WO2019077623A1

WO2019077623A1 - An improved disposable bioreactor

Info

Publication number: WO2019077623A1
Application number: PCT/IN2018/050660
Authority: WO
Inventors: Bhadreshkumar Mohanlal Pankhaniya
Original assignee: Shree Biocare Solutions Pvt. Ltd.
Priority date: 2017-10-17
Filing date: 2018-10-15
Publication date: 2019-04-25

Abstract

An improved disposable bioreactor (1) comprises a pre-sterilized flexible bag (3) for housing and processing biomaterials being configured to be removably supported within SS housing (2) through bag holding means (35), seed culture bag (5) supported upon SS housing. The bag holding means (35) comprises a support plate (36), a dish end plate (38) having grooves for receiving gasket (37) and clamping means (9) for tightening support plate (36) and dish end plate (38). A bottom-driven fluid-agitating means (10) disposed within the bag (3) for agitating fluid comprises an angularly inclined agitator shaft (25) and a rotating impeller (26) attached to shaft (25). A drive unit (28) in sealed cooperation with the bottom surface (7) of the bag (3) is magneto- mechanically coupled to agitator-shaft (25). A perforated/porous sparger (14) being centrally disposed within the bag (3) adapted to permit the passage of air into the interior of the bag (3).

Description

AN IMPROVED DISPOSABLE BIOREACTOR

Field of invention

The present invention relates to a disposable bioreactor and more particularly it relates to an improved disposable bioreactor which comprises an efficient aeration system for uniform distribution of air to increase the oxygen transfer rate (OTR) and an inclined agitation system to generate an adequate vortex for homogeneous mixing of biological cultures; which will enhance the microbial growth rate as well as cell growth rate and reduces the chances of damage to cells and prevents the risk of cross contamination.

Background of invention

Bioreactors are commonly cylindrical shaped vessels/tanks which are manufactured or designed to carry out a biochemical process involving microorganisms and biochemically active substances derived from such organisms. The bioreactors are usually used for providing a controlled environment for the growth of biological cultures i.e. cells, bacteria, yeasts, fungi, antibiotics, antibodies, microorganisms, biomass, fluids of human or animal origin or organs and tissues of plant and or animal origin. The said cultures are used to produce a variety of biologically active substances or medicinal products, including pharmaceuticals, fragrances, fuel and the like.

Conventional bioreactors are designed as stationary pressurized vessels which are non-disposable. Few decades ago, the pharmaceutical industry uses stainless steel tanks in order to develop animal cell cultures which require fastidious and costly cleaning and sterilization. The main problem involved in conventional bioreactors is the rigorous process involved in cleaning and sterilizing stainless steel tanks. In conventional bioreactors, the absolute necessity for a sterile environment is a real constraint and the cleaning processes for tanks between two reactions are costly in terms of products, water, time and energy consumption. Further, a lengthy verification process must also be put in place to ensure the absence of debris or lack of sterility in even the tiniest areas.

Hence, the disposable bioreactors are being progressively viewed as serious alternatives from the performance and an economical point of view. In disposable bioreactor, in order to provide greater flexibility in manufacturing and to reduce the time needed to effect a valid regeneration of the equipment; disposable sterilized plastic bags are used once with a product batch and then disposed. The disposable bioreactors are also having the same peripheral equipment as conventional bioreactors with connections for the measurement probes for oxygen levels, pH, temperature, pressure, an entry points for regulating the environment inside and an agitation.

In disposable bioreactors, the mixing has been accomplished using rotating element (impeller/stirrer), which is required to be rotated at sufficient high speed for homogenous mixing of the biological cultures; but at such high speed of the impeller/stirrer, the possibility of damage to the cells tends to increase and which decreases the microbial growth rate as well as cell growth rate. In disposable bioreactor, the aeration device plays vital role for increasing the oxygen transfer rate as well as microbial growth rate. Due to conventional aeration device in current disposable bioreactor, the air dispersion is not sufficient which decreases the oxygen transfer rate and results in reduced microbial growth rate.

