Formulations and methods for maintaining cell suspension and viability

Abstract

Described herein are DMSO formulations and methods for cell therapy. Methods of treating a cell with these formulations are also provided. The embodiments provide a formulation that maintains the suspension and viability of a T-cell during cell therapy (CT) manufacturing and through a cryopreservation thaw cycle, including dimethyl sulfoxide (DMSO); sucrose or trehalose; human semm albumin (HSA); and dextran 40 or hydroxyethyl starch (HES). wherein the formulation maintains the suspension and viability of T-cells during CT manufacturing and through a cryopreservation thaw cycle. The embodiments provide a method for maintaining a suspension and cell viability, including subjecting a T-cell during a cell therapy manufacturing cryopreservation thaw cycle to a formulation including DMSO; sucrose or trehalose; human semm albumin (HSA); and polysaccharide or HES wherein the formulation maintains the suspension and viability of a T-cell during cell therapy manufacturing and through a cryopreservation thaw cycle.

Description

[0001] Docket No. BYP240186 WO

[0002] FORMULATIONS AND METHODS FOR MAINTAINING CELL SUSPENSION AND VIABILITY

[0003] FIELD

[0004] The present embodiments relate to cell therapy DMSO formulations and methods.

[0005] BACKGROUND

[0006] Human cells have a density greater than that of water and T-cells have a density of about 1.08 gm / mL. Formulations used in industry for processing and cryopreserving cells will typically have densities less than 1.08 gm / mL. Because of the difference in density between cells and formulation, cells can settle out of suspension during the cell therapy (CT) manufacturing process. Cell settling in the feed container during filling operations would have a negative effect on the quality attribute of content uniformity in the filled bags or vials. To ensure content uniformity, the filling process would require continuous mixing. However, this mixing operation can present manufacturing issues as cells are sensitive to shear stress requiring that the mixing be gentle and potentially not optimal for the filling process. In addition, cells settling in bags or vials prior to freezing may have a negative effect on cell viability before and after freezing. The cell settling rate is primarily determined by three physicochemical factors: 1) Difference between solution and cell densities: the larger the difference, the faster the cell settling rate; 2) Formulation viscosity: the higher the viscosity, the slower the cell settling rate 3) Cell diameter: the larger the cell diameter or aggregation of cells, the faster the cell settling rate.

[0007] Further, there is a need for cell therapy formulations and methods to maintain cell suspension and viability during the freezing and thawing process of manufacturing.

[0008] These and other problems present in the art have been addressed by the present embodiments. Docket No. BYP240186 WO

[0009] SUMMARY

[0010] The embodiments provide a formulation that maintains the suspension and viability of a T-cell during cell therapy (CT) manufacturing and through a cryopreservation thaw cycle, comprising 10% dimethyl sulfoxide (DMSO); 5% sucrose or trehalose; 5% human serum albumin (HSA); and 2.5-10% polysaccharide or 5-7.5% hydroxyethyl starch (HES) wherein the formulation maintains the suspension and viability of a T-cell during CT manufacturing and through a cryopreservation thaw cycle.

[0011] The embodiments provide a formulation wherein the T-cell comprises a Jurkat cell. The embodiments provide a formulation wherein the T-cell comprises a Primary T-cell. The embodiments provide a formulation wherein the 5% HSA is in Plasma-Lyte A and substantially maintains cell suspensions for about 4-24 hrs.

[0012] The embodiments provide a formulation wherein the formulation kept cells suspended and maintained T-cell viability for up to 24 hrs. while stored at room temperature. Further, the cell viability post freezing (freezing initiated after a 3-4 hr. hold time) was maintained close to the level of cell viability prior to freezing for up to 3 hrs. post thaw, as compared to a DMSO control formulation.

[0013] The embodiments provide a method for maintaining a suspension and cell viability, comprising subjecting a T-cell during a cell therapy manufacturing cryopreservation thaw cycle to a formulation comprising 10% dimethyl sulfoxide (DMSO); 5% sucrose or trehalose; 5% human serum albumin (HSA); and 2.5-10% dextran 40 or 5-7.5% hydroxyethyl starch (HES) wherein the formulation substantially maintains the suspension and viability of a T-cell during cell therapy manufacturing and through a cryopreservation thaw cycle.

[0014] The embodiments provide a method wherein the T-cell comprises a Jurkat cell.

[0015] The embodiments provide a method wherein the T-cell comprises a Primary T-cell. The embodiments provide a method wherein the 5% HSA is in Plasma-Lyte A and substantially maintains cell suspensions for about 4-24 hrs.

[0016] The embodiments provide a method wherein the formulation kept cells suspended and maintained T-cell viability for up to about 24 hrs. while stored at room temperature. Further, the Docket No. BYP240186 WO

[0017] cell viability post freezing (freezing initiated after a 3-4 hr. hold time) was maintained close to the level of cell viability prior to freezing for up to 3 hrs. post thaw, as compared to a DMSO control formulation.

[0018] These and other features of the present teachings are set forth herein. Docket No. BYP240186 WO

[0019] Brief Description of the Drawings

[0020] The skilled artisan will understand that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings or claims in any way.

[0021] FIG. 1. shows Group 1 Dextran 40 / Trehalose / Sucrose Formulations Effect on Distribution of Total Jurkat T-Cell Count by Position over Time.

[0022] FIG. la shows Group 1 Total Cell Count Distribution by Layer - 1 hr.

[0023] FIG. lb shows Group 1 Total Cell Count Distribution by Layer - 6 hr.

[0024] FIG. 1c shows Group 1 Total Cell Count Distribution by Layer - 24 hr.

[0025] FIG. 2 shows Group 2 Dextran 40 / Sucrose and HES / Sucrose Formulations Effect on Distribution of Total Primary T-Cell Count by Position over Time.

[0026] FIG. 2a shows Group 2 Total Cell Count Distribution by Layer - 0.5 hr.

[0027] FIG. 2b shows Group 2 Total Cell Count Distribution by Layer - 4 hr.

[0028] FIG. 3 shows Group 3 Dextran 40 / Sucrose Formulations Effect on Distribution of Total Primary T-Cell Count by Position over Time.

[0029] FIG 4 shows Group 1 Formulation Effect on Jurkat T-Cell Viability over 24 hrs at RT. FIG. 5 shows Group 2 Formulation Effect on Primary T-Cell Viability over 24 hrs at RT. FIG. 6 shows Group 3 High Concentration Primary T-Cell Viability over 24 hr RT Hold Time.

