DESC:
FORM 2
THE PATENTS ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)

1. TITLE OF THE INVENTION: “An Automated System for Producing Antigen Specific Immune Cells and Method Thereof”
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

PROVISIONAL

The following specification describes the invention. þ COMPLETE

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

Field of invention

The present invention relates to fully integrated, automated system for producing antigen specific immune cells and method thereof. More specifically, it relates to a system and method for ex-vivo separation, activation, expansion, and formulation of antigen specific and/or non-modified cells products with closed loop automation system.
Background of invention

A cell and gene therapy represent overlapping fields of research with similar therapeutic goals developing a treatment that can correct the underlying cause of a disease, often a rare inherited condition that can be life-threatening or debilitating and has limited treatment options. Cell and gene therapies are focused on diseases with precise genetic origins and offering breakthrough treatments for cancer, diabetes, and numerous other diseases impacting patients globally. For example, overactive biological pathways are difficult to inhibit with mainstream drugs. Cell and gene therapies could step into this space and inhibit these pathways at the biological level. A powerful example is the chimeric antigen receptor (CAR) T-cell therapies, which have been approved for treating certain blood cancers and also work in progress on solid tumors. The approach involves genetically modifying a patient’s T cells in the laboratory before reintroducing them into the body to fight their disease.
Over the last decade, research has accelerated and resulted in several approvals for cell and gene therapies for a broad variety of indications. More recently, these include the FDA approvals for tisagenlecleucel for acute lymphoblastic leukemia (ALL; Kymriah®), axicabtagene ciloleucel for B-cell lymphoma, (BCL; Yescarta®), including diffuse large B-cell lymphoma (DLBCL) (LBCL, DLBCL; Breyanzi™), brexucabtagene autoleucel for mantle cell lymphoma (MCL; Tecartus™), lisocabtagene maraleucel for large B-cell lymphoma (LBCL), Idecabtagene vicleucel for relapsed or refractory multiple myeloma (MM, Abecma™), ciltacabtagene autoleucel for relapsed or refractory multiple myeloma (MM; Carvykti®). But manufacturing such highly complex products, it is highly important to acknowledge that any change, regardless of how minor in the culture environment as determined by the manufacturing process may result in the alteration of product quality which is a key determinant of its safety and efficacy (Morrow et al. 2017). In order to comply with these requirements, it is suggested that the manufacturing process should be simple enough to allow reproducibility and of a short duration to minimize costs associated with resources and labour (Masri et al. 2017).
Moreover, the biological variation is difficult to tackle due to the complexity of these products, in-process variation occurring from human handling is another persistent issue that can impact product quality. Even when stringent protocols are used, variation is observed between different handlers as a result of minor imprecisions in protocols (e.g. slight deviations in incubation times, variation in pipetting etc.).
Antibodies against CD3 are a critical component in many polyclonal T cell stimulation protocols. It was first demonstrated by Dixon et al., that immobilized anti-CD3 could mediate human T cell activation and expansion in the absence of cognate antigen recognition by the T cell receptor. Anti-CD3 initiates the activation and proliferation signalling cascade by crosslinking the components of the T cell receptor complex on the surface of T cells; thus, their requirement for immobilization. It was subsequently shown by Baroja et al., that a second signal from either an immobilized or soluble anti-CD28 stimuli was required for full T cell activation in combination with immobilized anti-CD3. Additional co-stimulatory signals provided through adhesion ligands such as CD2, LFA-1 and other TNF family members such as CD137 (4-1BB) can provide additional proliferative or survival signals to the T cells.

Commercial products for T cell activation using tissue culture plates coated with immobilized anti-CD3 antibodies are available from CORNING (BioCoat™ T cell activation plates, Cat #354725) and are widely prepared by researchers using standard methods known to those familiar with the art.
WO 2023/161961 A1 described as the system and method for T cell activation and immunomodulation. Plasma chemical vapour deposition (PCVD) technique is used for the grafting of peptide- Histocompatibility Molecules (pMHCs) and co-stimulatory / co-regulatory molecules on the polymeric surfaces (E) of plates. This system and method is reproducible because it takes less time in production and is fully robotic so the human error is minimized and the results are reproducible. It is also robust, scalable and less time consuming method for grafting molecules. It provides a fast track approach to activate T cells and immunomodulation for treating various types of diseases like cancer, autoimmune disorder, infectious diseases, allergies, inflammatory condition.
US11773363B2 described configurable method and systems of growing and harvesting in a hollow fiber Bioreactor system through a user interface, a user may configure display settings, system settings, and settings associated with protocols for loading, growing and/or harvesting cells.
US 2022/0282214 A1 discloses an automated method of producing genetically modified immune cells, including chimeric antigen receptor T (CAR T) cells, utilizing a fully - enclosed cell engineering system. This instrument provides a flexible platform for cell processing applications enabling the magnetic separation of different cell types as well as customized cell processing protocols. For example, June et al. (Pilot study of redirected autologous T cells engineered to contain humanized anti-CD19 in patients with relapsed or refractory CD19+ leukemia and lymphoma previously treated with cell therapy (2015) Clinicaltrials.gov) specified final product release criteria in the IND included the specifications that the number of anti-CD3/anti-CD28-coated paramagnetic beads should not exceed 100 per 3x106 cells and that cell viability should be greater than 70%. However, minimizing the number of beads represents a formidable obstacle in the clinical translation of such therapies, as most antibody-coated magnetic-bead based products lack the ability to release bound cells readily from capture molecules in a manner that does not alter the viability and phenotype of the isolated cells. Also, this platform is only applicable to suspension cells and has not been tested to date for its suitability on isolating and expanding adherent cells. To this extent, it is limited in its applicability and not highly versatile. Additionally, this platform is limited to a patient-specific (i.e. autologous) approach, mainly due to the processing volumes of up to 400 mL.
US 11268066B2 describes the culturing cells for cell therapy applications that includes growing desired cells in the presence of antigen-presenting cells and/or feeder cells and with medium volume to surface area ratio of up to 1 ml/cm2 if the growth surface is not comprised of gas permeable material and up to 2 ml/cm2 if the growth surface is comprised of gas permeable material. The desired cells are at a surface density of less than 0.5×106 cells/cm2 at the onset of a production cycle, and the surface density of the desired cells plus the surface density of the antigen presenting cells and/or feeder cells are at least about 1.25×105 cells/cm2.
US 11365382B2 describes the bioreactor system and method thereof wherein support matrix (2) comprises at last one central shaft and plurality of peripheral shaft being radially surrounds central shaft. Arrays of discs (11) are mounted along the shaft by defining interspatial vicinities between two successive plates. Thus, discs mounted on peripheral shafts are rotated within the interspatial vicinity of discs of central shaft to ensures sufficient mixing and avoid stagnant fluidic zones which is created when discs are mounted closely apart from each other on shafts. Further, plurality of deflector vanes that are axially provided along the length of the central shaft to redirect substantially co-axial direction fluid flow into interior of culture vessel and more specifically towards the central axis. Thus, bioreactor system provides scalable and disposable bioreactor with efficient mixing and homogeneous conditions and thereby supports high density growth and maintenance of cells and other biological material.

US 2022/0282214 A1 Patent described, an automated method of producing genetically modified immune cells, including chimeric antigen receptor T ( CAR T ) cells, utilizing a fully - enclosed cell engineering system and a method comprising activating an immune cell culture with an activation reagent to produce an activated immune cell culture ; transducing the activated immune cell culture with a vector, to produce a transduced immune cell culture; expanding the transduced immune cell culture; concentrating the expanded immune cell culture of; and harvesting the concentrated immune cell culture of to produce a genetically modified immune cell culture, further comprising washing either or both the expanded immune cell culture and the concentrated immune cell culture, wherein all steps are performed in a fully enclosed cell engineering system and are optimized via a process to produce the genetically modified immune cell culture. Still, this automated product contains magnetic beads separation and magnetic separation is expensive, slow, and laborious. In cases requiring positive selection techniques, magnetic separation leaves residual magnetic beads attached to purified cells and can impede proliferation and reduce viability. These residual beads ultimately reduce cell efficacy in transplantation and engraftment applications. Also, this platform is only applicable to suspension cells and has not been tested to date for its suitability on isolating and expanding adherent cells.
Also, existing methods can carry out a series of processing steps for a single patient product on a single multi-functional processing tool. However, users are unable to define biological processes using current solutions. Conventional semi-automated instruments have instrument components that sit idle and are incapable of simultaneous parallel use.

