Method of purifying apo-2 ligand/trail usin crystallisation the cold

Abstract

The present invention relates generally to Apo2L/TRAIL purification involving crystallization.

1 . A method of recovering Apo2L/TRAIL from a mixture comprising
 (a) loading the mixture on a cation exchange column;   (b) washing the cation exchange column with an equilibration buffer whereby non-binding components present in the mixture are removed;   (c) eluting Apo2L/TRAIL bound to the cation exchange column with an elution buffer;   (d) gradually cooling the eluate to a temperature of about 2 to 4° C., whereby Apo2L/TRAIL is spontaneously precipitated in a crystalline form to yield a mixture of mother liquor and Apo2L/TRAIL crystals, and   (e) recovering Apo2L/TRAIL from the mixture obtained in step (d) in a purity of at least about 99%.   
     
     
         2 . The method of  claim 1  wherein the mixture loaded on the cation exchange column is a culture medium or cell lysate of Apo2L/TRAIL producing cells. 
     
     
         3 . The method of  claim 2  wherein said mixture is the cell lysate of Apo2L/TRAIL producing  E. coli  host cells. 
     
     
         4 . The method of  claim 3  wherein said lysate is clarified prior to loading on the cation exchange column. 
     
     
         5 . The method of  claim 1  wherein the eluate obtained in step (c) is subjected to the crystallization step of (d) without additional purification. 
     
     
         6 . The method of  claim 1  wherein the cation exchange column is an SP-Sepharose column. 
     
     
         7 . The method of  claim 6  wherein the pH of the mixture loaded on said column is or is adjusted to about 7.5. 
     
     
         8 . The method of  claim 6  wherein elution of Apo2L/TRAIL is performed in an elution buffer comprising 100-200 mM NaCl or 100-150 mM Na 2 SO 4  in a buffer adjusting the pH to 7.5-7.8. 
     
     
         9 . The method of  claim 1  wherein in step (d) the eluate is cooled from a temperature of about 15 to 30° C. to a temperature of about 2 to 8° C. in about 1 to 60 hours. 
     
     
         10 . The method of  claim 9  wherein in step (d) the eluate is cooled to a temperature of about 2 to 8° C. in about 1 to 8 hours. 
     
     
         11 . The method of  claim 9  wherein in step (d) the eluate is cooled to a temperature of about 2 to 8° C. in about 1 hour. 
     
     
         12 . The method of  claim 11  wherein in step (d) the eluate is cooled to a temperature of about 4° C. in about 1 hour. 
     
     
         13 . The method of  claim 1  wherein the pH of the eluate is or is adjusted to pH 7.0-8.0 prior to crystallization. 
     
     
         14 . The method of  claim 13  wherein the pH of the eluate is or is adjusted to about 7.3 prior to crystallization. 
     
     
         15 . The method of  claim 13  wherein the pH of the eluate is or is adjusted to about 7.5-8.0 after crystallization. 
     
     
         16 . The method of  claim 1  wherein in step (d) the temperature of about 2 to 4° C. is maintained until equilibrium solubility of Apo2L/TRAIL is achieved or nearly achieved. 
     
     
         17 . The method of  claim 16  wherein in step (d) solubility of Apo2L/TRAII is decreased by the addition of an anti-solvent. 
     
     
         18 . The method of  claim 17  wherein said anti-solvent is selected from the group consisting of a polyethylene glycol (PEG), MPD, ethanol, isopropanol, and dioxane. 
     
     
         19 . The method of  claim 18  wherein the molecular weight of the PEG is between about 400 and about 10,000 daltons. 
     
     
         20 . The method of  claim 19  wherein the molecular weight of the PEG is 400, 3,350 or 10,000 daltons. 
     
     
         21 . The method of  claim 1  wherein in step (e) Apo2L/TRAIL is recovered in the form of crystals separated from the mother liquor by filtration or centrifugation or a combination thereof. 
     
     
         22 . The method of  claim 21  wherein pH of the mother liquor is adjusted to about 8.0 prior to filtration to decrease solubility. 
     
     
         23 . The method of  claim 1  further comprising the steps of dissolving the Apo2L/TRAIL crystals obtained in step (d) and subjecting the solution obtained to a second chromatographic purification step. 
     
     
         24 . The method of  claim 23  wherein said second chromatographic purification step is hydrophobic interaction chromatography. 
     
     
         25 . The method of  claim 24  wherein hydrophobic interaction chromatography is performed on a Phenyl-Sepharose column. 
     
     
         26 . The method of  claim 23  wherein said second chromatographic purification step is cation exchange chromatography. 
     
     
         27 . The method of  claim 26  wherein said cation exchange chromatography is performed on an CM-Sepharose or SP-Sepharose column. 
     
     
         28 . The method of  claim 23  wherein Apo2L/TRAIL is recovered and formulated following said second chromatographic purification step by ultrafiltration-diafiltration. 
     
     
         29 . The method of  claim 1  wherein said purity is at least about 99.5%. 
     
     
         30 . The method of  claim 1  wherein said purity is at least about 99.9%.