous material interacting with membrane-bound sugars, proteins, or other macromolecules. Furthermore, current hMSC manufacturing practices result in increased cell surface material, including sugars, proteins, and other macromolecules (e.g., CD105 and CD166), present in harvested hMSC compositions. Certain macromolecules, whether endogenous or exogenous, increase the adhesive properties of MSCs. As MSCs become more adherent, they exhibit an increased tendency to aggregate with one another. Such aggregates may potentially increase the risk of pulmonary embolism in recipients of hMSC pharmaceutical compositions. For example, BSA forms noncovalent bonds with MSC cell membranes, increasing the immunogenicity of MSCs and also inhibiting MSC adhesiveness. As such, the present technology has identified and solved a previously unrecognized problem by providing compositions of MSCs that have a reduced tendency to aggregate.

[0039]

[0042] As such, mass and size can be simultaneously measured, such as in centrifugal filtration. Techniques for treating cells by selection are preferred over techniques for sequential selection for mass, then size, or vice versa. Some embodiments of the present technology provide pharmaceutical MSC compositions comprising a plurality of MSCs, including one or more mesenchymal stem cell aggregates, and D of the aggregates. 90 Some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, the compositions comprising one or more mesenchymal stem cell aggregates, and the D of the aggregates. 90 Some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, the compositions comprising one or more mesenchymal stem cell aggregates, and the D of the aggregates. 90Indeed, some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, wherein the compositions do not contain detectable mesenchymal stem cell aggregates.

[0040]

[0043] Some embodiments of the present technology provide a pharmaceutical MSC composition comprising about 55 μg / mL and residual BSA, and wherein the composition comprises one or more mesenchymal stem cell populations and the D of the populations. 90 In other embodiments related to this aspect of the technology, the composition comprises less than about 42 μg / mL residual BSA, and the composition comprises one or more mesenchymal stem cell aggregates, wherein the D of the aggregates 90 In yet other embodiments, the composition comprises less than about 25 μg / mL residual BSA, and the composition comprises one or more mesenchymal stem cell aggregates, wherein the D of the aggregates 90 In some embodiments, the composition comprises less than about 13 μg / mL residual BSA, and the composition comprises one or more mesenchymal stem cell aggregates, wherein the D of the aggregates 90 In some embodiments, the composition comprises less than about 10 μg / mL residual BSA, and the composition comprises one or more mesenchymal stem cell aggregates, wherein the D of the aggregates 90 is less than about 150 μm.

[0041]

[0044] Some embodiments of the present technology include a pharmaceutical MSC composition comprising a plurality of MSCs. , one or more mesenchymal stem cell aggregates, wherein the aggregates do not comprise more than 1,000 MSCs. Some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, comprising one or more mesenchymal stem cell aggregates, wherein the aggregates do not comprise more than 750 MSCs. Some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, comprising one or more mesenchymal stem cell aggregates, wherein the aggregates do not comprise more than 500 MSCs. Some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, comprising one or more mesenchymal stem cell aggregates, wherein the aggregates do not comprise more than 200 MSCs. Some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, comprising one or more mesenchymal stem cell aggregates, wherein the aggregates do not comprise more than 100 MSCs. Some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, the compositions comprising one or more mesenchymal stem cell aggregates, wherein the aggregates do not comprise more than 50 MSCs. Some embodiments of the present technology include pharmaceutical MSC compositions comprising a plurality of MSCs, the compositions comprising one or more mesenchymal stem cell aggregates, wherein the aggregates do not comprise more than 10 MSCs.

[0042]

[0045] After incubation of hMSCs in the medium for a certain period of time, extracellular molecules and cell membranes were analyzed. Numerous molecules, including molecules bound to hMSCs, may be present in the medium. For example, such molecules may include xenogeneic substances such as BSA and other non-human molecules. In addition, substances produced by the hMSCs themselves may be present in the medium after incubation for a period of time. For example, such molecules may include secreted proteins such as cytokines and growth factors, as well as molecules expressed on the cell surface of hMSCs. It may be desirable to purify the hMSCs after incubation in the medium for a period of time to remove molecules present in the medium, including extracellular molecules and molecules bound to the cell membrane. Such purification may reduce or prevent the tendency of hMSCs to aggregate, reduce the size of any hMSC aggregates that form, or completely inhibit the formation of hMSC aggregates.

[0043]

[0046] Without wishing to be bound by theory, further purification may reveal that the IL-16 receptor agonist expressed on the MSC cell surface It is undesirable to purify MSCs beyond the minimum safety limits disclosed herein, as this may reduce the amount of adhesion molecules, such as integrins, required for MSCs to exert their therapeutic effects. Over-purified MSCs lack the amount of adhesion molecules necessary for them to adhere to target sites within the body. In some embodiments, systemically administered MSCs home to inflammatory sites within the body. These inflammatory sites exhibit a higher expression profile of adhesion molecules and further induce conformational changes in adhesion molecules as a means of increasing the affinity of MSCs for inflamed tissue. If MSCs lack the corresponding adhesion molecules, they will not adhere to inflamed tissue and will continue to circulate until apoptosis. Therefore, it is desirable to reduce the expression of adhesion molecules on MSCs to the level necessary to prevent assembly, but not to the extent that they lose their ability to adhere to inflamed tissue sites.

[0044]

[0047] The desired removal of foreign substances, such as extracellular molecules and cell surface membrane molecules, , serum proteins such as BSA, and other non-human-derived reagents for hMSC culture, such as porcine trypsin. In some embodiments of the present technology, the amount of xenogeneic material in a composition comprising purified culture-expanded hMSCs is about 1 log less than the amount present in a comparable amount of unpurified culture-expanded hMSCs. In some embodiments, the amount of xenogeneic material after purification is about 2 log less than the amount present in a comparable amount of unpurified culture-expanded hMSCs. In some embodiments, the amount of xenogeneic material after purification is about 3 log less than the amount present in a comparable amount of unpurified culture-expanded hMSCs. In some embodiments, the amount of xenogeneic material after purification is about 4 log less than the amount present in a comparable amount of unpurified culture-expanded hMSCs. In some embodiments, the amount of xenogeneic material after purification is about 5 log less than the amount present in a comparable amount of unpurified culture-expanded hMSCs. In some embodiments, the culture-expanded hMSC composition is substantially free of xenogeneic material. In some embodiments, there is no detectable xenogeneic material in the composition comprising culture-expanded hMSCs.

[0045]

[0048] In an additional embodiment of the present technology, a composition comprising purified, culture-expanded hMSCs is provided. The amount of xenogeneic material in the purified hMSCs is about 10 to about 1,000-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of xenogeneic material after purification is about 25 to about 750-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of xenogeneic material after purification is about 50 to about 500-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of xenogeneic material after purification is about 100 to about 300-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of xenogeneic material after purification is about 200-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs.

[0046]

[0049] In another embodiment of the present technology, a composition comprising purified culture-expanded hMSCs The amount of BSA after purification is about 1 log less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of BSA after purification is about 2 logs less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of BSA after purification is about 3 logs less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of BSA after purification is about 4 logs less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of BSA after purification is about 5 logs less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. Furthermore, in some embodiments, culture-expanded hMSC compositions can be substantially free of BSA. In other embodiments, there is no detectable BSA in compositions comprising purified, culture-expanded hMSCs.

