Abstract:
A bioreactor including a support, a container, means to secure the container on the support and nutrients and cell in the container. The container has top and bottom walls joined to form a chamber having a portion of the top and bottom walls joined by side walls; and end walls connected to the top and bottom walls forming a three dimensional container. The support is pivotally mounted to a base and driven about a single axis and the end walls of the container are transverse to the single axis.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is a filing under 35 U.S.C. §371 and claims priority to international patent application number PCT/US2008/076372 filed Sep. 15, 2008, published on Apr. 2, 2009, as WO 2009/042432, which claims priority to U.S. provisional patent application No. 60/975,213 filed on Sep. 26, 2007; the disclosure of which is incorporated herein by reference in its entirety. 
     
    
     FIELD OF THE INVENTION 
       [0002]    The present disclosure relates to hermetically sealed bags containing products used in the pharmaceutical and biotechnology processing industries and, more particularly, to disposable cell bags or bioreactors. 
       BACKGROUND OF THE INVENTION 
       [0003]    The bio-processing industry has traditionally used stainless steel systems and piping in manufacturing processes for fermentation and cell culture. These devices are designed to be steam sterilized and reused. Cleaning and sterilization are however costly labor-intensive operations. Moreover, the installed cost of these traditional systems with the requisite piping and utilities is often prohibitive. Furthermore, these systems are typically designed for a specific process, and cannot be easily reconfigured for new applications. These limitations have led to adoption of a new approach over the last ten years—that of using plastic, single-use disposable bags and tubing, to replace the usual stainless steel tanks. 
         [0004]    In particular bioreactors, traditionally made of stainless-steel, have been replaced in many applications by disposable bags which are rocked to provide the necessary aeration and mixing necessary for cell culture. These single-use bags are typically provided sterile and eliminate the costly and time-consuming steps of cleaning and sterilization. The bags are designed to maintain a sterile environment during operation thereby minimizing the risk of contamination. 
         [0005]    Commonly used bags are of the “pillow style,” mainly because these can be manufactured at low cost by seaming together two flexible sheets of plastic. 
         [0006]    One of the successful disposable bioreactor systems uses a rocking table on to which a bioreactor bag is placed. The bioreactor bag is partially filled with liquid nutrient media and the desired cells. The table rocks the bag providing constant movement of the cells in the bag and also aeration from the turbulent air-liquid surface. The bag, typically, has a gas supply tube for the introduction of air or oxygen, and an exhaust gas tube to allow for the removal of respired gases. Nutrients can be added through other tubes. 
         [0007]    One possible limitation of this type of device is that it may be difficult to scale up beyond a few hundred liters because poor liquid circulation causes nutrient and waste gradients that inhibit cell performance. This is because the back and forth motion of the single-axis rocker used in these applications creates good liquid circulation in the direction perpendicular to the rocking axis, but relatively little mixing in the direction parallel to the rocking axis. In large volume bags (greater than 100 liters), or in bags with a large length to width ratio, this poor axial circulation can result in a long time to achieve homogeneity of the bag contents. This makes pH control in the bioreactor bag difficult, since additions of acid or base added to the bioreactor to modulate the pH can take a long time to disperse throughout the bag. Nutrients added to the bioreactor bag may not be distributed uniformly. Poor liquid circulation also limits the amount of oxygen that can be transferred from the head space, and thus the maximum concentration of cells that can not be cultured. 
         [0008]    Circulation flow can be improved by incorporating a second axis of rotation. By synchronizing the two axes it is possible to impart a gyratory motion that greatly improves mixing and mass transfer. However, the addition of second axis increases the cost tremendously, and the increase in mechanical complexity makes the rocker less reliable and more difficult to maintain. 
         [0009]    Therefore, there is a need for an apparatus that enables a user to scale up the mixing of nutrient media in a bioreactor bag. Also, there is need for a bioreactor bag that makes it simple to control the pH where the addition of acid or base to the bioreactor bag does not take a long time. Further, there is need for a bioreactor bag where the amount of oxygen is not limited so the maximum concentration of cultures can be cultured. 