[0030] FIG. 7 shows Group 1 Jurkat T-Cell Viability Post Freeze / Thaw.

[0031] FIG. 8 shows Group 2 Primary T-Cell Viability Post Freeze / Thaw.

[0032] FIG. 9: Group 3 High Concentration Primary T-Cell Viability Pre and Post Freeze / Thaw. Docket No. BYP240186 WO

[0033] DETAILED DESCRIPTION DEFINITIONS

[0034] For interpreting this specification, the following definitions will apply. If any definition set forth below conflicts with the usage of that word in any other document, including any document incorporated herein by reference, the definition set forth below shall always control for purposes of interpreting this specification and its associated claims unless a contrary meaning is clearly intended (for example in the document where the term is originally used).

[0035] Whenever appropriate, terms used in the singular will include the plural and

[0036] vice versa. The use of “a” herein means “one or more” unless stated otherwise or where the use of “one or more” is clearly inappropriate. The use of “or” means “and / or” unless stated otherwise. The use of “comprise,” “comprises,” “comprising,” “include,”

[0037] “includes” and “including” are interchangeable and are not limiting. The terms “such

[0038] as,” “for example,” and “e.g.” are not intended to be limiting. For example, the term “including” shall mean “including, but not limited to.”

[0039] As used herein, the term “cell viability” refers to a measure of the proportion of live, healthy cells within a population. Cell viability assays are used to determine the overall health of cells, optimize culture or experimental conditions, and to measure cell survival following treatment with compounds, such as during a drug screen. For example, only dead cells or cells with damaged membranes will absorb trypan blue dye — after introducing this dye to a sample, dead or damaged cells will uptake the dye and take on a blue appearance, while healthy, viable cells with intact cell membranes will remain uncolored. The tetrazolium reduction, resazurin reduction, and protease activity assays measure some aspect of general metabolism or an enzymatic activity as a marker of viable cells. The definition of cell viability here is based on cytolysis or membrane leakage assays. The final stages in most forms of cell death will cause membrane leakage, thus coupling a microscope, spectrophotometer / spectrofluorometer with dyes with capability to stain membrane compromised cells can indicate cell viability and death. In this case, membrane leakage assays were implemented based on K2 and NC202 cell counters.

[0040] (Adapted from Advanced in Biopreservation, Chapter 6: Viability and functional assays used to assess preservation efficacy. Docket No. BYP240186 WO

[0041] As used herein, the term “viscosity” refers to the resistance of a liquid formulation to flow, such as when injected through a syringe needle during administration to a patient.

[0042] Viscosity measurements can be determined by a cone and plate technique with a Peltier element set at a defined temperature as described herein. Typically, a well-defined shear stress gradient is applied to a liquid formulation and the resulting shear rate is measured. Viscosity is the ratio of the shear stress to the shear rate. As used herein, viscosity is expressed in units of mPa-s at 5°C to 25 °C wherein 1 mPa-s = 1 cP.

[0043] As used herein, the term “osmolality” refers to a measure of solute concentration, defined as the number of millimole of solute (both non-ionized and ionized forms) per kg of solution. A desired level of osmolality can be achieved by addition of one or more stabilizers such as sugar or sugar alcohol including, but not limited to, mannitol, dextrose, glucose, trehalose, and / or sucrose. Stabilizers that are suitable for providing osmolality are described in references such as the Handbook of Pharmaceutical Excipients (Fourth Edition, Royal Pharmaceutical Society of Great Britain, Science & Practice Publishers) or Remington’s: The Science and Practice of Pharmacy (Nineteenth Edition, Mack Publishing Company).

[0044] As used herein, the term “about” refers to + / -3°C of the unit value provided for temperature and + / - 10% of the value for other metrics.

[0045] As used herein, the term “substantially” refers to the qualitative condition of exhibiting a total or approximate degree of a characteristic or property of interest. One of ordinary skill in the biological arts will understand that biological and chemical phenomena rarely, if ever, achieve or avoid an absolute result. The term “substantially” is therefore used herein to capture the potential lack of completeness inherent in many biological and chemical phenomena.

[0046] As used herein, the term “salt” refers to inorganic salts, which include, but are not limited to. sodium chloride (NaCl), sodium sulfate (Na2SO4). sodium thiocyanate (NaSCN), magnesium chloride (MgCl). magnesium sulfate (MgSO4), ammonium thiocyanate (NH4SCN), ammonium sulfate ((NH4)2SO4), ammonium chloride (NH4CI), calcium chloride (CaCh), calcium sulfate (CaSCh), zinc chloride (Z11CI2) and the like, or combinations thereof.

[0047] As used herein, the term “pharmaceutically effective amount” of a cell therapy formulation refers to an amount of the formulation that provides therapeutic effect in an administration regimen. Docket No. BYP240186 WO

[0048] The embodiments provide formulations that maintain suspension and viability of T-cells during Cell Therapy (CT) manufacturing and through a cryopreservation / thaw cycle. The formulations comprise 10% dimethyl sulfoxide (DMSO), 5% sucrose or trehalose. 5% human serum albumin (HSA), and 2.5-10% dextran 40 or 5-7.5% hydroxyethyl starch (HES).

[0049] A formulation strategy was developed that comprises the addition of a disaccharide (sucrose or trehalose) and a thickener (hydroxyethyl starch or dextran 40) to increase density and viscosity of the formulation such that T-Cells (Jurkat and Primary T-cells from a donor) remain suspended during a sustained period. Using Jurkat and Primary T-cells, it was shown that inclusion of 2.5-10% dextran 40 (a polysaccharide) or 5-7.5% hydroxyethyl starch (HES) and 5% trehalose or sucrose to a base formulation of 10% DMSO and 5% HSA in Plasma-Lyte A created formulations that could maintain cells suspension for 4-24 hrs. Plasma-Lyte A is a solution comprising water, sodium chloride, potassium chloride, magnesium chloride hexahydrate, sodium acetate trihydrate, and sodium gluconate.

[0050] The mitigation of cell settling was achieved by increasing formulation density, increasing viscosity, and forming a structured liquid (shear thinning solution), which has a small yield stress that inhibits cell movement. In addition, it was surprisingly discovered that this formulation strategy yielded a cell therapy drug product benefit. For cells that that are stored in the frozen state, formulations often use 10% DMSO as a cryoprotectant which can be toxic to cells and thereby can lead to a limit of 2-6 hrs for manufacturing time even when cells are kept refrigerated. It was demonstrated that the same formulations described in the previous paragraph kept cells suspended and maintained T-cell viability for up to about 24 hrs while stored at room temperature. In addition, the cell viability post freezing (freezing initiated after a 3-4 hr hold time) was maintained close to the level of cell viability prior to freezing for up to 3 hrs post thaw, as compared to a DMSO control formulation. In summary, the inclusion of a polysaccharide such as dextran 40 and sucrose or trehalose to the CT formulation reduced or prevented T-cell settling for up to 24 hrs and provided significant cell stabilization based on cell viability test results.

