Title of Invention

"A REACTOR SYSTEM AND A METHOD OF PREPARING THE SAME"

Abstract A reactor system, the system comprising: a container (302) comprising a flexible bag and having an interior surface bounding a compartment; a rotational assembly (301, 401, 501, 601, 701) in sealed cooperation with an opening of the container, the rotational assembly comprising a casing (360, 460, 560, 660, 760) mounted to the flexible bag and a hub (320, 420, 520, 620, 720) rotatably mounted to the casing, the hub having a passageway (320a) extending therethrough; and a drive shaft (304, 704, 804, 904,1004,1104, 1304, 2004,2204,2304) removably received within the passageway of the hub so as to extend into the compartment of the container, the drive shaft engaging the hub so that rotation of the drive shaft facilitates rotation of the hub relative to the casing. Figure 1
Full Text The present invention relates to a reactor system and a method of preparing the same.
CROSS-REFERENCES TO BELATED APPLICATIONS
[0001] This application claims the benefit under 35 USC 119(e) of US, Provisional Patent Application No. 60/555,908, filed April 27,2004 (Attorney Docket No. 20695C-006800US), the entire disclosure of which is herein, incorporated by reference for all purposes.
FIELD OF THE INVENTION
[0002] The present invention relates to a stirred-tank reactor system and methods of preparing such systems. The present invention further encompasses the use of the stirred-tank reactor system as a disposable bioreactor and in kits with disposable elements.
BACKGROUND OFTHE INVENTION
[0003] Bioreaotors or fermenters include containers used for fermentation, enzymatic reactions, cell culture, biologtcals, chemicals, biopuarmaceuticals, tissue engineering, microorganisms, plant metabolites, food production and the like. Bioreactors vary in size from benehtop fermenters to stand-alone units of various sizes. The stringent asepsis requirements for sterile production in some bioreactors can require elaborate systems to achieve the desired product volumes, Consequently, the production of products in aseptic bioreactors can be costly which provides the motivation for pursuing improved systems.
[0004] Conventional bioreactors perfuse nutrient media through a single type of hollow fiber. The various disadvantages of such bioreactors may include heterogeneous cell mass, difficult procurement of representative cell growth samples, poor performance due to inefficient oxygenation and an inability to control oxygen levels, and problems with contamination of cell cultures, morover micro-environmental factors such as pH may not be effeotively controlled and a mixed cultured or co-culture of cells may not be possible.
Some known bioreactors include a reaction container, through which a central strand of porous hollow fibers extends, through which a nutrient solution is pumped. This central strand of hollow fibers is concentrically surrounded by a plurality of strands of hollow fibers, through which a gaseous medium is conveyed. The hollow fibers of these strands are also constituted in such a manner that the gaseous medium-for example oxygen or carbon dioxide—can at least partly emerge from these strands or enter into these strands respectively. This type of bioreactor can achieve enhanced nutrient media oxygenation as compared to other known devices. However, occasional contamination of cell cultures and an inability to control pH levels effectively may continue to present difSculties.
[0005] The expense of producing cells, biophannaceuticals, biologjcals and the Hke in aseptic bioreactors is often exacerbated by the required cleaning, sterilization and validation of the standard bioreactors (Le., stainless steel or glass reactors). Attempts have been made to solve this problem with the development of pre-sterilized disposable bioreactor systems that need not be cleaned, sterilized or validated by end users. The use of such disposable bioreactor systems could provide significant savings. Furthermore, plastics are lightweight, easyto transport, and rcquire less room mân stainless steel or glass reactors. Somehave reported the use of disposable elemente m bioreactors that include a reactor chamber with a support housing. The interior chamber of the support housing is lined with a disposable liner and sealed with a head plate attachedto the liner to form asealed chamber. As the liner is open at the top, it is typically used in a vertically oriented bioreactor to preveni the contamination of the head plate. Although this system provides a disposable liner, the head plate and the interior chamber may still require cleaning and sterilization.
[0006] Others have attempted to develop flexible, disposable plastic vessels that do not require cleaning or steriHzation and require onlynrinimal validation efforts. Such approaches can include a flexible, disposable, and gas permeable cell culture chamber that is horizontally rotated. The cell culture chamber is made of twosheets of plastic fused together. maddition, the culture chamber is made of gas permeable material and is mounted on a horizontally rotating disk drive mat supports the flexible culture chamber without blocking airflow over the membrane surfaces. The chamber is placed in an incubator and oxygen transfer is controlled by controlling the gas pressure ni the incubator accordingto the penneability coefficientofthebag. ITierotationofthebagaSsistBinttiixingthewmtentsofthebag. However, the cell culture chamber willoftobemnitedto use wimin a controlled gas environment Particularly, the cell culture chamber may have no support apparatus and may be limited to small volumes. Furthermore, the chamber may not provide an inlet and an outlet for media to be constantly pumped into and out of the chamber duringrotation.
[0007] Some companies have developed a range of pre-sterilc, disposable bioreactors that do not require cleaning or sterilizing. Such reactors are made of sheets of flexible, gas impermeable material to form a bag. The bag is partially filled with media and ţhen inflated with air that continuallypasses through the bag's headspace. The media is mixed and aerated
by rockiag the bags to increase the air-liquid interface. However, since there is typically no solid houaing that Bupport the bags, the bags may become cumbersome and difficult ta handle as they increase in size. Furmermore, the wave action witbin fhe rocking bag can create darnaging turbulent forces. Certain cell cultures, particularly human cell cultures, may benefit ftom more gentle condiţiona.
[0008] Thus, thete ia a ccmtinuing need to develop flexible, pre-sterilized, disposable bioreactors that are easyto handle and require little training to operate, yet provide tbe necessary gas transfer and nutrient mbdng reqoired for successful cell and tissue cultures. Snch disposable bioreactors would be equally useful for ihe productionofchemicals, biopharmaceuticals, biologicals, cells, microorganisms, plant metaboh'tes, foods and the like.
BRIEF SUMMARY OF THE INVENHON
[0009} In a firat aspect, tbe present invention provides a stirred-tank reactor system wim 'disposable elemente, such as a flexîble plastic bag with an attached bearing, shaft, and impeller assembly. The instant invention fttrfher rclates to the use of this novei stirred-tank reactor system as a disposable bioreactor and in kits with disposable elements. The advantages of me present invention are numerous. Particulariy, the stirred-tank reactor system may be pre-sterilized and does not require a steam-in-place (ŞIP) or clcan-in-place (CIP) euvironment far changing fiom batch to batch or product to product in a cultare or production system. As such, the system may require lessregulatory control by assuring zero batch-to-batch contamination and can, finiş, be operated at a considerable cost-advantage and with minimal or no preparation prior to use. M additiori,me«ystan «m bea trac stirred-tank reactor system unfike other disposable reactors systems. This provides tihe added advantage • thal the instant inveritioa can offerahydnxiyn^
sizes similar to conventional non-disposable reactor systems. As me system typically does not rcquh^cleaningOT8terittring,it(»mbines a flexible, easy-to-use, true sthred-tank reactor environmentwifli zero cTOSs-contaminationduring the c»^
[001 Oî One aspect of the present invention provides a stirred-tank reactor system,
. comprising a flexiblo bag with at least one opening, wherem the bag functions as a sterile
container fin* a fluidic mediem; a shaft situated wimin &e bag; an impeller attachable to tbe
ahaft, wherem toc impeller is used to agitate the flmd^cmediumto provide a jryurodynarrac
eavironmmt; and a bearmg attached to the shaft and totheThe bag may
be affixed to the shaft and the bearing through at least one seal or o-ring such that the inside of the bag remains sterile. The seals or o-rings can be affixed to the bag. Thesystemmaybe disposable and pre-sterilized. The bag may further inchide a pH scnsor and a dissolved-oxygen sensor, wherein the sensors are incoiporated into the bag. Ia addition, the system may include at least one internai pouch sealed to the bag, wherein the pouch has one end that can be opened to the outeide of the bag such that a probe (i.e., a temperature probe, a pH probe, a dissolved gas sensor, an oxygen sensor, a carbon dioxide (CO2) sensor, a cell mass sensor, a nutrient sensor, an osmometer, and the like) can be inserted into the reactor. The system may also include at least one port m the bag allowing for the connectionofadevice such as a tube, afilter, a sampler, aprobe, or a connection device to the port. Aport allows for sampling; gas fiow in and out of the bag; liquid or media flow in and out of the bag; inoculation; titration; adding of chemostat reagents; sparging; and the like.
[0011] Another aspect of the prescmtinventionprovides a stirred-tank reactor systen^ comprising a flexible bag wim at least one opening, wherenl the bag functions as a sterile container for a fluidic medium; a shaft situated within the bag; an impeller attachable to the shaft, wherein the impeller is used to agitate the fluidic medium to provide a hydrodynamic environment; and a bearing attachcdto the shait and tome opening of the bag. The system may further inchide a housing, such as a reactor housing, on the outside of the bag, wherein the housing includes at least one support that holds the bearing and a motor, and wherein the bag is contained within the housing. The housing may further include a plurality of baffles such that the bag foldsaround the baffles. Optionally, ihe system further encompasses a heater (e.g., a heating pad, a steam jacket, a circulating fluid or water heater, etc.) that can be located berween the bag and the housing. Altematively, the heater may be incoiporated into the housing (&£., a pcnnanent reactor hxmsin^
[0012] foaiwmer aspect of rnemvention, the stirr^-t^
pcnnanenthoushigwimaproducrtlc>opwimflowpastapH8en8effandadissolved-oxygen sensor, wherein the sensors are incorporated into the housing. The permanent housing includes, but is not limited to, a metal hâtrei, a plastic barrel, a wood banei, a glass banei, and the like.
[0013] The mvention also contemplates a method for preparing a stirred-tank reactor system, comprisnigproviding a flexible bag with at least one opening, wherein the bag functions as a sterile container for a fluidic medium; inserting a shaft with an impeller
attachable to the shaft into the bag, wherein the impeller is used to agitate the fluidic medium to provide a hydrodynamic environment; attaching a bearing to the shaft and to the opening of the bag; and sealing the bag to the shaft and the bearing such that the inside of the bag remains sterile. The stirred-tank reactor system prepared by thls method includea at least one disposable element including, but not limited to, the bag, the shaft, the impeller, and the bearing.
[0014] The invention forther encompasses a kit comprising a stirred-tank reactor system andinstructionsforuse. The kit includea a disposable stirred-tank reactor system. The kit may also include a stirred-tank reactor system with at least one disposable element such as the bag, the shaft, the impeller, or the bearing. The bag may be affixed to the shaft and the bearing tbrough at least one seal or o-ring such that the inside of the bag remains sterile. Furthermore, the bag may include a pH sensor and a dissohred-oxygen sensor, wherein the sensors are incorporated into the bag. The Idt may also include at least one internai pouch sealed to the bag, wherein the pouch includes one end that canbe opened to the outside of the bag such that a probe canbeinserted into the reactor. In addition, the system may include at least one port in the bag allowing for the connection of a dcvice to the port, wherein the device includes, but is not limited to, a tobe, a filter, a sampler, and the Hke.
[0015] Another aspect of the invention provides a bag for use in a stirred-tank reactor system. The bag may bea disposable, ftatible, plastic bag. The bag may also include at least one disposable element including, but not limited to, a seal, an o-ring, a port, a pouch, a tube, a filter, a sampler, a probe, a sensor, a connection device, or the like.
[0016] In one aspect, the preseot invention provides a reactor system that includes a container and a rotational assembly. The rotational assembly can be in sealed cooperation wim an openmg of a container. THe rotational assembly can inoludp arotatable hub adapted to receive and releasably couple with a dnve shaft, such that when the drive shaft is operatively coupled with the rotatable hub, rotation of the drive shaft facilftates a (X)n«spondingrotationoftherotatablehub. m a related aspect, the system can further include nn impeller coupled with the rotatable hub, such mat the impeller is disposed within the container and adapted to couple with a distal end of the drive shaft. In other aspects, the rotational assembly can include a casing, whereby the rotational assembly is in sealed cooperation with the opening of mc container via the casing, Similarly, the system can include a drive shaft, wherein the rotatable hub and the drive shaft are disposed to rotate
relative to the casing. M still a related aspect, the rotational assembly can include a bearing assembly disposed between the casing and the rotatable hub. The rotational assembly may further include a sealing arrangement disposed circumferentially to the rotatable hub, between the rotatable hub and the casing. Relatedly, the bearing assembly can include a plurality of race bearings, and the sealing arrangement can închide a rotating disk coupled with the rotatable hub, a wear plate coupled with the casing, and a dynamic seal disposed between the rotating disk and the wear plate. In other aspects, a seal can include two or more seal subunits disposed in co-planar arrangement Relatedly, a bearing assembly can include a joumal bearing, and the sealing arrangement can include a wear plate coupled with the rotatable hub, and a dvnamic seal disposed between the casing and the wear plate. In a similar aspect, the impeller can include a spline adapted to couple with the drive shaft Often, the container can comprise a flexible bag. In another aspect, the rotatable hub can be coupled with the impeller via a flexible tube.
[0017] m one aspect, tbârfl^entmventionprovides a reactor syBtemthatincludes a container and a sparger assembly. The sparger assembly can be disposed within the container, and can include a flexible sheet of permeable material and a sparger conduit In a related aspect, the sheet of permeable material can include a vapor-penneable and water-resistant material. In some aspects, the sheet of permeable material can include a high density polyethylene fiber. In related aspects, the sparger assembly can be in fluid communication with aport of the container. Similarly, the reactor system may include a rotational assembly in sealed cooperation with an opening of the container, and an impeller disposed within the container and coupled with the rotational assembly. The sparger body may be aochored to an interior surface of the container, and in some cases, the sparger body ofmesrwgerassemblytMnbemasubstantiallysphericalshape.
[0018] hi another aspect, the present invention provides a bioreactor system that includes a frame support coupled with a drive motor; a flexible bag disposed within a housing of the frame support. The flexible bag «îanmcludeone or more ports for mtroducmg a (jellralture and a medium into fhe flexible bag; a rotational assembly coupled with a bracket of the frame support and in sealed cooperation with an opening of fhe flexible bag. The rotational assembly can include a hub adapted to house and couple with a drive shaft of the drive motor. The system can also include an impeller coupled with fhe hub for agitating fhe cell culture and medium. The impeller can be disposed within the flexible bag and adapted to couple with the drive shaft In one aspect, the bioreactor system can include a probe assembly. The
probe asscmbly can include a port couplcd with the flexible bag, a Pali connector coupled with the port, a sleeve coupled with the Pali connector, a coupler coupled with the sleeve, and a probe configured to be coupled with the coupler and inserted through the sleeve, Pali connector, and port, and partially into the flexible bag.
[0019] In one aspect, the present invention providcs a method for manufacturing a reactor system. The method can include coupling a container with a rotational assembly. The rotational assembly can be in sealed cooperation with an opening of the container. The rotational assembly can include a hub adapted to house and couple with a drive shaft. The method may also include coupling an impeller with the hub, where the impeller is disposed within the container. The method may further include sterilizing the reactor system. In a related aspect, mc sterilizing step can include treating the system with gamma radiation.
[0020] In another aspect, the present invention provides a method for preparing a reactor system. The method can închide coupling a casing of a rotational assembly of the reactor system to a frame bracket. The method can also include placing a container of me reactor system at least partially within a frame housing, and inserting a drive shaft into a hub of the rotational assembly. The hub can be disposed within the casing of the rotational assembly between a bearing and the casing. The method can further include coupling a distal end of the drive shaft to an impeller. The impeller can be disposed within the container and coupled with me hub. The method can also include introducing a reaction component into the container via aport
[0021] rn one embodiment, the present invention provides a reactor system kft. Thekitcan have a reactor system mat includea a container. The reactor system can also inchide a rotational assembly in sealed cooperation with an opening of me container. The rotational
coupled with me hub. The impeller can be disposed within the container and adapted to couple with me drive shaft. The kit also includea instructions fac use.
BRIEF DESCRIPTION OF THE DRAWINGS
[0022] The present invention isbestunderstoodwhenread hi conjunctionwimthe accompanymg figures which serve to illustrate me preferred embodnnents. H is understood, however, that me invention is not limited to the specific embodiments disclosed in the figures.[0023] Figure l depicts a longitudinal cross-section of one embodiment of the stirred-tank reactor system, whercin the stirred-tank reactor system is placedlnto a permanent housing.
[0024] Flgnre 2 depicts one embodiment of a probe connectkm in order to illustrate that a probe can be attached to the stirred-tank reactor system via a sterile or aseptic connection.
[0025] Fignres 3A and 3B illustrate cross-section views of a reactor system according to one embodiment of the present invention.
[0026] Plgnre 4A illustrates a cross-section view of a rotational assembly according to one embodiment of the present invention.
[0027] Figure 4B illustrates a cross-section view of a rotational assembly according to one embodiment of the present invention.
[0028] Figure 5 illustrates a cross-section view of a rotational assembly according to one embodiment of the present invention.
[0029] Figure 6 illustrates a pârtia! cross-section view of a rotational assembly according to one embodiment of the present invention.
[0030] Figure 7 illustrates a perspective view of a rotational assembly according to one embodiment of the present invention.
