A stirred bioreactor based on mechanical seal shaft drive

Abstract

This invention provides a stirred bioreactor based on a mechanical seal shaft drive, proposing a highly integrated mechanical seal shaft drive scheme. The main shaft, bearings, and sealing gaskets are pre-assembled as a shaft seal assembly and directly welded to a disposable reaction bag, forming a pre-sterilized rigid sealing interface. During use, the drive motor is directly and rigidly connected to the main shaft via a hexagonal interface and mechanically secured by TC clamps. This design not only completely eliminates the easily failed flexible intermediate links, achieving zero torque loss and high-precision transmission, but also simplifies the installation operation into a plug-and-play standardized procedure. While ensuring an absolute sterile barrier, it effectively overcomes the radial vibration problem caused by the load on the distal impeller, providing a more stable and uniform stirring process, improving cell culture results. With a simpler structure, it solves the industry challenge of high-torque, high-intensity stirring in ultra-large-capacity bioreactors, significantly improving the system's reliability and ease of operation.

Description

Technical Field

[0001] This invention relates to cell culture devices in the field of biotechnology, specifically to a stirred bioreactor based on mechanical seal shaft drive. Background Technology

[0002] In the field of biotechnology, especially in the research and production of biopharmaceuticals such as cell therapy, gene-engineered drugs, and monoclonal antibodies, bioreactors, as core equipment, play a crucial role in providing a suitable growth environment for cells and promoting their rapid proliferation and metabolism. Among them, stirred bioreactors have become one of the most widely used culture devices due to their advantages such as uniform mixing, high mass transfer efficiency, and flexible operation.

[0003] With the widespread adoption of single-use technology in the biopharmaceutical industry, single-use bioreactors are gradually replacing traditional stainless steel reactors and becoming the mainstream choice due to their advantages such as no need for cleaning and sterilization, reduced risk of cross-contamination, and ease of operation. The single-use stirred culture bag, as its core component, has a built-in stirring paddle that achieves mixing of the culture medium and gas exchange through external drive.

[0004] The core component of a disposable stirred bioreactor is the flexible culture bag and its built-in stirring mechanism. How to effectively transmit external driving power to the stirring paddle inside the bag without compromising the bag's aseptic sealing has always been a technical challenge in this field. An ideal transmission scheme must meet the following requirements: reliable aseptic sealing, efficient torque transmission, simple structure for easy single-use, and compatibility with existing culture processes.

[0005] In the existing technology, the driving schemes for disposable stirred bioreactors are mainly divided into two categories.

[0006] One type is magnetic coupling drive, which uses magnetic cores placed inside and outside the bag to transmit torque through the bag wall. For example, for small to medium-sized culture bags (below 2000L), magnetic stirring drive is commonly used. In this case, an external driving magnet is coupled to an internal driven magnet through the culture bag, thereby driving the stirring paddle to rotate. This shaftless design is considered to best reflect the advantages of disposable technology. However, as the industry develops towards larger scales (such as 5000L, 10000L or even larger) and higher cell densities, the inherent shortcomings of magnetic drive solutions are becoming increasingly apparent. Magnetic coupling suffers from low torque transmission efficiency, easy decoupling, and high energy loss under high torque and high speed conditions, making it difficult to meet the needs of high-density cell culture or high-viscosity culture systems.

[0007] Another type is mechanical shaft drive, where the stirring shaft passes through the bag wall and is directly connected to an external drive shaft. The core challenge of this type of solution lies in the dynamic sealing design of the passage section. Traditional mechanical seals or packing seals are complex, prone to wear and leakage after long-term operation, and struggle to balance sterility with reliable torque transmission. To address these issues, the industry has proposed several improved solutions. For example, patent CN1997730B (Baxter International, etc.) discloses a composite sterile transmission structure combining a flexible tube and an integrated sealing assembly. This solution inserts the drive shaft inside the flexible tube, transmits torque through the flexible tube, and maintains sterility with the help of a seal at the hub. However, this solution still has the following limitations in practical applications: First, the structure has high redundancy, requiring multiple components such as hubs, bearings, flexible tubes, and blades to be pre-installed on one side of the bag, increasing manufacturing costs and the difficulty of sterilization verification; Second, during installation, the long drive shaft needs to be inserted into the flexible tube as a whole, requiring high alignment accuracy and limiting operational convenience; Third, as a long-term dynamic torque transmission element, the flexible tube is prone to material fatigue under repeated torsional stress, and its connection interface with the hub and blades are potential leakage points, causing the reliability of the sterile barrier to rely excessively on the synergistic effect of multiple physical seals, which to some extent increases the risk of contamination.

[0008] In summary, existing agitator drive systems for disposable stirred bioreactors suffer from problems such as low torque transmission efficiency, complex structure, inconvenient assembly, insufficient sealing reliability, poor structural stability, and unsuitability for large-scale cultivation. Therefore, developing a mechanical stirring drive system that is structurally simple, reliably sealed, efficiently transmits torque, and is suitable for disposable culture bags remains a pressing technical challenge in this field. Summary of the Invention

[0009] To overcome the shortcomings of existing technologies, this invention provides a stirred bioreactor based on a mechanical seal shaft drive, proposing a highly integrated mechanical seal shaft drive scheme. The main shaft, bearings, and sealing gaskets are pre-assembled as a shaft seal assembly and directly welded to a disposable reaction bag, forming a pre-sterilized rigid sealing interface. During use, the drive motor is directly and rigidly connected to the main shaft via a hexagonal interface and mechanically secured by TC clamps. This design not only completely eliminates the easily failed flexible intermediate links, achieving zero torque loss and high-precision transmission, but also simplifies the installation operation into a plug-and-play standardized procedure. While ensuring an absolute sterile barrier, it effectively overcomes the radial vibration problem caused by the load on the distal impeller, providing a more stable and uniform stirring process, improving cell culture results. With a simpler structure, it solves the industry challenge of high-torque, high-intensity stirring in ultra-large-capacity bioreactors, significantly improving the system's reliability and ease of operation.

[0010] On one hand, the present invention provides a stirred bioreactor, including a stirring drive system and a flexible bag; the flexible bag is provided with a stirring paddle, and the main shaft of the stirring paddle has a first end extending out of the flexible bag; the flexible bag is provided with a mechanical seal assembly that cooperates with the main shaft, and the mechanical seal assembly seals the connection between the main shaft and the flexible bag to form a sterile and closed system; the stirring drive system is provided with a stirring drive shaft; the stirring drive shaft can be mechanically connected to the first end of the main shaft, thereby driving the stirring paddle to rotate within the flexible bag.

[0011] The mechanical connection refers to the rigid connection between the stirring drive shaft and the first end achieved solely through a mechanical structure, without requiring any other force (such as magnetic force) for auxiliary connection. Since the first end is located outside the flexible bag, meaning the stirring drive shaft is connected to the first end on the outside of the flexible bag, this is a completely different connection method from the existing method of inserting the drive shaft into the flexible tube of the flexible bag.

[0012] The disposable bioreactor solution provided by the present invention adopts mechanical seal shaft drive. By extending the stirring main shaft directly out of the flexible bag body and achieving a rigid mechanical connection (such as internal hexagonal fit) with the drive shaft of the drive motor, it abandons the traditional magnetic coupling transmission method and the method of the drive shaft extending into the flexible tube of the flexible bag. However, this design faces the following technical challenges: (1) Due to the short drive shaft, the center of gravity of the drive shaft is a certain distance away from the impeller hub inside the flexible bag. If the drive shaft and the main shaft are not fully aligned or the stability is insufficient after connection, radial vibration under high-speed rotation is very likely to occur, which will lead to increased seal wear, loose connection or even separation. This places extremely stringent requirements on the coaxiality accuracy of the connection structure, the rigidity of the support bearing and the reliability of the installation positioning; (2) The main shaft must achieve a reliable dynamic seal at the point where it passes through the bag body. It is necessary to ensure that the sterile barrier is not damaged during long-term rotation and to avoid leakage of culture medium. This places extremely high requirements on sealing materials, bearing lubrication and assembly process; (3) The rigid shaft seal assembly needs to be welded to the flexible bag body membrane material with high strength and no leakage to ensure that it does not fall off or crack under transportation, installation and stirring vibration; In addition, the entire pre-assembled component needs to be subjected to irradiation sterilization. The sealing parts and bearing materials must maintain their original elasticity and dimensional stability after irradiation to prevent failure after sterilization.

[0013] This invention introduces precision guidance and alignment design (such as mechanical connection structure, bearing double support structure, positioning step fit, etc.) in the connection structure, and successfully overcomes the above-mentioned problems through the selection of pre-assembled mechanical seal components and special materials, thereby gaining significant technical advantages: (1) Rigid connection realizes high torque and non-slip strong stirring, which can meet the needs of large-scale, high-density and high-viscosity cell culture; (2) The drive unit is simplified and moved to the outside of the bag body, and the installation operation is intuitive and reliable, eliminating the uncertainty of magnetic alignment and the risk of vibration caused by poor alignment; (3) The sterile isolation of the drive motor and consumables reduces the equipment cost and maintenance difficulty.

[0014] Furthermore, the top of the first end is provided with a drive groove, and the bottom of the drive groove is located outside the flexible bag body; the stirring drive shaft is inserted into the drive groove to achieve mechanical connection.

[0015] In the mechanical connection structure between the stirring drive shaft and the first end of the main shaft, the method of inserting the stirring drive shaft into the drive groove has multiple advantages: First, the drive groove is located at the top of the first end and its bottom is outside the flexible bag body, which is equivalent to building a closed force-bearing chamber at the end of the main shaft. This allows the stirring drive shaft to be completely accommodated within the groove for torque transmission, thus completely separating the mating surface between the drive groove and the stirring drive shaft from the flexible bag body. This avoids the situation where the mating surface is inside the bag (which is not only cumbersome to operate and difficult to align and insert, but also poses a risk of bacterial contamination and leakage), increasing the probability of bacterial contamination. At the same time, the drive groove can also completely wrap the mating surface, preventing it from being contaminated by the external environment. Firstly, the drive groove's inner wall (such as the internal hexagonal hole) forms a precise surface fit with the drive shaft's shape, achieving not only zero or minimal torque transmission but also automatically correcting minor radial deviations during insertion through the groove wall's guiding action, thus improving coaxiality after connection. Secondly, the bottom of the drive groove can serve as an axial positioning reference, limiting the drive shaft's insertion depth and ensuring consistent installation position each time. Furthermore, this grooved connection shifts the force point inward to the main shaft's interior, allowing the drive reaction force to act more evenly on the main shaft cross-section, which helps suppress radial vibration caused by the load on the distal impeller, thereby improving the stability and seal life of the rotating system.

