DESC:FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
1. TITLE OF THE INVENTION : A CELL CULTURE BIOREACTOR WITH CELL HARVESTOR AND METHOD OF CELL HARVESTING
2. APPLICANT:
(a) NAME : OMNIBRX BIOTECHNOLOGIES PRIVATE LIMITED
(b) NATIONALITY : India
(c) ADDRESS : B-202, Royal Residency,
Nr. Shukan,
Opp. Vandematram Arcade,
New S.G. Road,Gota,
Ahmedabad – 382481,
Gujarat, India

3. PREAMBLE TO THE DESCRIPTION
PROVISIONAL

The following specification describes the invention. ?COMPLETE

The following specification particularly describes the invention and the manner in which it is to be performed.

The present invention is a combination of Provisional Patent Application No. 202221058526 Filed on October 13, 2022 and Provisional Patent Application No. 202221076880 Filed on December 29, 2022.

Field of Invention
The present invention is generally relates to a method and system for harvesting cells grown in culture and more particularly, it relates to a cell culture bioreactor with cell harvester for harvesting cells grown in culture by applying jarring/jerky rotation motion to release the cells from the cell carrier matrix so they may be recovered with high yield, and high cell viability and vitality.
Background of Invention
Generally, Living cells are good host for production of therapeutics such as monoclonal antibodies, vaccines, cell & gene therapy medicines etc. To culture these cells, an apparatus termed as bioreactor are used which facilitate the growth of these cells and ultimately the production of the desired product.
Populations of living cells are becoming increasingly important in medicine as biologic agents useful for treating a variety of different medical conditions. For example, there has been considerable interest focused on the therapeutic potential of human cells for various medical applications including tissue repair of damaged organs such as the brain, heart, bone and liver, and to support bone marrow transplantation (BMT). One class of human cells, adult stem cells, have been evaluated for treating and curing various conditions such as hematopoietic disorders, heart disease, Parkinson's disease, Alzheimer's disease, stroke, burns, muscular dystrophy, autoimmune disorders, diabetes, and arthritis.
Currently, the growing of cells on a large scale is carried out in apparatus comprising a vessel for the growth medium and arranged within the vessel a plurality of surfaces upon which the cells grow, these surfaces being discs or plates spaced apart from one another.
Now, in order to harvest the cells from the surface of the cell-carriers or macro-carriers arranged inside the bioreactor, conventionally particular process steps are carried out like removal of spent media from bioreactor, addition of washing solution in bioreactor, removal of washing solution from bioreactor, addition of cell detachment solution (Enzyme), removal of cell detachment solution (Enzyme), addition of media for cell collection, removal of media containing cells etc.
For some bio-manufacturing applications, the cells grown in the fixed bed are required to be harvested from the bioreactor after the growth phase. This may be when the cell harvest is used as a seed culture to expand the cells to inoculate another (e.g. production) bioreactor or when the cells themselves are the product of interest (e.g. to produce a cell bank or for cell therapy application). To harvest living cells from a fixed bed, chemical reagents such as trypsin can be used. However, this alone usually results in a limited amount of cell separation, often due to the densely packed nature of the fixed bed material in a typical bioreactor, which makes it more difficult to circulate chemicals throughout the bed and decrease the yield of cells harvested.
One more technique prevailed for harvesting cells is vibration based harvesting method. In this method, the cells are harvested from the vessel by applying vibration to the adherent material to dislodge the cells from the surface of the discs. One such vibration-based harvesting method is described in the patent application US2014/0030805 A1. In said patent application, apparatus comprising an adherent material in a container, and a vibrator for imparting a reciprocating motion to the adherent material, the vibrato comprising one or more controls for adjusting amplitude and frequency of the reciprocating motion, wherein the vibrator is configured to vibrate in a manner causing cells attached to the adherent material to detach from the adherent material.
One more patent application US2022/0033751Al describes the method for harvesting cells by oscillating the basket. According to said patent application, basket is oscillated within culture vessel, along longitudinal axis of culture vessel to harvest the cells.
Cells are attached to the fixed bed by small, cable-like proteins called integrins. In addition, cells produce other proteins, like collagen and glycosaminoglycans, which form an extracellular matrix that resembles a mesh. Even when separation solutions such as trypsin are used to achieve the separation of cells from structures, these proteins may cause the harvested cells to clump together. This may be undesirable for recovery purposes and may result in a lower than desired cell yield.
However, after carrying out above given process, only 20-40% cells are recovered from the surfaces of the cell carrier discs arranged inside the bioreactor. Further, such process of recovering cells is tedious, time and cost consuming.
Hence, it is desperately needed to invent a system and method that substantially and effectively recover/harvest the cells from the cell carrier surfaces over which these cells are attached and grow.
Object of the Invention
The main object of present invention is to provide a vibro-rotating system and method of cell harvesting that effectively and substantially recover cells from the vessel without damaging the cells.
One more object of the present invention is to provide stably recover the cultured or grown cells on cell matrix assembly.
Further object of the present invention is to provide system (bioreactor) which is beneficial in terms of efficiency, cost, regulatory friendly etc.
Another object of the present disclosure is to provide a cell harvesting system that provides ease of operation.
One more object of the present invention is to provide a system that delivers mechanical stress to the cell carrier matrix assembly inside the bioreactor to dislodge and detach the grown cells more efficiently from the cell carrier matrix in more healthy state.
Yet another object of the present invention is to recover 80-95% cells from the cell carrier matrix assembly.
Summary of Invention
The present invention is relates to a system and method for harvesting of cells by detaching the cells from the cell carrier of the cell culture bioreactor. In present invention, the cell carrier matrix assembly being disposed within the hollow interior of the vessel and comprises a one or multiple stacked and spaced apart discs centrally and longitudinally loaded on the central shaft and rotates along with central shaft. A rotating member of a bottom ring is rotatably and secured at free end of respective extended member. A bottom hub is formed with a ridge is formed with downwardly extended legs. During rotation of central shaft, the rotating members of the bottom ring roll over the ridge of the bottom hub. Said rotating members make the cell carrier matrix assembly to experience jerk while moving over the projections on the bottom hub. Due to said configuration, the cell carrier matrix assembly causing cells attached to the discs to detach from the discs. Thus, the cells are effectively recovered from the surface of the discs.
Brief Description of Drawings
Fig 1: illustrates transparent view of the system for cell harvesting according to the present invention.
Fig 2: illustrates perspective view of cell carrier matrix assembly of the system according to the present invention.
Fig 3: illustrate exploded view of a bottom plate with first and second magnetic ring according to the present invention.
Fig 4: illustrate exploded view of a system according to the present invention.
Fig 5A: illustrates exploded view of bottom ring according to the present invention.
Fig 5B: illustrates assembled perspective view of bottom ring according to the present invention.
Fig 6: illustrates perspective view of a bottom hub assembly according to the present invention.
Fig 7: illustrates perspective view of a vessel with impeller and central hub according to the present invention.
Fig 8A: illustrates perspective view of a bottom ring assembly according to another embodiment.
Fig 8B: illustrates perspective view of a bottom hub assembly according to another embodiment.
Fig 9: illustrates assembling of bottom ring and bottom hub assembly illustrated in Fig. 8a and 8b.
Fig. 10: illustrate perspective view of bioreactors connected through the tubing according to the present invention.