[0031] Flgnre 8 illustrates a cross-section view of a rotational assembly according to one embodiment of the present invention.
[0032] Flgnre 9 illustrates a cross-section view of a rotational assembly according to one embodiment of the present invention.
[0033] Fîgure 10 mustratee a cross-section view of an impeHOT according to one embodiment of the present invention.
[0034] Fîgure 11 illustrates a parţial cross-section view of an impeller according to one embodiment of the present invention.
[0035] Ffgnre 12 illustrates a perspective view of drive shaft core according to one embodiment of the preseiit invention.
[0036] Figurel3iUuBtratesacross-sectipnviewofanimp embodiment of the present invention.
[0037] Figure 14 A illustrates a perspective view of an impeller according to one embodimcnt of the present invention.
[0038] Figure 14B illustrates a perspective view of an impeller according to one embodiment of the present invention.
[0039] Figure 15 illustrates a cross-section view of a sparger body according to one embodiment of the present invention.
[0040] Figure 16 ilhistrates a cross-section view of a sparger assembly according to one embodiment of the present invention.
[0041] Figure 17 illustrates a cross-section view of a sparger assembly according to one embodiment of the present invention.
[0042] Figure 18 illustrates a cross-section view of a sparger assembly according to one embodiment of the present invention.
[0043] Figure 19 illustrates a cross-section view of a sparger assembly according to one embodiment of the present invention.
[0044] Figure 20 illustrates a pârtia! perspective view of a reactor system according to one embodiment of the present invention.
[0045] Rgure 21 ilhisteates a partid perspexrâve view embodiment of the present invention,
[0046] Figure 22 ilhistrates a partial perspective vicrw of a reartor system embodimdht of the present invention.
[0047] V^ism23^ateawAmsK^Kcd [0048] F^ure 24 iUustrates a perspective view of a lector system aMordingto one embodiment of the present invention,
[0049] Figure 25 illustrates a perspective view of a reactor system according to one embodiment of the present invention.
[0050] Figiire26iUustratesaprobcasaen]foly2600a [0051] Fignre 27A provides a ilhistration of a probe port subassembly of a probe assembly according to one embodiment of the present invention.
[0052] Fignre 27B illustrates a probe kit subassembly of a probe assembly according to one embodiment of the present invention.
[0053] Fignre 27C illustrates an antoclave subassembly of a probe assembly according to
»
one embodiment of the present invention.
[0054] Fignre 28 A illustrates a probe assembly according to one embodiment of the present invention.
[0055] Fignre 28B illustrates aprobe assembly according to one embodiment of the present invention.
[0056] Fignre 29 provides a graph of data mat wasgeneratedusing a reactor system according to one embodiment of the present invention.
[0057] Fignre 30 provides a graph of datathat wasgeneratedusing a reactor system according to one embodiment of the present invention.
[0058] Fignre 31 provides a gtaph of datathatwasgeneratedusmg a reactor system according to one embodiment of the present invention.
[0059] Fignre 32 provides a graph of data that wasgeneratedusing a reactor system according to one embodiment of the present invention.
[0060] Figure 33 provides a graph of data that wasgeneratedusing a reactor system according to one embodiment of the present invention.
[0061] FJgtu^ 34 provides a gra^ of data that wwgerMjratedusirtg a react^ according to one embodiment of me present invention.
DETAJLED DESCRIPTION OF THE INVENTION
[0062] Insomeembodments,thetejm^erablebag"canre fluidic medhun. The bag may include one or more layer(s) of flexible or semi-flexible waterproof material a^>endmg c» size,strmgth and volume requirements. The inside surface of Ihe bag may be smooth and provide a sterile environment (&g„ far culturing cells or other organism, for food production, etc.). The bag may închide one or more openings, pouches (e.g., for inserting one or more probes, devices, etc.), ports (e.g., fac the cormection of one or
more probes, devices, e/c.) or the like. Furthermore, the bag can provide a disposable alternative to a solid vessel in a convenţional stined-tank bioreactor. The flexible bag may further include a shaft, an impeUer, a bearing and seals or o-rings, and may be entirely disposable.
[0063] In some embodiments, the term "fluidic medium" can refer to any biologica! fluid, cell culture medium, tissue culture medium, culture of microorganisms, culture of plant metabolites, food production, chemical production, biopharmaceutical production, and the like. The fluidic medium is not limited to any particular consistency and its viscosity may vary from high to medium to low. When the fluidic medium is a cell culture medium the system may be operated in, for example, batch mode, semi-batch mode, fed-batch mode, or continuous mode.
[0064] to some embodiments, the term "impeller" can refer to a device that is used for agitating or mixing the contents of a stiired-tank reactor system (e.g., bioreactor). The impeller may agitate the fluidic medium by stirring or other mechanical motion. The impeller of the instant invention includes, but is not limited to, a Rushton, a marine, a ' hydrofbil, a pitched blade, and any other commercially available impeller.
[0065] to some embodiments, a 'Tiydrodynamic" environment of the instant invention may refer to an environment that is influenced by the motion of fluids and the forces acting on solid bodies immersed in these fluids within the stirred-tank reactor system.
[0066] The present invention includes single use bioreactors, stirred tank reactors, and the
like. Such reactors have a variety of applications, such as for the production of merapeutic
proteins via batch cell culture. Relatedly, these systems can be used to provide for cell
growra and anhTx>dy production for CBO and o^ %ehydrodynatnic
environment withifl me reactors can be well diaracterized,and,assuch,maybescaledto other stirred tank bioreactors.
[0067] Single use bioprocess containers can be used for thestorage of biopharmaceutical media, buffers, and omerproducts. Using mese storage container systems, several mixing systems for preparation of media and buffers can be developed, often to commeroial scale up to 10,000 liters or more. Such mixing systems and bioreactors can use various means for mixing the reactor contenta, such as apulsating disk, apaddle mixer, a rockingplatfbnn, an impeller, and the like. These systems are well suited for use in chemical processing. The
operating characteristics of the reactors can be well defined, and can be readily predicted and scaled to various sizes. In the biopharmaceutical industry, such stirred tank biorcactors can be established as a means far manufacture of biologic products from a wide range of biological systems, including animal cell culture. Processes for biological systems can be developed using stirred tank bioreactors at the bench scale and transferred to stirred tank bioreactors at the commercial scale, up to 10,000 liters or greater, using well established scale-up methodologies. For a stirred tank bioreactor, design parameters such as tip speed, power input, Reynolds number, and oxygen transfer coefficient can be readily determined and used for scale-up.
[0068] A single use portion of the system can include a flexîble plastic container with the following single use integrated components: a bearing, shaft, and impeller assembly, a sparger assembly, ports for sterile attachment of sensor probes; and various ports for inlet and outlet of liquids and gases. A single use bioreactor can be manufactured using medical grade film. In some cases, other components of the single use bioreactor can be manu&ctured from readily machined materials that are not necessarily TJSP Class VI materials. The impeller can be a pitched-blade impeller that is attached to a bearing assembly by a flexible sheath. The impeller and sheath can rotate along with an inner bearing assembly, which is isolated from the exterior bearing assembly using various seal assemblies. An outer bearing assembly can be directly affîxed to the single use container. A sparger can include a porous membrane mat is sealed to the bottora of the single use container. Sparge gas can be introduced to the space between the porous membrane and bottom of me container tfarough a port after passing through a pre-attached sterilization filter. The pH and dO2 sensors may or may not be part of the single osc container and can connected to me bioreactor using Pali Kleenpack® cormectors. lodiistry-standard 12 mm sensors can be cah^rated, men steam sterilized with onehalfofthe(XJnnector. Theomerhalfofmew>nnectorcanbeprc-attachedtothe container, allowmg the sensor to be inserted in direct contact with the reactor contenta. Ports and tubing for headspace gas, thermo well, media inlet, titrant, aainpţing, harvest, and various puise feeds can be pre-attached and pre-storilized with the container.
[0069] A permanent swpportvessel mat contams a motor and drive shaft assembly, heat jacket, and openings for inlets, outiets, and probes can hold a single use container. A drive shaft can fit through the single-use bearing, through the flexible sheath, and lock into the impeller. This shaft can be driven using a standard bioreactor mixer motor of suffîcient power. Heat can be provided to the bioreactor contenta, for example, by electric heat banda
that are in direct contact with sides of the single-use container. The pennanent support vessel can be mobile, and can be placed on a weigh scale for control of reactor volume.
[0070] The system can beoperatedusing standard sensors and controllers that have industry-accepted track recorda of performance. In some embodiments, no control system may be required for steam sterilization or clean in place, and a controller commonly used for bench-scale bioreactors may be sufficient for control of the pH, dO2 concentration, and temperature of the single ase bioreador. A single ase bioeactor oâen requires no cleaning or sterilization in-place. As such, the capital and operating costs of control systems and utilities, such as clean steam, required for steam sterilzation of a large pressure vessel may be eliminated. The st for febrication of a rigid-waUed pressure vessel designedtohandle the stresses exerted during steam-in-place sterilization may also be eliminated Likewise, the capital and operating costs for clean-in-place control systems and utilities may be urmecessary. The design elements of tradiţional stainlcss steel vessels dictated by cleanabih'ty requirements may similarly be eliminated.
[0071] In some embodiments, a single use bioreactor can be a closed system mat is discardedafteruse. This may eliminate the need for cleaning validation studies. The potenţial for cross contamination between production batches may also be reduced. In some embodiments, the capital expenditure required to accommodate multiple products simultaneously in single use bioreactors can be low compared to the cost of the fîxed assets and utilities required to segregate traditional bioreactor systems. A single use bioreactor can be manufactured using medical grade film, and regulatory documentation for the film may be currently available. Other product contact componente of a single use bioreactor canbe manufactured ftom USP Qass VI materials. Current appfican'ons of bioprocess containers manufectured from thaw inat»^ storage of bulk intermediate and fi*»1 product
[0072] As note above, a stkred tank single use bioreactor according to the present invention can provide a well-characterized hydrodynamic environment for cell growth, Mîxing characteristics can be readily calculated and can be translatedtolargersthTedtankreactors. Thus, processes developed at the lab or pilot scale may be scalod up directly to commercial scale, either in larger single use bioreactors or larger traditional stkred tank bioreactors. Scale-up parameters such as power input per unit volume, tip speed, oxygen transfer coefficient, or geometric similarity may be mamtained at the larger scale, m some
emDooimems, me preseni invennon proviaes a suirea rame reactor wiin a oesign mar inciuaes a rotating impcllcr driven by a drive shaft isolated through a series of rotating seals. Such designa can provide efFcctivc and efficient means of transmitting tbe energy required for mixing and mass transfer to the reactor contents.
[0073] Thepresentinvention can also include or be compatible with industry-standard sensor and controller tecbnology. A standard tfaat has developed in the industry is the use of 12 mm diameter pH and dQ2 sensors inserted through DN25 (Inglold-style) ports in direct contact with the reactor contents. Systems such as a single use bioreactor can incorporate the same 12 mm diameter pH and dO2 sensors in direct contact with the reactor contents. Calibration and standaraization procedures for mese sensors can be readily perfbrmed during operation of the bioreactor. m addition, outputs firom these sensors can be compatible with current controllers used by industry. The use of PID controllere to maintain pH, dO2 concentration, and temperature can be used in such biorcactors. As a stirred tank bioreactor with standard sensors, mese control strategica can be directly translatable to a single use bioreactor. Because it can be a stand-alone unit, the single use bioreactor may be controlled using whichever controller type that is preferred by a given facility.
A. The Stirred-Tank Reactor System
[0074] In some embodiments, the stirred-tank reactor system of the present invention provides a flexible and disposable bag for a variety of purposes, mcmding culturing cells, microorganisms, or plant metabolites as well as processing foods, chemicals, biopharmaceutical and biologicals. The disposable bag may include disposable elements such as a shaft, impeller andbearing andis designedto fit into a permanent housing such as a reactor housing. Thebagn^further include one or moreopeaings,pouches, ports or the like. The stirred-tank rea(to system aUows a us^
relative ease and little training. m particular, the disposable system may not require cleaning or sterUizing. Furthennore, the system may not need contmuous validation between production runs. Thus, it combines a flexible, easy-to-use, true stirred-tank reactor environment with little or no cross-contamination during the production process.
[0075] Referring to the drawings, Flgnre l depicts a flexible bag 104 with at least one opening and an agitation shaft 112 with an attachable impeller 113. As shown, the agitation shaftll2andattachedimpeUerll3are8ituatedwithinthebagl04. Further, the agitation shaft 112 is connectable to a bearing 105, wherein the bearing 105 can be sealed to the bag by
beat welding to tbe bag and/or through seal(s) or o-ring(s) 6. The bag 104, agitation shaft 1 12, impeller 1 13, and bearing 105» incloding seals or o-rings 106 are optionally disposable. The disposable bag can be a flexible, plastic bag. The bag 104 can be afBxed to the agitation shaft 1 12 and the bearing 105 through at least one seal or o-ring 106 such that the inside of the bag remains sterile. The seals or o-rings can be further afBxed to the bag as is shown in Fîgure 1. Additkmally, the disposable stirred-tank reactor system may be connected to a support or oneormorebracket(s) 103 that hold the bearing 105 and motor 101. Ia one embodiment (as shown in Fîgure 1), the support 103 is a motor and bearing support 103, wherein the upper end of the agitation shaft 1 12 is further connected to a motor coupling 102. The motor coupling 102 is cormected to the motor 101 which drives the stirring motion of me agitation ahaft 12 and impeller 113 leading to a hydrodynamic environment witbin the bag 14. Thebagl4iadesignedtofitintoahousinglll suchasabarrelorchamber. The housingmaybe a metal barrel, a plastic barrel, a wood barrel, a glass barrel, or any other banei or chamber made firom a solid material, Ih one embodiment of the invention, the housing further includea a plurality of bafBes, wherein the bag folds around the baffles. Ia another embodiment, the flexible bag 104 further includes a top port (single or multiple) 108, a bottom port (single or multiple) 109, and a aide port (single or multiple) 110, wherein flexible tubing 107 can be connected to one or more of these porte.
[0076] The stirred-tank reactor system may optionally include a heater such as a heating
pad, a steam jacket, or a circulating fluid or water heater. M one embodiment, the heater is
located between the bag 104 and the housing 1 1 1. m another embodiment, the heater is
incorporated into the housing 111 (e.g., into a double wall between the reactor housing and
the bag). In yct anomer embodoment, the stirred-tank reactor system isplaced inside an
incubator.This isparticularlymiportant for cellcnidtures which are often
[0077] In one embodiment of the instant invention, the bag 104, the bearing 105, the seal(s) or o-ring(s) 106, the tubing 107, the top port(s) 108, the bottom port(s) 109, the side port(s) 1 10, the shaft 1 12, and the impeller 1 13 are disposable. The motor 101, the motor coupling 102, the bracket(8) or motor and bearing support 103, and the housing 1 1 1 are permanent
B. Devices and Porte
[0078] The stirred-tank reactor system may also include sensors and other devices. Ia one embodiment, the bag includes a pH sensor and a dissolved-oxygen sensor, wherein the
sensors are incorporated into the bag. As such, thc sensors are disposablo with the bag. In another embodiment, the sensors are attachable to the bag and are separate unite. Such sensors may optionally be reusable after sterilization. In another embodiment, the system includea a product loop with flow past a pH sensor and dissolved-oxygen sensor, wherein Ine sensors are incorporated into Ine reactor housing. The system is flexible and provides alternative ways of supplying opţional equipment of various kinds (e.g.. sensors, probes, devices, pouches, ports, etc.). The system may also include one or more internai pouches tbat
are SCaled to the bag. poneh Vina at least one emA that cm
be opened to the outside of the bag to insert aprobe into the reactor (Le., the bag) while remainmg on the exterior of the bag. The probe may be, for exemple, a ternperature probe, a pH probe, a dissolved gas sensor, an oxygen sensor, a carbon dioxide sensor, a cell mass sensor, a nutrient sensor, an osmometer or any other probe that allows for testing or checking the culture or production. In another preferred embodiment, the system includes at least one port in the bag allowing for me connection of a device to the port. Such a device includes, but is not limited to, a tube, a filter, a connector, a probe, and a sampler. The inoorporation of various ports into the bag allows for gas flow in and out of the bag as well as liquid flow m and out of the bag. Such ports also allow fixr sampling or testing the media or culture inside the bag. Tubing, filters, connectors, probes, samplers or other devices can be connected to the ports by using any desirable tubing connection technology. Pouches and ports that are sealed or affixed to the bag are disposable with the bag. The bag may also include a sparger (i.e.t the component of a reactor mat sprays air into the medium) sealed to the bag wbich can be disposed offwith the bag.
[0079] Particulady, ports may be incorporated at any place on the flexible bag to accommodate the following:
Headspace gas in
Headspace gas out
Temperaturc probe pH probe
Dissolved oxygen probe Other desired probes S ample apparatus Media in
Titrantin Inoculumin Nutrient feeds in Harvest out
[0080] Each port may have floxible tubing attached to the port, to which media bags, sample devices, filtera, gas lines, or harvest pumps may be attached with sterile or aseptic connections. In one embodiment, the ports are sealed onto the flexible bag during bag manufacture, and are sterilizedwith the bag assembly.
[0081] Devices that may be used to make aseptic connections to the flexible tubing are the following:
WAVE sterile tube fuser
TERUMO sterile tubing welder
PALL KLEENPAK connector
Connection mado under & laminar flow hood, using aseptic techniques
BAXTER Hayward proprietary "HEAT-TO-HRAT" connection using metal tubing
and an induction heater
