The present invention relates to the field of bioprocessing technology. More specifically, the present invention relates to bioreactors with one or more filters disposed therein.
Culturing of cells for producing regenerative medicine is done with the aim of harvesting cells which can subsequently be injected into a patient. The health and viability of the cells is of paramount importance. The cells need to be reproduced under controlled conditions and fed with nutrients to grow. One commercially successful disposable bioreactor system uses a flexible cellbag bioreactor placed on a rockable platform. The bioreactor is partially filled with liquid cell culture medium and cells of interest are introduced into the bioreactor. The culture medium and cells contact only a presterile, disposable chamber that is positioned on the rocking platform. The rocking motion of the platform induces waves in the culture fluid and thereby provides continual mixing and oxygen transfer, resulting in a robust environment for cell growth. The Bioreactor requires no cleaning or sterilization and provides ease of operation and protection against cross-contamination.
A perfusion bioreactor grows cells by continuously feeding the bioreactor with fresh cell culture medium so as to replace the spent cell culture medium all while keeping the volume of the cell culture medium constant in the bioreactor. The cells reach a steady state of reproduction and can be maintained in that state for a few weeks until the required cell density is achieved. Perfusion bioreactors typically employ a filter within the bioreactor for filtering out of the bioreactor both the spent cell culture medium and the toxic cell metabolites that inhibit cell growth while retaining the healthy and viable cells within the bioreactor.
One type of perfusion bioreactor is disclosed in U.S. Pat. No. 9,017,997B2, where the filter is affixed to the inner surface of a bottom wall of the bioreactor and thus does not float on the surface of the culture medium. This placement of the filter prevents the filter from getting damaged due to twisting or sticking to the walls of the bioreactor. However, the filter can get easily clogged and fouled. Similarly, WO2012/158108A1 discloses a perfusion bioreactor for cultivation of cells on microcarriers where the filter is fixed to the inner surface of a wall of the bioreactor. Further, WO2015/034416A1 discloses a bioreactor with internal dialysis modules suitable for dialysis cultivation of cells. The dialysis compartments are formed as either a freely movable bundle of hollow fiber membranes, a pouch attached to an inner wall of the bag, a pouch freely moving, or a sheet of membrane fixed to an inner wall of the bag.
Another perfusion bioreactor is disclosed in WO2017/055059A1, where the filter is held by a filter holding device. The filter holding device is attached to an inner wall of the bioreactor such that there is a limited space between the filter and the inner wall of the bioreactor and the liquid medium provided in the bioreactor can flow on both sides of the filter. While this arrangement of the filter holding device causes a cross flow filtration effect, it is quite limited in reducing the clogging and fouling of the filter due to limited space between the filter and the inner wall of the bioreactor.
In the perfusion bioreactor disclosed in U.S. Pat. No. 6,544,788, the perfusion filter is constructed to move freely on top of the liquid cell culture medium during the rocking motion of the bioreactor.
The present invention provides a stacked filter design which incorporates a porous membrane at both major surfaces of the filter stack rather than only a single side. The design allows for reduction of the overall footprint of the filter, as waste can be pumped through from both top and bottom membrane surfaces, i.e., waste can be drawn from both above and below the filter. In addition, by maintaining both membranes in a wetted state, the filter design mitigates the potential for air to be pumped through, as any wetted area would pull fluid into the filter stack and then out of the cellbag bioreactor.
During a cell expansion operation in which cells are perfused, the filter of the present invention can allow more fluid exposure to the membrane surface and therefore increased volume of waste to be admitted into the filter pumped from the bioreactor. Additionally, optimization of the filter size can also reduce the overall footprint of the filter in the bioreactor. Moreover, by placing a port on the bottom of the filter stack rather than the top, the filter design reduces the potential for air to be pumped through, as any wetted area would pull fluid into the filter stack and to the bottom side port.
The present invention further provides a perfusion filter tethered from the bottom surface of the cellbag bioreactor, thus providing an anchor point for the filter that is within the fluid volume. Controlling the length of the tether will allow the filter to remain wetted throughout the culture process, which mitigates the risk of the filter floating to the surface and parts of the filter not being exposed to fluid or otherwise wetted out.
