DESC:

FILED OF INVENTION
The present invention relates for an apparatus for cell retention and method thereof. More specifically, it relates to an apparatus which separates the cells from the media during the cell culture process and improves the batch longevity which increases the batch yield and overall process output.
BACKGROUND OF INVENTION
Cell separation is the operational step required for the perfusion process. The perfusion process is an important technique used in the biopharmaceutical industry for various purposes, primarily related to the production of biopharmaceuticals such as monoclonal antibodies, vaccines, and other therapeutic proteins. There are several reasons which suggest need of the cell separation and perfusion during the upstream batch execution of the bio-molecules.
Perfusion culture is a continuous cell culture system that maintains optimal growth conditions by continuously supplying fresh medium and removing spent medium. This dynamic approach facilitates the achievement of high cell densities and consistent product quality, reduced production time, reduced facility footprint & operations, and improved product consistency. A key component of this system is cell retention, which involves physically separating cells from the culture medium through techniques like tangential flow filtration or membrane filtration. By retaining cells within the bioreactor, continuous cultivation is enabled, leading to increased productivity and consistent product quality.
Cell retention devices remain in the core of the perfusion process as the function of the cell retention device is to retain the cells into the vessel/bioreactor. Other mechanisms such as addition of fresh media, removal of spent media, etc., are basic operation of pumps (i.e.: harvest pump, media addition pump) which manage fresh media addition and spent media removal through pre-defined logic & recipe of perfusion.
Cell death, a spontaneous process during cell culture, leads to cell lysis, releasing cellular debris into the culture medium. This debris, composed of cellular components, can negatively impact the culture by hindering cell growth, reducing product yield, and compromising product quality. Cells also produce waste by-products from their metabolism such as lactate, ammonia etc which are harmful for the cell growth and expression of desired product and quality of desired product. Cell separations techniques helps to remove these cell culture by-products through harvest line such as cell debris, lactate, ammonia and leading to a more efficient process which delivers higher-quality product.
Retained cells are separated and maintained in the same environment such as bioreactor which are supplied with fresh media to volumetrically compensate the harvested spent media. This continuous removal of by-products and addition of fresh media leads to increase the cell count (growth) & batch longevity compared to batch culture process into the bioreactor. The concept of more cells produces more products leading to produce more amount of product into the bioreactor.
In traditional batch processes, cells may experience stress due to fluctuations in nutrient availability and waste product accumulation. Perfusion minimizes these fluctuations, reducing cell stress and improving overall cultural longevity and productivity.
Hence, cell separation and perfusion are essential techniques in the biopharmaceutical industry because they enable the isolation and maintenance of cells, ensures the availability of fresh culture media to the separated and progressively concentrated cells, facilitate continuous production and improve operational productivity, product quality. These cell separation techniques are crucial for meeting the high-quality standards and production demands of the biopharmaceutical industry.
There are several tools currently being used as cell retention devices. Current cell separation devices for biopharmaceuticals are described in below table along with their bottlenecks:
Sr. No. Name of product Make Problems
1 ATF (Alternate Tangential flow filtration) Repligen • Reduced filtration efficiency over time due to membrane fouling, membrane blockage.
• Tedious & Complex set up of ATF with the bioreactor involves lot of manual interventions & connections, leads to error-prone set-up which may result in contamination & other experimental failures.
• Rupture in the septum may lead to batch failure.
• Possible operation range is 2 L to 500L. No module is available for below 2L and above 500L scale.
• Costly Capex and consumable material.
2 HF TFF (Hollow fibre Tangential flow filtration) Cytiva, Repligen • Reduced filtration efficiency over time due to membrane fouling, membrane blockage.
• Complex set up of TTF with the bioreactor involves lot of manual interventions & connections, leads to error-prone set-up which may result in contamination & other experimental failures.
• Generate lot of shear stress, negatively impact cell health & viability
3 Inclined Settler NA • Slow & less efficient, not suitable for high density culture and fast-growing cells
• Not suitable for cells with small size/diameter.
• Complex set up involves lot of manual interventions & connections, leads to error-prone set-up which may result in contamination & other experimental failures.
4 Spin filter Eppendorf etc • Spin filters clogged frequently with cells and debris, resulting in process failure.
• Complex set up involves lot of manual interventions & connections, leading to error-prone set-up which may result in contamination & other experimental failures.
• Generate a lot of shear stress, negatively impact cell health & viability
5 Acoustic filter (BioSep) Applicon • Complex and skill fullness are required for the setup and prone to error, contamination & ultimately experiment failure.
• Effective at lower scale, pilot scale & more than pilot scale is troublesome.
6 Xcellerex APS (Automated Perfusion System) Cytiva • Works on standard TFF based principle. Reduced filtration efficiency over time due to membrane fouling, membrane blockage.
• Complex set up involves lot of manual interventions & connections with the bioreactor, leads to error-prone set-up which may result in contamination & other experimental failures.
• Costly Capex and consumable material.

Hence, it is needed to invent an apparatus for Cell retention that overcomes the difficulties as described above.
OBJECTIVE OF INVENTION
The principle object of the present invention is to provide an apparatus for the cell retention and method thereof.
Another objective of the present invention is to provide the apparatus for cell retention and method thereof to enable the isolation and maintenance of cells.
Another objective of the present invention is to provide the apparatus for cell retention and method thereof to improve the product purity.
Another objective of the present invention is to provide the apparatus for cell retention and method thereof to facilitate the continuous production and increase the yield efficiency of the current production process.
Further, objective of the present invention is to provide the apparatus for cell retention and method thereof to provide the simple construction, total containment and cost effective.
Yet another objective of the present invention is to provide the apparatus for cell retention and method thereof in which no involvement of manual interventions and connections and provides the error free set-up.
Another objective of the present invention is to provide the apparatus for cell retention and method thereof which increase the efficiency of the bioreactor cell culture operation.
Yet another objective of the present invention is to provide the apparatus for cell retention and method thereof to use in small scale or pilot scale application or commercial scale.
Further, objective of the present invention is to provide the apparatus for cell retention and method thereof to eliminate the shear stress and negative impact on the cell heath and viability.
Further, objective of the present invention is to provide the apparatus for cell retention and method thereof to eliminate the problems associated with the conventional technologies and prior arts.
SUMMARY OF INVENTION
The present invention is an apparatus for cell retention and method thereof. The apparatus comprising at least a pair of vertically extended hollow shafts suspended on vessel of bioreactor, each hollow shaft is connected with the at least one cell compartments at lower end thereof, each said cell compartment is fluidly connected at lower end of respective hollow shaft, each hollow shaft is extended vertically, the hollow shafts are fluidly connected with each other at their upper end through external circulating tube, a draining tube fluidly connected to the external circulation tube. Bidirectional pump is placed over or in line with the external recirculation tube and a drain pump is place over or in line with the drain tube line. This set up is a closed loop system that controlled by pumps and inlet/outlets as per required time and quantity. The present invention enables automation of process and non-human intervention process.

