System for filtering and associated method
阅读说明：本技术 用于过滤的系统和其相关联的方法 (System for filtering and associated method ) 是由 A·巴尔加夫 P·保尔 P·H·莫翰 A·高雷 P·库马尔 V·约瑟 S·阿拉古尔 M 于 2019-08-20 设计创作，主要内容包括：一种方法包括引起第一量的供给流体(28)沿第一方向(72)从生物反应器(12)流向切向流过滤器(16),以使第一量的供给流体(28)分离为渗透流体(42)和渗余流体(48)。此外,控制单元(66)操作成控制至少一个供给流控制装置(24,26)来抑制第一量的供给流体(28)的流动。此外,操作控制单元(66)以控制至少一个供给流控制装置(24,26),以经由切向流过滤器(16)沿与第一方向(72)相反的第二方向(74)引导下者中的至少一个：来自生物反应器(12)的第二量的供给流体(28)的另一流,渗透流体(42)的一部分,以及营养物流体的一部分。(A method includes causing a first quantity of a feed fluid (28) to flow in a first direction (72) from a bioreactor (12) to a tangential flow filter (16) to separate the first quantity of the feed fluid (28) into a permeate fluid (42) and a retentate fluid (48). Further, the control unit (66) is operative to control the at least one supply flow control device (24,26) to inhibit flow of the first amount of supply fluid (28). Furthermore, the control unit (66) is operated to control the at least one feed flow control device (24,26) to direct, via the tangential flow filter (16), at least one of: another stream of a second quantity of feed fluid (28) from the bioreactor (12), a portion of the permeate fluid (42), and a portion of the nutrient fluid.)
1. A method of operation of a system (10), the method comprising:
causing a first amount of feed fluid (28) to flow from the bioreactor (12) via the feed flow path (14) in a first direction (72) to the tangential flow filter (16);
controlling at least one feed flow control device (24,26) in the feed flow path (14) by a control unit (66) for controlling the flow;
allowing the tangential flow filter (16) to separate the first quantity of the feed fluid (28) into a permeate fluid (42) and a retentate fluid (48) as a result of the flow in the first direction (72);
operating the control unit (66) to control the at least one supply flow control device (24,26) to inhibit or stop the flow of the first amount of the supply fluid (28); and
operating the control unit (66) to control the at least one feed flow control device (24,26) to direct at least one of the following in a second direction (74) opposite to the first direction (72) via the tangential flow filter (16) for a predefined duration to clean the tangential flow filter (16): a) a further stream of a second quantity of the feed fluid (28) from the bioreactor (12), b) a portion of the osmotic fluid (42), and c) a portion of nutrient fluid from a source.
2. The method of claim 1, wherein controlling the at least one feed control device (24,26) comprises controlling a feed pump (24) and a feed control valve (26).
3. The method according to claim 1, comprising feeding a first predetermined amount of the permeate fluid (42) to a permeate collection unit (30) via a permeate flow path (32) by operating at least one permeate flow control device (34,40) using the control unit (66).
4. The method of claim 3, wherein operating the at least one permeate stream control device (34) comprises operating a permeate pump (34) and closing a permeate stream control valve (40).
5. The method of claim 4, comprising stopping the permeate pump (34) and opening the permeate control valve (40) by the control unit (66) to direct a portion of the permeate fluid (42) to a fluid storage unit (38) via the permeate flow path (32) and a transfer path (36).
6. The method according to claim 5, comprising determining, by the control unit (66), a time required to fill the amount of the portion of the osmotic fluid (42) in the fluid storage unit (38) based on the flow rate of the osmotic fluid (42) and the volume of the fluid storage unit (38).
7. The method of claim 5, comprising closing the permeate control valve (40) by the control unit (66) after directing the portion of the permeate fluid (42) to the fluid storage unit (38).
8. The method according to claim 7, comprising controlling the feed control valve (26) by the control unit (66) and opening the permeate control valve (40) to direct a portion of the permeate fluid (42) from the fluid storage unit (38) to a waste storage unit (76) via the transfer path (36), the permeate flow path (32), the tangential flow filter (16), and the feed flow path (14) in the second direction (74) opposite the first direction (72).
9. The method according to claim 8, comprising controlling a membrane (88) of the fluid storage unit by the control unit (66) to direct a portion of the permeate fluid (42) from the fluid storage unit to the waste storage unit (76) via the transfer path (36), the permeate flow path (32), the tangential flow filter (16), and the feed flow path (14) in the second direction (74) opposite the first direction (72).
10. The method according to claim 8, comprising controlling a piston (96) of the fluid storage unit by the control unit (66) to direct a portion of the permeate fluid (42) from the fluid storage unit to the waste storage unit (76) via the transfer path (36), the permeate flow path (32), the tangential flow filter (16), and the feed flow path (14) in the second direction (74) opposite the first direction (72).
11. The method according to claim 3, comprising operating the at least one permeate flow control device (40,80) by:
closing, by the control unit (66), a first permeate control valve (40) located downstream of a fluid storage unit (38);
controlling a second permeate control valve (80) downstream of a permeate pump (34) and upstream of the fluid storage unit (38) by the control unit (66); and
operating the permeate pump (34) by the control unit (66) to direct a portion of the permeate fluid (42) to the fluid storage unit (38) via the permeate flow path (32).
12. The method of claim 11, comprising stopping the permeate pump (34) by the control unit (66) after directing the portion of the permeate fluid (42) to the fluid storage unit (38).
13. The method according to claim 12, comprising controlling the feed control valve (26) by the control unit (66) and opening the second permeate control valve (80) to direct a portion of the permeate fluid (42) from the fluid storage unit (38) to a waste storage unit (76) via a transfer path (36), the permeate flow path (32), the tangential flow filter (16), and the feed flow path (14) in the second direction (74) opposite the first direction (72).
14. The method according to claim 12, comprising controlling the feed control valve (26) by the control unit (66) and changing a rotational direction of the permeate pump (34) to direct a portion of the permeate fluid (42) from the fluid storage unit (38) to a waste storage unit (76) via a transfer path (36), the permeate flow path (32), the tangential flow filter (16), and the feed flow path (14) in the second direction (74) opposite the first direction (72).
15. The method according to claim 1, wherein operating the control unit (66) to control the at least one feed flow control device (24,26) comprises directing via the tangential flow filter (16) at least one of the following in the second direction (74) opposite to the first direction (72) at predetermined time intervals for the predefined duration: a further flow of a second quantity of the feed fluid (28) from the bioreactor (12), and a portion of the permeate fluid (42).