       SUMMARY OF THE INVENTION 
       [0010]    The present invention has been accomplished in view of the above-mentioned technical background, and it is an object of the present invention to provide a bioreactor bag that enables a user to scale up the mixing of nutrient media and efficiently mix the nutrient media in the bioreactor bag. 
         [0011]    A bioreactor including a support, a container, means to secure the container on the support and nutrients and cell in the container. The container has top and bottom walls joined to form a chamber having a portion of the top and bottom walls joined by side walls; and end walls connected to the top and bottom walls forming a three dimensional container. The support is pivotally mounted to a base and driven about a single axis and the end walls of the container are transverse to the single axis. 
         [0012]    The container walls are flexible sheets seamed together. The top and bottom walls are seamed together and portions of the top and bottom wall form the side walls of the chamber. The end walls, which are transverse to the single axis, are panels seamed to the top, bottom and side walls. Alternatively, The container may be a modeled structure. 
         [0013]    The juncture of the side and end walls are non-orthogonal so as to induce a swirling motion of liquids in the chamber when the container is rocked about the single axis. A first pair of opposed junctures are obtuse angles and a second pair of opposed junctures are acute angles. The junctures may be arcuate. The junctures may include a linear wall of the chamber connecting and being oblique to both a side and an end wall. A first pair of opposed junctures may have a first length and a second pair of opposed junctures have a second length shorter than the first length so as to produce a single direction of swirling during rocking. 
         [0014]    Baffles may be connected to the top and bottom walls and displaced from the side and end walls to induce a swirling motion of liquids in the chamber when the container is rocked about the single axis. 
     
    
     
       BRIEF DESCRIPTION OF DRAWINGS 
         [0015]    These and other advantages of the present invention will become more apparent as the following description is read in conjunction with the accompanying drawings, wherein: 
           [0016]      FIG. 1  shows a perspective view of a pillow style two dimensional bag construction according to the prior art. 
           [0017]      FIG. 2  shows a perspective view of the bag of  FIG. 1  secured to rocking bioreactor based on prior art. 
           [0018]      FIG. 3  shows the liquid flow pattern of the bag of  FIG. 1  resulting from a single axis of rocking based on prior art. 
           [0019]      FIG. 4  shows a perspective of a three-dimensional bag construction according to another embodiment of the invention. 
           [0020]      FIG. 5  shows a perspective view of the embodiment shown in  FIG. 4  depicting details of construction according to an embodiment of the invention. 
           [0021]      FIG. 6  shows a baffled bag and the single liquid flow pattern resulting from a single axis of rocking according to a first embodiment of the invention. 
           [0022]      FIG. 7  shows a baffled bag and the single liquid flow pattern resulting from a single axis of rocking according to a second embodiment of the invention. 
           [0023]      FIG. 8  shows a baffled bag and the dual liquid flow pattern resulting from a single axis of rocking according to a third embodiment of the invention. 
           [0024]      FIG. 9  shows a baffled bag with linear baffles and the liquid flow pattern resulting from a single axis of rocking according to a fourth embodiment of the invention. 
           [0025]      FIG. 10  shows a trapezoidal shaped baffled bag and the liquid flow pattern resulting from a single axis of rocking according to a fifth embodiment of the invention. 
           [0026]      FIG. 11  shows another embodiment with internal flow diversion baffles. 
       
    
    
     DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS 
       [0027]    The presently preferred embodiments of the invention are described with reference to the drawings, where like components are identified with the same numerals. The descriptions of the preferred embodiments are exemplary and are not intended to limit the scope of the invention. 