[0082] In another embodiment, flexible tubing mat is attached to an appropriate stainless-• steel valve assembly may be sterilized separately (e.g., via atrtoclave), and then used as a way to cormect the disposable bioreactor to tradiţional reactors or process piping. The valve assembly is used to make a tradiţional steam-in-place (ŞIP) connection to a traditional reactor or other process, and the flexible tubing is used to make a sterile or aseptic cormection to a port on thadisposable reactor.
[0083] Rcforrmgto the drawings,Mgure 2 dc^cts a probe corneei employed with me Btirred-tank reactor system accordhlg to one embodiment of the instant invention. As shown in Hgnre 2, me probe 201 can be connected to a flexible sleeve 202 or bag which extends to one half of a PALL connector 203. The PALL cormector 203 can be connected to the other half of the PALL connector 205 to provide far a sterile cormection between the probe and the stirred-tank reactor system. The PALL connectors 203,205 include covers 204 and filters 207 to keep the connection site sterile. Sterile tubing 206 extends from the other half of the PALL connector 205 to a reactor port 208 of the reactor vessel 209 of the stirred-tank reactor system. m ordertoattach the probe, the PALL connection is made by removing the covers 204, mating the connectors 203,205, removing
the filters 207, and sliding the movable part of the connector into position. The probe sensor tip 212 is then pushed into the reactor as the flexible sleeve or bag bunches or compresses 210. The probe senor tip 212 is tfaen in direct contact with the inside of the reactor vessel 209. A clamp 211 is placed around the probe and tubing to seal the reactor contents from the PALL connection assembly. Thus, when a sterile connection is made between the two halves of the PALL connectors 203,205, the flexible sleeve 202 or bag becomes compressed 210 and the probe is in contact with the culture or production media.
[0084] Ia one embodiment, the probes may be sterilized separately (e.g., via autoclave) then attached to the reactor via a sterile or aseptic connection. For example, a probe assembly may be made by inserting a probe 201 into one half of a PALL KLEENPAK connector 203 and sealing the probe to the connector using a flexible sleeve or bag 202 as described abovc and shown in Fignre 2. The sleeve extends from the outside end of the probe to the barb of the PALL connector. Tbis assembly is steriUzed separately. Theother half of the PALL connector 205 is connected to a port 208 on the reactor 209 via flexible tubing 206 that will accommodate the probe. Tbis assembly is steriUzed as part of the reactor. The PALL connector is described in detail in U.S. Patent No. 6,655,655, the content of which is incorporated herein by reference in its entirety.
[0085] Fignres 3 A and 3B illustrate cross-section views of a reactor system 300 according to one embodiment of the present invention. Reactor system 300 can include a rotational assembly 301 coupled with a container 302. Optionally, reactor system 300 may include an impeller 340. In some embodiments, rotational assembly 301 is in sealed cooperation with an opening or aperture in container 302. Similarly, rotational assembly 301 may include a casing 360 that is coupled with the opening or aperture m (X)ntamer 302. Typically, impeller 340 is disposedwttfain the interior of container 302. Rotationalassembly 301 canbc supported or held by bradoet 308.
[0086] m some embodiments, rotational assembly 301 mayinchideahub320thatis coupled with impeller 340, and hub 320 may be coupled with impeller 340 via a connector 390. OptionaUy, hub 320 may bedirectly coupled with impeller 340. m some embodiments, hub 320 is tabular k shape and includes an interior surfâce which bounds apassageway 320a longitudinally extending therethrough. In one embodiment an annular barb 321 radialty encircles and outwardly projects flom the exterior surface of hub 320. Barb 321 can be used for creating a sealed connection with connector 390.
[0087] Connector 390 can be tabular in shape, and can include an interior surface which bounds a passageway 390a extending longitudinally therethrough. Ia some embodiments, connector 390 includcs a flexible tube having a first end connected in sealed engagement with hub 320 and an opposing second end connected in sealed engagement wilh impeller 340. Hub 320, either atone or in cooperation with connector 390, can provide a sealed channel in which drive shaft 304 can be received and removably coupled wilh impeller 340. Consequently, drive shaft 304 can be used repeatedly without sterilizmg because it does not directly contact the contents of container 302. Furthermore, by usmg a flexible tube as connector 390, a flexible container 302 such as a bag assembly can be easily rolled up or folded for easy transport, storage, or processing.
[0088] Often, rotational assembly 301 will include a bearing assembly 370 disposed between hub 320 and casing 360. Bearing assembly 370 can include a joumal bearing, which may be in fixed relation with casing 360, and hub 320 can rotate relative to the joumal bearing and casing 360. Hub 320 may include a giride 324 for receiving a snap ring or retaining ring, which can hehp maintain hub 320 in place, relative to the joumal bearing.
[0089] Rotational assembly 301 may also include a sealing arrangement 380, which can be disposed between hub 320 and casing 360. Sealing arrangement 380 can închide, for example, a wear plate 382 and one or more seals 384, which may be, for example, dynamic seals. Wear plate 382 cianbe disposed circumferentiallyto, and coupled with, hub 322. Seal(s) 384 can be disposed between wear plate 382 and casing 360. Rotational assembly 301 may also include one or more seals 392 disposed between wear plate 382 and hub 322, wherein seals 392 may be, for exemple, static seals. m some embodiments, seal(s) 384 include one or more V-rings and seals(s) 392 include one or more O-rings. Ia the embodiments shown ia ftgnre SA* seal(S) 384 inehide *wo V-imgs, and seal(s) 392 include one O-ring. An annular flange 322 may also radially, outwardly project from the exterior surface of hub 320 and be disposed against scai 392.
[0090] Muse,hub320is(xmfiguredtoreceiveorhouseadriveshaft304thatisselectively coupled with a motor (not shown). m some embodiments, hub 320 may be confîgured to couple with one or more ears 306 located at an upper end of drive shaft 304 vk one or more hub notches 322 formed on hub 322. Impeller 340 may include a spline 342 confîgured to couple with a lower end of drive shaft 304. Drive shaft 304 can be placed in hub 322, and coupled with hub 322 and impeller 340. For example, drive shaft 304 may extend fhrongh
passageway320a. Similarly, drive shaft 304 mayextendthrbughpassageway390a. Drive shaft 304 can be rotated by a motor, thereby rotating hub 320, connector 390, and impeller 340. In tuni, impeller 340 agjtates the contente of container 302. As hub 320 is rotated by drive shaft 304, seal(s) 392 provide a seal between wear plate 382 and hub 320 as they both rotate in unison, relative to casing 360. As casing 360 remains stationary, seal(s) 384 provide a seal between wear plate 382 and casing 360, where wear plate 382 rotates relative to casing 360. Li some embodiments, seal(s) 384 provide a hermetic seal between wear plate 382 and casmg 360. As shown bere, seal(s) 384 can be in co-planar arrangement with one anofher.
[0091] hi same embodiments, hub 320 may be removably engagable with drive shaft 304 such that annular rotation of drive shaft 304 facilitates ammlar rotation of hub 320. Although the embodiment depicted in Flgnre 3A shows drive shaft ears 306 coupled with hub notches 322, the present invention contemplates any of a variety of coupling means for accomplishing tbis function. M yet other alternative embodiments, clamps, pins, collets, meshing teeth, or other fasteners can be used to removably secure drive shaft 304 to fhe hub 320 when the drive shaft 304 is coupled with hub 320. Similarly, the present invention contemplates any of a variety of coupling means for removably engaging drive shaft 304 to impeller 340, including the coupling means described above, such that rotation of drive shaft 304 facilitates rotation of impeller 340.
[0092] Acccnrimgtooiie embodiment of the present invention, reactor systemSOOcanbe
manufactured by coupling container 302 with rotational assembly 301, such mat container
302 and rotational assembly 301 are in sealed coopcration with one another. For example,
rotational assembly 301 can be coupled with an opening of container 302. Rotational
assembly 301 can be manufactured to închide hub 320, and hub 320 can be coupled withimpeUer 340 such tbatimpdte 3401Further, reactor Bystem examplebygamniaradiation.
[0093] According to another embodiment of the present invention, reactor system 300 can be prepared for use by coupfing casing 360 of rotational assembly 301 to frame bracket 308, and placing container 302 at least partiafly within a frame or container housmg (not shown). Drive shaft 304 can be inserted into hub 320, and a distal end of drive shaft 304 can be coupled with impeller 340. Further, reaction componente such as cells and culture media can be introduced mto container 302 via aport 310.
[0094] Container 302 can include any of a variety of materials. Ia some embodiments, container 302 inclodes a flexible bag of water impermeable material such as a low-density polyethylene or other polymeric sheets having a thickness in a range between about 0.1 mm to about 5 mm, or between about 0.2 mm to about 2 mm. Other thicknesses can also be used. The material can be comprised of a single ply material or can oomprise two or more layers which are either sealed together or separated toform a double wall container. Wherethe layers are sealed together, the material can oomprise a latninated or extruded material The laminated material can include two or more separatoly formed layers mat are subsequently secured together by an adhesive. The extruded material can include a single integral sheet having two or more layers of diffexmt material that are each separated by a contact kyer. AII of the layers can be simultaneously co-extruded. One example of an extraded material that can be used in the present invention is the HyQ CX3-9 fihn available from HyClone Laboratories, Inc. out of Logan, Utah. The HyQ CX3-9 fihn is a three-kyer, 9 mii cast fihn produced in a cGMP facility. Theouterlayerisapolyesterelastcmer(x>extnidedwithan ultra-low density polyethylene product contact layer. Another example of an extruded material mat can be osed in the present invention is the HyQ CX5-14 cast fihn also available from HyClone Laboratories, Inc. The HyQ CX5-14 cast fihn comprises a polyester elastomer outer layer, an ultra-low density polyethylene contact kyer, and an EVOH bamer kyer disposed therebetween. In another example, a muM-web fihn produced from three independent webs of blown film can be used. The two inner webs are each a 4 mii monokyer polyethylene fihn (which is referred to by HyClone as the HyQ BM1 fihn) while the outer bamer web is a S .5 mii thick 6-kyer coextrusion fihn (which is referred to by HyClone a the HyQ BX6 fihn).
[0095] Etgnre 4A illustrates a oross-section view of a rotatwnal asscrably 401 accordmg to one embodiment ofme present invention. Flgnre 4B illustrates a cross-section view of me rotational assembly 401 depicted in Flgure 4A coupled with a connector 490 and an impeller 440. Rotational assembly 401 may inchide a bearing assembly 470 disposed between a hub 420 and a casing 460. As shownhere, bearing assembly 470 inchides two race bearings, which are in fixed relation with casing 460. Hub 420 can rotate relative to the race bearings. Hub 420 may include guides 424,424a for receiving a snap ring or retaining ring, which can help maintain hub 420 in place, relative to race bearings.
[0096] Rotational assembly 401 may also inchide a sealing arrangement 480, which can be disposed between hub 420 and casing 460. Sealing arrangement 480 can include, for
example, a wear plate 482, one or more seals 484, and a rotating disk 450. Rotating disk 450 can be disposed circumferentially to, and coupled wira, hub 420. Seal(s) 484 can be disposed between rotating disk 450 and wear plate 482. Wear plate 482 can be coupled with casing 460 via screws or bolts inserted through casing columns 428. Rotational assembly 401 may also include one or more seals 492 disposed between rotating disk 450 and hub 422. In some embodiments, seal(s) 484 include one or more V-rings and seals(s) 492 include one or more O-rings. In the embodiment shown in Figures 4A and 4B, seal(s) 484 include three V-rings, and seal(s) 492 include one O-ring. Rotational assembly 401 may also include one or more seals 426 toprovide a seal between hub 420 and the top of casing 460, and one or more seals 462 to provide a seal between casing 460 and wear plate 482. As shown bere, seal(s) 426 include one V-ring and seal(s) 462 include one O-ring.
[0097] Inuse, hub420isconfiguredtoreceiveorhouseadriveshaft(notshown). In some embodiments, hub 420 may be confîgured to couple with an ear of drive shaft via hub notch 422. As hub 420 is rotated by drive. shaft, seal 492 provide a seal between rotating disk 450 and hub 420 as theyboth rotate in unison, relative to casing 460. As casing 460 remains stationary, seal(s) 484 provide a seal between rotating disk 450 and wear plate 482, where rotating disk 450 rotates relative to wear plate 482 and casing 460. In some embodiments, seal(s) 484 provide a hermetic seal between rotating disk 450 and wear plate 482. As shown here, seal(s) 484 can be m co-planar arrangement with one another.
[0098] Flgure S illustrates a cross-section view of a rotational assembly 501 according to one embodiment of the present invention. Rotational assembly 501 may include a bearing assembly 570 disposed between a hub 520 and an inner casing 560. As shown here, bearing assembly 570 includes two race bearings, which are m fixed relation with inner casing 560. Hub 520 can rotate relative to the race bearings. Htlb 520 may include girides 524,524a for recerving snap rings or retajning rings, which can help înaintam hub 520 m place, relative to race bearings.
[0099] Rotational assembly 501 may also include a sealing arrangement 580. SeaJing arrangement 580 can include, for example, a bottom plate 583 and one or more seals 584. Seal(s) 584 can be disposed between hub 520 and inner casing 560. A top plate 587 can be coupled with inner casing 560 via screws or bolts inserted through casing columna 528. Rotational assembly 501 may also include one or more seals 591 disposed between top plate 587 and an outer casing 561. m some embodiments, seal(s) 584 include one or more V-rings
and seals(s) 591 include one or more O-rings. In the cmbodiment shown ia Figure 5, seal(s) 584 include three V-rings, and seal(s) 591 include one O-ring. Rotational assembly 501 may also include one or more seals 526 to provide a seal between hub 520 and the top plate 587. As shown here, seal(s) 526 include one V-ring.
[0100] In use, hub 520 is configured to receive or house, and couple with, a drive shaft (not shown). As hub 520 is rotated by drive shaft, seal(s) 584 provide a seal between hub 520 and inner casing 560 as hub 520 rotates relative to inner casing 560. In some embodiments, seal(s) 584 provide a hennetic seal between hub 520 and inner casing 560. As shown here, seal(s) 584 can be in co-planar arrangement with one another.
[0101] Figure 6 illustrates a parţial cross-section view of a rotational assembly 601 according to one embodiment of fee present invention. Rotational assembly 601 may include a bearing assembly 670 disposed between a hub 620 and an inner casing 660. As shown here, a lower race bearing of the bearing assembly 670 is in fixed relation with inner casing 660. Hub 620 can rotate relative to the race bearing. Hub 620 may include a guide 624a for receiving snap rings or retaining rings, wbich can help maintain hub 620 in place, relative to race bearing.
[0102] Rotational assembly 601 may also include a sealing arrangement 680. Sealing arrangement 680 can include, for example, one or more seals 684. Seal(s) 684 can be disposed between hub 620 and inner casing 660. In some embodiments, seal(s) 684 include one or more V-rings. In the embodiment shown in Figure 6, seal(s) 684 include three V-rings.
[0103] In use, hub 620 ia configured to receive or house, and couple with, a drive shaft (not shown). ÂthtiN^iBJ®^
inner casing 660, a» hub 620 rotates relative to inner casing 660. In some embodiments, seal(s) 684 provide a hennetic seal between hub 620 and inner casing 660. As shown here, seal(s) 684 can be m a tiered-planar arrangement with one another.
[0104] Figore 7 illustrates a perspective view of a rotational assembly 701 according to one embodiment of the present invention. Rotational assembly 701 can include a hub 720 having one or more hub notches 722. Ia use, hub 720 is configured to receive or house, and couple with, a drive shafi 704. Hub notch(es) 722 are configured to couple with one or more drive shaft ears 706. A top plate 787 can be couplcd with casing 760 via screws or bolts inserted mrough top plate apertores 787a. As hub 720 is rotated by drive shaft 704, hub 720
rotates relative to top plate 787 and casing 760. Rotational assembly 701 may also include one or more seals 726 to provide a scai between hub 720 and the top plate 787. As shown bere, s«al(s) 726 include one V-ring.
[0105] Figure 8 illustrates a cross-section view of a rotational assembly 801 according to one embodiment of the present invention. Rotational assembly 801 can include a hub 820 having one or more hub notches 822. As shown here, a bearing assembly 870 is in fîxed relation with a housing 823. In use, hub 820 is configured to receive or house, and couple with, a drive shaft 804. Hub notch(es) 822 are configured to couple with one or more drive shaft ears 806, which may be at opposing ends of a drive shaft spindle 806a. As hub 820 is rotated by drive shaft 804, hub 820 rotates relative to housing 823, bearing assembly 870, and casing 860.
[0106] Rotational assembly 801 may also include a sealing arrangement 880, which can be disposed between hub 820 and housing 823. Sealing arrangement 880 can include, for example, one or more outer seals 884 and one or more inner seals 886. Seal(s) 884 can be disposed between an outer surface of hub cup 820a and housing 823, and seal(s) 886 can be disposed between an inner surface of hub cup 823 and housing 823. Housing 823 can be fixed with casing 860. In some embodiments, seal(s) 884 include one or more V-rings and seals(s) 886 include one or more oii seals. In the embodiment shown in Figure 8, seal(s) 884 include one V-ring, and seal(s) 886 include one oii seal. Hub 820 can be coupled with a flexible tube 890.
[0107] Figure 9 illustrates a cross-section view of a rotational assembly 901 according to one embodiment of the present invention. Rotational assembly 901 can include a hub 920 configured toreleasably couple with a drive shaft 904. Asshownhere,twobearingsofa bearing assembly 970 are in fixed relation wim a housing 923. hi usc, hub 920 is configured to receive or house, and couple with, a drive shaft 904. As hub 920 is rotated by drive shaft 904, hub 920 rotates relative to housing 923, bearing assembly 970, and casing 960.
[0108] Rotational assembly 901 may also include a sealing arrangement 980, which can be disposed between hub 920 and inner housing 923a. Sealing arrangement 980 can include, for example, one or more outer seals 984 and one or more inner seals 986. Seal(s) 984 can be disposed between hub 920 and seal(8) 986, and seal(s) 986 can be disposed seal(s) 984 and inner housing 923a. Housing 923 can be fixed with casing 960, and in sealed relation with casing 960 via one or more seal(s) 962. In some embodiments, seal(s) 984 include one or
more V-rings, seals(s) 986 include one or more oii seals, and seal(s) 962 include one or more O-rings. In the embodiment shown in Figure 9, seal(s) 984 include two V-rings, seal(s) 986 include two oii seals, and seal(s) 962 include two O-rings. Hub 920 can be coupled with a flexible tube 990.
[0109] Figure 10 illustrates a cross-section view of an impeller 1040 according to one embodiment of the present invention. Impeller 1040 can be coupled with connector 1090, which can be couple with hub (not shown). Impeller 1040 con închide an impeller spline 1042 which can couple with a spline 1005 of drive shaft 1004.
