A single-use bioreactor and method for preparing an adenovirus harvest

Abstract

The application discloses a kind of disposable bioreactor and method for preparing adenovirus harvest, belong to biological preparation technical field, the bioreactor includes reaction bag and magnetic stirring mechanism, the magnetic stirring mechanism includes stirring shaft, stirring vane, the number of the stirring vane is greater than 1 and is installed on stirring shaft in layers, the stirring vane includes second stirring vane and first stirring vane, the second stirring vane is fixed on stirring shaft, the first stirring vane is installed on stirring shaft by collar, further include linkage module and shear force transmission device, the linkage module is used to drive shear force transmission device action, realize shear force transmission device and collar connection and disengage.The method is realized based on the bioreactor.Adopting the present scheme, the stirring consistency of disposable bioreactor can be effectively guaranteed in the process of being used for expansion culture.

Description

Technical Field

[0001] This invention relates to the field of bioproduct preparation technology, and in particular to a disposable bioreactor and method for preparing adenovirus harvest. Background Technology

[0002] Adenoviruses, commonly used viral vectors in gene therapy and vaccines, are characterized by high safety, genetic stability, and the ability to insert large target fragments. Adenoviruses can infect both dividing and non-dividing cells. Modified replication-deficient adenoviruses specifically proliferate only in 293 cells, further enhancing the safety of the viral vector.

[0003] Currently, conventional adenovirus culture methods primarily involve culturing adherent 293 cells in flasks or cell factories. However, this method suffers from low cell density, low total virus yield, difficulty in scale-up, and the risk of animal-derived contamination (serum), making it unsuitable for large-scale, high-quality, rapid vaccine production. Serum-free suspension culture of 293 cells is an emerging cell culture method that allows for large-scale, rapid scale-up of 293 cells. Due to its ease of operation, low cost, and absence of animal-derived contamination, this technology is gaining popularity among vaccine manufacturers.

[0004] In the prior art, disposable bioreactors are widely used in cell culture due to their simplicity, easy pollution control, and low initial investment. More specifically, patent application CN202210281775.5 discloses a method for perfusion culture of Expi293F cells. In particular, with decades of development since the application of disposable bioreactors in production, existing perfusion culture technology can maintain high cell density while also effectively maintaining the cell growth environment, thereby extending the culture period and increasing the yield of the target product.

[0005] A typical structure of a single-use bioreactor includes a reaction bag and a stirrer housed inside the bag. The power unit for the stirrer is located outside the bag, and power transmission between the power unit and the stirrer is often provided magnetically. To allow the stirrer's effective area to change with the liquid level inside the bag, existing technologies have developed single-use bioreactors with adjustable stirrer blade positions. In this specific design, the stirring shaft is connected to a lifting device. Utilizing the flexibility of the reaction bag, as the liquid level rises, the lifting device simultaneously raises the upper part of the bag and the stirrer blades, allowing the stirrer blades to change position with the liquid level.

[0006] This solution takes the application of a disposable bioreactor to the working conditions involved in perfusion culture as its starting point, and provides a disposable bioreactor and method for preparing adenovirus harvest. Summary of the Invention

[0007] This solution, based on the operating conditions involved in the application of a disposable bioreactor to perfusion culture, provides a disposable bioreactor and method for preparing adenovirus harvest. Using this solution, the mixing consistency of the disposable bioreactor during scale-up culture can be effectively ensured.

[0008] To address the above problems, the present invention provides a disposable bioreactor and method for preparing adenovirus harvest, which solves the problems through the following technical points: The disposable bioreactor for preparing adenovirus harvest includes a reaction bag and a magnetic stirring mechanism disposed inside the reaction bag. The magnetic stirring mechanism includes a stirring shaft, on which stirring blades are mounted. The number of stirring blades is greater than one and they are installed in layers on the stirring shaft. The stirring blades include a second stirring blade at the bottom and a first stirring blade located above the second stirring blade. The second stirring blade is fixed to the stirring shaft. The first stirring blade rotates synchronously with the stirring shaft when the stirring shaft rotates. It is mounted on the stirring shaft via a collar, which is fixed in position on the axis of the stirring shaft and can rotate around the axis of the stirring shaft. The system also includes a linkage module and a shear force transmission device, both of which are mounted on the stirring shaft. The linkage module drives the shear force transmission device to connect and disconnect the shear force transmission device from the collar. In the connected state, the shear force is transmitted through the shear force transmission device, causing the first stirring blade to rotate under the action of the stirring shaft. In the disconnected state, the collar and the stirring shaft remain in a state where they can rotate relative to each other.

[0009] The method of using this reactor is as follows: In the process of preparing adenovirus harvest, the reactor is used for 293 cell amplification culture and virus culture. Specifically, the reaction bag is used to contain the culture medium in the above process, and the magnetic stirring mechanism is used to stir the culture medium in the reaction bag. The magnetic stirring mechanism is powered by a rotating magnet placed outside the reaction bag to drive the stirring shaft to rotate, and the stirring blades are used to agitate the culture medium.

[0010] The difference between this solution and existing technologies is that the multi-layered stirring blades installed on the stirring shaft are not all fixed to the stirring shaft. Instead, the second stirring blade at the bottom is fixed to the stirring shaft, while the first stirring blade above it, as needed, establishes a transmission relationship between the first stirring blade and the stirring shaft through a shear force transmission component under the action of the linkage module. Specifically, when the first stirring blade needs to work, the connection relationship is established, at which time the first stirring blade can agitate the culture medium. In the disengaged relationship, the stirring shaft rotates independently of the first stirring blade, and the stirring shaft does not provide torque to the first stirring blade. The control rules for the connection and disengagement relationships are: the usage mode of the stirring blades is determined according to the liquid level of the culture medium in the bioreactor. Specifically, the second stirring blade is always located in the culture medium as the stirring blade at the bottom of the culture medium. When the first stirring blade can act on the culture medium, the state of the shear force transmission device is changed through the linkage module, so that the stirring shaft transmits torque to the first stirring blade, and the first stirring blade agitates the culture medium. The shear force transmission device can adopt key transmission, gear transmission, or friction transmission widely used in existing technologies, and the linkage module can adopt control components widely used in existing technologies that can control the corresponding transmission relationships.

[0011] The design concept and effects of this scheme are as follows: In the existing technology, adenovirus harvesting can be efficiently completed using suspended cells. However, during the culture of suspended cells and the culture of cells after infection, there are situations where the culture medium needs to be stirred. Taking the scale-up culture process of suspended cells as an example, there is generally a step-by-step scale-up culture process. In the complete process, from primary culture to passage culture or subculture, the specific culture medium used can range from tens of milliliters to thousands of liters. In this process, it is necessary to consider the problem of transferring the optimal reaction results in small reactors to industrial-scale (larger scale) bioreactors for reproduction. The similarity theory is the basic theory of bioreactor methods. The objects that need to be considered in similarity include fluid dynamics similarity, mass concentration similarity, biochemical similarity, etc. Therefore, when implementing stirring, the stirring speed range is generally a narrow range. In existing technologies, magnetic stirring mechanisms are used in disposable bioreactors for their effective isolation. However, the stirring blades on the stirring shaft are typically all fixed to the shaft. During subculture, different blade configurations are used for each stage of the planned scale-up culture. This is not conducive to the structural design and simplification of the adenovirus harvester preparation system. If perfusion culture (nutrient replenishment) is involved in the preparation process, changes in the culture medium level may occur during single-stage culture. A current solution is to raise the stirring mechanism as the liquid level rises. Although the mechanism changes the overall agitation range of the culture medium by the stirring blades, the agitation situation changes for the culture medium at a specific depth when the viscosity of the culture medium and the rotation speed of the stirring blades are stable. For example, taking the bottom stirring blade as an example, if the stirring blade is a propeller impeller and is installed at one-third of the initial liquid layer height, the liquid layer height on the bottom side of the stirring blade increases when the stirring blade is lifted as a whole. With the rotation speed of the stirring blade remaining unchanged, the agitation of the liquid layer on the bottom side of the stirring blade changes, and even bottom sedimentation may occur. Increasing the rotation speed of the stirring blade to expand the area of ​​influence of the stirring blade will change the consistency of the culture medium agitation.In this scheme, firstly, the relative positions of each stirring blade on the stirring shaft axis are fixed. The second stirring blade rotates with the stirring shaft to agitate the culture medium at the bottom of the reaction bag. When the first stirring blade is not needed, the shear force transmission device is disconnected from the collar via the linkage module and shear force transmission device. This reduces vibration caused by the first stirring blade idling on the stirring shaft, preventing issues such as shaft bending, premature bearing damage, and reaction bag seal failure. Then, as the culture medium level rises, when the first stirring blade needs to operate, the shear force transmission device is reconnected to the collar via the linkage module and shear force transmission device. At this point, the first stirring blade, under the action of the stirring shaft, contacts the culture medium for damped rotation (the culture medium acts as a damping medium). This not only controls the oscillation of the magnetic stirring mechanism but also... Each of the first and second stirring blades is responsible for agitating the liquid layer at its respective location, preventing significant changes in the agitation of liquid layers far from the stirring blades due to lifting the stirring mechanism. Furthermore, since the position of each stirring blade within the reaction bag is fixed, the main agitation range of each blade covers a specific liquid layer height through blade selection. Therefore, whether the culture medium level is raised during perfusion culture or the original culture medium level is selected within a certain range, a balance can be achieved between agitation and anti-vibration purposes by controlling the operation of the first stirring blade. Thus, with a relatively stable stirring shaft rotation speed, a high degree of similarity in agitation can be achieved for culture media at different depths. With a constant rotation speed, the number of stirring blades participating in the agitation work is selected based on the culture medium depth, utilizing the characteristics of the first stirring blade's arrangement, to maintain the consistency of agitation in the single-use bioreactor during scale-up culture.

[0012] Understandably, if the bottom surface of the reaction bag is taken as the reference plane, the second stirring blade is located at position L1, and the first stirring blade on the top side is located at L... N The location, the point where the original culture medium was added, or the range of the culture medium level rise can be from near L1 to L... N The upper part of the position (from the action area of ​​the second stirring blade to the action area of ​​the first stirring blade at the top) means that: with this scheme, in the application of non-perfusion nutrient supplementation during the cultivation process, the amount of culture medium added to the disposable bioreactor with a specific volume design can be selected, and it is easy to obtain a stirring condition with high consistency with the previous or subsequent stage under different addition amounts; in the application of perfusion nutrient supplementation during the cultivation process, the liquid level of the culture medium in the disposable bioreactor can be changed, and it is easy to obtain a stirring condition with high consistency with the previous or subsequent stage and before the liquid level change under different liquid levels.