Alternatively, the present invention can tether the filter to the bottom of the bioreactor bag by selectively tethering the waste conduit to the bottom surface of the bioreactor bag, allowing a portion of the conduit proximate the filter to rise up from the bottom surface so as to maintain the filter within a predefined constrained volume of the bioreactor chamber such that the membranes of the filter remain wetted.
Desirably, the filter and tethers of the present invention will be used in cell therapy and bioprocessing applications so as to minimize the risk of a filter floating on the top surface of a culture volume and sucking in air. The present invention reduces risk and the need for manual manipulation of the bioreactor throughout the process. Moreover, as the present invention obviates the need to manually manipulate the bag to ensure wetting, or to change unit operations requiring the bioreactor be filled with liquid before inflating, user processes can be streamlined.
Each of membrane 112 and 116, mesh 114, port 118, and conduit 120 are formed of a biologically-compatible hydrophilic material suitable for bioprocessing operations as are known in the art. The perimetrical bonding of membranes 112 and 116, as well as the bonding of port 118 and conduit 120 are by means suitable for bioprocessing operations and compatible for pharmaceutical operations, as is known in the art. By way of illustration and not of limitation, membranes 112 and 116 may be formed of Ultra-high-molecular-weight polyethylene (UHMWPE), Nylon or Polyethersulfone (PE), mesh 114 may be formed of Polyethylene terephthalate (PETE), while port 118 and conduit 120 may be formed of any suitable plastic or rubber material. Filter 110 may be used in cell therapy and bioprocessing operations to reduce the risk for air to be pumped to waste during perfusion processes rather than waste material. The perfusion filter design of the present invention, with both top and bottom membranes, provides more surface areas for cells to migrate through to waste and when used in conjunction with other features, can resolve any air being pumped into the waste bag. Openings 114a formed in mesh 114 are larger than the pores 112a and 116a of membranes 112 and 116, respectively. It is contemplated that mesh 114 may approximate an interwoven lattice structure or any other structure which provides separation between membranes 112 and 116 while also allowing for the permeate which has passed through either membrane 112 or 116 to traverse filter cavity 115 to be conducted through aperture 119 and out conduit 120.
Filter 110 is located within chamber 158 so that waster material may be conducted through conduit 120 out of bioreactor 150. Platform 140 is urged to rock back and forth in the directions of Arrows A and B so as to impart counter-flowing waves of the cell media 151 within bioreactor 150 in the respective directions of Arrows C and D. The necessary oxygen and nutrients are provided to for cell growth and productivity. Top layer 152 and bottom layer 154 of the bioreactor 150 are formed from a suitable material for bioprocessing such as, by way of illustration but not of limitation, a multilayer laminated clear film of EVA. Top layer 152 and bottom layer 154 are further typically formed from a transparent or semitransparent multilayer laminated film which allows for an operator to generally observe bioreactor chamber 158. Top layer 152 supports multiple access ports 160 which when properly connected provide access to the bioreactor chamber 158 for needed nutrients, oxygen, or sensors. For example, one port 160 could be used to transfer fresh liquid media from the external space of the bioreactor 150 to the bioreactor chamber 158 while another port 160 could be connected to an oxygen level sensor. Bioreactor 150 further supports a perfusion port 162 through which conduit 120 transits top from chamber 158. The present invention contemplates that both membranes 112 and 116 of filter 110 are properly wetted before drawing waste from media 164 through filter 110 into conduit 120. With additional reference to
Referring to
Tethers 168 constrain the movement of filter 110 but allow some movement of filter 110 vertically, i.e., towards and away from bottom layer 154, laterally, towards and away from edge portion 155a, and longitudinally towards and away from longitudinal ends 150a and 150b such that the movement of the filter is within a constrained volume 159 of the overall chamber 158 which can be predefined by the amount of slack, flexibility or elasticity of each tether. Ideally constrained volume 159 is spaced from the inner surface of a cell bag in use to avoid the filter rubbing on the cell bag in use. Constrained volume 159 will thus change in use as the amount and volume of liquid in chamber 158 changes and in accordance with the length, positioning, and flexibility of the tethers 168 and conduit 120. Thus, at early stages of cell culture process when the cell bag is relatively empty, the constrained volume may be closely coincident with the inner surface of the cell bag. But, as the liquid volume increases, the cell bag inflates, and the resultant wave motion of that liquid attains more energy during rocking motion of the cell bag, so then the constrained volume of the filter avoids the inner surface of the cell bag to prevent rubbing of the filter with the cell bag at that higher energy phase of the cell culture. Desirably, the tethers have a length such that there is always some clearance between the filter and the inner surface of the bag, for example at least 10 mm clearance. In practice this can be achieved by multiple tethers working in combination, where at least one will be taut while another is slack at the extremities of the filter's permitted range of movement. Additionally, in embodiments of a bioreactor of the present invention where conduit 120 extends between the filter and bottom layer 154, it is contemplated that conduit 120 will also have minimal contact with bottom layer 154 while also helping to support the filter in spaced separation from layer 154.