[0047]

[0050] In some embodiments of the present technology, compositions containing purified culture-expanded hMSCs are The amount of BSA in the composition is about 10 to about 1,000-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of BSA after purification is about 25 to about 750-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of BSA after purification is about 50 to about 500-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of BSA after purification is about 100 to about 300-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of BSA after purification is about 200-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs.

[0048]

[0051] In some embodiments of the present technology, compositions containing purified culture-expanded hMSCs are The amount of extracellular nucleic acid in the composition is about 1 log less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of extracellular nucleic acid after purification is about 2 logs less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of extracellular nucleic acid after purification is about 3 logs less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of extracellular nucleic acid after purification is about 4 logs less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of extracellular nucleic acid after purification is about 5 logs less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In other embodiments, the culture-expanded hMSC composition is substantially free of extracellular nucleic acid. In some embodiments, there is no detectable extracellular nucleic acid in the composition comprising culture-expanded hMSCs after purification.

[0049]

[0052] In some embodiments of the present technology, compositions containing purified culture-expanded hMSCs are The amount of BSA and extracellular nucleic acid in the composition is each about 10 to about 1,000-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments of the present technology, the amount of BSA and extracellular nucleic acid in the composition comprising purified, culture-expanded hMSCs is each about 25 to about 750-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments of the present technology, the amount of BSA and extracellular nucleic acid in the composition comprising purified, culture-expanded hMSCs is each about 50 to about 500-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments of the present technology, the amount of BSA and extracellular nucleic acid in the composition comprising purified, culture-expanded hMSCs is each about 100 to about 300-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments of the present technology, the amounts of BSA and extracellular nucleic acids in compositions comprising purified culture-expanded hMSCs are each greater than or equal to a comparable amount of unpurified hMSCs. This is approximately 200-fold less than the amount present in culture-expanded hMSCs.

[0050]

[0053] Furthermore, in some embodiments of the present technology, the purified culture-expanded hMSCs can be The amount of extracellular nucleic acid in a composition comprising the purified hMSCs is about 10 to about 1,000-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of extracellular nucleic acid after purification is about 25 to about 750-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of extracellular nucleic acid after purification is about 50 to about 500-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of extracellular nucleic acid after purification is about 100 to about 300-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In some embodiments, the amount of extracellular nucleic acid after purification is about 200-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs. In other embodiments, the amount of extracellular nucleic acid after purification is about 1000-fold less; alternatively, about 900-fold less; alternatively, about 800-fold less; alternatively, about 700-fold less; alternatively, about 600-fold less; alternatively, about 500-fold less; alternatively, about 400-fold less; alternatively, about 300-fold less; alternatively, about 200-fold less; alternatively, about 100-fold less; alternatively, about 50-fold less; or alternatively, about 25-fold less than the amount present in a comparable amount of unpurified, culture-expanded hMSCs.

[0051]

[0054] In some embodiments of the present technology, the purified MSCs are stored in a suitable cryopreservation medium. For example, the pharmaceutical MSC composition may contain MSCs and about 20% DMSO. In other embodiments, the pharmaceutical MSC composition may contain MSCs and about 10% DMSO. In other embodiments, the pharmaceutical MSC composition may contain MSCs and about 3.8% DMSO. In some embodiments, DMSO can be added to purified MSCs.

[0052]

[0055] As used herein, the term "treating" refers to treating a disease, disorder, or condition. "Treating" as used herein means reversing, preventing, alleviating, or inhibiting the progression of a disease, disorder, or condition, or one or more symptoms of the disease, disorder, or condition. As used herein, "treating" may also mean reducing the incidence or number of occurrences of a disease, disorder, or condition in a mammal compared to an untreated control population, or compared to the same mammal prior to treatment. For example, "treating" as used herein may mean preventing a disease, disorder, or condition, and may include delaying or preventing the onset of a disease, disorder, or condition, or delaying or preventing symptoms associated with a disease, disorder, or condition. As used herein, "treating" may also mean reducing the severity of a disease, disorder, or condition, or symptoms associated with such a disease, disorder, or condition, before the disease, disorder, or condition is afflicted. Such prevention or reduction of the severity of a disease, disorder, or condition before afflicted relates to administration of the compositions of the technology described herein to a subject who, at the time of administration, is not afflicted with the disease, disorder, or condition. As used herein, "treating" may also mean preventing the recurrence of a disease, disorder, or condition, or one or more symptoms associated with such disease, disorder, or condition. The terms "therapy," "treatment," and "therapeutically," as used herein, refer to the act of treating as defined above.

[0053]

[0056] The term "culture-expanded" with respect to hMSCs refers to cells grown under standard cell growth conditions. means that hMSCs are (i) essentially free of cells of hematopoietic origin; and (ii) passaged one or more times in minimal essential medium supplemented with 10% FBS (by volume) to generate increased numbers of undifferentiated MSCs.

[0054]

[0057] The term "pharmaceutical composition" refers to the final pharmaceutically acceptable product and any It refers to a composition at any stage of a manufacturing process, including any in-process intermediates.

[0058] The pharmaceutical composition of the present technology comprises contacting a preparation containing mesenchymal stem cells with a washing solution, The preparation and washing solution can be agitated, and purified mesenchymal stem cells can be collected.

[0055]

[0059] In some embodiments, the wash solution comprises one or more ionic or The irrigation solution can include an electrolyte solution containing an ionizable compound. Such compounds include, but are not limited to, sodium chloride, sodium gluconate, sodium acetate trihydrate, potassium and magnesium chloride, and sodium and potassium phosphate. The irrigation solution can comprise a balanced electrolyte solution, which comprises sodium, potassium, chloride, or a combination thereof, at concentrations appropriate to maintain normal osmolality. Balanced electrolyte solutions include known salt solutions used in a variety of settings, including, for example, fluid electrolyte replacement therapy, tissue and cell irrigation solutions, and diluents for cells and other factors.

[0056]

[0060] In one embodiment, for example, the irrigation solution can be a non-pyrogenic isotonic solution, It contains approximately 526 mg of sodium chloride, 502 mg of sodium gluconate (CH 11 There are 368 mg of sodium acetate trihydrate (C2H3NaO2·3H2O), 37 mg of potassium chloride, and 30 mg of magnesium chloride. One such commercially available isotonic electrolyte solution is sold as PlasmaLyte A, a product of Baxter Healthcare Corporation, Deerfield, Illinois. There are.

[0057]

[0061] In another embodiment, the mesenchymal stem cells are stored in a bag, e.g., a cell source bag, a washing solution bag, The cells are washed in an apparatus that includes a recirculating wash bag, a rotating membrane filter with an inlet and an outlet, a filtrate bag, a mixing compartment, a final product bag for the washed cells, and appropriate tubing. The apparatus is a closed system, thereby reducing the possibility of contamination.

[0058]

[0062] Unwashed MSCs from the cell source bag are mixed with the wash solution in a centrifugal filter device. The resulting suspension of mesenchymal stem cells in the wash solution is then fed to a rotating membrane filter via an inlet. The filtrate containing the wash solution is withdrawn from the rotating membrane filter via a first outlet, and a concentrated suspension of MSCs is withdrawn from the rotating membrane filter via a second outlet and fed into a recirculating wash bag. The MSCs are then withdrawn from the recirculating wash bag, mixed with additional wash solution, and sent back to the rotating membrane filter. Once the recirculating wash of the MSCs is complete, the washed MSCs are sent to a product bag.