Detail Description of Invention
Before explaining the present invention in detail, it is to be understood that the invention is not limited in its application to the details of the construction and arrangement of parts illustrated in the accompanied drawings. The invention is capable of other embodiments, as depicted in different figures as described above and of being practiced or carried out in a variety of ways. It is to be understood that the phraseology and terminology employed herein is for the purpose of description and not of limitation.
It is to be inserted that with reference to the Figures, exemplary embodiments of the invention will now be described. The exemplary embodiments are described primarily with reference to drawings. It should be appreciated that not all components necessary for a completed implementation of a practical system are illustrated or described in detail. Rather, only those components necessary for a thorough understanding of the invention are illustrated and described. Furthermore, components which are conventional in accordance with the teachings provided herein are not described in detail.
As used herein, “Interior” means the inner volume of the container volume, which is enclosed by the wall.
As used herein, "cells" refers to any mammalian cell capable of being cultured in vitro. In certain embodiments, the cells are human.
As used herein the phrase "adherent cells" refers to a homogeneous or heterogeneous population of cells which are anchorage dependent, that is, require to attach with the surface in order to grow in vitro.
As used herein the phrase "harvesting" means removing cells from a 2-dimensional or a 3- dimensional carrier.
It is to be understood that the bioreactor system according to present invention is used for cell growth. It is configured to provide an optimal environment for cell growth allowing for precise control of temperature, PH, dissolve oxygen, and other factors. Said system is also configured for providing an efficient system for monitoring cell growth as well as the ability to scale up the production when necessary. In the present invention, the cells in culture are grown on the adherent material by any conventional method. Hence, in the description below, the method for growing cells is not described in detailed since it is well very understood by the person skilled in the art.
In particular, the cells are grown on an adherent Surface, which in some cases is a 2 or 3-dimensional matrix. After the cells have been cultured in the bioreactor, they must be harvested for further processing. The harvesting process typically involves removing the cells from collecting them in a sterile container. The cell carrier is used for their intended applications.
The present invention provides a system (100) and method for detachment and harvesting of cells grown in the culture by applying intermittent jarring/jerky rotational motion to the matrix (7). The cells that grow attached to the adherent material are detached with high efficiency, resulting in recovery of greater quantities of cells compared to existing methods for harvesting cells that are known in the art.
The system according to present invention may be, but not limited to, a cell culture bioreactor system which is generally used for culturing cells from animal origin. Now as shown in preferred embodiment of Figs. 1 and 4, the system (100) comprised of a vertically disposed vessel body (1) includes a cylindrical outer shell (2), a bottom plate (3)to seal the bottom of the outer shell and a head plate (4) that seals the top opening of the outer shell. Said configuration of vessel (1) forms a hollow interior within the outer shell. Further, said vessel (1) may equipped with an inlet port for introducing nutrient (culture) medium and/or biological cells into and an outlet port for discharging the nutrient (culture) medium from the vessel (1).
The system (100) according to present invention may further comprises a fluid pumping means for driving the nutrient medium through the vessel (1), a gas exchange module (26) for dissolving gases into and removing waste gases from the nutrient medium and a main conduit fluidly and externally connects said inlet port and outlet port to form a closed external loop for circulation of nutrient medium and being extended through the gas exchange module (26) and fluid pumping means. The gas exchanger module (26) is also internally placed and not need to connect externally with the main bioreactor vessel.
It is within the scope of present invention to employ pressure indicator and regulator, kinetic energy sources for rotation of discs (7) loaded within the cell carrier matrix assembly (6) and baffling means, one of more sensing elements, process control means, variable speed pump and/or fixed speed pump (not shown) in the fluid recirculation system. The nutrient fluid is discharged through the fluid outlet port and passes from the gas exchange means through the fluid pumping means and then fed into the culture vessel through the inlet port to form a closed circulation loop through the main conduit. Said gas exchange means is capable of transferring process gases such as oxygen into and removing waste gases such as carbon dioxide from the nutrient medium when the Gas exchanger module (26) is connected with the main bioreactor externally. Then the gas exchanger module (26) is placed inside the bioreactor, it is placed in between inside wall of the vessel and curved vans/impellers.
Now as shown in Fig. 1, said vessel (1) is equipped with a central shaft (5) being extended within the interior of the vessel (1), means for rotating being mounted on the head plate and engaged with said central shaft (5) for providing rotary motion to the shaft, a cell carrier matrix assembly (6) being disposed within the hollow interior of the vessel (1) and comprises a one or multiple stacked and spaced apart discs (7) centrally and longitudinally loaded on the central shaft (5), a bottom ring (8) being secured along the central shaft (5) just below the cell carrier matrix assembly (7), a central hub (9) being disposed on the upper surface of the bottom plate (3) and a bottom hub (10) being snugly fitted over the central hub (9). Said central shaft (5) being in connection with the rotating means at its first end and is secured within the central hub (9) at its distal end.
Now as shown in Fig. 2, said central shaft (5) is formed with a first region (22) and a second region (23). Said first region (22) of said shaft (5) is preferably kept in square shape and the second region (23) is preferably square or cylindrical. Said first region (22) and the second region (23) are separated at a retaining member (17). Said cell carrier matrix assembly (6) is disposed along the second region (23) of the central shaft (5).
Referring to Fig. 3, the center of the head plate having a predefined diameter hole where a central protrusion (32) is positioned. Said protrusion is in the form of cylindrical shape where the upper portion is covered through the plain surface and define a headspace toward the interior of the vessel (1). A downwardly extended pin (33) is placed at the center of the plain surface of protrusion (32).
In present embodiment, means for rotating the central shaft (5), as shown in Fig. 3 and 4, includes a first magnetic ring (11) being suspended and supported through the pin (33) within the headspace and a second magnetic ring (12) being disposed opposite to the first magnetic ring (11) on the outer surface of the protrusion (32). Said second magnetic ring (12) is connected with driving means i.e. motor (29) through a drive shaft (30). The distal end of the drive shaft (30) of the motor (29) is drivably fitted in the center opening of the second magnetic ring (12).
Referring continuous with to Figs. 3 and 4, said first magnetic ring (11) includes a center bearing (31) being fitted with the pin (33), a plurality of magnets (13) being arranged surrounding to the bearing and an elongated receiving member (15) being extended downwardly within the elongated space from the center of the first magnetic ring (11). Said second magnetic ring (12) includes a plurality of magnets (13) being arranged surrounding the central opening. Each magnet (13) includes a pole N and a pole S. The poles of each magnet (13) of the first (11) and second (12) magnetic rings are opposite to each other and have opposite magnetic polarities, so as to create a magnetic attraction between them. The magnets (13) of the first magnetic ring (11) and the second magnetic ring (12) are positioned such that the pole (for example S) of the magnets of second magnetic ring (12) faces toward the opposite pole (for example N) of the corresponding magnets (13) of the first magnetic ring (11). Hence, the magnetic field lines of pole N of the first magnetic ring (11) are in the opposite direction relative to those entering the pole S of the second magnetic ring (12). From that result, the magnets (13) of the first (11) and second (12) magnetic rings are magnetically attracted to each other, and the first (11) and second (12) magnetic rings come into contact through the magnetic force.
Now, as shown in Fig. 2, a resilient member (16) like, but not limited to, spring or bellow is disposed along the first region (22) of the central shaft (5). Said resilient member (16) is rested between the retaining member (17) of the central shaft (5) and a bottom end of the receiving member (15) of the first magnetic ring (11). Said resilient member (16) is prevented from dropping off and is limited in its downward motion through the retaining member (17). Said receiving member (15) is configured to slidably receive the top portion of the first region (22) of the shaft (5) formed above the resilient member (16).
It is within the scope of present invention to employ means for rotating the shaft other than described herein.
Now Figs. 2 and 4 illustrate an arrangement of discs (7) on the central shaft (5). The disc (cell carrier) (7) having a surface on which the cells are grown and cultured. According to Fig.2, the (permeable) discs (7) are centrally and longitudinally mounted on the shaft (5) by maintaining predetermined space between two successive discs (7) through a spacer (not shown) to define an interspatial space. Said spacer may be disposed between the discs (7) maintains substantially equidistant separation between the discs (7). Preferably spacers can be made of a similar material which is used for the construction of discs (7) or spacers can be made of silicon rubber. Ratio of spacer diameter and disc diameter is to be optimized according to the process scale. Other means of supporting and separating the discs (7) may be employed; for example, but not limited to, each disc (7) have a ridge or spacer formed integrally during its construction at the central portion. This ridge or spacer then rests on the spacers of the discs immediately adjacent to it. The presence of cylindrical spacers between each disc essentially ensures that the discs mounted on a shaft are in a separated state throughout the operation.
The discs (7) according to present invention are preferably constructed from, but not limited to, a non-woven fibrous material. cell attachment can occur on either side of the disc, thereby providing a very large surface area for attachment and growth of cells within a small space or volume. Typically, a thin monolayer or film of the cell growth is observed on disc surfaces and generally has a thickness of from a few µm, e.g. 1 µm, to about 1 mm, i.e. 100 µm. In case of where applications demand for multilayered or structured growth of cells, the discs (7) are molded in desired shape and the surfaces can be created by treating them physically, chemically or biologically.
The cell carrier matrix assembly is the structure of different arrangements of cell carrier discs (7) over the central shaft (5). The cell carrier matrix assembly (9) may be covered with the curved vane structure which delivers the impeller like function inside the vessel (1) and it is specifically incorporated to create an inward flow of the fluid content within the bioreactor whereby the fluid can penetrate within the internal vicinity of the disc loaded on the shaft and thereby provides sufficient nutrients and gaseous requirement of the cells growing inside the disc within the cell carrier matrix.
Referring Figs. 2 and 5, the bottom ring (8) mainly comprises a outer ring that diametrically confirms central shaft (5), extended members (18) being radially extended from the outer ring and a plurality of rotating members (19) such as roller, bearing, hose etc. Said rotating member (19) is freely rotatably and secured at a free end of respective extended member (18). The rotating member (19) rotates about the axis orthogonal to the vertical axis of the central shaft (5). The lower end of the central shaft is further extended through the bottom hub (10) and is rotatably secured within the central hub (9).
Now as shown in Fig. 6, said bottom hub (10) is formed with a ridge that defines a hole through which the lower end of the shaft (5) is extended downwardly. Said ridge of the bottom hub is formed with downwardly extended legs (20).
The ridge of the bottom hub (10) is formed with “triangle” shaped projections (14) at regular interval on said ridge (10). Each said projection (14) is formed with a first surface (14.1) formed at an angle (a) of 10-80 degree with respect to plane A of the bottom hub (10) and a second surface (14.2) formed in continuation with first surface (14.1)and forming an angle (ß) of 80-90 degree with respect to the plane A of the bottom hub (10). Each rotating member (19) is configured to roll from a lower end of the first surface (14.1) toward its second end during the rotation of the central shaft (5).
Now shown in Figs 7A and 7B, said central hub (9) is formed with vertical grooves. The legs (20) of the bottom hub (10) are confirmed within the grooves of central hub (9) so that said bottom hub (10) is firmly fitted with the central hub (9). A plurality of impeller (21) are radially extends within the vessel (1) with respect to central hub (9).
In operation, the cells are cultured and grown on the adherent material i.e. surface of discs (7) within the vessel (1) of bioreactor in conventional manner. Now, in the process for harvesting adherent cells from the surface of discs (7) according to present invention, firstly, the motor (29) rotates the second magnetic ring (12) through the drive shaft (30). Due to magnetic connection between the first magnetic ring (11) and the second magnetic ring (12), when the second magnetic ring (12) is driven to rotate in predetermined direction, the first magnetic ring (1) is also rotated in the same manner that of the second rotating ring (12).The rotation of the first magnetic ring (11) causes the rotating of the central shaft (5) and accordingly entire cell carrier matrix assembly (6) rotates along with the central shaft (5). Due to the rotation of central shaft (5), the rotating members (19) of the bottom ring roll over the ridge of bottom hub (10). In this situation, each rotating member (19) pass over the projections (14) formed on the ridge. Each rotating member (19) is uphill from the first surface (14.1) of its respective projection (14). In this condition, as much the rotating member (19) incline on the first surface of the projection (14), the central shaft (5) moves upward by sliding within the receiving member (15) and the resilient member (16) gets compressed. After termination of first surface (14.1) of the projection (14), the resilient member (16) releases the pressure and the rotating member (19) hits down on the surface of the ridge of the bottom hub (10). This process will be repeated during the rotation of the central shaft (5). When the rotating member (19) hit down, the cell carrier matrix assembly (6) experiences the jarring/jerky rotational motion during rotation of rotating member (19).
Here, the resilient member (16) will push the whole cell carrier matrix assembly (6) down with continual tension so that the bottom ring (8) will ease the rotation through rolling over the bottom hub (10). Due to continual tension applied by the resilient member (16)and rotation of rotating member (19) on the bottom hub (10), the cell carrier matrix assembly (6) experiences the jerk during rotation of rotating member (19) of the bottom ring (8) onto the bottom hub (10) that will generate and deliver mechanical disruption to the cell carrier matrix assembly (6).This mechanical motion generated by rotational and descend drop of the cell carrier matrix assembly (6) will dislodged & detach the adherent cells inside the cell carrier matrix assembly (6) efficiently.