Object of invention
The principal object of the present invention is to provide an automated system for producing antigen specific immune cells and method thereof.
Another objective of the present invention is to provide the method of cell sorting and CAR-T cell manufacturing.
Further, object of the present invention is to provide fully integrated and automated device for autologous, allogenic CAR-T, CAR-NK, CAR-M cells, TIL manufacturing wherein vessel with immobilized antibodies which would help in efficacious isolation of CD4+ and CD8+ cells.
Another object of the present invention is to immobilize Anti-CD3 on substrate and enrich CD3+ T cells which helps the researchers to easily transduce them for further steps of manufacturing unlike CliniMACS Prodigy® and Cocoon®.
Yet another object of the present invention is to provide fully integrated, automated and non-human intervention manufacturing platform for highly scalable, cost effective and GMP friendly production of CAR-T, CAR-NK, CAR-M and other gene therapies.
Another object of the present invention is to provide closed and continuous loop for autologous and allogenic gene therapy manufacturing which ascertains batch failure occur due to the contamination.
Yet object of the present invention is to provide expansion of activated CAR-T cells in the same vessel.
Yet another object of the present invention is to provide fully automated and integrated manufacturing platform which provides higher ready-to-scale index.
Another object of the present invention is to provide antibody coated discs in the vessel used for the enrichment of the desired cells.
Further, our invention is determined the plasma treatment used for the immobilization of the antibodies or any biomolecules on the discs or solid surface which are used for the activation of the cells.
Yet another object of the invention is used the vibration technology for separation of the sorted cells.
Further, object of the present invention is to provide closed loop manufacturing platform for CAR-T cells manufacturers, which is least cumbersome, scalable, smallest footprint and economic.

Summary of invention
The present invention relates to an automated system for producing antigen specific immune cells and method thereof. In the present invention, a automated closed loop system with non-human intervened sterile enabled system for the producing antigen specific immune cells. The present invention for the ex vivo enrichment, activation, transduction, expansion, and formulation of the desired antigen specific cells.
The system of the present invention comprises single or multiple chambers through which enriched immune cells are produced. The targeted cells are enriched through immobilization of the biological molecules (for example, Anti-CD3 antibody) on the disc matrix. By means, only desired cells are attached to the immobilized antibodies which are specific for the desired cells. For the further processing, desired cells are activated through specific activator biomolecules (for example Anti-CD3 antibody and Anti-CD28 antibodies) and transduction process is performed using specific transducing reagents in the specific chamber and after that the expansion of the cell is carried out in the same chamber or other specific chamber/s. The system of the present invention comprises a closed loop control system that controls material inlets, desired cell sorting and processing chamber, closed tubing sets, pumps, outlets for the products as well as waste collection bottle. The present invention enables the manufacturing of modified immune cells with complete process automation and without human interventions (no manual interventions).

Brief description of drawings
Fig.1 illustrates a schematic diagram of an automated system for producing specific immune cells in single chamber assembly according to present invention.
Fig.1A illustrates a schematic diagram of a primary chamber of the automated system for producing specific immune cells according to present invention.
Fig.1B illustrates a schematic diagram of a primary chamber having disc matrix submerged in processing fluid according to present invention.
Fig.1C illustrates a schematic diagram of a primary chamber having disc matrix up lifted to start cultivation of cells in same chamber according to present invention.
Fig.2 illustrates a flow diagram of method for producing specific immune cells in single chamber assembly according to present invention.
Fig.3 illustrates a schematic diagram of the automated system for producing specific immune cells in double chamber assembly according to present invention.
Fig.4 illustrates a flow diagram of method for producing antigen specific immune cells in double chamber assembly according to present invention.
Fig.5 illustrates a schematic diagram of the automated system for producing antigen specific immune cells in triple chamber assembly according to present invention.
Fig.6 illustrates a flow diagram of method for producing antigen specific immune cells in triple chamber assembly according to present invention.
Fig.7 illustrates % CD3 positive cells (purity) recovered from healthy donor’s PBMC pool.
Fig.8a illustrates % CD4 and % CD8 positive cells present in total CD3+ T cells.
Fig. 8b(1) & 8b(2) illustrate flow cytometry images of CD3+, CD4+ and CD8+ cells before & after enrichment without labelling.
Fig.9a illustrate % purity of CD4+ve cells in our system's enrichment procedure.
Fig.9b illustrates % purity of CD8+ve cells in our system's enrichment procedure.
Fig.10 illustrates flow cytometry data of stimulated CD3+ve cells via CD25 and CD69 marker gating in flow cytometry.
Fig.11 illustrates % transduction efficiency of the CD4+ve T-cells and CD8+ve T cells via GFP protein.
Fig.12 illustrates % transduced T cells with GFP protein via flow cytometry.
Fig.13 illustrates comparison of expanded transduced cells in our system and well plate.