[0110] Figure 11 illustrates a parţial cross-section view of an impeller 1140 according to one embodiment of the present invention. Impeller 1104 can include an impeller barb fitting 1141 that can couple with a rotational assembly hub (not shown) via a connector 1190. Drive shaft 1104 can be attachedto impeller 1140 byplacing drive shaft 1104 into impeller aperture 1142. When drive shaft 1104 is inserted into impeller aperture 1142, end cap 1107 can reach the distal end of impeller base 1143. As shown here, drive shaft 1104 is hollow and adapted toreceiveacore 1108. Drive shaft 1104 is coupled with an end cap 1107. Core 1108 includes a ball dent 1102 which operatively associates with a ball 1103. m a first ball configuration 1103a, ball 1103 is disposed at ball dent 1102. As core 1108 is advanced along the inside of hollow drive shaft 1104 toward the distal end of impeller aperture 1142, spring 1109 is compressed, and ball 1103 moves into opening 1104a in drive shaft opening 1104a and impeller base opening 1143a, thus adopting a second ball configuration 1103b. Distal end of core 1108 can căuşe end cap 1107 to separate from drive shaft 1104. In some embodiments, core 1108 is in threaded engagement wim end cap 1107, which can prevent spring 1109 from pushmg core 1108 back out of hollow drive shaft 1104.
embodiment of the present invention. Drive shaft core 1208 includes ball dent 1202, end cap 1207, spring 1209, and ball 1203. As shown here, ball 1203 can adopt a first ball configuration 1203a and a second ball configuration 1203b.
[0112] Sigure 13 illustrates a cross-section view of an impeller 1340 according to one embodiment of the present invention. Impeller 1340 can include a square spline 1342 for coupling wim a square spline 1305 of drive shaft 1304. Impeller 1340 can be coupled with hub (not shown) via a connector 1390. For the sake of clarity, the impeller blades are not shown in tbis figure.
[0113] Flgure 14A illustrates a perspective view of an impellcr 1440a according to oue embodiment of the present invention. Impeller 1440a can închide one or more impeller blades 1445a coopled with an impeller body 1446a. In some embodiments, impeller blades 1445a can be machined separately fiom impeller body 1446& Impeller blades 1445a may be constracted fiom a variety of materials, including Dehin, HDPE, and the like. Impeller body 1446a may be constracted fiom a variety of materials, including HDPE and the like.
[0114] Fig0re 14B illustrates a perspective view of an impeller 1440b according to one embodiment of me present invention. Impeller 1440b can închide one or more impeller blades 1445b and an impeller body 1446b. In some embodiments, impeller 1440b can be molded as a single piece. Impeller 1440b may be constracted fiom a variety of materials, including medram low density polyethylene, low density polyethylene, Dow Engage® polyolefin elastomers, and me like.
[0115] FifporelSilhistratesacxDSs-secticmviewofasp
embodiment of the present invention. Sparger body 1500 can închide a sheet of penneable material. In some embodiments, sparger body 1500 includea a vapor-permeable and water-resistant material. In related embodiments, sparger body 1500 includes a high density polyethylene fiber. For example, sparger body 1500 can include Tyvek® material. Sparger body 1500 can be in fluid communication with a port of a contamer (not shown) via a sparger conduitlSlO. As shown in Flgure 15, sparger body 1500 can be in the shq» of a donut or ring. Relatedly, sparger body 1500 can include abase 1502 whichis adapted to anchorto an interior surface of a container (not shown.). The base may or may not include a gas penneable material. In omor embodiments, one or more sheets of gas permeable material can be directly sealed with me interior of the container, whereby the interior of the sparger body 1500 includea a gas penneable matorial on one ride (e.g. top side of body), and a corresponding portion of the container on me other side (e.g. bottom side of body).
[0116] m someembodmimts,mepermeabiUty of me sparger body issuch mat flm^ prevented fiom flowmginto the sparger when not in use. Similarfy, the sparger may be constructed so as to only altow gas to pass through the permeable material whenitissubject tosufficiently high gas pressure. Often, a sparger body will închide a soft, flexible material. Li some embodiments, sparger body 1500 may be welded directly onto container so as to ensure proper placement and alignment When coupled with a flexible container such as a flexible bag, sparger body 1500 can effoctively be folded up wim me bag for storage and
transport, sterilized simultaneously with the bag, and disposed of so as to eliminate subsequent cleaning. Sparger body 1 500 can provide for minute gas bubbles which can increase diffusion of gas into the fluid. It is appreciated that other types of spargere can be used with the present system.
[0117] A variety of materials or assemblies can be used to provide gas transfer into growth chambers. These include, for example, porous materials in the farm of tubing made of Teflon® (PTFE), polysulfbne, polypropylene, silicone, Kynar® (PVDF), and the like. In some embodiments, used to provide gas transfer into growth chambers. As noted above, sparger body 1500 can include Tyvek® material, which can be usedmabioreactorfbrme use of active gas diffiision. Similarly, this material can be used in a growth cham
passive gas transfer. Penneability of Tyvek® film can be measured using the quantitative property of Qurley Hill Porosity. In some embodiments, such materials range in values between about 6 to about 30 (sec/lOOcc IN2). Permeabih'ty rated according to the methods of
*.
Bendtsen Air Permeabih'ty are often in a range between about 400 to about 2000 (ml/mm).
[0118] In some embodiments, a permeable material will have bigh permeability wbile maintaming hydrophobicity, strength, weldability, biocompatibility, and gamma stability. Often, it is desirable to have a flexible material mat welds readily to common materials used in the film or port configurations, often found in the manufacture of bioprocessing containere (BPCs). For example, the flexible nature of a soft or paper bice film can allow it to be folded during manufacturing, packaging, loading, and use of the bioreactor. It may also be desirous to allow for the surface area and shape of the sparge material to easily be modified or changed according to weld or cut pattern. Optionally, instead of providing a sparger body to be immersed in the contents of a container, a permeable envetope could be used encapsulate the liquid contenta of thd oiorMkâtor» thus proviânig'a%toad area for difEusion.
[0119] Welding me sparger body on a port or container surface can provide for a bigh level of surface area wbile providing a low-profile sparge. In some embodiments, this can reduce turbulence near the impeller and/or reduce the possibih'ty of cells accumulating in cracks, seams, or crevices. Often, convenţional sparge configurations rely on the use of spargmg rings mat have small hole perfbrations mat are placed bellow the impeller. Spargere can also include the use of extremely cr"ftH pore sizes. Such porous materials are commonly seen as sintered metal or ceramic matmals. Using a single use disposable material such as Tyvek® maybehdpfUmavoidingOTreducingcontaniina
associated with some convenţional spargere, which sometimes involve cleaning numerous holes, pores, and crevices of such units. For example, small void areas in some spargere may present areas for cell debris to lodge and accumulate leading to increased occurrence of contamination. In some cases, this may carry over in subsequent cell nins.
[0120] One purpose of a sparge unit in a cell culture is to aid in the mass transfer of oxygen (kLa), which is often necessary for the respiration of the growing cells. An advantage of a sparge approach used in a single use bioreactor is that the tortuous porc structure of a gas permeable membrane such as Tyvek® can allow for a beneficia! effect on mass transfer of oxygen fiom the bulk gas introducedthroughtbesparger. Iu some embodiments, it is desirable to have small bubbles introduced into the bioreactor as they can benefit mass transfer. Mass transfer across a permeable membrane can occur independent of mass transfer resulting fiom a gas bubble. Relatedly, a long gas retention tune within me fluid column and a higher surface to volume ratios are often desirable effects. It is generally accepted that the bubble size can be dominated by surface tension effects, rnherently related to the component ratio of salts, proteins, sugara, and micro and macro component» of the nutrient media. Experimentally calculated kLa values, visual observation, and data fiom bioreactor nms often indicate that bubble size and perhaps improved mass transfer are qualities of the present sparge approaches. The composition and rheological properties of the liquid, mixing intensity, tumover rate of the fluid, bubble size, presence of cell chnnping, and interfacial absorption characteristics all influence mass transfer of gas such as oxygen to the cells. Main driving forces of mass transfer include surface arca and concentration gradient hi many cases, a main source of resistance of oxygen mass transfer in a stirred tank bioreactor can be the liquid •fikn surrovmding the gas bubble.
[0121] A spargiag matmial such «B Tyvek® can provide forthe transfer of gas across the membrane. Relatedly, by incorporatnig Tyvek® and similar gas permeable membranes, the surfiacearea can easilybe increased. In some embodiments, the oxygen gradient betweenthe membrane and the liquid interface can be maintained at a Mghlevelthrough constant replenishmentdirectlythrough a sparge inlet Fur&er, a rapid mixing intensity can also benefit mass transfer as the impellerpumps media directly down onto a sparger surface. The use of a membrane can allow for mass transfer of oxygen across the bulk film surface, which can be in addraontothefonnation of bubbles uiatrise within the fluid column. In many cases, small bubbles can leadto greaţa foammg atme top of a bioreactor, which can have negative effects oncell viability andkLaatxx>rdingtoHenry's law andthe solubility of gases
related to parţial pressures. Ibis boundary layer often results in a reduced ability to control dissolved oxygen levels within the bulk liquid. Typically, it is desirable to avoid or mitigate the presence of foam, as excessive amounts can result in exhaust fîlter blocking and nm failure. The novei sparger approaches described herein can providc the desired mass transfer properties, often with reduced levels of foam generated as compared to convenţional systems. This may be due to greater efficacy and less gas being introducea through the sparger to a target oxygen solubility.
[0122] Tyvek®is similar is some aspecte to the material Gore-Tex®inthatithas hydrophobic qualities but will still allow water vapor to pass through. For medical grades of Tyvek® a large relative pore size can be about 20 (micrometers) and the surface energy can be about 25 to about 32 (dynes/cm). As mentioned elsewhere herein, it may be beneficia! to use a check valve in a gas inlet stream near a sparger to reduce undesirable transfer of water vapor through the membrane when the sparger is submerged while not in use. Actual moisture transmission rates may vary largely with the media used and the particular appfication. Moisture Vapor Transmission Rates (MTVR) often range ftom about 1500 to about 1640 (g/m2/24hrs). The present invention also contemplates the use of these sparger approaches in the form of a replaceable retrofît kit, which may be adapted for use with convenţional bioreactors. Such kits can improve kLa and replace a piece of hardware commonly used in steam sterilized bioreactors that may be difficult to sterilize or clean.
[0123] It is appreciated that any of a variety of permeable membranes may be used as a spargjng material. In some embodiments, such membranes may be comprised of high density polyethylene fîbers mat are beat sealed into a web having a thickness in a range between about 50 mlcrons to about 250 microns. The fîbers typically have a diameter in a range between about 2 microns toaboiit8mi(3ronsandoanbeproducedb othermethods.
[0124] In other embodiments, the sparging material may include a perforated film sheet, such as a sheet of low density PE film with small perforated holes. This may be in the form of a plastic tubing, molded plastic, shaped film, or flat film. The small perforated holes can be, forexample,puncb.ed,molded,orembossedhitothefihn. As described above, such sparging materials or constructions can be fîxed to the container. In some embodiments, a sparging mechamsm may include a combination of a permeable membrane and a perforated film.
[0125] Figure 16 ittustzates a cross-section view of a sparger assembly 1600 according to one embodiment of the present invention. Sparger assembly 1600 can include a sheet of penneable material 1605 and a sparger conduit 1610. As shown bere, sheet of penneable material 1605 is ww\w in shapc. Sparger assembly 1600 can be in fluid communication with aport of a container (now shown) via sparger conduit 1610. An inner ring 1603 and an outer ring 1604 of sheet 1605 can eachbe anchored to the interior surface of a container 1602, such that the sheet of penneable material 1605, as coupled with container 1602, definea a donut-shaped space.
[0126] Flgure 17 illustrates a cross-section view of a sparger assembly 1700 according to one embodiment of the present invention, Sparger assembly 1700 can include any number of shcets of penneable material 1705, a sparger tube 1730, and a sparger conduit 1710. Sparger assembly 1700 can be in fluid communication with a port 1720 of a container 1702 via a sparger conduit 1710. As shown bere, sparger assembly 1700 can include a sparger body 1706 that is constracted of two sheets of penneable material 1705 which are coupled together along their outer rings 1704. It is appreciated that sparger body 1706 can be configured in any of a variety of shapes, including spheres, cylinders, boxes, pyramids, irregular shapes, and the lik», and may include any combination of penneable and non-penneable materials or surfaces.
[0127] Fignre 18 illustrates a cross-section view of a sparger assembly 1800 according to one embodiment of the present invention. Sparger assembly 1800 can include a sheet of penneable material 1805 and a sparger conduit 1810. Sparger assembly 1800 can be in fluid communication with a port 1820 of a container 1802 via sparger conduit 1810. As shown here, sheet of penneable material 1805 is circular in shape. An outer ring 1804 of sheet 1805 can eachbe anchored tou^ interior surface of acxmtainer
penneable material 1805, as coupled with container 1802, definea a dome-shaped space. Sparger assembly configurations such as those described heredn can allow the surface area and corresponding gas flow rate requirements of, for example, the penneable material 1805 tobeadjustedbyutifizingâifferentsize shapes such as the dome shown here. Some embodiments of the present invention may include a check valve inline coupled with a tubing mat is attached to the sparger conduit 1810, which can prevent fluid backflow.
[0128] flgure 19 illustrates a cross-section view of a sparger assembly 1900 according to one embodiment of the present invention. Sparger assembly 1900 can include a sheet of
permeable material 1905 and a sparger conduit 1910. Sparger assembly 1900 can be in fluid communication with a port of a container (not shown) via sparger conduit 1910. As shown bere, sheet of permeable material 1905 is circular in shape. .An outer ring 1904 of sheet 1905 can be coupled with sparger conduit 1910, such that the sheet of permeable material 1905, as coupled with sparger conduit 1910, defînes a dome-shaped space.
[0129] FIgnre 20 illustrates a parţial perspective view of a reactor system 2000 accordmg to one embodiment of the present invention. Reactor system 2000 can include a drive motor 2095 coupled with a drive shaft 2004. Reactor system 2000 can also include a fiame support 2097 coupled with drive motor 2095. In use, drive shaft 2004 can be coupled with a rotational assembly 2001 to mix or agitate the contents of a container (not shown) which is coupled with rotational assembly 2001. In some embodiments, rotational assembly 2001 is coupled with fiame support 2097 via a bracket (not shown). Fignre 21 illustrates a partial perspective view of a reactor system 2100 according to one embodiment of the present invention. Reactor system 2100 can include a drive motor (not shown) coupled with a drive shaft 2104. Reactor system 2100 can also include a frame support 2197 coupled with the drive motor. Drive shaft 2004 may închide or be in operative association with a drive shaft ear 2006 mat is configured to couple with a notch of a rotational assembly hub (not shown). Drive shaft ear 2006 is often used to transmit torque from the drive motor to the rotational assembly hub.
[0130] Fignre 22 illustrates a partial perspective view of a reactor system 2200 according to one embodiment of the present invention. Reactor system 2200 can mclude a drive motor 2295 coupled with a drive shaft 2204. In use, drive shaft 2204 can be coupled with a rotational assembly 2201 to mix or agitate the contents of a container (not shown) which is coupled wim rotational a«s«fflbly 2201. A clamp 2205 nîay also be coupled widi rotational assembly 2201. In thiş embodiment, drive motor 2295 includea a right angle geannotor, which can allow an operator to pass drive shaft 2204 through drive motor 2295 without moving the drive motor 2295. Embodinients that include right angle gear motors, parallel shaft gear motors, and hollow shaft motors can piovide enhanced aligmnent and ease of connection between drive motor 2295 and rotational assembly 2201. Fignre 23 illustrates a cross-section view of a reactor system 2300 accordmg to one embodiment of the present invention. Reactor system 2300 can include a drive motor 2395 coupled with a drive shaft 2304. Drive shaft 2304 may mclude or be coupled with a tapered element 2304a that is configured to associate with a corresponding receiving element 2395a of motor 2395.
Tapered element 2304a can provide enhanced aligmnent between drive shaft 2304 and drive motor 2395.
[0131] Flgure 24 illustrates a perspective view of a reactor system 2400 according to one embodiment of the present invention. Reactor system 2400 can include a container housing 2411 coupled with a support shelf 2413. Support shclf 2413 may be adapted for supportmg sensmgpn)be8.(notshown) and other clementa of a reactor system. Container housing 2411 can be coupled with a drive motor 2495 via a support frame 2497. Figare 25 illustrates a perspective view of a reactor system 2500 according to ane embodiment of the present invention. Reactor system 2500 can include a container housing 2511 coupled with a support shelf 2513. Container homang 2511 can be coupled with a drive motor 2595 via a support frame 2597.
[0132] Figare 26 illustrates a probe assembly 2600 according to one embodiment of the present invention. As seen here, probe assembly 2600 is in a retracted configuration, prior to engagement with a reactor container. Probe assembly 2600 can include a dissolved oxygen and pH probe 2610 and Pali Kleenpak connectors 2620 for providing an aseptic cormection. Probe assembly 2600 can also închide a port 2630, a sleeve 2640, and a coupler 2650, and these three components can facilitate fhe mtegration of probe 2610 into the reactor utilizing Pali connectors 2620. Ia same embodiment, port 2630 and female Pali cormector 2620f can be part of or integral with the reactor container (not shown). Sleeve 2640, coupler 2650, and male Pali cormector 2620m can be manufactured or provided to the user as a separate subassembly. The user caninstallthedcsired probe into such a subassembly and thencan sterilize the complete probe assembly. Port 2630, sleeve 2640, and coupler 2650 can facilitate mtegration of probe 2610 into a bioreactor using Pali connector 2620.
[0133] Flgure27Aprovid^esamustrati(mofaprobeportBubassanbly2702ofaim)bc assembly according to one embodiment of fhe present mvention. Probe port subassembly 2702 can include a bioprocessing container port 2730 coupled with female Pali connector 2620f. Port 2730 may be, for exemple, heat welded into a container (not shown) via flange plane 2734. Port 2730 may also include a lip seal 2732 mat can prevent backflow of fluid or material from container into probe assembly or beyond flange 2734 plane. Insome embodiments, port 2730 and female Pali cormector 2620f are constructed integrally with me conismcr.

