US 20070094926 A1
A bioreactor assembly of the present invention for holding a media and supporting growth of a plurality of plants. The assembly includes a light source and a container having a light transmissive wall structure and defining a reservoir. A major axis of the reservoir is substantially horizontal allowing the reservoir to be filled with media to a partial level and to define a relatively large surface area for support of the plants. The assembly may also include clamps to secure and seal separate wall structure portions of the container together, and end caps to the wall portions, to define an aseptic environment. As another option, the clamps may define openings therethrough that allow passage of various devices for measuring and controlling bioreactor function such as a gas supply nozzle, a gas exit nozzle, an air temperature probe, a pH probe, a sampling drain, a gas composition probe and a media temperature probe.
1. An assembly for holding a media and supporting growth of a biological material requiring light for proliferation, said assembly comprising:
at least one light source; and
at least one container positioned adjacent the light source, said container having a flexible light transmissive wall structure defining an elongate, aseptic reservoir, said elongate reservoir having a major axis with a substantially horizontal orientation wherein said reservoir is capable of being partially filled with media so as to create a media surface on which the biological material is supported.
2. An assembly according to
3. An assembly according to
4. An assembly according to
5. An assembly according to
6. An assembly according to
7. An assembly according to
8. An assembly according to
9. An assembly for holding a media and supporting growth of a biological material requiring light for proliferation, said assembly comprising:
a support rack;
a plurality of elongate laterally extending containers carried by said support rack and arranged in a stack spaced apart vertically from one another, each container having a flexible light transmissive wall structure defining an elongate, aseptic reservoir, said elongate reservoir having a major axis with a substantially horizontal orientation wherein the reservoir is capable of being partially filed with media so as to create a media surface on which the biological material is supported; and
at least one light source carried by said support rack and mounted adjacent to said containers for illuminating the containers.
10. An assembly according to
11. An assembly according to
12. An assembly according to
13. An assembly according to
14. A method of growing in a liquid media a biological material requiring light for proliferation, said method comprising:
providing at least one flexible light transmissive container defining a reservoir having a major axis with a substantially horizontal orientation;
filling the reservoir with the liquid media until a partial fill level is reached so as to define a top surface of the media extending along a length of the reservoir;
adding the biological material to the reservoir and supporting the biological material on the top surface of the media; and
exposing the container to a light source so as to promote growth of the biological material via photosynthesis.
15. A method of
16. A method of
17. A method of
18. A method of
19. A method of
20. A method of
21. A method of
22. A method of
23. A method of
24. A method of
25. A method of
26. A method of
27. A method of
This application is a continuation of copending U.S. patent application Ser. No. 10/845,914, filed on May 14, 2004, which is hereby incorporated herein in its entirety by reference.
1. Field of the Invention
The present invention is related to the use of bioreactors for holding media and promoting the growth of biological materials, and in particular transparent bioreactors for growing biological materials requiring a light source, such as aquatic plants.
2. Description of Related Art
Photo-bioreactors are devices that allow photosynthetic microorganisms to grow in a controlled manner. U.S. Pat. No. 5,846,816 to Forth (“Forth”) discloses a biomass production apparatus including a transparent chamber 10 which has an inverted, triangular cross-section, as is shown in
The passage of air entering through the conduit establishes a distinctive flow pattern that causes the liquid in the chamber to circulate up through a central region of the chamber, across the upper portion of the chamber below a cover 16, and down along the chamber sidewalls 20 back to the conduit, as is shown in
Although the bioreactor disclosed by Forth promotes the growth of biological matter, it is generally not useful for applications requiring a sterile growth environment. The vents are open to external air which may include airborne contaminants. Such contaminants are especially troublesome for pharmacological applications wherein strict Food and Drug Administration guidelines for avoiding contamination must be met.
In addition, the constant circulation of the liquid required by Forth interferes with the growth of some types of biological matter. For instance, fully differentiated aquatic plants from the lemnaceae or “duckweed” family are fresh-water plants that grow best on the surface of the water. Such surface growing plants typically prefer relatively still water to support and promote optimal growth.
Therefore, it would be advantageous to have a photo-bioreactor system for promoting the growth of plant biological materials in a relatively sterile environment isolated from contaminants. It would be further advantageous if the system were capable of promoting growth of surface growing aquatic plants, such as the duckweed family of plants.
