Purification of Biological Molecules

Abstract

The present invention relates to improved processes and systems for purification of biological molecules, where the processes can be performed in a continuous manner.

What is claimed is: 
     
         1 . A process for the purification of a target molecule comprising the steps of:
 a) providing a sample comprising the target molecule and one or more impurities;   b) adding at least one precipitant to the sample and removing one or mom impurities, thereby to recover a clarified sample;   c) subjecting the clarified sample from step (b) to a bind and elute chromatography step comprising at least two separation units, thereby to obtain an eluate comprising the target molecule; and   d) subjecting the eluate to flow-through purification comprising use of two or more media;   wherein at least two steps are performed concurrently for at least a portion of their duration and wherein the process comprises only one bind and elute chromatography step.   
     
     
         2 . The process of  claim 1 , wherein the process is a continuous process. 
     
     
         3 . The process of  claim 1 , comprising a virus inactivation step between steps (c) and (d). 
     
     
         4 . The process of  claim 3 , wherein the virus inactivation step comprises use of a virus inactivating agent selected from acid, detergent, solvent and temperature change. 
     
     
         5 . The process of  claim 3 , wherein virus inactivation step comprises use of one or more in-line static mixers. 
     
     
         6 . The process of  claim 3 , wherein virus inactivation comprises use of one or more surge tanks. 
     
     
         7 . The process of  claim 1 , wherein the target molecule is an antibody. 
     
     
         8 . The process of  claim 7 , wherein the antibody is selected from a monoclonal antibody or a polyclonal antibody. 
     
     
         9 . The process of  claim 1 , wherein the precipitant in step (b) is a stimulus responsive polymer. 
     
     
         10 . The process of  claim 9 , wherein the stimulus responsive polymer is a modified polyallylamine polymer. 
     
     
         11 . The process of  claim 1 , wherein the precipitant in step (b) is selected from the group consisting of an acid, caprylic acid, a flocculant and a salt. 
     
     
         12 . The process of  claim 1 , wherein removing impurities in step (b) comprises use of one or more depth filters. 
     
     
         13 . The process of  claim 1 , wherein removing impurities in step (b) comprises use of centrifugation. 
     
     
         14 . The process of  claim 1 , wherein the bind and elute chromatography step in (c) employs continuous multi-column chromatography. 
     
     
         15 . The process of  claim 1 , wherein the bind and elute chromatography step in (c) is selected from the group consisting of affinity chromatography, cation exchange chromatography and mixed-mode chromatography. 
     
     
         16 . The process of  claim 1 , wherein the bind and elute chromatography step in (c) employs Protein A affinity chromatography. 
     
     
         17 . The process of  claim 16 , wherein Protein A affinity chromatography employs a Protein A ligand coupled to a matrix selected from the group consisting of rigid hydropbilic polyvinylether polymer, controlled pore glass and agarose. 
     
     
         18 . The process of  claim 1 , wherein the sample in step (a) is a cell culture. 
     
     
         19 . The process of  claim 18 , wherein the cell culture is provided in a bioreactor. 
     
     
         20 . The process of  claim 19 , wherein the bioreactor is a single use bioreactor. 
     
     
         21 . The process of  claim 18 , wherein the cell culture is provided in a vessel other than a bioreactor. 
     
     
         22 . The process of  claim 1 , wherein the precipitant in step (b) is added to a bioreactor comprising a cell culture. 
     
     
         23 . The process of  claim 22 , wherein the precipitant is added using a static mixer. 
     
     
         24 . The process of  claim 1 , wherein the precipitant in step (b) is added to a vessel other than a bioreactor which comprises the sample comprising the target molecule. 
     
     
         25 . The process of  claim 1 , wherein the flow-through purification in step (d) employs two or more media selected from activated carbon, anion exchange chromatography media and cation exchange chromatography media. 
     
     
         26 . The process of  claim 25 , wherein the flow-through purification in step (d) further comprises use of a virus filtration membrane. 
     
     
         27 . The process of  claim 25 , wherein the cation exchange chromatography media is in the form of a membrane, a bead or a fiber. 
     
     
         28 . The process of  claim 1 , wherein the process comprises use of one or more surge tanks and does not employ any pool tanks between process steps. 
     
     
         29 . The process of  claim 1 , further comprising a formulation step. 
     
     
         30 . The process of  claim 29 , wherein formulation comprises diafiltration, concentration and sterile filtration. 
     
     
         31 . A flow-through process for purifying a target molecule from a Protein A eluate comprising the steps of:
 (a) contacting the eluate recovered from a Protein A chromatography column with activated carbon;   (b) contacting the flow-through sample front step (a) with an anion exchange chromatography media; and   (c) contacting the flow-through sample from step (b) with a cation exchange cinematography media; and   (d) obtaining the flow-through sample from step (b) comprising the target molecule,   wherein the eluate flows continuously through steps (a)-(c) and wherein level of one or more impurities in the flow-through sample in (d) is lower than the level in the eluate in step (a).   
     