         [0028]    A prior art bag  20 , as shown in  FIG. 1 , is a flat, rectangular, “pillow-style” cell culture bag  20  commonly used in rocking bioreactor applications, for example in the system of U.S. Pat. No. 6,190,913 entitled “Method for Culturing Cells Using Wave-Induced Agitation” filed Aug. 12, 1998, which is hereby incorporated by reference. The bag  20  is formed by seaming together top sheet  22  to bottom sheet  24 . The outline seam  49  formed by sealing the two sheets together at all four edges  52 ,  54 ,  56 , and  58  which bounds the inside chamber in which the culture fluids  32  are contained. Ports  26  on top sheet  22  are used for the introduction and exhaust of gases. 
         [0029]      FIG. 2  shows the bag  20  secured to a support  10  of a rocking bioreactor using clamps  12  on side edges  52  and  54 . The support  10  is pivotable mounted to a base  14  and is rocked about a single axis  15 . The fluid flow induced by the rocking is depicted in  FIG. 3  as streamlines  40 . As shown, the rocking motion generates fluid motion  40 A mainly along the Y-axis (perpendicular to rocking axis  15 ). Very little fluid motion  40 B in the X-direction (parallel to rocking axis  15 ) is generated in the substantially orthogonal corners  30 - 33 . It can take considerable time to mix the contents of mixing bag  20  to homogeneity in the structure of bag  20 . The mixing time can be reduced by increasing the rocking speed, but this puts more stress on the bag leading to possible breakage and also increases the energy requirements for mixing. 
         [0030]    As shown in  FIG. 2  for the two dimensional bag  20 , creases and wrinkles  90 ,  91 ,  92  may form on upper surface  22  of each corner  30 - 33 , and also  93 ,  94  on the underside  24  of each corner  30 - 33  of the bag  20 . Excess material may develop in corners  30 - 33  because the inflation pulls in unrestrained end edges  56  and  58 , and pushes out corners  30 - 33 . This excess material cannot be inflated to rigidity, and may flop around during rocking, which could lead to premature fatigue failure. The bag  20  is stressed when inflated and this stress is transmitted through top sheet  22  and bottom sheet  24  to clamped edges  52  and  56 . The stress is distributed along edges  52  and  56 , but not at corners  30 - 33  which are not pulled taut into support  10  due to the excess material present at corners  30 - 33 . Consequently, failures can occur at corners  30 - 33  where the bag cannot be maintained taut and rigid. 
         [0031]      FIG. 4  depicts a three-dimensional cell culture bag  20 H, or container or bioreactor formed by forming the end walls  54 H and  58 H as gussets on side walls  52  and  56 . Culture bag  20 H is formed from a multiplicity of flat flexible panels  22 ,  24 ,  54 H, and  58 H as depicted the exploded view shown in  FIG. 5 . This figure shows one way in which culture bag  20 H can be formed by seaming together two flexible sheets  22  and  24 , folding in two smaller panels  54 H and  58 H, and closing them off by a cross or curved seam  49  which bounds the inner volume of culture bag  20 H. The segments of the seam  49  which joins the gusseted end walls  54 H and  58 H to the top sheet  22  and the bottom sheet  24  is arcuate so as to form the baffles in  FIG. 4 . The segments of the seam  49  which joins the top sheet  22  and the bottom sheet  24  forms the side walls  52  and  56 . 
         [0032]    When culture bags  20 H are restrained at edges  52  and  56 , and inflated, top sheets  22  and bottom sheet  24  are able to separate at the gusseted end walls  54 H and  58 H. The culture bag  20 H conforms to the inflated three-dimension shape without wrinkles, creases, or excess corner material. Corners  100 - 103  are now pulled taut and provide additional structural elements that distribute stress from the high points  110  and  112  of culture bag  20 H to the clamped edges  52  and  56 . These edges are clamped along their entire length to holder  10 , and form anchor points to restrain the bag from over inflating. The corner sections  100 - 103  also function as a reinforcing structure to support the bag during rocking. Rocking towards edge  56  about axis  15  causes culture bag  20 H to be pulled up from edge  52 . This movement is resisted by corners  100  and  101  that serve to hold culture bag  20 H down. The additional tendency for the bag to slide towards edge  56  is resisted by corners  100  and  101 . In the reverse stroke the same functionality is provided by corners  102  and  103 . 