BRIEF DESCRIPTION OF DRAWINGS
Fig. 1 illustrates a schematic diagram of an apparatus for cell retention, in accordance with an embodiment of the present invention.
Fig.2 illustrates a schematic diagram of the cell compartments of the cell retention apparatus, in accordance with an embodiment of the present invention.
Fig.3 illustrates a schematic diagram of the cell compartments of the cell retention apparatus while the pressure increased in the cell compartment, in accordance with an embodiment of the present invention.
Fig.4 illustrates a schematic diagram of the cell compartments of the cell retention apparatus while the vacuum increased in the cell compartment, in accordance with an embodiment of the present invention.
Fig.5A illustrates a side view of the flexible filter of the cell compartments of the cell retention apparatus, in accordance with an embodiment of the present invention.
Fig.5B illustrates a side view of the supportive mesh of the cell compartments of the cell retention apparatus, in accordance with an embodiment of the present invention.
Fig. 6 illustrates a graphical representation of viable cell density and percentage viability in different variant of apparatus, in accordance with an embodiment of the present invention.
DETAILED 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 accompany 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.
In the present document, the word "exemplary" is used herein to mean "serving as an example, instance, or illustration." Any embodiment or implementation of the present subject matter described herein as "exemplary" is not necessarily to be construed as preferred or advantageous over other embodiments.
While the disclosure is susceptible to various modifications and alternative forms, specific embodiments thereof has been shown by way of example in the figures and will be described in detail below. It should be understood, however that it is not intended to limit the disclosure to the forms disclosed, but on the contrary, the disclosure is to cover all modifications, equivalents, and alternative falling within the scope of the disclosure.
The terms “comprise”, “comprising”, or any other variations thereof, are intended to cover a non-exclusive inclusion, such that a method that comprises a list of acts does not include only those acts but may include other acts not expressly listed or inherent to such method. In other words, one or more acts in a method proceeded by “comprises… a” does not, without more constraints, preclude the existence of other acts or additional acts in the method.
The term “flexible filter” or “membrane” or “flexible membrane” or “cell retention membrane” may be used interchangeably and can be understood as a structure for filtering or serving as selecting barriers, irrespective of material composition, configuration or specifications. The terminology should not be construed as imposing any constrains on the scope of the invention.
The term “hollow shaft” or “tubular shaft” or “tubular element(s)” may be used interchangeably and can be understood as a structure that configured to flow the material, media or fluid. The terminology should not be construed as imposing any constrains on the scope of the invention.
The term “batch” or “perfusion culture batch” refers to a single lot of cells that are cultivated in the bioreactor from the inoculation to harvest.
The present invention discloses an apparatus (100) for cell retention and method thereof. Fig. 1 shows the schematic diagram of the apparatus (100) for cell retention according to the present invention. The apparatus (100) for cell retention may be configured with a bioreactor having a vessel (101). Said vessel (101) having a head plate contained with a flexible adapter containing with at least one shaft on which at least one impeller (103) is loaded. It is to be understood that there can be different combinations such as number of shafts, number of impeller (103) mounted over the shaft, arrangement of the shaft such as vertical, horizontal and size, shape & number of flexible adapter through which the shaft in enter the vessel (101) or bioreactor, mechanism to deliver mechanical motion to the shaft, type-shape location & number of impeller (103) and like.
In an embodiment, the impeller (103) is operated through the motor and provides a liquid flow of the fluid content within the chamber and provides sufficient mixing of the content inside the bioreactor vessel (101). A required gas enters into the bioreactor through sparger (any type: Micro, open pipe, ring etc) which is placed underneath the impeller (103). Upon introduction of gas, motion of impeller (103) disperses gas bubbles and mix content of the vessel (101) thoroughly.
It is to be understood that the bioreactor may be other type of a vessel (101) or a vessel (101) capable of performing equivalent functions, and the invention is not limited to the use of a bioreactor specifically.
In an embodiment, the apparatus (100) may comprise, at least a one pair of vertically extended hollow shaft (107). Each hollow shaft (107A, 107B) of pair of hollow shaft (107) may be fabricated from the various materials, for instance, the metal, or plastic composition or alloy or any other material which suitable for the application in the bioreactor.
In an embodiment, said pair of the hollow shaft (107) having first hollow shaft (107A) and second hollow shaft (107B). Each hollow shaft (107A, 107B) is connected with at least one cell compartment (105) at lower end thereof. Each said cell compartment (105) is fluidly connected at lower end of respective hollow shaft (107A, 107B), each hollow shaft (107A, 107B) is extended vertically. Said pair of hollow shaft (107) are suspended in the vessel (101) and mounted through the head plate of the vessel (101) such that the upper end of the hollow shaft (107A, 107B) extended above the head plate of the vessel and the lower end of the pair of the hollow shaft (107A, 107B) remain in the vessel (101).
Said pair of the hollow shaft (107) is preferably placed in the parallel orientation to the impeller (103) of the vessel (101) of the bioreactor. Said each hollow shaft (107A, 107B) is connected with the external circulating tube (113) at the upper end and said each external circulating tube (113) is configured with the sensors (117) to sense and measure the pressure and vacuum in the hollow shaft (107), external circulating tube (113) and the cell compartment (105). As shown in Fig. 1, the bidirectional pump is configured with the external circulating tube as per the specification of bidirectional pump. Function of said bidirectional pump (109) is to generate the vacuum and pressure in the cell compartments (105). Said bidirectional pump (109) re-circulates the media in both direction and pass the media alternatively in the hollow shaft (107). In an embodiment, the bidirectional pump (109) may include, but not limited to, a peristaltic pump, magnetic pump and like. Said bidirectional pump (109) may generate, but not limited to, the pressure and vacuum in range of 0.1 bar to 3 bar. Said pressure and vacuum may vary according to the size and capacity of the apparatus (100) associated with the cell retention.
In some embodiments, a single bidirectional pump (109) may be configured with the multiple pairs of the hollow shaft (107) having attached cell compartment (105) at the lower end of each hollow shaft (107A, 107B). In some embodiments, the multiple cell compartments (105) may be connected in series or in sequential manner along the single hollow shaft (107).
In an embodiment, a drain pump (111) may be fluidly connected to the external circulation tube (113) through a draining tube (115) and across the bidirectional pump (109) as shown in Fig. 1. Said drain pump (111) may configure to drain the media from the vessel (101) of the bioreactor.
Fig. 2 illustrates a schematic diagram of the cell compartment (105) of the cell retention apparatus (100) according to present invention.