     
         32 . The flow-through process of  claim 31 , further comprising subjecting the flow-through sample from step (c) to virus filtration. 
     
     
         33 . The flow-through process of  claim 31 , further comprising use of an in-line static mixer and/or a surge tank between steps (b) and (c) to change pH. 
     
     
         34 . The flow-through process of  claim 31  or  32  or  33 , wherein the process employs a single skid. 
     
     
         35 . The flow-through process of  claim 31 , wherein the eluate from the Protein A chromatography column is subjected to virus inactivation prior to contacting with activated carbon. 
     
     
         36 . The process of  claim 31 , wherein steps (a)-(c) may be performed in any order. 
     
     
         37 . A flow-through purification process for purifying a target molecule front a Protein A eluate comprising contacting the eluate with two or more media selected from activated carbon, anion exchange media, cation exchange media and virus filtration media, wherein the flow of the eluate is continuous. 
     
     
         38 . A system for use in a purification process comprising the following devices:
 a) a bioreactor:   b) a filtration device comprising one or more depth filters:   c) a single bind and elute chromatography apparatus; and   d) a flow-through purification system comprising at least a flow-through anion exchange device, wherein the devices in (a)-(d) are connected to be in fluid communication with each other, such that a sample can flow continuously through the system.   
     
     
         39 . The system according to  claim 38 , wherein the bioreactor in (a) is a single-use bioreactor. 
     
     
         40 . The system according to  claim 38 , wherein the system is enclosed in a sterile environment. 
     
     
         41 . The system according to  claim 38 , wherein the bind and elute chromatography apparatus in (c) comprises at least two separation units, with each unit comprising the same chromatography media, and wherein the two separation units are connected so a sample can flow front one to the next. 
     
     
         42 . The system according to  claim 38 , wherein the bind and elute chromatography apparatus in (c) comprises three or mote separation units having the same chromatography media, wherein the three of more separation units are connected so that liquid can flow from one separation unit to the next and from the last to the first separation unit. 
     
     
         43 . The system according to  claim 38 , wherein the flow-through purification system in (d) further comprises a device selected from an activated carbon device, a cation exchange chromatography device and a virus filtration device. 
     
     
         44 . The system according to  claim 38 , wherein the flow-through purification system in (d) employs a single skid. 
     
     
         45 . A process for purifying a target molecule from a sample, the process comprising the steps of:
 (a) providing a bioreactor comprising a cell culture;   (b) adding a precipitant to the bioreactor and removing one or more impurities, thereby resulting in a clarified sample;   (c) continuously transferring the clarified sample to a Protein A affinity chromatography step, which employs at least two separation units, thereby to obtain an eluate;   (d) continuously transferring the eluate from step (c) to a in-line static mixer or a surge tank for mixing the eluate with one or more virus inactivating agents;   (e) continuously transferring the eluate after step (d) into a flow-through purification operation comprising contacting the eluate in flow-through mode with activated carbon followed by an anion exchange chromatography media followed by an in-line static mixer and/or a surge tank to change pH followed by a cation exchange chromatography media followed by a virus filtration media; and   (f) formulating the flow-through sample from step (e) at a desired concentration in a desired buffer,   
       wherein process steps are connected to be in fluid communication with each other, such that a sample can flow continuously from one process step to the next, and where at least two process steps (b)-(f) are performed concurrently during at least a portion of their duration. 
     
     
         46 . A process for purifying a target molecule from a sample, the process comprises the steps of:
 (a) providing a bioreactor comprising a cell culture;   (b) adding a precipitant to the bioreactor and removing one or more impurities, thereby resulting in a clarified sample;   (c) adding one or more additives selected from the group consisting of a salt, a detergent, a surfactant and a polymer to the clarified sample;   (d) subjecting the clarified sample to a Protein A affinity chromatography step, which employs at least two separation units, thereby to obtain an eluate;   (e) subjecting the eluate from step (d) to a virus inactivating agent using an in-line static mixer or a surge tank;   (f) contacting the eluate after virus inactivation to a flow-through purification operation comprising contacting the eluate in flow-through mode with activated carbon followed by an anion exchange chromatography media followed by an in-line static mixer and/or a surge tank to change pH followed by a cation exchange chromatography media followed by a virus filtration media; and   (g) formulating the flow-through sample from step (f) at a desired concentration in a desired buffer,   
       wherein the process steps are connected to be in fluid communication with each other, such that a sample can flow continuously from one process step to the next, and where at least two process steps (b)-(g) are performed concurrently during at least a portion of their duration. 
     
     
         47 . The process of  claim 46 , wherein the additive is a salt. 
     
     
         48 . The process of  claim 47 , wherein the salt is 0.5 M NaCl.