Abstract:
A method for promoting biological activity uses a filter system to increase cell production of a fed batch bioreactor. The filter system cycles bioreactor fluid through a hollow fiber tangential flow filter which separates metabolic wastes (as well as proteins) from cells produced in bioreactor and returned to fed batch bioreactor, improving cell production in the fed batch bioreactor. The filter system includes a disposable pump and filter, and a reusable control system. The pump is a low shear gamma stable pump gently cycling bioreactor fluid through the filter with minimal damage to the cells produced in the bioreactor. The pumphead and hollow fiber tangential flow filter are disposable. The pump motor is part of the control system and is reusable. The pumphead and filter are provided as an assembled and pre-sterilized unit allowing simple and quick attachment to the fed batch bioreactor, and simple and quick disposal.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    The present application is a Continuation In Part of U.S. patent application Ser. No. 13/633,272 filed Oct. 2, 2012 which application is incorporated in its entirety herein by reference. 
     
    
     BACKGROUND OF THE INVENTION 
       [0002]    The present invention relates to bioreactors and in particular to an improved bioreactor system and method including a disposable pumphead and tangential flow filter. 
         [0003]    Bioreactor systems provide an environment supporting biological activity and known bioreactor systems build up metabolic waste in the bioreactor. The buildup of build up metabolic waste limits the amplification or cell growth within the bioreactor. As a result, known high capacity bioreactor systems require either a very large and expensive bioreactor or require filtering biological material in the bioreactor to continue the biological activity. Known pump and filter systems used for such filtering require a sterile environment. Components in the pump include parts which in some instances require replacement between each run. Mechanical pump components which wear or tear may give off debris into the bioreactor filter. Unfortunately, when pumps or filters of known systems require service or replacement, the required procedures can be time consuming due to the requirement to maintain the sterile environment. 
       BRIEF SUMMARY OF THE INVENTION 
       [0004]    The present invention addresses the above and other needs by providing a method for promoting biological activity uses a filter system to increase cell production of a fed batch bioreactor. The filter system cycles Bioreactor fluid through a hollow fiber tangential flow filter which separates metabolic wastes (as well as proteins) from cells produced in Bioreactor and returned to fed batch bioreactor, improving cell production in the fed batch bioreactor. The filter system includes a disposable pump and filter, and a reusable control system. The pump is a low shear gamma stable pump gently cycling bioreactor fluid through the filter with minimal damage to the cells produced in the bioreactor. The pumphead and hollow fiber tangential flow filter are disposable. The pump motor is part of the control system and is reusable. The pumphead and filter are provided as an assembled and pre-sterilized unit allowing simple and quick attachment to the fed batch bioreactor, and simple and quick disposal. 
         [0005]    In accordance with one aspect of the invention, there is provided a tangential flow filter system including a disposable, low shear pumphead. The disposable, low shear pumphead allows filtering bioreactor fluid without contamination or damage to cells. A preferred disposable pumphead includes no mechanical interaction between parts, and includes magnet elements which rotate in the presence of a rotating magnetic field, effectively as a rotor in an electric motor. An example of a preferred pump is made by Levitronix in Switzerland (Zurick) with offices in Waltham, Mass. The time to set-up, flush and sterilize known perfusion systems which do not include a disposable pumphead is extensive in comparison to the pumphead of the present invention. The disposable low shear recirculating pumphead, tangential flow filter, and associated tubing are pre-sterilized and aseptically connectable to the bioreactor. 
         [0006]    In accordance with another aspect of the invention, there is provided a tangential flow filter system including either manual or automatic control of a perfusion process. Some modes of operation are designed for seed reactors, continuous perfusion reactors, concentrated fed batch perfusion as well as cell or cell debris clarification (post transfection or Cell Lysis). 
         [0007]    In accordance with still another aspect of the invention, there is provided a disposable perfusion tangential flow filter system which decreases existing bioreactors size requirements. Systems one tenth the size of known fed-batch processing systems can provide protein productivity equivalent or better extracellular proteins as well as overall concentration of material. 