Thus, the cells are forced to detach from the surface and eventually the cell carriers. Due to the jarring/jerky rotational motion of the central shaft (5) and cell carriers, the cells will be dislodged mechanically from the cell carriers and it comes out in the solution and it can be subsequently harvested. Once the solution is having enough sufficient number of cells detected, one can harvest these cells in the same enzymatic solution. So, the content is then can be drained or collected in a separate container whereby this content the cell suspension harvested. Cell suspension can be used further for a particular process or particular intended use wherever it is required.

The cell detachment is visibly seen by increase in the substantial increase in the turbidity of the solution and the cell numbers can be measured by sampling the suspension and performing the cell count by normal cell counting methods such as Trypan blue cell counting. The cell suspension is collected in a container where it can be then utilized for its intended use.

Now, Fig. 8 and Fig.9 illustrate another exemplary embodiment of the bottom ring (8) and bottom hub (10) of the present invention.

As shown in Fig. 8A, the bottom ring (8) comprises an outer ring that diametrically confirms central shaft (5) and extended members (18) being radially extended from the outer ring. Said extended members are preferably formed in square shape or any other suitable shape. Unlike bottom ring (8) illustrated in previous embodiment shown in Fig.2 and Fig.5, the requirement of rotating member (19) is eliminated in said embodiment.

Now as shown in Fig.8B, the bottom hub (10) according to said embodiment is formed with a ridge that defines a hole through which the lower end of the shaft (5) is extended downwardly. Said ridge of the bottom hub is formed with downwardly extended legs (20).Further, a plurality of trapezoidal shaped projections (14) is distributed circumferentially over the ridge at equal distance by forming a gap (34) between subsequent projection (14) wherein the extended member (18) is received in such manner that the extended member (18) can freely movable between subsequent projections (14) as shown in Fig. 9.

Now, in preferred embodiment, the motor (29) is configured to provide To & fro motion to the center shaft (5) shall that will result into the to and fro motion of the extended member (18). During said to and fro movement, the extension member (18) collides with the side surfaces of the projections (14) of the bottom hub (10) repeatedly. This motion will create vibration in the whole cell carrier matrix (6) inside which grown cells are attached. The vibration shall dislodge the cells.
In accordance with further embodiment, after harvesting of cells, the cells are transferred from one bioreactor to another for scaling up the culturing of cells. Now in order to transfer the harvested cells from one bioreactor to another bioreactor, such SUB (single use bioreactor) of 1L, 5L, 10L and 50 L (27) are connected in series through tubing which is managed (open & close) by the pinch valves (24) as shown in Fig. 10A and 10B. The pinch valve (24) manipulates operation of trypsinization process from the one bioreactor (27) to another bioreactor (27). Upon achieving sufficient cell growth, the SeedBRx 1L SUB (27) is trypsinised automatically by the integrated program in the controller unit (28). The program run the sequence of process steps such as removal of media (Pump rotated to harvest the media and related pinch valve (24) open, other pinch valve (24) are closed during the operation), addition of buffer (pump rotated to add the buffer into the bioreactor, related pinch valve (24) open, other pinch valves (24) are closed during the operation), Buffer incubation, Removal buffer, Addition of enzyme (Trypsin), Enzyme incubation*, Enzyme removal with cells, Addition of fresh media, media incubation*, removal of media with the cells.
Referring continues in Figs. 10A and 10B, the SeedBRx 1L SUB (27) is connected to the SeedBRx 5L SUB (27) through the tubing which is managed (open & close) by pinch valve, the SeedBRx 5L SUB (27) is connected to SeedBRx 10L SUB (27) through the tubing which is managed (open & close) by pinch valve (24) and the SeedBRx 10L SUB (27) is connected to CellBRx 50L SUB (27)through the tubing which is managed (open & close) by pinch valve () for transferring harvested cells from 1L SUB (27)to 5L SUB (27), 5L SUB (27)to 10L SUB (27), 10L SUB (27) to 50L SUB (27).
Method provided in these steps, Rotation of the cell carrier matrix & physical stress to the cell carrier matrix to dislodge/remove the attached cells into the surrounded liquid. The harvested cells are inoculated in next stage of the SUB (27) (SeedBRx 5L) which is pre connected. One tap in the controller unit run the while sequence of the process steps also called as process recipe starting from media removal to harvested cells transfer to the next scale SUB.
Now, SeedBRx 5L (27) is inoculated, it is allowed to growth the cells for 3-6 days. Upon achieving sufficient cell growth, the SeedBRx 5L SUB (27) is trypsinized automatically by the integrated program in the controller unit (28). During the growth phase & Trypsinization process, the SUB (27) is managed by the controller unit (28). This is series of events from 1 L, to 5 L, to 10L to 50L scale production SUB (27). In 50L scale CellBRx SUB (27), cell growth phase and production phase (i.e.: vaccine, viral vector, biologics etc) are executed.
The present invention is experimented and illustrated more in details in the following example. The example describes and demonstrates embodiments within the scope of the present invention. This example is given solely for the purpose of illustration and is not to be construed as limitations of the present invention, as many variations thereof are possible without departing from spirit and scope.

EXAMPLES:

A. Detailed description of the common process steps and procedures for process variables
Three different cell types were used in this exercise which are,
A) VERO: Vero cells are a continuous line of monkey kidney cells that have been widely used in the biopharmaceutical industry and in scientific research for various purposes such as production of vaccines, Viral vectors, monoclonal antibodies.
B) HEK: HEK 293 cells, a derivative of Human Embryonic Kidney (HEK) cells, are of significant importance in the biopharmaceutical and biomedical research industries such as production of vaccines, Viral vectors, monoclonal antibodies.
C) ADSC: Adipose-derived stem cells (ADSCs) have gained significant attention in the biopharmaceutical industry due to their potential therapeutic applications and several advantages such as abundance & easy accessibility, multipotency, potential for regenerative therapies etc.