         [0033]    The improved bags  20  can be a molded three-dimensional structure or fabricated by seaming flexible sheets. The edges and gusset may be curved seams, or manufactured as a series of straight line seam segments as shown herein. 
         [0034]    Referring first to the embodiment shown in  FIG. 6  a cell culture bag  20 A bounded by seams  49 , containing components, at least one of which is a liquid, to be mixed. Cell culture bag  20 A is placed on a rocking platform  10 , which pivots about rocking axis  15 . Corner  42 A of the cell culture bag  20 A, is contoured in the same manner as the diagonally opposite corner  46 A, an arc that forms these corners can have a radius ranging from ¼ to ½ the width of the cell culture bag  20 A but is different from adjacent corner  44 A which is contoured in the same manner as its diagonally opposite corner  48 A with another arc radius ranging from 1/20 to ¼ the width of the cell culture bag  20 A. The corners  42 A and  46 A are baffles or flow directors formed at the juncture of the side walls  52 ,  54  and the end walls  56 ,  58 . These baffles or flow directors force the liquid in the bag to follow the contour as the liquid cannot pass through liquid-tight seam  49  formed by joining top sheet  22  to bottom sheet  24 . The corners form a juncture of the side and end walls, which is transverse to the single axis of rocking  15  to induce a circulatory swirling motion of the liquid in the chamber when the cell culture bag is rocked. The liquid may consist of soluble powders in liquid, low or high viscous liquids that are designed to be mixed or blended together. In bag  20 A, the junctures are oblique and arcuate. The oblique junctures have obtuse angles of 90 degrees plus while the arcuate junctures have angles of less than 90 degrees. 
         [0035]    The effect of this asymmetry of cell culture bag  20 A is that as it is tipped towards  100  by tilting support  10 , liquid flows due to gravity from edge  54  towards edge  52 . As the liquid approaches edge  52  it is diverted right towards the center of the cell culture bag  20 A by corner  42 A. Liquid on the opposite side flows into corner  44 A which not shaped so as to divert flow to the center. This imbalance of flow velocities forces flow from the left end  58  to the right end  56 , and flow from the right end is inhibited from entering the left end. On the reverse stroke, support  10  tilts towards  101 , and the liquid in cell culture bag  20 A flows from edge  52  towards edge  54 . Liquid entering corner  46 A is diverted to the center of container  20 A due to the shape of corner  46 A, while liquid entering corner  48 A is not diverted towards the center. After 2 to 5 rocking strokes a self sustaining motion develops, and is sustained as long as the bag is rocked, as shown by the fluid streamlines  40 C with the liquid in the bag circulating counter-clockwise. This self-sustaining motion persists as long as the rocking motion is continued. This circulatory motion is superposed on the back and forth motion and is very effective at mixing fluid parallel to the rocking axis  15 , a major limitation with prior art. The circulatory motion can easily be reversed to the clockwise direction by interchanging the geometry of the corners. 
         [0036]      FIG. 7  shows a cell culture bag  20 B which produces a greater circulation than in cell culture bag  20 A. This is because the radius of the corners  42 B and  46 B is larger that their counterparts  42 A and  46 A ( FIG. 6 ). The radius of these corners can range from ½ to 2× the width of the mixing bag  20 B. Corners  44 B and  48 B also have a larger radius (¼ to ½ the mixing bag width) than their counterparts  44 A and  48 A ( FIG. 6 ). These larger radius arcs provide a more gentle flow pattern, reducing some of the turbulence caused by the sharp corners  44 A and  48 A. The resulting circulation is shown as  40 D. It is critical that asymmetry of the adjacent corners be maintained. For example, the symmetrical cell culture bag  20 C shown in  FIG. 8  has small equal arcuate corners  42 C through  48 C. The resulting circulation  40 E has very little fluid circulation parallel to the rocking axis  15  and is therefore similar to prior art cell culture bags with relatively poor mixing. 