         [0008]    In accordance with yet another aspect of the invention, there is provided a disposable perfusion tangential flow filtering system which facilitates implementing or changing a pre-assembled, pre-sterilized perfusion tangential flow processing system without impacting the bioreactor sterility both during the operation or upon start up. The disposable perfusion tangential flow filtering system completely eliminates the need for autoclaving components. The disposable perfusion tangential flow filtering system is designed to connect to disposable, glass, and stainless steel bio reactors. The disposable perfusion tangential flow filtering system includes the disposable pumphead, hollow fiber filter and associated connections which is designed to be gentle on cells or other biological material without impacting viability and is scalable. 
         [0009]    In accordance with still another aspect of the invention, there is provided a hollow fiber perfusion tangential flow filter system providing quick assembly to a bioreactor processing flow path Module (hollow fiber), Bag and Tubing (MBT) assembly. The MBT assembly may include a bag containing media feeding the reactor and/or a permeate bag collecting metabolic wastes. In some instances the bioreactor vessel may be a bag. The pre-sterilized processing MBT assembly includes low shear a re-circulation pumphead, automatic control of filtration sequences of operation including: the operation of seed reactors; continuous tangential flow; concentrated cell tangential flow; concentrated fed-batch tangential flow; as well as cell clarification (post transfection or cell lysis). 
         [0010]    In accordance with another aspect of the invention, there is provided a disposable MBT assembly allowing a simple procedure for changing of the filtering loop without impacting sterility. The disposable MBT assembly eliminates the need for autoclaving components and is designed to connect to either disposable reactors, re-usable glass and stainless steel reactors. The MBT assembly is pre-sterilized providing an easily changed tangential flow processing loop without impacting sterility, is designed to connect to either disposable reactors, glass, and stainless steel reactors, without impacting viability. 
         [0011]    In accordance with another aspect of the invention, there is provided a method for proliferation of cells within a bioreactor using tangential flow perfusion filtering. The method includes, providing a bioreactor vessel containing bioreactor fluid, providing a pre-sterilized, disposable, assembled bioreactor fluid filtering system, aseptically connecting the bioreactor vessel to the filtering system, engaging a pumphead of the filtering system with a reusable pump motor element of a control system, priming the pumphead, circulating bioreactor fluid from the bioreactor vessel through the filter system, separating the bioreactor fluid into a flow of filtered bioreactor fluid and a flow of waste fluid, returning the filtered bioreactor fluid to the bioreactor vessel, carrying the waste fluid from the filter system for disposal, disconnecting the filtering system from the bioreactor vessel, and disposing of the filtering system. 
     
    
     
       BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING 
         [0012]    The above and other aspects, features and advantages of the present invention will be more apparent from the following more particular description thereof, presented in conjunction with the following drawings wherein: 
           [0013]      FIG. 1  is a bioreactor system according to the present invention. 
           [0014]      FIG. 2A  is a disposable portion of a tangential flow perfusion filtering system according to the present invention. 
           [0015]      FIG. 2B  is a reusable control system according to the present invention. 
           [0016]      FIG. 3A  shows a detailed view of a first tangential flow perfusion filtering system according to the present invention. 
           [0017]      FIG. 3B  shows a detailed view of a second tangential flow perfusion filtering system according to the present invention. 
           [0018]      FIG. 3C  shows a detailed view of a third tangential flow perfusion filtering system according to the present invention. 
           [0019]      FIG. 3D  shows a detailed view of a third tangential flow perfusion filtering system according to the present invention. 
           [0020]      FIG. 3E  shows a detailed view of a third tangential flow perfusion filtering system according to the present invention. 
           [0021]      FIG. 4A  shows a cross-sectional view of a hollow fiber tangential flow filter according to the present invention. 
           [0022]      FIG. 4B  shows a cross-sectional view of a wall of a thick wall hollow fiber tangential flow filter according to the present invention. 
           [0023]      FIG. 5  shows a method according to the present invention. 
       
    
    
       [0024]    Corresponding reference characters indicate corresponding components throughout the several views of the drawings. 
       DETAILED DESCRIPTION OF THE INVENTION 
       [0025]    The following description is of the best mode presently contemplated for carrying out the invention. This description is not to be taken in a limiting sense, but is made merely for the purpose of describing one or more preferred embodiments of the invention. The scope of the invention should be determined with reference to the claims. 
         [0026]    Where the term “generally” is associated with an element of the invention, it is intended to describe a feature&#39;s appearance to the human perception, and not a precise measurement. 