[0134] Figure 27B ilfastrates a probe kit subassembly 2704 of a probe assembly according to one embodiment of the present invention. Probe kit subassembly 2704 can include a coupler 2750, a sleevo 2740, and a male Pali connector 2620m. Probe kit subassembly 2704 may be supplied to an end user as a separate kit. Sleeve 2740 may be coupled with coupler 2750 via a barb fitting (not shown) of coupler 2750. Similarly, sleeve 2740 may be coupled with male Pali connector 2620m via a barb fitting (not shown) of male Pali connector 2620m.
[0135] Fignre 27C iliustrates an autoclave subassembly 2706 of a probe assembly according to one embodiment of the present invention. Autoclave subassembly 2706 can include a probe 2710, coupler 2750, sleeve 2740, and male Pali connector 2620m. An end user can install the desired probe 2710 into a probe kit subassembly 2704 as descrîbe above, and sterilize the resulting autoclave assembly 2706. Afier sterilization, the user can join the male Pali connector 2620f and the female Pali connector 2620f, and complete the probe engagement into the fluid stream. In some embodiments, sleeve 2740 is a flexible member that can collapse and allow probe 2710 to be displăcea, and coupler 2750 can provide an interface between sleeve 2740 and probe 2710.
[0136] Bîgure 28A illustrates a probe assembly 2800 according to one embodiment of the present invention. Probe assembly 2800 includes probe 2810, coupler 2850, sleeve 2850, male Pali connector 2820m, female Pali connector 2820& and port 2830. Probe assembly 2800 is shown in a firet connected configuration, wherein probe assembly is engaged with container, but the probe is not yet mtroduced mto the fhud stream. Figure 28B iliustrates a probe assembly according to one embodiment of the present invention, wherein probe assembly 2800 is in a second connected configuration such that sleeve 2840 is collapsed and
w
a distal end of probe 2710 is introduced into the fluid stream of the container. C. Cultares
[0137] The stured^ank reactor system cm bededgnedto ho
biologica! fluid, a cell culture medium, a cutare of microorganisms, a food production, or the
like. WhenmefhudicmediumisaceUcutturemesyste^
batch-mc a large scale cell culture in which a cell inoculum is cultured to a maximum density in a tank
orfermenter, andharvestedandprocessedasabatch. A fed-batch culture can bea batch
culture which is supplied with either ftesh nutrients (e.g„ growm-tirniting substrates) or
additives (e.g., precursors to products). A continuous culture can be a suspension culture that
is continuously supplied with nutrients by the inflow of fresh medium, wherein the culture volume is usually constant. Similarly, continuous fermentation can refer to a. process in which cells or micro-organisms are mamtained in culture in the exponenţial growth pbase by the continuous addition of fresh medium that is exactly balanced by the removal of cell suspension fiom the bioreactor. Furthermore, the stirred-tank reactor system can be used for suspension, perfusion or microcanier cultures. Generally, the stirred-tank reactor system can be operated as any convenţional stirred-tank reactor with any type of agitator such as a Rushton, hydrofoil, pitched blade, or marine. With reference to Figure l, the agitation shaft 112 can be mounted at any angle or position relative to the housing 111, such as upright centered, upright oflset, or 15° ofisct. The control of the stirred-tank reactor system can be by convenţional means without me nced for steam-in-place (ŞIP) or clean-in-place (CIP) control, hi fact, the system of the instant mvention is not limited to sterile bioreactor operation, but can be used in any operation in which a clean product is to be mixed using a stirred tank, for example, food production or any clean-room mixing without the need for a clean-room.
D. TheKit
[0138] The mvention encompasses a kit that includea a stirred-tank reactor system and instructions for use. In one embodiment, the kit includes a disposable stirred-tank reactor system. Accordingly, the kit includes at least one disposable element such as the bag, Ihe shaft, the impeller, or the bearing. The kit can be entirely disposable. The flccrible, disposable bag may be affixed to Ihe shaft and the bearing through at least one seal or o-ring such that Ihe inside of the bag remains sterile. In addition, the bag may include a pH sensor and a dissolved-oxygen sensor, wherein the sensors are incorporated into the bag and are disposable with the bag. The Mt may alao Mode o^^^
sealedtothebag. The pouch has one end that can beopenedtonieoutside of the bag such that a probe can be inserted into me reactor. Theprobemaybeatemperatureprobe, apH probe, a dissolved gas sensor, an oxygen sensor, a carbon dioxide (CO2) sensor, a cell mass sensor, a nutrient sensor, an osmometer, and the like. Furthennore, the system may include at least one port in the bag allowing for the connection of a device to me port, wherein the device includes, but is not limited to, a tube, a filter, a sampler, a probe, a connector, and the like. The port allows for sampling, titration, adding of chcmostat reagents, spargmg, and the like. The advantage of this kit is that it is optionally entirely disposable and easy-to-use by following the attached instructions. This Mt comes in diflferent sizes depending on the
preferred culture volume and can be employed with any desired reaction chamber or barrel. This kit is pre-sterilized and requires no validation or cleaning. The kit can be used for cell culture, culture of microorganisms, culture of plant metabolites, food production, chemical production, biopharmaceutical production, and others.
[0139] In another embodiment the kit includes a housing or barrel that holds the disposable bag. Such a housing or barrel can be supplied with the bag or provided separately.
E. Examples
[0140] The following specific examples are intended to ilhistrate the invention and should not be construed as limiting the scope of the claims.
[0141] (1) A Dfeposable Bioreactor
[0142] One example of a stirred-tank reactor aystem of the instant invention is a disposable bioreactor, or single use bioreactor (SUB). The bioreactor is similar to a 250 liter media bag with built-in agitatum and attachable sensors (e.g., pH sensors, temperature sensors, dissolved oxygen (dO2) sensors, etc.). The reactor is operated via convenţional controllere. The agitator (e.g., agitation shaft and impeller) and bearing are disposable and built into the bag. The motor attaches to a support (e.g., motor and bearing support) or bracket(s) on the 250 liter barrel that holds the bag. Insize, shape, and operation, this bioreactor appears similar to a stamless steel reactor wim a sterile liner, however, the bioreactor of tins invention provides a multitude of advantages compared to a convenţional atainless steel reactor. It can be appreciated mat the size and volume of such media bags can be scaled both upward and downward^accontingtoindustryneeds.
Most importantiy, theneadfbr cleaniog and steam sterilization is eliminated. The bag is I>re-sterili7^by iffadiatiOtt and, tiius, ready foruse. In fect, no cleaning, sterilization, validation or tcsting is required at culture start-up or between culture runs. Consequently, the bioreactor provides a culture environment of zero cross-contamination between nins. In convenţional systems, the majority of costs are related to clean-in-progress (CIP) and steam-in-progress (ŞIP) as well as fee design of a sMd and control system to oversee these functions. These costs are eliminated in the disposable bioreactor and multiple producte may be cultured or manu&ctnred simultaneousiy and with much greater ease.
[0143] The disposable bioreactor can be easily scaled-up by using larger culture bags and larger barrels to hold the bags. Multiple bioreactors can be operated at the same time without
any need for extensive engjneering or cleaning. The bioreactor is a truc stiired tank with wcll characterized mixing. As such, the bioreactor has the added advantage that it can be scaled and its contents transferred to a stainless steel reactor if desired. Notably, the bioreactor combines ease of usc with low cost and flexibility and provides, thus, a new technical pktform for cell culture.
[0144] (2) CeU Culture
[0145] The disposable bioreactor of the instant invention can be used for a batch culture in which cells are inoculated into fresh media. As the cells grow, they consume the nutrients in the media and waste producte accumulate. For a secreted product, when me culture has run its course, cells are separated from the product by a filtration or centrifugation step. For viral-vector production, cells are infected with a virus during the growth phase of the culture, allowing expression of the vector followedbyharvest Smce mere is zero cross-contamination in the bioreactor it works well with batch cultures.
[0146] The bioreactor can also be used for perfusion cultures, wherein product and/or waste media is continuously removed and the volume removed is replaced with fresh media. The constant addition of fresh media, while elmiinating waste products, provides the cells with the nutrients they require to achieve higher cell concentrations. Unlike the consfantly changing conditions of a batch culture, the perfusion method ofiers the means to achieve and maintain a culture in a state of equiHbrium in which cell concentration and productivity may be maintained in a steady-state condition. This can be accomplished in me disposable bag as easily as in any convenţional stainless steel reactor. For viral- vector production, the perfusion process allows for an increase in the cell concentration and, thcreby the post-infection virus titer. For a secreted product, periMon m me bioreactor offere opportunity to increase flie productivity by sin^lyincreasmgttiesiaaofmeOTlturebag. Most importantly, there is no need for sterilization, validation, or cleaning because the system experiences zero cross-oraiamination during the production process.
[0147] (3) Batch Date l
[0148] Figure 29 provides a graph of data that was generated using a reactor system according to one embodiment of the present invention. Human embryonic Mdney (HEK) 293 cells m 200 Utere of CDM4 culture medium were incubated in a 250 h'ter capacity reactor system. Among other parameters shown in the graph, the viable cell density of me reactor system culture increased for about the first 14 days of the batch run.
[0149] (4) BatchDafa2
[0150] FIgures 30-34 illustrate data obtained fiom a single use bioreactor system for mammalian cell culture according to one embodiment of the present invention. The scaleable mass transfer characteristics of the single use stirred tank bioreactor are descrîbed. Cell growth and metabolism, antibody production, and antibody characterization data from batoh culture using a 250-liter ptototype system are presented and compared to results from a tradiţional stainless-steel bioreactor of similar scale.
[0151] Materials and Methods - Mixing Studies. Mixing time in the bioreactor was estimated at various agjtation rates by tracking the change in pH in the reactor over time in response to addition of a base solution. The reactor was filled to worldng volume of 250 h'tera with typical cell culture media. At time zero, 500 ml of IN NaOH was added at the top of the reactor, and a combined pH glass electrode was used to measure pH fiom time zero until the pH hâd stabilized. The pH versus time was plotted, and the time reqirired to reach 95% of the final pH was estimated from the graph.
[0152] Key scde-up parameters were determined using standard calculations that have been well established in the chemical and pharmaceutical industry.
[0153] The mixing Reynolds number, NRB is the ratio of fluid kmetic and inerţial forces and is used to determine the mixing regune, either laminar or turbulent:
[0154] NRB = NDj2p/ji
[0155] The energy input into the reactor, P0) per volume of the reactor, V, relates to the scale at wMch fluid mixing and mass transfer occurs and is dependent on the impeller power number, Np:
[0156] Po/V^NppNW/V
[0157] The impeller power number depends on the design of the impeller and is a fimction of number of blades, blade width, and blade pitch. Np is also a fimction of the clearance of the impeller fiom the sides and bottom of the reactor. For various impeller types, the power number is well documented.
[0158] Tip speed of the impeller, \\, relates to the fluid shear stress in the vicinity of the unpeller:
[0159]
[0160] In the above equations, N = impeller rotational speed, D{ = impeller diameter, p = fluid density, and \i = fluid viscosity.
[0161] Materials and Methods - Oxygen Transfer Studies. The volumetric oxygen transfer coefficient, kLa, was estimated at various agitation and spargjng rates by tracking the change in dissolved oxygen, dOz, concentration over tinie at the appropriate condition. The reactor was fîlled to me working volume of 250 litera with typical cell culture media, and a dCfe sensor was InstaUed in the reactor. To prepare for each experiment, nitrogen was sparged mrough the bioreactor until the dOz concentration dropped below approximately 20% saturation with air. For each experiment, the agitation rate was set, and then air was sparged at the desired rate. The dOj concentration was measured versus time until it reached approximately 80% saturation with air. The value of k^a can be estimated from a graph of CL versus dQ/dt, based on the following mass balance equation:
[0162] dCi/dt - kta(C -
[01 63] where CL is the concentration, and C is the equilibrium value for CL.
[0164] Materials and Methods - Cell Culture Procedures. A cell culture process that hâd been developed for a tradiţional stainless-steel reactor of 300-liter working volume was used to demonstrate the perfonnance of the single use bioreactor. The cell line, media, and process parameters that hâd been demonstrated m the tradiţional reactor wererepeated in the single use reactor.
[0165] ITiecellsusedwereCHOwllsexpressingahiimankedmonoclonalantibody. Cells were thawed and maintained m T-flasks using standard methods. Cells were then expanded fram T-flasks into custom 1-liter expansion baga prior to being introduced into a tradiţional stainless-steel 1 10-Kter inocula bioreaotor. Oace wllsreaoiied a concentration of 1.6 x ^ cells/ml, 45 litera from me traditional 1 1 0-h'ter bioreactor were used as inocula for the single use bioreactor. Thus, exponentially growing cells from a controlled bioreactor at a pre-determined cell concentration were provided as inocula for the single use bioreactor.
[0166] A standard, commercially available, chemically defined cell culture medium was used. At a spccified point in the batch culture, a commercially available nutrient feed that is of non-animal origin but is not chemically defined was added. Solutions of D-glucose and L-glutamine were added daily as required during the batch culture to a concentration
of D-glucose between l and 3 mg/Iitcr and a concentration of L-glutamine between l and 3 mMol/liter throughout the batch.
[0167] Control of the single use bioreactor was accomplished using standard, industry-acceptcd sensors and controllers. The temperature, pH, and dOi feedback controllere operated using proporţional, integral, and differential (PJD) control. Temperature was measured by a platinum resistance thermometer inserted in a thermo well in the reactor, and was controlled at 37 °C via a electric beat jacket. The pH was measured using a combined pH glass electrode that was in direct contact with the bioreactor contents. The pH was controlled at a value of 7. l via addition of CCfe into the headspace or addition of IM Na2CO3 to the culture. The dOj concentration was measured using a dQz sensor that was in direct contact with the bioreactor contents. The dOj concentration was controlled at 30% saturation with air via sparging of 62 at approximately 0.2 liters/min. Agitation was not controlled by feedback but was rnaintained at a single set point of 110 rpm and checked daily. Level in the bioreactor was measured using a weigh scale.
[0168] A sampling system was attached to the bioreactor using a sterile connection device, and was used to withdraw 10-ml samples as required during the batch culture. Samples were withdrawn at least once daily. Samples were immediately analyzed using a Nova BioProfîle 200 analyzer, which provided culture pH, dQz, dCOît D-glucose, and L-glutamine concentrations. The pH probe was standardized, as required, and D-glucose and L-glutamine solutions were added based on the Nova measurements. Viable and total cell concentrations were determined for each sample based on hemacytometer counts using trypan blue dye exclusion. Samples were fîltered through a 0.2 um fîlter and stored for later analysis using an Igen based assay for antibody titer.
[0169] Key cell culture paramcters wewcalciu^ted based on
Maximum viable cell concentration, cumulative cell timeatharvest, final antibody
concentration, and total glucose and glutamine consumed were calculated directly from the
sample data. As a batch culture, me specific growth rate of the cells, |i, was determined for
only the exponenţial phase of the culture. Specific growth rate was calculated from a
regression fit of viable cell concentration, Xy, fiom days one mrough fbur fbllowing
inoculation:
[0170]
[0171] Results fiom a series of batch culturcs using a tradiţional stainless-steel bioreactor of similar scale were available for comparison with the single ase results. The ranges of values tabulated for the tradiţional bioreactor are the 95% prediction intervals for a single future observation:
[0172] x™»* Wi • s v(l + (l/n))
[0173] wherc x^m = sample mean, s = sample standard deviation, n = sample size, and to^n-i is the appropriate Studenfs t-statistic.
[0174] The single ase bioreactor supematant was harvested, clarifîed by filtration and purifîed (protein A-based affiniry purification combined with ion exchange chromatography) using the procedures established for the tradiţional stamless bioreactor manufacturing process. The resultant purified antibody was characterized and compared to antibody derived fiom the tradiţional stamless steel process. Carbohydrate (CHO) profile, SDS-PAGB (reduced and non reduced), SEC-HPLC, SEC-MALS (Multi-Angle Light Scattering), BIACore Binding, RP-HPLC, Capillary Electrophoresis Isoelectric Focusing (CEIEF) and MALDI-TOF Mass Spectrometry assays were utilized to characterize Ine purified antibody derived fiom the single use bioreactor. The results obtained were compared to those seen for antibody produced in a tradiţional stainless steel bioreactor.
[0175] Results - Mîxing Studios. Thotimerequiredtoreach95%homogeneitydecreased with increasing agitation speed. Each experiment was repeated twice, and the average mixing times are shown in Table 1.
(Table Removed)