[0013] The linkage module can be understood as the driving device of the shear force transmission device. Although the linkage module can adopt common electric drive, pneumatic drive, and hydraulic drive schemes, conventional schemes have the following problems: supplying power, gas, or liquid to the inside of the reaction bag will complicate the structural design of the magnetic stirring mechanism, be detrimental to the sealing reliability of the reaction bag, and also affect the thermal similarity of the inside of the reaction bag. Preferably, the magnetic stirring mechanism also includes an isolation sleeve set at the bottom of the disposable bioreactor and a first permanent magnet installed in the isolation sleeve. The first permanent magnet is fixed to the side wall of the end of the stirring shaft that extends into the isolation sleeve; the stirring shaft is a hollow shaft; the linkage module includes a top rod set inside the stirring shaft and a second permanent magnet set inside the stirring shaft. The body is located in one end of the stirring shaft that extends into the isolation sleeve, and the second permanent magnet is fixedly connected to the push rod. The push rod extends along the length of the stirring shaft and is also provided with a boss. The stirring shaft and the collar are both provided with pin holes. The pin holes are all oblique holes, and the end of the pin hole near the axis of the stirring shaft is the lower end of the pin hole. The linkage module also includes a transmission pin set in the pin hole. The end of the transmission pin near the axis of the stirring shaft is supported on the outer surface of the boss or the outer surface of the push rod. When the push rod is in the lowest position in the stirring shaft, the transmission pin slides down under its own weight to the position where it is dislodged from the pin hole of the collar. During the process of the push rod sliding upward relative to the stirring shaft along the length of the stirring shaft, under the action of the guide surface on the top side of the boss, the guide surface can push the transmission pin so that the transmission pin is embedded in the pin hole of the collar.

[0014] In operation, the above scheme involves installing the isolation sleeve within the cavity of the external structural frame of the reaction system. A first permanent magnet, mounted at the bottom of the stirring shaft, rotates the shaft under the magnetic force of the isolation sleeve. A second permanent magnet is located at the bottom of the linkage module. A magnetic device, providing magnetic force to the second permanent magnet, is positioned on the outer bottom of the isolation sleeve. This allows the push rod to be lifted by the magnetic device, which can be either a height-adjustable permanent magnet or an electromagnet. When the push rod is in the released state, the boss allows the transmission pin to slide down under its own weight to the outside of the collar pin hole. At this time, the stirring shaft cannot provide torque to the first stirring blade through the transmission pin. When the push rod is lifted by the second permanent magnet, the boss pushes the transmission pin, causing it to engage in the pin hole of the collar. At this point, one end of the transmission pin is in the pin hole of the stirring shaft, and the other end is in the pin hole of the collar. The transmission pin is sheared, achieving torque transmission, and the first stirring blade rotates with the stirring shaft. The above solution has a simple structure. For example, by setting a sealing plate at the end of the stirring shaft, it does not affect the sealing reliability of the reaction bag, nor does it cause a change in the thermal similarity inside the reaction bag. The position of the second permanent magnet on the stirring shaft axis can be adjusted by adjusting the height of the height-adjustable permanent magnet in space, and the position of the second permanent magnet on the stirring shaft axis can be adjusted by adjusting the output power of the electromagnet. Therefore, it has the advantages of being able to actively control whether the first stirring blade is working and being convenient to achieve the control objective. As someone skilled in the art, when the transmission pin disengages from the pin hole of the collar and the stirring shaft continues to rotate, there is a situation where the pin hole on the collar and the pin hole on the stirring shaft are misaligned. In this case, the transmission pin may not be able to re-engage into the pin hole on the collar at all times. This situation causes the push rod to be unable to rise further smoothly. The rising height of the stirring shaft can be determined by the interaction force between the magnetic device and the second permanent magnet, or by using a sensor based on the magnetic field strength to detect the position of the second permanent magnet. This solution controls the magnitude of the force applied to the second permanent magnet, ensuring that the friction between the transmission pin and the collar is insufficient to force the first stirring blade to rotate with the stirring shaft. Furthermore, during the relative rotation of the stirring shaft and the first stirring blade, the transmission pin can transmit torque in a shearing manner after the two pin holes become coaxial. This solution uses the second permanent magnet as the driving component of the push rod and also features high torque transmission reliability and reduces the impact on the end of the transmission pin, thus ensuring its lifespan.

[0015] Preferably, the inner side of the collar has an annular groove coaxial with the stirring shaft, the pin hole on the collar is located at the bottom of the annular groove, and the top of the drive pin is spherical. With this structure, the groove surface of the annular groove is machined to a smooth surface with a set roughness, the top of the drive pin is machined to a smooth surface with a set roughness, and the smooth surfaces at both locations are surface-hardened. Thus, when the drive pin is located outside the pin hole of the collar and is pressed against the groove surface of the annular groove, the lower friction facilitates rapid alignment of the pin hole on the drive pin and the pin hole on the collar. The reduced friction, lower wear rate, and the hardened impact resistance contribute to maintaining the reliability and lifespan of the magnetic stirring mechanism.

[0016] Preferably, multiple drive pins are provided between the stirring shaft and the collar, and each drive pin is provided with a pin hole on both the stirring shaft and the collar; the boss is a columnar structure with an outer diameter larger than the outer diameter of the push rod and coaxial with the push rod; the drive pins are evenly arranged in the circumferential direction of the stirring shaft, and the guide surface is a spherical surface with a smooth transition between its lower end and the side of the boss, and the shape of the spherical surface and the relative position of the drive pins satisfy the following: when the boss rises and falls relative to the stirring shaft, the drive pins rise and fall synchronously under the action of the spherical surface on each drive pin; the relative relationship between the pin hole and the drive pin satisfies the following: as the drive pin is further embedded in the pin hole in the collar, the push rod can rise to the position where the lower end of the drive pin acts on the side of the boss. The design principles of this scheme include addressing the problem of unilateral force on the first stirring blade, which is detrimental to the fit accuracy between the collar and the stirring shaft: by setting multiple transmission pins, the corresponding torque transmission is multi-point action and evenly distributed in the circumferential direction of the collar, which helps to reduce the vibration caused by centrifugal force when the rotating body rotates; it also includes solving the problems of transmission pin response speed and lifting rod displacement: by setting the rod to a position where the lower end of the transmission pin acts on the side of the boss, the length of the boss axis occupied by the guide surface can be set to be shorter, and the rod can complete the lifting and releasing of the transmission pin within a smaller stroke range; the above-mentioned smoothly transitioned spherical surface is used to reduce the impact that the rod and transmission pin may be subjected to during operation. The above-mentioned limitation on the synchronous rise and fall of the transmission pin is actually a limitation on the form of the guide surface, the design position of the pin hole, etc.: if the rotation of the rod is not considered or the rotation of the rod is solved, different transmission pins are acted on by different positions of the spherical surface, and the transmission pins do not necessarily have to be set at the same height, and the corresponding different positions of the spherical surface do not need to be consistent in form. Therefore, the above limitation is merely a functional expression to limit the many implementation methods. In practical implementation, for ease of processing, the boss shape is machined by turning, and the pin holes on the stirring shaft and the collar are at the same height. When there are multiple first stirring blades, and the lower first stirring blade establishes a torque transmission relationship with the stirring shaft before the upper first stirring blade, it is easier to ensure that all the first stirring blades rotate with the stirring shaft.

[0017] Preferably, the number of the first stirring blades is greater than one, and each first stirring blade is equipped with a boss, a pin hole, and a drive pin. As the push rod rises in the stirring shaft, the first stirring blades arranged sequentially along the length of the stirring shaft have the following relationship: the lower first stirring blade is connected to the stirring shaft via the drive pin inserted into the pin hole of the collar before the upper first stirring blade. When the topmost first stirring blade is connected to the stirring shaft via the drive pin, the other first stirring blades maintain their connection with the stirring shaft. The purpose of this design is to allow for a larger number of first stirring blades to accommodate a wider range of liquid levels, and the different first stirring blades establish their connection with the stirring shaft asynchronously: the lower first stirring blade establishes its connection with the stirring shaft before the upper first stirring blade to adapt to specific liquid level changes or specific liquid levels, avoiding the situation where the first stirring blades rotate idly with the stirring shaft simultaneously. As those skilled in the art know, when all the first stirring blades, the collar on the first stirring blades, and the pin holes on the collars have the same design parameters, the above objective can be achieved by controlling the position of the bosses acting on different first stirring blades on the push rod: for any two adjacent first stirring blades in any height direction, the relative position between the bosses satisfies the following condition: the lower boss acts on the corresponding transmission pin, so that after the transmission pin enters the pin hole of the collar, the transmission pin corresponding to the upper first stirring blade is still in the case of being externally placed in the corresponding collar pin hole. As the push rod rises further, the lower transmission pin remains in the state of shear torque transmission, and the upper transmission pin enters the corresponding collar pin hole under the action of the boss, and the transmission pin changes to the state of shear torque transmission.

[0018] The above provides a technical solution that controls the relationship between the first stirring blade and the stirring shaft by placing a magnetic device outside the reaction bag and acting on the shear force transmission device through a linkage module. The advantages of this solution are that it simplifies the internal structure of the isolation sleeve and facilitates the control of heat introduction inside the isolation sleeve.

[0019] Preferably, to improve fitting accuracy, performance stability, and reduce wear, a rolling bearing is provided between the stirring shaft and the isolation sleeve, and a sliding bearing is provided between the push rod or the second permanent magnet and the stirring shaft. The collar is rotatably connected to the stirring shaft through the sliding bearing. According to the general structure of existing disposable bioreactors, the first stirring blade is designed to have two or three layers.