         [0027]    A bioreactor system  10  according to the present invention is shown in  FIG. 1 . The bioreactor system  10  includes a bioreactor vessel  11  containing bioreactor fluid  13 , a pre-assembled and pre-sterilized, disposable, tangential flow perfusion filtering system  14 , and a reusable control system  20 . The filtering system  14  is connected between a bioreactor outlet  11   a  and bioreactor inlet  11   b  to receive a bioreactor material flow (or a first flow of the bioreactor fluid)  12  (see  FIG. 3A ) through bioreactor tubing  15  from the bioreactor  11  and return a filtered flow (or a return flow of the bioreactor fluid)  16  (see  FIG. 3A-3C ) through return tubing  17  to the bioreactor  11 . The bioreactor system  10  cycles bioreactor fluid through the filtering system  14  which separates out metabolic wastes and/or protein waste material and thereby allows the reaction in the bioreactor vessel  11  to continue to completion allowing higher cell densities within the same bioreactor which provides greater proteins to be expressed due to this increased density of viable cells. 
         [0028]    The bioreactor tubing  15  is preferably connected to the lowest point/dip tube on the bioreactor  11  below the sparger ring and the return tubing  17  is preferably connected to the bioreactor in the upper ¼ of the bioreactor volume and submerged in the bioreactor fluid  13 . 
         [0029]    The bioreactor vessel  11  receives the return flow of the bioreactor fluid  16  through a pre-sterilized assembly comprising a pump  26 , hollow fiber filter  30 , and associated fittings and connections. The pump  26  preferably includes a low shear, gamma stable, disposable, levitating pumphead  26   a , for example, a model number MPD-200 low shear re-circulation pump manufactured by Levitronix in Waltham, Mass. The MPD-200 includes a magnetically levitated rotor inside a disposable pumphead, and stator windings in the pump body, allowing simple removal and replacement of the pumphead. 
         [0030]    The flow of bioreactor material  12  passes from the bioreactor vessel  11  to the filtering system  14  and the return flow of the bioreactor fluid  16  passes from the filtering system  14  back to the bioreactor vessel  11 . A permeate flow  24  (for example, a metabolic waste material flow) (see FIG.) is stripped from the flow of bioreactor material  12  by the hollow fiber perfusion filtering system  14  and away carried by waste material tubing  19 . The metabolic waste, as well as associated proteins, are drawing from the hollow fiber perfusion tangential flow system  14  by a permeate pump  22  into a waste container  23 . 
         [0031]    The filtering system  14  is shown in  FIG. 2A . The filtering system  14  includes a disposable pumphead  26   a , which simplifies initial set up and maintenance. The pumphead  26   a  circulates the bioreactor fluid  12   a  through the hollow fiber tangential flow filter  30  and back to the bioreactor vessel  11 . A non-invasive transmembrane pressure control valve  34  in line with the flow  16  from the hollow fiber tangential flow filter  30  to the bioreactor vessel  11 , controls the pressure within the hollow fiber tangential flow filter  30 . The permeate flow  24  is continually removed from the bioreactor fluid  13  which flows through the tangential flow hollow fiber filter  30 . The pumphead  26   a  and the permeate pump  22  are controlled by the control system  20  to maintain the desired flow through the hollow fiber tangential flow filter  30 . 
         [0032]    A detailed view of a first pre-sterilized, pre-assembled, disposable, tangential flow perfusion filtering system  14   a  is shown in  FIG. 3A . The filtering system  14   a  receives the bioreactor material flow  12  through a male sanitary connector  38  on first bioreactor fluid flow tubing  15   a  connected to the bioreactor  11 . The connector  38  is connected to a female connector  40  on second bioreactor fluid flow tubing  15   b  after removing a cap  41 , and the tubing  15   b  is connected to the pumphead  26   a . The flow  12  passes through a non-invasive ultrasonic flow meter  42  and then through the disposable pumphead  26   a  to provide a controlled bioreactor material flow  12   a  through third bioreactor tubing  15   c  to the hollow fiber tangential flow filter  30 . The tubing  15   c  is connected to the hollow fiber tangential flow filter  30  through a sanitary connection  50 . Another sanitary connector  50  connects return tubing  17   b  to the hollow fiber tangential flow filter  30 . A second female connector  40  on the end of the tubing  17   b  connects to a second male connector  38  on the end of the tubing  17   a . The non-invasive TMP pressure control valve  34  located on the tubing  17   b  back to the bioreactor  11  can be used to maintain the correct pressure within the hollow fiber tangential flow filter  30 . The flow  16  passes through aseptic connectors  38 ,  40  and returns to the bioreactor vessel  11 . 