The apparatus (100) for the cell retention may comprise a cell compartment (105). Said cell compartment (105) may comprise a supportive mesh (201). Over the supportive mesh (201), the flexible filter (203) is mounted. The supportive mesh (201) acts as a resting platform for flexible filter (203) during the generation of vacuum in such cell compartment (105). Said supportive mesh (201) also provides the strength to the cell compartment (105). Said flexible filter (203) is mounted with the cell compartment (105) or hollow shaft (107) through the process of sealing, welding, merging or any conventional method. The flexible filter (203) having porous structure configuration which allows the media to flow through and impedes cell migration, causing cell retention at the outer ends of the pores. The thickness of the flexible filter (203) is between 50-200 micron and the size of pores present in the said flexible filter (203) is between 0.2 to 10 micron. The size of the mesh (pores) of the supportive mesh (201) is higher than the size of pores in the flexible filter (203).
In some embodiments, the apparatus (100) for the cell retention may comprise, a stiff filter (not shown in Figures) having porous structure configuration which allows the media to flow through and impedes cell migration, causing cell retention at the pores. In an embodiment, as shown in Fig. 2 at the lower end of the each hollow shaft (107A, 107B) of the pair of hollow shaft (107) suspended in the vessel (101), the cell compartment (105) is connected. The multiple cell compartments (105) may configure in series sequence and mounted in line with each hollow shaft (107A, 107B) of the pair of hollow shaft (107) and the numbers of the cell compartment (105) is not limited thereto.
Fig.3 illustrates a schematic diagram of the cell compartments (105) of the cell retention apparatus (100) while the pressure increased in the cell compartment (105) according to present invention. In similar manner, Fig.4 illustrates a schematic diagram of the cell compartments (105) of the cell retention apparatus (100) while the in the vacuum increased in the cell compartment (105) according to present invention.
In an embodiment, the flexible filter (203) is expandable and retractable while the pressure and vacuum are generated in the cell compartment (105) respectively as show in Fig. 3 and 4. As shown in Fig. 1 and Fig. 3, when the bidirectional pump (109) rotates in the clockwise direction, the culture media in the vessel (101) flows from first hollow shaft (107A) to second hollow shaft (107B) through the cell compartments (105) and connected external circulating tube (113) as show in Fig. 1. Said flow of culture media generates the pressure in the cell compartment (105) associated with the second hollow shaft (107B) and create the vacuum in cell compartment (105) associated with the first hollow shaft (107A). As vacuum generated in the first hollow shaft (107A) of the pair of the hollow shaft (107) and simultaneously a pressure generated in the second hollow shaft (107B) of the pair of the hollow shaft (107), the flexible filter (203) of the cell compartment (105) associated with the first hollow shaft (107A) may retract and simultaneously in the flexible filter (203) of the cell compartment (105) associated with the second hollow shaft (107B) may expand as shown in Fig. 3 and Fig. 4. The flexible filter (203) of the cell compartment (105) associated with the first hollow shaft (107A) may retract by suction of the media from the vessel (101) through the pores of the flexible filter (203) of the cell compartment (105). The sizes of the pores of the flexible filter (203) are smaller than the size of the cells so that the cells are prohibited to pass through the flexible filter (203) and deposited on the outer surface of the flexible filter (203) and only cell free culture media flows into the cell compartment (105) associated with the first hollow shaft (107A) and flown to the cell compartment (105) associated with the second hollow shaft (107B) because of function of bidirectional pump configured with the external circulating tube (113).
On reaching the predetermined pressure or vacuum in the cell compartment (105) or after the certain time duration, the bidirectional pump may be operated in reverse mode and rotate in anti-clockwise direction.
When the bidirectional pump (109) rotates in the anti-clockwise direction, the culture media in the vessel (101) flows from second hollow shaft (107B) to first hollow shaft (107) through the cell compartments (105) and connected external circulating tube (113) as show in Fig. 1. Said flow of culture media may generate the pressure in the cell compartment (105) associated with the first hollow shaft (107A) and may create the vacuum in cell compartment (105) associated with the second hollow shaft (107B). As vacuum generated in the second hollow shaft (107B) and simultaneously a pressure generated in the first hollow shaft (107A), the flexible filter (203) of the cell compartment (105) associated with the second hollow shaft (107B) may retract and simultaneously in the flexible filter (203) of the cell compartment (105) associated with the first hollow shaft (107A) may expand as shown in Fig. 3 and Fig. 4. The flexible filter (203) of the cell compartment (105) associated with the second hollow shaft (107B) retract by sucking of the culture media from the vessel (101). The sizes of the pores of the flexible filter (203) are smaller than the size of the cells so that the cells are prohibited to pass through the flexible filter (203) and deposits on the outer surface of the flexible filter (203) and only cell free culture media flows into the cell compartment (105) associated with the second hollow shaft (107B) and flown to the cell compartment (105) associated with the first hollow shaft (107A) through external circulating tube (113). At the same instant, as the pressure is generated at the cell compartment (105) associated with the first hollow shaft (107A) and the culture media flows through the cell compartment (105) associated with the first hollow shaft (107A), release the cells which were stuck & deposited externally over the pores of the flexible filter (203) of the cell compartment (105) associated with the first hollow shaft (107A) during the first cycle, to the culture media of the vessel (101).
The rotation of bidirectional pump (109) in both the directions will continuously generate and release pressure and vacuum into each cell compartment (105) where cell retention happens into the bioreactor. The continuous expansion & retraction and media flow direction in both to-and-from ways through the flexible filter (203) will not allow the filter to get blocked.
It is to be understood that the number, size and shape of the cell compartment (105) is depending upon the operation scale and type of application. The present invention is possible to be implemented in smallest (i.e.: 2ml) to largest (i.e.: 10000 L) scale bioreactor with linear scalability.
In an embodiment, the drain pump (111) may remove the cell free spent media from the bioreactor as per set perfusion rate. Level sensor may install in the vessel (101) of the bioreactor. Said sensor may be a contactless sensor which installed in the mounting station whose role is to sense the level of liquid. Said level sensors may sense the level of the culture media in the vessel (101) of the bioreactor. Perfusion pump (not shown) may be configured to add fresh culture media upon accounting the low level of the culture media in the vessel (101) of the bioreactor and stop adding the culture media until the set level is achieved. Said operation may also be performed by the other means such as flow rate & time dependent pump rotation to achieve pre-defined perfusion rate.
Fig. 5A and Fig. 5B illustrates a side view of the flexible filter (203) and supportive mesh (201) of the cell compartments (105) of the cell retention apparatus (100) according to present invention.