Moreover, currently used disposable bioreactors do not include a total disposable system-probes, sensors and other components are generally used again and required sterilization prior to repeated use. Such disposable bioreactor system has been disclosed in US 7629167 which lacks an efficient aeration system for uniform distribution of the air to increase the oxygen transfer rate for increasing the microbial growth rate as well as cell growth rate. One such disposable bioreactor system is also disclosed in US 9550157 in which the spargers are utilized to introduce a specific gas or air into a liquid in order to agitate and/or dissolve the air or gas into the liquid; but there is no provision of aerating device to increase the oxygen transfer rate for increasing the microbial growth rate as well as cell growth rate. Further, due to vertically positioned agitator shaft and motor shaft, the stirring impeller can not generate sufficient vortex for homogenous mixing of the biological cultures.

Thus, current state of the art disposable bioreactors are less efficient, especially when it comes to mixing, and have a lag time between uses (batches) so that probes, sensors and/or other components may be sterilized prior to another use. Though, the presently available disposable bioreactors provide numerous advantages, there is still scope for auxiliary improvement to the existing disposable bioreactor systems.

Hence, it is necessary to approach an improved disposable bioreactor system that is an efficient in terms of time and energy consumption and cost effective in terms of maintenance and installation. Objects of Invention

The main object of the present disclosure is to ameliorate one or more drawbacks and shortcomings of the prior art bioreactors or to at least provide a useful alternative.

Another object of the present invention is to provide an improved disposable bioreactor which can generate adequate vortex which leads to homogenous O2/C02 gas mixing for enhancing the microbial growth rate as well as cell growth rate.

Still another object of the present invention is to provide an improved disposable bioreactor having an improved aeration device for increasing the oxygen transfer rate which will enhance the cell growth rate as well as microbial growth rate. Yet, another object of the present invention is to increase the efficiency of the disposable bioreactor in terms of time and energy consumption.

Yet, further object of the present invention is to provide an improved disposable bioreactor which is cost effective in terms of maintenance and installation.

Still, further object of the present invention is to provide an improved disposable bioreactor which reduces the cleaning and sterilization demands. Further, more object of the present invention is to provide an improved disposable bioreactor which reduces the risk of cross contamination and enhances the biological and process safety.

Further, one more object of the present invention is to provide an improved disposable bioreactor which reduces the shear force as well as mechanical force and thereby reduces the chances of damage to the cells and increases the cell growth rate.

Brief description of drawings

Other objects, advantages and novel features of the present invention will become apparent from the following detailed description of the present embodiment when taken in conjunction with the accompanying drawings. Fig. 1 illustrates a schematic perspective view of disposable bioreactor according to the present invention. Fig. 2 illustrates a schematic perspective view of SS housing according to the present invention.

Fig. 3 illustrates a schematic perspective view of flexible and disposable 3D plastic bag according to the present invention.

Fig. 4A shows a detailed view of an aeration system comprising perforated hollow pipes according to one embodiment of the present invention. Fig. 4B shows a detailed view of an aeration system comprising star-shaped pipes having radially outwardly extending tubular arms having porous holes according to another embodiment of the present invention.

Fig. 4C shows a detailed view of an aeration system comprising a circular sparger disc having perforated holes for supplying air according to one another embodiment of the present invention.

Fig. 5 shows a detailed view of an agitation system according to an embodiment of the present invention.

Fig. 6 shows an exploded view of the bioreactor which illustrates a bag holding means through clamping means according to the present invention.