[0020] As another method of setting up linkage modules and shear force transmission devices with a simple internal structure of the isolation sleeve that does not require an additional power source:

[0021] Preferably, the linkage module includes a floating platform, and the shear force transmission device includes a flat key and a sliding platform. The flat key is parallel to the stirring shaft and is disposed on the outer wall of the stirring shaft. The sliding platform is slidably connected to the flat key through a groove on it, and the floating platform is fixed on the sliding platform. The floating platform, flat key, and sliding platform are all disposed below the first stirring blade. During the process of the floating platform driving the sliding platform to slide upward along the length direction of the flat key under the action of buoyancy, the top surface of the sliding platform can contact the bottom surface of the collar. The force applied to the collar by the sliding platform causes the stirring shaft to drive the first stirring blade to rotate.

[0022] The working principle of this scheme is as follows: the slide and the key maintain a sliding connection. Under the buoyancy of the culture medium in the isolation sleeve, the buoyancy of the float forces the slide to slide upwards along the key relative to the stirring axis. The interaction between the top of the slide and the bottom surface of the collar provides torque to the first stirring blade, enabling the first stirring blade to agitate the culture medium. This scheme is a technical solution that allows the first stirring blade to adapt to the culture medium level. Depending on the arrangement of the float, the shape of the top surface of the slide, and the shape of the bottom surface of the collar, the above forces can originate from friction, the shear force provided by the strip edge, and other alternative forms. In specific implementation, to maintain the constant engagement between the slide and the key, it is preferable to use a limiting device on the stirring shaft to restrict the lowest position of the slide, while the highest position of the slide's upward movement can be limited by the collar above it.

[0023] Preferably, there are multiple flat keys, which are evenly arranged in the circumferential direction of the stirring shaft to form a splined shaft segment on the stirring shaft. The slide is annular, and its inner surface has multiple grooves, the same number as the number of flat keys and in the same relative position as the flat keys. Each flat key is slidably connected to one groove. The float is an annular structure coaxial with the stirring shaft, and its top surface is lower than that of the slide. This solution is a preferred method for driving the first stirring blade using flat keys, a slide, and a float. This solution not only facilitates the counterweighting of the stirring shaft and reduces vibration during rotation, but also avoids issues such as reduced sliding shaft fit accuracy and reduced fit accuracy between the slide and the collar due to uneven wear caused by the fit between the flat keys and the grooves. It is understood that the splined shaft segment mentioned above refers to the stirring shaft segment containing multiple flat keys. This segment is splined, and the grooves on the slide match the distribution of the flat keys. Each groove mutually constrains the movement direction of the slide to maintain a stable contact state between the slide and the collar. The purpose of setting the position of the floating platform is to ensure its lifespan.

[0024] As mentioned above, as long as the floating platform can generate sufficient buoyancy, the friction between the slide and the collar can be used to drive the first stirring blade. However, this method results in an excessively large floating platform, a significant impact of the slide's mass on driving the first stirring blade, increased wear, and the introduction of culture medium contamination. Consequently, this driving method is also unstable. Therefore, preferably, one of the bottom surface of the collar and the top surface of the slide has a strip-shaped ridge extending radially along the stirring shaft, and the other has a strip-shaped groove extending radially along the stirring shaft. The strip-shaped ridge can be embedded in the strip-shaped groove. The design purpose of this scheme is that during the upward movement of the slide under the action of the floating platform, the insertion of the strip-shaped ridge into the strip-shaped groove allows the torque transmission of the stirring shaft to the first stirring blade to sequentially pass through the shearing of the flat key and the shearing of the strip-shaped ridge. This maintains the reliability of power and torque transmission to the first stirring blade. The purpose of setting the extension direction of the strip-shaped ridges and grooves is as follows: Since the first stirring blade and the stirring shaft rotate relative to each other, when the slide rises and contacts the collar, there is a high probability that the strip-shaped ridges and grooves will misalign. Having specified the extension direction of the strip-shaped ridges and grooves, a simple scheme is proposed to achieve the following mating relationship: Specifically, as the slide and collar rotate relative to each other, when the strip-shaped ridges and grooves rotate to the same vertical direction, the strip-shaped ridges can be embedded in the grooves, making the slide and collar mesh in a gear-driven state. To control the center position of the slide and collar, one side has multiple strip-shaped ridges, and the other side has the same number of grooves as the number of strip-shaped ridges. Furthermore, the strip-shaped ridges and grooves are evenly arranged in a ring on the top surface of the slide or the bottom surface of the collar relative to the axis of the stirring shaft.

[0025] The following describes the application of the single-use bioreactor proposed above in the preparation of adenovirus harvest.

[0026] Specifically, it is a method for preparing adenovirus harvest, comprising the following steps performed sequentially: 293 cell resuscitation, 293 cell amplification culture, adenovirus inoculation, virus culture, and lysis harvest;

[0027] The 293 cell scale-up culture utilizes a disposable bioreactor as described above. During the 293 cell scale-up culture process, the usage of the stirring blades is determined based on the culture medium level in the bioreactor: the second stirring blade is located in the culture medium, and when the first stirring blade can act on the culture medium, the state of the shear force transmission device is changed through the linkage module, causing the stirring axis to transmit torque to the first stirring blade, thereby agitating the culture medium using the first stirring blade. This scheme uses the disposable bioreactor proposed above to prepare adenovirus harvest. During the preparation of adenovirus harvest, the activation of the first stirring blade and the activation method can be determined based on the actual culture medium level. This approach benefits the bioreactor's lifespan, allows for good adaptation to large-scale changes in the culture medium level within the reaction bag, and helps maintain consistent agitation during the scale-up culture process.

[0028] Preferably, the culture medium used in the 293 cell amplification process contains 0.1% to 1% by weight of dextran sulfate, wherein the molecular weight of the dextran sulfate is 5KD to 50KD. In this scheme, a commonly used anti-clustering agent is further selected, and the amount and type of dextran sulfate added are further limited. The main reason is that the addition of dextran sulfate can effectively improve the adhesion and clumping phenomenon of 293 cells during culture, which is beneficial to the activity, viability, and proliferation rate of 293 cells. It can effectively prevent cell clumping or foam formation, ensure stable production at a production scale of 2000L, maintain a cell viability of over 90% throughout the amplification process, prevent significant cell clumping, and eliminate the need for defoaming agents. Currently, the molecular weight range of dextran sulfate used for cell culture is generally 3-2000 kDa. Dextran sulfate with excessively large molecular weight or excessive concentration can affect the concentration and viability of 293 cells (generally appearing in the later stages of 293 cell culture). Using dextran sulfate with a molecular weight of 50 kDa at a concentration of 0.1% to 1% can effectively reduce the aggregation of 293 cells and increase the cell concentration. When adding dextran sulfate with a molecular weight of 5 kDa at a weight percentage of 0.1% to 1%, the growth of 293 cells, the basic charge isomer, and the viability can all be significantly improved, and it has a certain impact on ammonium cations, etc. The above methods of adding dextran sulfate can increase cell concentration by 1.2 to 1.5 times. Comparing the amounts and molecular weights of dextran sulfate mentioned above, when the amount added and the molecular weight of dextran sulfate are lower, the increase in cell concentration is less than 1.2 times. When the amount added and the molecular weight of dextran sulfate used are higher, the cell concentration can be increased by 1.2 to 1.5 times or more within a certain range. However, this will make it more difficult to remove dextran sulfate from the protein during the subsequent purification process. When using the above-mentioned amounts and molecular weights of dextran sulfate, the recovery rate of dextran sulfate can be greater than 99.5% when precipitating proteins with general TCA reagent.

[0029] Preferably, in order to avoid contamination from animal-derived substances and improve toxin production efficiency and product quality, the culture medium used is Dynamis serum-free medium.

[0030] The present invention has the following beneficial effects:

[0031] The main improvement to the disposable bioreactor provided in this solution is the magnetic stirring mechanism, adopting the above solution:

[0032] First, the relative positions of each stirring blade on the stirring shaft axis are fixed. The second stirring blade rotates with the stirring shaft to agitate the culture medium at the bottom of the reaction bag. When the first stirring blade is not needed, the shear force transmission device is disconnected from the collar through the linkage module and the shear force transmission device. At this time, the vibration caused by the first stirring blade spinning idling to the stirring shaft can be reduced, so as to avoid problems such as the stirring shaft bending, the bearing being damaged too quickly, and the reaction bag sealing failure.

[0033] Simultaneously, as the culture medium level rises, when it reaches a point where the first stirring blade needs to operate, the linkage module and shear force transmission device establish a connection between the shear force transmission device and the collar. At this time, the first stirring blade, under the action of the stirring shaft, contacts the culture medium for damped rotation (the culture medium serves as the damping medium). This not only controls the oscillation of the magnetic stirring mechanism but also ensures that the first and second stirring blades each agitate the liquid layer at their respective locations, preventing significant changes in the agitation of liquid layers far from the stirring blades due to raising the stirring mechanism. Furthermore, since the positions of each stirring blade within the reaction bag are fixed, the selection of the stirring blade type... Each stirring blade's main agitation range covers a specific liquid layer height. Therefore, whether it's during perfusion culture where the culture medium level rises or when the original culture medium level is selected within a certain range, controlling the operation of the first stirring blade allows for a balance between agitation and anti-vibration purposes. Thus, with a relatively stable stirring shaft rotation speed, it can achieve a high degree of similarity in agitation for culture media at different depths. By utilizing the characteristics of the first stirring blade's arrangement and selecting the number of stirring blades involved in the agitation work according to the culture medium depth, the consistency of agitation during the scaling-up process in a single-use bioreactor can be maintained, ensuring uniformity in agitation.

[0034] This scheme also provides a method for preparing adenovirus harvest using the proposed disposable bioreactor. Attached Figure Description

[0035] Figure 1 This is a schematic diagram of the bottom structure of a specific embodiment of the disposable bioreactor for preparing adenovirus harvest according to the present invention;

[0036] Figure 2 This is a cross-sectional view of the stirring shaft portion of a specific embodiment of the disposable bioreactor for preparing adenovirus harvest according to the present invention;

[0037] Figure 3 This is a cross-sectional view of the stirring shaft portion of a specific embodiment of the disposable bioreactor for preparing adenovirus harvest according to the present invention, and... Figure 2 Distinguished, Figure 2 The push rod is driven by magnetic force. Figure 3 The sliding table is driven by a floating platform.