[0051] Cell settling in the feed container during filling operations has a negative effect on the quality attribute of content uniformity in the filled bags or vials. To ensure content uniformity, the filling process requires continuous mixing. However, this mixing operation can present manufacturing issues as cells are sensitive to shear stress, requiring that the mixing be gentle and Docket No. BYP240186 WO

[0052] potentially not optimal for the filling process. Cells settling in bags or vials, prior to freezing may have a negative effect on cell viability before and after freezing. The settling rate is primarily determined by physicochemical factors:

[0053] 1) Difference between solution and cell densities: the larger the difference, the faster the cell settling rate;

[0054] 2) Formulation viscosity: the higher the viscosity, the slower the cell settling rate;

[0055] 3) Cell diameter: the larger the cell diameter or aggregation of cells, the faster the settling rate will be;

[0056] 4) Structured liquid (shear thinning solution), or yield stress, can inhibit cell movement and aid in maintenance of cell suspensions.

[0057] A formulation strategy was developed to tune formulation density and viscosity such that the T-Cells (Jurak and Primary T-Cells from a donor) remain suspended during a sustained period. Using Jurkat and Primary T-cells, it was shown that formulations with densities between 1.05-1.08 g / cm3, viscosities between 4-10 cP, and a yield-stress between 1-1.5 were able to maintain cell suspensions between about 4-24 hrs at concentrations between 2-24x10 6 cells / mL. Formulation excipients comprised 2.5-10% dextran 40 or 5-7.5% hydroxyethyl starch (HES), (both thickeners and non-penetrating cryoprotecting agents (nCPA) and 5% trehalose or sucrose (both nCPAs and membrane stability enhances synergistically with dextran / HES) to a base formulation of 10% DMSO (CPA) and 5% HSA membrane stabilizer in Plasma-Lyte A.

[0058] In addition, it was unexpectedly discovered that this formulation strategy yielded a cell therapy drug product storage benefit. For cells that that are stored in the frozen state, formulations often use 10% DMSO as a cryoprotectant that can be toxic to cells and thereby can lead to a limit of 2-6 hrs for manufacturing time even when cells are kept refrigerated. It was demonstrated that the same formulations described in the previous paragraph kept cells suspended and maintained T-cell viability for up to 24 hrs while stored at room temperature. In addition, the cell viability post freezing (freezing initiated after a 3-4 hr hold time) was maintained close to the level of cell viability prior to freezing for up to 3 hrs post thaw compared to the DMSO control formulation.

[0059] In summary, solutions with the desired physical properties of density, viscosity, yieldstress could maintain cell suspensions for up to 24 hours; provide cryoprotection and maintain Docket No. BYP240186 WO

[0060] cell viability for up to 24 hours even at Room Temperature (RT). Moreover, based on formulation strategy and design, it is possible to tune the physical properties of the formulations to meet other manufacturing considerations such as mixing and filling product.

[0061] Component Use Range (%w / w)

[0062] HSA Stabilize cell membrane Non1-5%

[0063] penetrating CPA (nCPA)

[0064] Dextran 40 Thickening agent / increase 2.5-20%

[0065] density & viscosity stabilize

[0066] membrane nCPA

[0067] HES Thickening agent / increase 2.5-20%

[0068] density & viscosity stabilize

[0069] membrane nCPA

[0070] Sucrose / Trehalose nCPA (osmolyte pulls water out 1-7.5%

[0071] of cell to prevent intracellular

[0072] ice formation). Help stabilize

[0073] cell membrane and membrane

[0074] proteins

[0075] DMSO CPA (replace water). Comes 10%

[0076] with other toxicity / chemical

[0077]

[0078] leaching problems.

[0079] Physical parameters Use Range

[0080] Density Reduces eliminates settling 1.03-1.08 g / cm3

[0081] Viscosity Slows settling 2-20 cP

[0082] Yield Stress Creates structured liquid that 0.4-2 mPa

[0083]

[0084] slows / reduces aggregation

[0085] FORMULATION EMBODIMENTS

[0086] In some embodiments disclosed herein are stable liquid pharmaceutical formulations comprising cell therapy formulations.

[0087] In some embodiments, the cells comprise primary T-cells and Jurkat cells.

[0088] With these embodiments novel formulation strategies are developed using various components to optimize the density and viscosity of cell formulations to slow or prevent cells from settling in the formulation. Disaccharides (e.g., sucrose and trehalose) and polymers (e.g.. dextran 40 and hydroxyethyl starch) were added at various levels to increase the density and viscosity of cryopreservative formulations that contain DMSO, HSA, and Plasma-Lyte A. The study descriptions and BioBook references are summarized in Table 1. Eleven formulations (Group 1) including different excipient compositions were used (Table 2) to increase the density Docket No. BYP240186 WO

[0089] and viscosity of the solution and assess Jurkat T-cell settling over a 24 hr hold time.

[0090] Formulations F1J-F11J comprised a base solution containing about 5% Human Serum Albumin (HSA),10% DMSO, and 85% Plasma- Lyte A (formulation # F1J) to which dextran 40, trehalose, and sucrose were added at varying concentrations as detailed in Table 2. These formulations had cell concentrations of 0.8-1.4 million cells / mL. Cell settling was measured for formulations F1J-F11J after holding at room temperature for 1 hr, 6 hr, and 24 hr. Six formulations (Group 2) comprising different excipient compositions were prepared to increase the density of the solution and assess Primary T-cell settling over a 24 hr hold time. Formulations F1T-F6T comprising a base solution containing approximately 5% Human Serum Albumin (HSA),10% DMSO, and 85% Plasma-Lyte A (Formulation # FIT) to which sucrose and dextran 40 or hydroxyethyl starch (HES) were added at varying concentrations as detailed in Table 3. These formulations had cell concentrations of 1.1 - 1.2 million cells / mL. Cell settling was evaluated after 0.5 hr, 4 hr and 24 hr hold times. Density and viscosity of Group 2 formulations were measured. Three formulations (Group 3) comprising different excipient compositions were prepared to increase the density of the solution and assess Primary T-cell settling over a 24 hr hold time using a high cell concentration (16-24 million cells / mL). Formulations F0H-F2H comprising a base solution containing about. 5% Human Serum Albumin (HSA),10% DMSO, and 85% Plasma-Lyte A (Formulation # F0H) to which sucrose and dextran 40 were added at varying concentrations as detailed in Table 4. Cell settling was evaluated after 0 hr, 5 hr, 8 hr, and 24 hr hold times. Density and viscosity of Group 3 formulations were measured. The cell counts and viability of Jurkat T-cells (Group 1) and Primary T-cells (Groups 2 and 3) were measured after holding vials for up to 24 hr at room temperature (RT). The cell count and viability of all formulations were measured for vials that were held at RT for 3-4 hrs, frozen to -130°C with a controlled-rate freezer and stored in a liquid nitrogen freezer for one week.