[0016] Furthermore, the first end of the main shaft extends out of the flexible bag body from any one or more directions, including the top, bottom, left, right, front, and rear sides.

[0017] It is understood that the mechanical seal transmission method provided by the present invention allows the main shaft direction of the stirring paddle inside the flexible bag to be set not only in the traditional top-to-bottom mode, but also in any direction such as bottom, front-to-back, left-to-right, etc. It can be connected to the stirring drive shaft in the corresponding direction provided by the motor, thereby providing a stirring function for cell culture inside the flexible bag.

[0018] Furthermore, the cross-section of the drive groove is any one or more of a circle, triangle, or polygon.

[0019] Theoretically, any shape of drive groove can be used to achieve mechanical connection and mechanical seal transmission, as long as it can be connected to a stirring drive shaft of the corresponding shape. However, the transmission effect may vary.

[0020] Furthermore, the insertion end of the stirring drive shaft is an external hexagonal prism structure with a regular hexagonal cross-section; the drive groove is an internal hexagonal hole with a regular hexagonal cross-section, matching the shape of the insertion end of the stirring drive shaft.

[0021] The use of an external hexagonal prism with an internal hexagonal hole as the drive connection method offers multiple technical advantages. The hexagonal structure transmits torque through six evenly distributed contact surfaces, resulting in more dispersed driving force and more uniform stress distribution. This allows it to withstand high torque output while effectively preventing deformation or fatigue failure caused by localized stress concentration. Simultaneously, the hexagonal fit exhibits excellent self-centering characteristics, automatically correcting minor radial deviations during insertion to ensure high alignment between the rotation axes of the stirring drive shaft and the main shaft. This is crucial for suppressing radial vibration caused by the load on the distal impeller and maintaining the stability of the rotation system. Furthermore, precise tolerance design allows for zero-clearance or minimal-clearance fits, eliminating transmission backlash and enabling more accurate speed control, preventing impact loads from being transmitted to the mechanical seals. The hexagonal connection also boasts engineering advantages such as mature processing technology, controllable cost, and intuitive operation (similar to the principle of an internal hex wrench). The bottom of the drive groove can serve as an axial positioning reference, ensuring consistent installation depth each time. Therefore, while achieving efficient power transmission, it significantly improves the operational reliability and seal life of the disposable bioreactor under high-intensity stirring conditions.

[0022] Furthermore, the insertion end of the stirring drive shaft is a tapered hexagonal prism, with an axial taper of 0.5 to 2 degrees from the root to the end.

[0023] The insertion end of the stirring drive shaft is designed as a tapered hexagonal prism (axial shrinkage of 0.5~2 degrees). This tapered structure provides a progressive guide during insertion, allowing for smooth sliding even with slight angular deviations, significantly reducing the difficulty of bag installation. Furthermore, with the axial locking force, the tapered surface gradually weaves into the inner hexagonal hole wall, adaptively eliminating radial clearance caused by machining tolerances and achieving a near-zero clearance rigid connection. This effectively suppresses fretting and vibration caused by clearance. Simultaneously, the tapered design allows for control of the tightness through insertion depth, automatically compensating for wear from repeated insertions and removals, extending the spindle's lifespan. In addition, the tapered contact ensures uniform axial transmission of driving force, avoiding stress concentration at the root of straight edges. This better protects the drive groove edges under high torque conditions, thus significantly improving the connection rigidity, vibration resistance, and durability of the rotating system while ensuring smooth insertion and removal.

[0024] In some embodiments, the taper is preferably 1 degree.

[0025] A taper that is too small (less than 0.5 degrees) will make its performance approach that of a right hexagonal fit, making it difficult to effectively eliminate radial clearance caused by machining tolerances, and lacking progressive guidance during insertion, easily leading to jamming or misalignment. Conversely, a taper that is too large (greater than 2 degrees) will shorten the contact length of the mating surfaces, drastically increase the pressure per unit area, accelerate edge wear, and the excessive wedging force may trigger self-locking, making disassembly difficult, and even causing bending deformation of the spindle or agitator drive shaft due to excessive axial clamping force. A 1-degree taper achieves the optimal balance: it can adaptively eliminate clearance through moderate wedging, ensuring vibration-free rigid transmission, while retaining the engineering convenience of smooth insertion and removal, and the contact stress distribution is uniform, balancing torque carrying capacity and service life.

[0026] Furthermore, the root of the insertion end is in interference contact with the drive groove.

[0027] Building upon the tapered hexagonal fit, an interference contact is introduced between the root of the insertion end and the drive groove, further enhancing the connection rigidity. When the drive shaft is fully inserted, the interference fit at the root forms a localized prestressed contact, equivalent to adding an auxiliary support point at the fixed end of the cantilever beam, effectively suppressing micro-oscillations caused by the load on the distal impeller. Simultaneously, the gradually tightening wedging of the interference section and the tapered section together constitute a dual locking mechanism, making the torque transmission path more continuous and preventing loosening of the connection due to wear or tolerance fluctuations on a single mating surface. In addition, the tight fit at the root also acts as a damper, absorbing minor vibrations that may be generated during high-speed rotation and preventing vibration from being transmitted to the mechanical seal. The interference contact also provides a defined axial thrust position, ensuring that the relative height of the main shaft and the stirring drive shaft is consistent after each installation, thereby improving the overall stability of the rotation system and extending its service life.

[0028] Furthermore, the connection ends of the stirring drive shaft and the main shaft are respectively provided with mutually matching positioning structures; the positioning structures are mutually matching concave and convex structures.

[0029] A matching concave-convex positioning structure is installed at the connection end between the stirring drive shaft and the main shaft. Its core function is to significantly improve the accuracy and stability of the connection system through a physical alignment mechanism. The concave-convex structure forms a precision fit before torque transmission. The contact between the convex and concave parts forcibly corrects the rotation axes of the drive shaft and the main shaft, making them highly coincident. This effectively suppresses radial runout caused by machining tolerances or installation deviations, and prevents vibration caused by the load of the distal impeller from being transmitted to the mechanical seal. The concave-convex mating surfaces form a rigid support point after connection, which can withstand the shear force generated during high-speed rotation and prevent relative slippage or fretting wear at the connection end. At the same time, the concave-convex structure can serve as a dual positioning reference for axial and radial directions, ensuring that the relative position of the stirring drive shaft and the main shaft is consistent after each installation, improving assembly repeatability. In addition, the concave-convex fit concentrates the force on the positioning surface, avoiding the connection bolts or clamps bearing impact loads alone, thereby improving the overall rigidity and reliability of the rotation system.

[0030] Furthermore, the positioning structure includes a first positioning step located at the upper end of the drive groove and a second positioning step located at the corresponding position of the stirring drive shaft.

[0031] A preferred design is to provide a first positioning step at the upper end of the drive slot and a second positioning step at the corresponding position of the drive shaft. This design provides precise axial and radial limits for the insertion depth and radial position of the drive shaft through end-face contact, ensuring that the relative height of the spindle and drive shaft is consistent after each installation, significantly improving the repeatability of positioning during consumable replacement. Simultaneously, the positioning steps separate the axial load from the torque transmission path, allowing the hexagonal mating surface to focus on torque transmission, preventing axial forces caused by operational collisions or thermal expansion and contraction from directly acting on the bottom of the drive slot, thus protecting the torque transmission surface from improper clamping or wear. Furthermore, the contact of the step end faces provides the operator with clear feedback on proper installation, and when locked with clamps, it effectively suppresses micro-oscillations caused by the load on the distal impeller, enhancing the overall rigidity of the rotating system. This design achieves precise positioning, protects core transmission components, and improves assembly reliability without significantly increasing manufacturing complexity; therefore, it is the preferred structure in this invention that balances functionality and engineering economy.

[0032] In this invention, the axial direction refers to the length direction of the main shaft, and the radial direction refers to the direction perpendicular to the main shaft, that is, the diameter or radius direction of the main shaft.

[0033] Furthermore, a polymer bushing is provided outside the insertion end, and a self-lubricating friction pair is formed between the polymer bushing and the drive groove.

[0034] A polymer bushing is installed on the outside of the insertion end of the stirring drive shaft, forming a self-lubricating friction pair with the inner wall (or ribs) of the drive groove. The low coefficient of friction and self-lubricating properties of the polymer material significantly reduce frictional resistance and wear between the insertion end and the drive groove, improving transmission efficiency and extending component lifespan. Simultaneously, the bushing absorbs certain assembly tolerances and vibrations, resulting in smoother transmission and lower noise. It also effectively suppresses micro-oscillations caused by the load on the distal impeller. Furthermore, the self-lubricating friction pair eliminates the need for additional lubricant, avoiding the risk of lubricant contamination of the sterile environment and meeting the cleanliness requirements of disposable bioreactors. The polymer bushing is also easy to replace and maintain, reducing operating costs.

[0035] In some methods, a polymer bushing can be used to fill the gaps formed by the contraction taper at the insertion end, thereby maintaining the hexagonal prism shape of the insertion end of the agitator drive shaft. After prolonged use, the gaps caused by wear can be further eliminated by replacing the polymer bushing.

[0036] Furthermore, the polymer bushing is made of any one or more of PEEK, PTFE, and medical-grade POM.

[0037] PEEK (polyetheretherketone), PTFE (polytetrafluoroethylene), or medical-grade POM (polyoxymethylene) all possess excellent self-lubricating properties, wear resistance, chemical corrosion resistance, and biocompatibility. They can ensure long-term stable operation in dry friction or sterile environments, avoiding the risk of contaminating the culture system due to the addition of additional lubricants. At the same time, they can withstand the harsh conditions of steam sterilization or irradiation sterilization, maintaining dimensional stability and mechanical strength, thereby ensuring transmission accuracy and sealing reliability.