In some embodiments, the cell compartments (105) may be configured to rotate relative to the hollow shaft (107A, 107B) during the cell retention. The cell compartment (105) has a spear shape and mounted with the each of the hollow shaft (107A, 107B) through the bearing or related means. During the rotation of impeller (103) of bioreactor, angular momentum of culture media, and during sucking of culture media, the cells may equally spread on the outer surface of the flexible filter (203). In similar manner, during the rotation of impeller (103) of bioreactor, angular momentum of culture media, and during discharging the flow of culture media, the cell compartment (105) may rotate and deliver the centrifugal force to the cells stuck or deposited on the outer surface of flexible filter (203) of the cell compartment (105) which provides easy removal of the cells from the outer surface of flexible filter of the cell compartment (105).
In addition to that, the process of the perfusion and cell retention may be driven automatically by the rotation of bidirectional pump (109), drain pump (111) and perfusion pump (not shown). For production concentration mechanism, ultra filtration technique is employed. An ultra filter (119) may place in-line with the drain tube (115). The size of the filter is to be selected based upon size of the molecule to be concentrated. For instance, the molecule with less than 50 kDa (kilo Dalton) are to be concentrated in the bioreactor, an ultra-filter with pore cut off size of 50 kDa is used which will allow all the molecules more than 50 kDa size to be passed through harvest/drain line and as the size of the product to be concentrated is less than 50 kDa which will keep recirculating in the loop of external circulating tube (113) & ultimately in the bioreactor vessel (101).
The method for the retention of the cells using the cell retention apparatus (100) according to the present invention comprises the following steps:
a) suspending at least pair of hollow shaft (107) having a first hollow shaft (107A) and a second hollow shaft (107B) in a vessel (101);
b) connecting at least one cell compartment (105) at lower end of each hollow shaft (107);
c) connecting the first hollow shaft (107A) and the second hollow shaft (107B) at their upper end through external circulating tube (113) and configuring the bidirectional pump (109) with the external circulating tube;
d) generating, by operating a bidirectional pump (109) in clockwise direction, a vacuum in the cell compartment (105) connected with lower end of the first hollow shaft (107A);
e) retracting a flexible filter (203) of the cell compartment (105) connected with lower end of the first hollow shaft (107A);
f) sucking a culture media from a vessel (101) to the cell compartment (105) connected with lower end of the first hollow shaft (107A) though the flexible filter (203) and sticking the cells on an outer surface of the flexible filter (203) of the cell compartment (105) connected with lower end of the first hollow shaft (107A);
g) flowing the sucked culture media from the cell compartment (105) connected with lower end of the first hollow shaft (107A) into the cell compartment (105) connected with lower end of the second hollow shaft (107B) till the predetermined pressure generated in later cell compartment (105) or till the predetermined vacuum generated in earlier cell compartment;
h) disposing the culture media from the cell compartment (105) connected with lower end of the second hollow shaft (107B) by passing through the flexible filter (203);
i) generating, by operating a bidirectional pump (109) in anti-clockwise direction, a vacuum in the cell compartment (105) connected with lower end of the second hollow shaft (107B);
j) retracting a flexible filter (203) of the cell compartment (105) connected with lower end of the second hollow shaft (107B);
k) sucking a culture media from a vessel (101) to the cell compartment (105) connected with lower end of the second hollow shaft (107B) though the flexible filter (203) and sticking the cells on an outer surface of the flexible filter (203) of the cell compartment (105) connected with lower end of the second hollow shaft (107B);
l) flowing the sucked culture media from the cell compartment (105) connected with lower end of the second hollow shaft (107B) into the cell compartment (105) connected with lower end of the first hollow shaft (107A) till the predetermined pressure generated in later cell compartment (105) or till the predetermined vacuum generated in earlier cell compartment;
m) disposing the culture media from the cell compartment (105) connected with lower end of the first hollow shaft (107A) by passing through the flexible filter (203);
n) releasing the cells from the outer surface of the flexible filter (203) of the cell compartment (105) connected with lower end of the first hollow shaft (107A);
o) repeating, steps (d) to (n) till reaching optimum cell density or till batch termination; and
p) drawing, by a drain pump (111), a cells free spent media from the vessel (101) through the at least one hollow shaft (107), during or after performing the above method step (a)-(o).
In an embodiment, during the execution of the method for cell retention and perfusion, the level sensor simultaneously detect the level of the culture media in the vessel (101) and adding the fresh culture media from the culture media feed line on detection of low level, by perfusion pump., until the set level is achieved. Spent media is removed via a drain tube (115) at a controlled flow rate regulated by a drain pump (111). Simultaneously, fresh media is added through a feed line (not shown) at a controlled flow rate regulated by a media addition pump (not shown). The perfusion rate determines relative flow rates of these pumps, which is the rate of media exchange within the vessel (101) of bioreactor.
In an embodiment, the predetermined pressure or vacuum of the cell compartment (105) are between the 0.1 bar to 3 bar and on reaching of such pressure or vacuum, the bidirectional pump (109) rotates to opposite side.
The invention will now be illustrated by means of the following example. However, the following example is only for explaining the present invention in more detail, but scope of the present invention is not limited to the particular embodiments only.
EXAMPLE
• Control Cell retention system using Alternate Tangential flow filtration (ATF) (Control Variant):
In the experiment, the process for the control batch was executed by the following steps. Commercial cell culture media (Name: ActiPro, Make: Cytiva) was used in this study. Culture media preparation was performed as the vendor recommended hydration protocol. Different scale shake flasks were utilized for the initial stages of cell propagation as mentioned in the below flow chart. Cell bank vial was removed from liquid nitrogen (LN2) container and thawed as per standard procedure. Cell bank vial was revived in 125 mL shake flask and shake flask was incubated at 37°C, 5% CO2 and 160 RPM in shaker incubator for 3 to 4 days. Cell expansion (Seed development) was carried out as per the number of cells required to inoculate 5L bioreactor scale as per standard procedure. Standard STR (Stirred Tank Reactor) 5L scale single use bioreactor (SUB) was installation, followed by culture media fill activity in the same SUB as per standard procedure. Bioreactor was inoculated with n-1 seed culture. Inoculation criteria for the study experiments was 0.4 to 0.6 million cells/mL. Process parameters were monitored and maintained as per the standard process specifications. The experimental duration for both control and test groups were 20 days. The temperature was maintained at 37 ± 1.0 °C throughout batches execution. pH was maintained between 7.1 ± 0.3 with 7% filter sterilized bicarbonate or CO2 gas. Dissolved oxygen concentration was maintained at set value of 40 % by a combination of aeration and agitation strategy. Sampling of the culture media was performed as per the standard procedure. Cell growth was measured by dye exclusion method which derives cell density (million cells/ml) and % viability. Spent media sample was used to analyze offline pH, concentration of Glucose, lactate & other metabolites. Residual glucose concentration of NLT (not less than) 2.0 gm/L was maintained by the addition of 20% w/v filter sterilized glucose solution.