Detailed description of the 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.
The current approaches to producing adoptive cell therapies are laborious and costly, which has created a significant obstacle to the broad adoption of cell and gene therapy in medical settings. These and other issues with the production of cell- and gene-based therapeutics are resolved by the composition and procedures. The fully automated, integrated system and technique for manufacturing immune cells specific to antigens are the subject of the current invention. It is more especially related to a closed loop automation system and method for the ex-vivo separation, activation, expansion, and formulation of products containing antigen-specific and/or non-modified cells.
In various embodiments, methods for manufacturing adoptive cellular therapies, methods for expanding immune cells manufacturing platforms are provided.
Further, it is to be also understood that the phrase as used herein, "biological material" mean, but are not limited to, any particle(s), substance(s), extract(s), mixture, and/or assembly derived from or corresponding to one or more organisms, cells, and/or viruses. It will be apparent to one skilled in the art that cells which may be cultured in an automated cell management system comprise one or more cell types including, but not limited to, animal cells, insect cells, mammalian cells, human cells, transgenic cells, genetically engineered cells, transformed cells, cell lines, plant cells, anchorage-dependent cells, anchorage-independent cells, and other cells capable of being cultured in vitro as known in the art. The biological material also may include additional components to facilitate analysis, such as fluid (e.g., water), buffer, culture nutrients, salt, other reagents, dyes, etc. Accordingly, the biological material may include one or more cells disposed in a culture medium and/or another suitable fluid medium. As used herein the phrase, "Discs or plates" describes, but are not limited to, any geometrical shaped material capable of providing surface area for attachment, entrapment or encapsulation of particles like, but are not limited to, cells, proteins and other biochemical and chemical substances.
The automated system and methodology for the production of specific immune cells are disclosed in the invention. The therapies that are manufactured here can be used to treat or prevent a variety of illnesses, such as cancer, autoimmune diseases, infectious diseases, inflammatory diseases, and immunodeficiency.
The techniques are repeatable, scalable, and compatible with cGMP manufacturing procedures, and they provide less patient-to-patient variability in the production of adoptive cellular therapies. As a result, as compared to the current automated cell therapy manufacturing process, the techniques and compositions considered here are better.
In the most recent state of the art, there is a closed loop automation system for the production of specific immune cells and a fully integrated, automated system for the ex-vivo separation, activation, expansion, and formulation of antigen-specific and/or non-modified cell products. The manual approach requires a lot of steps. Furthermore, robust generation of a significant number of highly viable T cells can be achieved in less than 15 days. The primary benefit of our technology is that it does not require the use of beads for T cell enrichment. Here, every step is carried out in a closed, single-use vessel with a sterile set of tubing and a control unit that manages the entire operation. Surprisingly, this technique selects T cells with a higher enrichment efficiency. Also, include highly maintained environments during overall process for the cell growth.
The process of creating a therapeutic composition of T cells is referred to as "T cell manufacturing or immune cell modification" or "methods of manufacturing" T cells or comparable terms. These terms can refer to one or more iterations of a population of cells that comprise T cells or a population of purified T cells; enrichment, isolation, washing, stimulation, activation, modification, expansion, final formulation, cryopreservation and thawing for the therapeutic purpose, or any appropriate combination thereof.
Thymocytes, naïve T lymphocytes, immature T lymphocytes, mature T lymphocytes, resting T lymphocytes, or activated T lymphocytes are all included in the accepted definitions of "T cells" and "T lymphocytes." The helper T cell population (CD4+ T cells), cytotoxic T cell population (CD8+ T cells), CD4+ CD8+ T cells, CD8-CD4- T cells, and any other subset of T cells are illustrative populations of T cells that can be used in certain implementations. T cells that exhibit one or more of the following markers—CD3, CD4, CD8, CD27, CD28, CD45RA, CD45RO, CD62L, CD127, CD197, and HLA-DR —are among the other illustrated populations of T cells that may be used in specific embodiments. If desired, these cells can also be further isolated using positive or negative selection procedures.
Thus, an Automated system of present invention typically includes primary chamber and/or secondary chamber and/or tertiary chamber. It is also noted that a single chamber assembly having a primary chamber. A double chamber assembly comprises a primary chamber and the secondary chamber. A triple chamber assembly comprises a primary chamber, secondary chamber and tertiary chamber. The combined processing enrichment, activation and expansion system of the invention includes a closed system, that programmed automatically for various steps including, cell selection and/or enrichment via immobilized antibody, activation or stimulation, transduction and expansion, washing or final product. So, all process controlled via control unit consisting software. Also our invented fully automatic system of the cell and gene therapeutics which delivers cells, cell materials like cytokines, DNA, RNA, Viruses and other factors used in manufacturing process.
The invention is a system that reduces operator exposure when handling infectious material, maintains sterility with minimal to no manual labor, and is easy to handle in terms of cell complexity.
In the one embodiment, the present invention provides a fully automatic system provide for the producing specific immune cells and method. The method consisting of following steps: (a)Providing prepared a pool of leukocytes or blood or PBMC cells. (b)Cell selection using specific immobilized antibodies to aid in cell enrichment processing step. (c) Selected cell detachment process in which cells are detached in the given media. (d)Activation of the enriched immune cells using activators. (e)Transduction of the specific immune cells using viral vector. (f)Expansion of the cells in either same activation chamber or secondary chamber or tertiary chamber. (g)Washing of the cultured T cells and final formulation of specific immune cell therapy.
Immune cell subsets, dendritic cells, T cells, NK cells, macrophages and/or immune cell progenitors can all be subjected to immobilized antibody separation using antibody binding molecules specific for a surface marker, such as markers CD2, CD3, CD4, CD8, CD25, CD27, CD45RA, CD45RO, CD62L, CD95, CD127, CD137, CD19, CD20, CD11, CD13, CD14, CD206, CD68, alpha/beta TCR, gamma/delta TCR, CCR7, PD-1, or Lag3. However, the process is not restricted to defined specific markers.
A peripheral blood mononuclear cell (PBMC) is any blood cell (lymphocyte, monocyte, macrophage, or immune cell) having a circular nucleus. These blood cells are essential for the immune system to combat infection and adjust to outside influences. The lymphocyte population is made up of basophils, neutrophils, eosinophils, dendritic cells, CD3+, CD4+, CD8+ T cells, B cells, and natural killer cells, as well as CD14+ monocytes. Frequently, leukopaks—a hydrophilic polysaccharide that divides blood layers—are used to isolate these cells from whole blood. Monocytes and lymphocytes create a buffy coat beneath a layer of plasma. "PBMCs" can refer to a population of cells that includes antigen-presenting cells and NK cells in addition to T cells, which are the minimum number of cells in the population.
A “stimulatory molecule” refers to a molecule on a T cell that specifically bind with cognate stimulatory ligand.
The term, “activation” refers to the state of T cell that has been sufficiently stimulated to induce detectable cellular proliferation. In particular embodiments, activation can also be associated with induced cytokine production, and detectable effector function.
The term “activated T cells” refers to among other things, t cells that are proliferating. Signals generated through the TCR alone are insufficient for full activation of the T cell and one or more secondary or costimulatory signals are also required. Thus, T cell activation comprises a primary stimulation signal through the TCR/CD3 complex and one or more secondary or costimulatory signals. Co-stimulation can be evidenced by proliferation and/or cytokine production by T cell that have received a primary activation signal, such as stimulation through the CD3/TCR complex or through CD2.
A variety of substances, including agonistic antibodies, cytokines, recombinant costimulatory molecules, and small drugs inhibitors, can be chosen as such modulatory agents. These modulatory agents are primarily anti-CD-3 and/or anti-CD-28 antibodies or their fragments coupled to solid surfaces, that stimulate or modulate the activities of the corresponding cells when cells comes around them bound with antibodies or biological molecules, which are strongly bound to the solid surface so, those are not removed from the solid surface and antibodies bind only selected cells and other non-target cells remain in media un-bound.
Following machine-assisted density-based separation, whole blood processing, or leukopak processing, the aforementioned cells are subjected to further processing for cell washing and enrichment of the selected cells utilizing our invented targeted antibody-based cell selection technology. Cell culture, including enrichment, cell washing, cell selection, activation/stimulation, differentiation, re-differentiation, transduction, cell expansion, cell washing, and final product manufacturing, which can be adherent or suspended, multicellular, multilayer, or mixed, are additional processing steps/techniques that can be carried out with our creative method. Blood, leukapherised cells, bone marrow, liposuction, any bodily fluid containing cells, tumor cells, single cells, and cell clumps are examples of input material, but they are not the only ones.
The current specification, including definitions, will take precedence in the event of a dispute.
All technical and scientific terms used herein, unless otherwise stated, have the same meaning as is generally known by one of skill in the field to which the subject matter pertains. To aid in comprehending the current invention, the following definitions are provided as utilized herein.
The word "comprise" usually refers to incorporate, meaning allowing the existence of one or more characteristics or components.
Unless the context makes it obvious otherwise, the singular terms "a," "an," and "the" as used in the specification and claims include references to plurals.
In this context, "about" or "approximately" refers to a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that differs from a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length by as much as 50, 45,40,35,30,25, 20,10,5,1,%. When used before a numerical value in specific embodiments, the terms "about" or "approximately" denote the number plus or minus a range of 100%, 90%, 80%, 70%, 60%, 50%, 40%, 30%, 20%, 10%, 5%, or 1%. yet not restricted to this range.
When a quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length is 80%, 85%, 90%,91%, 92%,93%,94%,95%,96%,97%, 98%,99%, or more of a reference quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length is referred to as "substantially" in this context. A quantity, level, value, number, frequency, percentage, dimension, size, amount, weight, or length that has an impact is referred to as "substantially the same" in some embodiments.
When terms like "one embodiment," "an embodiment," "a particular embodiment," "a related embodiment," "or further embodiment," or any combination of these terms are used throughout this specification, it indicates that at least one embodiment of the current invention contains a specific feature, structure, or characteristic that is described in relation to the embodiment. As a result, not all of the instances where the aforementioned phases appear in this specification are necessarily referring to the same embodiments. Additionally, in one or more implementations, the specific features, structures, or qualities may be combined in any way that makes sense.
As used herein the term, processing chamber, without or proceeded with any combination of the term rotating, concentration vessel and hollow refers to the same object.
As used herein the phrase, " disposable" mean, but are not limited to, any process suitable material once used for the purpose essentially be discarded and not to be reused for the same of other purpose. As used herein, the term "disposable material or disposable film" refers to a polymeric films, including for example, multilayer polymeric films and thermoplastic film made using a film extrusion and/or foaming process, such as a cast film or blown film extrusion process. For the purposes of the present invention, the term includes nonporous films as well as microporous or macroporous films. Films may be vapor permeable or impermeable, and function as liquid barriers and/or gas barriers under normal use conditions. As used herein, the term "polymers" or "polymeric material" includes, but is not limited to, homopolymers, copolymers, such as for example, block, graft, random and alternating copolymers, terpolymers, etc and blends and modifications thereof. Furthermore, unless otherwise specifically limited, the term "polymer" shall include all possible geometrical configurations of the material. These configurations include, but are not limited to, isotactic, syndiotactic and atactic symmetries. The polymers used in the present invention can be natural, synthetic, biocompatible and/or biodegradable. The term "natural polymer" refers to any polymers that are naturally occurring, for example, silk, collagen-based materials, chitosan, hyaluronic acid and alginate. The term "synthetic polymer" means any polymers that are not found in nature, even if the polymers are made from naturally occurring biomaterials. Examples include, but are not limited to aliphatic polyesters, poly(amino acids), copoly(etheresters), polyalkylenes, oxalates, polyamids, tyrosine derived polycarbonates, poly(iminocarbonates), polyorthoesters, polyoxaesters, polyamidoesters, polyoxaesters containing amino groups, poly(anhydrides), polyphosphazenes and combinations thereof. The term "biocompatible polymer" refers to any polymer which when in contact with the cells, tissues or body fluid of an organism does not induce adverse effects such as immunological reactions and/or rejections and the like. The term "biodegradable polymer" refers to any polymer which can be degraded in the physiological environment such as by proteases. Examples of biodegradable polymers include, collagen, fibrin, hyaluronic acid, polylactic acid (PLA), polyglycolic acid (PGA), polycaprolactone (PCL), polydioxanone (PDO), trimethylene carbonate (TMC), polyethyleneglycol (PEG), alginate, chitosan or mixtures thereof.
The chamber may comprise or may be made of various materials, In a preferred embodiment, transparent materials are used like plastics, polystyrol(PS), polysterene, polyvinylchloride, polycarbonate, glass, polyacrylate, polyacrylamide, polymethymethacrylate (PMMA), and/or Polyethylenterephalate(PET). Polytetrafluorethylene (PTFE) and/or thermoplastic polyurethane(TPU), silicone or compositions comprising one or more of the above mentioned materials. The chamber can aslo be made of collagen, chitin, aginate, and/or hyaluronic acid derivatives. Possible are also possible are also polyactide(PLA), olyglycolida(PGA) and their copolymers, which are biodegradable. Alternatively, non-biodegradable materials can be used, such as polystyrol(PS), polysterene, polycarbonate, polyacrylate, polyethylene(PE), poymethy-methacrylate(PMMA) and/or polyethylenterephtala(PET). Polytetrafluorethylen(PTFE) and/or thermoplastic polyurethane(TPU) can also be used. Other alternatives include ceramics and glass materials,like hydroxylspstite(HA)or calcium phosphate. The layers in the chamber can be solid or porous materials.
Here using the chamber is not limited to the word, it may consist of the single use vessel, single use bioreactor, primary chamber, secondary chamber and/or tertiary chamber.
As used herein the term, stopper or plug or actionable plug or pinch valve with the purpose of obstructing the flow of a liquid.
As use herein the term, temperature compatible with cell processing standard, in between 30 to 40°C when referring to cell culture in terms of expansion or incubation. But not limited to this range.
As used herein the terms, low speed rotation of whole chamber or only antibody immobilized disc or shaft consisting of the immobilized disc rotation speed between 1 to 100 rpm but not limited to 100, 1-300, 3-500, 4-600, 5-800, 6-900,7-2000 rpm; are rotation between 1 to 50,000 seconds.
Said genetic modification of T cells or immune cells, T cell subsets or immune cell subsets and or T cell progenitors and/or immune cells progenitors may be performed by transduction, transfection or electroporation.
Most preferably, transduction is performed with lentiviruses, adeno viruses, adeno associated viruses, gamma-retroviruses, alpha retroviruses or with electroporation or transfection by nucleic acids (DNA, mRNA, ODNs, antagomirs, miRNA), proteins, diseases specific antigens, site specific nucleases (zinc finger nucleases, TALENs, CRISPER, piggibag), self replicating RNA viruses or integration-deficient lentiviral vectors also not limited to defined procedures or molecules or biomolecules or techniques.
A vector is a nucleic acid molecules that is capable of transporting another nucleic acid. Vector may be, eg., viruses, phages, a DNA vector, a RNA vector, a viral vector, a bacterial vector, a plasmid vector, a cosmid vector, and an artificial chromosome vector. An “expression vector” is any type of vector that is capable of directing the expression of a protein encoded by one or more genes carried by the vector when it is present in the appropriate environment.
More preferentially, said genetic modification of T cells, T cell subsets and or T cell progenitors may be performed by transducing said cells with 3rd generation lentiviral vector but not limited to this.
Said expansion of genetically modified T cells, T cell subsets and/or T cell progenitors may be performed by adding a suited GMP grade cell medium for cell culture expansion to said cultivation chamber or vessel.
Said enrichment, cells are selected based on the antibodies or biomolecules which are immobilized on the solid surface said disc which are many of numbers would be stacked in the shaft for cells attachment and make a bond with immobilized antibody. Preferentially the shaking is performed during enrichment and also incubate the cells for said and defined time for the cell attachment and detachment.
Said activation, genetic modification and/or said expansion of T cells, T cell subset and/or T cell progenitors or immune cell, immune cell subset may be performed by agitation. Preferentially the agitation is performed during expansion of defined cells. But not limited to this only stage for the expansion, which may be used in the enrichment, activation, washing or any defined processing step in the invention.
As a part of the expansion of T cells, other conditions such as temperature, pH, Oxygen concentration, carbon dioxide concentration, waste concentration, metabolite concentrate may also be controlled in the chamber.
Suitably, the method further includes removing the activation reagent from the activated immune cell culture, which can also include removing the vector following the transducing step. The activation reagent is suitably removed from the immune cell culture by washing, draining or physically removing the cells or the activation reagent. The vector can be removed by washing, or by binding the vector to a surface and then transferring the cells to a different chamber or expand the cells in to same chamber.
Said activation may be performed by using cell densities between 0.1 x 106 / ml cells to 5 x 108 cells to be activated and preferably between 0.3 x 106 cells/ml to 2.5 x 108 cells /ml and most preferably 1 x 106 cells/ml. Alternatively, said activation may be performed by using high cell density 2x 106 cells/ml to 4 x107 cells/ml. but not limited to these criteria.
As used herein the term, sufficient time, when referring to cell expansion is between 1 day to 7 days but not limited to 7 days it may go to 20 days for the definite cell type, 1 to 50 minutes when referring to solid resuspension; 5 min to 10 hours when referring to viral incubation for transduction process.
As used herein the term, satisfactory cell count, refers to a number of cells between 10¬5 and 1010 cells but not limited to this range. It might be less or more number of the cells taken for the processing.
In addition sterile processes for the cell culturing is possible which is important feature for the long term T cell in vitro expansion under conditions which are compliant with rigorous cGMP standard. That means in as closed and sterile cell culture system.
It is to be noted that the components like pinch valve, pump, inlet, outlet, sensors and others are very well understood by the person skilled in art and hence, such elements are not described in detail.
Fig. 1 and 1A shows the first embodiment of the automated system for producing specific immune cells in the single chamber assembly (A) according to the first embodiment of the present invention. The single chamber assembly (A) comprises a primary chamber (1), where cell enrichment, activation, transduction, expansion, and formulation of the desired antigen specific cells process are occurred in the same chamber.
The primary chamber (1) comprises a top plate (11), a cylindrical body (11A) and a bottom plate (11B) forming an hollow interior and having contained with a silicon bellow type adapter (12) in the center of the top plate (11) and containing with at least one shaft (13) throughout the silicon bellow type adapter (12). Said shaft (13) of the primary chamber (1) containing at least one or multiple spaced apart circular discs (14) (cell attachment matrix/ disc matrix) which is coated with a biomolecules (i.e. antibodies against CD3+ve T-cells). Said arrangement of disc matrix (14) coated with bio-molecules enables a target cell sorting. The target cells are attached/bound to the coated biomolecules on the disc surfaces and non-target cells are washed-out (removed) from the primary chamber.
The shaft(s) (13) is mounted entering through the top plate (11) using the flexible silicon bellow type adapter (12) or any other type & means which is flexible of any other mean to maintain close contained environment within the primary chamber (1) as well as giving the flexibility to the shaft (13) for its motion so that the shaft (13) when it is mounted over vibration/motion means (15) such as servo meter at top end thereof, it allows the motion in vertical & horizontal orientation. The shaft (13) is radially connected with a single vibration shaft which comes out from the top plate or the body of the primary chamber. The vibration is delivered to shaft. Said vibration means helps to detach transduced cells from the disc matrix (14).
Referring Figure 1B, the disc matrix (14) of the shaft (13) of primary chamber (1) is submerged in the fluid media for the cell enrichment, activation, transduction process. The shaft (13) is configured with the lifting means. Said lifting means lift the discs (14) toward the top plate (11) by the lifting the shaft of the primary chamber (1) and said configuration of the primary chamber act as a bio-reactor as shown in Fig. 1C.
Surrounding of the disc matrix (14), the curved vane structure of the impeller (16) is configured as show in Fig. 1A. The impeller (16) within the primary chamber (1) along with the gassing means and the process sensor elements like pH, temperature & %DO sensors inside of vessel act as a bioreactor when the disc matrix (14) is uplifted to the top plate (11) of the primary chamber (1) through the lifting means as shown in Fig. 1C. The impeller (16) is operated through the motor and provides an inward flow of the fluid content towards the centre of the primary chamber and provides sufficient nutrients and gaseous requirement for the efficient cell growth.
In addition, the primary chamber (1) having at least one port for fluid inlet, at least one port for fluid outlet, at least one sensor Port, at least one sensor element, sensors for different process parameters such as pH, temperature, dissolve oxygen, carbon dioxide and other process analytics which are required for a bioprocessing.