[0038] Figure 4 A proliferation curve of Expi293 suspension cells associated with Example 6 is provided;

[0039] Figure 5 The nutritional metabolism of Expi293 suspension cells during proliferation is provided in the associated Example 6;

[0040] Figure 6 A flowchart of the adenovirus preparation process associated with Example 6 is provided.

[0041] Figure 7 A comparison chart of virus titers before and after process optimization in Example 6 is provided.

[0042] The markings in the diagram are as follows: 1. Pressure plate, 2. Reaction bag, 3. Isolation sleeve, 4. First permanent magnet, 5. Sliding bearing, 6. Rolling bearing, 7. Stirring shaft, 8. Top rod, 9. Second permanent magnet, 10. First stirring blade, 11. Second stirring blade, 12. Boss, 13. Drive pin, 14. Collar, 15. Flat key, 16. Strip ridge, 17. Slide table, 18. Floating platform, 19. Pin hole. Detailed Implementation

[0043] The present invention will be further described in detail below with reference to the embodiments, but the present invention is not limited to the following embodiments:

[0044] Example 1:

[0045] like Figures 1 to 3 As shown, a disposable bioreactor for preparing adenovirus harvest includes a reaction bag 2 and a magnetic stirring mechanism disposed inside the reaction bag 2. The magnetic stirring mechanism includes a stirring shaft 7, on which stirring blades are mounted. The number of stirring blades is greater than one and they are installed in layers on the stirring shaft 7. The stirring blades include a second stirring blade 11 at the bottom and a first stirring blade 10 above the second stirring blade 11. The second stirring blade 11 is fixed to the stirring shaft 7 and rotates synchronously with the stirring shaft 7 when the stirring shaft 7 rotates. The first stirring blade 10... The collar 14 is mounted on the stirring shaft 7 and its position on the axis of the stirring shaft 7 is fixed. The collar 14 can rotate around the axis of the stirring shaft 7. The system also includes a linkage module and a shear force transmission device. Both the linkage module and the shear force transmission device are mounted on the stirring shaft 7. The linkage module is used to drive the shear force transmission device to operate, so as to connect and disconnect the shear force transmission device from the collar 14. In the connected state, the shear force is transmitted through the shear force transmission device, so that the first stirring blade 10 rotates under the action of the stirring shaft 7. In the disconnected state, the collar 14 and the stirring shaft 7 remain in a state of relative rotation.

[0046] The method of using this reactor is as follows: In the process of preparing adenovirus harvest, the reactor is used for 293 cell amplification culture and virus culture. Specifically, the reaction bag 2 is used to contain the culture medium in the above process, and the magnetic stirring mechanism is used to stir the culture medium in the reaction bag 2. The magnetic stirring mechanism obtains power by using a rotating magnet placed outside the reaction bag 2 to drive the stirring shaft 7 to rotate, and the stirring blades are used to stir the culture medium.

[0047] The difference between this solution and existing technologies is that the multi-layered stirring blades installed on the stirring shaft 7 are not all fixed to the stirring shaft 7. Instead, the second stirring blade 11 at the bottom is fixed to the stirring shaft 7, while the first stirring blade 10 above it establishes a transmission relationship with the stirring shaft 7 through a shear force transmission device under the action of the linkage module, as needed. Specifically, when the first stirring blade 10 needs to work, the connection relationship is established, and the first stirring blade 10 can stir the culture medium. In the disengagement relationship, the stirring shaft 7 rotates independently of the first stirring blade 10, and the stirring shaft 7 does not provide torque to the first stirring blade 10. The control rules for the connection and disengagement relationships are: the usage mode of the stirring blades is determined according to the liquid level of the culture medium in the bioreactor. Specifically, the second stirring blade 11 is always located in the culture medium as the bottom stirring blade. When the first stirring blade 10 can act on the culture medium, the state of the shear force transmission device is changed through the linkage module, so that the stirring shaft 7 transmits torque to the first stirring blade 10, and the first stirring blade 10 stirs the culture medium. The shear force transmission device can adopt key transmission, gear transmission, or friction transmission, which are widely used in the prior art, and the linkage module can adopt control components that can control the corresponding transmission relationship, which are widely used in the prior art.

[0048] The design concept and effects of this scheme are as follows: In the existing technology, adenovirus harvesting can be efficiently completed using suspended cells. However, during the culture of suspended cells and the culture of cells after infection, there are situations where the culture medium needs to be stirred. Taking the scale-up culture process of suspended cells as an example, there is generally a step-by-step scale-up culture process. In the complete process, from primary culture to passage culture or subculture, the specific culture medium used can range from tens of milliliters to thousands of liters. In this process, it is necessary to consider the problem of transferring the optimal reaction results in small reactors to industrial-scale (larger scale) bioreactors for reproduction. The similarity theory is the basic theory of bioreactor methods. The objects that need to be considered in similarity include fluid dynamics similarity, mass concentration similarity, biochemical similarity, etc. Therefore, when implementing stirring, the stirring speed range is generally a narrow range. In existing technologies, magnetic stirring mechanisms are used in disposable bioreactors for their effective isolation. However, the stirring blades on the stirring shaft 7 are generally fixed to the shaft. During subculture, different blade configurations are used for each stage of the planned scale-up culture. This is not conducive to the structural design and simplification of the adenovirus harvester preparation system. If perfusion culture (nutrient replenishment) is involved in the preparation process, changes in the culture medium level may occur during single-stage culture. A current solution is to raise the stirring mechanism as the liquid level rises. Although the mixing mechanism changes the overall agitation range of the culture medium by the mixing blades, the agitation situation changes for the culture medium at a specific depth when the viscosity of the culture medium and the rotation speed of the mixing blades are stable. For example, taking the bottom mixing blade as an example, if the mixing blade is a propeller impeller and is installed at one-third of the initial liquid layer height, the liquid layer height at the bottom of the mixing blade increases when the mixing blade is lifted as a whole. With the rotation speed of the mixing blade remaining unchanged, the agitation of the liquid layer at the bottom of the mixing blade changes, and even sedimentation at the bottom may occur. Increasing the rotation speed of the mixing blade to expand the area of ​​influence of the mixing blade will change the consistency of the culture medium agitation.In this scheme, firstly, the relative positions of each stirring blade on the axis of the stirring shaft 7 are fixed. The second stirring blade 11 rotates with the stirring shaft 7 to agitate the culture medium at the bottom of the reaction bag 2. When the first stirring blade 10 is not needed, the shear force transmission device is disconnected from the collar 14 through the linkage module and shear force transmission device. At this time, the vibration caused by the first stirring blade 10 spinning idling to the stirring shaft 7 can be reduced, thus avoiding problems such as bending of the stirring shaft 7, premature bearing damage, and failure of the seal of the reaction bag 2. Then, as the culture medium level rises, when the first stirring blade 10 needs to work, the shear force transmission device is connected to the collar 14 through the linkage module and shear force transmission device. At this time, the first stirring blade 10 contacts the culture medium under the action of the stirring shaft 7 and rotates with damping (the culture medium is the damping medium). This not only controls the vibration of the magnetic stirring mechanism, but also... Simultaneously, the first stirring blade 10 and the second stirring blade 11 are each responsible for stirring the liquid layer at their respective positions, avoiding significant changes in the stirring of liquid layers far from the stirring blades due to lifting the stirring mechanism. At the same time, since the position of each stirring blade in the reaction bag 2 is fixed, the main stirring range of each stirring blade covers a specific liquid layer height through the selection of stirring blades. Therefore, whether it is the raising of the liquid level of the culture medium during perfusion culture or the selection of the liquid level of the original culture medium within a certain range, a balance can be achieved between the stirring purpose and the anti-vibration purpose by controlling the working condition of the first stirring blade 10. Thus, when the rotation speed of the stirring shaft 7 is relatively stable, it can provide a high degree of similarity in the stirring state of the culture medium at different depths. With the rotation speed unchanged, by utilizing the setting characteristics of the first stirring blade 10, the number of stirring blades participating in the stirring work can be selected according to the depth of the culture medium, so as to maintain the stirring consistency of the disposable bioreactor during the scale-up culture process.

[0049] Understandably, if the bottom surface of reaction bag 2 is taken as the reference plane, the second stirring blade 11 is located at position L1, and the topmost first stirring blade 10 is located at position L. N The location, the point where the original culture medium was added, or the range of the culture medium level rise can be from near L1 to L... N The upper part of the position (from the working area of ​​the second stirring blade 11 to the working area of ​​the topmost first stirring blade 10) means that: with this scheme, in the application of non-perfusion nutrient supplementation during the cultivation process, the amount of culture medium added to the disposable bioreactor with a specific volume design can be selected, and it is easy to obtain a stirring condition with high consistency with the previous or subsequent stage under different addition amounts; in the application of perfusion nutrient supplementation during the cultivation process, the liquid level of the culture medium in the disposable bioreactor can be changed, and it is easy to obtain a stirring condition with high consistency with the previous or subsequent stage and before the liquid level change under different liquid levels.

[0050] exist Figure 1In this case, the pressure plate 1 and the isolation sleeve 3 can adopt the split structure shown. For ease of operation, the pressure plate 1 and the isolation sleeve 3 can also adopt an integrated structure: the top structure of the isolation sleeve 3 has an annular groove with an outer opening, and the reaction bag 2 is embedded under the pressure plate 1 through the annular groove.

[0051] Example 2:

[0052] This embodiment further refines the scheme based on Embodiment 1: The linkage module can be understood as the driving device of the shear force transmission device. Although the linkage module can adopt common electric drive schemes, pneumatic drive schemes, and hydraulic drive schemes, conventional schemes have the following problems: supplying power, gas, or liquid to the inside of the reaction bag 2 will complicate the structural design of the magnetic stirring mechanism, and will also be detrimental to the sealing reliability of the reaction bag 2, and will also affect the thermal similarity of the inside of the reaction bag 2. Preferably, the magnetic stirring mechanism also includes an isolation sleeve 3 set at the bottom of the disposable bioreactor and a first permanent magnet 4 installed in the isolation sleeve 3. The first permanent magnet 4 is fixed to the side wall of the end of the stirring shaft 7 that extends into the isolation sleeve 3; the stirring shaft 7 is a hollow shaft; the linkage module includes a top rod 8 set inside the stirring shaft 7 and a second permanent magnet 9 set inside the stirring shaft 7. The second permanent magnet 9 is located on the stirring shaft 7. The second permanent magnet 9 is fixedly connected to the push rod 8 at one end of the isolation sleeve 3. The push rod 8 extends along the length of the stirring shaft 7 and is also provided with a boss 12. The stirring shaft 7 and the collar 14 are both provided with pin holes 19. The pin holes 19 are all oblique holes, and the end of the pin hole 19 near the axis of the stirring shaft 7 is the lower end of the pin hole 19. The linkage module also includes a transmission pin 13 disposed in the pin hole 19. The end of the transmission pin 13 near the axis of the stirring shaft 7 is supported on the outer surface of the boss 12 or the outer surface of the push rod 8. When the push rod 8 is at the lowest position in the stirring shaft 7, the transmission pin 13 slides down under its own weight to the position where it is dislodged from the pin hole 19 of the collar 14. During the process of the push rod 8 sliding upward relative to the stirring shaft 7 along the length of the stirring shaft 7, under the action of the guide surface on the top side of the boss 12, the transmission pin 13 can be pushed by the guide surface to embed into the pin hole 19 of the collar 14.