In control variant, ATF-2 (Alternate tangential flow) was used for perfusion process and the culture media perfusion strategy (RV/day) and other process parameters were followed as mentioned in below table 1.
Sr No. Process parameters Growth phase set point
1 Temperature 37 ± 1 ° C
2 pH 7.1 ± 0.3
3 %DO 40%
4 Agitation 80 to 200 RPM
5 Perfusion rate 0.5 to 4 RV/day from day 2 onwards
Table 1

Through this arrangement and process, the results achieved in the Control Cell retention system (ATF) shown in the below table 2 and Fig. 6.

Growth days Control Variant
Cell density (million cells / mL) Cell viability (%)
0 0.5 98.9
2 1.8 98.9
4 8.1 98.8
6 27.8 98.2
8 51.4 97.0
10 84.6 95.6
12 81.3 96.8
14 79.2 93.6
16 69.3 89.2
18 57.3 78.3
20 48.3 65.2
Table 2

• Comparative case study of novel apparatus for cell retention:

Control batch was executed through the strategy discussed in above section. The apparatus (100) for cell retention was tested for its functional operation in comparison with control process. There were different variables of present invention which are mentioned in table 3. All the other aspects of the bioprocess such as seed expansion, type of culture media, seeding density, process analysis (cell count, glucose, lactate), bioreactor parameters (pH, temperature, DO, RPM etc), perfusion rate, where kept constant as mentioned in above section of the control batch.

Details of attributes Variant 1 Variant 2 Variant 3 Variant 4 Variant 5 Variant 6 Variant 7 Variant 8 Variant 9
Number of cell compartment/ number of impeller/ location of cell compartment A single pair cell compartment /single impeller/parallel to impeller A single pair cell compartment /single impeller/middle of SUB A two pair of sequentially arranged two cell compartments/dual impeller/parallel to impellers A single pair cell compartment /single impeller/parallel to impeller A single pair cell compartment /single impeller/parallel to impeller A single pair cell compartment /single impeller/parallel to impeller A single pair cell compartment /single impeller/parallel to impeller A single pair cell compartment /single impeller/parallel to impeller Control
Recirculation pump Peristaltic Peristaltic Peristaltic Magnetic Peristaltic Peristaltic Peristaltic Peristaltic
Shape of cell container Square Square Square Square Round Round Square Square
Rotation of cell compartment NO NO NO NO NO Yes NO NO
Vacuum threshold for reversing culture flow 1 bar 1 bar 1 bar 1 bar 1 bar 1 bar 3 bar 3 bar
Porosity of flexible membrane 5 micron 5 micron 5 micron 5 micron 5 micron 5 micron 5 micron 10 micron