The above advantages are achieved and other needs addressed by a bioreactor assembly of the present invention for holding a media and supporting growth of a plurality of plants. The assembly includes a light source and a container having a light transmissive wall structure allowing light to pass therethrough and defining a reservoir filled with media and biological material. A major axis of the reservoir is substantially horizontal allowing the reservoir to be filled with media to a partial level and to define a relatively large surface area for support of plants and other biological materials that prefer such support. The assembly may also include clamps to secure and seal separate wall structure portions of the container together and end caps secured to the wall portions to maintain an aseptic environment. As another option, the clamps may define openings therethrough that allow passage of various devices for measuring and controlling bioreactor function such as a gas supply nozzle, a gas exit nozzle, an air temperature probe, a pH probe, a sampling drain, a gas composition probe and a media temperature probe.
In one embodiment, the present invention includes an assembly for holding a media and supporting growth of a plurality of plants. The assembly includes at least one light source and at least one container positioned adjacent the light source. A wall structure of the container has light transmissive properties to allow passage of light from the light source and in combination with other components defines a reservoir closed to the ambient environment. The reservoir has an elongate shape that defines a major axis generally extending in the direction of its longest dimension. In the assembly, the container is oriented so that the major axis of the reservoir is in a substantially horizontal plane with respect to gravity. In this manner, the reservoir, when partially filled with the media, creates a relatively large media surface on which the plants are supported.
In one aspect, the wall structure has an extruded shape with a constant cross-section. For instance, the wall structure may have a cylindrical, oval or rectangular cross-section. Preferred dimensions for the pipe wall structure range between 10 and 50 feet in length and between 2 and 12 inches in diameter. In cross-sections with a major axis, the major axis is preferably aligned with the substantially horizontal plane to further maximize the media surface area. For instance, two opposite corners of the rectangular cross-section could be positioned closer to the substantially horizontal plane than the remaining two corners.
In another aspect, a plurality of the containers may be used wherein the containers are arranged in a vertical stack with spacing between each of the containers. The vertical stack may be combined with electrically powered lights, such as light-emitting diodes or fluorescent lights as the light source. Light is supplied to the vertical stack by positioning the lights on both sides of the stack and potentially in the space between the containers.
Also included in the container may be one or more clamps for holding multiple portions of the wall structure together. The clamp may include one or more openings for the insertion of various sampling and control devices, such as a gas supply nozzle, a gas exit nozzle, an air temperature probe, a pH probe, a sampling drain, a gas composition probe and a media temperature probe extending into the reservoir through an opening defined in the clamp.
In another aspect, the clamp defines an opening that is sized and shaped to receive adjacent ends of the wall structure portions. For instance, the clamp may include a central band sized to extend around the clamp ends. Optionally, the clamp may have a pair of inwardly directed flanges spaced apart on opposite ends of the clamp wherein the flanges are configured to grip the ends of the wall structure portions. To facilitate gripping, the ends of the wall structure portions may flare outwardly to engage the inwardly directed flanges. Preferably, each of the clamps is constructed of an FDA approved composite material and includes a silicone seal to block contaminates from entering the container.
In an alternative embodiment, the container wall structure may define a closed reservoir having at least two spaced-apart portions each having a major axes. The major axes of the spaced apart portions lie in a common, substantially horizontal plane. In this manner, partially filling with media at one of the portions also partially fills the remaining portions and creates a media surface on which the plants are supported.
The present invention has many advantages. Overall, the bioreactor assembly allows the production of clinical and commercial scale quantities of biopharmaceuticals from genetically modified plants in a controlled, sterile and clean environment. For example, the use of containers defining reservoirs for partial filling with media provides a relatively large surface for the large-scale production of surface-borne biological materials, such as duckweed plants. In addition, use of the clamps having seals to interconnect the various portions of the container wall structure and sealed openings for insertion of various measurement and supply devices ensures a clean and aseptic environment to promote the growth of the biological materials for medical uses. The clamping system also allows for easy assembly and disassembly of the containers for maintenance and modification. The measurement and supply devices ensure that the environment within the reservoir is tightly controlled to maximize growth and expression of the biological materials therein.