It is to be noted that there can be different combinations such as shaft, impellers, number of shafts, number of discs mounted over the shaft, arrangement of the shaft such as vertical, horizontal and size, shape & number of flexible adapter through which the shaft enter the primary chamber, mechanism to deliver mechanical motion (vibration) to the shaft, type-shape location & number of impeller, rotation-number & shape of disc.

Referring Fig. 2 shows the flow diagram of the method for producing antigen specific immune cells in single chamber assembly according to present invention. The method for producing antigen specific immune cells in single chamber assembly according to the present invention comprises the following steps:

i. Peripheral blood mononuclear cells (PBMC) are inserted from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) of a system through a pinch valve and a pump.
ii. The CD3+ T-cells are attached to the immobilized anti-CD3 antibodies of a disc matrix (14) mounted on a shaft (13) of the primary chamber (1).
iii. The unwanted cells are removed with a bulk media from said primary chamber (1) using the pump and pinch valve.
iv. Isolated CD3+T-cells are washed by inserting a wash buffer through an inlet of primary chamber (1) by the pinch valve and pump and said buffer is removed from an outlet of primary chamber.
v. An activation reagent is supplied to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump.
vi. The CD3+ T-cells are incubated for the desired time for effective activation of said cells.
vii. After the incubation, the activation reagent is taken out from the outlet of primary chamber (1) through the pinch valve and pump and collected in a waste collection bottle.
viii. Post activation, the activated cells in the primary chamber (1) are washed by inserting the wash buffer through the inlet of primary chamber by the pinch valve and pump and said buffer is removed from the outlet of primary chamber and collected in the waste collection bottle.
ix. A transduction reagent is supplied to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump.
x. Transduction of CAR vector is carried out and is incubated for desired time.
xi. The transduction reagent is taken out from the outlet of primary chamber (1) through the pinch valve and pump.
xii. After taken out the transduction reagent, the transduced cells in the primary chamber (1) are washed by inserting the wash buffer through the inlet of primary chamber by the pinch valve and pump and said buffer is removed from the outlet of primary chamber and collected in the waste collection bottle.
xiii. The appropriate cell culture media with required cell growth promoting process additives are added in the primary chamber (1) through the pinch valve and pump.
xiv. The transduced cells in the primary chamber (1) are detached from the disc matrix (14) mounted on the shaft (13) of the primary chamber (1) through a mechanical vibration means (15).
xv. After the detachment of the cells, the disc matrix (14) is up lifted or moved toward the head plate (11) of the primary chamber (1) operated by lifting means and the primary chamber (1) is worked as a bioreactor.
xvi. All process additive connections are made to the primary chamber (1) which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth.
xvii. A fresh media is added in the primary chamber (1) through the inlet by the pinch valve and pump to support the cell growth.
xviii. Through a process of perfusion, the spent media is filtered out and the cells are concentrated in the primary chamber (1) through appropriate cell separation device (for example, the hollow-fiber filter (HF-TFF) attached in the recirculation loop) and the volume of the cells is further expanded.
xix. After achieving the desired quantity of the cells, the culture media is exchanged with formulation buffer using the perfusion setup consisting of appropriate cell separation device and recirculation loop operation. Complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient).
xx. The drug product is taken out in the sterile bag and frozen for its therapeutic use.