Table 3
• Variant 1:
A single-impeller bioreactor was employed for this example. A single cell compartments (105) connected with each hollow shaft (107A, 107B) of the pair of the hollow shaft (107), namely first hollow shaft (107A) and second hollow shaft (107B) was suspended in parallel with the single impeller bioreactor. The cell compartment (105) associated with the first hollow shaft (107A) and second hollow shaft (107B) can be considered as first cell compartment (105) and second cell compartment (105) respectively. Both cell-compartments (105) associated with each hollow shaft (107A, 107B) of the pair of the hollow shaft (107) were static and square-shaped. Recirculation was facilitated by a bidirectional peristaltic pump, with a vacuum threshold of 1 bar triggering flow reversal. The flexible filter (203) of the cell compartment (105) having a thickness of 200-micron with a 5-micron pore size, separated the cell compartments (105) from the bulk culture.
A bidirectional pump (109) was employed to induce pressure and vacuum within the cell compartments (105). During clockwise operation of the bidirectional pump (109), culture medium was propelled from the first cell compartment (105) to the second compartment. This flow generates the pressure within the second cell compartment (105) and a corresponding vacuum in the first cell compartment (105). Upon reaching the pressure or vacuum threshold, bidirectional pump (109) operates in anti-clockwise direction and reverses the flow direction. During anti-clockwise operation of the bidirectional pump (109), culture medium was propelled from the second cell compartment (105) to the first cell compartment (105). This flow generated the pressure within the first cell compartment (105) and a corresponding vacuum in the second cell compartment (105). The apparatus (100) comprising hollow shafts and an external circulating tube (113), facilitated the transfer of culture medium between the first and second cell compartment (105) and vice-versa.
The second cell compartment (105), equipped with a flexible filter (203), expanded upon generation of pressure by operating the bidirectional pump (109) in clockwise direction. Simultaneously, the first compartment experienced a vacuum due to the rheological properties of the recirculating culture media. This vacuum facilitated the deposition of cells onto the pores of the flexible filter (203) of the first cell compartment (105). As culture media continued to circulate, the first side vacuum intensified, eventually triggering the pressure or vacuum threshold & bidirectional pump (109) to reverse the flow direction. The reversal in the flow direction triggered when the preset vacuum of the first cell compartment (105) was reached or when the preset pressure of the second cell compartment (105) was reached. This flow reversal dislodges cells from the first cell compartment’s (105) flexible filter (203) pores, propelling them into the bulk culture. Subsequently, the first cell compartment (105) pressurized, while the second cell compartment (105) developed a vacuum. This continuous process persisted throughout the batch duration, ensuring efficient cell detachment and re-suspension.
The perfusion rate, a critical process parameter, was adjusted by the user to optimize nutrient supply and waste removal. The cell free culture media was drain continuously from the bioreactor and was transferred to a collection vessel (not shown in figure).
Through said process, cell density within the bioreactor was increased. This rise in cell density leads to a corresponding increase in transmembrane vacuum (TMV) and transmembrane pressure (TMP) across the flexible membranes (203). Consequently, increasing cell density reflects in decrease of time interval between two predefined vacuum thresholds, resulting in a higher frequency of culture flow direction reversals in the later stages of the culture. The result of achieved in the variable 1 having the viable cell density and the viability shown in the below table 4 and Fig. 6.

Growth days Variable 1
Cell density (million cells / mL) Cell viability (%)
0 0.5 99.3
2 1.8 99.2
4 7.9 99.2
6 29.2 98.6
8 54.2 98.2
10 93.6 98.4
12 112.2 98.2
14 109.2 97.3
16 107.7 94.7
18 91.5 88.9
20 72.0 80.3
Table 4
• Variable 2:
The experiment was executed at 5L bioreactor with CHO cell line. Process steps and procedure followed for the experiment were identical to the process followed for Variable 1. The variable 2 exhibits the modification relative to variable 1, i.e. cell compartments (105) were placed in the middle area of the bioreactor.

Through this arrangement and process, the results achieved in the variable 2 shown in the below table 5 and Fig. 6.

Growth days Variable 2
Cell density (million cells / mL) Cell viability (%)
0 0.5 99.2
2 1.8 99.2
4 7.2 98.9
6 25.8 98.1
8 49.8 98.0
10 87.6 98.3
12 99.9 97.9
14 104.7 97.8
16 101.4 93.6
18 89.4 89.2
20 68.1 79.1
Table 5

• Variable 3:
The experiment was executed at 5L bioreactor with CHO cell line. Process steps and procedure followed for the experiment were identical to the process followed for variable 1. The variable 3 exhibits the modification relative to variable 1, i.e., a bioreactor equipped with two impellers, a bottom impeller and a top impeller mounted on a central shaft was utilized. In addition to, two pair of the hollow shaft (107) having sequentially arranged two cell compartments (105) on each hollow shaft (107A, 107B), in which the four cell compartments (105) attached to the lower end of the each of the hollow shaft (107A, 107B) was positioned parallel to the bottom impeller and remaining four cell compartments (105) was positioned parallel to the top impeller. Said pairs were arranged in a 90-degree angle apart.

Through this arrangement and process, the results achieved in the variable 3 shown in the below table 6 and Fig. 6.

Growth days Variable 3
Cell density (million cells / mL) Cell viability (%)
0 0.5 99.2
2 1.8 99.2
4 8.4 99.1
6 31.4 98.7
8 56.8 97.9
10 93.3 97.2
12 117.9 97.3
14 114.3 96.2
16 109.5 95.5
18 93.3 90.1
20 77.1 77.9
Table 6

• Variable 4:
The experiment was executed at 5L bioreactor with CHO cell line. Process steps and procedure followed for the experiment were identical to the process followed for variable 01. The variable 4 exhibits the modification relative to variable 1, i.e. the type of bidirectional pump (109). Magnetically driven single use pump as a bidirectional pump (109) was placed in the apparatus (100).

Through this arrangement and process, the results achieved in the variable 4 shown in the below table 7 and Fig. 6.

Growth days Variable 4
Cell density (million cells / mL) Cell viability (%)
0 0.5 99.2
2 1.9 99.0
4 8.8 98.9
6 33.7 98.0
8 60.2 97.4
10 103.5 97.5
12 127.2 97.9
14 109.2 96.0
16 98.4 93.4
18 87.9 89.1
20 65.4 79.6
Table 7
• Variable 5:
The experiment was executed at 5L bioreactor with CHO cell line. Process steps and procedure followed for the experiment were identical to the process followed for variable 1. The variable 5 exhibits the modification relative to variable 1, i.e. the shape of cell compartment (105). Instead of square shaped, round shaped cell compartments (105) were employed during the experiment.

Through this arrangement and process, the results achieved in the variable 5 shown in the below table 8 and Fig. 6.

Growth days Variable 5
Cell density (million cells / mL) Cell viability (%)
0 0.5 99.2
2 1.7 98.8
4 7.0 98.3
6 26.4 98.1
8 51.2 97.7
10 90.0 97.4
12 119.1 97.1
14 110.4 96.6
16 101.1 95.2
18 93.9 88.7
20 82.5 79.9
Table 8

• Variable 6:
The experiment was executed at 5L bioreactor with CHO cell line. Process steps and procedure followed for the experiment were identical to the process followed for variable 5. The variable 6 exhibits the modification relative to variable 5, i.e. dynamic nature of the cell compartment (105). While Variable 5 employed a static, fixed cell compartment (105), Variable 6 incorporated a rotating cell compartment (105) mounted on the bearings. This design enabled the compartment to rotate on its axis in response to changes in liquid flow direction. Specifically, upon initiation of liquid flow into the compartment, the combined forces of the incoming flow and the inertial flow induced by the impeller triggered the rotational movement. This rotational motion potentially improved cell detachment and decreases the frequency of cell deposition on the pores of cell compartment (105).

