Apo-2 ligand/trail formulations

Abstract

The inventions include Apo2L/TRAIL formulations and methods of using such formulations. Lyophilized and crystal formulations of Apo-2L/TRAIL which are stable and have improved Apo2L/TRAIL trimer formation are provided. Methods of making Apo-2L/TRAIL formulations, as well as devices and kits containing such formulations are also provided.

1 . A stable formulation of Apo-2 ligand, comprising Apo-2 ligand and about 0.2M to about 0.5M salt, wherein said formulation has a pH of about 6 to about 9.  
     
     
         2 . The formulation of  claim 1  wherein said salt is arginine salt.  
     
     
         3 . The formulation of  claim 2  wherein the concentration of said arginine salt in the formulation is about 0.4M to about 0.5 M.  
     
     
         4 . The formulation of  claim 2  wherein the arginine salt is selected from the group consisting of arginine succinate, arginine sulphate, arginine malate, arginine citrate, arginine tartrate, and arginine phosphate.  
     
     
         5 . The formulation of  claim 2  wherein the arginine salt is arginine succinate.  
     
     
         6 . The formulation of  claim 1  wherein the salt is sodium sulphate.  
     
     
         7 . The formulation of  claim 1  or  6  wherein the Apo-2 ligand comprises crystallized protein.  
     
     
         8 . The formulation of  claim 1  wherein said formulation is lyophilized.  
     
     
         9 . The formulation of  claim 1  wherein the pH of said formulation is about 6.5 to about 8.5.  
     
     
         10 . The formulation of  claim 9  wherein the pH of said formulation is about 7 to about 7.5.  
     
     
         11 . The formulation of  claim 1  wherein the concentration of Apo-2 ligand is about 1 mg/ml to about 20 mg/ml.  
     
     
         12 . The formulation of  claim 1  wherein said Apo-2 ligand comprises amino acids 114 to 281 of  FIG. 1 .  
     
     
         13 . The formulation of  claim 12  wherein said Apo-2 ligand is not linked or fused to an epitope tag.  
     
     
         14 . The formulation of  claim 1  wherein said formulation further comprises surfactant.  
     
     
         15 . The formulation of  claim 14  wherein said surfactant is a polysorbate or poloxamer.  
     
     
         16 . The formulation of  claim 14  wherein the concentration of said surfactant in the formulation is about 0.005% to about 0.2%.  
     
     
         17 . The formulation of  claim 1  wherein said formulation further comprises buffer.  
     
     
         18 . The formulation of  claim 17  wherein said buffer is Tris buffer.  
     
     
         19 . The formulation of  claim 18  wherein the pH of the formulation is about 7 to about 7.5.  
     
     
         20 . The formulation of  claim 1  wherein said formulation further comprises one or more divalent metal ions.  
     
     
         21 . The formulation of  claim 20  wherein said one or more divalent metal ions is zinc.  
     
     
         22 . The formulation of  claim 1  further comprising a preservative.  
     
     
         23 . The formulation of  claim 1  wherein said formulation is storage-stable for at least 12 months.  
     
     
         24 . The formulation of  claim 23  wherein said formulation is storage-stable for at least 24 months.  
     
     
         25 . A stable, lyophilized formulation of Apo-2 ligand, comprising about 1 mg/ml to about 20 mg/ml Apo-2 ligand, about 0.2 M to about 0.5M arginine salt, buffer, and surfactant, wherein said formulation has a pH of about 6 to about 9.  
     
     
         26 . The formulation of  claim 25 , wherein said arginine salt is arginine succinate.  
     
     
         27 . The formulation of  claim 26 , wherein the concentration of said arginine succinate is about 0.4M to about 0.5M.  
     
     
         28 . The formulation of  claim 25 , wherein said buffer is Tris buffer.  
     
     
         29 . The formulation of  claim 25 , wherein said surfactant is a polysorbate.  
     
     
         30 . The formulation of  claim 25 , wherein said Apo-2 ligand comprises amino acids 114 to 281 of  FIG. 1 .  
     
     
         31 . The formulation of  claim 25 , wherein said formulation further comprises one or more divalent metal ions.  
     
     
         32 . A stable formulation of Apo-2 ligand, comprising about 1 mg/ml to about 20 mg/ml Apo-2 ligand, about 0.2M to about 0.5 M salt, buffer, and surfactant, wherein said Apo-2 ligand comprises crystallized protein and said formulation has a pH of about 6 to about 9.  
     
     
         33 . The formulation of  claim 32 , wherein said salt is sodium sulphate.  
     
     
         34 . The formulation of  claim 32 , wherein said buffer is Tris buffer.  
     
     
         35 . The formulation of  claim 32 , wherein said surfactant is polysorbate.  
     
     
         36 . The formulation of  claim 32 , wherein said formulation has a pH of about 7 to about 7.5.  
     
     
         37 . A stable formulation of Apo-2 ligand, comprising about 0.1 mg/ml to about 2 mg/ml Apo-2 ligand, sugar, and surfactant, wherein said formulation has a pH of about 6 to about 9.  
     
     
         38 . The formulation of  claim 37  wherein said sugar is trehalose.  
     
     
         39 . The formulation of  claim 37  wherein the concentration of the sugar in the formulation is about 1% to about 8%.  
     
     
         40 . The formulation of  claim 37  wherein said formulation is lyophilized.  
     