The predefined constrained volume 159 is thus generally depicted to represent an area within chamber 158 in which the filter 110 is constrained to remain within and where media fluid 151 is able to maintain the membranes 112 and 114 wetted desirably throughout operation of bioreactor 150, but at least while the waste contents are being evacuated from filter cavity 115 out conduit 120. Predefined constrained volume 159 is desirably defined to have separation from layers 152 and 154 while remaining below the surface of the rocking media 151. Tethers 168 desirably loosely maintains filter 110 under the surface of media 151 in that they do not fully constrain filter 110 against, e.g., layer 154 as filter clogging may in then arise. The present invention thus maintains the membranes of the filter fully wetted by the media to avoid bubble forming and filling of Waste bag with air which require customer to interrupt the process to add a new waste bag.
The present invention contemplates that while portion 120a of conduit 120 is depicted as naturally flexing so as to turn towards the predefined constrained volume of chamber 158, conduit 120 may be formed with either an imparted bend about portion 120a or include one or more elbow segments arranged to turn the conduit body as shown and described. Such elbow segments may be bonded together by a suitable means of the prior art so as to be compatible with bioprocessing operations. Additionally, while port 118 and conduit 120 are shown to extend substantially normal to the planar filter 110, the present invention further contemplates that port 118 may provide for acute connection of conduit 120 to filter 110 so as to minimize the angle formed between conduit 120 and membrane 112. Such angled connection to conduit 120 may further reduce the minimum spacing between filter 110 and lower layer 154 of bioreactor 150.
Desirably, the tethers and/or conduit are positioned and sized so as to maintain the filter in a submerged state when the bioreactor includes its operation fill of media, thus maintaining the filter membranes below the surface of the media and mitigating the risk of bubbling while also providing space to either above and below the portion of the conduit which is tethered so as to permit media flow past the conduit as the bioreactor bag is rocked. The present invention also contemplates that the positions and sizes of the tethers and conduit allow the filter to maintain the membranes as wetted so as to avoid bubbling while waste is evacuated from filter cavity 115 through conduit 120.
Moreover, the present invention further contemplates a bioreactor 550 as shown in
The fitment body 219 of port 218 will thus be bonded to both membrane 212 and membrane 216. Fitment body 219 is known as a ‘boat fitment’ includes symmetrical, or opposed counter-tapering, surfaces 219a and 219b extending between opposed lateral edges 221a and 221b. Surfaces 219a and 219b are shaped to minimize the risk of any gaps forming between membranes 212 and 216 adjacent to edges 221a and 221b. Fitment body 219 defines an elongate open fitment passageway 290 extending therethrough in fluid communication so as to open on opposed fitment surfaces 292a and 292b. Passageway 290 further opens at the free end of a cylinder 294 protruding from surface 292b. When bonded together as shown, membranes 212 and 216 and port 218 thus define a filter cavity 215 within which is positioned a planar open mesh 214. Mesh 214 is shaped to allow for fluid flow from the pores 212a, 216a of membranes 212 and 216 through filter cavity 215 to and out of port 218.