         [0033]    Feed and retentate pressure sensors  44  and  49  reside in communication with the flows  12   a  and  16  respectively before and after the hollow fiber tangential flow filter  30 . A connector (or retentate port)  46   a  provides access to the return flow of the bioreactor fluid  16  and specifically provides a port for priming the pumphead  26   a . The connector  46   a  is preferably a self-closing needle free, sterile connector, for example, a CLAVE® needle free connector. The fittings  50  include nipples for attachment of the pressure sensors  44  and  49 . Clamps  51  attach the fittings  50  to the filter  30 . The clamps  51  are preferably a sanitary two piece clamp compressing a gasket over the connection, commonly called a TC clamp. The hollow fiber tangential flow filter  30  includes one or two ports  30   a  and  30   b  (also see  FIG. 4A ) in fluid communication with the permeate flow  24 , for example, for the release of waste material, expressed proteins, or viruses of interest separated from the flow  12   a . Pressure sensor  48  resides in communication with the metabolic waste material flow  24 . Pinch clamps  36  reside over tubing  15   b ,  17   b , and  19 . A second connector  46   b  allows access to the permeate flow  24 . 
         [0034]    The hollow fiber tangential flow filter  30  is preferably a hollow fiber filter which may be either a microporous or ultrafilter pore size. Further, pore size can be selected depending on the operation to be performed. The pore size can be selected concentrate up expressed product in the bioreactor while passing metabolic waste, or the membrane pore size may be selected to pass products of interest the cells are expressing (for example, express proteins or viruses of interest) as well as metabolic waste. The hollow fiber tangential flow filter  30  is pre-sterilized with the associated sensors and connections, and manufactured with no biocides, and only animal free glycerine is present within the pores of the hollow fiber tangential flow filter  30 . The hollow fiber tangential flow filter  30  eliminates the need for autoclaving prior to using. Preferably, a very low protein binding chemistry is used, however, polysulfone (PS) as well as other chemistries can be utilized. Preferably, a very low protein binding chemistry Modified Polyether Sulfone (mPES) membrane is used. The perfusion hollow fiber can be either a 0.5 mm lumen or 1.0 mm lumen with scaleable hollow fiber elements to accommodate varying bioreactor sizes. An example of an acceptable hollow fiber tangential flow filter  30  is a hollow fiber filter such as the KrosFlo Filter Module manufactured by Spectrum Labs in Rancho Dominguez, Calif. 
         [0035]    The valve  34  is preferably a non-invasive valve which resides outside tubing carrying the return flow  16 . The valve “squeezes” the tubing to restrict and control the flow. Such a valve  34  is non-invasive and provides a commercial advantage since the return line to the reactor is situated thru the valve to regulate the applied pressure on the membrane. 
         [0036]    The pumphead  26   a  and hollow fiber tangential flow filter  30  in the filtering system  14   a  are preferably connected by flexible tubing allowing easy changing of the elements. Such tubing allows aseptically replacement of the hollow fiber tangential flow filter  30  during a run in case the hollow fiber pore becomes plugged, over-loaded with material which therefore provides easy exchange to a new perfusion hollow fiber assembly. 
         [0037]    A second pre-sterilized, pre-assembled, tangential flow perfusion filtering system  14   b  is shown in  FIG. 3B . The filtering system  14   b  replaces the connectors  38  and  40  with a first disposable aseptic connector  54 . Filter systems according to the present invention may further include tube welding or aseptic connectors manufactured by GE, Pall, Millipores and other, and filtering systems according to the present invention including any aseptic connectors is intended to come within the scope of the present invention. The filtering system  14   b  is otherwise similar to the filtering system  14   a.    
         [0038]    A third pre-sterilized and pre-assembled tangential flow perfusion filtering system  14   c  is shown in  FIG. 3C . The filtering system  14   c  replaces the connectors  38  and  40  with a second disposable aseptic connector  56 . The filtering system  14   c  is otherwise similar to the filtering system  14   a.    