16. The method of claim 1, wherein operating the control unit (66) comprises directing, for the predefined duration, at least one of a determined pressure differential across the tangential flow filter (16) and a determined transmembrane pressure of the tangential flow filter (16) in the second direction (74) opposite the first direction (72) via the tangential flow filter (16) based on at least one of: a further flow of a second quantity of the feed fluid (28) from the bioreactor (12), and a portion of the permeate fluid (42).
17. A system (10), the system (10) comprising:
a bioreactor (12);
a tangential flow filter (16), the tangential flow filter (16) being coupled to the bioreactor (12) via a feed flow path (14) and at least one feed flow control device (24,26), wherein the tangential flow filter (16) is for separating a feed fluid (28) into a permeate fluid (42) and a retentate fluid (48); and
a control unit (66), the control unit (66) communicatively coupled to the at least one flow control device (24,26), wherein the control unit (66) is configured to:
causing a first amount of the feed fluid (28) to flow from the bioreactor (12) via the feed flow path (14) to the tangential flow filter (16) in a first direction (72);
controlling the at least one feed flow control device (24,26) in the feed flow path (14) for controlling the flow;
allowing the tangential flow filter (16) to separate the first quantity of the feed fluid (28) into a permeate fluid (42) and a retentate fluid (48) as a result of the flow in the first direction (72);
controlling the at least one supply flow control device (24,26) to inhibit or stop the flow of the first amount of the supply fluid (28); and
controlling the at least one feed flow control device (24,26) to direct, for a predefined duration, at least one of the following in a second direction (74) opposite to the first direction (72) via the tangential flow filter (16) to clean the tangential flow filter (16): a) a further stream of a second quantity of the feed fluid (28) from the bioreactor (12), b) a portion of the osmotic fluid (42), and c) a portion of nutrient fluid from a source.
18. The system (10) of claim 17, wherein the system (10) comprises a fluid storage unit (38), the fluid storage unit (38) being coupled to the tangential flow filter (16) via a transfer path (36) and a permeate flow path (32), wherein the fluid storage unit (38) is for storing a portion of the permeate fluid (42).
19. The system (10) of claim 18, wherein the system (10) comprises at least one permeate flow control device comprising a permeate control valve (40) coupled to the transfer path (36), wherein the permeate control valve (40) is configured to direct a portion of the permeate fluid (42) to and from the fluid storage unit (38) via the transfer path (36).
20. The system (10) of claim 18, wherein the at least one feed flow control device (24,26) comprises a feed control valve (26) and a feed pump (24) coupled to the feed flow path (14).
21. The system (10) of claim 20, wherein the system (10) comprises a waste storage unit (76), the waste storage unit (76) being coupled to the feed flow path (14) via the feed control valve (26), wherein the feed control valve (26) is configured to direct a portion of the permeate fluid (42) from the fluid storage unit (38) to the waste storage unit (76) via the transfer path (36), the permeate flow path (32), the tangential flow filter (16), and the feed flow path (14) in the second direction (74) opposite the first direction (72).
22. The system (10) of claim 21, wherein the fluid storage unit comprises a membrane (88) disposed within a container (90), and wherein the membrane (88) is to direct a portion of the permeate fluid (42) to the waste storage unit (76) via the transfer path (36), the permeate flow path (32), the tangential flow filter (16), and the feed flow path (14) in the second direction (74) opposite the first direction (72).
23. The system (10) of claim 21, wherein the fluid storage unit comprises a piston (96) disposed within a container (98), and wherein the piston (96) is to direct the portion of the permeate fluid (42) to the waste storage unit (76) via the transfer path (36), the permeate flow path (32), the tangential flow filter (16), and the feed flow path (14) in the second direction (74) opposite the first direction (72).
24. The system (10) of claim 18, wherein the system (10) includes at least one permeate flow control device comprising:
a permeate pump (34), the permeate pump (34) coupled to the permeate flow path (32);
a first permeate control valve (40), the first permeate control valve (40) coupled to the transfer path (36), wherein the first permeate control valve (40) is located downstream of the fluid storage unit (38); and
a second permeate control valve (80), the second permeate control valve (80) coupled to the permeate flow path (32), wherein the second permeate control valve (80) is located downstream of the permeate pump (34);
wherein the first permeate control valve (40), the second permeate control valve (80), and the permeate pump (34) are configured to direct a portion of the permeate fluid (42) to the fluid storage unit (38).
25. The system (10) according to claim 17, wherein the system (10) comprises: a first pressure sensor (60), the first pressure sensor (60) coupled to a permeate flow path (32) and configured to measure a flow pressure of the permeate fluid (42); a second pressure sensor (62), the second pressure sensor (62) coupled to the feed flow path (14) and configured to measure a flow pressure of the feed fluid (28); and a third pressure sensor (64), the third pressure sensor (64) coupled to the retentate flow path (46) and configured to measure a flow pressure of the retentate fluid (48); wherein the control unit (66) is communicatively coupled to the first pressure sensor (60), the second pressure sensor (62), and the third pressure sensor (64) and configured to determine at least one of a determined pressure difference across the tangential flow filter (16) and a determined transmembrane pressure of the tangential flow filter (16) based on outputs from the first pressure sensor (60), the second pressure sensor (62), and the third pressure sensor (64).
Technical Field
Embodiments of the present description relate generally to filtration systems, and more particularly to perfusion systems having tangential flow filters and methods for cleaning tangential flow filters of perfusion systems.
Background
The presence of many substances as solutions or mixtures creates a need for a process to separate solutions or mixtures to be developed. The need to purify, recover, separate, and remove materials from process streams in chemical, pharmaceutical, food, petroleum, healthcare, and wastewater treatment applications has driven the need for separation techniques.
The most common filtration processes are Microfiltration (MF), Ultrafiltration (UF) and Reverse Osmosis (RO). This filtration process is pressure driven and is used to separate macromolecules from fluids using filters. The filter acts as a selective barrier by allowing certain components of the mixture to pass while retaining other components of the mixture. The filtration process results in two stages: a permeate stage and a retentate stage.
For bioprocessing applications, continuous processing is a growing trend because smaller bioreactors can be used to produce the desired amount of product as compared to using larger bioreactors operated in a batch feed mode. Perfusion may allow for continuous processing by allowing for a continuous supply of nutrients and removal of waste. Continuous processes result in better product yield, product quality, process intensification, reduced capital expenditure, and reduced operational expenditure.