[0053] In operation, the above scheme allows the isolation sleeve 3 to be installed inside the cavity of the external structural frame of the reaction system. The first permanent magnet 4, installed at the bottom of the stirring shaft 7, drives the stirring shaft 7 to rotate under the action of the external magnetic force of the isolation sleeve 3. The lowest end of the linkage module is the second permanent magnet 9. A magnetic device is set on the bottom outer side of the isolation sleeve 3 to provide magnetic force for the second permanent magnet 9, so that the push rod 8 can be lifted by the second permanent magnet 9 under the action of the magnetic device. The magnetic device can be a permanent magnet or an electromagnet with adjustable spatial height. Thus, when the push rod 8 is in the released state, the boss 12 is in a state that allows the transmission pin 13 to slide down to the outside of the pin hole 19 of the collar 14 under its own weight. At this time, the stirring shaft 7 cannot provide torque to the first stirring blade 10 through the transmission pin 13. When the push rod 8 is lifted by the second permanent magnet 9, the boss 12 pushes the transmission pin 13 so that the transmission pin 13 is embedded in the pin hole 19 of the collar 14. At this time, one end of the transmission pin 13 is located in the pin hole 19 of the stirring shaft 7, and the other end is located in the pin hole 19 of the collar 14. At this time, the transmission pin 13 is sheared to achieve the purpose of torque transmission, and the first stirring blade 10 rotates with the stirring shaft 7. The above scheme has a simple structure. For example, by setting the end of the stirring shaft 7 to have a sealing plate, it does not affect the sealing reliability of the reaction bag 2, nor does it cause a change in the internal thermal similarity of the reaction bag 2. The position of the second permanent magnet 9 on the axis of the stirring shaft 7 can be adjusted by adjusting the height of the height-adjustable permanent magnet in space, and the position of the second permanent magnet 9 on the axis of the stirring shaft 7 can be adjusted by adjusting the output power of the electromagnet. Therefore, it has the characteristics of being able to actively control whether the first stirring blade 10 is working and being convenient to achieve the control purpose. As someone skilled in the art, when the transmission pin 13 is disengaged from the pin hole 19 of the collar 14 and the stirring shaft 7 continues to rotate, there is a situation where the pin hole 19 on the collar 14 and the pin hole 19 on the stirring shaft 7 are misaligned. At this time, the transmission pin 13 may not be able to be inserted into the pin hole 19 on the collar 14 at any time. In this case, the push rod 8 cannot rise further smoothly. The rising height of the stirring shaft 7 can be judged by the interaction force between the magnetic device and the second permanent magnet 9, or by using a sensor based on the magnetic field strength to detect the position of the second permanent magnet 9. This solution controls the magnitude of the force applied to the second permanent magnet 9, ensuring that the friction between the transmission pin 13 and the collar 14 is insufficient to force the first stirring blade 10 to rotate with the stirring shaft 7. Furthermore, during the relative rotation of the stirring shaft 7 and the first stirring blade 10, the transmission pin 13 can transmit torque in a shear manner after the two pin holes 19 become coaxial. This solution uses the second permanent magnet 9 as the driving component of the push rod 8, and also features high torque transmission reliability and reduces the impact on the end of the transmission pin 13 to ensure its lifespan.

[0054] Preferably, the inner side of the collar 14 has an annular groove coaxial with the stirring shaft 7, the pin hole 19 on the collar 14 is located at the bottom of the annular groove, and the top of the drive pin 13 is spherical. With this structure, the groove surface of the annular groove is machined to a smooth surface with a set roughness, the top of the drive pin 13 is machined to a smooth surface with a set roughness, and the smooth surfaces at both locations are surface-hardened. Thus, when the drive pin 13 is located outside the pin hole 19 of the collar 14 and is pressed against the groove surface of the annular groove, the smaller frictional force facilitates rapid alignment of the pin hole 19 on the drive pin 13 and the pin hole 19 on the collar 14. The reduced frictional force, lower wear rate, and the hardened impact deformation resistance contribute to maintaining the reliability and lifespan of the magnetic stirring mechanism.

[0055] Preferably, multiple drive pins 13 are provided between the stirring shaft 7 and the collar 14, and each drive pin 13 is provided with a pin hole 19 on both the stirring shaft 7 and the collar 14; the boss 12 is a columnar structure with an outer diameter larger than the outer diameter of the push rod 8 and coaxial with the push rod 8; the drive pins 13 are evenly arranged in the circumferential direction of the stirring shaft 7, and the guide surface is a spherical surface with a smooth transition between the lower end and the side of the boss 12, and the shape of the spherical surface and the relative position of the drive pins 13 satisfy the following: when the boss 12 rises and falls relative to the stirring shaft 7, the drive pins 13 rise and fall synchronously under the action of the spherical surface on each drive pin 13; the relative relationship between the pin hole 19 and the drive pin 13 satisfies the following: as the drive pin 13 further embeds into the pin hole 19 in the collar 14, the push rod 8 can rise to the position where the lower end of the drive pin 13 acts on the side of the boss 12. The design principle of this scheme includes solving the problem that the first stirring blade 10 is subjected to force on one side, which is not conducive to the fitting accuracy between the collar 14 and the stirring shaft 7: by setting multiple transmission pins 13, the corresponding torque transmission is carried out in a multi-point mode and is evenly distributed in the circumferential direction of the collar 14, which helps to reduce the vibration problem caused by centrifugal force when the rotating body rotates; it also includes solving the problems of the response speed of the transmission pin 13 and the displacement of the lifting rod 8: by setting the lifting rod 8 to a position where the lower end of the transmission pin 13 acts on the side of the boss 12, the length of the axis of the boss 12 occupied by the guide surface can be set to be shorter, and the lifting rod 8 can complete the lifting and releasing of the transmission pin 13 within a small stroke range; the above-mentioned smoothly transitioned spherical surface is used to reduce the impact that the lifting rod 8 and the transmission pin 13 may be subjected to during operation. The above limitations on the synchronous rise and fall of the transmission pins 13 are actually limitations on the form of the guide surface and the design position of the pin holes 19. If the rotation of the push rod 8 is not considered or is solved, different transmission pins 13 are acted upon by different positions of the spherical surface. The transmission pins 13 do not necessarily need to be set at the same height, and the different positions of the spherical surface do not need to be in the same form. Therefore, the above limitations are merely functional expressions that limit many implementation methods. In specific implementation, for ease of processing, the outer shape of the boss 12 is machined by turning, and the pin holes 19 on the stirring shaft 7 and the pin holes 19 on the collar 14 are at the same height. The above lifting displacement of the push rod 8 is easier to achieve when there are multiple first stirring blades 10, and the first stirring blade 10 at the bottom establishes a torque transmission relationship with the stirring shaft 7 before the first stirring blade 10 at the top.

[0056] Preferably, the number of the first stirring blades 10 is greater than one, and each first stirring blade 10 is equipped with a boss 12, a pin hole 19, and a transmission pin 13. As the push rod 8 rises in the stirring shaft 7, the first stirring blades 10 arranged sequentially along the length of the stirring shaft 7 have the following relationship: the lower first stirring blade 10 is connected to the stirring shaft 7 via the transmission pin 13 inserted into the pin hole 19 of the collar 14 before the upper first stirring blade 10. When the topmost first stirring blade 10 is connected to the stirring shaft 7 via the transmission pin 13, the other first stirring blades 10 maintain their transmission connection with the stirring shaft 7. The purpose of this design is to allow for a larger number of first stirring blades 10 to accommodate a wider range of liquid levels, and the transmission relationship between different first stirring blades 10 and the stirring shaft 7 is not synchronous: the lower first stirring blade 10 establishes its transmission relationship with the stirring shaft 7 before the upper first stirring blade 10 to adapt to specific liquid level changes or specific liquid levels, avoiding the situation where the first stirring blades 10 rotate idly synchronously with the stirring shaft 7. As those skilled in the art know, when all the first stirring blades 10, the collar 14 on the first stirring blade 10, and the pin hole 19 on the collar 14 have the same design parameters, the above objective can be achieved by controlling the position of the boss 12 acting on different first stirring blades 10 on the push rod 8: for any two adjacent first stirring blades 10 in any height direction, the relative position between the bosses 12 is satisfied that the lower boss 12 acts on the corresponding transmission pin 13, so that after the transmission pin 13 enters the pin hole 19 of the collar 14, the transmission pin 13 corresponding to the upper first stirring blade 10 is still in the case of being externally placed in the pin hole 19 of the corresponding collar 14. As the push rod 8 rises further, the lower transmission pin 13 remains in the state of shearing torque transmission, and the upper transmission pin 13 enters the pin hole 19 of the corresponding collar 14 under the action of the boss 12, and the transmission pin 13 changes to the state of shearing torque transmission.

[0057] The above provides a technical solution that controls the relationship between the first stirring blade 10 and the stirring shaft 7 by placing a magnetic device outside the reaction bag 2 and acting on the shear force transmission device through a linkage module. The feature of this solution is that it can simplify the internal structure of the isolation sleeve 3 and facilitate the control of heat introduction inside the isolation sleeve 3.