Having thus described the invention in general terms, reference will now be made to the accompanying drawings, which are not necessarily drawn to scale, and wherein:
The present invention now will be described more fully hereinafter with reference to the accompanying drawings, in which some, but not all embodiments of the invention are shown. Indeed, the invention may be embodied in many different forms and should not be construed as limited to the embodiments set forth herein; rather, these embodiments are provided so that this disclosure will satisfy applicable legal requirements. Like numbers refer to like elements throughout.
A bioreactor assembly 10 of one embodiment of the present invention is shown in
The term “media” as used herein refers to any liquid, gel, partially liquid-partially solid, or otherwise flowable supply of compounds, chemicals or nutrients that are used to promote the growth, testing, modification or manipulation of the biological matter housed within the reservoir 15. Media therefore, can be water alone, a combination of water with fertilizer, soil, an agar gel, mud or other combination of components, with or without water, as long as some type of flow and manipulation of the components can be induced using the devices described herein.
The term “biological materials” or “biological matter” as used herein describe any material that requires light and a supply of media in order to support proliferation or expression. Preferably, the biological materials are plants that require or thrive on liquid surfaces, such as plants within the duckweed family. Other preferred aquatic plants include Giant Salvinia, Kariba weed, Aquarium watermoss, Water Fern, Carolina mosquito fern, water hyacinth, jacinthe d'eau, Variable-leaf Pondweed, Waterthread Pondweed, Hydrilla, American Water-Plantain, Marsh Pennywort, and Creeping Rush. These plants and other biological material may be either wild plants, or transgenic plants for the production of vaccines, therapeutic proteins and peptides for human or animal use, neutraceuticals, small molecule pharmaceuticals, research and production reagents (growth factors and media additives for cell culture) or excipients for pharmaceuticals.
The rack 12 of the bioreactor assembly 10 includes a base 16 for supporting the remaining portions of the rack, as is shown in
Three vertical support members 17 are connected to and extend upwards from each lateral side and the center of the horizontal support rail 24. Preferably, the vertical support members 17 are supported nearer a rear edge of the base 16 so as to provide additional stability for the containers 11 which are supported on cantilevered support members 19 that extend towards a front edge of the base.
Each of the cantilevered support members 19 is mounted to a front edge of the vertical support member at regularly spaced intervals. In addition, each of the cantilevered support members includes a mounting plate 20 attached to the vertical support member 17 on its rear surface and a retaining member 21 on its front surface. As is shown in
In the embodiment illustrated in
Supported at the top ends of each of the vertical support members 17, 25 is another horizontal support rail 26 that is equal in length and extends parallel to the bottom support rail 24. Both the top and bottom horizontal support rails support a plurality of light mounts 27. The light mounts are positioned in corresponding pairs extending along the rails 24, 26 at regular, spaced intervals. In this manner, each pair of light mounts can support a vertically extending one of the lights 13.
The lights 13 are preferably artificial lights that are electrically powered. For instance, lighting can be supplied by light-emitting diodes, fluorescent lights, incandescent lights, sodium vapor lights, metal halide lights or various combinations of these, and other, types of lights. Alternatively, the artificial lights may also be aided by, or replaced with, direct and indirect sunlight. However, artificial lights are preferred due to their ease of control and positioning so that all of the duckweed, or other biological material, contained in the reservoir 15 is supplied a sufficient amount of light to promote growth. Supplying power to the various types of lights can be done via wiring, or other manner that is conventional in the art and therefore not described herein in additional detail.
As noted above, the lights 13 of the embodiment illustrated in
Various different configurations of the lights 13 are possible depending upon different factors such as the intensity of the lighting need to support growth, the positions of the containers 11, or the desired temperature of the media in the reservoir 15. For instance, an alternative configuration for the lights 13 is shown in
Another alternative configuration for the lights 13 is shown schematically in
The containers 11 in the embodiment illustrated by
The relative arrangement of the containers 11 in
Another advantage of the side-by-side positioned vertical stacks of containers 11 shown in
It should be noted that separate versions of the rack 12 are not described in additional details herein for the remaining embodiments because the aspects of the rack illustrated in
It should also be noted that the relative positions of the lights 13 and the containers 11, as well as the number of lights and containers, may be modified to suit a particular application. For instance, larger numbers of lights could be used to accelerate growth of the biological material, or larger numbers of containers stacked in a tighter arrangement may be used to grow larger amounts of biological material. Therefore, the combinations of lights and containers are not necessarily restricted to the above-listed configurations and would still fall within the scope of the present invention.
