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 - 46 . (canceled) 
     
     
         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 2° C. to about 8° 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 0.1 M 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 . 
     
     
         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 , 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 . 
     
     
         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 .