According to another embodiment of the method for producing antigen specific immune cells in single chamber assembly, the detachment of the cell is performed before the activation process. Said process comprises following steps:
i. inserting Peripheral blood mononuclear cells (PBMC) from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) through a pinch valve and a pump;
ii. attaching CD3+ T-cells to immobilized anti-CD3 antibodies of a disc matrix (14) mounted on a shaft (13) of the primary chamber (1);
iii. removing unwanted cells with a bulk media from said primary chamber (1) using the pump and pinch valve;
iv. washing Isolated CD3+T-cells by inserting a wash buffer through an inlet of the primary chamber (1) by the pinch valve and pump and said buffer is removed from an outlet of primary chamber (1);
v. detaching the isolated cells in the primary chamber from the disc matrix mounted on the shaft of the primary chamber through a mechanical vibration means (15);
vi. After the detachment of the cells, lifting the disc matrix (14) toward the head plate (11) of the primary chamber (1) operated by lifting means and the primary chamber (1) works as a bioreactor;
vii. supplying an activation reagent to said primary chamber (1) through the inlet of primary chamber (1) by the pinch valve and pump;
viii. incubating the CD3+ T-cells for the desired time for effective activation of said cells;
ix. after the incubation, taking out the activation reagent from the outlet of the primary chamber (1) through the pinch valve and pump and collected in a waste collection bottle;
x. post activation, washing the activated cells in the primary chamber (1) by inserting the wash buffer through the inlet of primary chamber (1) by the pinch valve and pump and said buffer is removed from the outlet of primary chamber and collected in the waste collection bottle;
xi. supplying a transduction reagent to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump;
xii. carrying out transduction of CAR vector and incubating for desired time;
xiii. taking out the transduction reagent from the outlet of primary chamber (1) through the pinch valve and pump;
xiv. after taken out the transduction reagent, washing the transduced cells in the primary chamber (1) by inserting the wash buffer through the inlet of primary chamber by the pinch valve and pump and said buffer is removed from the outlet of primary chamber and collected in the waste collection bottle;
xv. adding the appropriate cell culture media with required cell growth promoting process additives in the primary chamber (1) through the pinch valve and pump;
xvi. making all process additive connections to the primary chamber (1) which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth;
xvii. adding the fresh media in the primary chamber (1) through the inlet by the pinch valve and pump to support the cell growth;
xviii. through a process of perfusion, filtering out the spent media and the cells are concentrated in the primary chamber (1) through appropriate cell separation device and the volume of the cells is further expanded;
xix. after achieving the desired quantity of the cells, exchanging the culture media with formulation buffer using the perfusion setup consisting of appropriate cell separation device and recirculation loop operation; complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient);
xx. taking out the drug product in the sterile bag and frozen for its therapeutic use.

Referring Figure 3 shows the schematic diagram of the automated system for producing antigen specific immune cells in double chamber assembly (3) according to another embodiment of the present invention. The double chamber assembly (3) comprises a primary chamber (1) where cell enrichment, activation, transduction of the desired antigen specific cells process are occurred and a secondary chamber (2) where cell expansion, and formulation of the desired antigen specific cells process are occurred. The configuration of the primary chamber is similar to the chamber as discussed above and disclosed in the Fig.1A, Fig.1B and Fig. 1C of the present invention. The Secondary chamber is a bioreactor.
The secondary chamber (2) comprises the impeller which is configured through the shaft and rotates through the motor assembly. Said configuration of the secondary chamber (2) is connected with the ports for process additives and culture media addition/removal which establish the condition in the secondary chamber for cell growth (E.g. pH, Temperature, dissolved oxygen etc.). Further, said secondary chamber (32) is connected with the perfusion mechanism.
The secondary chamber (2) having at least one port for fluid inlet, at least one port for fluid outlet, at least one sensor Port, at least one sensor element, sensors for different process parameters such as PH, dissolve oxygen, carbon dioxide and other process analytics which are required for a bio processing. In said secondary chamber, the cells are grown and expand as per the desired quantity of the cell.
There can be different combinations such as shaft, impellers, arrangement of the gassing means, and size, shape & number of flexible adapter through which the shaft enter the chamber, mechanism to deliver rotational motion to the impeller, type-shape location & number of impeller.

Referring Fig. 4 shows the flow diagram of method for producing antigen specific immune cells in double chamber assembly (3) according to present invention. The method for producing antigen specific immune cells in double chamber assembly according to the present invention comprises the following steps:

i. Peripheral blood mononuclear cells (PBMC) are inserted from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) of a system through a pinch valve (31) and a pump (31).
ii. The CD3+ T-cells are attached to the immobilized anti-CD3 antibodies of a disc matrix (14) mounted on a shaft (13) of the primary chamber (1).
iii. The unwanted cells are removed with a bulk media from said primary chamber (1) using the pump and pinch valve.
iv. Isolated CD3+ T-cells are washed by inserting a wash buffer through an inlet of primary chamber (1) by the pinch valve and pump and said buffer is removed from an outlet of primary chamber (2).
v. An activation reagent is supplied to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump.
vi. The CD3+ T-cells are incubated for the desired time for effective activation of said cells.
vii. After the incubation, the activation reagent is taken out from the outlet of primary chamber through the pinch valve and pump and collected in a waste collection bottle.
viii. Post activation, the activated cells in the primary chamber (1) are washed by inserting the wash buffer through the inlet of chamber by the pinch valve and pump and said buffer is removed from the outlet of primary chamber and collected in the waste collection bottle.
ix. A transduction reagent is supplied to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump.
x. Transduction of CAR vector is carried out and is incubated for desired time.
xi. The transduction reagent is taken out from the outlet of primary chamber (1) through the pinch valve and pump.
xii. After taken out the transduction reagent, the transduced cells in the primary chamber (1) are washed by inserting the wash buffer through the inlet of primary chamber by the pinch valve and pump and said buffer is removed from the outlet of primary chamber (1) and collected in the waste collection bottle.
xiii. The appropriate cell culture media with required cell growth promoting process additives is added in the primary chamber (1) through the pinch valve and pump.
xiv. The transduced cells in the primary chamber (1) are detached from the disc matrix (14) mounted on the shaft (13) of the primary chamber (1) through a mechanical vibration means (15).
xv. After the detachment of the cells, a detached cell culture pool is transferred in the secondary chamber (2) for the expansion.
xvi. All process additive connections are made to the secondary chamber which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth.
xvii. A fresh media is added in the secondary chamber (2) through the inlet by the pinch valve and pump to support the cell growth.
xviii. Through a process of perfusion, the spent media is filtered out and the cells are concentrated in the secondary chamber (2) through appropriate cell separation device (for example, the hollow-fiber filter (HF-TFF) attached in the recirculation loop) and the volume of the cells is further expanded.
xix. After achieving the desired quantity of the cells, the culture media is exchanged with formulation buffer using the perfusion setup comprising appropriate cell separation device and recirculation loop operation. Complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient).
xx. The drug product is taken out in the sterile bag and frozen for its therapeutic use.