Summary of invention

An improved disposable bioreactor comprises a pre-sterilized flexible and disposable plastic bag for housing biomaterials for processing and having a flat top surface and a bottom surface and being configured to be removably supported within a SS housing through a bag holding means, the peripheral edges of top surface and the bottom surface are covered with gasket. The bag holding means comprises a support plate, a dish end plate having grooves for receiving the gasket and clamping means for tightening support plate and dish end plate. The gasket is received within the groove of the dish end plate while tightening the dish end plate and support plate through the clamping means. Said bioreactor further comprises a bottom driven fluid-agitating means disposed within the flexible bag for agitating the fluid comprises an agitator shaft and at least one rotating impeller attached to the shaft. The agitator shaft is extended within the bag at the angle of 25° with respect to vertical axis A-A of the bioreactor. A drive unit in sealed cooperation with the bottom surface of the bag and is magneto-mechanically coupled to the agitator shaft. Said bioreactor also comprises a perforated or porous sparger in the form of circular disc, hollow circular pipe and a star- shaped pipe (23) having radially outwardly extending tubular arms (24) being centrally disposed within the bag adapted to permit the passage of air into the interior of the bag. Detailed description of invention

The nature of the invention and the manner in which it works is clearly described in the complete specification. The invention has various embodiments and they are clearly described in the following pages of the complete specification. Before explaining the present invention, it is to be understood that the phraseology and terminology employed herein is for the purpose of description and not of limitation.

Before explaining the present invention the term "microorganism" refers to microscopic organisms which include all bacteria, archaea, most protozoa, microalgae, cyanobacteria, fungi, algae, and some micro-animals such as rotifers as etc. Further, the term "microbiological culture" or "microbial culture" or "microorganism culture" refer to a method or system for multiplying microorganisms through reproduction in a predetermined culture medium, including under controlled environmental conditions. The term "culture medium" refers to a solid, liquid or semi-solid designed to support the growth of microorganisms or cells, or small plants like the moss Physcomitrella patens. In liquid culture medium, the term microbiological, microbial, or microorganism culture generally refers to the entire liquid medium and the microorganisms in the liquid medium regardless of the vessel in which the culture resides. A liquid medium is often referred to as "media", "culture medium", or "culture media".

The term "impeller" refers to a device that is used for agitating or mixing the contents of a bioreactor. The impeller may agitate the fluidic medium by stirring or other mechanical motion. The impeller of the instant invention includes, but is not limited to, a rushton, a marine, a hydrofoil, a pitched blade, and any other commercially available impeller.

Now as shown in Fig. 1, a disposable bioreactor (1) according to present invention mainly includes a SS housing (2) preferably made from, but not limited to, a stainless steel, a pre-sterilized flexible and disposable flexible plastic bag (3) having a flat top surface (6) and a torispherical bottom surface (7) with diameter larger than the outer diameter of the flexible plastic bag (3) for a variety of purposes, including culturing cells, microorganism, or plant metabolites as well as processing foods, chemicals, biopharmaceutical and biological. Said plastic bag (3) capable of receiving and stirring culture medium is configured to be removably and supportably housed within a SS housing (2) through a bag holding means (35) [discussed in detail below]. Said disposable bioreactor (1) further includes a bottom driven agitation system (10) [as shown in Fig. 4] being angularly extended within the plastic bag (3) from the bottom (7) thereof, a seed culture bag (5) for holding and supplying contamination free seeds and being supported through a supporting stand (8) upon the SS housing (2), a plurality of sampling bags (39) for collecting samples of medium from the plastic bag (3) through a sampling outlet pipe (40) and a base supporting means (4) for firmly holding the SS housing (2) in upright position. Said flexible plastic bag (3) is made of high density polyethylene, polysulphone, polyetylene-LD type, ethylene vine acetate, polyacrylate, polycarbonate, polystyrene, polyesters or combination of ploymers foam of multi film.

As shown in Fig. 2, the SS housing (2) is equipped with hinged arms (45) supported through hinge joints (46) at circumferential bottom surface of the SS housing (2) that allows hinged arms (45) to radially move outside so that sensor ports (13) equipped on the flexible plastic bag (3) be extended outside from the SS housing (2) while said flexible bag (3) is disposed within the SS housing (3).