1.1 The experiment was executed with different variables to study growth profile, metabolite profile, cell viability post-harvest and cell recovery from cell carrier.
1.2 DMEM high glucose commercial cell culture media was used in these studies.
1.3 Surface treated tissue culture flasks for adherent cells were used for propagation of cell numbers in initial seed expansion stages.
1.4 Cell bank vial was removed from LN2 container and thawed as per standard procedure.
1.5 Cell bank vial was revived in treated tissue culture flask and flask was incubated at 37°C and 5% CO2 in static incubator for 3 to 5 days.
1.6 Cell expansion (Seed development) was carried out as per the number of cells required to inoculate desired bioreactor scale as per standard procedure.
1.7 Installation, media fill and other bioreactor related operations were executed as per standard procedure.
1.8 Bioreactor was inoculated with n-1 seed. Inoculation criteria for cells growth study experiments (HEK & VERO) was 0.02 to 0.03 million cells per cm2 surface area. Whereas stem cells experiment inoculation criteria was 0.004 to 0.006 million cells per cm2 surface area. Process parameters were monitored and maintained as per the standard process specifications.
1.9 Temperature was maintained 37 ± 0.5 °C throughout batch execution in all the scales of the bioreactor. pH was maintained between 7.2 ± 0.3 with 7% filter sterilized bicarbonate or CO2 gas. Dissolved oxygen concentration was maintained at 40 ± 10 % by a combination of aeration and agitation strategy. Dissolved oxygen concentration was maintained at 80 ± 10 % for stem cell growth study experiment.
1.10 For the cell counting purpose, sampling of cell carrier was performed as per manufacturer’s recommendation. Sterile sampling of the cell carrier was performed by picking the cell carrier from the SUB. Cell growth was measured by nuclei count method. Spent media was sampled through sampling port located at bottom of the SUB. Spent media sample was used to analyze offline pH, concentration of Glucose, lactate & other metabolites.
1.11 Residual glucose concentration was maintained by the addition of 20% filter sterilized glucose solution or by increasing media perfusion rate.
1.12 The media perfusion strategy (RV/day) was designed based on growth profile and residual glucose concentration data of in-process sample.
1.13 Cells were recovered using different process variables. Different enzymes were used as cell dissociation agents which are described below,
A) Trypsin: Trypsin is an animal origin enzyme being widely used for cell detachment in cell culture. It is used with a concentration of 0.25 % w/v.
B) Recombinant Trypsin: Recombinant trypsin was used because of its benefits over trypsin such as consistency in quality, animal source free, regulatory friendly & mild proteolytic activity compared to trypsin. TrypLE Express was used as recombinant trypsin in this experiment.
C) Accutase: Accutase™ is a natural animal (crabs origin) enzyme mixture with proteolytic and collagenolytic enzyme activity. It is used for sensitive cell types, such as stem cells or primary cells, that may be more prone to damage or clumping when exposed to stronger enzymes like Trypsine or TrypLE.
1.14 Integrated Seed train (IST):
The Integrated seed train (IST) is a process designed to generate an adequate number of cells required for the inoculation of large-scale production bioreactor. It involves serial integration of more than one SeedBRx (A bioreactors with innovative design of the vibro-rotation, generally used when cell harvesting is to be performed) with each other in a chronological series, scale wise. The IST technology delivers maximum cell recovery from the cell carrier matrix at every scale of SeedBRx as compared to conventional methods.
1.15 The whole study has been divided into six case studies on the basis of its application. Study matrix with these five case studies is illustrated in table below.

Case study 01 Processes
Enzyme Vibration duration (min) Scale (L) Cells Process output
Rec.Trypsin 1-1 1 HEK Cell growth
Accutase 3-1 5 VERO % Recovery
Trypsin 10 % Viability

Case study 02 IST (Integrated seed train)
Enzyme Vibration duration (min) Scale Cells Process output
Rec.Trypsin 3-1 1L=>5L=>10L=>50L (with std. Surface area) HEK Cell growth
VERO % Recovery
% Viability

Case study 03 Vibration Vs Vibro-Rotation
Enzyme Vibration duration (min) Scale Cells Process output
Rec.Trypsin 3-1 1 HEK Cell growth
5 VERO % Recovery
10 % Viability
Variables: Vertical vibration & Vibro-Rotation

Case study 04 Second embodiment (horizontal)
Enzyme Vibration duration (min) Scale Cells Process output
Rec.Trypsin 3-1 5 HEK Cell growth
% Recovery
% Viability

Case study 05 Stem cells (Adipose tissue derived)
Enzyme Vibration duration (min) Scale Cells Process output
Accutase 3-1 1L (0.2m2) =>1L (1m2) =>5L (5m2) =>10L (25m2) Stem cells Cell growth
% Recovery
% Viability
Stemness markers

Case study 06 Microcarrier Vs Vibro Rotation
Enzyme Vibration duration (min) Scale Cells Process output
Rec.Trypsin 3-1 1 HEK Cell growth
5 VERO % Recovery
% Viability post recovery

In said experiment, the study was executed at three different scales (SeedBRx 1L, SeedBRx 5L and SeedBRx 10L) with HEK and VERO cell line. Process steps and procedure were performed as per section 1.1 for batches execution. 2.1.3 Two vibration patterns (1:1, 3:1) and three enzymes (Rec. Trypsine, Accutase and Trypsin) were utilized to dislodge cells from carrier.
Table 1: List of variables of process parameters to study cell growth profile
Enzyme Vibration duration (minutes) Scale (L) Cell line Process Output
-Rec. Trypsine
-Accutase
-Trypsin -1:1
-3:1 -1
-5
-10 -HEK
-VERO -Cell growth
-% Recovery
-% Viability post recovery
Three replicative batches were executed at 1L, 5L and 10L scale with HEK and VERO cell line to study the cell harvest efficiency with innovative vibro-rotation concept with different enzymes (Rec. Trypsin, Accutase and Trypsin) which are widely used in the bioprocess industry. The vibration pattern (1:1 and 3:1) was employed to evaluate the process output (Cell harvest) with different vibration duration.
Cells were inoculated and grown in the bioreactor as per standard procedure. After reaching sufficient cell count, cells were harvested. Sterile enzyme was added into the SUB, and incubated as per suppliers’ recommendations (I.e.: 15 minutes in the case of Accutase). Vibro-rotation was given at time period of 1 minutes followed by pause of 1 minute during the incubation of the cell carrier matrix with the enzyme as well as with the media. This is called 1:1 vibration duration strategy. In 3:1 vibration duration strategy, the vibro-rotation time is three times longer than the pause period.
Growth profile data (cell density and Recovered cell viability) and recovered cells data were summarized in below mentioned experiments.
EXEPRIMENT: 1.1
[Variables process parameters data for cell recovery with HEK cell line]
In said experiment, growth profilecell recovery and cell viability data were measured for different enzymes and 1:1 vibration pattern at 1L scale with HEK cells as shown in table 1.
Table 2
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
SeedBRx 1L 0 290 280 274
24 510 350 492
48 1017 1121 908
72 1785 1970 1885
Recovered Cell 1725 1932 1650
Cell recovery (%) 96.64 98.07 87.53
Cell Viability of Recovered Cells (%) 94.30 93.80 87.60

Below graph shows the graphical presentation of cell Growth profile, Cell recovery and cell viability data with different enzymes and 1:1 vibration pattern at 1L scale as per experiment 1.1.

EXEPRIMENT: 1.2
In said experiment, growth profile, cell recovery and cell viability data were measured for different enzymes and 1:1 vibration pattern at 5L scale with HEK cells as shown in table 1.
Table 3
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
5L BRX 0 2900 2800 2740
24 5100 4500 3500
48 9800 10745 9080
72 19888 19700 17865
Recovered Cell 18065 19078 15325
Cell recovery (%) 90.83 96.84 85.78
Cell Viability of Recovered Cells (%) 94.80 93.70 85.40
Below graph shows the graphical presentation of Growth profile, Cell recovery and cell viability data with different enzymes and 1:1 vibration pattern at 5L scale as per experiment 1.1.

EXEPRIMENT: 1.3
In said experiment, growth profile and cell recovery data were measured for different enzymes and 1:1 vibration pattern at 10L scale with HEK cells as shown in table 1.
Table 4
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
10L BRX 0 12050 12710 12500
24 18500 15525 17585
48 32525 31585 35858
72 65065 72554 69545
Recovered Cell 62080 71350 60085
Cell recovery (%) 95.41 98.34 86.40
Cell Viability of Recovered Cells (%) 93.80 92.40 83.80
Below graph shows the graphical presentation of Growth profile and Cell recovery data with different enzymes and 1:1 vibration pattern at 10L scale with HEK cells.