         [0037]    The flow contours can be molded in the bag as curved surfaces or fabricated by seaming sections of plastic. The contours may be curved seams, or manufactured as a series of straight line seam segments. The seams are made by welding together the top and bottom sheet. Various methods—heat sealing, ultrasonic etc are commonly used. Straight seams can be easily made by inexpensive thermal bar sealers. Curved seams are much more difficult and are typically made using heated platens. These are expensive and designed for specific bag sizes. The laser method has the advantage that any shape seam, bag geometry or size can be made by just changing the software. 
         [0038]    Manufacturing curved seams in bags is difficult, and requires complex equipment. Straight line seams can be easily made using commercial bar type heat sealers. The embodiment shown in  FIG. 9  illustrates a cell culture bag made using straight or linear seams that achieves circulation flow. The outer seam  49  forms portions  52 ,  54 ,  56 ,  58  of the inner chamber of the mixing bag  20 E that contains the liquid and components to be mixed. Seam  51  defines the baffles  72 ,  74 ,  76 ,  78  as linear segments at the corners and are connected to the top, bottom, side and ends walls. These baffles are typically oriented at 45 degrees (angles from 30 to 60 degrees can be used) to the rocking axis  15 . The longer baffles  42 E and  46 E can extend from ¼ to ⅓ of the length of the side of bag  20 E and the shorter baffles  44 E and  48 E are typically ⅕ to ½ the length of the longer baffles. The resulting circulation of the asymmetrical baffles is shown as  40 G. The corners may be removed where they extend beyond the baffles. Also the seam  49  need not extend past the juncture of the baffles, the inner seam  51 , to the side and end walls. 
         [0039]    The embodiment shown in  FIG. 10  illustrates a cell culture bag made using straight or linear seams that achieves circulation flow by changing the shape of cell culture bag  20 F from an essentially rectangular form into a trapezoidal shape. The end walls  54  and  58  of the cell culture bag  20 F are parallel to each other, but are not perpendicular to the side walls  52  and  56 . This creates a first pair of opposed junctures and baffles  42 F and  46 F that are obtuse angles ranging from 100 to 130 degrees and a second pair of opposed junctures and baffles  44 F and  48 F that are corresponding acute angles. By setting the obtuse and acute angles automatically fixes the shapes as a parallelogram. When the cell culture bag  20 F is rocked about axis  15 , the fluid circulates in the direction shown by the flow streamlines  40 H effectively mixing the contents of cell culture bag  20 F. 
         [0040]    With a two and three dimensional bags, the baffles formed by the intersection of the side and end walls may not have sufficient height when the bag is inflated for the liquid level and rocking motion to produce the desired amount of circulation. The embodiment shown in  FIG. 11  illustrates a cell culture bag  20 G wherein the baffles are separated and displaced from the intersection of the side and end walls. The baffles  82 ,  84 ,  86 ,  88  are adjacent to the corners  42 G,  44 G,  46 G,  48 G. The baffles are connected to the top wall  22 , the bottom wall  24 , the side walls  52 ,  56  and the end walls  54 G,  58 G. The baffles may be connected to the top and bottom walls first and then joined to the side and ends walls when they are formed or joined to the top and bottom walls. 
         [0041]    This invention provides an apparatus that enables a user to scale up the mixing of nutrient media in a bioreactor bag. This apparatus makes it simple to control the ph where the addition of acid or base to the bioreactor bag does not take a long time to obtain. Thus, this invention provides the user with a simple method to scale up the mixing of nutrient media in a bioreactor bag. 
         [0042]    Although the present bag has been described and illustrated in detail, it is to be clearly understood that this is done by way of illustration and example only and is not to be taken by way of limitation. The scope is to be limited only by the terms of the appended claims.