[0091] In some embodiments, disclosed herein are stable liquid pharmaceutical formulations comprising cell therapy formulations.

[0092] In some embodiments, the cells comprise primary T-cells and Jurkat cells.

[0093] In some embodiments, the stable liquid pharmaceutical formulation comprises about 1% weight to volume (w / v) DMSO, about 2% weight to volume (w / v) DMSO, about 3% weight to volume (w / v) DMSO, about 4% weight to volume (w / v) DMSO, about 5% weight to volume (w / v) DMSO, about 6% weight to volume (w / v) DMSO, about 7% weight to volume (w / v) Docket No. BYP240186 WO

[0094] DMSO, about 8% weight to volume (w / v) DMSO, about 9% weight to volume (w / v) DMSO, or about 10% weight to volume (w / v) DMSO.

[0095] In some embodiments, sugars and inorganic salts are commonly used as stabilizers in cell therapy formulations with increased stability, and reduced tendency of aggregation and precipitation. Sugar or sugar alcohols (such as mannitol, dextrose, glucose, trehalose, and / or sucrose) are used separately or in combination, both as cryo-protectants and as stabilizers.

[0096] In some embodiments, the formulations comprise about 6% weight to volume (w / v) to about 10% (w / v) sucrose or trehalose; to about 7% (w / v) to about 10% (w / v) sucrose or trehalose; to about 8% (w / v) sucrose or trehalose; or to about 10% (w / v) sucrose or trehalose. Unless otherwise indicated, all percentage ranges provided herein for sucrose refer to weight over volume percentage.

[0097] In some embodiments, the formulation comprises about 10%, 15%, 20%, 25%, 30%, 35%, 40%, 45% or 50% (w / v) sucrose or trehalose.

[0098] In some embodiments, the formulation comprises dextran 40. Dextran 40 is a linear glucose polymer (polysaccharide) chemically designated (C6H10O5)n. Dextrose, USP is chemically designated D-glucose monohydrate (C6H12O6• H2O), a hexose sugar freely soluble in water.

[0099] In some embodiments, the formulation comprises about 1% weight to volume (w / v) dextran 40, about 2% weight to volume (w / v) dextran 40, about 3% weight to volume (w / v), dextran 40, about 4% weight to volume (w / v) dextran 40, about 5% weight to volume (w / v) dextran 40, about 6% weight to volume (w / v) dextran 40, about 7% weight to volume (w / v) dextran 40, about 8% weight to volume (w / v) dextran 40, about 9% weight to volume (w / v) dextran 40, or about 10% weight to volume (w / v) dextran 40.

[0100] In some embodiments, the formulation comprises about 10%, 15%, 20%, 25%, 30%, 40%, or 50% weight to volume (w / v) dextran 40.

[0101] In some embodiments the formulation can comprise about 0.1%, 1%, 2%, 3%, 4% and 5% weight to volume (w / v) Human Seram Albumin (HSA) in Plasma- Lyte A solution. Further, recombinant forms of HSA can be employed with the present embodiments.

[0102] In some embodiments the formulation can comprise about 6%, 7%, 8%, 9%, 10%, 11%, 12%, 13%, 14%, 15%, 16%, 17%, 18%, 19%. or 20% weight to volume Human Seram Albumin (HSA) in Plasma-Lyte A solution. Docket No. BYP240186 WO

[0103] The presently described formulations in some embodiments can be characterized by prolonged stability. In some embodiments, the stability can be assessed upon storage of the liquid pharmaceutical composition under suitable storage conditions.

[0104] In some embodiments, the formulations described herein can be characterized by comparably low viscosity. In some embodiments, the viscosity of the formulation is in range of from about 1 to about 50 mPa-s, about 1 to about 45 mPa-s, about 1 to about 40 mPa-s, about 1 to about 35 mPa-s, about 1 to about 30 mPa-s, about 1 to about 25 mPa-s, or about 1 to about 20 mPa-s, about 1 to about 15 mPa-s, about 1 to about 10 mPa-s, about 1 to about 5 mPa-s, about 1 to about 4 mPa-s, about 1 to about 3 mPa-s, or about 1 to about 20 mPa-s, between 5°C to 25°C. For example, in some embodiments, the formulation comprises a viscosity of about 1-2 mPa-s at about 20° C. In some embodiments, the formulation composition can be isotonic.

[0105] In some embodiments, the analysis is conducted on a formulation upon storage under suitable storage conditions.

[0106] In some embodiments, the analysis is conducted on a formulation reconstituted under suitable storage conditions.

[0107] CELL THERAPY CELL TYPES

[0108] It is within the scope of the embodiments that various T-cells can be employed with the present embodiments. For instance, the T-cells can comprise immortalized T-cell lines, primary T-cells, Jurkat cells, and other similar type cells.

[0109] SYSTEMS, KITS, AND METHODS OF PREPARATION Provided are systems, kits, and methods of preparation of the formulations described herein.

[0110] Provided are kits comprising any one of the formulations described herein. The kits can be useful for any of the methods of treatment described herein.

[0111] In some embodiments, there is provided a kit comprising a cell therapy cell, such as a primary T-cell or Jurkat cell.

[0112] In some embodiments, the kit further comprises a device configured for delivering the formulation to an individual. One type of device, for applications such as parenteral delivery, is a syringe used to inject the composition into a subject's body. Docket No. BYP240186 WO

[0113] In some embodiments, the kit further comprises a therapeutic agent for treating a disease or condition, e.g., cancer or hypertension.

[0114] The kits of the present embodiments can be in suitable packaging. Suitable packaging comprises vials, bottles, jars, flexible packaging (e.g., sealed Mylar or plastic bags), and the like. Kits can optionally provide components such as buffers and interpretative information.