[0176] In addition, key scale-up parameters for the single use bioreactor could be readily 0|lculated. The single use bioreactor was designedusmgdesigncriteriaforatypicalfitirred tank bioreactor, and me impeller was a typical pitehed-blade design, as shown in Table 2. In Ihe âbsence of bafQes, vortex formation in tbe reactor was avoided by mounting the impeller at an offeet fiom center and at a 20° angle fiom vertical
(Table Removed)
[0177] Using the power number fiom Table 2, characteristic scale-up parameters can be readily calculated for various agitation speeds, as listed in Table 3.

(Table Removed)
[0178] Results-Oxygen Transfer Studios. The volumetric oxygen transfer coefficient, kta was determined for various flowrates of air througb the sparger and for various agitation speeds, shown in Figure 30. As expected, kLa increased with increasing air flowrate and wim increasing agitation speed, with one exception. At 200 rpm, kua was lower than fhat at 100 rpm. Tbis discrepancy may be due to an increased surface effect on kta at the higher agitation rate. (Due to the experimental procedure, the beadspace contained a mixture of nitrogen and air.) Futtfaer experimenta are rarairedtoqura
[0179] These results are oomparable, as expected, wim oxygen transfer characteristics of tradiţional stinedta^Woreactors of me same geometry. Atypicalliteramrevahiefisrthe equilibrium oxygen concentration in cell culture media is 0.18 mMol/liter, and specific oxygen uptake rate fac typical animal cell culture is 0.15 mMol/109 cells/hr. pperated in the middle of the range fiom fhe above chart (agitation = 100 rpm; sparge rate =1.0 literAnin; kLa «10 hr"1) the smgle usc bioreactor is calculated to be capable of maintaming cell concentrations greater than lOx IO6 cells/ml using air as the sparge gas and greater than 50 x IO6 cells/ml using oxygen as the sparge gas.
[0180] Results -BatchCell Culture. To demonstrate the suitability of mesingleuse bioreactor for cell culture production, CHO cells producing a hnmanized monoclonal
antibody were grown in batch culture and compared to historical results firom the same cell line and process carried out in a tradiţional stainless steel bioreactor of similar scale. This process has been repeated five times in a 300-liter Abec tradiţional stainless steel reactor that is specifically designed for cell culture. Key cell culture parameters firom the two reactors are compared in Table 4.