     
         41 . A method of making a stable formulation of Apo-2 ligand, comprising steps of (a) providing about 1 mg/ml to about 20 mg/ml Apo-2 ligand, about 0.2 M to about 0.5M arginine salt, buffer, and surfactant, (b) combining or mixing the ingredients of step (a) to make a formulation, and (c) adjusting the pH of the formulation of step (b) to about 6 to about 9.  
     
     
         42 . The method of  claim 41 , wherein said arginine salt is arginine succinate.  
     
     
         43 . The method of  claim 42 , wherein the concentration of said arginine succinate is about 0.4M to about 0.5M.  
     
     
         44 . The method of  claim 41 , wherein said buffer is Tris buffer.  
     
     
         45 . The method of  claim 41 , wherein said surfactant is a polysorbate.  
     
     
         46 . The method of  claim 41 , wherein said Apo-2 ligand comprises amino acids 114 to 281 of  FIG. 1 .  
     
     
         47 . A method of making crystallized Apo-2 ligand, comprising steps of (a) providing Apo-2 ligand, buffer, and monovalent cationic salt, (b) combining or mixing the ingredients of step (a) to make a formulation at a temperature of about 20° C. to about 30° C, and (c) lowering the temperature of the formulation of step (b) to about 20 C to about 80 C; wherein Apo-2 ligand crystallization occurs as the temperature of the formulation of step (b) is lowered.  
     
     
         48 . The method of  claim 47 , wherein said salt is sodium sulphate or sodium chloride.  
     
     
         49 . The method of  claim 48  wherein the concentration of the salt is 0.1M to about 0.15M.  
     
     
         50 . The method of  claim 47 , wherein the formulation of step (b) is agitated as the temperature is lowered in step (c).  
     
     
         51 . The method of  claim 47 , wherein the method further comprises a step (d) in which the Apo-2 ligand crystals are dried.  
     
     
         52 . The method of  claim 51 , wherein prior to said step (d), the Apo-2 ligand crystals are washed.  
     
     
         53 . A method of making Apo-2 ligand, comprising the steps of: (a) providing host cells comprising a vector containing DNA encoding Apo-2 ligand; (b) culturing the host cells in culture medium under conditions sufficient to express Apo-2 ligand; (c) obtaining said expressed Apo-2 ligand from the host cells and culture medium; (d) formulating said Apo-2 ligand into a solution containing sodium chloride or sodium sulphate to make a formulation at a temperature of about 20° C. to about 30° C., and (e) lowering the temperature of said formulation of step (d) to about 2° C. to about 8° C., wherein Apo-2 ligand crystals form when the temperature of step (e) is lowered.  
     
     
         54 . The method of  claim 53  wherein prior to said step (d), the Apo-2 ligand protein is concentrated.  
     
     
         55 . The method of  claim 54  wherein the Apo-2 ligand protein is concentrated by centrifugation, column chromatography or ultrafiltration.  
     
     
         56 . The method of  claim 53  wherein step (d) is conducting by applying the Apo-2 ligand to a chromatographic column and eluting the Apo-2 ligand into a sodium chloride or sodium sulphate containing buffer solution.  
     
     
         57 . The method of  claim 56  wherein said chromatographic column is a cation exchange column.  
     
     
         58 . The method of  claim 56  wherein said cation exchange column comprises SP-Sepharose fast flow, CM-Sepharose fast flow, or Macro-prep ceramic HS.  
     
     
         59 . The method of  claim 56  wherein said buffer solution contains 50 mM Hepes, 50 mM Tris, 50 mM triethanolamine, 0.05% Triton X 100, 1 mM DTT, pH 7.5-8.0.  
     
     
         60 . The method of  claim 53  wherein the formulation is agitated during step (e).  
     
     
         61 . The method of  claim 53  wherein the pH of the formulation in step (d) is about 6.5 to about 8.5.  
     
     
         62 . The method of  claim 53  wherein said host cells are prokaryote cells.  
     
     
         63 . The method of  claim 62  wherein said prokaryote cells are  E. coli.    
     
     
         64 . A device for administering a formulation of Apo-2 ligand to a mammal, comprising a container holding at least one dosage unit of the Apo-2 ligand formulation of  claim 1 ,  25 ,  32 , or  37 .  
     
     
         65 . The device of  claim 64  wherein said device is a pen injector device.  
     
     
         66 . The device of  claim 64  wherein the container is a cartridge.  
     
     
         67 . An article of manufacture, comprising a container which includes the Apo2L/TRAIL formulation of  claim 1 ,  25 ,  32 , or  37 , and printed instructions for use of said Apo-2L/TRAIL formulation.  
     
     
         68 . The article of manufacture of  claim 67  where said container is a bottle, vial, syringe, or test tube.  
     
     
         69 . The article of manufacture of  claim 67  which comprises a second container which includes water-for-injection, saline, Ringer's solution, or dextrose solution.  
     
     
         70 . A method of inducing apoptosis in mammalian cells, comprising exposing mammalian cells to an effective amount of the Apo-2 ligand formulation of  claim 1 ,  25 ,  32 , or  37 .  
     
     
         71 . The method of  claim 70  wherein said mammalian cells are cancer cells.  
     
     
         72 . A method of treating cancer in a mammal, comprising administering to a mammal diagnosed as having cancer an effective amount of the Apo-2 ligand formulation of  claim 1 ,  25 ,  32 , or  37 .