A disadvantage associated with the use of a filter separation process is a phenomenon known as filter fouling. Fouling refers to the deposition of material, called fouling, on the membrane or pore surfaces of a filter, resulting in a change in the filter performance, or even in complete clogging of the filter. As a result, filter efficiency is reduced due to filter clogging, which in turn affects filtration quality and increases overall process time. In the case of continuous processing using disposable tangential flow filters, filter clogging can limit the duration that the process can be run without interruption. In particular, filter clogging limits the perfusion duration and limits the cell density achievable at the end of the process.
There is a need for an enhanced priming system having a tangential flow filter and a method for cleaning the tangential flow filter of the priming system.
Disclosure of Invention
According to one aspect of the present description, a method of operation of a system is disclosed. The method includes causing a first amount of a feed fluid to flow from the bioreactor via the feed flow path in a first direction to the tangential flow filter. The method further comprises controlling by a control unit at least one feed flow control device in the feed flow path for controlling said flow and allowing the tangential flow filter to divide a first amount of feed fluid into a permeate fluid and a retentate fluid due to said flow in said first direction. Further, the method includes operating the control unit to control the at least one supply flow control device to inhibit or stop the flow of the first amount of supply fluid. Further, the method comprises operating the control unit to control the at least one feed flow control device to direct at least one of the following in a second direction opposite to the first direction via the tangential flow filter for a predefined duration to clean the tangential flow filter: a) another stream of a second quantity of feed fluid from the bioreactor, b) a portion of the permeate fluid, and c) a portion of the nutrient fluid from the source.
According to one aspect of the present description, a system is disclosed. The system includes a bioreactor and a tangential flow filter coupled to the bioreactor via a feed flow path and at least one feed flow control device. Tangential flow filters are used to separate a feed fluid into a permeate fluid and a retentate fluid. The system also includes a control unit communicatively coupled to the at least one flow control device. The control unit is configured to cause a first amount of feed fluid to flow from the bioreactor to the tangential flow filter in a first direction via the feed flow path. The control unit is further configured to control at least one feed flow control device in the feed flow path for controlling said flow. Further, the control unit is configured to control the at least one supply flow control device to inhibit or stop the flow of the first amount of supply fluid. Further, the controller is configured to control the at least one feed flow control device to direct, for a predefined duration, at least one of the following in a second direction opposite to the first direction via the tangential flow filter to clean the tangential flow filter: a) another stream of a second quantity of feed fluid from the bioreactor, b) a portion of the permeate fluid, and c) a portion of the nutrient fluid from the source.
Drawings
These and other features, aspects, and advantages of the present disclosure will become better understood when the following detailed description is read with reference to the accompanying drawings in which like characters represent like parts throughout the drawings, wherein:
FIG. 1 is a block diagram of a system that utilizes reverse flow of permeate fluid through a tangential flow filter, according to embodiments herein;
FIG. 2 is a block diagram of a system utilizing reverse flow of permeate fluid through a tangential flow filter according to another embodiment of the present description;
FIG. 3 is a block diagram of a system including a fluid storage unit having a septum disposed in a container according to an embodiment of the present description;
FIG. 4 is a block diagram of a system including a fluid storage unit having a piston disposed in a container according to an embodiment of the present description;
FIG. 5 is a block diagram of a system that utilizes reverse flow of a feed fluid through a tangential flow filter according to embodiments herein.
Detailed Description
Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. The terms "first," "second," and the like, as used herein do not denote any order, quantity, or importance, but rather are used to distinguish one element from another. Furthermore, the terms "a" and "an" do not denote a limitation of quantity, but rather denote the presence of at least one of the referenced item. The use of "including," "comprising," or "having" and variations thereof herein is meant to encompass the items listed thereafter and equivalents thereof as well as additional items. The terms "communicatively coupled" and "coupled" are not limited to physical or mechanical connections or couplings and may include direct or indirect electrical connections or wireless couplings.
According to an embodiment of the present specification, a method is disclosed. The method includes causing a first amount of a feed fluid to flow from the bioreactor via the feed flow path in a first direction to the tangential flow filter. The method further comprises controlling by the control unit at least one feed flow control device in the feed flow path for controlling said flow. In addition, the method includes allowing the tangential flow filter to separate the first quantity of the feed fluid into a permeate fluid and a retentate fluid. The method also includes operating the control unit to control the at least one supply flow control device to inhibit or stop the flow of the first quantity of supply fluid. Further, the method comprises operating the control unit to control the at least one feed flow control device to direct at least one of the following in a second direction opposite to the first direction via the tangential flow filter for a predefined duration to clean the tangential flow filter: a) another stream of a second quantity of feed fluid from the bioreactor, b) a portion of the permeate fluid, and c) a portion of the nutrient fluid from the source.
According to another embodiment, an associated system is disclosed. According to embodiments of the present description, the exemplary systems and methods enable reduced filter clogging, thereby allowing for enhanced bioprocesses and extended life of tangential flow filters. The entire filter cleaning process is completely sterile and automated, as no user intervention is required. Furthermore, since the cleaning of the filter can be performed during the biological process, a higher density of cell cultures may be produced.
Referring to FIG. 1, a block diagram of a system 10 in accordance with embodiments of the present description is shown. In the illustrated embodiment, the system 10 includes a bioreactor 12 coupled to a tangential flow filter (e.g., a hollow fiber filter) 16 via a feed flow path 14. Tangential flow filter 16 has an inlet 18, a first outlet 20, and a second outlet 22.
The system 10 also includes a feed pump 24 and a feed control valve 26 coupled to the feed flow path 14. The feed pump 24 is located upstream of the feed control valve 26. For example, bioreactor 12 is used to store a feed fluid 28 associated with cell culture. In particular, feed pump 24 is used to feed fluid 28 from bioreactor 12 to inlet 18 of tangential flow filter 16 via feed flow path 14 at a predetermined flow rate. Feed control valve 26 is used to control the flow of feed fluid 28 through feed flow path 14.