[0038] While medical-grade POM is less expensive in some applications, it is slightly inferior in terms of long-term heat resistance. Considering performance, cost, and processability, the optimal choice is a composite material of PEEK and PTFE, which exhibits exceptional self-lubricating properties. For example, by adding 10% PTFE to a PEEK matrix for modification, PEEK provides skeletal support and wear resistance, while PTFE further reduces the coefficient of friction, achieving a synergistic effect of strength and super-lubricity. This is particularly suitable for demanding transmission scenarios involving extremely high speeds or frequent start-stop operations.

[0039] Furthermore, the first end of the main shaft corresponds to the top of the drive groove, and its shape gradually expands downwards, and after passing the bottom of the drive groove, its shape quickly shrinks back.

[0040] The "gradually enlarging then rapidly retracting" shape design at the spindle end offers significant benefits in eliminating radial chatter and enhancing torque transmission stability. Furthermore, this design utilizes shape changes during spindle rotation to further improve torque transmission stability and contributes to a better seal at the shaft seal, thereby further enhancing the reliability and aseptic safety of the transmission system.

[0041] Furthermore, the first end of the main shaft extends out of the flexible bag body from above, and the second end of the main shaft is located inside the flexible bag body; a first sealing component is provided between the first end and the flexible bag body; the bottom of the drive groove is higher than the first sealing component.

[0042] The mechanical seal assembly described in this invention is the first sealing component, which enables the spindle to maintain a good sealing effect and torque transmission effect even when rotating at high speed.

[0043] Furthermore, the first sealing component includes a disc seat, a sealing gasket, and a bearing arranged sequentially from bottom to top; the disc seat and / or the sealing gasket form a low-friction pair with the main shaft; the bearing includes a first bearing and a second bearing; the inner rings of the first bearing and the second bearing are tightly fitted with the main shaft of the agitator.

[0044] The mechanical seal structure used in this design is a contact-type end-face seal, where a certain clamping force is maintained between the sealing gasket and the disc seat to achieve a reliable seal. Although this clamping force generates frictional torque, due to the use of low-friction materials, high-smoothness mating surfaces, and optimized clamping force design, the frictional torque is far less than the rated output torque of the drive motor and will not affect the normal operation of the stirring system. Furthermore, since the reaction bag is for single use, the wear of the sealing gasket is extremely low within a single batch culture cycle, and it does not affect the sealing performance or rotational stability at all.

[0045] The low-friction pair formed by the disc seat and the sealing gasket can reduce frictional resistance and heat generation of the spindle at high speeds, ensuring sealing performance without affecting transmission performance and extending seal life.

[0046] The dual-bearing design (first and second bearings) and its tight fit with the main shaft ensure the rotational stability of the agitator, preventing seal failure or uneven torque transmission due to radial runout.

[0047] Furthermore, the first sealing component also includes a disc cover plate extending circumferentially from the main shaft; the disc cover plate is matched and engaged with the disc seat, and a cavity is provided in the middle; the sealing gasket is placed in the cavity between the disc cover plate and the disc seat.

[0048] The sealing gasket completely fills all cavities between the disc cover and the disc seat to ensure a tight seal and prevent leakage and contamination risks.

[0049] The disc cover plate on the main shaft helps improve the dynamic sealing effect. During assembly, the disc seat is directly welded and sealed to the flexible bag body. The main shaft is inserted into the flexible bag body from the center of the disc seat, and the disc cover plate is combined with the disc seat. The cavity in the middle is sealed with a sealing gasket.

[0050] The first sealing component has a compact and highly integrated structure, which not only achieves reliable sealing of the flexible bag, but also ensures transmission accuracy and aseptic safety under high torque and high speed conditions.

[0051] Furthermore, the disc seat is made of a composite material of PEEK and PTFE, and the sealing gasket is filled with polytetrafluoroethylene to form a bio-inert low-abrasion friction pair; the disc cover plate is provided with a downwardly extending leak-proof protective layer, which is perpendicular to the disc surface of the disc cover plate.

[0052] The first sealing component uses a combination design of PEEK and PTFE composite material for the disc seat and PTFE-filled gasket for the sealing gasket. The main advantages are: both contain PTFE components, which can form a synergistic effect of homologous lubrication during relative rotation, significantly reducing the coefficient of friction and the generation of wear particles at the contact interface, thereby constructing a bio-inert low-wear friction pair; at the same time, the PEEK matrix provides the disc seat with excellent mechanical strength and high-temperature sterilization resistance, making up for the shortcomings of insufficient stiffness and easy creep of pure PTFE material, while the PTFE-filled sealing gasket maintains good compliance and sealing performance; this combination does not require additional coatings or lubricants, simplifies the process, controls costs, and fully meets the stringent requirements of single-use bioreactors for material biocompatibility, low exudates, and aseptic safety.

[0053] The leak-proof protective layer is perpendicular to the surface of the disc cover and extends downwards, which significantly improves the dynamic sealing effect. If culture medium seeps into the first sealing component, or even into the disc seat, the culture medium must leak outwards and upwards due to the centrifugal force of the stirring action to truly detach from the first sealing component. The downward-extending isolation plate of the leak-proof protective layer effectively prevents the liquid from penetrating outwards and upwards, thus making the dynamic sealing effect more reliable.

[0054] Furthermore, the sealing end face between the disc seat and the main shaft is provided with a micron-level dynamic pressure groove for achieving fluid dynamic pressure lubrication and sealing; the micron-level dynamic pressure groove is one or more combinations of spiral groove, herringbone groove or arc groove.

[0055] Micron-level dynamic pressure grooves (such as spiral grooves, herringbone grooves, or arc grooves) are set on the sealing end faces of the disc seat and the main shaft. When the main shaft rotates, the dynamic pressure grooves pump the culture medium or gas into the space between the sealing end faces through the hydrodynamic effect, forming an extremely thin bearing liquid film. This enables non-contact operation of the end faces, thereby significantly reducing the coefficient of friction and end face wear, and avoiding contamination from abrasive debris caused by solid contact. At the same time, this liquid film also plays a dynamic sealing role, effectively preventing external contaminants from entering and internal culture medium from leaking, thus improving sterility reliability. Different groove designs can also adapt to bidirectional rotation or enhance the pumping effect, ensuring a stable lubricating film is maintained over a wide speed range, thereby significantly extending the seal life, reducing maintenance requirements, and meeting the stringent requirements of single-use bioreactors for high-speed and high-torque conditions.

[0056] Different hydrodynamic groove types play different roles in the hydrodynamic effect of fluids. Spiral grooves have strong unidirectional pumping capacity, continuously pressurizing fluid into the sealing end face to form a stable liquid film, suitable for high-efficiency sealing in a single rotation direction. Herringbone grooves consist of two sets of symmetrical spiral grooves, generating a centripetal pumping effect during bidirectional rotation, automatically balancing end face pressure and preventing liquid film instability, making them particularly suitable for conditions with frequent start-stop cycles or uncertain rotation directions. Arc-shaped grooves reduce fluid resistance through streamlined design, lowering temperature rise and enhancing liquid film stiffness during high-speed rotation. Since the disposable bioreactor provided by this invention typically operates unidirectionally for extended periods with a wide rotational speed range, spiral grooves are preferred due to their highest pumping efficiency and best liquid film stability in a single rotation direction.

[0057] Furthermore, the first sealing component also includes a housing, the bottom of which is welded to the flexible bag body, and a first chuck is provided at the top of the housing; the stirring drive shaft is also provided with a second chuck; the first chuck and the second chuck are used in combination with clamps to realize the fixed connection between the stirring drive shaft and the main shaft.

[0058] In some configurations, the first and second chucks are TC chucks (Tri-Clamp). The TC chuck simultaneously performs three functions: motor mount fixation, gasket clamping, and transmission alignment—a significant functional integration innovation. This highly integrated solution achieves synergistic unity of structure, sealing, and transmission through a single interface, greatly simplifying the overall assembly hierarchy and the number of parts. This reduces manufacturing costs and potential leakage paths and failure points. Operationally, users only need to tighten one clamp to simultaneously complete the rigid connection between the drive motor and the spindle, pre-tighten the mechanical seal assembly, and achieve alignment, enabling "one-click" quick installation and greatly improving ease of use. Furthermore, as a standard sanitary connector, the TC chuck's high-precision centering characteristics ensure the coaxiality of the spindle and drive shaft, effectively preventing seal failure or vibration caused by uneven wear. Thus, while ensuring aseptic safety, it meets the stringent requirements for transmission stability under high torque and high speed conditions, truly demonstrating the comprehensive advantages of functional integration: simplified operation, enhanced performance, and improved reliability.

[0059] Furthermore, a second sealing component is provided below the flexible bag body; the second sealing component is provided with a support seat for supporting the second end.

[0060] Furthermore, the support base is provided with a third bearing, and the second end is tightly fitted with the outer ring of the third bearing and can rotate freely; the second sealing component also includes a second disc for welding and sealing with the flexible bag body.

[0061] Because the second end of the main shaft is far from the first end, this length extension causes the center of gravity of the stirring drive to be separated from the impeller hub by a long distance. Once misalignment occurs, it can quickly lead to vibration and eventually disengagement, especially at high speeds, where the impact is more severe.

[0062] This invention, by incorporating a second sealing component including a support base and a third bearing beneath the flexible bag (still within the flexible bag), provides reliable support to the second end of the agitator shaft via the third bearing. This effectively suppresses radial vibration and swaying that may occur during high-speed rotation of the long cantilever agitator shaft, significantly improving the stability of high-torque transmission and the smoothness of agitator operation. Simultaneously, since the second end of the shaft does not need to extend beyond the bag body, a complex rotary dynamic seal is no longer required. A simple static sealing structure constructed from welded discs is sufficient to ensure the aseptic integrity of the bag, thus completely eliminating the traditional leakage risk point of the bottom dynamic seal and significantly reducing the possibility of seal failure. This multi-bearing layout, with dynamic sealing above and static sealing below, ensures the mechanical reliability of large reaction bags under high-intensity stirring conditions while simplifying the bottom sealing structure, making overall aseptic assurance more reliable and assembly more convenient.

[0063] Furthermore, the support base is provided with a receiving disc and a support frame, and the second end is provided with a support structure that cooperates with the support frame; after the support structure is combined with the support frame, the second end of the support structure, the support structure and the support frame form an integral structure, and can rotate freely under the support of the support base.