EXEPRIMENT: 1.4
In said experiment, growth profile and cell recovery data were measured for different enzymes and 3:1 vibration pattern at 1L scale with HEK cells as shown in table 1.
Table 5
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
1L BRX 0 275 288 298
24 435 498 540
48 852 945 1054
72 1852 1959 2252
Recovered Cell 1750 1895 1951
Cell recovery (%) 94.49 96.73 86.63
Cell Viability of Recovered Cells (%) 95.30 92.56 88.40
Below graph shows the graphical presentation of Growth profile, Cell recovery and cell viability data with different enzymes and 3:1 vibration pattern at 1L scale with HEK cells.

EXEPRIMENT: 1.5
In said experiment, growth profile, cell recovery and cell viability data were measured for different enzymes and 3:1 vibration pattern at 5L scale with HEK cells as shown in table 1.
Table 6
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
5L BRX 0 2750 2880 2980
24 4350 4980 5400
48 8520 9450 10540
72 18520 19590 22520
Recovered Cell 17022 19245 19565
Cell recovery (%) 91.91 98.24 86.88
Cell Viability of Recovered Cells (%) 96.20 93.40 87.68

Below graph shows the graphical presentation of Growth profile, Cell recovery and cell viability data with different enzymes and 3:1 vibration pattern at 5L scale with HEK cells.

EXEPRIMENT: 1.6
In said experiment, growth profile, cell recovery and cell viability data were measured for different enzymes and 3:1 vibration pattern at 10L scale with HEK cells as shown in table 1.
Table 7
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
10L BRX 0 12400 12800 12100
24 19845 18553 15011
48 31585 35850 38125
72 65054 68502 62580
Recovered Cell 62005 64000 53656
Cell recovery (%) 95.31 93.43 85.74
Cell Viability of Recovered Cells (%) 94.80 92.40 89.10

Below graph shows the graphical presentation of Growth profile, Cell recovery and cell viability data with different enzymes and 3:1 vibration pattern at 10L scale with HEK cells.

Different variables process parameters data with VERO cell line.
EXEPRIMENT: 1.7
In said experiment, growth profile,cell recovery and cell viability data were measured for different enzymes and 1:1 vibration pattern at 1L scale with VERO cells as shown in table 1.
Table 8
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
1L BRX 0 250 300 270
24 335 489 412
48 724 987 789
72 1521 1902 1627
Recovered Cell 1400 1789 1489
Cell recovery (%) 92.04 94.06 91.52
Cell Viability of Recovered Cells (%) 91.50 92.60 86.30

Below graph shows the graphical presentation of Growth profile, Cell recovery and cell viability data with different enzymes and 1:1 vibration pattern at 1L scale with VERO cells.

EXEPRIMENT: 1.8
In said experiment, growth profile, cell recovery and cell viability data were measured for different enzymes and 1:1 vibration pattern at 5L scale with VERO cells as shown in table 1.

Table 9
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
5L BRX 0 2509 3000 2700
24 4892 3350 4120
48 9873 5879 7890
72 23500 18561 19856
Recovered Cell 21585 16573 17858
Cell recovery (%) 91.85 89.29 89.94
Cell Viability of Recovered Cells (%) 92.60 91.80 82.80

Below graph shows the graphical presentation of Growth profile,Cell recovery and cell viability data with different enzymes and 1:1 vibration pattern at 5L scale with VERO cells.

EXEPRIMENT: 1.9
In said experiment, growth profile, cell recovery and cell viability data were measured for different enzymes and 1:1 vibration pattern at 10L scale with VERO cells as shown in table 1.
Table 10
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
10L BRX 0 12258 11085 11858
24 19635 14565 17525
48 34251 31252 31854
72 63254 56858 61252
Recovered Cell 58961 53254 54585
Cell recovery (%) 93.21 93.66 89.12
Cell Viability of Recovered Cells (%) 91.10 89.80 83.80
Below graph shows the graphical presentation of Growth profile, Cell recovery and cell viability data with different enzymes and 1:1 vibration pattern at 10L scale with VERO cells.

EXEPRIMENT: 1.10
In said experiment, growth profile and cell recovery data were measured for different enzymes and 3:1 vibration pattern at 1L scale with VERO cells as shown in table 1.
Table 11
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
1L BRX 0 280 250 321
24 412 308 469
48 989 908 784
72 1687 1897 1935
Recovered Cell 1500 1680 1785
Cell recovery (%) 88.92 88.56 92.25
Cell Viability of Recovered Cells (%) 92.70 91.80 84.60

Below graph shows the graphical presentation of Growth profile,Cell recovery and cell viability data with different enzymes and 3:1 vibration pattern at 1L scale with VERO cells.

EXEPRIMENT: 1.11
In said experiment, growth profile,cell recovery and cell viability data were measured for different enzymes and 3:1 vibration pattern at 5L scale with VERO cells as shown in table 1.
Table 12
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
5L BRX 0 2897 2978 3128
24 3258 3987 4387
48 8546 7198 8254
72 16870 15854 17854
Recovered Cell 16000 14542 16898
Cell recovery (%) 94.84 91.72 94.65
Cell Viability of Recovered Cells (%) 91.10 90.70 84.80

Below graph shows the graphical presentation of Growth profile, Cell recovery and cell viability data with different enzymes and 3:1 vibration pattern at 5L scale with VERO cells.

EXEPRIMENT: 1.12
In said experiment, growth profile,cell recovery and cell viability data were measured for different enzymes and 3:1 vibration pattern at 10L scale with VERO cells as shown in table 1.
Table 13
Enzyme used for dissociation of cells Rec.Trypsin (TrypLE) Accutase Trypsin
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
5L BRX 0 12487 12145 11985
24 15985 19897 17912
48 28954 34626 36254
72 53254 62525 68525
Recovered Cell 50254 59654 59254
Cell recovery (%) 94.37 95.41 86.47
Cell Viability of Recovered Cells (%) 90.30 91.10 85.40
Below graph shows the graphical presentation of Growth profile,Cell recovery and cell viability data with different enzymes and 3:1 vibration pattern at 10L scale with VERO cells.

CONCLUSION:
Cell growth trend was similar irrespective of scale. It shows the robustness of process, system, and clone phenotypic stability. Selected parameters played a critical role in cell recovery procedure whereas they did not affect cell growth trend. Three different enzymes and two different vibration patterns were used to dislodge cells. No significant differences were observed in % cell recovery data. HEK cells recovery with different enzymatic treatment and vibration pattern was about 90 to 95% whereas in case of Vero cell line it was about 85 to 90%. The percentage cell viability of recovered cells was comparable except trypsin enzyme treated cells. Cell viability of trysin-recovered cells was ~80 to 90% whereas in case of accutase and trypLE enzyme-recovered cells viability was more than 90%.
Thus, it is seen that significantly high cell recovery and cell viability is observed.
EXAMPLE: 2
[Integrated seed train (IST) study with HEK and VERO cell line]
The Integrated seed train (IST) is a process designed to generate an adequate number of cells required for the inoculation of large-scale production bioreactor. It involves more than one SeedBRx (A bioreactors with innovative design of the vibro-rotation, generally used when cell harvesting is to be performed) connected with each other in a chronological series, scale wise.

The IST technology delivers maximum cell recovery from the cell carrier matrix at every scale of SeedBRx as compared to conventional methods. Different scales of SeedBRx (i.e.: SeedBRx 1L to SeedBRx 5 L to SeedBRx 10) are employed to generate and recover sufficient number of cells to inoculate large scale bioreactor (i.e.: CellBRx 50L). Integration and automation of all these bioreactors (Seed expansion=SeedBRx 1L, SeedBRx 5 L, SeedBRx 10L, Production = CellBRx 50L) makes the IST set up most efficient, affordable, robust & regulatory compliant solution for large scale bioprocessing operations.
The study was executed with HEK and VERO cell line. Vibration patterns of 3:1 and Recombinant Trypsine (TrypLE) enzyme was utilized to dislodge cells from carrier in this study.
Table 14: List of variables process parameters to study cell growth profile
Enzyme Vibration duration (minutes) Scale (L) Cell line Plots
Rec.Trypsin 3:1 SeedBRx 1L(1 m2)=> SeedBRx 5L(6.5 m2)=>SeedBRx 10L (40 m2)=>CellBRx 50L (250 m2) HEK
VERO Cell growth
% Recovery
% Viability post recovery