[0115] The present application provides articles of manufacture. The article of manufacture can comprise a container and a label or package inserted on or associated with the container. Suitable containers include vials (such as sealed vials), bottles, jars, flexible packaging, and the like. The container holds a composition and can have a sterile access port (for example the container can be an intravenous solution bag or a vial having a stopper pierceable by a hypodermic injection needle). The label or package insert indicates that the composition is used for imaging, diagnosing, or treating a particular condition in an individual. The label or package insert will further comprise instructions for administering the composition to the individual and for imaging the individual. The label can indicate directions for reconstitution and / or use. The container holding the composition can be a multi-use vial, which allows for repeat administrations (e.g., from 2-6 administrations) of the reconstituted formulation. Package insert refers to instructions customarily included in commercial packages of diagnostic products that contain information about the indications, usage, dosage, administration, contraindications, and / or warnings concerning the use of such diagnostic products. The article of manufacture can further comprise a second container comprising a pharmaceutically acceptable buffer, such as bacteriostatic water for injection (BWFI), phosphate-buffered saline, Ringer's solution, and dextrose solution. It can further include other materials desirable from a commercial and user standpoint, including other buffers, diluents, filters, needles, and syringes.

[0116] The kits or article of manufacture can include multiple unit doses of the compositions and instructions for use, packaged in quantities sufficient for storage and use in pharmacies, for example, hospital pharmacies and compounding pharmacies.

[0117] Those skilled in the art will recognize that several embodiments are possible within the scope and spirit of this invention. The embodiments will now be described in greater detail by reference to the following nonlimiting examples. The following examples further illustrate the embodiments but should not be construed as limiting its scope. Docket No. BYP240186 WO

[0118] EXAMPLES

[0119] Example 1 - Experimental Formulations Materials and Methods: The results from three cell therapy formulation experiments are summarized in this example.

[0120] • Group 1 Formulations: eleven formulations were prepared for a Jurkat T-Cell formulation experiment using lower cell concentrations (0.8-1.4xl0A6 cells / ml) as summarized in Table 2.

[0121] • Group 2 Formulations: six formulations were prepared for a Primary T-Cell formulation experiment using lower cell concentrations (l.l-1.6xlOA6 cells / ml) as summarized in Table 3.

[0122] • Group 3 Formulations: three formulations were prepared for a Primary T-Cell formulation experiment using higher cell concentrations (16-23xlOA6 cells / ml) as summarized in • Table 4. Each formulation number has an associated formulation code for use in figures and tables. Variance of % HSA and % DMSO in the Group 1 and Group 2 formulations was due to the way each formulation was prepared by combining base formulations to achieve the target excipient levels of dextran 40, sucrose, HES, and trehalose.

[0123] Table 1: Group 1 Formulation Number, Code and Composition (Used for Jurkat T-Cells) F# Formulation Code Target Formulation Composition

[0124] F1J F1J 5% HSA, 10.0% DMSO, 85% Plasma-Lyte A

[0125] F2J F2J7.5%Dex 5% HSA, 10% DMSO, 77.5% Plasma-Lyte A, 7.5% Dextran 40 F3J F3J 10%Dex 5.0% HSA, 10% DMSO, 75% Plasma-Lyte A, 10% Dextran 40 F4J F4J 7.5%Dex5%Tre 5% HSA, 10% DMSO, 72.5% Plasma-Lyte A, 7.5% Dextran 40,

[0126] 5.0%

[0127] Trehalose

[0128] F5J F5J 10%Dex5%Tre 5% HSA. 10% DMSO, 70% Plasma-Lyte A, 10.0% Dextran 40,

[0129] 5.0%

[0130] Trehalose

[0131] F6J F6J 2.5%Tre 5% HSA, 10% DMSO, 82.5% Plasma-Lyte A, 2.5% Trehalose F7J F7J 5 %Tre 5% HSA, 10% DMSO, 80% Plasma-Lyte A, 5.0% Trehalose

[0132]

[0133] Docket No. BYP240186 WO

[0134] F8J F8J 10%Tre 5% HSA, 10% DMSO, 75% Plasma-Lyte A, 10.0% Trehalose F9J F9J 7.5%Dex5%Suc 5% HSA. 10% DMSO, 72.5% Plasma-Lyte A, 7.5% Dextran 40,

[0135] 5.0%

[0136] Sucrose

[0137] F1OJ F1OJ 10%Dex5° / oSuc 5% HSA, 10% DMSO, 70% Plasma-Lyte A, 10.0% Dextran 40, 5.0% Sucrose

[0138] F11J F11 J 1 %Dex5%T re 5% HSA, 10% DMSO, 79% Plasma-Lyte A, 1.0% Dextran 40, 5.0%

[0139] Trehalose

[0140]

[0141] Table 2: Group 2 Formulation Number, Code and Composition (Used for Primary T-Cells)

[0142] F# Formulation Code Target Formulation Composition

[0143] FIT FIT 5% HSA, 10.0% DMSO, 85% Plasma-Lyte A

[0144] 5% HSA, 10% DMSO, 77% Plasma-Lyte A, 2.5% Dextran 40, 5.0% F2T F2T 2.5%Dex5%Suc

[0145] Sucrose

[0146] 5% HSA, 10% DMSO, 75.0% Plasma-Lyte A, 5% Dextran 40, 5.0% F3T F3T 5%Dex5%Suc

[0147] sucrose

[0148] 5% HSA, 10% DMSO, 72.5 % Plasma-Lyte A, 7.5% Dextran 40, F4T F4T 7.5%Dex5%Suc

[0149] 5.0%

[0150] sucrose

[0151] 5% HSA, 10% DMSO, 70% Plasma-Lyte A, 10% Dextran 40, 5.0% F5T F5T 10%Dex5%Suc

[0152] sucrose

[0153] F6T F6T 5% HSA, 10% DMSO, 72.5% Plasma-Lyte A, 7.5% HES, 5.0%

[0154]

[0155] 7.5%HES5%Suc sucrose

[0156] Table 3: Group 3 Formulation Number, Code and Composition (Used for High Concentration Primary T-Cells)

[0157] Formulation Code Target Formulation Composition

[0158] F0H F0H 5.0% HSA, 10.0% DMSO, 85.0% Plasma-Lyte A

[0159] 5.0% HSA, 10.0% DMSO, 77.0% Plasma-Lyte A, 3.0% Dextran 40, F1H F1H 3%Dex5%Suc

[0160] 5.0%

[0161] sucrose

[0162] 5.0% HSA, 10.0% DMSO. 75.0% Plasma-Lyte A, 5.0% Dextran 40, F2H F2H 5%Dex5%Suc

[0163] 5.0%

[0164]

[0165] sucrose Docket No. BYP240186 WO

[0166] Formulation Density and Viscosity

[0167] A formulation strategy was developed that involves addition of a disaccharide (sucrose or trehalose) and a thickener (hydroxyethyl starch or dextran 40) to increase density and viscosity of the formulation such that cells remain suspended during the manufacturing process and prior to product administration.