[0064] The addition of a support base at the second end of the stirring shaft provides stable radial and axial support for the end of the main shaft extending into the reaction bag, significantly improving the operational stability of the main shaft under high-speed rotation or high-torque conditions. The receiving disc plays a crucial role in ensuring stability and alignment, effectively suppressing radial oscillation and axial movement of the main shaft, thereby reducing the risk of wear on the mechanical seals and ensuring smooth rotation of the stirring paddle, preventing radial oscillation and axial movement.

[0065] The support structure at the second end, after being combined with the support frame, maintains its ability to rotate freely along with the support base, without affecting the rotation of the spindle. A first spring piece with a first barb and a connecting groove are provided above the support frame to engage the support structure at the second end of the spindle and rotate together. The connecting groove ensures a stable connection between the support structure and the support frame, and the first spring piece with the first barb prevents the support structure from detaching from the support frame during rotation. A second spring piece with a second barb is provided below the support frame, which can be fitted onto the outer surface of the third bearing to connect and allow rotation. The inner surface of the third bearing is fixed by a protruding support column on the support base. In other words, the third bearing has a fixed inner surface and a freely rotating outer surface, unlike the first and second bearings. The first and second bearings, which fix the first end of the spindle, have fixed outer surfaces and their inner surfaces are connected to the first end, allowing free rotation, while the third bearing is the opposite.

[0066] This design, which swaps the functions of the inner and outer rings of the third bearing, provides rigid support at the end of the main shaft. The increased size of the rotating chassis makes rotation more stable and effectively prevents radial chatter. It also cleverly mitigates the potential threat to the bag's sealing and structural stability posed by the bearing's own rotation. Specifically, by fixing the inner ring of the bearing and rotating the outer ring synchronously with the support structure on the main shaft, stable radial constraints are provided to the extended cantilever shaft end, making the area below the main shaft more stable and better at suppressing swaying and vibration under high-speed rotation. This also avoids introducing an additional rotational sealing point at the bottom of the bag. Because the rotating components (support structure and bearing outer ring) are entirely located inside the bag and integrated with the main shaft, the stationary support can pass through from inside the bag to the outside, achieving dynamic isolation through the functional separation of the inner and outer rings of the bearing. This design not only significantly enhances the rigidity and durability of the stirring system under high torque conditions but also ensures that the welded interface at the bottom of the bag is not affected by rotational friction, maximizing transmission stability while effectively guaranteeing the integrity of the sterile barrier of the disposable bioreactor.

[0067] This design also enhances the rigidity of the entire transmission system, enabling it to withstand stronger stirring requirements and meet the demanding process conditions of high-viscosity, high-density cell culture. At the same time, it avoids vibration or seal failure caused by excessively long spindle cantilever, further improving the reliability and safety of disposable bioreactors in large-scale production.

[0068] On the other hand, the present invention provides a method for culturing cells, wherein the method employs a stirred bioreactor as described above.

[0069] The present invention has the following beneficial effects: 1. Overcoming the limitations of magnetic drive to achieve high torque and high speed stirring: The mechanical seal shaft drive replaces the traditional magnetic coupling, eliminating the risk of slippage and torque limitation, which can meet the high-intensity stirring requirements of ultra-large capacity (such as 5000L and above) and high cell density culture, and significantly improve mass transfer and mixing efficiency.

[0070] 2. Pre-assembled and pre-sterilized design to ensure aseptic safety: The stirring shaft, mechanical seal and disposable reaction bag are pre-assembled into a whole and delivered to the user after irradiation sterilization. There is no need to assemble the sealing parts on site during use, which completely avoids the risk of contamination during the installation process.

[0071] 3. One-click quick connection, convenient and reliable operation: The drive motor and main shaft are rigidly connected by the stirring drive shaft and drive groove, as well as the TC chuck and clamp. At the same time, the sealing pre-tightening, centering and positioning and motor base fixing are completed. The user operation is simple, the installation time is short and human error is reduced.

[0072] 4. Multi-bearing support structure to improve operational stability: The double bearings at the top and the support bearings at the bottom work together to effectively suppress radial vibration of the long main shaft when rotating at high speed, ensuring smooth operation of the agitator, extending the seal life, and making it suitable for harsh working conditions such as high viscosity and high speed.

[0073] 5. Static bottom seal enhances sterility reliability: The lower end of the main shaft does not need to extend outside the bag to obtain stable support. The bottom adopts only a static seal structure, which completely avoids the most difficult leakage point to guarantee in traditional designs, such as bottom rotation dynamic seal, and greatly improves sterility reliability.

[0074] 6. Functional integration and innovation, simplifying system structure: The TC chuck performs three functions simultaneously: fixing, sealing, and centering, reducing the number of parts and assembly layers, lowering manufacturing costs, and improving the overall reliability and ease of maintenance of the system.

[0075] 7. Bio-inert friction pair design to ensure a clean culture environment: The low-abrasion friction pair is composed of a PEEK / PTFE composite disk and a PTFE-filled sealing gasket, or a micron-level dynamic pressure groove is added to achieve hydrodynamic lubrication, minimizing particle generation and ensuring that the culture medium is not contaminated.

[0076] 8. The precise fit between the drive groove and the insertion end ensures transmission accuracy and assembly reliability: The drive groove adopts an internal hexagonal hole design, which forms a non-circular cross-section fit with the tapered hexagonal prism-shaped insertion end of the stirring drive shaft, enabling the transmission of large torque without relative rotation; the tapered structure makes the fit tighter the deeper the insertion, effectively eliminating radial clearance and ensuring centering accuracy.

[0077] 9. Interference contact and positioning step design to achieve precise axial positioning and anti-dislodgement: The root of the insertion end is in interference contact with the drive groove. Combined with the mutual abutment of the first and second positioning steps, it can achieve precise axial positioning and prevent insertion from being too deep or loosening and dislodging. At the same time, the interference fit enhances the connection rigidity and avoids fretting wear under high torque.

[0078] 10. The shrinking taper and the polymer bushing work together to reduce friction and wear and extend service life: The shrinking taper of the stirring drive shaft and the polymer bushing at the insertion end form an effective transmission structure, which not only ensures the reliability of torque transmission, but also reduces the coefficient of friction and wear through the self-lubricating properties of the polymer material, optimizes the contact stress distribution, prevents stress concentration, and improves the centering and positioning accuracy.

[0079] 11. The gradually expanding and contracting shape of the spindle end achieves integrated guidance, locking and sealing: The first end of the spindle adopts a shape design that gradually expands and then quickly retracts, which plays a guiding role during insertion and reduces assembly resistance; the structure that quickly retracts after passing the bottom of the drive groove forms a wedge-shaped locking effect, effectively eliminating axial and radial clearances and preventing shaking during operation.

[0080] 12. Multi-stage anti-loosening structure to ensure connection reliability under extreme working conditions: Through the combined effects of tapered fit, interference contact, positioning step limit, and gradually expanding and contracting shape, a multi-stage axial and circumferential anti-loosening mechanism is formed to ensure that the connection between the drive shaft and the main shaft is always stable and reliable under high speed, high torque, or vibration conditions, and to improve the centering and positioning accuracy.

[0081] 13. Wide range of applications, expanding the application boundaries of disposable reactors: enabling disposable bioreactors to enter the field of high-intensity stirring applications that were previously limited to stainless steel reactors, and promoting the industrial application of disposable technology in larger-scale and higher-density cultivation. Attached Figure Description

[0082] Figure 1 This is an overall view of a stirred bioreactor; Figure 2 This is a cross-sectional view of a stirred bioreactor; Figure 3 This is a schematic diagram of the combined structure of the stirring drive system and the stirring paddle inside the bag (partial explosion of the mechanical seal assembly). Figure 4 Exploded view of the stirring drive system and mechanical seal assembly; Figure 5 This is a schematic diagram of the stirring drive shaft and drive tank structure; Figure 6 This is a schematic diagram of the stirring drive shaft structure; Figure 7 This is a schematic diagram of the drive slot structure; Figure 8 A schematic diagram of a stirring drive shaft with a polymer bushing. Figure 9 A simplified cross-sectional schematic diagram of the stirring drive system and mechanical seal assembly; Figure 10 This is a partial cross-sectional schematic diagram of the mechanical seal assembly; Figure 11 This is a schematic diagram of the structure of the agitator and the second sealing component (partial explosion of the second sealing component). Figure 12 Exploded view of the second sealing component; Figure 13 This is a structural diagram of the support frame; Figure 14 This is a diagram of the second sealing component assembly. Detailed Implementation

[0083] To describe the present invention more specifically, the technical solution of the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments. These descriptions are merely illustrative of how the present invention is implemented and do not limit the specific scope of the invention. All materials used in this experiment are commercially available. The scope of the present invention is defined in the claims.

[0084] Example 1: A stirred bioreactor based on mechanical seal shaft drive provided by the present invention. The stirred bioreactor 1 provided in this embodiment, such as Figures 1-3As shown, the system includes a stirring drive system 2 and a flexible bag 3. A stirring paddle 4 is housed within the flexible bag 3, and the main shaft 5 of the stirring paddle 4 has a first end 6 extending outside the flexible bag 3. A mechanical seal assembly is provided on the flexible bag 3 to cooperate with the main shaft 5, sealing the connection 7 between the main shaft 5 and the flexible bag 3, forming a sterile and closed system. The stirring drive system 2 includes a stirring drive shaft 8. The stirring drive shaft 8 can be mechanically connected to the first end 6 of the main shaft 5, thereby driving the rotation of the stirring paddle 4 within the flexible bag 3. Mechanical connection means that the stirring drive shaft 8 and the first end 6 are rigidly connected solely through a mechanical structure, without requiring any other force (such as magnetic force) for connection. Of course, the stirred bioreactor 1 may also include a rigid reaction vessel for placing or supporting the flexible bag 3, located outside the flexible bag 3. This structure is more conventional and is therefore not shown in the figure.