In addition, the system 10 includes a permeate collection unit 30 coupled to the first outlet 20 via a permeate flow path 32. The system 10 also includes a permeate pump 34 coupled to the permeate flow path 32. In addition, the system 10 includes a transfer path 36 extending from the permeate flow path 32 to a fluid storage unit 38 at a location upstream of the permeate pump 34. In addition, the system 10 includes a permeate control valve (also alternatively referred to as a first permeate control valve) 40 coupled to the transfer path 36. The tangential flow filter 16 is used to separate the permeate fluid 42 from the feed fluid 28 by utilizing a pressure differential across the tangential flow filter 16. The permeate pump 34 is operable to supply a first predetermined amount of permeate fluid 42 to the permeate collection unit 30 via the permeate flow path 32 at a predetermined flow rate. The permeate control valve 40 is used to control the flow of a portion of the permeate fluid 42 through the transfer path 36 to the fluid storage unit 38.
In addition, bioreactor 12 is coupled to second outlet 22 of tangential flow filter 16 via retentate flow path 46. Retentate fluid 48 flows through second outlet 22 of filter 16 via tangential flow and through retentate flow path 46 to bioreactor 12. Retentate fluid 48 is the remainder of feed fluid 28 after separation of permeate fluid 42. A retentate valve 49 is coupled to the retentate flow path 46 and is used to control the flow of the retentate fluid 48 through the retentate flow path 46. In addition, source 50 is coupled to bioreactor 12 via flow path 52. The source 50 is for storing nutrient fluid 51. A delivery pump 54 is coupled to flow path 52 and is used to deliver nutrient fluid 51 from source 50 to bioreactor 12 via flow path 52. Whenever needed, the transfer pump 54 is operated to replenish the bioreactor 12 with nutrient fluid 51. It should be noted herein that the illustrated system 10 is an exemplary embodiment and should not be construed as limiting. The configuration of the system 10 may vary depending on the application. Feed pump 24 and feed control valve 26 are referred to as a feed flow control device, permeate control valve 40 and permeate pump 34 are referred to as a permeate fluid flow control device, and retentate valve 49 is referred to as a retentate flow control device of system 10.
In another embodiment, instead of using feed pump 24, pressurized gas may be fed from a gas source to bioreactor 12 via a filter for feeding feed fluid 28 from bioreactor 12 to tangential flow filter 16 via feed flow path 14. In such embodiments, the permeate pump 34 may not be required.
In the illustrated embodiment, the system 10 also includes a control system 56 having a flow sensor 58 and a first pressure sensor 60 coupled to the permeate flow path 32. A flow sensor 58 is located downstream of the permeate control valve 40 and upstream of the permeate pump 34. The first pressure sensor 60 is located upstream of the permeate control valve 40. The flow sensor 58 is used to measure the flow rate of the permeate fluid 42 flowing out of the tangential flow filter 16 into the permeate flow path 32. In one embodiment, the flow sensor 58 may output a signal representative of the flow rate of the permeate fluid 42 flowing through the permeate flow path 32. In another embodiment, the flow sensor 58 may output a signal indicative of a parameter (e.g., volume or velocity) of the osmotic fluid 42 to calculate the flow rate of the osmotic fluid 42. In another embodiment, the flow sensor 58 may be disposed downstream of the permeate pump 34. Any type of flow sensor that may be used to measure the flow rate of the osmotic fluid 42 is contemplated. The first pressure sensor 60 is used to sense the pressure of the permeate fluid 42 flowing through the permeate flow path 32.
In another embodiment, control system 56 may have a flow sensor (not shown) coupled to feed flowpath 14. Such flow sensors may be used to measure the flow rate of the feed fluid 28 flowing through the feed flow path 14.
Control system 56 also includes a second pressure sensor 62 coupled to supply flow path 14. The second pressure sensor 62 is located downstream of the supply control valve 26. Second pressure sensor 62 is used to sense the pressure of supply fluid 28 flowing through supply flow path 14. The control system 56 also includes a third pressure sensor 64 coupled to the retentate flow path 46. The third pressure sensor 64 is for sensing the pressure of the retentate fluid 48 flowing through the retentate flow path 46.
Further, in the illustrated embodiment, the control system 56 includes a control unit 66 having a processor 68 and a memory unit 70 coupled to the processor 68. In some embodiments, control unit 66 is used to control at least one function of system 10. In certain embodiments, the control unit 66 may include more than one processor that cooperate with each other to perform the intended functions. The control unit 66 is also configured to store content in the storage unit 70 and to retrieve content from the storage unit. In one embodiment, the control unit 66 is configured to initiate and control functions of the system 10.
In one embodiment, the control unit 66 includes at least one of a general purpose computer, a Graphics Processing Unit (GPU), a digital signal processor, and a controller. In other embodiments, the control unit 66 includes custom processor elements, such as, but not limited to, Application Specific Integrated Circuits (ASICs) and Field Programmable Gate Arrays (FPGAs). In some embodiments, the control unit 66 may be communicatively coupled with at least one of a keyboard, a mouse, and any other input device, and configured to receive commands and/or parameters from an operator via a console.
In one embodiment, memory unit 70 is Random Access Memory (RAM), Read Only Memory (ROM), flash memory, or any other type of computer-readable memory accessible to processor 68. Additionally, in certain embodiments, the storage unit 70 may be a non-transitory computer readable medium encoded with a program having a plurality of instructions to instruct the processor 68 to perform a series of steps to operate the system 10.
In the illustrated embodiment, control unit 66 is communicatively coupled to flow sensor 58. In one embodiment, control unit 66 is configured to receive an output signal from flow sensor 58 representative of the flow rate of permeate fluid 42. In another embodiment, the control unit 66 is configured to receive output signals from the flow sensor 58 representative of a parameter (e.g., volume or velocity) of the permeate fluid 42 to calculate the flow rate of the permeate fluid 42 according to known techniques.
The control unit 66 is also coupled to the feed pump 24 and the permeate pump 34, and is configured to control the operation of the feed pump 24 and the permeate pump 34. The control unit 66 may also be coupled to the delivery pump 54 and configured to control the delivery pump 54. Control unit 66 is also coupled to supply control valve 26, permeate control valve 40, and retentate valve 49, and is configured to control actuation of supply control valve 26, permeate control valve 40, and retentate valve 49. Additionally, the control unit 66 is communicatively coupled to the first pressure sensor 60, the second pressure sensor 62, and the third pressure sensor 64. In one embodiment, control unit 66 is communicatively coupled to first pressure sensor 60, second pressure sensor 62, and third pressure sensor 64, and is configured to determine a transmembrane pressure (TMP) of tangential flow filter 16 based on outputs from first pressure sensor 60, second pressure sensor 62, and third pressure sensor 64. It should be noted here that TMP represents the pressure required to pass the water through the filter. In another embodiment, the control unit 66 is configured to determine a pressure differential across the tangential flow filter 16 based on the outputs from the second pressure sensor 62 and the third pressure sensor 64.