Through this arrangement and process, the results achieved in the variable 6 shown in the below table 9 and Fig. 6.

Growth days Variable 6
Cell density (million cells / mL) Cell viability (%)
0 0.5 99.2
2 1.7 99.1
4 7.8 99.0
6 31.4 98.1
8 54.0 97.5
10 101.4 97.1
12 104.7 97.2
14 98.4 95.9
16 96.3 92.2
18 86.1 89.4
20 74.1 72.4
Table 9

• Variable 7:
The experiment was executed at 5L bioreactor with CHO cell line. Process steps and procedure followed for the experiment were identical to the process followed for variable 1. The variable 7 exhibits the modification relative to variable 1, i.e. the threshold value for flow direction reversal. Apparatus was facilitated by a peristaltic pump, with a vacuum threshold of 3 bar triggering flow reversal. Variable 7 extends the time interval between vacuum-triggered flow reversals compared to Variable 1. In this configuration, the system requires a 3-bar vacuum within the cell compartment (105) to initiate a flow reversal, resulting in a longer duration between flow reversal cycles.

Through this arrangement and process, the results achieved in the variable 7 shown in the below table 10 and Fig. 6.

Growth days Variable 7
Cell density (million cells / mL) Cell viability (%)
0 0.5 99.2
2 1.9 98.9
4 8.1 99.0
6 30.4 97.7
8 55.2 97.2
10 98.4 97.3
12 99.9 97.1
14 96.3 95.8
16 92.1 90.6
18 88.2 86.7
20 71.4 70.2
Table 10

• Variable 8:
The experiment was executed at 5L bioreactor with CHO cell line. Process steps and procedure followed for the experiment were identical to the process followed for variable 7. The variable 8 exhibits the modification relative to variable 7, i.e. the pore size of the flexible membrane. In this experiment, flexible membrane with pores size of 10 micron was employed. Variable 8 extends the time interval between vacuum-triggered flow reversals compared to Variable 07. In this configuration, the system requires a 3-bar vacuum within the cell compartment (105) to initiate a flow reversal, resulting in a longer duration between cycles compared to variable 07 as comparatively lager pore size of the flexible membrane allows small sized debris to pass through the membrane which exits from the bioreactor through harvest line.

Through this arrangement and process, the results achieved in the variable 8 shown in the below table 11 and Fig. 6.

Growth days Variable 8
Cell density (million cells / mL) Cell viability (%)
0 0.5 99.2
2 1.8 99.2
4 7.5 98.7
6 29.2 98.0
8 52.6 97.0
10 94.8 97.2
12 102.3 97.4
14 104.7 96.1
16 95.7 91.9
18 89.4 88.2
20 73.2 71.1
Table 11
CONCLUSION:
The key process output parameters for these experiments were cell density and cell viability. These metrics were used to assess the overall performance of the bioreactor system. To isolate the impact of the experimental variables, all input parameters were maintained constantly across both control and test conditions. These controlled parameters included seed expansion methodology, culture media composition, initial cell seeding density, process analytics (cell count, glucose, lactate), bioreactor operating conditions (pH, temperature, dissolved oxygen,), and perfusion rate.
The experimental data presented in the following section illustrates the cell density and viability profiles for both the cell retention apparatus (100) and the control process. Notably, the test experiments employing the apparatus (100) demonstrated superior performance in terms of cell density and viability compared to the conventional control process. These results suggest that further optimization and intensification of the process could lead to even greater improvements in cell culture outcomes.
The apparatus (100) for cell retention demonstrated superior performance in enhancing both cell density and viability. This increased cellular proliferation directly translates to a significant boost in the production of the target protein synthesized by these cells.
The whole apparatus (100) may configure with a close loop control system and automation system through which all pumps of the system may be operated. Said pumps through the automation, transmit/drain the culture media from the vessel (101) as per desired quantity and time.
• Advantages of the embodiments of the present invention are illustrated herein:
In an embodiment, using the rotation of the bidirectional pump (109) in alternative rotation and using the cell compartments (105) of the pair of the hollow shaft (107) to suck and discharge the culture media helps to retain the cell and separates the cell from the circulating culture media. This further helps in perfusion process of the cell and improve the density of the culture.
In an embodiment, the apparatus (100) suspended in the vessel (101) of the bioreactor, facilitate the continuous production and increase the yield efficiency of the current production process. Further, there is no involvement of manual interventions and connections and provides the error free set-up.
All of the disclosed and claimed apparatus and methods can be made and executed without undue experimentation in light of the present disclosure. While the system, apparatus and methods of this invention have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations can be applied to the methods, system and apparatus and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the invention.

Reference Numeral
Reference Number Description
100 Apparatus for cell retention
101 Vessel of bioreactor
103 Impeller
105 Cell Compartment
107 Pair of Hollow Shaft
107A First Hollow Shaft
107B Second Hollow Shaft
109 Bidirectional pump
111 Drain pump
113 External circulating tube
115 Draining tube
117 Sensor
119 Ultra filter
201 Supportive mesh
203 Flexible Filter

,CLAIMS:We Claim:

1. An apparatus for cell retention, the apparatus comprising:
at least a pair of vertically extended hollow shafts (107);
each hollow shaft (107A, 107B) is connected with the at least one cell compartment (105) at lower end thereof;
each said cell compartment (105) is fluidly connected at lower end of respective hollow shaft, each hollow shaft (107A, 107B) is extended vertically, the hollow shafts (107A, 107B) are fluidly connected with each other at their upper end through an external circulating tube (113) and a bidirectional pump (109); a draining tube (115) fluidly connected with the external circulating tube (113).
2. The apparatus as claimed in claim 1, wherein the bidirectional pump (109) is configured over or in-line with the external circulating tube (113).
3. The apparatus as claimed in claim 1, wherein the drain pump (111) connected with the draining tube (115).
4. The apparatus as claimed in claim 1, wherein the cell compartment (105) having a wall of supportive mesh (201).
5. The apparatus as claimed in claim 4, wherein a flexible filter (203) is disposed over the wall of supportive mesh (201).
6. The apparatus as claimed in claim 1, wherein the lower end of each hollow shaft (107A, 107B) with the cell compartment (105) is suspended within a vessel (101) filled with a culture media.
7. The apparatus as claimed in claim 6, wherein the vessel (101) comprising at least one central shaft provided with at least one impeller (103).
8. The apparatus as claimed in claim 7, wherein the vessel (101) is provided with a level sensor to detect the level of the culture media in the vessel (101).
9. The apparatus as claimed in claim 7, wherein each hollow shaft (107A, 107B) with the cell compartment (105) disposed within the parallel orientation of the central shaft.
10. The apparatus as claimed in claim 1, wherein a sensor (117) is provided along the external circulating tube (113) to sense and measure the pressure or vacuum within the circulating tube (113) and the cell compartment (105).
11. The apparatus as claimed in claim 1, wherein the flexible filter (203) having porous structure configured to prohibit penetration of cells from the culture media into the cell compartment (105).
12. The apparatus as claimed in claim 1, wherein the size of pores in the flexible filter (203) is between 0.2 to 10 micron.
13. The apparatus as claimed in claim 11, wherein the flexible filter is expandable and retractable.
14. The apparatus as claimed in claim 11, wherein the thickness of the flexible filter is between 50 to 200 micron.
15. The apparatus as claimed in claim 1, wherein the supportive mesh (201) is made from the plastic material or metal material.
16. The apparatus as claimed in claim 1, wherein the size of the mesh of the supportive mesh (201) is higher than the size of pores in the flexible filter (203).
17. The apparatus as claimed in claim 1, wherein the multiple cell compartments (105) are connected in series through hollow shaft (107A, 107B).
18. The apparatus as claimed in claim 1, wherein each cell compartment (105) is rotatably connected with respective hollow shaft (107A, 107B).
19. The apparatus as claimed in claim 2, wherein the bidirectional pump (109) is peristaltic pump.
20. The apparatus as claimed in claim 2, wherein the bidirectional pump (109) is magnetic pump.
21. The apparatus as claimed in claim 1, wherein the single bidirectional pump (109) is mounted across the multiple pair of the hollow shaft (107) connected with the at least one cell compartment (105).
22. The apparatus as claimed in claim 1, wherein an ultra filter (119) is placed in-line with the draining tube (115).
23. The apparatus as claimed in claim 1, wherein the shape of the cell compartment (105) is round, square, rectangular or any other shape.
24. A method for cell retention, the method comprising:
a. suspending at least a pair of hollow shaft (107) having a first hollow shaft (107A) and a second hollow shaft (107B) in a vessel (101);
b. connecting at least one cell compartment (105) at lower end of each hollow shaft (107A, 107B);
c. connecting the first hollow shaft (107A) and the second hollow shaft (107B) at their upper end through an external circulating tube (113) and a bidirectional pump (109);
d. generating, by operating the bidirectional pump (109) in clockwise direction, a vacuum in the cell compartment (105) connected with lower end of the first hollow shaft (107A),
e. retracting a flexible filter (203) of the cell compartment (105) connected with lower end of the first hollow shaft (107A);
f. sucking a culture media from the vessel (101) to the cell compartment (105) connected with lower end of the first hollow shaft (107A) though the flexible filter (203) and depositing the cells on an outer surface of the flexible filter (203) of the cell compartment (105) connected with lower end of the first hollow shaft (107A);
g. flowing the sucked culture media from the cell compartment (105) connected with lower end of the first hollow shaft (107A) into the cell compartment (105) connected with lower end of the second hollow shaft (107B) till a predetermined pressure generated in later cell compartment (105) or predetermined vacuum generated in the earlier cell compartment (105);
h. disposing the culture media from the cell compartment (105) connected with lower end of the second hollow shaft (107B) by passing through the flexible filter (203);
i. generating, by operating a bidirectional pump (109) in anti-clockwise direction, a vacuum in the cell compartment (105) connected with lower end of the second hollow shaft (107B),
j. retracting the flexible filter (203) of the cell compartment (105) connected with lower end of the second hollow shaft (107B);
k. sucking the culture media from the vessel (101) to the cell compartment (105) connected with lower end of the second hollow shaft (107B) though the flexible filter (203) and sticking the cells on an outer surface of the flexible filter (203) of the cell compartment (105) connected with lower end of the second hollow shaft (107B);
l. flowing the sucked culture media from the cell compartment (105) connected with lower end of the second hollow shaft (107B) into the cell compartment (105) connected with lower end of the first hollow shaft (107A) till a predetermined pressure generated in later cell compartment (105) or till a predetermined vacuum generated in earlier cell compartment (105);
m. disposing the culture media from the cell compartment (105) connected with lower end of the first hollow shaft (107A) by passing through the flexible filter (203);
n. releasing the cells from the outer surface of the flexible filter (203) of the cell compartment (105) connected with lower end of the first hollow shaft (107A); and
o. repeating, steps (d) to (n) till reaching optimum cell density or till batch termination.
25. The method, as claimed in claim 24, further comprising,
drawing, by a drain pump (111), cells free spent media from the vessel (101) through at least one hollow shaft (107A, 107B) during or after performing the steps (a)-(o).
26. The method, as claimed in claim 24, further comprising,
detecting, by a level sensor, the level of the culture media in the vessel (101); and
adding, by a perfusion pump or media addition pump, a fresh culture media from a culture media feed line on detection of low level, until the set level is achieved.
27. The method, as claimed in claim 24, further comprising,
drawing, by a drain pump (111), cells free spent media from the vessel (101) through at least one hollow shaft (107A, 107B) at a controlled flow rate; and simultaneously,
adding, by a perfusion pump or media addition pump, a fresh culture media from the culture media feed line at a controlled flow rate.

28. The method, as claimed in claim 24, wherein the predetermined vacuum & pressure of the cell compartments (105) are between the 0.1 to 3 bar.
29. The method as claimed in claim 24, further comprising, rotating, the cell compartment (105) for easy removal of the cell from the pores of the flexible filter (203) of the cell compartment (105).

Dated this on 18th December, 2024