[0085] like Figures 3-5 As shown, the top of the first end 6 is provided with a drive groove 9, and the bottom 10 of the drive groove 9 is located outside the flexible bag body 3; the stirring drive shaft 8 is inserted into the drive groove 9 to achieve mechanical connection. The drive groove 9 is set at the top of the first end 6 and its bottom 10 is located outside the flexible bag body 3, which is equivalent to building a closed force-bearing chamber at the end of the main shaft 5. It can completely accommodate the stirring drive shaft 8 in the drive groove 9 for torque transmission, so that the mating surface of the drive groove 9 and the stirring drive shaft 8 (including the bottom 10 and the groove wall 11) is completely separated from the flexible bag body 3, avoiding the situation where the mating surface is inside the bag (not only is the operation troublesome and the alignment difficult, but the mating surface also has the risk of bacterial contamination and leakage), which increases the probability of bacterial contamination. At the same time, the drive groove 9 can also completely wrap the mating surface to avoid external environmental contamination or operational bumps. The inner wall of the drive groove 9 (such as the internal hexagonal hole) forms a precise surface fit with the outer shape of the stirring drive shaft 8, which not only achieves torque transmission with no or minimal clearance, but also automatically corrects minor radial deviations during insertion through the guiding effect of the groove wall 11, helping to improve the coaxiality after connection. Furthermore, the bottom 10 of the drive groove 9 can serve as an axial positioning reference, limiting the insertion depth of the stirring drive shaft 8 and ensuring consistent installation position each time. This groove-type connection moves the force point inward to the inside of the main shaft 5, allowing the driving reaction force to act more evenly on the cross-section of the main shaft 5, which helps to suppress radial vibration caused by the load on the distal impeller 20, thereby improving the stability of the rotating system and the seal life.

[0086] It is understood that in the mechanical seal transmission scheme provided in this embodiment, the direction of the main shaft 5 of the stirring paddle 4 inside the flexible bag 3 can not only be set in the traditional top-to-bottom mode, but also in any direction such as below, front, back, left, or right. It can be connected to the stirring drive shaft 8 provided by the motor 47 in the corresponding direction, thereby providing a stirring function for cell culture inside the flexible bag 3. That is to say, the first end 6 of the main shaft 5 can extend out of the flexible bag 3 from any one or more directions, such as above, below, left, right, front, or back.

[0087] The cross-section of the drive groove 9 can be any one or more of the following shapes: circular, triangular, or polygonal. Theoretically, any shape of drive groove 9, as long as it can be connected to a matching stirring drive shaft 8, can be used to achieve a mechanical connection and for mechanical seal transmission; however, the transmission effect may differ. For example... Figure 5 and Figure 7 As shown, preferably, the insertion end 12 of the stirring drive shaft 8 provided in this embodiment is an external hexagonal prism structure with a regular hexagonal cross-section; the driving groove 9 is an internal hexagonal hole with a regular hexagonal cross-section, matching the shape of the insertion end 12 of the stirring drive shaft 8. Using an external hexagonal prism with a regular hexagonal cross-section and an internal hexagonal hole as the driving connection method, torque is transmitted through six evenly distributed contact surfaces, making the driving force more dispersed and the stress distribution more uniform. This not only can bear high torque output but also effectively avoids deformation or fatigue failure caused by local stress concentration. While achieving efficient power transmission, it significantly improves the operational reliability and sealing life of the disposable bioreactor under high-intensity stirring conditions.

[0088] Preferably, the insertion end 12 of the stirring drive shaft 8 is a tapered hexagonal prism with a 0.5-2 degree taper from the root to the end along its axial direction. This tapered structure provides a progressive guide during insertion, allowing for smooth sliding even with slight angular deviations, significantly reducing the difficulty of bag installation. Furthermore, with the axial locking force, the tapered surface 13 gradually weds into the inner hexagonal hole wall (groove wall 11), adaptively eliminating radial clearance caused by machining tolerances and achieving a near-zero clearance rigid connection, thereby effectively suppressing micro-movements and vibrations caused by clearances. Simultaneously, the tapered design allows for control of the tightness through insertion depth, automatically compensating for wear caused by repeated insertions and removals, extending the spindle's service life. In addition, the tapered surface contact ensures uniform axial transmission of driving force, avoiding stress concentration at the root of the straight edges, and better protecting the edges of the drive groove 9 under high torque conditions. Thus, while ensuring smooth insertion and removal, it significantly improves the connection rigidity, vibration resistance, and durability of the rotating system. In this embodiment, the shrinkage taper is preferably 1 degree, which can eliminate gaps through appropriate wedging and ensure vibration-free rigid transmission, while retaining the engineering convenience of smooth insertion and removal. At the same time, the contact stress distribution is uniform, which can take into account both torque carrying capacity and service life.

[0089] Preferably, the root 14 of the insertion end 12 is in interference contact with the drive groove 9, which further strengthens the connection rigidity. When the drive shaft 8 is fully inserted, the interference fit of the root 14 forms a local prestressed contact, which is equivalent to adding an auxiliary support point at the fixed end of the cantilever beam, effectively suppressing the slight sway caused by the load of the distal impeller 20; at the same time, the gradually tightening wedge of the interference section and the tapered section together constitute a double locking mechanism, making the torque transmission path more continuous and avoiding connection loosening due to wear of a single mating surface or tolerance fluctuations; in addition, the tight fit of the root 14 can also play a damping role, absorbing the small vibrations that may be generated during high-speed rotation and preventing vibration from being transmitted to the mechanical seal; the interference contact also provides a defined axial thrust position, ensuring that the relative position height of the main shaft 5 and the stirring drive shaft 8 is consistent after each installation, thereby improving the overall stability of the rotation system and extending its service life.

[0090] like Figure 6 and Figure 7 As shown, the connection ends of the stirring drive shaft 8 and the main shaft 5 are respectively provided with mutually matching positioning structures 15. The positioning structure 15 is a mutually matching concave-convex structure, the core function of which is to significantly improve the accuracy and stability of the connection system through a physical alignment mechanism. The concave-convex structure forms a precision fit before torque transmission. The fit between the convex and concave parts forces the correction of the rotation axes of the stirring drive shaft 8 and the main shaft 5, making them highly coincident. This effectively suppresses radial runout caused by machining tolerances or installation deviations, and avoids the transmission of radial vibration to the mechanical seal. The concave-convex mating surfaces form a rigid support point after connection, which can withstand the shear force generated during high-speed rotation and prevent relative slippage or fretting wear at the connection end. At the same time, the concave-convex structure can serve as a dual positioning reference for the axial and radial directions, ensuring that the relative positions of the stirring drive shaft 8 and the main shaft 5 are consistent after each installation, improving assembly repeatability. In addition, the concave-convex fit concentrates the force on the positioning surface, avoiding the connection bolts or clamps bearing the impact load alone, thereby improving the overall rigidity and reliability of the rotation system.

[0091] Preferably, the positioning structure 15 includes a first positioning step 16 located at the upper end of the drive groove 9 and a second positioning step 17 located at the corresponding position of the stirring drive shaft 8. This design provides precise axial and radial limits for the insertion depth and radial position of the stirring drive shaft 8 through end-face contact, ensuring that the relative position height between the main shaft 5 and the stirring drive shaft 8 is consistent after each installation, significantly improving the repeatability of positioning accuracy during consumable replacement. Simultaneously, the positioning steps separate the axial load from the torque transmission path, allowing the hexagonal mating surface to focus on torque transmission, preventing axial forces generated by operational collisions or thermal expansion and contraction from directly acting on the bottom of the drive groove 9, thereby protecting the torque transmission surface from improper clamping or wear. Furthermore, the contact of the step end faces provides the operator with clear feedback on the installation being in place, and when locked with clamps, it effectively suppresses slight oscillations caused by the load on the distal impeller 20, enhancing the overall rigidity of the rotating system. This design achieves precise positioning, protects core transmission components, and improves assembly reliability without significantly increasing manufacturing complexity, making it a preferred structure that balances functionality and engineering economy.

[0092] In this embodiment, the axial direction refers to the length direction of the main shaft 5, and the radial direction refers to the direction perpendicular to the main shaft 5, that is, the diameter or radius direction of the main shaft 5.

[0093] like Figure 8 The insertion end 12 is provided with a polymer bushing 18, which forms a self-lubricating friction pair with the drive groove 9. Through the low coefficient of friction and self-lubricating properties of the polymer material, the frictional resistance and wear between the insertion end 12 and the drive groove 9 can be significantly reduced, improving transmission efficiency and extending the service life of the components. At the same time, the polymer bushing 18 can absorb certain assembly tolerances and vibrations, making the transmission smoother and the noise lower. It can also effectively suppress the micro-oscillation caused by the load on the distal impeller 20. In addition, the self-lubricating friction pair does not require the addition of additional lubricant, avoiding the risk of lubricant contamination of the sterile environment and meeting the cleanliness requirements of disposable bioreactors. The polymer bushing 18 is also easy to replace and maintain, reducing the cost of use.

[0094] Preferably, a polymer bushing 18 can be used to fill the gap formed by the contraction taper of the insertion end 12, thereby maintaining the insertion end 12 of the stirring drive shaft 8 in a regular hexagonal prism shape. After prolonged use, the gap caused by wear can be further eliminated by replacing the polymer bushing 18.

[0095] Preferably, the polymer bushing 18 is made of any one or more of PEEK, PTFE, and medical-grade POM. PEEK (polyetheretherketone), PTFE (polytetrafluoroethylene), or medical-grade POM (polyoxymethylene) all possess excellent self-lubricating properties, wear resistance, chemical corrosion resistance, and biocompatibility, ensuring long-term stable operation in dry friction or sterile environments and avoiding the risk of contaminating the culture system due to the addition of additional lubricants. Simultaneously, they can withstand the harsh conditions of steam sterilization or irradiation sterilization, maintaining dimensional stability and mechanical strength, thereby ensuring transmission accuracy and sealing reliability. While medical-grade POM has a lower cost, it is slightly inferior in long-term heat resistance. Considering performance, cost, and processability, the polymer bushing 18 is most preferably made of a composite material of PEEK and PTFE, exhibiting exceptional self-lubricating properties. In this embodiment, a polymer bushing 18 is prepared by adding 10% PTFE to a PEEK matrix. PEEK provides skeletal support and wear resistance, while PTFE further reduces the coefficient of friction, achieving a synergistic effect of strength and super lubrication. It is particularly suitable for harsh transmission scenarios with extremely high speeds or frequent start-stops.

[0096] like Figure 5 The outer surface 401 of the first end 6 of the spindle 5, corresponding to the first position 402 of the top 19 of the drive groove 9, gradually enlarges downwards and then rapidly retracts after passing the second position 403 corresponding to the bottom 10 of the drive groove 9. This "gradually enlarging and then rapidly retracting" shape design of the end of the spindle 5 has certain benefits in eliminating radial chatter and enhancing torque transmission stability due to the enlarged size at this position. It can also enhance the stability of torque transmission by utilizing shape changes when the spindle 5 rotates. At the same time, this arc-shaped design is more convenient to hold and grip, which is conducive to the quick connection and disconnection of the first end 6 and the drive groove 9, and facilitates replacement.