[0168] Formulation density and viscosity was measured for the Group 2 and 3 formulations to determine when the solution density and viscosity start mitigating cell settling and to confirm that viscosity is within an acceptable level for administration of drug product at the clinic.

[0169] Materials and methods: viscosity was measured on a Rheosense VROC-Initium using a shear-rate sweep protocol (50-4,000 RPM) at 23°C. Density was measured on an Anton Paar DMA 500 density meter.

[0170] Experimental results summary: Viscosity of Groups 2 and 3 formulations ranged from 1.8 mPa*s to 10.1 mPa*s and the calculated yield stress ranged from 0.7-1.5 mPa. Density of formulations ranged from 1.031-1.085 g / cm3 forgroup 2 and 3 formulations. Data are summarized in Table 4 and Table 5. A yield stress can inhibit flow under the relatively low stresses induced by gravity and thus having a yield stress in the formulation should help to mitigate cell settling in addition to the effect of increasing density and viscosity.

[0171] Table 4: Group 2 Formulation Viscosity and Density as a Function of Dextran 40 or HES and Sucrose Concentration

[0172] Viscosity Density Formulation1

[0173] (g / cm3)

[0174] Water 0.9 0.0 0.998

[0175] FIT 2.3 0.7 1.048

[0176] F2T 2.5%Dex5%Suc 3.6 0.8 1.057

[0177] F3T 5.0%Dex5%Suc 5.7 1.2 1.066

[0178] F4T 7.5%Dex5%Suc 6.6 0.4 1.075

[0179] F5T 10.0%Dex5%Suc 10.1 1.2 1.085

[0180]

[0181] F6T 7.5%HES5%Suc 8.4 1.5 1.075

[0182] 1 Properties of the formulations were measured without cells present Docket No. BYP240186 WO

[0183] Table 5: Group 3 Formulation Viscosity and Density as a Function of Dextran 40 and Sucrose Concentration

[0184] Densit Formulation

[0185] (g / cm3) iilililililil

[0186] F0H 1.8 0.7 1.031

[0187] F1H 3%Dex5%Suc 4.4 1.4 1.063

[0188]

[0189] F2H 5%Dex5%Suc 5.3 0.7 1.072

[0190] 1Properties of the formulations were measured without cells present

[0191] Effect of formulation components and density on cell settling

[0192] Materials and methods: Group 1, 2, and 3 formulations, as shown in Table 1, Table 2, and Table 3 were used to resuspend Jurkat T-Cells (Group 1) and Primary T-Cells (Group 2 and 3). Cells were received after cell expansion, spun down and decanted, and then resuspended in one of the designated formulations at a concentration of 0.8-1.6xl0A6 cells / mL for Groups 1 and 2 and a concentration of 18-27xlOA6 cells / mL for Group 3. The formulated cells were aliquoted into vials that were then stored at RT for up to 24 hr.

[0193] The total cell counts per mL and viability of the top, middle, and bottom layer of each vial were measured with a K2 Cellometer. The percentage of total cell count per mL in each layer was calculated by dividing the total cell count per mL of each layer by the sum of total cell counts per mL of the three layers in each vial.

[0194] Experimental results summary: From the Group 1 formulations, the data showed that the addition of 7.5-10% Dextran 40 to a formulation was a factor in maintaining Jurkat T-Cells in suspension after 6 hr and 24 hr hold times, see FIG. 1 and Table 6. Group 1 formulations that contained 5-10% Trehalose but formulations with no dextran 40 showed significant cell settling after a 6 hr hold time. From the Group 2 formulations, the data showed that the control formulation (F1T) and those containing dextran 40 concentrations ranging from 2.5%- 10% with 5% sucrose (minimum density of 1.057 g / mL and viscosity of 3.6 mPa*s) had no significant Primary T-Cell settling after a 3 hr hold time. Formulations with 7.5%-10% dextran 40 or 7.5% HES (minimum density of 1.075 g / mL and viscosity of 7 mPa*s) experienced less Primary T-Cell settling after a 24 hr hold time compared to the other formulations, see FIG. 2 and Table 7. From the Group 3 formulations, the data showed Docket No. BYP240186 WO

[0195] that the control formulation (FIT) had all Primary T-cells settling after a 5 hr hold time. The 3% dextran 40 formulation had slower cell settling compared to formulation FIT, but most cells settled by 8 hrs. The 5% dextran 40 formulation had the slowest cell settling that stabilized at about 60% at 5-8 hrs. Almost all cells settled for the three formulations by 24 hrs. See FIG. 3 and Table 8.

[0196] Table 6: Summary of Group 1 Formulation Effect on Distribution of the Total Count of Jurkat T-Cells by Position over Time

[0197] Group 1 Total Jurkat T-Cell Count

[0198] Distribution (%)

[0199] Formulation fiBli 24 hr

[0200] Bottom RT 39% 100% 100%

[0201] F1J Middle RT 36% 0% 0%

[0202] Top RT 25% 0% 0%

[0203] Bottom RT 29% 30% 49%

[0204] F2J 7.5Dex Middle RT 35% 30% 23%

[0205] Top RT 36% 40% 28%

[0206] Bottom RT 30% 34% 24%

[0207] F3J lODex Middle RT 30% 34% 38%

[0208] Top RT 40% 31% 38%

[0209] Bottom RT 37% 37% 44%

[0210] F4J 7.5Dex5%Tre Middle RT 33% 33% 36%

[0211] Top RT 30% 31% 20%

[0212] Bottom RT 31% 28% 38%

[0213] F5J 10%Dex5%Tre Middle RT 38% 30% 28%

[0214] Top RT 31% 42% 34%

[0215] Bottom RT 37% 79% 97%

[0216] F6J 2.5%Tre Middle RT 19% 14% 3%

[0217] Top RT 44% 7% 0%

[0218] Bottom RT 22% 69% 95%

[0219] F7J 5%Tre Middle RT 42% 9% 3%

[0220] Top RT 37% 22% 2%

[0221] Bottom RT 31% 72% 99%

[0222] F8J 10%Tre Middle RT 36% 17% 1%

[0223] Top RT 33% 12% 0%

[0224] Bottom RT 34% 39% 49%

[0225] F9J Middle RT 33% 31% 17% 7.5%Dex5%Suc Top RT 33% 30% 34%

[0226] Bottom RT 35% 32% 47%

[0227] F10J Middle RT 32% 27% 27% 10%Dex5%Suc Top RT 34% 41% 26%

[0228] Bottom RT 31% 85% 98%

[0229] Fl 1J l%Dex5%Tre Middle RT 37% 8% 0%

[0230]