(Table Removed)
[0181] The single use bioreactor was an iniţial prototype. Asaprototypebeingusedforthe firet tune, adjustments to the controller PID parameters were made several times during the batch culture. Temporary excursions in pH, dO2 Concentration, sparger flowrate, and agitation speed occurred at times during the batch duc to these adjustments. Despite these excursions, results fixnn this bioreactor are equivalent to results from the tradiţional stainless steel bioreactor. Graphs of the pH,d02, and dCO2 Concentration firom off-linesamples measured-by the Nova analyzer are shown in Fignre 31.
[0182] Detailedreaultsfixm the single use bi(^^
The single use bioreactor was inoculated at 0.33 x IO6 cells/mL and reached a mffirWm cell density of 7.6 x IO6 cells/mL. Viability remained above 90% during the growth portion of the batch curve. Total and viable cell Concentration and percentviability are shown in Fignre 32.
[0183] Antibody titer over tune, as a percent of final titer at harvest, is shown in Figure 33. As is typical for this cell line, approximately 50% of the antibody was produced in the second half of the batch as the cell Concentration was declining.
[0184] Cumulative glucose and glutamine consumption is shown in Flgure 34. Glucose and glutamine consumption for the single use bioreactor was comparable to historical results from the tradiţional stirred tank bioreactor.
[0185] A summary of the assay results is contained in Table 5. In all cases, the antibody derived fiom the single use bioreactor showed equivalent results to that produced in the tradiţional stainless steel bioreactor.

