Depth Filters For Disposable Biotechnological Processes

Abstract

A process for the primary clarification of feeds, including chemically treated flocculated feeds, containing the target biomolecules of interest such as mAbs, mammalian cell cultures, or bacterial cell cultures, using a primary clarification depth filtration device without the use of a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step. The primary clarification depth filtration device contains a porous depth filter having graded porous layers of varying pore ratings. The primary clarification depth filtration device filters fluid feeds, including chemically treated flocculated feeds containing flocculated cellular debris and colloidal particulates having a particle size distribution of approximately about 0.5 μm to 200 μm, at a flow rate of about 10 litres/m 2 /hr to about 100 litres/m 2 /hr. Kits and methods of using and making the same are also provided.

1 . A process for the clarification of a feed containing a target biomolecule of interest and a plurality of cellular debris and/or colloidal particulates by depth filtration without the use of a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step, the process comprising:
 a) providing a depth filtration device having a porous depth filter media;   b) providing a feed containing a target biomolecule of interest and a plurality of cellular debris and/or colloidal particulates;   c) contacting the depth filter media with the feed; and   d) separating the target biomolecule of interest from the cellular debris and the colloidal particulates in the feed without the use of a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step.   
     
     
         2 . The process of  claim 1 , further comprising adding a chemical flocculant to the feed in step (b), forming a chemically flocculated feed including a plurality of flocculated cellular debris and colloidal particulates. 
     
     
         3 . The process of  claim 1 , wherein the porous depth filter media is anisotropic, the pores have a nominal pore size rating >about 25 μm, and the filter flocculated feed have >about 3% solids resulting in a turbidity output <about 20 NTU. 
     
     
         4 . The process of  claim 1 , wherein the clarification process is a primary clarification process, and the depth filter comprises at least 2 graded layers of non-woven fibers. 
     
     
         5 . The process of  claim 4 , wherein the graded layers have a total thickness of about 0.3 cm to about 3 cm. 
     
     
         6 . The process of  claim 1 , wherein the depth filter comprises at least 3 graded layers of non-woven fibers. 
     
     
         7 . The process of  claim 6 , wherein the graded layers have a total thickness of about 0.3 cm to about 3 cm. 
     
     
         8 . The process of  claim 1 , wherein the cellular debris and the colloidal particulates have a particle size distribution from about 0.5 μm to about 200 μm, and a mean particle size greater than about 10 μm. 
     
     
         9 . The process of  claim 2 , wherein the depth filter media comprises a composite of graded layers of non-woven fibers, cellulose, and diamatoceous earth having an open nominal pore size rating sufficient to filter the chemically flocculated feedstock. 
     
     
         10 . The process of  claim 1 , wherein the target biomolecule of interest includes monoclonal antibodies (mAbs), polyclonal antibodies, and biotherapeutics. 
     
     
         11 . The process of  claim 1 , wherein the chemical flocculant is a polymer or an acid. 
     
     
         12 . The process of  claim 1 , wherein the chemical flocculant is a smart polymer. 
     
     
         13 . The process of  claim 12 , wherein the smart polymer is a modified polyamine. 
     
     
         14 . The process of  claim 11 , wherein the acid is acetic acid. 
     
     
         15 . The process of  claim 4 , wherein the non-woven fibers comprise polypropylene, polyethylene, polyester, or nylon. 
     
     
         16 . The process of  claim 2 , wherein the depth filter provides throughputs >about 100 L/M 2 /hr and removes flocculated cellular debris and colloidal particulates having a particle size distribution of about 0.5 μm to about 200 μm. 
     
     
         17 . A process according to  claim 1 , wherein the feed is a cell culture located in a bioreactor and is directly loaded from the bioreactor into the depth filtration device for primary clarification processing. 
     
     
         18 . A process according to  claim 17 , wherein the clarified cell culture effluent is directly loaded onto a protein A bind and elute chromatography column for chromatographic processing. 
     
     
         19 . A process for the primary clarification of a flocculated feed including a bimolecular species of interest and a plurality of cellular materials by depth filtration without the use of a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step, the process comprising:
 a) providing a depth filtration device having a porous depth filter media;   b) providing a chemical flocculant;   c) providing a feed containing a target biomolecule of interest and a plurality of cellular materials and/or colloidal particulates;   d) adding the chemical flocculant to the feed;   e) forming a chemically flocculated feed including flocculated cellular materials and/or colloidal particulates;   f) contacting the depth filter media with the chemically flocculated feed; and   g) separating the target biomolecule of interest from the flocculated cellular materials and/or flocculated colloidal particulates in the feed without the use of a primary clarification centrifugation step or a primary clarification tangential flow microfiltration clarification step.   
     
     
         20 . The process of  claim 19 , wherein the porous depth filter media is anisotropic, the pores have a nominal pore size rating >about 25 μm, and the flocculated feed has >about 3% solids resulting in a turbidity output <about 20 NTU. 
     
     
         21 . The process of  claim 19 , wherein the depth filter comprises at least 2 graded layers of non-woven fibers. 
     