[0059]

[0063] Figure 1 shows a representative device for washing or purifying MSCs. An example of such a device is further described in U.S. Pat. No. 6,251,295. The device shown in U.S. Pat. No. 6,251,295 may include, for example, a recirculation bag 5 having a top port 2 and a bottom port 1; a rotary membrane filter 6 having an inlet 11 for a dilute suspension of MSCs, an outlet 14 for a concentrated suspension of MSCs, and an outlet 24 for filtrate; and a filtrate bag 30 having an inlet 29. It may further include one or more of a washed cell bag 46 having an outlet 47, an unwashed cell bag 44 having an outlet 45, and a wash solution bag 7 having an outlet 21. Top port 2 of bag 5 is connected to connector 49 by tubing 8. Port 21 of wash solution bag 7 is connected to Y-connector 55 by tubing 15, which is connected to connector 49 by tubing 20 having clamp C1. The mouth 45 of the unwashed cell bag is connected by tubing 43 with clamp C3 to Y-connector 53 and then by tubing 51 to connector 49. Connector 49 serves as a mixing compartment for the unwashed cells in wash solution from bag 44 and the unwashed cells in wash solution from bag 5. The cells in the wash solution and the wash solution from bag 7 are recirculated. Connector 49 is connected by tubing 10 to inlet 11 of rotating membrane filter 6. Filtrate outlet 24 of spinner 6 is connected by tubing 23 to Y-connector 54 and by tubing 26 to inlet 29 of filtrate bag 30. Connector 55 is connected by tubing 52 with clamp C2 to connector 54. Connector 54 is connected by tubing 41 to pressure transducer 50. Outlet 14 of spinner 6 is connected by tubing 13 to bottom port 1 of bag 5. Y-connector 53 is connected by tubing 48 with clamp C4 to inlet 47 of washed cell bag 46.

[0060]

[0064] During recirculation washing, the MSC suspension in washing solution is drawn from the bag 5 through the top port 2. and flows via tube 8 to mixing compartment 49. MSC suspension is drawn from bag 44 via port 45 and (with clamp C3 open and clamp C4 closed) via tube 43 to Y-connector 53 and then by transfer pump P2 via tube 51 to mixing compartment 49. Wash solution is drawn by buffer pump P2 from bag 7 via port 21 and tube 15 to connector 55. With clamp C1 open, wash solution flows via tube 20 to mixing compartment 49. The MSC suspension in wash solution flows from mixing compartment 49 via tube 10 to inlet 11 of spinner 6. A concentrated suspension of MSCs in wash solution flows via outlet 14 of spinner 6, via tube 13 and inlet 1 into bag 5 by recirculation pump P3. Filtrate flows through outlet 24 and tubing 23 of spinner 6 to connector 54 and, with clamp C2 closed, through tubing 26 and inlet 29 into filtrate bag 30 by pump P4. Recirculation washing continues until the desired amount of target components has been removed from the MSCs. Clamps C1, C2, and C3 are then closed, clamp C4 is opened, and the direction of pump P1 is reversed so that the washed MSC suspension flows from bag 5 through tubing 8, 51, and 48 and port 47 into washed cell bag 46. The lines, bag, and spinner are then rinsed by closing clamps C1 and C3, opening clamps C4 and C2, and using pump P2 to sequentially pump buffer with pumps P1 and P3 to rinse the spinner, bag, and tubing.

[0061]

[0065] The purification step may include sequential or simultaneous centrifugation and filtration.

[0066] Centrifugal filters containing rotating membranes remove platelets and antibodies from blood products. Representative centrifugal filtration devices include those disclosed in, for example, U.S. Pat. Nos. 5,034,135; 5,053,121; 5,234,608; 5,536,475; and 6,251,295, each of which is independently incorporated by reference in its entirety.

[0062]

[0067] In one or more embodiments, the spin membrane filter has a pore size of about 3 μm. In another embodiment, the membrane filter has a pore size of about 4 μm.

[0063]

[0068] In some embodiments of the present technology, the post-wash viability of the cells is greater than about 60%. In other embodiments, the post-wash viability of the cells is greater than about 70%. In further embodiments, the post-wash viability of the cells is greater than about 80%. In still further embodiments, the post-wash viability of the cells is greater than about 90%. In still further embodiments, the post-wash viability of the cells is greater than about 95%.

[0064]

[0069] A particular embodiment of the present technology is a purified pharmaceutical MSC composition comprising approximately 55 μg / m L residual BSA; wherein the composition comprises one or more mesenchymal stem cell aggregates and D of the aggregates. 90 is less than about 150 μm; and wherein the post-purification viability of the MSCs is greater than about 80%. In other embodiments related to this aspect, the composition comprises less than about 42 μg / mL residual BSA; the composition comprises one or more mesenchymal stem cell populations, and the D 90 and the post-purification viability of the MSCs is greater than about 80%. In yet other embodiments, the composition comprises less than about 25 μg / mL residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates 90 In some embodiments, the composition comprises less than about 13 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates is greater than about 80%. 90In some embodiments, the composition comprises less than about 10 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates is greater than about 80%. 90 and the post-purification viability of the MSCs is greater than about 80%. In some embodiments, the composition comprises between about 7 μg / mL and about 15 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates 90 and the post-purification viability of the MSCs is greater than about 80%. In other embodiments, the composition comprises about 8 μg / mL to about 12 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates 90 are less than about 150 μm; and the post-purification viability of MSCs is greater than about 80%.

[0065]

[0070] A particular embodiment of the present technology is a purified pharmaceutical MSC composition comprising approximately 55 μg / m L residual BSA; wherein the composition comprises one or more mesenchymal stem cell aggregates and D of the aggregates. 90 In other embodiments of this aspect of the present technology, the composition comprises less than about 42 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates is less than about 150 μm; and wherein the post-purification viability of the MSCs is greater than about 70%. ... 90 In yet another embodiment, the composition comprises less than about 25 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates is greater than about 70%. 90In some embodiments, the composition comprises less than about 13 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates is greater than about 70%. 90 In some embodiments, the composition comprises less than about 10 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates is greater than about 70%. 90 and the post-purification viability of the MSCs is greater than about 70%. In some embodiments, the composition comprises between about 7 μg / mL and about 15 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates is greater than about 150 μm. 90 In another embodiment, the composition comprises about 8 μg / mL to about 12 μg / mL of residual BSA; the composition comprises one or more mesenchymal stem cell aggregates, and the D of the aggregates is greater than about 70%. 90 are less than about 150 μm; and the post-purification viability of MSCs is greater than about 70%.

[0066]

[0071] The techniques described herein will now be described with reference to the following examples; however, The scope of the present technology is not thereby limited. It should be understood that the scope of the present technology should not be limited to the specific embodiments described above. The present technology can be practiced other than as specifically described and still be within the scope of the claims. [Example]

[0067] Example 1: Residual Heterologous Protein Limits in Compositions for IV Administration

[0072] The Absorption, Distribution, Metabolism, Excretion and Toxicity ("ADMET") properties of MSC pharmaceutical compositions are demonstrated. reported sporadic and unpredictable deaths in rodent test populations receiving MSCs. When studying multiple parameters, residual amounts of xenogeneic proteins, residual amounts of other debris resulting from expansion in the medium, and the degree of cell aggregation were identified as significant contributing factors to the adverse events observed in preclinical experiments, and later proven to be significant contributing factors.