[0058] Preferably, to improve fitting accuracy, performance stability and reduce wear, a rolling bearing 6 is provided between the stirring shaft 7 and the isolation sleeve 3, and a sliding bearing 5 is provided between the push rod 8 or the second permanent magnet 9 and the stirring shaft 7. The collar 14 is rotatably connected to the stirring shaft 7 through the sliding bearing 5. According to the general structure of existing disposable bioreactors, the first stirring blade 10 is designed to have two or three layers.

[0059] Example 3:

[0060] This embodiment further refines the scheme based on embodiment 1: as another way to set up the linkage module and shear force transmission device with a simple internal structure of the isolation sleeve 3 that does not require an additional power source:

[0061] Preferably, the linkage module includes a floating platform 18, and the shear force transmission device includes a flat key 15 and a sliding platform 17. The flat key 15 is parallel to the stirring shaft 7 and is disposed on the outer wall of the stirring shaft 7. The sliding platform 17 is slidably connected to the flat key 15 through a sliding groove thereon. The floating platform 18 is fixed on the sliding platform 17. The floating platform 18, the flat key 15, and the sliding platform 17 are all disposed below the first stirring blade 10. During the process of the floating platform 18 driving the sliding platform 17 to slide upward along the length direction of the flat key 15 under the action of buoyancy, the top surface of the sliding platform 17 can contact the bottom surface of the collar 14. The force applied to the collar 14 by the sliding platform 17 causes the stirring shaft 7 to drive the first stirring blade 10 to rotate.

[0062] The working principle of this scheme is as follows: the slide 17 and the flat key 15 always maintain a sliding connection. Under the buoyancy of the culture medium in the isolation sleeve 3, the buoyancy of the float 18 forces the slide 17 to slide upward relative to the stirring shaft 7 along the flat key 15. Under the interaction between the top of the slide 17 and the bottom surface of the collar 14, torque is provided to the first stirring blade 10, enabling the first stirring blade 10 to agitate the culture medium. This scheme is a technical solution that allows the first stirring blade 10 to adapt to the liquid level of the culture medium. Depending on the arrangement of the float 18, the top surface form of the slide 17, and the bottom surface form of the collar 14, the above forces can come from friction, the shear force provided by the strip rib 16, and other alternative forms. In specific implementation, to maintain the constant engagement between the slide 17 and the flat key 15, it is preferable to use a limiting device on the stirring shaft 7 to limit the lowest position of the slide 17, while the highest position of the upward movement of the slide 17 can be limited by the collar 14 above it.

[0063] Preferably, there are multiple flat keys 15, which are evenly arranged in the circumferential direction of the stirring shaft 7 to form splined shaft segments on the stirring shaft 7; the slide table 17 is annular, and the inner surface of the slide table 17 is provided with multiple grooves, the same number and relative positions as the flat keys 15, and each flat key 15 is slidably connected to a groove; the floating platform 18 is an annular structure coaxial with the stirring shaft 7, and the top surface of the floating platform 18 is lower than the top surface of the slide table 17. This scheme is a preferred solution for driving the first stirring blade 10 using flat keys 15, slide table 17, and floating platform 18. This scheme not only facilitates the counterweight of the stirring shaft 7 and reduces the vibration during the rotation of the stirring shaft 7, but also avoids problems such as reduced fitting accuracy of the slide table 17 and affected fitting accuracy between the slide table 17 and the collar 14 due to uneven wear through the matching relationship between each flat key 15 and the groove. Understandably, the spline shaft segment mentioned above refers to the section of the stirring shaft 7 containing multiple flat keys 15. This section is spline-shaped, and the grooves on the slide table 17 are distributed and matched with the flat keys 15. Each groove mutually constrains the movement direction of the slide table 17 to ensure that the slide table 17 and the collar 14 maintain a stable contact state. The purpose of the floating platform 18 position setting is to ensure the service life of the floating platform 18.

[0064] As described above, as long as the floating platform 18 can generate sufficient buoyancy, the friction between the sliding platform 17 and the collar 14 can be used to drive the first stirring blade 10. However, this method results in an excessively large volume of the floating platform 18, a significant impact of the mass of the sliding platform 17 on driving the first stirring blade 10, increased wear, and the introduction of culture medium contamination. Consequently, this driving method is also unstable. In this context, preferably, one of the bottom surface of the collar 14 and the top surface of the sliding platform 17 is provided with a strip-shaped rib 16 extending radially along the stirring shaft 7, and the other is provided with a strip-shaped groove extending radially along the stirring shaft 7. The strip-shaped rib 16 can be embedded in the strip-shaped groove. The design purpose of this scheme is that during the upward movement of the sliding platform 17 under the action of the floating platform 18, the strip-shaped rib 16 is embedded in the strip-shaped groove, so that the torque transmission from the stirring shaft 7 to the first stirring blade 10 is sequentially sheared by the flat key 15 and the strip-shaped rib 16. In this way, the reliability of power and torque transmission to the first stirring blade 10 can be maintained. Regarding the setting of the extension direction of the strip ridge 16 and the strip groove, the purpose is as follows: Since the first stirring blade 10 and the stirring shaft 7 rotate relative to each other, when the slide 17 floats and contacts the collar 14, there is a high probability that the strip ridge 16 and the strip groove will be misaligned. Having specified the extension direction of the strip ridge 16 and the strip groove, a simple scheme is proposed to achieve the following mating relationship. Specifically: As the slide 17 and the collar 14 rotate relative to each other, when the strip ridge 16 and the strip groove rotate relative to each other to the same vertical direction, the strip ridge 16 can be embedded in the strip groove, so that the slide 17 and the collar 14 are in a gear-driven state. To control the center position of the slide 17 and the collar 14, one side has multiple strip ridges 16, and the other side has the same number of strip grooves as the number of strip ridges 16. Furthermore, the strip ridges 16 and the strip grooves are evenly arranged in a ring on the top surface of the slide 17 or the bottom surface of the collar 14 relative to the axis of the stirring shaft 7.

[0065] Example 4:

[0066] This embodiment, based on Embodiment 1, provides an application of the disposable bioreactor proposed above in the preparation of adenovirus harvest.

[0067] Specifically, it is a method for preparing adenovirus harvest, comprising the following steps performed sequentially: 293 cell resuscitation, 293 cell amplification culture, adenovirus inoculation, virus culture, and lysis harvest;

[0068] The 293 cell amplification culture utilizes a disposable bioreactor as described above. During the 293 cell amplification culture process, the usage of the stirring blades is determined based on the liquid level of the culture medium in the bioreactor: the second stirring blade 11 is located in the culture medium, and when the first stirring blade 10 can act on the culture medium, the state of the shear force transmission device is changed through the linkage module, causing the stirring shaft 7 to transmit torque to the first stirring blade 10, thereby agitating the culture medium using the first stirring blade 10. This scheme uses the disposable bioreactor proposed above to prepare adenovirus harvest. During the preparation of adenovirus harvest, the activation of the first stirring blade 10 and the activation method of the first stirring blade 10 can be determined based on the actual liquid level of the culture medium. This approach benefits the bioreactor's lifespan, allows for good adaptation to large-scale changes in the liquid level of the culture medium within the reaction bag 2, and helps maintain consistent agitation during the amplification culture process.

[0069] Example 5:

[0070] This embodiment further refines the scheme based on Embodiment 1: Preferably, the culture medium used in the 293 cell amplification process contains 0.1% to 1% by weight of dextran sulfate, wherein the molecular weight of the dextran sulfate is 5KD to 50KD. In this embodiment, a commonly used anti-clustering agent is further selected, and the amount and type of dextran sulfate added are further limited. The main reason is that the addition of dextran sulfate can effectively improve the adhesion and clumping phenomenon of 293 cells during culture, which is beneficial to the activity, viability, and proliferation rate of 293 cells. It can effectively prevent cell clumping or foam formation, ensuring stable production at a scale of 2000L. The entire amplification process can maintain a cell viability of over 90%, with no obvious cell clumping and no foam formation in the solution, eliminating the need for antifoaming agents. Currently, the molecular weight range of dextran sulfate used for cell culture is generally 3-2000 kDa. Dextran sulfate with excessively large molecular weight or excessive concentration can affect the concentration and viability of 293 cells (generally appearing in the later stages of 293 cell culture). Using dextran sulfate with a molecular weight of 50 kDa at a concentration of 0.1% to 1% can effectively reduce the aggregation of 293 cells and increase the cell concentration. When adding dextran sulfate with a molecular weight of 5 kDa at a weight percentage of 0.1% to 1%, the growth of 293 cells, the basic charge isomer, and the viability can all be significantly improved, and it has a certain impact on ammonium cations, etc. The above methods of adding dextran sulfate can increase cell concentration by 1.2 to 1.5 times. Comparing the amounts and molecular weights of dextran sulfate mentioned above, when the amount added and the molecular weight of dextran sulfate are lower, the increase in cell concentration is less than 1.2 times. When the amount added and the molecular weight of dextran sulfate used are higher, the cell concentration can be increased by 1.2 to 1.5 times or more within a certain range. However, this will make it more difficult to remove dextran sulfate from the protein during the subsequent purification process. When using the above-mentioned amounts and molecular weights of dextran sulfate, the recovery rate of dextran sulfate can be greater than 99.5% when precipitating proteins with general TCA reagent.

[0071] Preferably, in order to avoid contamination from animal-derived substances and improve toxin production efficiency and product quality, the culture medium used is Dynamis serum-free medium.

[0072] Example 6:

[0073] This embodiment, based on Embodiment 4 or 5, provides a more detailed method for preparing adenovirus harvest:

[0074] This embodiment uses serum-free medium to culture suspension 293 cells at high density. When the cell density is appropriate, genetically modified adenovirus is inoculated. The virus is prepared using shake flasks or disposable bioreactors (using disposable bioreactors for scale-up culture). The preparation process includes: stepwise cell scale-up, virus inoculation, virus culture, lysis and harvesting, with a preparation scale of 2000L. The core of the process lies in optimizing various procedures and parameters in the cell and virus culture process. The magnetic stirring mechanism simplifies large-scale production, reduces the risk of contamination, and ensures consistency of process parameters. The entire process uses serum-free medium with clearly defined components for high-density culture of 293 suspension cells, avoiding contamination from animal-derived substances and improving toxin production efficiency and product quality. The addition of appropriate multi-component nutrient supplements during virus culture can effectively increase the adenovirus titer in the harvested product, thereby improving economic benefits.