The wall structure 14 of each of the containers 11 is constructed of a light transmissive material which allows the passage of sufficient light to promote growth of the biological material stored in the reservoir 15 defined therein. For instance, the wall structure 14 may be constructed of a glass, such as a borosilicate or flint glass, or a plastic, such as a polycarbonate, polyvinylchloride, polystyrene, TEFLON, silicone, nylon or polyethylene. These materials may be either flexible or relatively rigid. Preferably, the light transmissive material not only allows the passage of some light, but is completely transparent to promote full passage of the light necessary to support growth. However, translucent materials may be used to screen out certain wavelengths or light intensities depending upon such factors as the needs of a biological material or the need to reduce the accumulation of heat in the reservoir 15.
The term “wall structure” herein refers to any member or collection of members that at least partially defines the reservoir 15. The wall structure 14 illustrated in the embodiment of
Other shapes could also be used for the wall structure 14 including shapes with, and without, a constant cross-section. For instance the wall structure may have a teardrop shape, or some arbitrary or irregular shape constructed to fit lighting needs or available space. Preferably, however, the shape is chosen to maximize the surface area of the portion of a cross-section of the reservoir 15 formed by the wall structure in a plane that is orthogonal to the pull of gravity (i.e., a horizontal plane). For instance, a wall structure 14 having a 6 inch diameter circular, cylindrical cross-section (the embodiment illustrated in
An increase to a 10 inch diameter cross-section of a circular, cylindrical cross-section would result in an increase in media surface area to 42 square feet, as is shown by another embodiment illustrated in
In another embodiment, the cross-section of the wall structure 14 is an oval which has a major axis (i.e., its widest diameter) and a minor axis (i.e., its narrowest diameter), as is shown in
In yet another embodiment, the wall structure 14 has a rectangular cross-section with four corners, as is shown in
Referring again to
The clamp 40 includes an outer band portion 42 as shown in
The clamp 40 also includes an inner sealing portion 46, as shown in
Recessed within each of the chamfered edges 47 is preferably a seal 48, which in the illustrated embodiment is an O-ring having a circular cross-section. Such positioning of the seal 48 is advantageous because it is interposed and compressed between the end of the wall structure 14 or the edge of the end cap 41 when the outer band portion 42 is tightened thereon, as shown in the separate embodiment of
Different seal configurations may be employed for different shapes and materials of the wall structure 14, end cap 41 or clamp 40. For instance, a gasket-type seal formed of a circular blank defining a hole therethrough could be employed between a wall structure having flat edge defining its end and an end cap that is a flat circular blank held onto the wall structure end using a latch and lever type clamp. In such a case, the seal 48 may be constructed of a polymeric or metal material that need not be compressed as much to form a gas and liquid-tight seal due to the increased surface area over which it is applied.
In another embodiment of the present invention, the wall structure 14 of each of the containers 11 is split up into separate portions 31 joined together at adjacent ends by clamps 40, as shown in
As is shown in
During assembly, the flared edge of the wall structure portion 31 is seated against the seal 48 and its respective chamfered edge of the sealing portion 46. Then, the inwardly directed flange at one end of the band portion 42 is extended over the outwardly directed flared edge 32 of the wall structure portion. As the band portion 42 is tightened, the inwardly directed flange 49 extends downwards onto the outer surface of the wall structure 14 at the base of the flared edge 32. This holds the flared edge by forming an inner diameter smaller than the diameter of the flared edge, thereby restraining the wall structure portion against axial movement.
In order to facilitate providing an aseptic environment while still allowing control of the environment within the reservoir 15, a plurality of threaded openings are defined through the inner band portion 42 and/or the end cap 41 to allow passage therethrough of various sampling, measurement and supply devices. Referring to
In addition, the nozzles 51 are attached to elbows and main fill and drain conduits 52 that extend downwards from the end cap 41. As is shown in
As another option, the bioreactor assembly 10 may further include a disposable container liner that is configured to extend around the inside of each of the containers 11. For instance, such a liner could be inflatable to overly the portions of the container defining the reservoir 15 and translucent to allow light therethrough, or could be a translucent polymeric sleeve that slides into the wall structure 14. Advantageously, the container liner would promote cleaning procedures by being removable and disposable, allowing insertion of another container liner. Cleaning may also be facilitated through use of a special end cap 41 having particularly large openings defined therethrough for connection of large media extraction nozzles. Attachment of the end cap facilitates drawing of a vacuum in the reservoir 15 during media extraction.