[0068]

[0073] This technique is based on the clinical response thresholds established for oral administration of antigens. However, it has been revealed and understood that the threshold for IV exposure to antigens is still unknown. As such, the present technology recognized that the problem of understanding the threshold for IV exposure to antigens needed to be resolved before safe MSC pharmaceutical compositions and manufacturing processes could be designed accordingly. To understand adverse reactions associated with cellular and animal-derived products, such as anaphylaxis and serum sickness-like illness, in the case of IV administration of compositions containing MSCs, animal models were used to confirm the possibility of xenogeneic residual antigens.

[0069]

[0074] Ovalbumin ("OVA") is structurally related to BSA and BSA was chosen as the representative heterologous protein because it is significantly more immunogenic than trypsin and OVA, and has been documented in a significant number of published reports. BSA has significantly lower allergenic potential than OVA (Hilton J. et al., Food Chem Toxicol, 1997, 35:1209).

[0070]

[0075] OVA was administered by systemic non-mucosal intraperitoneal (IP) injection, which This route was considered to be most relevant to IV administration, as IP and IV routes of antigen administration in animals have been shown to produce similar results (Shepard et al., Infection and Immunity, 1982, 38: 673).

[0071]

[0076] For the calculation of BSA and trypsin limit resistance in MSC pharmaceutical compositions, detection The lowest cumulative dose of OVA that did not trigger a possible IgE response was selected. The lowest cumulative OVA dose that did not trigger sensitization when delivered IP was 10 μg / mouse, which corresponds to 500 μg / kg, based on an average mouse body weight of 20 g. Therefore, the safe cumulative dose of animal protein residues in the MSC pharmaceutical composition for non-susceptible patients undergoing MSC treatment is 500 μg / kg. According to this safety limit, a 100 kg patient could be safely administered an MSC pharmaceutical composition containing less than about 50 mg of animal protein; a 40 kg patient could be safely administered an MSC pharmaceutical composition containing less than about 20 mg of animal protein; or a 5 kg pediatric patient could be safely administered an MSC pharmaceutical composition containing less than about 2.5 mg of animal protein.

[0072]

[0077] Recognize and resolve issues related to limited IV exposure to antigens to ensure safe MSC therapy. Suitable manufacturing process parameters for producing the pharmaceutical composition are contemplated herein. 6 For a regimen consisting of two IV infusions of MSCs / kg body weight, the residual BSA and trypsin limits per MSC pharmaceutical composition (830 μg, equivalent to 55 μg / mL) were calculated as shown in Table 1.

[0073] [Table 1]

[0074] Example 2: Purity of MSC pharmaceutical compositions

[0078] The possible effects of culture-expanded MSCs on possible changes in lung function were investigated. To evaluate the purity of SC pharmaceutical compositions, we used: (1) MSC populations; and (2) vehicle, consisting of 85% PlasmaLyte A, 10% DMSO, and 5% FBS (by volume). A pharmaceutical hMSC composition was prepared consisting essentially of:

[0075]

[0079] The experiments shown in this Example 2 were carried out using a vehicle (purchased as a sterile component). that there was no certification of the strength, purity, and composition of the Plasma-Lyte A ingredients; With the exception of the above, the test articles were manufactured in accordance with the US Food and Drug Administration's Good Laboratory Practice Regulations, as codified in 21 C.F.R. 58. Manufacturing practices were followed. Species selection and number of animals tested were supported by the FDA guidelines for Expanded Acute Studies, the ICH Harmonized Tripartite Guidelines, the FDA Guidance for Human Somatic Cell Therapy and Gene Therapy, and generally accepted procedures for preclinical pharmaceutical testing.

[0076]

[0080] A population of MSCs derived from rat bone marrow was cultured as follows: The collected bone marrow was The cells were pooled, counted, and centrifuged at 100g for 10 minutes. The cells were then placed in a flask containing 10% FBS, 45% Ham's solution, and flushed with Hank's balanced solution. Cells were plated at 120x10^6 cells / cm^2 in F-12, 45% α-Minimum Essential Medium ("α-MEM") supplemented with 100 U / ml penicillin G and 100 μg / ml streptomycin sulfate. Flasks were incubated at 37°C in 10% CO2. After 8 days, nonadherent cells were removed, and remaining cells were detached with 0.05% porcine trypsin and 0.53 mM EDTA. Adherent cells were replated at 2,000 cells / cm^2. Two (2) subsequent passages were performed at 4-day intervals.

[0077]

[0081] After expansion, an aliquot of the cell suspension was obtained to assess residual BSA levels. After lysing the cells in the aliquots, an ELISA was performed to determine the residual BSA, and the results are shown in Table 2. The MSCs in α-MEM were then frozen in cryovials containing known numbers of MSCs.

[0078]

[0082] To prepare the dosage formulation for each concentration, the estimated final suspension volume was adjusted to 9 The desired cell concentration per cryovial was calculated from the estimated cell count and the desired cell concentration, assuming 0% MSC viability. The amount of vehicle required to obtain the estimated final suspension volume was placed in a conical tube. The cryovials containing the MSC population were transferred from liquid nitrogen storage to a water bath until thawed to a semi-liquid state. Approximately 0.5 mL of vehicle was placed into each cryovial to complete the suspension. The MSC population was transferred to a conical tube, at which point the population was purified by either (A) Basic Centrifugation; or (B) Centrifugal Filtering.

[0079]

[0083] (A) Basic centrifugation. The cell / vehicle suspension was centrifuged at approximately 500 g force at 4°C. The mixture was centrifuged for 10 minutes (1,480-1540 3,000 RPM in a Beckman GS-6R rotor GH3.8). The supernatant was removed and kept in a separate vial. The pelleted cells were resuspended in vehicle, if necessary. The MSCs were counted and viability was confirmed. Based on the total cell count, the amount of vehicle necessary to obtain the desired final suspension volume was added to provide the hMSC pharmaceutical composition. The composition purified by basic centrifugation was assayed by ELISA to determine residual BSA; the results are shown in Table 2.

[0080]

[0084] (B) Centrifugal filtration. The cell / vehicle suspension is filtered through a rotating membrane with a pore size of approximately 4 μm. In a Cytomate Cell Processing System (sold by Baxter in 2007) with a thrombus filter and using a Residual Fold Reduction (RFR) setting of 150, The supernatant was then washed with PBS for approximately 25 minutes at room temperature. The MSCs were counted and viability was confirmed. The compositions purified by centrifugal filtration were assayed by ELISA to determine residual BSA, and the results are shown in Table 3.

[0081]

[0085] Based on the total cell count, calculate the amount of vehicle needed to achieve the desired final suspension volume. Additionally, hMSC pharmaceutical compositions were administered. All hMSC pharmaceutical compositions were prepared less than 4 hours prior to administration. The animals were administered via a femoral catheter. As shown in Table 2 below, the MSC pharmaceutical composition purified by centrifugal filtration did not cause any deaths, while the MSC pharmaceutical composition purified by traditional centrifugation resulted in 80% mortality. The cause of death was confirmed to be pulmonary embolism.

[0082] [Table 2]

[0083]

[0086] Among the several parameters subsequently investigated were the residues present in the MSC composition. A second analysis comparing the amount of residual BSA to the No Observed Adverse Events Level ("NOAEL") limit, the results of which are presented in Table 3, gave the most surprising results.