[0075] The specific process includes:

[0076] Suspension cells were seeded into shake flasks and progressively scaled up to a bioreactor for culture, with a cell passage density of 4 × 10⁶ cells / year. 5 1 x 10^1 ml 6 Cells / ml, cell harvest density 6×10⁶ 6 7 x 10 cells / ml 6 Cells were cultured at a density of 0.1%–1% dextran sulfate, with a molecular weight of 5–50 kDa. The suspension cells used were Expi293F cells (purchased from Thermo Fisher Scientific, catalog number 14527CN). The temperature for suspension cell culture in shake flasks was set at 35–38°C, the shaker speed at 100–130 rpm / min, the amplitude at 30–50 mm, 4–8% CO2 was introduced, and the humidity was controlled at 60%–90%. For large-scale culture of suspension 293F cells in a single-use reactor using serum-free medium, the cell culture temperature was set at 37–38°C, dissolved oxygen (DO) at 40%–60%, pH at 7.2–7.4, the reactor speed at 30–60 rpm / min, and the glutamine concentration at 6–8 mM. The culture medium was Dynamis (Gibco, catalog number A26175-03) serum-free medium with clearly defined components. The cell density at virus inoculation was 3–4 × 10⁶ cells / ml. 6The virus culture rate is 100 cells / ml, and the virus culture time is 48-60 hours. The adenovirus is a replication-defective adenovirus with the E1 gene knocked out, including but not limited to adenovirus types 7, 26, or 68. The virus can specifically proliferate in 293 cells. During the adenovirus culture process, a suitable multi-component nutrient supplement is added to supplement nutrients, promote virus proliferation, and increase virus titer. The components are characterized by containing one or more of the following components: 1-10 μg / ml of vitamin C, 0.1-0.5 mM of sodium butyrate, 0.1-0.8 mM of non-essential amino acids, 5-10 mM of glucose, 2-8 mM of glutamine, and 0.05%-1% of dextran sulfate. The lysis buffer added at virus harvest consists of: 500 mM Tris-HCl, 10% Tween 20, 20 mM MgCl2, and pH 8.0. The pyrolysis treatment time is 2 to 4 hours. The reactor parameters during pyrolysis are set as follows: temperature 35 to 38℃, rotation speed 60 to 120 rpm / min, and bottom aeration 0.1 to 0.5 L / min.

[0077] More detailed:

[0078] A. Cell resuscitation: Prepare Dynamis complete medium, adding 6-8 mM glutamine and 0.1%-1% dextran sulfate. Remove cells from liquid nitrogen and thaw in a 37°C water bath. Transfer the thawed cell suspension to 29 ml of Dynamis complete medium. Culture the cells in 125 ml shake flasks in a CO2 shaking incubator. Set the incubator speed to 100-130 rpm / min, amplitude to 30-50 mM, culture temperature to 35-38°C, and CO2 concentration to 4%-8%.

[0079] B. Cell amplification: Cells in shake flasks grow to 5~7*10 6 When the cell count is 1 cell / ml (cell viability > 90%), passage the cells at a ratio of 1:7 to 1:10. Scale up the cells to a single-use bioreactor with the following parameters: culture temperature 35–38°C, dissolved oxygen (DO) 40%–60%, pH 7.2–7.4, reactor rotation speed 30–60 rpm / min, and bottom aeration 0.1–0.5 L / min.

[0080] C. Virus culture: Cells in the reactor grow to a density greater than 6*102 6 When the cell density is 3-4 cells / ml (cell viability > 90%), dilute the cells to 3-4 × 10⁻⁴ cells / ml using Dynamis complete medium. 6Approximately [number] cells / ml were added, along with 5 μg / ml of vitamin C, 0.1 mM sodium butyrate, 1X non-essential amino acid solution (NEAA), 10 mM glucose, and a final glutamine concentration of 8 mM. The reactor culture parameters were set as follows: culture temperature 35℃, DO 30%, pH 7.2, and reactor rotation speed 40 rpm / min. Virus was inoculated at an MOI (copy number) of 100-400, and after 48-60 hours of virus culture, lysis buffer was added to lyse the cells and harvest the virus.

[0081] D. Lysis and Harvesting: When the virus culture is complete, add lysis buffer at 1 / 9 of the total volume, and add toleranced nuclease (Shanghai Yisheng) at a final concentration of 10 U / ml for treatment. The treatment time is 2-4 hours, the treatment temperature is 35.0℃, and the reactor speed is 60-120 rpm / min. After treatment, the virus is transferred to downstream purification.

[0082] By employing the above process and optimizing various conditions and parameters for cell and virus culture, and using the disposable bioreactor described above, the cell culture density and growth status are improved, enabling large-scale harvesting of high-titer virus products. The entire production process does not add any animal-derived components, ensuring vaccine safety; and the use of suspension 293 cells to prepare adenovirus meets the needs of rapid scale-up for industrial production.

[0083] Figure 4 The accompanying Expi293 suspension cell proliferation curve is provided, wherein the cell proliferation curve during large-scale culture of Expi293 suspension cells is as follows: Figure 4 As shown, 4*10 were inoculated. 5 Cells per ml proliferated rapidly to a plateau phase after 96 hours, reaching a density of 7*102 cells / ml. 6 Cells / ml, up to 9*10 6 The cell count was reduced to 10 cells / ml, achieving nearly 20-fold proliferation. Throughout the culture process, there was no large-scale cell aggregation, and the cell viability remained above 90%, indicating good cell condition.

[0084] Figure 5 The nutritional metabolism of Expi293 suspension cells during proliferation is provided in connection with this embodiment, wherein the detection of metabolic-related indicators during the proliferation of Expi293 suspension cells is as follows: Figure 5 As shown, without the need for fresh culture medium perfusion support, the glucose and glutamine contents in the culture medium gradually decreased during cell proliferation, reaching their limit at 96 h; the metabolites lactic acid and ammonia gradually increased, but their concentrations were controlled at relatively reasonable levels, and they did not seriously inhibit cell proliferation, indicating that the overall nutrient supply of this high-density culture mode of 293 suspension cells was relatively balanced.

[0085] Small-scale process comparison:

[0086] Preparation of recombinant adenovirus from adherent cells (control group):

[0087] 293A adherent cells were passaged into T225 cell culture flasks at a ratio of 1:7. The culture medium was DMEM high-glucose medium (Gibco) containing 10% fetal bovine serum (FBS). The culture volume was 60 ml. After about 3 days, when the cells reached 100% confluence and formed a dense monolayer, the cell density was approximately 2*102. 5 pcs / cm 2 Adenovirus can be inoculated. Before inoculation, discard the original culture medium and replace it with DMEM high glucose medium (Gibco) containing 2% fetal bovine serum (FeBCO). Inoculate with replication-defective adenovirus (Adc68 or Adc7) at an MOI of 100-400 (in copy number). Incubate the virus at 35°C for 48 hours. After two freeze-thaw cycles in the culture flask, lyse the cells thoroughly. Take 1 ml of the virus suspension and use qPCR to detect the virus copy number.

[0088] Preparation of recombinant adenovirus from suspension cells (experimental group):

[0089] Suspended 293 cells (Expi293) were seeded at a density of 4-5 x 10⁻⁵. 5 Cells were seeded at a density of 60 ml into 250 ml shake flasks. The culture medium was Dynamis serum-free Gibco medium supplemented with 8 mM glutamine and 1% dextran sulfate. The suspension cells were then placed in a CO2 shaker at 37°C with a shaking speed of 130 rpm and an amplitude of 50 mm. 5% CO2 was introduced, and humidity was controlled at 80%–90%. After 3 days of culture, the cell density reached 6–7 × 10⁻⁶ cells / ml. 6 Cells / ml, up to 9*10 6 Cells / ml were diluted to a density of 3-4 × 10⁶ cells / ml using Dynamis serum-free medium. 6Cells were counted at 1 / ml, excess cells were discarded, and cells were supplemented with 5 μg / ml vitamin C (Sigma), 0.1 mM sodium butyrate (Sigma), 1x non-essential amino acid NEAA (Sigma), 10 mM glucose, 8 mM glutamine, and 1% dextran sulfate as the experimental group. Recombinant adenovirus (Adc68 or Adc7) was inoculated at an MOI of 100–400 (copy number). The virus was cultured at 35°C in a shaker for 48–60 h with a shaker speed of 130 rpm / min, an amplitude of 50 mm, 5% CO2, and humidity controlled at 80%–90%. At virus harvest, lysis buffer was added to lyse the cells at 1 / 9 of the total virus volume, and a totipotent nuclease (Shanghai Yisheng) was added to a final concentration of 10 U / ml to degrade host nucleic acid. All parameters of the shaker remained unchanged during cell lysis. After 2–4 h of treatment, 1 ml of the virus suspension was collected for qPCR to detect viral copy number. The virus detection results for the experimental and control groups are shown in Table 1 and [Table data would be inserted here]. Figure 7 As shown:

[0090] Table 1. Results of Process Comparison (Virus Yield)

[0091] Group Cell types MOI Training time Culture volume Virus production control group 293A Wall-mounted 400 48h 60ml <![CDATA[1.24~3.04*10 11 copy / ml]]> experimental group Suspension 293 400 48~60h 60ml <![CDATA[7.53~11.40*10 11 copy / ml]]>

[0092] From Table 1 and Figure 7 It is known that, using suspension cells to prepare adenovirus, under the same culture volume and MOI conditions, the virus content obtained by suspension cell preparation is about 3 to 4 times higher than that of adherent cells, which greatly improves the total virus yield and has significant economic benefits. The suspension 293 cells are cultured in serum-free medium throughout the process, without any animal-derived components, which reduces the contamination of exogenous substances and improves the safety of the vaccine product. Compared with adherent cells, suspension 293 cells are easier to scale up for high-density culture, are simpler to operate, do not require trypsin digestion, and reduce the risk of contamination.

[0093] To meet the needs of industrial vaccine production and achieve the rapid preparation of large-scale adenovirus vector vaccines, the scale was gradually increased based on small-scale trials, and scaled up and validated at a production scale of 2000L.

[0094] Process scale-up and verification:

[0095] use Figure 6 The provided adenovirus preparation process flow chart.