[0097] like Figure 2The first end 6 of the main shaft 5 extends from above the flexible bag body 3, and the second end 21 of the main shaft 5 is located inside the flexible bag body 3. A first sealing component 22 (i.e., a mechanical seal assembly, which enables the main shaft 5 to maintain good sealing and torque transmission effects even when rotating at high speed) is provided between the first end 6 and the flexible bag body 3. The bottom 10 of the drive groove 9 is higher than the first sealing component 22. The first sealing component 22 includes a disc seat 23, a sealing gasket 24, and a bearing 25 arranged sequentially from bottom to top. A low-friction pair is formed between the disc seat 23, the sealing gasket 24, and the main shaft 5. The bearing 25 includes a first bearing 26 and a second bearing 27. The inner rings of the first bearing 26 and the second bearing 27 are tightly fitted with the main shaft 5 of the stirring paddle 4. The mechanical seal structure used in this scheme is a contact end face seal, and a certain clamping force is maintained between the sealing gasket 24 and the disc seat 23 to achieve a reliable seal. Although the clamping force generates frictional torque, due to the use of low-friction materials, high-smoothness mating surfaces, and optimized clamping force design, the frictional torque is far less than the rated output torque of the drive motor and will not affect the normal operation of the stirring system 2. Simultaneously, since the flexible bag 3 is for single use, the wear of the sealing gasket 24 is extremely low within a single batch culture cycle, completely unaffected by sealing performance or rotational stability. The low-friction pair formed by the disc seat 23 and the sealing gasket 24 reduces frictional resistance and heat generation on the main shaft 5 during high-speed rotation, ensuring both sealing effectiveness and transmission efficiency, and extending seal life. The dual-bearing design (first bearing 26 and second bearing 27) and its tight fit with the main shaft 5 ensure the rotational stability of the stirring paddle 4, preventing seal failure or uneven torque transmission due to radial runout.

[0098] Preferably, the first sealing component 22 further includes a disc cover plate 28 extending circumferentially from the main shaft 5. The disc cover plate 28 has a downwardly extending leak-proof protective layer 58, which is perpendicular to the disc surface 59 of the disc cover plate 28. The disc cover plate 28 is matched and engaged with the disc seat 23, with a first cavity 29 in the middle; the sealing gasket 24 is placed in the first cavity 29 between the disc cover plate 28 and the disc seat 23. The sealing gasket 24 completely fills all cavities between the disc cover plate 28 and the disc seat 23, ensuring a sealing effect and strictly preventing leakage and the risk of contamination. Figure 9 As shown, there is a second cavity 30 between the disc cover plate 28 and the second bearing 27, and a third cavity 61 between the disc seat 23 and the outer shell 33, both of which need to be filled with sealing gaskets 24 to ensure the sealing effect.

[0099] The disc cover plate 28 on the main shaft helps improve the dynamic sealing effect. During assembly, the disc seat 23 is directly welded and sealed to the flexible bag body 3. The main shaft 5 is inserted into the flexible bag body 3 through the central cutout 31 of the disc seat 23. The disc cover plate 28 is combined with the disc seat 23, and the middle cavity 29 and the second cavity 30 are sealed with sealing gaskets 24. The overall structure of the first sealing component 22 is compact and highly integrated, which not only achieves reliable sealing of the flexible bag body 3, but also ensures transmission accuracy and aseptic safety under high torque and high speed conditions. The disc seat 23 is made of a composite material of PEEK and PTFE, and the sealing gasket 24 is made of polytetrafluoroethylene, forming a bio-inert low-abrasion friction pair. Both the disc seat 23 and the sealing gasket 24 in the first sealing component contain PTFE components, which can form a synergistic effect of homologous lubrication during relative rotational motion, significantly reducing the coefficient of friction and the generation of wear particles at the contact interface, thereby constructing a bio-inert low-wear friction pair. At the same time, the PEEK matrix provides the disc seat 23 with excellent mechanical strength and high-temperature sterilization resistance, making up for the shortcomings of insufficient stiffness and easy creep of pure PTFE material, while the PTFE-filled sealing gasket maintains good compliance and sealing performance. This combination does not require additional coatings or lubricants, simplifies the process, controls costs, and fully meets the stringent requirements of single-use bioreactors for material biocompatibility, low exudates, and aseptic safety.

[0100] The leak-proof protective layer 58 and the disc surface 59 of the disc cover plate 28 are perpendicular to each other and extend downwards, which can significantly improve the dynamic sealing effect. Once the culture medium enters the first sealing component 22, or even enters the disc seat 23, due to the stirring and centrifugal action, the culture medium must leak outwards and upwards to truly detach from the first sealing component 22. The downward-extending isolation plate of the leak-proof protective layer 58 prevents the liquid from penetrating outwards and upwards, thus making the dynamic sealing effect more reliable.

[0101] like Figure 10 The disc seat 23 has a micron-level dynamic pressure groove 31 on the sealing end face between it and the main shaft 5, which is used to achieve fluid dynamic pressure lubrication and sealing; the micron-level dynamic pressure groove 31 is one or more combinations of spiral groove, herringbone groove or arc groove.

[0102] Micron-level dynamic pressure grooves 31 are provided on the sealing end faces of the disc seat 23 and the main shaft. When the main shaft 5 rotates, the micron-level dynamic pressure grooves 31 pump the culture medium or gas into the space between the sealing end faces through the hydrodynamic pressure effect, forming an extremely thin bearing liquid film. This enables the end faces to operate in a non-contact manner, thereby significantly reducing the friction coefficient and end face wear, and avoiding contamination from wear debris caused by solid contact. At the same time, this liquid film can also play a dynamic sealing role, effectively preventing external contaminants from entering and internal culture medium from leaking, thus improving sterility reliability. Different groove designs can also adapt to bidirectional rotation or enhance the pumping effect, ensuring that a stable lubricating film is maintained within a wide speed range, thereby significantly extending the seal life, reducing maintenance requirements, and meeting the stringent requirements of disposable bioreactors for high-speed and high-torque operating conditions. Different hydrodynamic groove types play different roles in the hydrodynamic effect of fluid. Spiral grooves have strong unidirectional pumping capacity, continuously pressurizing fluid into the sealing end face to form a stable liquid film, suitable for high-efficiency sealing in a single rotation direction. Herringbone grooves consist of two sets of symmetrical spiral grooves, generating a centripetal pumping effect during bidirectional rotation, automatically balancing end face pressure and preventing liquid film instability, making them particularly suitable for conditions with frequent starts and stops or uncertain directions. Arc-shaped grooves are annular grooves, each parallel to the others, reducing fluid resistance through streamlined design, lowering temperature rise during high-speed rotation, and enhancing liquid film stiffness. In this embodiment, spiral groove 32 is preferred because it has the highest pumping efficiency and best liquid film stability in a single rotation direction, suitable for long-term rotation in one direction and achieving a stable sealing effect.

[0103] like Figure 9 and Figure 10 The first sealing component 22 also includes a housing 33. The bottom 34 of the housing 33 is welded to the flexible bag body 3. The top 35 of the housing 33 is provided with a first chuck 36. The housing 33 is also provided with a cover plate 49, which is threaded to the housing 33. The cover plate 49 can be opened to insert the sealing gasket 24 and then tightened. The stirring drive shaft 8 is also provided with a second chuck 37. The first chuck 36 and the second chuck 37 are used in combination with the clamp 48 to realize the fixed connection between the stirring drive shaft 8 and the main shaft 5. In this embodiment, the first chuck 36 and the second chuck 37 are TC chucks. The TC chuck simultaneously undertakes three major functions: fixing the motor base, pressing the sealing gasket, and centering the transmission. This is a functional integration innovation. In addition, at the contact position 50 between the main shaft 5 and the cover plate 49, the size of the main shaft 5 is reduced, so that there is a gap between it and the cover plate 49, avoiding friction between the main shaft 5 and the cover plate 49 during the rotation of the main shaft 5, which would affect the transmission effect.

[0104] like Figure 11 A second sealing component 38 is provided below the flexible bag body 3; a support seat 39 is provided on the second sealing component 38 to support the second end 21 of the main shaft 5.

[0105] Preferably, the support base 39 is provided with a third bearing 40, and the second end 21 is tightly fitted with the outer ring of the third bearing 40 and can rotate freely; the second sealing component 38 includes a second disc 41 for welding and sealing with the flexible bag body 3. Since the second end 21 of the main shaft 5 is far from the first end 6, this length extension causes the center of gravity of the stirring drive to be separated from the impeller hub by a long distance. Once misalignment occurs, it can quickly lead to vibration and eventually disengagement, especially at high speeds, where the impact is more severe. This embodiment incorporates a second sealing component 38, including a support base 39 and a third bearing 40, located below the flexible bag body 3 (still inside the flexible bag body 3). The third bearing 40 reliably supports the second end 21 of the main shaft 5, effectively suppressing radial vibration and swaying that may occur when the long cantilevered stirring shaft 4 rotates at high speed. This significantly improves the stability of high-torque transmission and the smooth operation of the stirring paddle 4. Simultaneously, since the second end 21 of the main shaft 5 does not need to extend outside the bag body, a complex rotary dynamic seal is no longer required. A simple static sealing structure, formed by welding the second disc 41 to the flexible bag body 3 (which can be used for pipelines conveying culture medium without requiring a rotary dynamic seal), is sufficient to ensure the aseptic integrity of the bag body. This completely eliminates the traditional leakage risk point of the bottom dynamic seal, significantly reducing the possibility of seal failure. This multi-bearing layout, with dynamic seals at the top and static seals at the bottom, ensures the mechanical reliability of the large reaction bag under high-intensity stirring conditions and simplifies the bottom sealing structure, making overall aseptic assurance more reliable and assembly more convenient.