[0084] [Table 3]

[0085]

[0087] Table 2. Basic centrifugation-purified MSC composition injected into 10 Of the rats, seven died shortly after administration, one died the day after administration, and two survived 14 days until scheduled terminal necropsy. Before proceeding with additional preclinical or clinical phase studies, it was desirable to increase the safety margin provided by the basic centrifugation-purified MSC composition.

[0086]

[0088] Additional toxicology testing was performed on MSC pharmaceutical compositions purified by centrifugal filtration. In this experiment, 120 rats were given 10x10 6 pieces / kg, 40x10 6 pcs / kg or 75x10 6 Cells were administered at a dose of 120 / kg. Of the 120 rats studied, only two died during the study. Further supporting the entirely binary outcome of this experiment (i.e., complete survival or complete lethality), animals receiving the centrifugally filtered MSC pharmaceutical composition were just as likely to die (75x10%) as those purified by basic centrifugation. 6 It was also noted that even at a dose level of 2 (84 cells / kg), few clinical symptoms were observed.

[0087]

[0089] After this experiment was performed, all cell processing protocols were adapted for centrifugal filtration.

[0090] As shown in Table 3, the MSC composition purified by basic centrifugation was 1 was found to have residual BSA of 0.29 μg / mL, and6 In comparison, an MSC composition purified by centrifugal filtration was found to have residual BSA of 0.13 μg / mL, resulting in a NOAEL of 40×10 cells / kg. 6 This resulted in a NOAEL of 10 cells / kg, thus representing an approximately 10-fold increase in the safety margin. The approximately 20-fold decrease in residual BSA between the unpurified MSC composition and the MSC composition purified by centrifugation places the NOAEL limit at a clinically significant dose (i.e., 10x10 6 pieces / kg, 40x10 6 pcs / kg or 75x10 6 cells / kg) and still 37.5x10 6 Since 10 cells / kg resulted in complete mortality, it was quite surprising to realize that a further 10-fold increase in purity would increase the NOAEL limit by another log and still result in complete survival of the test population.

[0088]

[0091] When analyzing combined beef and pork residues, centrifugal filtration is used to separate animal proteins. The residual levels of β-glucan were reduced by approximately 1,000-fold relative to basic centrifugation. This 1,000-fold reduction reduces the maximum tolerated dose for bolus injection by 20x10 6 Individual cells / kg~65x1 0 6 The dose was increased to 100 cells / kg.

[0089]

[0092] Unpurified MSC composition (Figure 2), MSC composition purified by centrifugation (Figure Photographs taken under 10x magnification of MSC composition purified by centrifugal filtration (Figure 3) and centrifugal filtration (Figure 4) show the reduced tendency of MSC composition purified by centrifugal filtration to form aggregates.

[0090]

[0093] We now describe additional aspects of the technology described herein; however, The scope is not intended to be limited thereby. It should be understood that the scope of the present technology should not be limited to the specific embodiments described below. The present technology can be practiced other than as specifically described and still be within the scope of the claims. Additional embodiments of the present technology include the following.

[0091]

[0094] A pharmaceutically acceptable composition comprising purified mesenchymal stem cells, comprising one or and D of those populations. 90 is less than about 150 μm.

[0092]

[0095] The collective D 90 A pharmaceutically acceptable composition according to paragraph

[0094] , wherein the particle size is less than about 100 μm.

[0096] The collective D 90 A pharmaceutically acceptable composition according to paragraph

[0095] , wherein the particle size is less than about 50 μm.

[0093]

[0097] A pharmaceutically acceptable composition described in any one of paragraphs

[0094] to

[0096] , wherein the viability of the purified mesenchymal stem cells is greater than about 70%.

[0098] A pharmaceutically acceptable composition described in paragraph

[0097] , wherein the viability of the purified mesenchymal stem cells is greater than about 80%.

[0094]

[0099] Any of paragraphs

[0094] to

[0098] further containing dimethyl sulfoxide (DMSO) A pharmaceutically acceptable composition according to any one of the preceding claims. [000100] A pharmaceutically acceptable composition described in paragraph

[0099] , comprising about 10% DMSO.

[0095] [000101] The pharmaceutically acceptable composition of paragraph

[0099] , comprising about 3.8% DMSO. . [000102] A pharmaceutically acceptable composition comprising purified mesenchymal stem cells, comprising less than about 55 μg / mL of residual bovine serum albumin, and wherein the mesenchymal stem cells have a D of about 18 μm to about 30 μm. 90 A pharmaceutically acceptable composition comprising:

[0096] [000103] The composition of paragraph [000102], wherein the composition contains less than about 42 μg / mL of residual bovine serum albumin. [000104] The composition of paragraph [000103], wherein the composition contains less than about 25 μg / mL of residual bovine serum albumin.

[0097] [000105] The composition of paragraph [000104], wherein the composition contains less than about 13 μg / mL of residual bovine serum albumin. [000106] The composition of paragraph [000105], wherein the composition contains less than about 10 μg / mL of residual bovine serum albumin.

[0098] [000107] The composition of paragraph [000102], wherein the composition comprises from about 7 μg / mL to about 15 μg / mL of residual bovine serum albumin. [000108] The composition of paragraph [000107], wherein the composition comprises about 8 μg / mL to about 12 μg / mL of residual bovine serum albumin.

[0099] [000109] The mesenchymal stem cells have a D of about 18 μm to about 25 μm. 90 The composition according to any one of paragraphs [000102] to [000108], [000110] The mesenchymal stem cells are grafted to a D 90 The composition of paragraph [000109],

[0100] [000111] The composition described in any of paragraphs [000102] to [000110], wherein the mesenchymal stem cells are cultured and expanded in a medium containing bovine serum albumin. [000112] The composition described in any of paragraphs [000102] to [000110], wherein the mesenchymal stem cells are cultured and expanded in a medium containing animal serum.

[0101] [000113] The composition of paragraph [000112], wherein the animal serum is human serum. [000114] The composition of paragraph [000112], wherein the animal serum is non-human serum. [000115] The composition of paragraph [000114], wherein the non-human serum is bovine serum.

[0102] [000116] The composition of paragraph [000114], wherein the non-human serum is porcine serum. [000117] The composition of any one of paragraphs [000102] to [000116], wherein the viability of the purified mesenchymal stem cells is greater than about 70%.

[0103] [000118] The composition of paragraph [000117], wherein the viability of the purified mesenchymal stem cells is greater than about 80%. [000119] The composition of any one of paragraphs [000102] to [000118], further comprising dimethyl sulfoxide (DMSO).

[0104] [000120] The pharmaceutically acceptable composition of paragraph [000119], comprising about 10% DMSO. [000121] The pharmaceutically acceptable composition of paragraph [000119], comprising about 3.8% DMSO.

[0105] [000122] A method of producing a purified mesenchymal stem cell composition, comprising: (i) obtaining a preparation containing ex vivo cultured mesenchymal stem cells; (ii) contacting the preparation with a wash solution to form a mixture; (iii) agitating the mixture in a centrifugal filter; and (iv) recovering the purified mesenchymal stem cell composition. A method including the steps of:

[0106] [000123] The mesenchymal stem cells cultured ex vivo are cultured in a medium containing bovine serum albumin. The method of paragraph [000122], wherein the cells are cultured in a [000124] The ex vivo cultured mesenchymal stem cells are cultured in a medium containing animal serum. The method described in paragraph [000122].