EXEPRIMENT: 2.1
For HEK cells, 3.2 Growth profile data (cell density and Recovered cell viability) and cell recovery data were summarized in below mentioned tables and figures.
Table 15: Growth profile and cell recovery data with Rec. Trypsin enzyme and 3:1 vibration pattern for IST with HEK cells
Scale of bioreactor Hrs. of Growth HEK B#1 HEK B#2 HEK B#3 Average Count
Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
SeedBRx 1L (1m2)
0 290 280 310 293
24 480 328 509 439
48 858 1040 1091 996
72 1868 1989 1852 1903
Cell Recovery 1825 1835 1803 1821
Cell recovery (%) 97.70 92.26 97.35 95.77
Cell Viability of recovered cells (%) 95.30 94.50 93.70 94.50
SeedBRx 5L (6.5 m2)
0 1800 1800 1800 1800
24 3100 2458 3258 2939
48 4865 5821 6325 5670
72 12092 12985 13080 12719
Cell Recovery 12056 12200 12800 12352
Cell recovery (%) 99.70 93.95 97.86 97.17
Cell Viability of recovered cells (%) 96.20 94.60 92.80 94.53
SeedBRx 10L (40 m2)
0 12000 12000 12000 12000
24 18595 19258 14525 17459
48 38665 31258 36854 35592
72 78521 76208 77865 77531
Cell Recovery 75251 75025 75098 75125
Cell recovery (%) 95.84 98.45 96.45 96.90
Cell Viability of recovered cells (%) 94.80 93.80 95.10 94.57
CellBRx 50L (250 m2) 0 75000 75000 75000 75000
24 111800 90852 110900 104517
48 189562 238525 212581 213556
72 398254 354256 425352 392621
96 825625 965854 908652 900044

Below graph shows the graphical presentation of average growth profile and cell recovery data with Rec. Trypsin (TrypLE) enzyme and 3:1 vibration pattern for IST with HEK cells.

EXEPRIMENT: 2.2
For VERO cells, Growth profile data (cell density and Recovered cell viability) and cell recovery data were summarized in below mentioned tables and figures.
Table 16: Growth profile and Cell recovery data with Rec. Trypsin (TrypLE) enzyme and 3:1 vibration pattern for IST with VERO cells
Number of batches Vero B#1 Vero B#2 Vero B#3 Average Count
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
SeedBRx 1L (1m2)
0 280 340 320 313
24 508 403 514 475
48 986 1182 1098 1089
72 2098 2015 2258 2124
Cell Recovery 1950 1960 2010 1973
Cell recovery (%) 92.95 97.27 89.02 93.08
Cell Viability of recovered cells (%) 92.70 93.80 89.90 92.13
SeedBRx 5L (6.5 m2)
0 1950 1950 1950 1950
24 2458 3521 2980 2986
48 5086 6125 6354 5855
72 12856 12965 13586 13136
Cell Recovery 12208 12081 12985 12425
Cell recovery (%) 94.96 93.18 95.58 94.57
Cell Viability of recovered cells (%) 91.10 90.70 91.52 91.11
SeedBRx 10L (40 m2) 0 12000 12000 12000 12000
24 21254 16525 14523 17434
48 38652 32087 34265 35001
72 76254 78526 76328 77036
Cell Recovery 75009 75052 74900 74987
Cell recovery (%) 98.37 95.58 98.13 97.36
Cell Viability of recovered cells (%) 90.30 89.70 91.20 90.40
CellBRx 50L (250 m2) 0 75000 75000 75000 75000
24 91254 121523 109525 107434
48 198544 169852 225452 197949
72 325452 458652 405235 396446
96 898652 825452 989521 904542
Below graph shows the graphical presentation of average growth profile and cell recovery data with Rec. Trypsin (TrypLE) enzyme and 3:1 vibration pattern for IST with VERO cells.

Conclusion of IST validation study:

From aforementioned experimental data, it is seen that adequate cell growth was achieved across all scales of SeedBRx SUBs (1L, 5L & 10L) on day 3 which was sufficient to inoculate production bioreactor for HEK and VERO cell line. Results showed robustness of both cell line and IST. The proprietary design of SeedBRx support significantly high healthy cell recovery post trypsinization. Cell recovery was 90 to 95 % with HEK 293 cells and 85 to 90 % with Vero cells in un-optimized process. Fully automated integrated seed train (IST) makes the whole seed expansion and large-scale production activity cost-effective, robust and immune to contamination failure.

EXAMPLE: 3
The experiment was conducted to compare efficiency of vertical vibration (conventional technology for detachment of cells by vibration) and upgraded vibro-rotation technology for cell detachment (present invention).
The study was executed at three different scales with HEK and VERO cell line. Vibration patterns (3:1), Rec. Trypsin (TrypLE) enzyme and two vibration parameters (Vertical vibration and Vibro-rotation) were utilized to dislodge cells from carrier.
Vertical vibration is the cell detachment process which involves generating and transferring mechanical stress in the form of vibration to the cell carrier matrix to dislodge the gown cells in the 3-D geometry of cell carrier matrix. Whereas Vibro Rotation is the technology of generating and delivering mechanical stress to the cell carrier matrix in a more efficient and healthier way.
In the table below, variables process parameter to study cell growth profile of both technologies is listed.
Table 17
Enzyme Vibration duration (minutes) Variable parameters Scale (L) Cell line Plots
Rec. Trypsin
3:1 Vertical vibration and
Vibro-rotation -1L
-5L
-10L -HEK
-VERO -Cell growth
-% Cell Recovery
-% Viability post recovery
Process step and procedure were performed as per section 1.1 for batches execution. Batches were executed at 1L, 5L and 10L scale of SeedBRx with HEK and VERO cell line to study vertical vibration and Vibro rotation impact for cells dislodgement from carrier. Growth profile data (cell density and Recovered cell viability) and recovered cells data were summarized in below mentions tables and figures.
EXEPRIMENT: 3.1
In said experiment, the Growth profile, Cell recovery and cell viability data were measured for vertical vibration and vibro-rotation parameters at 1L scale bioreactor with HEK and VERO cells. The process parameters and comparative results achieved are listed in the table below.
Table 18
Parameters Vertical vibration Vibro rotation Vertical vibration Vibro rotation
Cell line HEK 293 HEK 293 VERO VERO
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
1L BRX 0 280 300 290 280
24 565 560 402 512
48 1250 1185 945 1180
72 2089 2152 1842 1650
Cell Recovery 1685 2089 1458 1589
Cell recovery (%) 80.66 97.07 79.15 96.30
Cell Viability of recovered cells 86.90 95.80 86.20 93.40
Below graph shows the graphical presentation of growth profile, cell recovery and cell viability data with vertical vibration and vibro-rotation parameters at 1L scale bioreactor with HEK and VERO cells.

EXEPRIMENT: 3.2
In said experiment, the Growth profile, Cell recovery and cell viability data were measured for vertical vibration and vibro-rotation parameters at 5L scale bioreactor with HEK and VERO cells. The process parameters and comparative results achieved are listed in the table below.
Table 19
Parameters Vertical vibration Vibro rotation Vertical vibration Vibro rotation
Cell line HEK 293 HEK 293 VERO VERO
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
5L BRX 0 3000 3000 3000 3000
24 3985 5600 4125 5120
48 8952 11525 7869 9054
72 18965 20358 15085 16858
Cell Recovery 15085 19125 11525 15985
Cell recovery (%) 79.54 93.94 76.40 94.82
Cell Viability of recovered cells 89.85 96.60 91.10 92.80

Below graph shows the graphical presentation of growth profile and cell recovery data with vertical vibration and vibro-rotation parameters at 5L scale bioreactor with HEK and VERO cells.

EXEPRIMENT: 3.3
In said experiment, the Growth profile, Cell recovery and cell viability data were measured for vertical vibration and vibro-rotation parameters at 10L scale bioreactor with HEK and VERO cells. The process parameters and comparative results achieved are listed in below table.
Table 20
Parameters Vertical vibration Vibro rotation Vertical vibration Vibro rotation
Cell line HEK 293 HEK 293 VERO VERO
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells) Cell count (Total million cells)
10L BRX 0 12000 12000 12000 12000
24 19800 16251 18523 14526
48 34525 38525 31025 27856
72 67526 72021 62325 59362
Cell Recovery 53254 68652 47659 56452
Cell recovery (%) 78.86 95.32 76.47 95.10
Cell Viability of recovered cells 87.56 95.80 89.60 91.20
Below graph shows the graphical presentation of growth profile, cell recovery and cell viability data with vertical vibration and vibro-rotation parameters at 10L scale bioreactor with HEK and VERO cells.