As shown in Fig. 3, the flexible bag (3) is equipped with a raw inlet port (11) being connected to the seed culture bag (5) for receiving contamination free seeds from the seed culture bag (5) and a media inlet port (12) being connected to a media container [not shown in figure] for contamination free seed transfer into the flexible bag (3). The flexible bag (3) is also equipped with sensor ports (13) for receiving pre-sterile sensors for monitoring and testing the conditions of culture medium within the flexible bag (3). Each sensor port (13) includes at least one end that can be opened to the outside of the flexible plastic bag (3) to insert a sensor probe into the plastic bag (3). The probe may be, for example, a temperature probe, a pH probe, a dissolved gas sensor, an oxygen sensor, a carbon dioxide sensor, a cell mass sensor, a nutrient sensor, an osmometer or any other probe that allows for testing or checking the culture medium. Such sensors may optionally be reusable after sterilization. Thus, the system is flexible and provides alternative ways of supplying optional equipment of various kinds (e.g. sensors, probes, devices, pouches, ports etc).

Now referring to Fig.3 and Fig. 4A, the flexible bag (3) is further equipped with an aeration system which comprises a central air sparger (14) being vertically and downwardly extended within the bag (3) through an air influent pipe (15) and having porous holes of 0.5 mm to 10 mm for forming bubbles disposed into the flexible bag (3); said air influent pipe (15) being connected to an air inlet filter (16) through an empty sterile bag (17) disposed outside the SS housing (2) for supplying air to the central sparger (14) through the air influent pipe (15); an air effluent pipe (18) being connected to an air outlet filter (19) for discharging air from the flexible plastic bag (3) and an inoculation port (20) for introducing inoculants into the flexible bag (3).

Now, Fig. 4B shows another embodiment of the aeration system according to present invention. According to this embodiment, said aeration system includes the air influent pipe (15) being vertically and downwardly extended within the bag (3). At the free end of the air influent pipe (15), a circular sparger disc (21) having perforated holes (22) of 0.5 mm to 10 mm is mounted. Here, the aeration process is carried out by supplying air through the perforated holes (22) of the sparger disc (21). According further embodiment of the aeration system as shown in Fig.

4C, said aeration system include a star-shaped pipes (23) having radially outwardly extending tubular arms (24) having porous holes that discharge air into the flexible bag (3). As shown in Fig. 5, said bottom driven agitation system (10) disposed into the flexible bag (3) comprises an angularly extended agitator shaft (25) [preferably at 25° with respect to vertical axis A-A of bioreactor] having an upper end configured with a dual impellers (26) and a lower end coupled to an inclined impeller hub (27) being positioned inside the flexible bag (3) at the torispherical bottom surface (7) and a magneto-mechanical drive system or drive unit (28) situated within the base supporting means (4) and outside the flexible bag (3).

The magneto-mechanical drive system or drive unit (28) includes a Teflon cover (41), a rotatable drive head (43) housed within the Teflon cover (41), at least two driven magnets (44) being located within the drive head (43), at least two impeller magnets (34) that rotates the agitator shaft (25) in response to attraction with drive magnet (44), a motor shaft (30) being drivably connected with a motor-gear assembly (29) and being rotatably supported through the shaft bearing (32) within a casing (31) and terminates into an impeller thrust bearing (33) for rotation of the agitator shaft (25) along with the impeller (26). Said casing (31) and the Teflon cover (41) are tightly connected through TC clamps (42). Here, said motor shaft (30) is axially aligned relative to the agitator shaft (25). Here, when motor shaft (30) rotates by the motor gear assembly

(29) , the drive head (43) is rotated along with the rotation of the motor shaft

(30) . Said drive head (43) rotates the driven magnet (44) in response that the impeller magnets (34) are rotated through the magnetic force. Said impeller magnets (34) transfer the rotating motion to the agitator shaft (25) and hence to the impellers (26) through the impeller bearing (33).