[0107] [000125] The method of paragraph [000124], wherein the animal serum is human serum. [000126] The composition of paragraph [000124], wherein the animal serum is non-human serum. [000127] The composition of paragraph [000126], wherein the non-human serum is bovine serum.

[0108] [000128] The composition of paragraph [000127], wherein the non-human serum is porcine serum. [000129] The method of any one of paragraphs [000122] to [000128], wherein the purified mesenchymal stem cell composition contains less than about 55 μg / mL of residual bovine serum albumin.

[0109] [000130] The method of paragraph [000129], wherein the purified mesenchymal stem cell composition contains less than about 42 μg / mL of residual bovine serum albumin. [000131] The method of paragraph [000130], wherein the purified mesenchymal stem cell composition contains less than about 25 μg / mL of residual bovine serum albumin.

[0110] [000132] The method of paragraph [000131], wherein the purified mesenchymal stem cell composition contains less than about 13 μg / mL of residual bovine serum albumin. [000133] The method of paragraph [000132], wherein the purified mesenchymal stem cell composition contains less than about 10 μg / mL of residual bovine serum albumin.

[0111] [000134] The method described in any one of paragraphs [000122] to [000128], wherein the purified mesenchymal stem cell composition contains about 7 μg / mL to about 15 μg / mL of residual bovine serum albumin. [000135] The method of paragraph [000134], wherein the purified mesenchymal stem cell composition comprises about 8 μg / mL to about 12 μg / mL of residual bovine serum albumin.

[0112] [000136] The method described in any one of paragraphs [000122] to [000135], further comprising adding DMSO to the purified mesenchymal stem cell composition. [000137] The method of paragraph [000136], wherein the composition comprises about 10% DMSO.

[0113] [000138] The method of paragraph [000136], wherein the composition comprises about 3.8% DMSO. [000139] A purified mesenchymal stem cell composition, obtained by the method described in any one of paragraphs [000122] to [000138].

[0114] [000140] A purified mesenchymal stem cell composition comprising: (i) culturing mesenchymal stem cells in a medium containing serum; (ii) obtaining a preparation containing those mesenchymal stem cells; (iii) contacting the preparation with a wash solution to form a mixture; (iv) agitating the mixture in a centrifugal filter; and (v) recovering the purified mesenchymal stem cell composition. A purified mesenchymal stem cell composition produced by the method of claim 1.

[0115] [000141] Mesenchymal stem cells have a D of about 18 μm to about 30 μm 90 The composition of paragraph [000140], [000142] The mesenchymal stem cells have a D of about 18 μm to about 25 μm. 90 The composition of paragraph [000141],

[0116] [000143] The mesenchymal stem cells are 90 The composition of paragraph [000142], [000144] The composition of any one of paragraphs [000140] to [000143], wherein the serum is bovine serum and the composition contains less than about 55 μg / mL of residual bovine serum albumin.

[0117] [000145] The composition of paragraph [000144], wherein the composition contains less than about 42 μg / mL of residual bovine serum albumin. [000146] The composition of paragraph [000145], wherein the composition contains less than about 25 μg / mL of residual bovine serum albumin.

[0118] [000147] The composition of paragraph [000146], wherein the composition contains less than about 13 μg / mL of residual bovine serum albumin. [000148] The composition of paragraph [000147], wherein the composition contains less than about 10 μg / mL residual bovine serum albumin.

[0119] [000149] The composition of any one of paragraphs [000140] to [000143], wherein the serum is bovine serum and the composition contains from about 7 μg / ml to about 15 μg / ml of residual bovine serum albumin.

[0120] [000150] The composition of paragraph [000149], wherein the composition comprises about 8 μg / ml to about 12 μg / ml of residual bovine serum albumin. [000151] A pharmaceutically acceptable composition comprising purified mesenchymal stem cells, wherein the mesenchymal stem cells are expanded in culture in a medium comprising bovine serum albumin; and wherein the composition comprises less than about 55 μg / mL of residual bovine serum albumin.

[0121] [000152] The composition of paragraph [000151], wherein the composition contains less than about 42 μg / mL of residual bovine serum albumin. [000153] The composition of paragraph [000152], wherein the composition contains less than about 25 μg / mL of residual bovine serum albumin.

[0122] [000154] The composition of paragraph [000153], wherein the composition contains less than about 13 μg / mL of residual bovine serum albumin. [000155] The composition of paragraph [000154], wherein the composition contains less than about 10 μg / mL residual bovine serum albumin.

[0123] [000156] The composition of paragraph [000151], wherein the composition comprises from about 7 μg / ml to about 15 μg / ml of residual bovine serum albumin. [000157] The composition of paragraph [000156], wherein the composition comprises about 8 μg / ml to about 12 μg / ml of residual bovine serum albumin.

[0124] [000158] A pharmaceutically acceptable composition comprising purified mesenchymal stem cells, the composition comprising less than about 55 μg / ml of residual bovine serum albumin. [000159] The composition of paragraph [000158], wherein the composition contains less than about 42 μg / mL of residual bovine serum albumin.

[0125] [000160] The composition of paragraph [000159], wherein the composition contains less than about 25 μg / mL of residual bovine serum albumin. [000161] The composition of paragraph [000160], wherein the composition comprises less than about 13 μg / mL of residual bovine serum albumin.

[0126] [000162] The composition of paragraph [000161], wherein the composition contains less than about 10 μg / mL residual bovine serum albumin. [000163] The composition of paragraph [000158], wherein the composition comprises from about 7 μg / mL to about 15 μg / mL of residual bovine serum albumin.

[0127] [000164] The composition of paragraph [000163], wherein the composition comprises about 8 μg / mL to about 12 μg / mL of residual bovine serum albumin. [000165] The composition of any one of paragraphs [000158] to [000164], further comprising DMSO.

[0128] [000166] The composition of paragraph [000165], further comprising about 10% DMSO. [000167] The composition of paragraph [000165], further comprising about 3.8% DMSO. [000168] A pharmaceutically acceptable composition comprising purified mesenchymal stem cells, comprising one or more mesenchymal stem cell populations, and a D of the populations. 90 However, less than about 150 μm wherein the composition comprises residual bovine serum albumin of about 8 μg / mL to about 12 μg / mL; and wherein the mesenchymal stem cells have a D of about 18 μm to about 30 μm. 90 A composition showing

[0129] [000169] The aggregate D 90 is less than about 100 μm. [000170] The aggregate D 90 is less than about 50 μm.

[0130] [000171] The mesenchymal stem cells have a D of about 18 μm to about 25 μm. 90 A pharmaceutically acceptable composition according to any one of paragraphs [000168] to [000170], wherein [000172] The mesenchymal stem cells are grafted to a D 90 The pharmaceutically acceptable composition of paragraph [000171],

[0131] [000173] A method for producing purified mesenchymal stem cells, comprising: (i) obtaining a cell suspension containing a plurality of mesenchymal stem cells; (ii) simultaneously selecting mesenchymal stem cells from the suspension based on mass and diameter; A method including the steps of:

[0132] [000174] The method of paragraph [000173], further comprising contacting the suspension with a cleaning solution. [000175] The composition comprises one or more mesenchymal stem cell aggregates, and the aggregates have a D of less than about 150 μm. 90 The method according to paragraph [000174],

[0133] [000176] The aggregates have a D 90 The method according to paragraph [000175], [000177] The aggregates have a D 90 The method according to paragraph [000176],

[0134] [000178] The method of paragraph [000173], wherein the composition does not contain detectable mesenchymal stem cell populations. [000179] A pharmaceutically acceptable composition comprising purified mesenchymal stem cells, wherein the mesenchymal stem cells have a D of about 18 μm to about 30 μm. 90 A pharmaceutically acceptable composition comprising:

[0135] [000180] The mesenchymal stem cells have a D of about 18 μm to about 25 μm. 90 The pharmaceutically acceptable composition of paragraph [000179], [000181] The mesenchymal stem cells are grafted to a D 90 The pharmaceutically acceptable composition of paragraph [000180],

[0136] [000182] The composition of any one of paragraphs [000179] to [000181], further comprising DMSO. [000183] The composition of paragraph [000182], comprising about 10% DMSO.