During operation of system 10, control unit 66 operates feed pump 24 and controls feed control valve 26 to feed a first amount of feed fluid 28 from bioreactor 12 to tangential flow filter 16 via feed flow path 14 in a first direction 72. Tangential flow filter 16 separates a first quantity of feed fluid 28 into a permeate fluid 42 and a retentate fluid 48. In particular, the first quantity of feed fluid 28 traverses the tangential flow filter 16 tangentially at a positive pressure relative to the permeate side of the tangential flow filter 16. The control unit 66 operates the permeate pump 34 and the permeate flow control valve 40 to supply a first predetermined amount of permeate fluid 42 to the permeate collection unit 30 via the permeate flow path 32. Control unit 66 opens retentate valve 49 to supply retentate fluid 48 to bioreactor 12 via retentate flow path 46.
In some cases, the control unit 66 stops the permeate pump 34 and opens the permeate control valve 40 to direct a portion of the permeate fluid 42 to the fluid storage unit 38 via the transfer path 36. The control unit 66 determines the time required to fill the volume of the portion of the osmotic fluid 42 in the fluid storage unit 38 based on the determined flow rate of the osmotic fluid 42 and the volume of the fluid storage unit 38. As previously described, control unit 66 determines the flow rate of permeate fluid 42 based on the output of flow sensor 58.
Thereafter, the control unit 66 closes the permeate control valve 40 and stops the feed pump 24 to stop the flow of the first quantity of feed fluid 28 in the first direction 72 from the bioreactor 12 through the tangential flow filter 16. Thereafter, the control unit 66 controls the feed control valve 26, opens the permeate control valve 40, and closes the retentate valve 49 to direct the portion of the permeate fluid 42 under gravity in a second direction 74 opposite the first direction 72 from the fluid storage unit 38 to the waste storage unit 76 via the tangential flow filter 16 for a predefined duration to clean the tangential flow filter 16. The predetermined duration of cleaning the tangential flow filter 16 can vary depending on the application. In one embodiment, the cleaning of the tangential flow filter 16 is performed at predetermined time intervals. In one embodiment, control unit 66 may be used to determine the predetermined time interval based on the output of a flow sensor coupled to feed flow path 14. Here again, the predetermined time interval may vary depending on the application.
In one embodiment, cleaning of the tangential flow filter 16 is performed based on the TMP of the tangential flow filter 16 (which is determined by the control unit 66 based on the outputs from the first pressure sensor 60, the second pressure sensor 62, and the third pressure sensor 64). The control unit 66 calculates TMP based on the following relationship:
TMP=((p2+p3)/2)-p1
wherein p is1Is the output of the first pressure sensor 60, p2Is the output of the second pressure sensor 62, p3Is the output of the third pressure sensor 64. If the TMP is greater than the threshold limit, the control unit 66 determines that the tangential flow filter 16 is clogged and the cleaning process is performed as described above.
In another embodiment, cleaning of the tangential flow filter 16 is performed based on a pressure differential across the tangential flow filter 16 (which is determined by the control unit 66 based on the outputs from the second pressure sensor 62 and the third pressure sensor 64). If the pressure differential across the tangential flow filter 16 is greater than the threshold limit, the control unit 66 determines a blockage of the tangential flow filter 16 and performs the cleaning process as described above.
In yet another embodiment, the cleaning of the tangential flow filter 16 is performed based on the permeate flux rate of the tangential flow filter 16. The control unit 66 is configured to determine a permeate flux rate through the tangential flow filter 16 based on the measured flow rate of the permeate fluid 42. It should be noted herein that the permeate flux rate through the tangential flow filter 16 is defined as the measured flow rate of permeate fluid 42 per unit area of the tangential flow filter 16. In particular, if the permeate flux rate decreases, cleaning of the tangential flow filter 16 is performed.
In yet another embodiment, the cleaning of the tangential flow filter 16 is performed based on the detected pressure of the osmotic fluid 42. In particular, if a pressure drop of the permeate fluid 42 is detected, cleaning of the tangential flow filter 16 is performed.
After cleaning the tangential flow filter 16, the control unit 66 switches the system 10 from the cleaning state to the normal flow state. The volume of permeate fluid 42 used to clean the tangential flow filter 16 and the pressure used to reverse-flow the permeate fluid 42 through the tangential flow filter 16 can be controlled based on the type of tangential flow filter 16 and the density of the feed fluid 28.
Referring to FIG. 2, a block diagram of a system 78 according to another embodiment of the present description is shown. The system 78 is substantially similar to the system 10 shown in fig. 1. Similar to system 10, in the illustrated embodiment, system 78 includes a permeate collection unit 30 coupled to first outlet 20 via a permeate flow path 32. The system 78 also includes a permeate pump 34 coupled to the permeate flow path 32. In addition, the system 78 includes a transfer path 36 extending from the permeate flow path 32 to the fluid storage unit 38 at a location upstream of the permeate pump 34. In addition, the system 78 includes a first permeate control valve 40 coupled to the transfer path 36. In particular, a first permeate control valve 40 is located downstream of the fluid storage unit 38. The system 78 also includes a second permeate control valve 80 coupled to the permeate flow path 32 and located downstream of the permeate pump 34 and upstream of the fluid storage unit 38. Feed pump 24 and feed control valve 26 are referred to as a feed flow control device, first and second permeate control valves 40 and 80 and permeate pump 34 are referred to as a permeate flow control device, and retentate valve 49 is referred to as a retentate flow control device of system 78.
The permeate pump 34 is operable to supply a first predetermined amount of permeate fluid 42 to the permeate collection unit 30 via the permeate flow path 32 at a predetermined flow rate. In the illustrated embodiment, a first permeate control valve 40 is used to control the flow of the portion of permeate fluid 42 from the fluid storage unit 38 to the permeate flow path 32 via the transfer path 36. The second permeate control valve 80 is used to control the flow of the first predetermined amount of permeate fluid 42 through the permeate flow path 32 to the permeate collection unit 30 at a predetermined pressure. In addition, the second permeate control valve 80 is also used to control the flow of the portion of permeate fluid 42 to the fluid storage unit 38 via path 82. In the illustrated embodiment, the control unit 66 is also coupled to the first and second permeate control valves 40,80 and is configured to control actuation of the first and second permeate control valves 40, 80.