     
         22 . The process of  claim 21 , wherein the graded layers have a total thickness of about 0.3 cm to about 3 cm. 
     
     
         23 . The process of  claim 19 , wherein the depth filter comprises at least 3 graded layers of non-woven fibers. 
     
     
         24 . The process of  claim 23 , wherein the graded layers have a total thickness of about 0.3 cm to about 3 cm. 
     
     
         25 . The process of  claim 19 , wherein the cellular materials and colloidal particulates have a particle size distribution from about 0.5 μm to about 200 μm, and a mean particle size greater than about 10 μm. 
     
     
         26 . The process of  claim 19 , wherein the depth filter media comprises a composite of graded layers of non-woven fibers, cellulose, and diamatoceous earth having an open nominal pore size rating sufficient to filter the chemically flocculated feed. 
     
     
         27 . The process of  claim 19 , wherein the target biomolecule of interest includes monoclonal antibodies (mAbs), polyclonal antibodies, and biotherapeutics. 
     
     
         28 . The process of  claim 19 , wherein the chemical flocculant is a polymer or an acid. 
     
     
         29 . The process of  claim 19 , wherein the chemical flocculant is a smart polymer. 
     
     
         30 . The process of  claim 29 , wherein the smart polymer is modified polyamine. 
     
     
         31 . The process of  claim 28 , wherein the acid is acetic acid. 
     
     
         32 . The process of  claim 19 , wherein the depth filter provides throughputs >about 100 L/M 2  and removes the flocculated cellular debris and flocculated colloidal particulates. 
     
     
         33 . The process of  claim 19 , wherein the feed includes cell cultures, transgenic mammalian cell cultures, bacterial cell cultures, nontransgenic mammalian cell cultures, tissue cultures, microbial fermentation batches, plant extracts, biofuel, seawater cultures, freshwater cultures, wastewater cultures, treated sewage, untreated sewage, milk cultures, blood cultures, and combinations thereof. 
     
     
         34 . The process of  claim 19 , wherein the non-woven fibers comprise polypropylene. 
     
     
         35 . A process according to  claim 19 , wherein the feed is a cell culture located in a bioreactor and is directly loaded from the bioreactor into the depth filtration device for primary clarification processing. 
     
     
         36 . A process according to  claim 35 , wherein the clarified cell culture effluent is directly loaded onto a protein A bind and elute chromatography column for chromatographic processing. 
     
     
         37 . A depth filtration device for the primary clarification of a feed containing a target biomolecule of interest and a plurality of cellular debris and/or colloidal particulates without the use of a primary clarification centrifugation step or a primary clarification tangential flow microfiltration step, comprising a porous depth filter media having at least 2 graded layers of non-woven fibers having a total thickness of about 0.3 cm to about 3 cm, and the pores have a nominal pore size rating >about 25 μm. 
     
     
         38 . The device of  claim 37 , wherein the media is anisotropic. 
     
     
         39 . The device of  claim 37 , wherein the media comprises at least 3 graded layers of non-woven fibers. 
     
     
         40 . The device of  claim 37 , wherein the media comprises a composite of graded layers of non-woven fibers, cellulose, and diamatoceous earth having an open nominal pore size rating sufficient to filter the chemically flocculated feedstock. 
     
     
         41 . The device of  claim 37 , wherein the non-woven fibers comprise polypropylene, polyethylene, polyester, or nylon. 
     
     
         42 . The device of  claim 37 , wherein the cellular debris and the colloidal particulates have a particle size distribution from about 0.5 μm to about 200 μm, and a mean particle size greater than about 10 μm. 
     
     
         43 . The device of  claim 37 , wherein the media provides throughputs >about 100 L/M 2 /hr and removes flocculated cellular debris and colloidal particulates having a particle size distribution of about 0.5 μm to about 200 μm. 
     
     
         44 . The device of  claim 37 , wherein the filter flocculated feed has >about 3% solids resulting in a turbidity output <about 20 NTU when a chemical flocculant is added to the feed. 
     
     
         45 . The device of  claim 37 , wherein the target biomolecule of interest includes monoclonal antibodies (mAbs), polyclonal antibodies, and biotherapeutics. 
     
     
         46 . A process for purifying a target protein from a sample comprising:
 (i) providing a sample comprising a target protein and one or more impurities;   (ii) adding a precipitant to the sample and removing one of more impurities using a gradient density depth filter, thereby to obtain a clarified sample;   (iii) subjecting the clarified sample from (ii) to a bind and elute chromatography step, thereby to obtain an eluate;   (iv) contacting the eluate from step (iii) with one or more virus inactivating agents using one or more in-line static mixers or one or more surge tanks;   (v) subjecting the output of the virus inactivation step to a flow-through purification process comprising the use of two or more matrices selected from activated carbon, anion exchange chromatography media, cation exchange chromatography media and virus filtration membrane; and   (vi) formulating the output from step (v) at desired concentration and buffer conditions,   wherein the process is performed continuously such that at least two or more of the process steps are performed concurrently during at least portion of their duration.