[0231] Top RT 32% 7% 2%

[0232] RT = room temperature Docket No. BYP240186 WO

[0233] Table 7: Group 2 Formulation Effect on Distribution of the Total Count of Primary T-Cells by Position over Time

[0234] Group 2 % Cell count during hold

[0235] Location time

[0236] Formulation ■Oil 4 hr

[0237] Top RT 36% 46%

[0238] FIT Middle RT 32% 23%

[0239] Bottom RT 32% 31%

[0240] Top RT 31% 42%

[0241] F2T Middle RT 33% 27%

[0242] 2.5%Dex5%Suc Bottom RT 36% 31%

[0243] Top RT 28% 30%

[0244] F3T 5%Dex5%Suc Middle RT 39% 21%

[0245] Bottom RT 32% 49%

[0246] Top RT 32% 35%

[0247] F4T Middle RT 36% 27%

[0248] 7.5%Dex5%Suc Bottom RT 32% 38%

[0249] Top RT 29% 34%

[0250] F5T 10%Dex5%Sue Middle RT 35% 32%

[0251] Bottom RT 35% 34%

[0252] Top RT 28% 47%

[0253] F6T Middle RT 29% 25%

[0254] 7.5%HES5%Suc

[0255]

[0256] Bottom RT 43% 28%

[0257] RT = room temperature

[0258] Table 8: Summary of Group 3 Formulation Effect on Distribution of the Total Count of Primary T-Cells by Position over Time

[0259] Group 3 % Cell count during hold time Location

[0260] Formulation lllllilll iiiiliiiii 8 hr 24 hr Bottom RT 33% 0% 1% 0%

[0261] FOH Middle RT 34% 0% 3% 0%

[0262] Top RT 32% 99% 96% 100% Bottom RT 34% 1% 3% 0%

[0263] F1H 3%Dex5%Suc Middle RT 36% 27% 4% 2%

[0264] Top RT 30% 72% 93% 98% Bottom RT N / A 9% 11% 1% Middle RT N / A 22%

[0265] F1H 5%Dex5%Suc 30% 2%

[0266]

[0267] Top RT N / A 69% 59% 97%

[0268] RT = room temperature Docket No. BYP240186 WO

[0269] Formulation effect on cell viability of Jurkat and Primary T-cells.

[0270] Viability of Jurkat and Primary T-cells were evaluated for Group 1 and Group 2 formulations after storage at RT over a 24 hr period. Viability was measured for each location (top, middle, bottom) for vials stored at RT. In addition, a subset of vials was held at RT for 3-4 hrs, frozen to -130°C with a controlled-rate freezer and stored in a liquid nitrogen freezer for one week. Cell viability during RT hold time prior to freezing sets the acceptable processing time for the Fill / Finish manufacturing process. Cell stability post cryopreservation is a criterion for overall assessment of formulation performance.

[0271] Materials and methods: Cell viability was measured for all formulations with a K2 Cellometer. Frozen vials were thawed using a 37 °C water bath and vials were removed from the water as soon as the last ice crystals melted.

[0272] Experimental results summary: The formulation strategy of adding a disaccharide and Dextran 40 or HES to the base DMSO formulation not only maintained cells in suspension but enhanced the stability of Jurkat and Primary T-Cells while being stored at RT and after a cryopreservation / thaw cycle (See FIG. 8).

[0273] Viability was measured during a 24 hr hold time for each of the formulations: Group 1 Formulations: Jurkat T-Cell viability started at about 95% and was maintained above 90% for formulations that contained 7.5 - 10% dextran 40 and 5% of sucrose or trehalose. The formulations that contained no dextran 40 and 2.5% trehalose. See FIG. 4 and Table 10.

[0274] Group 2 Formulations: Primary T-Cell viability started at about 87% viability at 1 hr and most of the Group 2 formulations showed directionally higher cell viability compared to the DMSO control formulation (F1J) during the RT hold time of 24 hrs. See FIG. 5 and Table 11.

[0275] Group 3 Formulations: Jurkat T-Cell viability started at 81- 90% viability at 0 hr and there was no significant change in viability during the RT hold time of 24 hrs. See FIG. 6 and Table 11. Docket No. BYP240186 WO

[0276] Table 9: Group 2 Formulation Effect on Primary T-Cell Viability over 24 hrs at RT Group 2 lOiBii Primary T-Cell Viability (%)

[0277] Formulation 24 hr

[0278] (C°)

[0279] FIT RT 87% 82% 76%

[0280] F2T RT 92% 87% 82%

[0281] 2.5%Dex5%Suc

[0282] F3T 5%Dex5%Suc RT 86% 82% 82%

[0283] F4T RT 88% 88% 85%

[0284] 7.5%Dex5%Suc

[0285] F5T 10%Dex5%Suc RT 88% 87% 83%

[0286] F6T RT 88% 86% 80%

[0287]

[0288] 7.5%HES5%Suc

[0289] RT = room temperature

[0290] Table 10: Group 1 Formulation Effect on Jurkat T-Cell Viability over 24 hrs at RT Group 1 liHllll Jurkat T-Cell Via tility ( % )

[0291] Formulation liillli 6 hr 24 hr

[0292] F1J RT 99% 92% 68%

[0293] F2J 7.5%Dex RT 98% 90% 58%

[0294] F3J 10%Dex RT 99% 89% 72%

[0295] F4J 7.5%Dex5%Tre RT 100% 96% 97%

[0296] F5J 10%Dex5%Tre RT 100% 99% 94%

[0297] F6J 2.5%Tre RT 99% 100% 95%

[0298] F7J 5%Tre RT 99% 95% 89%

[0299] F8J 10%Tre RT 99% 99% 80%

[0300] F9J 7.5%Dex5%Suc RT 99% 97% 96%

[0301] F10 RT 99% 97% 97%

[0302] J10%Dex5%Suc

[0303]