It is to be understood that the motor (29) used in magneto mechanical drive system is typically an electric drive motor i.e. a variable speed electric motor, a pneumatic driven motor or a hydraulic drive motor. Further, the agitation system may include various kinds of impeller i.e. rushton impeller, pitched-blade impellers, marine-blade impellers etc.

According to the present invention said bag holding means (35) enables the plastic bag (3) to safely house within the SS housing (2). Now as shown in Fig. 6, said bag holding means (35) comprises a support plate (36) is being placed on gasket (37), a dish end plate (38) having grooves for receiving the gasket (37) and clamps (9) for clamping the dish end plate (38) and support plate (36). Here, firstly said plastic bag (3) is put on the dish end plate (38), said rubber gasket (37) is received within the groove of dish end plate (38). Thereafter, clamps (9) are tightened so that it creates pressure between said support plate (36) and said dish end plate (38) so that said gasket (37) is firmly received within the groove of dish end plate (38). Thus, the plastic bag (3) is firmly held within the SS housing (2). In order to remove said plastic bag (3) from SS housing (2), said clamps (9) are loosen so that said gasket (37) can easily be removed from the groove of dish end plate (38). Further, due to gasket (37) and groove, there is no chance of leakage in the bag (3) and user can easily fit and remove the plastic bag (3) from the SS housing (2). The invention has been explained in relation to specific embodiment. It is inferred that the foregoing description is only illustrative of the present invention and it is not intended that the invention be limited or restrictive thereto. Many other specific embodiments of the present invention will be apparent to one skilled in the art from the foregoing disclosure. All substitution, alterations and modification of the present invention which come within the scope of the following claims are to which the present invention is readily susceptible without departing from the spirit of the invention. The scope of the invention should therefore be determined not with reference to the above description but should be determined with reference to appended claims along with full scope of equivalents to which such claims are entitled.

