Sterile chromatography and manufacturing processes

Abstract

Provided herein are methods of performing chromatography with gamma-irradiated chromatography resin that include providing a chromatography column including a gamma-irradiated chromatography resin; performing a first cycle of chromatography through the column, where the cycle includes exposing the chromatography resin to a denaturing buffer; and performing at least one additional cycle of chromatography through the column. Also provided are integrated, closed or substantially closed, and continuous processes for manufacturing of a recombinant protein that include the use of at least one chromatography column including gamma-irradiated chromatography resin, where the gamma-irradiated chromatography resin is exposed to denaturing buffer during each cycle in the process, and reduced bioburden buffer is used in the process.

What is claimed is: 
     
         1 . A method of performing chromatography with gamma-irradiated chromatography resin, comprising:
 (a) providing a chromatography column containing a gamma-irradiated chromatography resin; and   (b) performing at least two cycles of chromatography with the chromatography column, wherein the chromatography column is exposed to a denaturing buffer during each of the at least two cycles of chromatography, and wherein during each exposure the flow rate, volume, and concentration of the denaturing buffer are selected to substantially recover binding capacity lost due to the gamma-irradiated chromatography resin.   
     
     
         2 . The method of  claim 1 , wherein the denaturing buffer comprises one or more of urea, guanidine hydrochloride, and 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol. 
     
     
         3 . The method of  claim 2 , wherein the denaturing buffer comprises 6 M to 9 M urea. 
     
     
         4 . The method of  claim 2 , wherein the denaturing buffer comprises 5 M to 7 M guanidine hydrochloride. 
     
     
         5 . The method of  claim 2 , wherein the denaturing buffer comprises 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol. 
     
     
         6 . The method of  claim 1 , wherein the denaturing buffer comprises one or more of a detergent, a reductant, an acid, a chaotropic agent, an organic solvent, and a crosslinking agent. 
     
     
         7 . The method of  claim 6 , wherein the denaturing buffer comprises one or more detergent(s) selected from the group consisting of: 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol, sodium dodecyl sulfate, and ethyl trimethyl ammonium bromide. 
     
     
         8 . The method of  claim 6 , wherein the denaturing buffer comprises one or more of the reductant(s) selected from the group consisting of: 2-mercaptoethanol, dithiothreitol, and tris(2-carboxyethyl)phosphine. 
     
     
         9 . The method of  claim 6 , wherein the denaturing buffer comprises one or more of the acid(s) selected from the group consisting of: acetic acid, trichloroacetic acid, and sulfosalicylic acid. 
     
     
         10 . The method of  claim 6 , wherein the denaturing buffer comprises one or more of the chaotropic agent(s) selected from the group consisting of: urea, thiourea, guanidine hydrochloride, lithium perchlorate, and lithium acetate. 
     
     
         11 . The method of  claim 6 , wherein the denaturing buffer comprises one or more of the organic solvent(s) selected from the group consisting of: ethanol, butanol, phenol, propanol, and methanol. 
     
     
         12 . The method of  claim 1 , wherein the denaturing buffer is 8 M urea, 1 M NaCl, 0.1 M citric acid, pH 2.5. 
     
     
         13 . The method of  claim 1 , wherein the denaturing buffer is 6 M guanidine hydrochloride, pH 2.5. 
     
     
         14 . The method of  claim 1 , wherein the denaturing buffer is 0.5% 2-[4-(2,4,4-trimethylpentan-2-yl)phenoxy]ethanol in 0.1 M acetic acid, pH 2.5. 
     
     
         15 . The method of  claim 1 , wherein the chromatography resin is an anion exchange chromatography resin or a cation exchange chromatography resin. 
     
     
         16 . The method of  claim 1 , wherein the chromatography resin is an anion exchange chromatography resin.