[0096] Expi293 cell amplification culture route as follows: Figure 6The process was scaled up gradually in shake flasks and disposable bioreactors to reach a final production scale of 1700-2000L. Cell passage in shake flasks followed the same method as the pilot-scale process. When culturing cells in a modified 200L or 2000L disposable bioreactor (manufacturer: PALL, model STR200 or STR2000) with a magnetic stirring mechanism, the following parameters were set: culture temperature 37℃, dissolved oxygen (DO) 60%, pH 7.2, reactor rotation speed 30-40 rpm / min, and bottom aeration 0.1-0.5 L / min. Cells were cultured to a density of 6-7 x 10⁻⁶ cells / min. 6 At a cell density of 4 x 10⁶ cells / ml, the cells were diluted to 4 x 10⁶ cells / ml using Dynamis medium. 6 Cells were cultured at a concentration of 100 cells / ml, supplemented with 5 μg / ml vitamin C (Sigma), 0.1 mM sodium butyrate (Sigma), 1x non-essential amino acid NEAA (Sigma), 10 mM glucose, 8 mM glutamine, and 1% dextran sulfate. Recombinant adenovirus (Adc68 or Adc7) was inoculated at an MOI of 100–400 copies / ml. During virus culture, the reactor temperature was set at 35°C, the rotation speed at 30–40 rpm / min, dissolved oxygen (DO) at 30%, pH at 7.2, and bottom aeration at 0.1–0.5 L / min. After approximately 56 hours of virus culture, lysis buffer was added to lyse the cells at 1 / 9 of the total volume. A totipotent nuclease at a final concentration of 10 U / ml was added to degrade host nucleic acid. After 2–4 hours of treatment, the harvested virus was transferred to the purification section.

[0097] Based on the virus content detection results of the 2000L lysed harvest, the copy number was 1.08~1.2*10. 12 The copy / ml reading indicates that this process can be easily scaled up for production, and the viral yield of the harvested material is consistent with that of the small-scale process. In summary, by optimizing the cell culture and adenovirus preparation process and related parameters, high-density, large-scale serum-free culture of 293 suspension cells was achieved, resulting in a higher adenovirus content without the risk of animal-derived contamination. This process is simple to operate, has low contamination risk, and offers significant economic benefits, meeting the needs for rapid scale-up production of adenovirus vector vaccines.

[0098] The above description, in conjunction with specific preferred embodiments, provides a further detailed explanation of the present invention. It should not be construed that the specific embodiments of the present invention are limited to these descriptions. For those skilled in the art, other embodiments derived without departing from the technical solution of the present invention should be included within the scope of protection of the present invention.

Claims

1. A disposable bioreactor for preparing adenovirus harvest, comprising a reaction bag (2) and a magnetic stirring mechanism disposed inside the reaction bag (2), the magnetic stirring mechanism comprising a stirring shaft (7), wherein stirring blades are mounted on the stirring shaft (7), the number of stirring blades being greater than 1 and arranged in layers on the stirring shaft (7), characterized in that, The stirring blades include a second stirring blade (11) at the bottom and a first stirring blade (10) above the second stirring blade (11). The second stirring blade (11) is fixed on the stirring shaft (7). The second stirring blade (11) rotates synchronously with the stirring shaft (7) when the stirring shaft (7) rotates. The first stirring blade (10) is installed on the stirring shaft (7) through a collar (14). The collar (14) is fixed on the axis of the stirring shaft (7). The collar (14) can rotate around the axis of the stirring shaft (7). The stirring blades also include a linkage module and a shear force transmission device. The linkage module and the shear force transmission device are both installed on the stirring shaft (7). The linkage module is used to drive the shear force transmission device to connect and disconnect the shear force transmission device from the collar (14). In the connected state, the shear force is transmitted through the shear force transmission device, so that the first stirring blade (10) rotates under the action of the stirring shaft (7). In the disconnected state, the collar (14) and the stirring shaft (7) remain in a state of relative rotation. The magnetic stirring mechanism also includes an isolation sleeve (3) at the bottom of the disposable bioreactor and a first permanent magnet (4) installed in the isolation sleeve (3). The first permanent magnet (4) is fixed to the side wall of the end of the stirring shaft (7) that extends into the isolation sleeve (3). The stirring shaft (7) is a hollow shaft. The linkage module includes a push rod (8) inside the stirring shaft (7) and a second permanent magnet (9) inside the stirring shaft (7). The second permanent magnet (9) is located in the end of the stirring shaft (7) that extends into the isolation sleeve (3). The second permanent magnet (9) is fixedly connected to the push rod (8). The push rod (8) extends along the length of the stirring shaft (7). A boss (12) is also provided on the push rod (8). Pin holes (19) are provided on both the stirring shaft (7) and the collar (14). The pin holes (19) are all oblique holes, and the pin holes (19) are close to the stirring shaft (7). One end of the axis is the lower end of the pin hole (19). The linkage module also includes a transmission pin (13) set in the pin hole (19). The end of the transmission pin (13) near the axis of the stirring shaft (7) is supported on the outer surface of the boss (12) or the outer surface of the push rod (8). When the push rod (8) is at the lowest position in the stirring shaft (7), the transmission pin (13) slides down under its own weight to the position where it is dislodged from the pin hole (19) of the collar (14). During the process of the push rod (8) sliding upward relative to the stirring shaft (7) along the length direction of the stirring shaft (7), under the action of the guide surface on the top side of the boss (12), the transmission pin (13) can be pushed by the guide surface to make the transmission pin (13) embed into the pin hole (19) of the collar (14). The inner side of the collar has an annular groove coaxial with the stirring shaft. The pin hole opening on the collar is located at the bottom of the annular groove. The top of the transmission pin is a spherical surface. Alternatively, the linkage module includes a floating platform (18), and the shear force transmission device includes a flat key (15) and a sliding platform (17). The flat key (15) is parallel to the stirring shaft (7) and is disposed on the outer wall of the stirring shaft (7). The sliding platform (17) is slidably connected to the flat key (15) through a sliding groove on it. The floating platform (18), the flat key (15), and the sliding platform (17) are all disposed below the first stirring blade (10). Under the action of buoyancy, the floating platform (18) drives the sliding platform (17) to slide upward along the length direction of the flat key (15). During this process, the top surface of the sliding platform (17) can contact the bottom surface of the collar (14). The force applied to the collar (14) by the sliding platform (17) causes the stirring shaft (7) to drive the first stirring blade (10) to rotate. A limiting device is provided on the stirring shaft to limit the lowest position of the sliding platform.

2. The disposable bioreactor for preparing adenovirus harvest according to claim 1, characterized in that, Multiple drive pins (13) are provided between the stirring shaft (7) and the collar (14), and each drive pin (13) is provided with a pin hole (19) on the stirring shaft (7) and the collar (14); the boss (12) is a columnar structure with an outer diameter larger than the outer diameter of the push rod (8) and coaxial with the push rod (8); the drive pins (13) are evenly arranged in the circumferential direction of the stirring shaft (7), and the guide surface is a spherical surface with a smooth transition between the lower end and the side of the boss (12), and the shape of the spherical surface is... The relative positions of the drive pins (13) satisfy the following: when the boss (12) rises and falls relative to the stirring shaft (7), the drive pins (13) rise and fall synchronously under the action of the spherical surface on each drive pin (13); the relative relationship between the pin hole (19) and the drive pin (13) satisfies the following: as the drive pin (13) is further embedded in the pin hole (19) in the collar (14), the push rod (8) can rise to the position where the lower end of the drive pin (13) acts on the side of the boss (12).

3. The disposable bioreactor for preparing adenovirus harvest according to claim 1, characterized in that, The number of the first stirring blades (10) is greater than 1. Each first stirring blade (10) is equipped with a boss (12), a pin hole (19) and a transmission pin (13). As the position of the push rod (8) rises in the stirring shaft (7), the first stirring blades (10) arranged in sequence along the length direction of the stirring shaft (7) have the following relationship: the first stirring blade (10) at the bottom is connected to the stirring shaft (7) through the pin hole (19) in the collar (14) via the transmission pin (13) before the first stirring blade (10) at the top. When the first stirring blade (10) at the topmost side is connected to the stirring shaft (7) via the transmission pin (13), the other first stirring blades (10) maintain the transmission connection relationship with the stirring shaft (7).

4. The disposable bioreactor for preparing adenovirus harvest according to claim 1, characterized in that, The number of the flat keys (15) is multiple, and the flat keys (15) are evenly arranged in the circumferential direction of the stirring shaft (7) to form a spline shaft segment on the stirring shaft (7); the slide (17) is annular, and the inner side of the slide (17) is provided with multiple sliding grooves, the number of which is the same as the number of flat keys (15) and the relative position of which is the same as the relative position of the flat keys (15), and each flat key (15) is slidably connected to a sliding groove; the floating platform (18) is an annular structure coaxial with the stirring shaft (7), and the top surface of the floating platform (18) is lower than the top surface of the slide (17).

5. The disposable bioreactor for preparing adenovirus harvest according to claim 1 or 4, characterized in that, On the bottom surface of the collar (14) and the top surface of the slide (17), one of them is provided with a strip rib (16) extending in the radial direction along the stirring shaft (7), and the other is provided with a strip groove extending in the radial direction along the stirring shaft (7). The strip rib (16) can be embedded in the strip groove.

6. A method for preparing adenovirus harvest, comprising the following steps performed sequentially: 293 cell resuscitation, 293 cell amplification culture, adenovirus inoculation, virus culture, and lysis harvest; Its features are, The 293 cell amplification culture uses a disposable bioreactor as described in any one of claims 1 to 5. During the 293 cell amplification culture process, the method of using the stirring blade is determined according to the liquid level of the culture medium in the bioreactor: the second stirring blade (11) is located in the culture medium. When the first stirring blade (10) can act on the culture medium, the state of the shear force transmission device is changed through the linkage module, so that the stirring shaft (7) transmits torque to the first stirring blade (10) and the culture medium is stirred by the first stirring blade (10).

7. The method for preparing adenovirus harvest according to claim 6, characterized in that, The culture medium used in the 293 cell amplification process contains 0.1% to 1% by weight of dextran sulfate, wherein the molecular weight of the dextran sulfate is 5KD to 50KD.

8. The method for preparing adenovirus harvest according to claim 6, characterized in that, The culture medium is Dynamis serum-free medium.

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