Conclusion:
Selected parameters played a critical role in cell recovery procedure, so they did not affect cell growth trend. Cell growth trend was comparable irrespective of scale, it shows the robustness of the single use bioreactor system and the process.
Different vibration patterns (Vertical vibration and Vibro-rotation) were used to dislodge cells with Rec. Trypsin (TrypLE) enzyme. HEK and VERO cells percentage harvest cell recovery with vertical vibration were around 80 % and with vibro-rotation it was about 95 %. Cell viability of harvested cells with vertical vibration was around 86 % and with vibro-rotation it was about 93%. Vibro-rotation had a positive impact on the percentage cell recovery and cell viability post recovery. Because of the innovative design of the vibro-rotation technique, cell health was maintained which was reflected in percentage viability and it also yield more cells compared with vertical vibration technique.
EXAMPLE: 4
The experiment was conducted to compare the consistency between first embodiment (Fig. 5) and second embodiment (Fig. 8 and 9) on cell growth and cell detachment.
The comparative study was executed at 5L bioreactor scale (data shown are average 3 three batches) with control design and second embodiment (Horizontal vibration). Vibration patterns (3:1), Rec. Trypsin (TrypLE) enzyme was utilized to dislodge cells from carrier.
Table 21: Cell growth profile study after second embodiment in system with HEK cells
Enzyme Vibration duration (minutes) Variable parameters Scale (L) Cell line Plots
Rec. Trypsin
3:1 Second embodiment of vibration (horizontal vibration) and improved vibro-rotation 5L HEK -Cell growth,
-% Cell Recovery,
-% Viability post recovery

EXEPRIMENT: 4.1
In said experiment, the Growth profile, cell recovery and cell recovery data of first embodiment (Fig. 5) and second embodiment (Fig. 8 and 9) were measured with HEK cell line at 5L scale as listed in below table.
Table 22
Parameters Standard embodiment Second embodiment
Cell line HEK 293 HEK 293
Scale of bioreactor Hrs. of Growth Cell count (Total million cells) Cell count (Total million cells)
5L BRX 0 3000 3000
24 3350 4532
48 8452 7652
72 18955 17526
Cell Recovery 17856 16058
Cell recovery (%) 94.20 91.62
Cell Viability of recovered cells 93.54 92.51

Below graph shows the graphical presentation of growth profile, cell recovery and cell viability comparison with Standard embodiment and second embodiment at 5L scale bioreactor with HEK cells.

Conclusion:
Growth profile was comparable in both control design and second embodiment design at 5L scale bioreactor in all the three batches. HEK cells recovery and cell viability of recovered cells were significantly high = 90% and comparable to each other so the second embodiment also delivered same process output compared to vibro-rotation design mentioned in this document.

EXAMPLE: 5
[Growth compatibility study of adipose derived stem cells with IST]
The Adipose derived stem cell expansion study was executed with integrated seed train. The Integrated seed train (IST) is a process designed to generate an adequate number of cells for which require for the inoculation of large-scale production bioreactor.
Vibration patterns (3:1) and Accutase enzyme were utilized to dislodge cells from carrier.
Table 23: Cell growth profile study with Adipose derived stem cell line
Enzyme Vibration duration (minutes) Scale (L) Cell line Plots
Accutase 3:1 1L(0.2 m2)=>1L(1 m2)=>5L (5 m2)=>10L (25 m2) Adipose derived stem cells Cell growth

% Recovery
% Viability post recovery
EXEPRIMENT: 5.1
In said experiment, 6.2, Growth profile data (cell density and Recovered cell viability) and recovered cells data were measured as shown in below mentioned tables and figures.

Table 24:
Integrated seed train with Stem cell (Adipose derived)
Scale of bioreactor
Details of bioreactor
Hrs of Growth
Stem cell B#1
Cell count (total million cells)
SeedBRx 1L
SeedBRx 1L (0.2m2)

0 10
24 12
48 18
72 29
96 44
Cell Recovered 41
Cell recovery (%) 93.18
Cell Viability of recovered cells 92.00
SeedBRx 1L
SeedBRx 1L (1 m2)

0 40
24 54
48 79
72 135
96 220
Cell Recovered 205
Cell recovery (%) 93.18
Cell Viability of recovered cells 93.00
SeedBRx
5L
SeedBRx 5L (5 m2)

0 201
24 286
48 448
72 652
96 1169
Cell Recovered 1050
Cell recovery (%) 89.82
Cell Viability of recovered cells 86.92
10 L BRX Cellbrx 10L (25 m2) 0 1050
24 1652
48 2164
72 3521
96 4986

Below graph shows the graphical presentation of growth profile, cell recovery and cell viability data with Rec. Trypsin (TrypLE) enzyme and 3:1 vibration pattern for IST with Adipose-derived stem cell line.

Conclusion:
The IST technology delivers maximum cell recovery from the cell carrier matrix at every scale of SeedBRx as compared to conventional methods. Different scales of SeedBRx (i.e.: SeedBRx 1L to SeedBRx 1 L to SeedBRx 5) are employed to generate and recover sufficient number of cells to inoculate large scale bioreactor (i.e.: CellBRx 10L). Integration and automation of all these bioreactors (Seed expansion=SeedBRx 1L, SeedBRx 1 L, SeedBRx 5L, Production = CellBRx 10L) makes the IST set up most efficient, affordable, robust & regulatory compliant solution for large scale bioprocessing operations.
Invitro growth of stem cell line (Adipose derived) at 3D platform was challenging task due to higher doubling time and lower doubling stability.
Successful scaling up of stem cell’s growth up to 10L bioreactor with 25 m2 area shows IST system’s robustness.
Stemness markers such as CD105, CD73 and CD90 were monitored at each stage of cell recovery which were found to be more than 90% for reach marker which indicated preserved cell health post cell recovery.
All substitution, alterations and modification of the present invention which come within the scope of the following claims are to which the present invention is readily susceptible without departing from the spirit of the invention. The scope of the invention should therefore be determined not with reference to the above description but should be determined with reference to appended claims along with full scope of equivalents to which such claims are entitled.
EXAMPLE: 6
The experiment was conducted to compare efficiency of cell growth and cell recovery with microcarrier technology and upgraded vibro-rotation technology for cell detachment (present invention).
The study was executed at two different scales with HEK and VERO cell line. Vibration patterns (3:1), Rec. Trypsin (tTrypLE) enzyme and two different cell growth & cell detachment parameters (microcarrier and Vibro-rotation) were utilized to dislodge cells from carrier.
Microcarrier technology is a widely used method in bioprocess industry for the growth and harvest of adherent mammalian cells. Cells are seeded onto the microcarrier surface in a stirrer tank bioreactor unlike packed bed/fixed bed/dynamic bed bioreactors. Cells adhere to the microcarrier surface and form a monolayer. When the desired cell density is reached, the cells were harvested from the microcarriers through enzymatic detachment along with mechanical agitation.
The Vibro Rotation is the technology of generating and delivering mechanical stress to the cell carrier matrix in the dynamic bed reactors, SeedBRx.
In the table below, variables process parameter to study cell growth profile of both technologies is listed.
Table : 25

Enzyme Vibration duration (minutes) Variable parameters Scale (L) Cell line Plots
Rec. Trypsin 03:01 Vibro- rotation SeedBRx 1L(1m2) HEK Cell growth
SeedBRx 5L (10m2) VERO % Cell Recovery
As per standard practice Microcarrier STR 1L (1.3 m2)
% Viability post recovery

STR 10L (10.3 m2)
Process step and procedure were performed as per section 1.1 for batches execution. In the case of micro carrier, batch in the stirred tank bioreactor was executed with standard process parameters. Batches were executed at different scales as mentioned in table 25. Growth profile data (cell density and Recovered cell viability) and recovered cells data were summarized in below mentions tables and figures.
EXEPRIMENT: 6.1
In said experiment, the Growth profile, Cell recovery and cell viability data were measured with microcarrier and vibro-rotation parameters at 1L scale bioreactor with HEK and VERO cells. The process parameters and comparative results achieved are listed in below table.
Table: 26
Parameters Microcarrier (HEK) Vibro rotation (HEK) Microcarrier (VERO) Vibro rotation (VERO)
Scale of bioreactor Hrs. of Growth Cell count ( million cells/cm2) Cell count (millioncells/cm2) Cell count (million cells/cm2) Cell count (million cells/cm2)
SeedBRx 1L (1 m2) vs STR 1L (1.3 m2) 0 0.03 0.03 0.03 0.02
24 0.06 0.05 0.04 0.03
48 0.13 0.11 0.08 0.09
72 0.21 0.22 0.18 0.16
Cell Recr 0.17 0.21 0.15 0.16
Cell recovery (%) 78.99 94.17 79.15 97.53
Cell Viability of recovered cells 84.35 95.80 86.72 93.40

Below graph shows the graphical presentation of growth profile and cell recovery data with micro carrier and vibro-rotation parameters at 1L scale bioreactor with HEK and VERO cells