[0137] [000184] The composition of paragraph [000182], comprising about 3.8% DMSO. [000185] A method of producing a pharmaceutical mesenchymal stem cell composition, comprising: (i) obtaining a mesenchymal stem cell suspension containing a plurality of mesenchymal stem cells and mesenchymal stem cell aggregates; (ii) contacting the suspension with a wash solution to form a mixed suspension; (iii) The mixed suspension is filtered using a centrifugal filter to separate the mesenchymal stem cell aggregates into particles of less than about 150 μm. D 90 Stir until (iv) Recovering the pharmaceutical mesenchymal stem cell composition. A method including the steps of:

[0138] [000186] The mesenchymal stem cell aggregates have a D of less than about 100 μm. 90 The method according to paragraph [000185], [000187] The mesenchymal stem cell aggregates have a D of less than about 50 μm 90 The method according to paragraph [000186],

[0139] [000188] The pharmaceutical mesenchymal stem cell composition has a D of about 18 μm to about 30 μm. 90 The method according to any one of paragraphs [000185] to [000187], comprising mesenchymal stem cells exhibiting the following: [000189] The pharmaceutical mesenchymal stem cell composition has a D of about 18 μm to about 25 μm. 90 The method of paragraph [000188], comprising mesenchymal stem cells exhibiting:

[0140] [000190] The pharmaceutical mesenchymal stem cell composition has a D of about 20 μm to about 25 μm. 90 The method of paragraph [000189], comprising mesenchymal stem cells exhibiting: [000191] A composition comprising a population of purified mesenchymal stem cells, obtained by the method described in any one of paragraphs [000185] to [000190], wherein the cell viability is greater than about 70%.

[0141] [000192] The composition of paragraph [000191], wherein the cell viability is greater than about 80%. [000193] The composition of any one of paragraphs [000185] to [000192], further comprising DMSO.

[0142] [000194] The composition of paragraph [000193], comprising about 10% DMSO. [000195] The composition of paragraph [000193], comprising about 3.8% DMSO. [000196] A pharmaceutically acceptable composition comprising purified mesenchymal stem cells, comprising one or more mesenchymal stem cell aggregates, wherein the aggregates have a D of less than about 150 μm. 90 and wherein the mesenchymal stem cells have a D of about 18 μm to about 30 μm. 90 A pharmaceutically acceptable composition comprising:

[0143] [000197] The aggregates have a D of less than about 100 μm 90 The pharmaceutically acceptable composition of paragraph [000196], [000198] The aggregates have a D of less than about 50 μm 90 The pharmaceutically acceptable composition of paragraph [000197],

[0144] [000199] The mesenchymal stem cells have a D of about 18 μm to about 25 μm. 90 A pharmaceutically acceptable composition according to any one of paragraphs [000196] to [000198], wherein [000200] The mesenchymal stem cells are grafted to a D 90 The pharmaceutically acceptable composition of paragraph [000199],

[0145] [000201] A pharmaceutically acceptable composition according to any one of paragraphs [000196] to [000200], wherein the composition comprises from about 7 μg / mL to about 15 μg / mL of residual bovine serum albumin. [000202] The pharmaceutically acceptable composition of paragraph [000201], wherein the composition comprises from about 8 μg / mL to about 12 μg / mL of residual bovine serum albumin.

[0146] [000203] A method for selecting a cell suspension containing at least one non-human protein for administration to a patient, comprising: (a) obtaining at least one representative sample of the cell suspension; (b) determining the level of the non-human protein present in the sample; and (c) identifying the cell suspension as suitable for administration to a patient if the sample contains less than about 42 micrograms / milliliter of the non-human protein. A method including the steps of:

[0147] [000204] The method of Paragraph [000203], wherein the human cells are mesenchymal stem cells. [000205] The method of Paragraph [000203], wherein the non-human protein is albumin. [000206] The method of Paragraph [000203], wherein the non-human protein is bovine serum albumin.

[0148] [000207] Step (b) i. contacting the sample with at least one anti-albumin antibody; and ii. quantitating the level of albumin in the sample The method of paragraph [000205] or [000206], comprising:

[0149] [000208] The method of Paragraph [000203], wherein the non-human protein is trypsin. [000209] The method of Paragraph [000203], wherein the non-human protein is porcine trypsin.

[0150] [000210] Step (b) i. contacting the sample with at least one substance that selectively binds trypsin; and ii. quantitating the level of trypsin in the sample The method of paragraph [000208] or [000209], comprising:

[0151] [000211] The method of paragraph [000210], wherein the at least one substance is a trypsin inhibitor. [000212] The method of Paragraph [000210], wherein the at least one substance is an antitrypsin antibody.

[0152] [000213] Step (b) i. contacting the sample with an immobilized trypsin inhibitor to form an immobilized trypsin inhibitor-trypsin conjugate; ii. contacting the immobilized conjugate with an anti-trypsin antibody to form an immobilized trypsin inhibitor-trypsin-antibody complex; and iii. detecting a signal generated by the complex. The method of paragraph [000203], comprising:

[0153] [000214] A pharmaceutical composition selected by the method described in any one of paragraphs [000203], [000208]-[000209] or [000211]-[000213]. [000215] A method of treating or preventing a disease or disorder in a subject, comprising: (a) incubating human cells in a medium containing a non-human protein; (b) selecting a suspension of incubated cells containing less than about 42 micrograms per milliliter of the non-human protein; (c) administering the cell suspension to the subject. A method including the steps of:

[0154] [000216] The method of Paragraph [000215], wherein the human cells are mesenchymal stem cells. [000217] The method of Paragraph [000215], wherein the non-human protein is albumin. [000218] The method of Paragraph [000215], wherein the non-human protein is bovine serum albumin.

[0155] [000219] Step (b) i. contacting the sample with at least one anti-albumin antibody; and ii. quantitating the level of albumin in the sample The method of paragraph [000217] or [000218], comprising:

[0156] [000220] The method of Paragraph [000215], wherein the non-human protein is trypsin. [000221] The method of paragraph [000215], wherein the non-human protein is porcine trypsin.

[0157] [000222] Step (b) i. contacting the sample with at least one substance that selectively binds trypsin; and ii. quantitating the level of trypsin in the sample The method of paragraph [000220] or [000221], comprising:

[0158] [000223] The method of paragraph [000222], wherein the at least one substance is a trypsin inhibitor. [000224] The method of Paragraph [000222], wherein the at least one substance is an antitrypsin antibody.