Beyond primary supply and removal of the media, the media can be sampled and measured by other devices. For instance, openings 53 defined in the band portion 42 and threaded openings 54 defined in the sealing portion 46 of each clamp 40 can allow insertion and securing of a sampling nozzle 55, a multiple-level temperature probe 56 and a pH probe 57, as is shown in
Extending from the threaded portion into the media is the probe (or nozzle) itself. For the probes, extending from the bolt head 58 are electrical leads 59 that connect to a conventional electronic measurement and control system 60. The sampling nozzle 55 has its own conduit 65 connected to a sampling and supply network 66. In this manner, the media can be measured for pH level, measured for temperature (at multiple levels in the media) or sampled for other measurements without opening of the containers 11 and the risk of contamination. Preferably, all of the threaded openings described herein are sealed against passage of contaminants, such as through use of polymeric tape, solder, a washer and seal combination, etc. As another alternative, once the threaded portion is secured within the threaded opening they could be welded, glued or otherwise permanently attached for a tight seal. Advantageously, for such permanent attachments threads may not even be required as long as the devices can be appropriately positioned during the welding or attachment process.
In addition to the supply, removal and measurement of media, air or other gasses in the reservoir 15 can be supplied, removed and measured using various other devices attached in a similar manner to the above-described devices. For example, additional threaded openings 50, 54 allow passage of an air temperature probe 61, a gas supply nozzle 62 and a gas exit nozzle 63. Each of these devices is secured in its respective threaded opening with its own bolt head 58 and threaded portion 64. The air temperature probe 61 allows the air temperature to be measured. Gas supply and exit nozzles 62, 63 allow control of the type, temperature, flow rate and other characteristics of the gasses in the reservoir 15. Preferably, the gas exit nozzle 63 is biased so as to allow flow in only a single direction, thereby preventing the infiltration of contaminants. At one end of the gas supply nozzle 62 is preferably a sparger 67 that diffuses the air supply so that it does not unduly disturb the media and biological matter within the reservoir 15. At the other end of the gas supply nozzle is a gas supply line 70.
It should be noted that other measurements within the reservoir 15 could also be made with a variation of other devices depending upon the information desired by the user. For instance, a gas composition probe could be used to sample the amount of CO2 which would be used as feedback to modify the composition of gasses being added or removed by the nozzles 62, 63. The measurement, supply and removal devices discussed herein may also have different sizes, configurations and placements depending upon the desired frequency, accuracy, speed and other qualities of their performance.
In addition, the devices could also be extended through the container at other locations and portions of the containers 11 depending upon various needs of the user such as accessibility, tolerability of the container materials to openings, the risk of leakage and contamination, etc. For instance, the devices could extend through openings defined in the wall structure 14 or other components of the bioreactor assembly 10, in addition to the end cap 41 of the clamps 40. As another alternative a short section of the wall structure 14 (e.g., 4 to 6 inches in length) could define the openings for various devices. For instance, the nozzles 62, 63 could be supported and extend through the short section of wall structure and the short section of wall structure could be secured to the remaining wall structure with a victualic-type clamp. The short section could be removed and reattached for easy maintenance and cleaning.
The gas supply and removal nozzles 62, 63 could also be employed with a pump having sufficient power to reduce the gas pressure within the reservoir 15 prior to harvest of the biological materials in order to increase protein production by the biological materials. Alternatively, during a growth phase the gas pressure can be increased to promote growth of the biological materials in the reservoir. Notably, this is particularly effective for the media surface-borne plants which have large portions exposed to the gasses in the reservoir. Also, the air-tight construction of the reservoir of the present invention facilitates manipulation of the gas pressure therein.
It should be further recognized that although the illustrated clamp 40 is preferred for the illustrated wall structure 14 configurations, other types of clamps may also be employed herein to connect different portions of the bioreactor assembly 10. Alternative clamp configurations can address various factors, as desired, such as easy application and removal, firm attachment (which would benefit from the above-mentioned lever-type variation or a lever and ratchet design that allows progressive tightening), a complementary seal design that ensures an air and liquid-tight seal to ensure an aseptic environment within the reservoir 15, corrosion resistance, biocompatibility, use of acceptable materials under FDA regulations for pharmacological manufacturing processes and ability to support various measurement and sampling devices while maintaining the aseptic reservoir environment.