According to another embodiment of the method for producing antigen specific immune cells in double chamber assembly the detachment of the cell is performed before the activation process. Said process comprises following steps:

a) inserting Peripheral blood mononuclear cells (PBMC) from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) through a pinch valve and a pump;
b) attaching CD3+ T-cells to immobilized anti-CD3 antibodies of a disc matrix (14) mounted on a shaft (13) of the primary chamber (1);
c) removing unwanted cells with a bulk media from said primary chamber (1) using the pump and pinch valve;
d) washing Isolated CD3+T-cells by inserting a wash buffer through an inlet of the primary chamber (1) by the pinch valve and pump and said buffer is removed from an outlet of primary chamber (1);
e) detaching the isolated cells in the primary chamber from the disc matrix mounted on the shaft of the primary chamber through a mechanical vibration means (15);
f) transferring a detached cell culture pool from the primary chamber in the secondary chamber (2) for the expansion;
g) supplying an activation reagent to said second chamber (1) through the inlet of the secondary chamber (2) by the pinch valve and pump;
h) incubating the CD3+ T-cells for the desired time for effective activation of said cells;
i) after the incubation, taking out the activation reagent from the outlet of the secondary chamber (2) through the pinch valve and pump and collected in a waste collection bottle;
j) post activation, washing the activated cells in the secondary chamber (2) by inserting the wash buffer through the inlet of the secondary chamber (2) by the pinch valve and pump and said buffer is removed from the outlet of the secondary chamber and collected in the waste collection bottle;
k) supplying a transduction reagent to said secondary chamber (2) through the inlet of the secondary chamber by the pinch valve and pump;
l) carrying out transduction of CAR vector and incubating for desired time;
m) taking out the transduction reagent from the outlet of the secondary chamber (2) through the pinch valve and pump;
n) after taken out the transduction reagent, washing the transduced cells in the secondary chamber (2) by inserting the wash buffer through the inlet of the secondary chamber by the pinch valve and pump and said buffer is removed from the outlet of the secondary chamber and collected in the waste collection bottle;
o) adding the appropriate cell culture media with required cell growth promoting process additives in the secondary chamber (2) through the pinch valve and pump;
p) making all process additive connections to the secondary chamber (2) which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth;
q) adding the fresh media in the secondary chamber (2) through the inlet by the pinch valve and pump to support the cell growth;
r) through a process of perfusion, filtering out the spent media and the cells are concentrated in the secondary chamber (2) through appropriate cell separation device and the volume of the cells is further expanded;
s) after achieving the desired quantity of the cells, exchanging the culture media with formulation buffer using the perfusion setup consisting of appropriate cell separation device and recirculation loop operation; complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient);
t) taking out the drug product in the sterile bag and frozen for its therapeutic use.
Referring Figure 5 shows the schematic diagram of the automated system for producing antigen specific immune cells in triple chamber assembly according to another embodiment of the present invention. The triple chamber assembly (5) comprises a primary chamber (1) where cell enrichment, activation, transduction of the desired antigen specific cells process are occurred, a secondary chamber (2) where cell expansion of the desired antigen specific cells occurs and a tertiary chamber (4) where the cell expansion and formulation of the desired antigen specific cells process are occurred in the large scale (E.g. 1 to 2 liter and it is not limited thereto). The configuration of the primary chamber (1) is similar to the chamber as discussed above and disclosed in the Fig.1A, Fig. 1B and Fig. 1C of the present invention. In similar way, the secondary chamber (2) and tertiary chamber are bioreactors as discussed above embodiment of the present invention.
The tertiary chamber (4) is configured in similar way of the secondary chamber (2) and said tertiary chamber (4) expands the cells in large scale. In the tertiary chamber (4), the impeller is configured with the shaft and rotates through the motor assembly. Said configuration of the tertiary chamber is connected with the ports for process additives and culture media addition/removal which establish the condition in the tertiary chamber for cell growth (E.g. pH, Temperature, dissolved oxygen etc.). Further, said tertiary chamber is connected with the perfusion mechanism.
The tertiary chamber (4) having at least one port for fluid inlet, at least one port for fluid outlet, at least one sensor Port, at least one sensor element, sensors for different process parameters such as PH, dissolve oxygen, carbon dioxide and other process analytics which are required for a bio processing. In said tertiary chamber, the cells are grown and expand on the larger scale (E.g. 1 to 2 liter and it is not limited thereto) as per the desired quantity of the cell.
There can be different combinations such as shaft, impellers, arrangement of the gassing means, and size, shape & number of flexible adapter through which the shaft enter the chamber, mechanism to deliver rotational motion to the impeller, type-shape location & number of impeller.

Referring Fig. 6 shows the flow diagram of method for producing antigen specific immune cells in triple chamber assembly (5) according to present invention. The method for producing antigen specific immune cells in triple chamber assembly according to the present invention comprises the following steps:

i. Peripheral blood mononuclear cells (PBMC) are inserted from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) of a system through a pinch valve and a pump.
ii. The CD3+ T-cells are attached to the immobilized anti-CD3 antibodies of a disc matrix mounted on a shaft of the primary chamber (1).
iii. The unwanted cells are removed with a bulk media from said primary chamber (1) using the pump and pinch valve.
iv. Isolated CD3+ T-cells are washed by inserting a wash buffer through an inlet of primary chamber by the pinch valve and pump and said buffer is removed from an outlet of primary chamber (1).
v. An activation reagent is supplied to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump.
vi. The CD3+ T-cells are incubated for the desired time for effective activation of said cells.
vii. After the incubation, the activation reagent is taken out from the outlet of primary chamber (1) through the pinch valve and pump and collected in a waste collection bottle.
viii. Post activation, the activated cells in the primary chamber (1) are washed by inserting the wash buffer through the inlet of chamber by the pinch valve and pump and said buffer is removed from the outlet of primary chamber (1) and collected in the waste collection bottle.
ix. A transduction reagent is supplied to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump.
x. Transduction of CAR vector is carried out and is incubated for desired time.
xi. The transduction reagent is taken out from the outlet of primary chamber (1) through the pinch valve and pump.
xii. After taken out the transduction reagent, the transduced cells in the primary chamber (1) are washed by inserting the wash buffer through the inlet of primary chamber (1) by the pinch valve and pump and said buffer is removed from the outlet of primary chamber (1) and collected in the waste collection bottle.
xiii. The appropriate cell culture media with required cell growth promoting process additives is added in the primary chamber (1) through the pinch valve and pump.
xiv. The transduced cells in the primary chamber (1) are detached from the disc matrix (14) mounted on the shaft (13) of the primary chamber (1) through a mechanical vibration means (15).
xv. After the detachment of the cells, a detached cell culture pool is transferred in the secondary chamber (2) for the expansion.
xvi. All process additive connections are made to the secondary chamber (2) which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth.
xvii. A fresh media is added in the secondary chamber (2) through the inlet by the pinch valve and pump to support the desired cell growth.
xviii. Said cultivated cell pool in secondary chamber (2) is transferred to the tertiary chamber (4) for the large scale expansion of the cells.
xix. Said tertiary chamber (4) is attached with the perfusion mechanism and through a process of perfusion, the spent media is filtered out and the cells are concentrated in the tertiary chamber (4) through appropriate cell separation device (for example, the hollow-fiber filter (HF-TFF) attached in the recirculation loop) and the volume of the cells is further expanded.
xx. After achieving the desired quantity of the cells, the culture media is exchanged with formulation buffer using the perfusion setup comprising appropriate cell separation device and recirculation loop operation. Complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient).
xxi. The drug product is taken out in the sterile bag and frozen for its therapeutic use.

According to another embodiment of the method for producing antigen specific immune cells in triple chamber assembly the detachment of the cell is performed before the activation process. Said process comprises following steps:
a) inserting Peripheral blood mononuclear cells (PBMC) from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) through a pinch valve and a pump;
b) attaching CD3+ T-cells to immobilized anti-CD3 antibodies of a disc matrix (14) mounted on a shaft (13) of the primary chamber (1);
c) removing unwanted cells with a bulk media from said primary chamber (1) using the pump and pinch valve;
d) washing Isolated CD3+T-cells by inserting a wash buffer through an inlet of the primary chamber (1) by the pinch valve and pump and said buffer is removed from an outlet of primary chamber (1);
e) detaching the isolated cells in the primary chamber from the disc matrix mounted on the shaft of the primary chamber through a mechanical vibration means (15);
f) transferring a detached cell culture pool from the primary chamber in the secondary chamber (2) for the expansion;
g) supplying an activation reagent to said second chamber (1) through the inlet of the secondary chamber (2) by the pinch valve and pump;
h) incubating the CD3+ T-cells for the desired time for effective activation of said cells;
i) after the incubation, taking out the activation reagent from the outlet of the secondary chamber (2) through the pinch valve and pump and collected in a waste collection bottle;
j) post activation, washing the activated cells in the secondary chamber (2) by inserting the wash buffer through the inlet of the secondary chamber (2) by the pinch valve and pump and said buffer is removed from the outlet of the secondary chamber and collected in the waste collection bottle;
k) supplying a transduction reagent to said secondary chamber (2) through the inlet of the secondary chamber by the pinch valve and pump;
l) carrying out transduction of CAR vector and incubating for desired time;
m) taking out the transduction reagent from the outlet of the secondary chamber (2) through the pinch valve and pump;
n) after taken out the transduction reagent, washing the transduced cells in the secondary chamber (2) by inserting the wash buffer through the inlet of the secondary chamber by the pinch valve and pump and said buffer is removed from the outlet of the secondary chamber and collected in the waste collection bottle;
o) adding the appropriate cell culture media with required cell growth promoting process additives in the secondary chamber (2) through the pinch valve and pump;
p) making all process additive connections to the secondary chamber (2) which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth;
q) adding the fresh media in the secondary chamber (2) through the inlet by the pinch valve and pump to support the cell growth;
r) Said cultivated cell pool in secondary chamber (2) is transferred to the tertiary chamber (4) for the large scale expansion of the cells.
s) Said tertiary chamber (4) is attached with the perfusion mechanism and through a process of perfusion, the spent media is filtered out and the cells are concentrated in the tertiary chamber (4) through appropriate cell separation device (for example, the hollow-fiber filter (HF-TFF) attached in the recirculation loop) and the volume of the cells is further expanded.
t) After achieving the desired quantity of the cells, the culture media is exchanged with formulation buffer using the perfusion setup comprising appropriate cell separation device and recirculation loop operation. Complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient).
u) The drug product is taken out in the sterile bag and frozen for its therapeutic use.