EXEPRIMENT: 6.2
In said experiment, the Growth profile, Cell recovery and cell viability data were measured for micro carrier and vibro-rotation parameters with HEK and VERO cells as mentioned in table 26. The process parameters and comparative results achieved are listed in below table.
Table : 27
Parameters Microcarrier (HEK) Vibro rotation (HEK) Microcarrier (VERO) Vibro rotation (VERO)
Scale of bioreactor Hrs. of Growth Cell count (106 cells/cm2) Cell count (106 cells/cm2) Cell count (106 cells/cm2) Cell count (106 cells/cm2)
SeedBRx 5L (10 m2) vs STR 10L (10.3 m2) 0 0.03 0.02 0.03 0.03
24 0.05 0.04 0.04 0.04
48 0.11 0.10 0.07 0.08
72 0.25 0.22 0.15 0.17
Cell Recr 0.19 0.21 0.12 0.16
Cell recovery (%) 75.92 95.07 77.12 94.19
Cell Viability of recovered cells 89.85 96.60 88.50 92.80
Below graph shows the graphical presentation of growth profile and cell recovery data with micro carrier and vibro-rotation parameters at 10L scale in case of micro carrier and at 5L scale in case of vibro rotation, with HEK and VERO cells

Conclusion:
Selected process platform played a critical role in cell recovery procedure, so they did not affect cell growth trend. Cell growth trend was comparable irrespective of scale and the production platform, it shows the robustness of the single use bioreactor system and the process.
Different platform (micro carrier in stirrer tank bioreactor and Vibro-rotation in SeedBRx bioreactor) were used to dislodge cells with trypLE enzyme. HEK and VERO cells percentage harvest cell recovery with micro carrier were around 80 % and with vibro-rotation it was about 95 %. Cell viability of harvested cells with micro carrier was around 87 % and with vibro-rotation it was about 95%. Vibro-rotation had a positive impact on the percentage cell recovery and cell viability post recovery. Because of the innovative design of the vibro-rotation technique, cell health was maintained which was reflected in percentage viability and it also yield more cells compared with conventional micro carrier platform.

? Reference Numerals
1. Vessel body
2. Side Wall
3. Bottom Plate
4. Head Plate
5. Rotatable Shaft
6. Cell carrier matrix assembly
7. Disc
8. Bottom ring
9. Central hub
10. Bottom hub
11. First magnetic ring
12. Second magnetic ring
13. Magnets
14. Projection
14.1. First surface
14.2. Second surface
15. Receiving member
16. Resilient member
17. Retaining member
18. Extended member
19. Rotating member
20. Legs
21. Impeller
22. First region
23. Second region
24. Pinch valve
25. Docking Station
26. Gas Exchange Module
27. SUB(1L, 5L, 10L, 50L)
28. Controller Unit
29. Motor
30. DriveShaft
31. Bearing
32. Protrusion
33. Extended Pin
34. Gap
100. Bioreactor System
,CLAIMS:We Claim:
1. A cell culture bioreactor with cell harvester comprising:
a vessel (1) having a cylindrical shell, a bottom plate (3) and a head plate (4) forming an hollow interior, a central shaft (5) being vertically disposed within the hollow interior of the vessel (1), a rotating means for rotating said central shaft, a cell matrix assembly (6) being disposed within the hollow interior of the vessel and comprises a one or multiple stacked and spaced apart discs (7) centrally and longitudinally loaded on the central shaft (5), a bottom ring (8) being secured along the central shaft (5) below the cell carrier matrix assembly (6), a central hub (9) being disposed on an inner surface of the bottom plate (3) and a bottom hub (10) being snugly fitted over the central hub (9);
said central shaft (5) being in connection with the rotating means at its first end and is secured within the central hub (9) at its distal end;
wherein the bottom ring (8) is configured to rotate over the bottom hub (10) in a manner to impart jarring/jerky rotational motion to the cell carrier matrix assembly (6) causing cells attached to the discs (7) to detach from the discs (7).
2. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the bottom hub (10) is formed with a ridge that define a hole through which a lower end of the central shaft (5) is extended downwardly.
3. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the ridge is formed with downwardly extended legs (20).
4. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the legs (20) of the ridge are confirmed within grooves of the central hub (9).
5. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein said ridge is formed with triangle shaped projection (14) at regular interval on an upper surface thereof.
6. The cell culture bioreactor with cell harvester as claimed in claim 5, wherein said projection (14) is formed with a first surface (14.1) and a second surface (14.2).
7. The cell culture bioreactor with cell harvester as claimed in claim 6, wherein the first surface (14.1) of said projection (14) is formed at an angle (a) of 10-80 degree with respect to a plane A of the bottom hub.
8. The cell culture bioreactor with cell harvester as claimed in claim 6, wherein the second surface (14.2) of said projection (14) formed in continuation with the first surface (14.1) and forming an angle (ß) of 80-90 degree with respect to the plane A of the bottom hub (10).
9. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the bottom ring (8) comprises an outer ring that diametrically confirms the central shaft (5), extended members (18) being radially extended from the outer ring and a rotating member (19) freely and rotatably secured at a free end of each extended member (18).
10. The cell culture bioreactor with cell harvester as claimed in claims 1 to 9, wherein each rotating member (19) is configured to roll over the ridge from a lower end of the first surface (14.1) toward its second end during the rotating of the central shaft (5).
11. The cell culture bioreactor with cell harvester as claimed in claim 9, wherein the rotating member (19) rotates about an axis orthogonal to a vertical axis of the central shaft (5).
12. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein a central protrusion (32) is formed in a center of the head plate to define a headspace within the vessel.
13. The cell culture bioreactor with cell harvester as claimed in claim 12, wherein a downwardly extended pin (33) is positioned at the center of a plain surface of the protrusion (32).
14. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein a plurality of trapezoidal shaped projections (14) are distributed circumferentially over the ridge of the bottom hub (10) by forming a gap (34) between subsequent projections (14).
15. The cell culture bioreactor with cell harvester as claimed in claim 14, wherein the extended member (18) is received in the gap (34).
16. The cell culture bioreactor with cell harvester as claimed in claim 14 and 15, wherein a to and fro motion to is given to the extended member (18) so that the extension member (18) collides with side surfaces of the projections (14) of the bottom hub (10).
17. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the central shaft (5) is formed with a first region (22) and a second region (23).
18. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the first region (22) and second region (23) of the central shaft (5) are separated through a retaining member (17).
19. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the rotating means comprises a first magnetic ring (11) being disposed within the headspace and a second magnetic ring (12) being disposed on an outer surface of the head plate (3).
20. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the first magnetic ring (1) includes a center bearing (31) being fitted with the pin (33), a plurality of magnets (13) being arranged surrounding to the bearing and an elongated receiving member (15) being extended downwardly from the center of the first magnetic ring (11).
21. The bioreactor with cell harvester as claimed in claim 1, wherein the elongated receiving member (15) is formed with an elongated space.
22. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein said second magnetic ring (12) is formed with a center opening and a plurality of magnets (13) being arranged surrounding the central opening.
23. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the magnets (13) of the first magnetic ring (11) and the second magnetic ring (12) are opposite each other in a manner that a pole of magnets (13) of the first magnetic ring (11) faces an opposite pole of the magnets (13) of the second magnetic ring (12) to create a magnetic attraction effect there between.
24. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein the second magnetic ring (12) is connected to a motor (29) through a drive shaft (30).
25. The cell culture bioreactor with cell harvester as claimed in claim 16, wherein a resilient member (16) is disposed along the first region (22) of the central shaft (5) and is rested between the retaining member (17) of the central shaft (5) and a bottom end of the receiving member (15).
26. The cell culture bioreactor with cell harvester as claimed in claim 16, wherein the receiving member (15) is configured to slidably confirm the top part of the first region (22) of the central shaft (5) within the elongated space.
27. The cell culture bioreactor with cell harvester as claimed in claim 1, wherein a central hub (9) formed with grooves and a plurality of impeller (21) being radially extends within the vessel (1) with respect to the central hub (9).
28. The cell culture bioreactors with cell harvester as claimed in claim 1 to 27 are connected with each other in a scale wise chronological series.
29. A method for harvesting cells grown in the culture comprising following steps;
a. growing the cells on discs (7), wherein the cells are attached to the discs (7);
b. rotating a drive shaft (30) of a motor (29) for rotating a second magnetic ring (12) through a drive shaft (30);
c. rotating a first magnetic ring (11) along with the rotation of the second magnetic ring (12) through magnetic attraction there between;
d. rotating a central shaft (5) along with the rotation of the first magnetic ring (11) which causes a rotation of a cell carrier matrix assembly (6);
e. rolling a rotating member of a bottom ring (8) over a ridge of a bottom hub (9) and imparting a jarring/jerky rotational motion while passing over projections (21) causing cells attached to the discs (7) to detach from the disc (7);
f. recovering the cells.
30. The method for cell harvesting as claimed in claim 29, wherein the resilient member (16) is configured to push the central shaft (5) downwardly.
31. The method for cell harvesting as claimed in claim 29, wherein said receiving member (15) is configured to allow the reciprocal movement of the central shaft (15) within the elongated space.

Dated this on October 12, 2023.