Cylinder 294 is bonded to an adaptor body 300 which defines an elongate adaptor passageway 302 therethrough. Passageways 302 and 290 are thus placed in fluid communication with each other and thus with the pores 212a and 216a of membranes 212 and 216. The exterior surface 304 of adaptor body 300 is contoured to provide a tapered annular rim 306 over which one open end of conduit 210 is connected so as to conduct fluid from filter cavity 215 and out of the bioreactor in which it is positioned. Adaptor body 300 further supports a radially-displaced elongate projection 325 having a distal end 325a which is at least partially positioned in spaced registry with membrane 216. Desirably, adaptor body 300 is bonded to fitment body 219 after membranes 212 and 216 are bonded thereto, bonding mesh 214 within filter cavity 215, so that projection 325 does not interfere with properly bonding the membranes to the fitment body.
With additional reference to
As shown in
Filter 210 may thus also be set to remain within predefined constrained volume 159 of bioreactor chamber 158 as has been described for the present invention. Mesh 214 of filter 210 is contemplated to be similar in design and construction as is suitable for mesh 114 of filter 110 and is desirably also formed to have some flexibility such that membranes 212 and 216 may deflect so as to remain submerged during rocking of bioreactor 650. Additionally, projection 325 protrudes a sufficient distance towards lower layer 154 to prevent the membranes of filter 210 to fully rest upon the lower layer 154 of bioreactor 150. Projection 325 desirably ensures that a minimum separation is maintained between the membranes of filter 210 and lower layer 154. The present invention further contemplates that projection 325 provides a rounded, blunt or non-sharp, shape so as to minimize risk of scratching or puncturing bioreactor 150 during transportation and storage, as well as during operation of bioreactor 150, should filter 210 touch upon lower layer 154 during rocking.
While the tethers of the present invention are each depicted as being used to properly position a stacked filter 110 of the present invention, it is also contemplated that the tethers of the present invention may be employed as shown and described with a single-membrane filter 10 of the prior art.
Additionally, the present invention further contemplates a combination of the rigidity of conduit 120 and the supporting arrangement for conduit 120 outside of a bioreactor of the present invention so as to maintain the filter within the predefined constrained volume of the present invention such that both membranes are wetted while waste is evacuated from filter cavity 115, 215.
In each embodiment of the present invention employing tethers, reference to a tether being bonded to a bottom layer 154 refers specifically to the tether being bonded to the surface of layer 154 facing bioreactor chamber 158. Additionally, for all embodiments, each bioreactor port 180 is bonded to both the bioreactor and to the conduit extending therethrough to prevent leaks and thus maintain fluid integrity of the bioreactor.
The invention is not to be seen as limited by the embodiments described herein, but can be varied within the scope of the appended claims as is readily apparent to the person skilled in the art. For example, in alternate embodiments, the filter could be attached to the inner surface of the bioreactor by two, three, four or more tethers. The filter could be of any suitable shape including but not limited to a square, a triangle or a circle. The filter could also have more than one port. The filter could be loosely tethered in various spatial orientations within the bioreactor chamber, with the aim that the filter can move within the bioreactor but not so much that the filter touches the inner surface of the bioreactor. Additionally, while the present invention has depicted conduit 120 as extending from filter 110 towards either upper layer 152 or lower layer 154 of the bioreactor, the present invention that in either case that conduit 120 may exit from either upper layer 152 or lower layer 154 of the bioreactor. Similarly, the conduit 120 extending from filter 210 is contemplated to exit the bioreactor through either upper layer 152 or lower layer 154 at a location suitable for the process to which the bioreactor is set to run, including but not limited to those locations described for bioreactors 150, 250, 350, 450, 550, or 650.
Moreover, while prior art mitigation techniques for mitigating the occurrence of bubbling may be employed with the present invention, the present invention further contemplates that the particular design and arrangement of the filter, conduit, and any tethers may be selected consistent with the present invention so as to mitigate bubbling apart from such prior art techniques.
While the particular embodiment of the present invention has been shown and described, it will be apparent to those skilled in the art that changes and modifications may be made without departing from the teachings of the invention. The matter set forth in the foregoing description and accompanying drawings is offered by way of illustration only and not as a limitation. The actual scope of the invention is intended to be defined in the following claims when viewed in their proper perspective based on the prior art.
Filing Document | Filing Date | Country | Kind |
---|---|---|---|
PCT/EP2019/082722 | 11/27/2019 | WO | 00 |
Number | Date | Country | |
---|---|---|---|
62772175 | Nov 2018 | US |