[0304] F11J 1%Dex5%Tre RT 97% 91% 85%

[0305] RT = room temperature Docket No. BYP240186 WO

[0306] Table 11: Group 2 Formulation Effect on Primary T-Cell Viability over 24 hrs at RT Group 2 Primary T-Cell Viability < % »

[0307] Formulation iiililii 24 hr

[0308] iiiiiii

[0309] FIT RT 87% 82% 76%

[0310] F2T RT 92% 87% 82%

[0311] 2.5%Dex5%Suc

[0312] F3T 5%Dex5%Suc RT 86% 82% 82%

[0313] F4T RT 88% 88% 85%

[0314] 7.5%Dex5%Suc

[0315] F5T 10%Dex5%Suc RT 88% 87% 83%

[0316] F6T RT 88% 86% 80%

[0317]

[0318] 7.5%HES5%Suc

[0319] RT = room temperature

[0320] Table 12: Group 3 High Concentration Primary T-Cell Viability over 24 hr RT Hold Time Primary T-Cell Viability (%)

[0321] Group 3

[0322] Formulation liiiiili Ohr lliilliliil 8 hr 24 hr

[0323] F0H RT 81% 84% 85% 77% 87% F1H 3%Dex 5% Sue RT 89% 88% 89% 90% 88%

[0324]

[0325] F2H 5%Dex 5% Sue RT 88% 90% 88% 89% 83% RT = room temperature

[0326] Viability was measured after cell formulations were held frozen for 1 week in an LN2 freezer and thawed using a 37°C water bath.

[0327] • Group 1 Formulations: Jurkat T-Cell viability of about 95% or greater was achieved with all formulations except for the DMSO control (F1J), formulations with dextran 40 but no disaccharide, and the formulation with 10% trehalose and no dextran 40. See FIG 7 and Table 12.

[0328] • Group 2 Formulations: Primary T-Cell viability for all formulations had

[0329] similar cell viability post thaw, between 73-80%. See FIG. 8 and Table 13.

[0330] • Group 3 Formulations: Formulations with dextran 40 and 5% sucrose had Docket No. BYP240186 WO

[0331] marginally better cell viability compared to the DMSO control formulation

[0332] (FOH) post thaw. See FIG. 9 and Table 14.

[0333] Table 13: Group 1 Jurkat T-Cell Viability Post Freeze / Thaw

[0334] Group 1 Formulation Post Thaw F reeze / Thaw

[0335] F1J 87%

[0336] F2J 7.5%Dex 91%

[0337] F3J 10%Dex 90%

[0338] F4J 7.5%Dex5%Tre 98%

[0339] F5J 10%Dex5%Tre 98%

[0340] F6J 2.5%Tre 95%

[0341] F7J 5%Tre 97%

[0342] F8J 10%Tre 89%

[0343] F9J 7.5%Dex5%Suc 99%

[0344] F10J 10%Dex5%Suc 98%

[0345]

[0346] F11J 1%Dex5%Tre 96%

[0347] Table 14: Group 2 Primary T-Cell Viability Post Freeze / Thaw

[0348] Group 2 Formulation Post Thaw F reeze / Thaw

[0349] FIT 79%

[0350] F2T 2.5%Dex5%Suc 73%

[0351] F3T 5%Dex5%Suc 78%

[0352] F4T 7.5%Dex5%Suc 79%

[0353] F5T 10%Dex5%Suc 79%

[0354]

[0355] F6T 7.5%HES5%Suc 80% Docket No. BYP240186 WO

[0356] Table 15: Group 3 Primary T-Cell Viability Pre and Post Freeze / Thaw

[0357] Group 3 3 hr RT Post

[0358] Formulation iiiii^iii^ii^iiiiiii Freeze / Thaw Hi OBBOi III

[0359] Thaw + 3 iiii? Oi?iiiiiiiiiiiiii^

[0360] FOH 84% 74% 69%

[0361] F1H 3%Dex 5% Sue 88% 86% 86%

[0362]

[0363] F2H 5%Dex 5% Sue 90% 85% 85%

Claims

1. Docket No. BYP240186 WO2.CLAIMS3.We Claim:

1. A formulation that maintains the suspension and viability of a T-cell population during cell therapy (CT) manufacturing and through a cryopreservation thaw cycle, comprising:5.(a) 10% dimethyl sulfoxide (DMSO);6.(b) 1-7.5% sucrose or trehalose;7.(c) 1-5% human serum albumin (HSA); and8.(d) 2.5-20% polysaccharide or 2.5-20% hydroxyethyl starch (HES).9.wherein the formulation maintains suspension and viability of the T-cell population during CT manufacturing and through a cryopreservation thaw cycle.

2. A formulation as recited in Claim 1, wherein the T-cell population comprises a Jurkat cells.

3. A formulation as recited in Claim 1, wherein the T-cell population comprises a Primary T-cells.

4. A formulation as recited in Claim 1, wherein the 5% HSA is in Plasma-Lyte A and maintains cell suspension and viability for 4-24 hrs.

5. A formulation as recited in Claim 1, wherein the formulation kept cells suspended and maintained T-cell viability for up to 24 hrs. while stored at room temperature and, cell viability post freezing (freezing initiated after a 3-4 hr. hold time) was maintained close to a level of cell viability prior to freezing for up to 3 hrs. post thaw, as compared to a DMSO control formulation.

6. A method for maintaining a suspension and cell viability, comprising subjecting a T-cell population during a cell therapy manufacturing cryopreservation thaw cycle to a formulation comprising 10% dimethyl sulfoxide (DMSO); 5% sucrose or trehalose; 5% human serum albumin (HSA); and 2.5-10% dextran 40 or 5-7.5% hydroxyethyl starch (HES) wherein theDocket No. BYP240186 WO15.formulation maintains substantial suspension and viability of T-cell during cell therapy manufacturing and through a cryopreservation thaw cycle.

7. A method as recited in Claim 6, wherein the T-cell population comprises Jurkat cells.

8. A method as recited in Claim 6, wherein the T-cell population comprises Primary T-cells.

9. A method as recited in Claim 6, wherein the 5% HSA is in Plasma- Lyte A and maintains a cell suspension for 4-24 hrs.

10. A method as recited in Claim 6, wherein the formulation kept cells suspended and maintained T-cell viability for up to 24 hrs. while stored at room temperature and, cell viability post freezing (freezing initiated after a 3-4 hr. hold time) was maintained close to a level of cell viability prior to freezing for up to 3 hrs. post thaw, as compared to a DMSO control formulation.

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