         [0039]    A fourth tangential flow perfusion filtering system  14   d  is shown in  FIG. 3D . The filtering system  14   d  is simplified to remove elements not necessary for all applications and is otherwise similar to the filtering system  14   a.    
         [0040]    A fifth tangential flow perfusion filtering system  14   e  is shown in  FIG. 3E . The filtering system  14   e  is also a simplified to remove elements not necessary for all applications and is otherwise similar to the filtering system  14   a , but includes a flow meter  58  for monitoring the permeate flow  24 . In some methods of use, it is desirable or necessary to match the permeate flow  24  to the bioreactor  11  in use. In other instances, the permeate is collected and weighed to monitor the system. 
         [0041]    A cross-sectional view of the hollow fiber tangential flow filter  30  is shown in  FIG. 4A . The hollow fiber tangential flow filter  30  includes hollow fibers  60  releasing the waste material (i.e., permeate) flow  24  into a permeate section  61  of the filter housing  31 . The waste material flow  24  travels to the ports  30   a  and  30   b  and is drawn from the filter housing  31  by the permeate pump  22  (see  FIG. 1 ). The filtered return flow (or retentate)  16  is released through retentate port  30   c.    
         [0042]    A cross-sectional view of a wall  70  of a thick wall hollow fiber tangential flow filter is shown in  FIG. 4B . The wall  70  includes tortuous paths  71  to capture certain elements of the flow through the thick wall hollow fiber tangential flow filter. Surface retained material  72  too large to enter the tortuous paths  71  is retained on an interior surface of the wall  70 . Setting zones  73  capture small particles which enter the tortuous paths  71 . Depth strained particles  74  enter the tortuous paths  71  but become lodged in narrowing channels. Some small particles  75  become lodged in walls of the tortuous paths  71 . 
         [0043]    The settling zones  73  as well as the narrowing channels cause a separation unlike filtering obtained by the skin or surface of common thin wall tangential flow filter membranes. The thick wall hollow fiber tangential flow filter is preferably made from Polyethylene (PE) which has a molecular structure of repeating —CH2-CH2 units where the wall thickness of the fiber is in the range between 1.2 mm to 1.7 mm, providing the depth filtration of the material passing thru the wall  70 . 
         [0044]    A method for proliferation of cells within a bioreactor using tangential flow perfusion filtering is described in  FIG. 5 . The method includes, providing a bioreactor vessel containing bioreactor fluid at step  100 , providing a pre-sterilized, disposable, assembled bioreactor fluid filtering system at step  102 , aseptically connecting the bioreactor vessel to the filtering system at step  104 , engaging a pumphead of the filtering system with a reusable pump motor element of a control system at step  106 , priming the pumphead at step  107 , circulating bioreactor fluid from the bioreactor vessel through the filter system at step  108 , separating the bioreactor fluid into a flow of filtered bioreactor fluid and a flow of waste fluid at step  110 , returning the filtered bioreactor fluid to the bioreactor vessel at step  112 , carrying the waste fluid from the filter system for disposal at step  114 , disconnecting the filtering system from the bioreactor vessel at step  116 , and disposing of the filtering system at step  118 . 
         [0045]    The pinch clamps  36  are used to block the tubing  15   b ,  17   b , and  19  during priming of the flow path as well as utilized during the shutdown of the flow path for discarding the filter system  14 . 
         [0046]    The feed and retentate pressure sensors  44  and  49  respectively monitor operating conditions based upon the flow and changes in viscosity/cell density as the process ages. The feed and retentate pressure sensors  44  and  49  are used during operation to monitor these changes in the cell conditions within the reactor at a give recirculation flow rate (fiber shear rate). The permeate pressure sensor  49  is used to ensure the permeate pump  22  is not running to fast. A high permeate pump speed results in pulling a vacuum on the hollow fibers  60  which may inadvertently foul the pores on the hollow fibers  60 . Process loop controls are put in place to ensure correct operation of the hollow fiber tangential flow filter  30  as well as pressure associate alarms/system shut down to ensure safe perfusion operations. 
         [0047]    While the invention herein disclosed has been described by means of specific embodiments and applications thereof, numerous modifications and variations could be made thereto by those skilled in the art without departing from the scope of the invention set forth in the claims.