[0159] [000225] Step (b) i. contacting the sample with an immobilized trypsin inhibitor to form an immobilized trypsin inhibitor-trypsin conjugate; ii. contacting the immobilized conjugate with an anti-trypsin antibody to form an immobilized trypsin inhibitor-trypsin-antibody complex; and iii. detecting a signal generated by the complex. The method of paragraph [000215], comprising:

[0160] [000226] A method of manufacturing a cell therapy product, comprising: (a) incubating human cells in a solution containing a non-human protein; (b) adding a volume of a liquid vehicle to the cells to obtain a pharmaceutically acceptable cell suspension; (c) obtaining at least one representative sample of said pharmaceutically acceptable cell suspension; (d) quantifying the amount of the non-human protein present in the sample; and (e) retaining the cell suspension for administration to the patient if the sample contains less than about 42 micrograms per milliliter of the non-human protein. A method including the steps of:

[0161] [000227] The method of paragraph [000226], further comprising retaining the cell suspension for administration to the patient if the sample contains less than about 30 micrograms / milliliter of the non-human protein.

[0162] [000228] The method of paragraph [000226], further comprising retaining the cell suspension for administration to the patient if the sample contains less than about 25 micrograms / milliliter of the non-human protein.

[0163] [000229] The method of paragraph [000226], further comprising retaining the cell suspension for administration to the patient if the sample contains less than about 13 micrograms / milliliter of the non-human protein.

[0164] [000230] The sample contains less than about 10 micrograms / milliliter of the non-human protein. The method of paragraph [000226], further comprising, if containing the substance, retaining the cell suspension for administration to the patient.

[0165] [000231] The method of paragraph [000226], further comprising retaining the cell suspension for administration to the patient when the sample contains about 7 to about 15 micrograms / milliliter of the non-human protein.

[0166] [000232] The method of paragraph [000226], further comprising retaining the cell suspension for administration to the patient when the sample contains about 8 to about 12 micrograms / milliliter of the non-human protein.

[0167] [000233] The method of paragraph [000226], wherein the human cells are adherent cells. [000234] The method of paragraph [000226], wherein the human cells are mesenchymal stem cells. [000235] The method of Paragraph [000226], wherein the non-human protein is albumin.

[0168] [000236] The method of Paragraph [000226], wherein the non-human protein is bovine serum albumin. [000237] Step (d) i. contacting the sample with at least one anti-albumin antibody; and ii. quantitating the level of albumin in the sample The method of paragraph [000235] or [000236], comprising:

[0169] [000238] The method of Paragraph [000226], wherein the non-human protein is trypsin. [000239] The method of Paragraph [000226], wherein the non-human protein is porcine trypsin.

[0170] [000240] Step (d) i. contacting the sample with at least one substance that selectively binds trypsin; and ii. quantitating the level of trypsin in the sample The method of paragraph [000238] or [000239], comprising:

[0171] [000241] The method of paragraph [000240], wherein the at least one substance is a trypsin inhibitor. [000242] The method of Paragraph [000240], wherein the at least one substance is an antitrypsin antibody.

[0172] [000243] Step (d) i. contacting the sample with an immobilized trypsin inhibitor to form an immobilized trypsin inhibitor-trypsin conjugate; ii. contacting the immobilized conjugate with an anti-trypsin antibody to form an immobilized trypsin inhibitor-trypsin-antibody complex; and iii. detecting a signal generated by the complex. The method of paragraph [000238] or [000239], comprising:

[0173] [000244] A method of manufacturing a cell therapy product, comprising: (a) incubating human cells in a solution containing non-human trypsin; (b) adding a volume of a liquid vehicle to the cells to obtain a pharmaceutically acceptable cell suspension; (c) obtaining at least two representative samples of the pharmaceutically acceptable cell suspension; (d) determining the level of trypsin present in the first sample; (e) incubating a second sample in a solution containing at least one substance that selectively binds trypsin to obtain a control; (f) determining the trypsin level in the control; (g) comparing the trypsin level in the first sample with the trypsin level in the control to obtain a trypsin level in the cell suspension; and (h) retaining the cell suspension of cells for administration to a patient when the cell suspension contains less than about 30 micrograms / milliliter of trypsin. A method including the steps of:

[0174] [000245] The method of paragraph [000244], further comprising retaining the cell suspension if the cell suspension contains less than about 25 micrograms / milliliter of trypsin.

[0175] [000246] The method of paragraph [000244], further comprising retaining the cell suspension if the cell suspension contains less than about 13 micrograms / milliliter of trypsin.

[0176] [000247] The method of paragraph [000244], further comprising retaining the cell suspension when the cell suspension contains less than about 10 micrograms / milliliter of trypsin.

[0177] [000248] The method of paragraph [000244], further comprising retaining the cell suspension when the cell suspension contains about 7 to about 15 micrograms / milliliter of trypsin.

[0178] [000249] The method of paragraph [000244], further comprising retaining the cell suspension when the cell suspension contains about 8 to about 12 micrograms / milliliter of trypsin.

[0179] [000250] The method of paragraph [000244], wherein the human cells are mesenchymal stem cells. [000251] The method of paragraph [000244], wherein the substance is a trypsin inhibitor. [000252] The method of paragraph [000244], wherein the substance is an antitrypsin antibody.

[0180] [000253] A cell therapy product comprising: A cell therapy product manufactured by a method according to any one of [000244] to [000252]. [000254] A cell therapy product, wherein the cell therapy product is manufactured by the method described in paragraph [000237].

[0181] [000255] A cell therapy product, wherein the cell therapy product is manufactured by the method described in paragraph [000240]. [000256] A cell therapy product, wherein the cell therapy product is manufactured by the method described in paragraph [000241].

[0182] [000257] A cell therapy product, wherein the cell therapy product is manufactured by the method described in paragraph [000242]. [000258] A cell therapy product, wherein the cell therapy product is manufactured by the method described in paragraph [000243].

[0183] [000259] A pharmaceutically acceptable carrier comprising human mesenchymal stem cells and at least one non-human protein. an acceptable cell suspension containing less than about 42 micrograms / milliliter of the non-human protein;

[0184] [000260] The cell suspension of paragraph [000259], further comprising retaining the cell suspension for administration to a patient when the sample contains less than about 30 micrograms / milliliter of the non-human protein.

[0185] [000261] The cell suspension of paragraph [000259], further comprising retaining the cell suspension for administration to the patient when the sample contains less than about 25 micrograms / milliliter of the non-human protein.

[0186] [000262] The cell suspension of paragraph [000259], further comprising retaining the cell suspension for administration to the patient if the sample contains less than about 13 micrograms / milliliter of the non-human protein.

[0187] [000263] The cell suspension of paragraph [000259], further comprising retaining the cell suspension for administration to a patient when the sample contains less than about 10 micrograms / milliliter of the non-human protein.

[0188] [000264] The cell suspension of paragraph [000259], further comprising retaining the cell suspension for administration to a patient when the sample contains about 7 to about 15 micrograms / milliliter of the non-human protein.

[0189] [000265] The cell suspension of paragraph [000259], further comprising retaining the cell suspension for administration to a patient when the sample contains about 8 to about 12 micrograms / milliliter of the non-human protein.

[0190] [000266] The method of Paragraph [000259], wherein the non-human protein is albumin. [000267] The method of Paragraph [000259], wherein the non-human protein is bovine serum albumin.

[0191] [000268] The method of Paragraph [000259], wherein the non-human protein is trypsin. [000269] The method of Paragraph [000259], wherein the non-human protein is porcine trypsin.

Claims

[Claim 1] The invention described in the specification.

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