Another embodiment of the invention discloses two chamber system where the first chamber is a primary chamber comprises the previously disclosed features including the arrangement of disc matrix coated with biomolecules (mainly the Anti-CD3 antibodies) which enables a target cell sorting. Another chamber is activation chamber comprises the arrangement of disc matrix similar to the primary chamber and coated with biomolecules mainly the Anti-CD3 antibodies which enable the attachment of CD3+ cells. The addition of activation reagents (mainly the anti-CD28 antibody, IL-2 and/or IL7 and/or IL15) with the desired cells are activated in primary chamber. The primary chamber works as a bioreactor vessel for cell expansion having configuration as described Figure.1A, 1B and 1C.

Another embodiment of the invention discloses the method for producing antigen specific immune cells in a single chamber assembly according to the present invention comprises the following steps:

i. Peripheral blood mononuclear cells (PBMC) are inserted from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) of a system through a pinch valve and a pump.
ii. The CD3+ T-cells are attached to the immobilized anti-CD3 antibodies of a disc matrix (14) mounted on a shaft (13) of the primary chamber (1).
iii. The unwanted cells are removed with a bulk media from said primary chamber (1) using the pump and pinch valve.
iv. Isolated CD3+ T-cells are washed by inserting a wash buffer through an inlet of primary chamber (1) by the pinch valve and pump and said buffer is removed from an outlet of primary chamber (1).
v. The appropriate cell culture media with required cell growth promoting process additives is added in the primary chamber (1) through the pinch valve and pump.
vi. The isolated cells in the primary chamber are detached from the disc matrix mounted on the shaft of the primary chamber through a mechanical vibration means (15).
vii. After the detachment of the cells, the disc matrix (14) is up lifted or moved toward the head plate (11) of the primary chamber (1) operated by lifting means and the primary chamber (1) works as a bioreactor.
viii. Appropriate amount of activation reagents are supplied to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump.
ix. The CD3+ T-cells are incubated for the desired time for effective activation of said cells.
x. Post activation, appropriate amount of transduction reagents are supplied to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump.
xi. Transduction of CAR vector is carried out and is incubated for desired time.
xii. The transduced cells in the primary chamber are cultured in the primary vessel and cell expansion occurs.
xiii. All process additive connections are made to the primary chamber which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth.
xiv. Said primary chamber is attached with the perfusion mechanism and through a process of perfusion, the spent media is filtered out and the cells are concentrated in the primary chamber through appropriate cell separation device (for example, the hollow-fiber filter (HF-TFF) attached in the recirculation loop) and the volume of the cells is further expanded.
xv. After achieving the desired quantity of the cells, the culture media is exchanged with formulation buffer using the perfusion setup comprising appropriate cell separation device and recirculation loop operation. Complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient).
xvi. The drug product is taken out in the sterile bag and frozen for its therapeutic use.

It is to be noted that the activation reagent is anti-CD3 antibody or anti-CD28 antibodies, IL-2 and/or IL7 and/or IL15 but it is not limited thereto.

It is to be noted that the use and operation of the device including primary, secondary, or tertiary chamber configuration of the device, is not limited by the sequence of addition and removal of the appropriate reagents. The sequence of process steps and the number / amount of reagents with their incubation time within the chambers can be sequenced or optimized by the researchers to get the best output and desired results from the device usage.

It is within the scope of the present invention to vary the sequence of the process steps.

The whole system of the present invention is a close loop system and automation system through which the pinch valves and pumps of the system are operated. Said pinch valve and pumps through the automation transmit the media, buffers, activation reagents, transduction reagent, waste media, spent media etc. in the different chambers through the inlet of the particular chamber in the desired quantity and time and carried out from the outlet of the particular chamber in the desired quantity and time through the pump and pinch valve operated through automation. Further, the automation system also controls the physical condition/environmental condition necessary in the chambers for the particular time according to the present invention.

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:

The final product was characterized in terms of percentage of CD3+ cells and therefore % CD3+ T cells (purity) were measured. Figure 7 represents purity of CD3+ve T cells in PBMC of various healthy donors.

Figure 8a and figure 8b show the results of the CD4 and CD8+ve total viable cells (%viability) present in leukapharesis product. This number denotes separation efficiency by number of viable cells in the leukapharesis product in our enrichment process. The cells are washed and fluorescent labeled with CD3, CD4 and CD8 antibody reagent. Labelled T cells are before and after enrichment process and analyzed by flow cytometry.

Figure 9a and Figure 9b represents %purity of the CD4 and CD8 cells which were present in the enriched CD3+ve cells after enrichment process. Enrichment of the cells done by present invention. In which CD3 antibodies were immobilized on the disc of the primary chamber. After incubation of the cells with the antibodies, unwanted cells were washed out from the chamber and retained cells were recovered in the further more process. That process is known as the detachment process. This process proceed with the vibration of the shaft with the automation technique and cells were detached from the disc and come in the fresh media filled in the chamber before the detachment process. So after detachment, media consisted the targeted cells which we were modified for the therapy. 2 x109 ± 0.5% lymphocyte cells in PBMC were taken for the processing. As given in the graphs in the figure 9a and 9b, different donor’s PBMC were proceed for the enrichment process. Average % purity before the enrichment process were 41% for CD4+ve cells and 39% for CD8+ve cells and after enrichment process, % purity of the CD4+ve cells and CD8+ve cells 93% and 92% respectively.

Figure 10 represent a T cell activation via our fully integrated, automated system on day 0, 1.5 x 108 enriched T cells in the reagent supplement consisting media present in the primary chamber were introduced with the activation reagent CD28 antibody. The, same day, T cells were sample out before activation and after incubation there after incubated with the CD69 and CD25 activation marker for the upregulation of the early activation. Given flow cytometry data represent an analysis of Live T cells before activation and 24hr after activation which shows a strongly upregulation of CD25 and CD69 in the cells.

The transduction effectiveness of CD4+ and CD8+ T cells via GFP protein is shown in Fig. 11. This indicates the quantity of GFP gene that would be transduced and result in fluorescence in T cells and which is determined by flow cytometry. Thus, our innovative method's transduction efficiency is comprehended.

A very small percentage of GFP-positive cells are shown in the graph, as described in the non-transduced, non-activated cells condition described here. According to another non-transduced cell, some cells have transduction activity but do not exhibit florescence under certain circumstances. According to the figure, 51.98% and 39.86%, respectively, of CD4+ and CD8+ T cells were GFP positive during the experiment's primary trial. According to our novel procedure, CGT compass device, GFP positive cells' florescence was seen.

There are five different types of the healthy donors, cells were leukapharised and process in the our automatic system CGT compass. The expansion time was 7 days for the T cells. Donor’s cells were culture for 7 days to determine optical culture period. All donors blood were proceed for leukapharised process. After process was done by apharesis machine, cells concentration and viability were measured. And % of lymphocytes, CD3+ve cells were measure. After measurement, cells were enriched and activated using reagents and media supplements, expand cell till 7 days. After 7 days of the expansion, Recovered CD3+ve cells were counted, checked viability, parallelly check the %CD4 and %CD8 cells recovered in the CD3+ve cells.


As per figure 13 describes; cell expansion of the cells in the well plate and our inventive system at different days. Which describes the how cells are expanded in wells compare to the system although shows that after 3 days of the culture, cells are counted per ml of the cell culture media.

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.
,CLAIMS:We Claim:

1. An automated system for producing antigen specific immune cells comprising a single chamber assembly, said single chamber assembly comprising a primary chamber (1) having a cylindrical body, a top plate and a bottom plate forming an hollow interior, a bellow type adapter (12) disposed in the center of the top plate (11), a shaft (13) extended through the bellow type adapter (12) within the hollow interior of the chamber (1), a bottom end of said shaft (13) is extended to the bottom plate and an upper end thereof is extended outside from the chamber (1), said shaft (13) is mounted with a vibration means (15) at the upper end thereof; a plurality of spaced apart circular discs (14) are mounted on the shaft (13) within the primary chamber (1);
Wherein the system is configured to carry out ex-vivo separation, activation, expansion, and formulation of antigen specific cells in the primary chamber (1);
wherein the discs (14) is coated with an antibodies against CD3+ve T-cells; CD3+ T-cells are attached to the immobilized anti-CD3 antibodies of a disc matrix (14);
wherein transduced cells in the primary chamber (1) are detached from the disc matrix (14) mounted on the shaft (13) of the primary chamber (1) through the mechanical vibration means (15);
wherein the disc matrix (14) is up lifted or moved toward the head plate (11) of the primary chamber (1) by lifting means and the primary chamber (1) is worked as a bioreactor.