It should also be noted that although the above-described embodiments each has a continuous reservoir 15 extending in along a single major axis (i.e., a length extending in its longest dimension) and having a constant cross-section, the present invention should not be limited to such shapes. The wall structure 14, end cap 41 and other portions of the containers 11 defining the reservoir 15 can have several twists, turns, bifurcations and deviations as long as the media within the reservoir can be filled to a level defining a relative large media surface area for the support of surface-borne biological materials, such as duckweed plants.
Generally, this reservoir will have one or more major portions that each have a primary axis wherein the axes of all or most of the portions share a common plane. In this manner, the reservoir can be oriented (by orienting the container) until it is substantially horizontal (i.e., orthogonal to the pull of gravity) so that the flowable media forms the relatively large surface area. The term “substantially horizontal” is used herein because some angle in the major axis or axes may be desired to induce flow for processing purposes. For instance, the containers 11 illustrated in
During initial use, the containers 11 are filled with the media using the main fill and drain nozzles 51 to supply relatively large volumes of the media. Biological materials can also be added using the main nozzles 51, or may be added when initially assembling the containers 11. Preferably, a surface-borne biological material is added such as plants from the duckweed family, or the aquatic plant species described above, that require light to proliferate via photosynthesis. As the reservoir 15 is filled it is monitored either visually, or automatically, to determine at which point the media reaches a level at which a maximized surface area is defined. In the case of the embodiment illustrated in
After the biological material and media are added, the power is supplied to the lights 13 (or the lights may have already been on) so as to cast light through the transparent wall structure 14 into the reservoir 15. Over time, the biological materials draw energy from the light and nutrients from the media and begin to proliferate. In the case of biological materials used for pharmacological purposes, the biological materials begin to secrete peptides and proteins into the surrounding media.
Also during this time, the various probes 56, 57, 61 are used to measure the properties (temperature, pH, CO2 composition, etc.) of the gaseous and media environment in the reservoir. In turn, this data is collected and used to control the intensity of the lights 13, the temperature and convection properties of the ambient air around the containers 11, the temperature and amounts of gasses and media supplied to the reservoir 15 through the gas supply nozzle 62 and fill and drain conduits 52. In addition, the sampling nozzle 55 can be used to take small samples to determine the progress of the secretions. Such progress may also be used to determine the various aforementioned conditions within the reservoir 15.
At a certain point, such as when the media is exhausted or a complete harvesting of the biological materials is desired, the entire contents of the reservoir 15 can be flushed out of the main fill and drain nozzles 51 and conduits 52. After such flushing, cleaning compounds can be run through the system using the same nozzles and conduits. Alternatively, some type of steady state can be established wherein the expressed products of the biological materials can be continuous sampled, or partially drained, and the media and gasses refreshed, so that the growth and expression process is continues almost indefinitely.
The present invention has many advantages. Overall, the bioreactor assembly 10 allows the production of clinical and commercial scale quantities of biopharmaceuticals from genetically modified plants in a contained, aseptic environment. For example, the use of containers 11 defining reservoirs 15 for partial filling with media provides a relatively large surface for the large-scale production of surface-borne biological materials, such as duckweed plants. In addition, use of the clamps 40 having seals to interconnect the various portions of the container wall structure 14 and sealed openings 50, 54 for insertion of various measurement and supply devices ensures a clean and aseptic environment to promote the growth of the biological materials for medical uses. The clamping system also allows for easy assembly and disassembly of the containers 11 for maintenance and modification. The measurement and supply devices ensure that the environment within the reservoir 15 is tightly controlled to maximize growth and expression of the biological materials therein.
Many modifications and other embodiments of the inventions set forth herein will come to mind to one skilled in the art to which these inventions pertain having the benefit of the teachings presented in the foregoing descriptions and the associated drawings. Therefore, it is to be understood that the inventions are not to be limited to the specific embodiments disclosed and that modifications and other embodiments are intended to be included within the scope of the appended claims. Although specific terms are employed herein, they are used in a generic and descriptive sense only and not for purposes of limitation.