[0106] like Figures 12-14 The support base 39 is equipped with a receiving disc 43 and a support frame 44. The second end 21 is equipped with a support structure 45 that cooperates with the support frame 44. When the support structure 45 and the support frame 44 are combined, the second end 21, the support structure 45, and the support frame 44 form an integrated structure, allowing free rotation under the support of the support base 39. The design of adding a support base 39 to the second end 21 of the main shaft 5 provides stable radial and axial support for the end of the main shaft 5 extending into the flexible bag body 3, significantly improving the operational stability of the main shaft 5 under high-speed rotation or high-torque conditions. The receiving disc 43 plays a crucial role in ensuring stability and alignment, effectively suppressing radial sway and axial movement of the main shaft 5, thereby reducing the risk of wear on the mechanical seal and ensuring smooth rotation of the agitator 4, preventing radial sway and axial movement. Simultaneously, a microchannel can be set inside the receiving disc 43 as an aeration disc for supplying gas from the outside to the flexible bag body 3. Existing aeration discs are usually fixed to the bottom of the bag. The receiving disc 43 in this embodiment can be used to improve the radial oscillation and axial movement of the stirring paddle 4, and can also be used as an aeration disc. By rotating, the aeration effect can be improved, which can be described as killing two birds with one stone.

[0107] After the support structure 45 on the second end 21 is combined with the support frame 44, it maintains its ability to rotate freely under the support of the support seat 39, without affecting the rotation of the main shaft 5. A first spring piece 52 with a first barb 51 and a connecting groove 61 are provided above the support frame 44. Figure 13 The connecting groove 61 has a hollow structure in the middle, used to connect the support structure 45 on the second end 21 of the main shaft and rotate together. The connecting groove 61 is used for the stable connection between the support structure 45 and the support frame 44. The first spring piece 52 with the first barb 51 can prevent the support structure 45 from separating from the support frame 44 during rotation. The support structure 45 has a narrowed annular groove 60, which can be embedded in the first barb 51 of the first spring piece 52 for fixation. The support frame 44 is provided with a second spring piece 54 with a second barb 53 below, which can be sleeved on the outer surface 55 of the third bearing 40 to achieve connection and fixation with the outer surface 55 of the third bearing 40 for rotation. The inner surface 56 of the third bearing 40 is sleeved on the protruding support column 57 on the support seat 39 for fixation. That is to say, the inner surface 56 of the third bearing 40 is fixed, while the outer surface 55 can rotate freely, unlike the first and second bearings. The first bearing 26 and the second bearing 27 of the first end 6 of the fixed spindle 5 are fixed on the outer surface and connected to the first end on the inner surface so that they can rotate freely, while the third bearing 40 is exactly the opposite.

[0108] This design, which swaps the functions of the inner and outer rings of the third bearing 40, provides rigid support at the end of the main shaft 5 while increasing the stability of the rotation due to the enlarged rotating chassis, effectively preventing radial vibration. It also cleverly mitigates the potential threat to the bag's sealing and structural stability posed by the bearing's own rotation. Specifically, by fixing the inner ring of the bearing and rotating the outer ring synchronously with the support structure 45 on the main shaft 5, a stable radial constraint is provided to the extended cantilever shaft end, making the area below the main shaft 5 more stable and better at suppressing swaying and vibration under high-speed rotation. This also avoids introducing an additional rotational sealing point at the bottom of the bag. Because the rotating components (support structure 45 and third bearing 40) are entirely located inside the bag and integrated with the main shaft 5, the stationary support seat 39 can pass through the bag from the inside to the outside. The two are dynamically isolated through the functional separation of the inner and outer rings of the bearing. This design not only significantly enhances the rigidity and durability of the stirring system under high torque conditions but also ensures that the welded interface at the bottom of the bag is not affected by rotational friction, maximizing transmission stability while effectively guaranteeing the integrity of the sterile barrier of the disposable bioreactor.

[0109] Meanwhile, a pipe 46 can also be installed on the support base 39. For example, the pipe 46 can extend from the outside of the bag through the support base 39 into the bag, thereby draining liquid or taking samples from the flexible bag 3. This design also enhances the rigidity of the entire transmission system, enabling it to withstand stronger stirring requirements and meet the demanding process conditions of high-viscosity, high-density cell culture. At the same time, it avoids vibration or sealing failure caused by excessive cantilever of the main shaft 5, further improving the reliability and safety of the disposable bioreactor in large-scale production. Of course, after the support base 39 extends out of the flexible bag 3, it needs to be fixed at the bottom of the reaction tank. Similarly, the position of the upper motor 47 also needs to be fixed to ensure the stable operation of the entire stirring drive system 2 and the stirring paddle 4.

[0110] Example 2: A stirred bioreactor The stirred bioreactor 1 provided in this embodiment, such as Figures 1-3 As shown, the system includes a stirring drive system 2 and a flexible bag 3. The flexible bag 3 contains a stirring paddle 4, and the main shaft 5 of the stirring paddle 4 has a first end 6 extending out of the flexible bag 3. The flexible bag 3 is provided with a mechanical seal assembly that cooperates with the main shaft 5. The mechanical seal assembly seals the connection 7 between the main shaft 5 and the flexible bag 3, forming a sterile and closed system. The stirring drive system 2 is provided with a stirring drive shaft 8. The stirring drive shaft 8 can be mechanically connected to the first end 6 of the main shaft 5, thereby driving the stirring paddle 4 to rotate inside the flexible bag 3.

[0111] like Figures 3-5 As shown, the top of the first end 6 is provided with a drive groove 9, and the bottom 10 of the drive groove 9 is located outside the flexible bag body 3; the stirring drive shaft 8 is inserted into the drive groove 9 to achieve mechanical connection. The insertion end 12 of the stirring drive shaft 8 is an external hexagonal prism structure with a regular hexagonal cross-section; the drive groove 9 is an internal hexagonal hole with a regular hexagonal cross-section, matching the shape of the insertion end 12 of the stirring drive shaft 8. Among them, the insertion end 12 of the stirring drive shaft 8 is a tapered hexagonal prism, and its axial direction has a 1-degree tapering taper from the root to the end. The root 14 of the insertion end 12 is in interference contact with the drive groove 9.

[0112] like Figure 6 and Figure 7 As shown, the connection ends of the stirring drive shaft 8 and the main shaft 5 are respectively provided with matching positioning structures 15, including a first positioning step 16 located at the upper end of the drive groove 9 and a second positioning step 17 located at the corresponding position of the stirring drive shaft 8.

[0113] like Figure 8The insertion end 12 is provided with a polymer bushing 18, which forms a self-lubricating friction pair with the drive groove 9. The polymer bushing 18 is prepared by adding 10% PTFE to a PEEK matrix. The polymer bushing 18 fills the gap formed by the contraction taper of the insertion end 12, thereby maintaining the insertion end 12 of the stirring drive shaft 8 into a regular hexagonal prism shape.

[0114] like Figure 5 The outer surface 401 of the first end 6 of the spindle 5 corresponds to the first position 402 of the top 19 of the drive groove 9. The shape gradually expands downwards and then quickly shrinks back after passing the second position 403 corresponding to the bottom 10 of the drive groove 9.

[0115] like Figure 2 The first end 6 of the main shaft 5 extends out of the flexible bag 3 from above, and the second end 21 of the main shaft 5 is located inside the flexible bag 3; a first sealing component 22 is provided between the first end 6 and the flexible bag 3; the bottom 10 of the drive groove 9 is higher than the first sealing component 22. The first sealing component 22 includes a disc seat 23, a sealing gasket 24 and a bearing 25 arranged sequentially from bottom to top; the disc seat 23, the sealing gasket 24 and the main shaft 5 form a low friction pair; the bearing 25 includes a first bearing 26 and a second bearing 27; the inner rings of the first bearing 26 and the second bearing 27 are tightly fitted with the main shaft 5 of the stirring paddle 4.

[0116] Preferably, the first sealing component 22 further includes a disc cover plate 28 extending circumferentially from the main shaft 5; the disc cover plate 28 is matched and engaged with the disc seat 23, and a first cavity 29 is provided in the middle; the disc cover plate 28 is provided with a downwardly extending leak-proof protective layer 58, the leak-proof protective layer 58 being perpendicular to the disc surface 59 of the disc cover plate 28; the sealing gasket 24 is placed in the first cavity 29 and the second cavity 30 between the disc cover plate 28 and the disc seat 23. The sealing gasket 24 completely fills all cavities between the disc cover plate 28 and the disc seat 23, ensuring a sealing effect and strictly preventing leakage and the risk of contamination. Figure 9 As shown, a second cavity 30 exists above the disc cover 28 and between it and the second bearing 27, which also needs to be filled with a sealing gasket 24 to ensure a sealing effect. During assembly, the disc seat 23 is directly welded and sealed to the flexible bag body 3. The main shaft 5 is inserted into the flexible bag body 3 through the central cutout 31 of the disc seat 23. The disc cover 28 is combined with the disc seat 23, and both the central cavity 29 and the second cavity 30 are sealed with sealing gaskets 24. The disc seat 23 is made of PEEK matrix with 5-10% PTFE added, and the sealing gasket 24 is made of polytetrafluoroethylene, forming a bio-inert low-abrasion friction pair.

[0117] like Figure 10The disc seat 23 has a micron-level dynamic pressure groove 31 on the sealing end face between it and the main shaft 5, which is used to achieve fluid dynamic pressure lubrication and sealing; the micron-level dynamic pressure groove 31 is a spiral groove. The spiral groove 32 has a groove depth of 3 microns, a spiral angle of preferably 15 degrees, and a groove width of 2 microns. Because it has the highest pumping efficiency and the best liquid film stability in a single rotation direction, it is suitable for long-term rotation in a single direction and can achieve a stable sealing effect.

[0118] like Figure 9 and Figure 10 The first sealing component 22 also includes a housing 33. The bottom 34 of the housing 33 is welded to the flexible bag body 3. The top 35 of the housing 33 is provided with a first chuck 36. The housing 33 is also provided with a cover plate 49, which is threaded to the housing 33. The cover plate 49 can be opened to insert the sealing gasket 24 and then tightened. The stirring drive shaft 8 is also provided with a second chuck 37. The first chuck 36 and the second chuck 37 are used in combination with the clamp 48 to realize the fixed connection between the stirring drive shaft 8 and the main shaft 5. In this embodiment, the first chuck 36 and the second chuck 37 are TC chucks. In addition, at the contact position 50 between the main shaft 5 and the cover plate 49, the size of the main shaft 5 is reduced, so that there is a gap between it and the cover plate 49, so as to avoid friction between the main shaft 5 and the cover plate 49 during the rotation of the main shaft 5, which would affect the transmission effect.