List of Reference Numerals

I. Bioreactor

2. SS Housing

3. Flexible 3D plastic bag

4. Base supporting means

5. Seed culture bag

6. Flat top surface of flexible plastic bag

7. Bottom torispherical surface of flexible plastic bag

8. Supporting stand

9. Clamping means

10. Agitating means

II. Raw inlet port

12. Media inlet port

13. Sensor ports

14. Sparger

15. Air influent pipe

16. Air inlet filter

17. Sterile bag

18. Air effluent pipe

19. Air outlet filter

20. Inoculation port

21. Circular sparger disc

22. Perforated holes

23. Star-shaped pipes

24. Tubular arms 25. Agitator shaft

26. Impellers

27. Impeller hub

28. Magneto-mechanical drive system (Drive unit) 29. Motor Gear Assembly

30. Motor shaft

31. Casing

32. Shaft bearing

33. Impeller thrust bearing

34. Impeller magnets

35. Bag holding means

36. Support plate

37. Gasket

38. Dish end plate

39. Sampling bags

40. Sampling outlet pipe

41. Teflon cover

42. TC clamp

43. Drive head

44. Driven Magnet

45. Hinged Arms

46. Hinge Joints

Claims

We claim,

1. An improved disposable bioreactor (1) comprises: a pre-sterilized flexible and disposable plastic bag (3) being removably disposed within a SS housing (2) for housing biomaterials for processing and having a flat top surface (6) and a bottom surface (7) with diameter larger than the outer diameter of the flexible plastic bag (3); peripheral edges of the top surface (6) and the bottom surface (7) covered with gasket (37); said flexible plastic bag (3) being configured to be removably supported within a SS housing (2) through a bag holding means (35); a base supporting means (4) for holding the flexible plastic bag (3) and SS housing (2) in upright position; a seed culture bag (5) for holding and supplying contamination free seeds into the flexible plastic bag (3) and being supported through a supporting stand (8) upon the SS housing (2); said bag holding means (35) comprises a support plate (36), a dish end plate (38) having grooves for receiving the gasket (37) and clamping means (9) for tightening the support plate (36) and dish end plate (38); a bottom driven fluid-agitating means (10) disposed within the flexible bag (3) for agitating the fluid and assisting in distributing the bubbles comprising an angularly extended agitator shaft (25) and at least one rotating impeller (26) attached to the shaft (25); a drive unit (28) in sealed cooperation with the bottom surface (7) of the bag (3) and is magneto-mechanically coupled to the agitator shaft (25); a perforated or porous sparger (21) being centrally disposed within the bag (3) adapted to permit the passage of air into the interior of the bag (3); wherein said SS housing (2) is equipped with hinged arms (45) supported through hinge joints (46) at circumferential bottom surface of the SS housing (2) allowing free radial movement of the hinged arms (45); wherein said sparger (14) is a circular disc (21) with pores (22) being suspended within the bag (3) through an air influent pipe (15); wherein said gasket (37) is received within the groove of the dish end plate (38) while tightening the dish end plate (38) and support plate (36) through the clamping means (9); wherein said agitator shaft (25) is extended within the bag (3) at the angle of 25° with respect to vertical axis A-A of the bioreactor (1); wherein said flexible plastic bag (3) is made of high density polyethylene, polysulphone, polyetylene-LD type, ethylene vine acetate, polyacrylate, polycarbonate, polystyrene, polyesters or combination of ploymers foam of multi film.

2. The improved disposable bioreactor as claimed claim 1, wherein optionally said sparger (14) is hollow circular pipe with perforated holes (22).

3. The improved disposable bioreactor (1) as claimed in claim 1, wherein optionally said sparger (14) is star-shaped pipe (23) having radially outwardly extending tubular arms (24) with perforated holes (22).

4. The improved disposable bioreactor (1) as claimed in claim 1, wherein the size of pores or perforated holes (22) of the sparger (14) is in the range of 0.5 mm to 10 mm.

5. The improved disposable bioreactor as claimed in claim 1, wherein said air influent pipe (15) is connected to an empty sterile bag (17) outside the flexible bag (3).

6. The improved disposable bioreactor as claimed in claim 4, wherein said sterile bag (17) is connected with an air inlet filter bag (16) having a filter of 0.2 micron.

7. The improved disposable bioreactor as claimed in claim 1, wherein said drive unit (28) comprising a Teflon cover (41), a rotatable drive head (43) housed within the Teflon cover (41), at least two driven magnets (44) being rotatably located within the drive head (43), at least two impeller magnets (34) that rotates the agitator shaft (25) in response to attraction with drive magnet (44), a motor shaft (30) being drivably connected with a motor-gear assembly (29) and being rotatably supported through the shaft bearing (32) within a casing (31) and terminates into an impeller thrust bearing (33) for rotation of the agitator shaft (25) along with the impeller (26).

8. The improved disposable bioreactor as claimed in claim 6, wherein said casing (31) and the Teflon cover (41) are tightly connected through TC clamps (42).

9. The improved disposable bioreactor as claimed in claim 1 and 6, wherein said agitator shaft (25) and the motor shaft (30) are axially aligned relative to each other.

10. The improved disposable bioreactor as claimed in claim 1, wherein the bottom surface (7) of the bag (3) is torispherical.

11. The improved disposable bioreactor as claimed in claim 1, further including a material addition conduit (11) in at least selective fluid communication with the interior of the flexible bag (3) and adapted to permit the addition of a material to the interior of the bag (3).

12. The improved disposable bioreactor as claimed in claim 1, further including a material extraction conduit (40) in at least selective fluid communication with the interior of the flexible bag (3) and adapted to permit the extraction of material from the interior of the flexible bag (3).

13. The improved disposable bioreactor as claimed in claim 1, further including at least one sensor for sensing a condition of the fluid in the flexible bag (3).

14. The improved disposable bioreactor as claimed in claim 12, wherein said sensor includes a temperature sensor, a pH sensor, a dissolved gas sensor, an oxygen sensor, a carbon dioxide sensor, a cell mass sensor, a nutrient sensor, an osmometer.