During operation of system 78, control unit 66 operates feed pump 24 and controls feed control valve 26 to feed a first amount of feed fluid 28 from bioreactor 12 to tangential flow filter 16 via feed flow path 14 in a first direction 72. Tangential flow filter 16 separates a first quantity of feed fluid 28 into a permeate fluid 42 and a retentate fluid 48. The control unit 66 then closes the first permeate control valve 40, controls the second permeate control valve 80, and operates the permeate pump 34 to supply the first predetermined amount of permeate fluid 42 to the permeate collection unit 30 via the permeate flow path 32. Control unit 66 opens retentate valve 49 to direct retentate fluid 48 to bioreactor 12 via retentate flow path 46. In some cases, the control unit 66 controls the second permeate control valve 80 to direct a portion of the permeate fluid 42 to the fluid storage unit 38 via a path 82. As previously described, the control unit 66 determines the time required to fill the volume of the portion of the osmotic fluid 42 in the fluid storage unit 38 based on the determined flow rate of the osmotic fluid 42 and the volume of the fluid storage unit 38. Control unit 66 determines the flow rate of permeate fluid 42 based on the output of flow sensor 58.
Thereafter, the control unit 66 stops the feed pump 24 to stop the flow of the first quantity of feed fluid 28 in the first direction 72 from the bioreactor 16 through the tangential flow filter 16. Thereafter, the control unit 66 stops the permeate pump 34, controls the feed control valve 26, closes the retentate valve 49, and opens the first permeate control valve 40 to direct the portion of the permeate fluid 42 under gravity from the fluid storage unit 38 to the waste storage unit 76 via the transfer path 36, the permeate flow path 32, and the tangential flow filter 16 in a second direction 74 opposite the first direction 72 for a predefined duration to clean the tangential flow filter 16. The predetermined duration of cleaning the tangential flow filter 16 can vary depending on the application.
In another embodiment, the control unit 66 controls the feed control valve 26, closes the first permeate control valve 40 and the retentate valve 49, controls the second permeate control valve 80, and changes the rotational direction of the permeate pump 34 to direct the portion of the permeate fluid 42 from the fluid storage unit 38 to the waste storage unit 76 via the path 82, the permeate flow path 32, and the tangential flow filter 16 in a second direction 74 opposite the first direction 72 for a predefined duration to clean the tangential flow filter 16. After cleaning the tangential flow filter 16, the control unit 66 switches the system 78 from the cleaning state to the normal flow state.
Referring to FIG. 3, a block diagram of a system 84 in accordance with embodiments of the present description is shown. The system 84 is substantially similar to the system 10 discussed with reference to fig. 1. In the illustrated embodiment, the system 84 includes a permeate collection unit 30 coupled to the first outlet 20 via a permeate flow path 32. The system 84 includes a permeate pump 34 coupled to the permeate flow path 32. In addition, the system 84 includes a transfer path 36 extending from the permeate flow path 32 to a fluid storage unit 86 at a location upstream of the permeate pump 34. The pump storage unit 86 includes a diaphragm 88 disposed within a container 90. The membrane 88 may be made of a material including at least one of, but not limited to: rubber (such as chloroprene rubber, nitrile rubber, ethylene propylene diene monomer rubber, etc.), thermoplastic elastomer (such as Sanifflex, wilflex ™)TMPolyurethane, geolastTMEtc.) and polytetrafluoroethylene. In addition, the system 78 includes a permeate control valve 40 coupled to the transfer path 36. The permeate pump 34 is operable to supply a first predetermined amount of permeate fluid 42 to the permeate collection unit 30 via the permeate flow path 32 at a predetermined flow rate. The permeate control valve 40 is used to control the flow of a portion of the permeate fluid 42 flowing through the transfer path 36 to the fluid storage unit 86.
In the illustrated embodiment, the control unit 66 is also coupled to an actuator (not shown) for actuating a diaphragm 88 disposed within the container 90. As previously described, during certain conditions, the control unit 66 stops the permeate pump 34 and opens the permeate control valve 40 to direct a portion of the permeate fluid 42 to the fluid storage unit 86 via the transfer path 36. Control unit 66 opens retentate valve 49 to direct retentate fluid 48 to bioreactor 12 via retentate flow path 46. Thereafter, the control unit 66 controls the feed control valve 26, closes the retentate valve 49, opens the permeate control valve 40, and actuates the membrane 88 for a predefined duration to direct a portion of the permeate fluid 42 from the fluid storage unit 86 to the waste storage unit 76 via the permeate flow path 32 and the tangential flow filter 16 in a second direction 74 opposite the first direction 72 to clean the tangential flow filter 16.
Referring to FIG. 4, a block diagram of a system 92 is shown, according to an embodiment of the present description. The system 92 is substantially similar to the system 10 discussed with reference to fig. 1. In the illustrated embodiment, the system 92 includes a permeate collection unit 30 coupled to the first outlet 20 via a permeate flow path 32. The system 92 includes a permeate pump 34 coupled to the permeate flow path 32. In addition, the system 92 includes a transfer path 36 extending from the permeate flow path 32 to a fluid storage unit 94 at a location upstream of the permeate pump 34. The fluid storage unit 94 includes a piston 96 disposed within a reservoir 98. In addition, the system 94 includes a permeate control valve 40 coupled to the transfer path 36. The permeate pump 34 is operable to supply a first predetermined amount of permeate fluid 42 to the permeate collection unit 30 via the permeate flow path 32 at a predetermined flow rate. The permeate control valve 40 is coupled to the transfer path 36 and is used to control the flow of a portion of the permeate fluid 42 flowing through the transfer path 36 to the fluid storage unit 94.
In the illustrated embodiment, the control unit 66 is also coupled to an actuator (not shown) for actuating a piston 96 disposed within a reservoir 98. As previously described, during certain conditions, the control unit 66 stops the permeate pump 34 and opens the permeate control valve 40 to direct a portion of the permeate fluid 42 to the fluid storage unit 94 via the transfer path 36. Control unit 66 opens retentate valve 49 to direct retentate fluid 48 to bioreactor 12 via retentate flow path 46. Thereafter, the control unit 66 controls the feed control valve 26, closes the retentate valve 49, opens the permeate control valve 40, and actuates the piston 96 for a predefined duration to direct a portion of the permeate fluid 42 from the fluid storage unit 94 to the waste storage unit 76 via the permeate flow path 32 and the tangential flow filter 16 in the second direction 74 opposite the first direction 72 to clean the tangential flow filter 16.