(Table Removed)
[0186] Various modifications and variations of the present invention will be apparent to those skilled in the art without depardng fixed the scope and spirit of the invention. Although the invention bas been described in connection wtth specific preferred embodiments, it should be understood mat the invention as clauned should not be unduly limited to to such specific embodiments. Jndeed, various modifications of fhe described modes for carrying out the invention which are obvious to those skilled in the art are intended to be within the scope of the claims








We Claim:
1. A reactor system comprising:
a container (302) comprising a flexible bag and having an interior surface bounding a
compartment; characterized by,
a rotational assembly (30 1, 401, 50 1, 60 1, 701) in sealed cooperation with an opening of
the container, the rotational assembly comprising a casing (360, 460, 560, 660, 760) mounted to
the flexible bag and a hub (320, 420, 520, 620, 720) rotatably mounted to the casing, the hub
having a passageway (320a) extending therethrough; and
a drive shaft (304, 704, 804, 904, 1004, 1104, 1304, 2004, 2204, 2304) removably
received within the passageway of the hub so as to extend into the compartment of the container,
the drive shaft engaging the hub so that rotation of the drive shaft facilitates rotation of the hub
relative to the casing.
2. The reactor system as claimed in claim 1, comprising an impeller (340, 440, 1040, 1040a,
1040b, 1140) coupled with the rotatable hub, the impeller being disposed within the container
and adapted to couple with a distal end of the drive shaft.
3. The reactor system as claimed in claim 1, wherein the rotational assembly comprises a
bearing assembly (370, 470, 570, 670, 870, 970) disposed between the casing and the rotatable
hub.
4. The reactor system as claimed in claim 3, wherein the rotational assembly comprises a
sealing arrangement (380, 480, 580, 680, 880, 980) disposed circurnferentially to the rotatable
hub, between the rotatable hub and the casing.
5. The reactor system as claimed in claim 4, wherein the bearing assembly comprises a
plurality of race bearings, and the sealing arrangement comprises a rotating disk (450) coupled
with the rotatable hub, a .wear plate (382, 482) coupled with the casing, and a dynamic seal
disposed between the rotating disk and the wear plate.
6. The reactor system as claimed in claim 5, wherein the dynamic seal comprises two or
more seal subunits disposed in co-planar arrangement.
7. The reactor system as claimed in claim 4, wherein the bearing assembly comprises a
journal bearing, and the sealing arrangement comprises a wear plate coupled with the rotatable
hub, and a dynamic seal disposed between the casing and the wear plate.
8. The reactor system as claimed in claim 7, wherein the dynamic seal comprises two or
more seal subunits disposed in co-planar arrangement.
9. The reactor system as claimed in claim 2, wherein the impeller comprises a spline (342,
1042, 1342) adapted to couple with the drive shaft.
10. The reactor system as claimed in claim 2, wherein the rotatable hub is coupled with the
impeller via a flexible tube (390, 490, 890, 990, 1090, 1190, 1390), the drive shaft being
disposed within the flexible tube and being coupled with the impeller.
1 1. A reactor system as claimed in claim 1, comprising:
an elongated tubular connector (390, 490, 890, 990, 1090, 1190, 1390) disposed within
the compartment of the flexible bag, the tubular connector having a first end and an opposing
second end, the first end of the tubular connector being secured to the hub;
an impeller (340,440, 1040, 1040a, 1040b, 1 140) disposed within the compartment of the
flexible bag and secured to the second end of the tubular connector; and
the drive shaft (304, 704, 804, 904, 1004, 1104, 1304, 2004, 2204, 2304) removably
received within the hub and the tubular connector such that rotation of the drive shaft facilitates
rotation of the impeller, hub, and connector.
12. The reactor system as claimed in claim 11, wherein the drive shaft directly engages the
hub.
13. The reactor system as claimed in claim 1 1, wherein the drive shaft is linear.
14. The reactor system as claimed in claim 11, wherein the tubular connector comprises a
flexible tube.
15. A method for preparing a reactor system as claimed in claim 1, the method comprising:
coupling the casing (360, 460, 560, 660, 760) of the rotational assembly (301, 401, 501,
601, 701) to a frame bracket (308), the rotational assembly comprising the casing secured to the
container (302) and the tubular hub (320,420, 520,620,720) rotatably secured to the casing, the
container comprising a flexible bag;
placing the container at least partially within a flame housing (1 1 1,24 1 1,25 1 1);
inserting the drive shaft (304, 704, 804, 904, 1004, 1104, 1304, 2004, 2204, 2304)
through the hub of the rotational assembly,
coupling a distal end of the drive shaft to ai impeller (340, 440, 1040, 1040a, 1040b,
1140), the impeller being disposed within the container and coupled with the hub;
introducing a reaction component into the container via a port; and
rotating the drive shaft so as to rotate the impeller and the hub, the impeller mixing the
reaction component within the container.
16. The method as claimed in claim 15, comprising passing the distal end of the drive shaft
through a flexible tube (390, 490, 890, 990, 1090, 1 190, 1390) extending between the hub and
the impeller before coupling the distal end of the drive shaft to the impeller.

Documents:

5012-DELNP-2006-Abstract-(23-04-2010).pdf

5012-delnp-2006-abstract.pdf

5012-DELNP-2006-Claims-(21-02-2012).pdf

5012-DELNP-2006-Claims-(23-04-2010).pdf

5012-delnp-2006-claims.pdf

5012-delnp-2006-Correspondence Others-(13-08-2012).pdf

5012-DELNP-2006-Correspondence Others-(21-02-2012).pdf

5012-delnp-2006-Correspondence Others-(23-07-2012).pdf

5012-DELNP-2006-Correspondence-Others-(09-12-2009).pdf

5012-DELNP-2006-Correspondence-Others-(23-04-2010).pdf

5012-delnp-2006-correspondence-others-1.pdf

5012-delnp-2006-correspondence-others.pdf

5012-DELNP-2006-Description (Complete)-(21-02-2012).pdf

5012-DELNP-2006-Description (Complete)-(23-04-2010).pdf

5012-delnp-2006-description (complete).pdf

5012-DELNP-2006-Drawings-(21-02-2012).pdf

5012-DELNP-2006-Drawings-(23-04-2010).pdf

5012-delnp-2006-Drawings-(23-07-2012).pdf

5012-delnp-2006-drawings.pdf

5012-DELNP-2006-Form-1-(23-04-2010).pdf

5012-delnp-2006-form-1.pdf

5012-delnp-2006-form-13-(09-12-2009).pdf

5012-DELNP-2006-Form-13-(21-02-2012).pdf

5012-delnp-2006-form-18.pdf

5012-DELNP-2006-Form-2-(23-04-2010).pdf

5012-delnp-2006-form-2.pdf

5012-delnp-2006-Form-3-(13-08-2012).pdf

5012-DELNP-2006-Form-3-(23-04-2010).pdf

5012-delnp-2006-form-3.pdf

5012-delnp-2006-form-5.pdf

5012-DELNP-2006-GPA-(23-04-2010).pdf

5012-delnp-2006-GPA-(23-07-2012).pdf

5012-delnp-2006-gpa.pdf

5012-delnp-2006-pct-101.pdf

5012-delnp-2006-pct-210.pdf

5012-delnp-2006-pct-220.pdf

5012-delnp-2006-pct-237.pdf

5012-delnp-2006-pct-304.pdf

5012-DELNP-2006-Petition-137-(23-04-2010).pdf


Patent Number 258956
Indian Patent Application Number 5012/DELNP/2006
PG Journal Number 08/2014
Publication Date 21-Feb-2014
Grant Date 18-Feb-2014
Date of Filing 31-Aug-2006
Name of Patentee BAXTER INTERNATIONAL INC.
Applicant Address ONE BAXTER PARKWAY, DEERFIELD, IL, 60015, U.S.A.
Inventors:
# Inventor's Name Inventor's Address
1 KURT T. KUNAS 1134 MATARO COURT, PLEASANTON, CA 94566, U.S.A.
2 ROBERT V. OAKLEY 1011 BROWN AVE., #17, LAFAYETTE, CA 94549, USA
3 FAUAD F. HASAN 3371 CABRILLO AVENUE, SANTA CLARA, CA 95051, USA.
4 MICHAEL E. GOODWIN 1355 RED ROX TRACE, LOGAN, UT 84321, USA
5 JEREMY K. LARSEN 327 NORTH HAMMOND LANE, PROVIDENCE, UT 84332M USA.
6 NEPHI D. JONES 162 SOUTH 400 WEST, NEWTON, UT 84327, USA
PCT International Classification Number C12M 1/12
PCT International Application Number PCT/US2005/013920
PCT International Filing date 2005-04-22
PCT Conventions:
# PCT Application Number Date of Convention Priority Country
1 60/565,908 2004-04-27 U.S.A.