2. The automated system for producing antigen specific immune cells as claimed in claim 1, wherein a second chamber (2) where cell expansion and formulation of the desired antigen specific cells is occurred is fluidly connected with the primary chamber (1).

3. An automated system for producing specific immune cells as claimed in claim and 1 and 4, wherein a tertiary chamber (4) is fluidly connected with the second chamber (2) for cell expansion of large scale.

4. A method for producing antigen specific immune cells using single chamber assembly comprising steps of:
i. inserting Peripheral blood mononuclear cells (PBMC) from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) through a pinch valve and a pump;
ii. attaching CD3+ T-cells to immobilized anti-CD3 antibodies of a disc matrix (14) mounted on a shaft (13) of the primary chamber (1);
iii. removing unwanted cells with a bulk media from said primary chamber (1) using the pump and pinch valve;
iv. washing Isolated CD3+T-cells by inserting a wash buffer through an inlet of the primary chamber (1) by the pinch valve and pump and said buffer is removed from an outlet of primary chamber (1);
v. detaching the isolated cells in the primary chamber from the disc matrix mounted on the shaft of the primary chamber through a mechanical vibration means (15);
vi. After the detachment of the cells, lifting the disc matrix (14) toward the head plate (11) of the primary chamber (1) operated by lifting means and the primary chamber (1) works as a bioreactor;
vii. supplying an activation reagent to said primary chamber (1) through the inlet of primary chamber (1) by the pinch valve and pump;
viii. incubating the CD3+ T-cells for the desired time for effective activation of said cells;
ix. after the incubation, taking out the activation reagent from the outlet of the primary chamber (1) through the pinch valve and pump and collected in a waste collection bottle;
x. post activation, washing the activated cells in the primary chamber (1) by inserting the wash buffer through the inlet of primary chamber (1) by the pinch valve and pump and said buffer is removed from the outlet of primary chamber and collected in the waste collection bottle;
xi. supplying a transduction reagent to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump;
xii. carrying out transduction of CAR vector and incubating for desired time;
xiii. taking out the transduction reagent from the outlet of primary chamber (1) through the pinch valve and pump;
xiv. after taken out the transduction reagent, washing the transduced cells in the primary chamber (1) by inserting the wash buffer through the inlet of primary chamber by the pinch valve and pump and said buffer is removed from the outlet of primary chamber and collected in the waste collection bottle;
xv. adding the appropriate cell culture media with required cell growth promoting process additives in the primary chamber (1) through the pinch valve and pump;
xvi. making all process additive connections to the primary chamber (1) which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth;
xvii. adding the fresh media in the primary chamber (1) through the inlet by the pinch valve and pump to support the cell growth;
xviii. through a process of perfusion, filtering out the spent media and the cells are concentrated in the primary chamber (1) through appropriate cell separation device and the volume of the cells is further expanded;
xix. after achieving the desired quantity of the cells, exchanging the culture media with formulation buffer using the perfusion setup consisting of appropriate cell separation device and recirculation loop operation; complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient);
xx. taking out the drug product in the sterile bag and frozen for its therapeutic use.

5. The method for producing antigen specific immune cells using single chamber assembly as claimed in claim 4, wherein the cell separation device is hollow-fiber filter (HF-TFF).

6. The method for producing specific immune cells as claimed in claim 4, wherein for cells expansion a secondary chamber is fluidly connected with the primary chamber, the method comprising the steps of, after performing steps (i) to (vi) of claim 4;
a) after step (vi), transferring a detached cell culture pool in the secondary chamber (2) for the expansion;
b) supplying an activation reagent to said second chamber (1) through the inlet of the secondary chamber (2) by the pinch valve and pump;
c) incubating the CD3+ T-cells for the desired time for effective activation of said cells;
d) after the incubation, taking out the activation reagent from the outlet of the secondary chamber (2) through the pinch valve and pump and collected in a waste collection bottle;
e) post activation, washing the activated cells in the secondary chamber (2) by inserting the wash buffer through the inlet of the secondary chamber (2) by the pinch valve and pump and said buffer is removed from the outlet of the secondary chamber and collected in the waste collection bottle;
f) supplying a transduction reagent to said secondary chamber (2) through the inlet of the secondary chamber by the pinch valve and pump;
g) carrying out transduction of CAR vector and incubating for desired time;
h) taking out the transduction reagent from the outlet of the secondary chamber (2) through the pinch valve and pump;
i) after taken out the transduction reagent, washing the transduced cells in the secondary chamber (2) by inserting the wash buffer through the inlet of the secondary chamber by the pinch valve and pump and said buffer is removed from the outlet of the secondary chamber and collected in the waste collection bottle;
j) adding the appropriate cell culture media with required cell growth promoting process additives in the secondary chamber (2) through the pinch valve and pump;
k) making all process additive connections to the secondary chamber (2) which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth;
l) adding the fresh media in the secondary chamber (2) through the inlet by the pinch valve and pump to support the cell growth;
m) through a process of perfusion, filtering out the spent media and the cells are concentrated in the secondary chamber (2) through appropriate cell separation device and the volume of the cells is further expanded;
n) after achieving the desired quantity of the cells, exchanging the culture media with formulation buffer using the perfusion setup consisting of appropriate cell separation device and recirculation loop operation; complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient);
o) taking out the drug product in the sterile bag and frozen for its therapeutic use.

7. The method for producing specific immune cells as claimed in claim 4 and 6, wherein for cells expansion on large scale a tertiary chamber is fluidly connected with the secondary chamber, the method comprising the steps of, after performing steps (a) to (l) of claim 6;

i) transferring said cultivated cell pool from the secondary chamber (2) to the tertiary chamber (4) for the large scale expansion of the cells;
ii) attaching said tertiary chamber (4) with the perfusion mechanism and through a process of perfusion, the spent media is filtered out and the cells are concentrated in the tertiary chamber (4) through appropriate cell separation device and the volume of the cells is further expanded;
iii) after achieving the desired quantity of the cells, exchanging the culture media with formulation buffer using the perfusion setup comprising appropriate cell separation device and recirculation loop operation; complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient).
iv) taking out the drug product in the sterile bag and frozen for its therapeutic use.

8. The method for producing antigen specific immune cells using single chamber assembly as claimed in claim 4 and 6, wherein optionally, the detachment of the cell is performed after the activation process.

9. A method for producing specific immune cells using single chamber assembly comprising steps of:
i. inserting peripheral blood mononuclear cells (PBMC) from a peripheral blood mononuclear cell (PBMC) pool to a primary chamber (1) of a system through a pinch valve and a pump;
ii. attaching the CD3+ T-cells to the immobilized anti-CD3 antibodies of a disc matrix (14) mounted on a shaft (13) of the primary chamber (1);
iii. removing the unwanted cells with a bulk media from said primary chamber (1) using the pump and pinch valve;
iv. washing Isolated CD3+ T-cells by inserting a wash buffer through an inlet of primary chamber (1) by the pinch valve and pump and said buffer is removed from an outlet of primary chamber (1);
v. adding the appropriate cell culture media with required cell growth promoting process additives in the primary chamber (1) through the pinch valve and pump;
vi. detaching the isolated cells in the primary chamber from the disc matrix mounted on the shaft of the primary chamber through a mechanical vibration means (15);
vii. After the detachment of the cells, lifting the disc matrix (14) toward the head plate (11) of the primary chamber (1) operated by lifting means and the primary chamber (1) works as a bioreactor;
viii. supplying appropriate amount of activation reagents to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump;
ix. incubating the CD3+ T-cells for the desired time for effective activation of said cells;
x. Post activation, supplying appropriate amount of transduction reagents to said primary chamber (1) through the inlet of primary chamber by the pinch valve and pump;
xi. carrying out transduction of CAR vector and incubating for desired time;
xii. culturing the transduced cells in the primary chamber in the primary vessel and cell expansion occurs;
xiii. making all process additive connections to the primary chamber which maintain the desired pH, temperature and dissolve oxygen level for efficient cell growth;
xiv. attaching said primary chamber with the perfusion mechanism and through a process of perfusion, the spent media is filtered out and the cells are concentrated in the primary chamber through appropriate cell separation device (for example, the hollow-fiber filter (HF-TFF) attached in the recirculation loop) and the volume of the cells is further expanded;
xv. After achieving the desired quantity of the cells, exchanging the culture media with formulation buffer using the perfusion setup comprising appropriate cell separation device and recirculation loop operation; complete exchange of culture media with formulation buffer is performed to generate the drug product (i.e. the cells ready for infusion in the patient);
xvi. taking out the drug product in the sterile bag and frozen for its therapeutic use.