Referring to fig. 1-4, in certain embodiments, the fluid storage units 38,86,94, the transfer path 36, the first and second permeate flow control valves 40,80, and the path 82 may not be required. In such embodiments, the control unit 66 controls the permeate pump 34 to rotate in the opposite direction to direct the portion of the permeate fluid 42 from the permeate collection unit 30 to the waste storage unit 76 via the permeate flow path 32, the tangential flow filter 16, and the feed flow path 14 in a second direction 74 opposite the first direction 72 for a predefined duration to clean the tangential flow filter 16.
In certain other embodiments, instead of osmotic fluid 42, fluid storage units 38,86,94 may be filled with nutrient fluid 51 from a source such as source 50 or an external source. In such embodiments, a portion of the nutrient fluid 51 is directed from the fluid storage unit 38,86,94 to the waste storage unit 76 via the permeate flow path 32, the tangential flow filter 16, and the feed flow path 14 in a second direction 74 opposite the first direction 72 for a predefined duration to clean the tangential flow filter 16.
Referring to FIG. 5, a block diagram of a system 100 according to embodiments of the present description is shown. In the illustrated embodiment, system 100 includes a bioreactor 102 coupled to a tangential flow filter 106 via a feed flow path 104. Tangential flow filter 106 has an inlet 108, a first outlet 110, and a second outlet 112.
The system 100 also includes a feed pump 114, a first feed control valve 116, and a second feed control valve 118 coupled to the feed flow path 104. The feed pump 114 is located upstream of the first feed control valve 116, and the second feed control valve 118 is located downstream of the first feed control valve 116. For example, bioreactor 102 is used to store feed fluid 119 associated with cell culture. In particular, the feed pump 114 is used to feed a feed fluid 119 from the bioreactor 102 to the inlet 108 of the tangential flow filter 106 via the feed flow path 104 at a predetermined flow rate. First and second supply control valves 114 and 116 are used to control the flow of supply fluid 119 through supply flowpath 104.
In addition, the system 100 includes a permeate collection unit 120 coupled to the first outlet 110 via a permeate flow path 122. The system 100 also includes a permeate pump 124 coupled to the permeate flow path 122, a first permeate control valve 126, and a second permeate control valve 128. The permeate pump 124 is located downstream of the first permeate control valve 126, and the second permeate control valve 128 is located downstream of the permeate pump 124. The tangential flow filter 106 is used to separate the permeate fluid 130 from the feed fluid 119 by utilizing a pressure differential across the tangential flow filter 106. The permeate pump 124 is used to supply permeate fluid 130 to the permeate fluid collection unit 120 via the permeate flow path 122 at a predetermined flow rate. A first permeate control valve 126 and a second permeate control valve 128 are used to control the flow of permeate fluid 130 flowing through the permeate flow path 122 to the permeate collection unit 120.
In addition, bioreactor 102 is coupled to second outlet 112 of tangential flow filter 106 via retentate flow path 132. The system 100 also includes a first retentate control valve 134 and a second retentate control valve 136 coupled to the retentate flow path 132. Retentate fluid 138 flows to bioreactor 102 via second outlet 112 of tangential flow filter 106 and through retentate flow path 132. Retentate fluid 138 is the remainder of feed fluid 119 after separation of permeate fluid 130. First retentate control valve 134 and second retentate control valve 136 are used to control the flow of retentate fluid 134 to bioreactor 102 via retentate flow path 132.
In the illustrated embodiment, system 100 includes a feed bypass path 140 that extends from feed flow path 104 at a location downstream of feed pump 114 to retentate flow path 132 at a location downstream of first retentate control valve 134 and upstream of second retentate control valve 136. A third supply control valve 142 and a first check valve 144 are coupled to supply bypass path 140. A first check valve 144 is disposed downstream of the third supply control valve 142. System 100 also includes a return path 146 that extends from supply flow path 104 at a location between first supply control valve 116 and second supply control valve 118 to retentate flow path 132 at a location downstream of second retentate control valve 136. Fourth supply control valve 148 and second check valve 150 are coupled to return path 146. A second check valve 150 is disposed downstream of fourth supply control valve 148.
System 100 also includes a waste path 152 that extends from a location that intersects supply flow path 104 and return path 146 to a waste storage unit 154. A waste control valve 156 is coupled to the waste path 152. The feed pump 114, first feed control valve 116, second feed control valve 118, third feed control valve 142 and fourth feed control valve 148, first check valve 144 and second check valve 150, and waste control valve 156 are referred to as a feed flow control device, the permeate pump 124 and first and second permeate control valves 126 and 128 are referred to as a permeate flow control device, and the first and second retentate control valves 134 and 136 are referred to as a retentate flow control device. In one embodiment, first feed control valve 116, second feed control valve 118, third feed control valve 142, and fourth feed control valve 148, first permeate control valve 126, and second permeate control valve 128, first retentate control valve 134, and waste control valve 156 are pinch valves. The configuration of the system 100 may vary depending on the application.
In another embodiment, instead of using feed pump 114, pressurized gas can be supplied to bioreactor 102 from a gas source via a filter for feeding feed fluid 119 from bioreactor 102 to tangential flow filter 106 via feed flow path 104. In such embodiments, the permeate pump 124 may not be required.
In the illustrated embodiment, the system 100 also includes a control system 158 having a flow sensor 160 and a first pressure sensor 162 coupled to the permeate flow path 122. A flow sensor 160 is located downstream of the first permeate control valve 126 and upstream of the permeate pump 124. The first pressure sensor 162 is located upstream of the first permeate control valve 126. In another embodiment, the flow sensor 160 is disposed downstream of the permeate pump 124. The flow sensor 160 is used to measure the flow rate of the permeate fluid 130 flowing through the tangential flow filter 106 to the permeate flow path 122. The first pressure sensor 162 is used to sense the pressure of the permeate fluid 130 flowing through the permeate flow path 122.
In another embodiment, control system 158 may have a flow sensor (not shown) coupled to feed flowpath 104. Such flow sensors may be used to measure the flow rate of the feed fluid 119 flowing through the feed flow path 104.