[0119] like Figure 11 A second sealing component 38 is provided below the flexible bag body 3; a support seat 39 is provided on the second sealing component 38 to support the second end 21 of the main shaft 5. A third bearing 40 is provided on the support seat 39, and the second end 21 is tightly fitted with the outer ring of the third bearing 40 and can rotate freely; the second sealing component 38 includes a second disc 41 for welding and sealing with the flexible bag body 3.

[0120] like Figures 12-14 The support base 39 is provided with a receiving disc 43 and a support frame 44. The second end 21 is provided with a support structure 45 that cooperates with the support frame 44. When the support structure 45 is combined with the support frame 44, the second end 21, the support structure 45, and the support frame 44 form an integral structure and can rotate freely under the support of the support base 39.

[0121] After the support structure 45 on the second end 21 is combined with the support frame 44, it maintains its ability to rotate freely under the support of the support seat 39, without affecting the rotation of the main shaft 5. A first spring piece 52 with a first barb 51 and a connecting groove 61 are provided above the support frame 44. Figure 13The support structure 45 on the second end 21 of the main shaft 5 is used for connection. The support structure 45 has a narrowed annular groove 60, which can be embedded in the first barb 51 of the first spring piece 52 for fixation. The connection groove 61 is used for the stable connection between the support structure 45 and the support frame 44. The second spring piece 54 with a second barb 53 is provided below the support frame 44, which can be sleeved on the outer surface 55 of the third bearing 40 to achieve connection and fixation with the outer surface 55 of the third bearing 40 for rotation. The inner surface 56 of the third bearing 40 is sleeved on the protruding support column 57 on the support seat 39 for fixation. That is to say, the inner surface 56 of the third bearing 40 is fixed, and the outer surface 55 can rotate freely, which is different from the first and second bearings. The first bearing 26 and the second bearing 27 that fix the first end 6 of the main shaft 5 have fixed outer surfaces and their inner surfaces are connected to the first end for free rotation, while the third bearing 40 is exactly the opposite. At the same time, a pipe 46 can also be provided on the support seat 39. For example, the pipe 46 can go from the outside of the bag through the support seat 39 and into the bag, thereby draining liquid or taking samples from the flexible bag 3. After the support base 39 extends out of the flexible bag body 3, it is fixed at the bottom of the reaction vessel, and the position of the motor 47 above is also fixed, thereby ensuring the stable operation of the entire stirring drive system 2 and the stirring paddle 4.

[0122] Experimental Example 3: A method for cell culture using a disposable bioreactor based on mechanical seal shaft drive. This embodiment uses the disposable stirred bioreactor provided in Example 2 for culturing, and the cultured cells are human embryonic kidney cells HEK-293. The specific culturing process is as follows: HEK-293 was inoculated into FreeStyle™ 293 Expression Medium (Thermo Fisher Scientific) at a density of 0.6 × 10⁻⁶. 6 The culture medium volume was 2L in a 5L fermenter, the temperature was 37℃, the CO2 aeration rate was 8%, the dissolved oxygen content was 40%, the pH was 7.1, the stirring speed was 100 rpm, and the culture time was 5 days. Cell viability and cell density were measured after culture. Cell viability was detected by flow cytometry using propidium iodide (PI) dye, and cell density was measured by cell counting. After 5 days of culture, the cell viability of HEK-293 was 95.9%, and the cell density was 33.5 × 10⁻⁶ cells / ml. 6 cells / ml.

[0123] Experiment Example 4: The Influence of Polymer Bushing on Stirring Effect This embodiment uses the disposable stirred bioreactor provided in Example 2 to culture HEK-293 cells according to the method provided in Example 3. The polymer bushing on the stirring drive shaft is made of several materials as shown in Table 1. Other conditions are kept the same, and each group is cultured 5 times to investigate the effect of the polymer bushing setting on the batch-to-batch effect of HEK-293 cell culture.

[0124] Table 1. Influence of the polymer bushing of the stirring drive shaft on the stirring effect As shown in Table 1, compared with the control group, adding polymer bushings helps improve batch-to-batch consistency of HEK-293 cell culture, significantly reduces CV value, and improves culture effect. At the same time, there are significant differences in the improvement of consistency when using polymer bushings made of different materials. Among them, the fourth group is the best choice, which helps improve batch-to-batch consistency of HEK-293 cell culture, reduces CV value to 0.21%, and has very high batch-to-batch consistency.

[0125] Experiment Example 5: The Influence of Sealing Gasket Material Selection on Mixing Effect This embodiment uses the disposable stirred bioreactor provided in Example 2 to culture HEK-293 cells according to the method provided in Example 3. The sealing gaskets are prepared using several materials as shown in Table 1. Other conditions are kept consistent. Each group is cultured 5 times and the average value is taken to investigate the effect of the choice of sealing gasket material on the HEK-293 cell culture effect.

[0126] Table 2. The Influence of Gasket Material Selection on Mixing Effect As shown in Table 2, the sealing gaskets prepared using different materials exhibit varying effects. A combination of PEEK and PTFE is preferred for preparing the gaskets, as it helps improve the sealing effect and also increases cell culture density. This may be related not only to the sealing effect but also to the cell compatibility of the gasket materials. The optimal method is to add 5-10% PTFE to PEEK, which achieves the best cell culture density while ensuring no leakage.

[0127] While the present invention has been disclosed above, it is not limited thereto. Any person skilled in the art can make various modifications and alterations without departing from the spirit and scope of the invention; therefore, the scope of protection of the present invention should be determined by the scope defined in the claims.

Claims

1. A stirred bioreactor, characterized in that, The utility model provides a stirring drive system and a flexible bag body, the flexible bag body is equipped with a stirring paddle, the main shaft of the stirring paddle has a first end extending out of the flexible bag body, the flexible bag body is equipped with a mechanical seal assembly matched with the main shaft, the mechanical seal assembly seals the joint of the main shaft and the flexible bag body, the stirring drive system is equipped with a stirring drive shaft, the stirring drive shaft can be mechanically connected with the first end of the main shaft to drive the rotation of the stirring paddle in the flexible bag body.

2. The stirred bioreactor of claim 1, wherein, The top of the first end is equipped with a drive groove, the bottom of the drive groove is located outside the flexible bag body, the stirring drive shaft is inserted into the drive groove to realize mechanical connection, the cross section of the drive groove is circular, triangular or polygonal.

3. The stirred bioreactor of claim 2, wherein, The first end of the main shaft extends out of the flexible bag body from any one or more of the following directions: above, below, left side, right side, front side and back side; and / or the insertion end of the stirring drive shaft is an outer hexagonal prism structure with a cross section in the shape of a regular hexagon; the drive groove is an inner hexagonal hole with a cross section in the shape of a regular hexagon, matching the shape of the insertion end of the stirring drive shaft.

4. The stirred bioreactor of claim 3, wherein, The insertion end of the stirring drive shaft is a tapered hexagonal prism, the shaft is tapered from the root to the end at an angle of 0.5-2 degrees; the root of the insertion end is in interference contact with the drive groove.

5. The stirred bioreactor of claim 4, wherein, The connection ends of the stirring drive shaft and the main shaft are respectively equipped with matching positioning structures; the positioning structures are matching concave-convex structures; the positioning structures include a first positioning step at the upper end of the drive groove and a second positioning step at the corresponding position of the stirring drive shaft.

6. The stirred bioreactor of claim 4, wherein, The insertion end of the stirring drive shaft is externally equipped with a polymer bushing, a self-lubricating friction pair is formed between the polymer bushing and the drive groove; the polymer bushing is made of any one or more of PEEK, PTFE and medical-grade POM.

7. The stirred bioreactor of claim 2, wherein, The first end of the main shaft corresponds to the position of the top of the drive groove, the shape gradually enlarges downward, and then rapidly shrinks after passing the corresponding position of the bottom of the drive groove; the first end of the main shaft extends out of the flexible bag body from above, and the second end of the main shaft is located inside the flexible bag body; a first sealing component is arranged between the first end and the flexible bag body; the bottom of the drive groove is higher than the first sealing component.

8. The stirred bioreactor of claim 7, wherein, The first sealing component includes a disc seat, a sealing gasket and a bearing arranged in sequence from bottom to top; a low-friction pair is formed between the disc seat and / or the sealing gasket and the main shaft; the bearing includes a first bearing and a second bearing; the inner rings of the first bearing and the second bearing are tightly matched with the main shaft of the stirring paddle; the first sealing component further includes a disc cover plate extending outward in the circumferential direction of the main shaft; the disc cover plate is matched and combined with the disc seat, and a cavity is arranged therebetween; the sealing gasket is arranged in the cavity between the disc cover plate and the disc seat; the disc seat is made of a composite material of PEEK and PTFE, the sealing gasket is filled with polytetrafluoroethylene, forming a biologically inert low-abrasive friction pair; the disc cover plate is equipped with a downwardly extending leakage-proof protective layer, which is perpendicular to the disc surface of the disc cover plate.

9. The stirred bioreactor of claim 8, wherein, The sealing end face between the disc seat and the main shaft is provided with micron dynamic pressure grooves for realizing hydrodynamic pressure lubrication sealing; the micron dynamic pressure grooves are one or more combinations of spiral grooves, herringbone grooves or arc grooves; and / or the first sealing component further comprises an outer shell, the bottom of the outer shell is welded integrally with the flexible bag body, and the top end of the outer shell is provided with a first chuck; the stirring driving shaft is further provided with a second chuck; the first chuck and the second chuck are used to realize fixed connection of the stirring driving shaft and the main shaft in combination with the clamp.

10. The stirred bioreactor of claim 7, wherein, The lower part of the flexible bag body is provided with a second sealing component; the second sealing component is provided with a support seat; the support seat is provided with a third bearing, and the second end is tightly matched with the outer ring of the third bearing and can freely rotate; the support seat is provided with a bearing disc and a support frame, and the second end is provided with a support structure matched with the support frame; the support structure freely rotates with the support seat after being combined with the support frame; the upper part of the support frame is provided with a first elastic sheet with a first barb and a combination groove for combining the support structure; the lower part of the support frame is provided with a second elastic sheet with a second barb, which can be sleeved on the outer surface of the third bearing to realize connection with the outer surface of the third bearing for rotation; the second sealing component further comprises a second disc for welding sealing with the flexible bag body.

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