The control system 158 also includes a second pressure sensor 164 coupled to the feed flowpath 104. The second pressure sensor 164 is located downstream of the first supply control valve 116. The second pressure sensor 164 is used to sense the pressure of the feed fluid 119 flowing through the feed flow path 104. The control system 158 also includes a third pressure sensor 166 coupled to the retentate flow path 132. A third pressure sensor 166 is used to sense the pressure of the retentate fluid 138 flowing through the retentate flow path 132.
Further, in the illustrated embodiment, the control system 158 includes a control unit 168 having a processor 170 and a memory unit 172 coupled to the processor 170. In some embodiments, control unit 168 is used to control at least one function of system 100. The control system 158 is similar to the control system 56 shown in FIG. 1.
In the illustrated embodiment, control unit 168 is communicatively coupled to flow sensor 160. In one embodiment, control unit 168 is configured to receive an output signal from flow sensor 160 representative of the flow rate of permeate fluid 130. In another embodiment, the control unit 168 is configured to receive output signals from the flow sensor 160 representative of a parameter (e.g., volume or velocity) of the permeate fluid 130 to calculate the flow rate of the permeate fluid 130 according to known techniques.
The control unit 168 is also coupled to the feed pump 114 and the permeate pump 124 and is configured to control the operation of the feed pump 114 and the permeate pump 124. Control unit 168 is also coupled to and configured to control first feed control valve 116, second feed control valve 118, third feed control valve 142, and fourth feed control valve 148, first permeate control valve 126, and second permeate control valve 128, first retentate control valve 134, and second retentate control valve 136, first check valve 144, and second check valve 150, and waste control valve 156. Additionally, the control unit 168 is communicatively coupled to the first pressure sensor 162, the second pressure sensor 164, and the third pressure sensor 166. In one embodiment, control unit 168 is communicatively coupled to first pressure sensor 162, second pressure sensor 164, and third pressure sensor 166 and is configured to determine a transmembrane pressure (TMP) of tangential flow filter 106 based on outputs from first pressure sensor 162, second pressure sensor 164, and third pressure sensor 166. In another embodiment, the control unit 168 is configured to determine a pressure differential across the tangential flow filter 106 based on the outputs from the second and third pressure sensors 164, 166.
During operation of the system 100, the control unit 168 operates the feed pump 114 and controls the first feed control valve 116 and the second feed control valve 118 to feed a first amount of feed fluid 119 from the bioreactor 102 to the tangential flow filter 106 in the first direction 174 via the feed flow path 104. Tangential flow filter 106 separates a first quantity of feed fluid 119 into a permeate fluid 130 and a retentate fluid 134. The control unit 168 operates the permeate pump 124 to supply the permeate fluid 130 to the permeate collection unit 120 via the permeate flow path 122. Control unit 168 opens retentate fluid 134 to supply retentate fluid 134 to bioreactor 102 via retentate flow path 132.
During certain conditions, the control unit 168 closes the first and second permeate control valves 126, 128 and stops the permeate pump 124 to stop the flow of the permeate fluid 130 to the permeate collection unit 120. The control unit 168 also closes the first and second feed control valves 116, 118, the second retentate control valve 136, and the waste control valve 156 to direct a second amount of the feed fluid 119 from the feed flow path 104 via the feed bypass path 140, the retentate flow path 132, and the tangential flow filter 106 in a second direction 176 opposite the first direction 174 for a predefined duration to clean the tangential flow filter 106. The predetermined duration of cleaning the tangential flow filter 106 can vary depending on the application. In one embodiment, the cleaning of the tangential flow filter 106 is performed at predetermined time intervals.
In one embodiment, cleaning of the tangential flow filter 106 is performed based on the TMP of the tangential flow filter 106 (which is determined by the control unit 168 based on the outputs from the first pressure sensor 162, the second pressure sensor 164, and the third pressure sensor 166). If the TMP is greater than the threshold limit, the control unit 168 determines that the tangential flow filter 106 is clogged and the cleaning process is performed as described above. In another embodiment, cleaning of the tangential flow filter 106 is performed based on a pressure differential across the tangential flow filter 106 (which is determined by the control unit 168 based on the outputs from the second pressure sensor 164 and the third pressure sensor 166). If the pressure differential across the tangential flow filter 106 is greater than the threshold limit, the control unit 168 determines a blockage of the tangential flow filter 106 and performs the cleaning process as described above.
In yet another embodiment, the cleaning of the tangential flow filter 106 is performed based on the permeate flux rate of the tangential flow filter 106. The control unit 168 is configured to determine a permeate flux rate of the tangential flow filter 106 based on the measured flow rate of the permeate fluid 130. In particular, if the permeate flux rate decreases, cleaning of the tangential flow filter 106 is performed.
In yet another embodiment, the cleaning of the tangential flow filter 106 is performed based on the detected pressure of the osmotic fluid 130. In particular, if a pressure drop of the permeate fluid 130 is detected, cleaning of the tangential flow filter 106 is performed.
Thereafter, a second amount of feed fluid 119 is supplied to the bioreactor 106 via the feed flow path 104, the return path 146, and the retentate flow path 132. In another embodiment, fourth feed control valve 148 is closed and waste control valve 156 is opened to direct a second quantity of feed fluid 119 from tangential flow filter 106 to waste storage unit 154 via feed flow path 104 and waste path 152.
After cleaning the tangential flow filter 106, the control unit 168 switches the system 100 from the cleaning state to the normal flow state. The volume of permeate fluid 42 used to clean the tangential flow filter 106 and the pressure used to reverse flow the permeate fluid 130 through the tangential flow filter 106 can be controlled based on the type of tangential flow filter 16 and the density of the feed fluid 119.
According to embodiments of the present description, the reverse direction of the feed fluid or the osmotic fluid or the nutrient fluid through the tangential flow filter creates turbulence, thereby reducing clogging of the tangential flow filter. Exemplary techniques can extend the life of a disposable tangential flow filter and also extend the duration over which a process can run uninterrupted. Because the tangential flow filter can be cleaned in a sterile manner during the process, higher cell densities can be achieved. In some embodiments, all permutations and combinations of fig. 1-5 are contemplated.
While only certain features of the specification have been illustrated and described herein, many modifications and changes will occur to those skilled in the art. It is, therefore, to be understood that the appended claims are intended to cover all such modifications and changes as fall within the true spirit of the specification.