DESC:FORM 2
THE PATENT ACT 1970
(39 of 1970)
&
The Patents Rules, 2003
COMPLETE SPECIFICATION
(See section 10 and rule 13)
TITLE OF THE INVENTION: A MULTISTAGE BIOREACTOR SYSTEM AND METHOD THEREOF

1. APPLICANT:
(a) NAME : OMNIBRX BIOTECHNOLOGIES PRIVATE LIMITED
(b) NATIONALITY : India
(c) ADDRESS : 5, Times corporate park, Opp. Copper stone, ShilajThaltej Road, Thaltej, Ahmedabad 380 059, Gujarat, INDIA.

3. PREAMBLE TO THE DESCRIPTION
PROVISIONAL

The following specification describes the invention. ?COMPLETE

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

FIELD OF INVENTION
[1] The present disclosure relates to bioreactor system for cell culture and bio-manufacturing, and more particularly to a multistage bioreactor with multiple integrated compartments that enables in-situ inoculation from one stage to the next without external transfers for the cultivation of biological materials and production of biologics, enabling high scaling cultivation with reduced foot print.

BACKGROUND OF INVENTION
[2] Bio-manufacturing processes for the production of therapeutic proteins, viral vectors, and vaccines rely on cell culture techniques to expand and cultivate biological materials. These processes typically involve a series of scale-up steps, known as a seed train, where cells are progressively grown in increasing volumes before reaching the final production stage. Traditional seed train approaches utilize multiple separate bioreactors of increasing size, with cells being transferred between vessels as they expand.
[3] Figure 1 illustrates a conventional serial seed-train bioreactor setup, highlighting the multiple standalone bioreactors (1) used in traditional upstream processing. The diagram shows a series of bioreactor vessels (1) increasing in size from left to right, with each vessel representing a distinct stage in the cell culture scale-up process. The setup begins with a small vial containing cells, followed by progressively larger vessels labeled as "Shake Flask" and then through multiple bioreactor stages. Each vessel is equipped with dedicated agitation systems represented by impeller assemblies. The vessels are connected in series, indicating the sequential nature of the process where cells are transferred from one vessel to the next as the culture expands. Each vessel has its own control systems and monitoring capabilities. The progression clearly illustrates how the working volume expands by approximately 10-fold between each stage, while maintaining consistent process parameters across the entire train. Such linear, stepwise approach necessitates discrete vessel operation and manual or semi-automated inter-stage transfers between bioreactors, contributing to increased process time, contamination risk, and facility footprint.
[4] This conventional seed-train architecture presents operational challenges including large facility footprint, extended turnaround time, risk of contamination, manual intervention, and elevated capital and consumable costs. Each scale-up step typically requires a dedicated bioreactor vessel, tubing connections, environmental control systems, and aseptic transfers, all of which contribute to process complexity and potential product loss.
[5] Various attempts have been made to improve upstream efficiency. For example, some approaches describe vertically extended variable-diameter bioreactors allowing for gradual scale-up within a single vessel using conical or stepped cylindrical geometries. While these designs intend to optimize aspect ratios through geometric tapering, they can introduce mechanical and operational challenges. Other designs disclose flexible cuboidal bio-containers or support totes with simplified installation features. Though effective for single-use deployment, these designs are often limited to single internal chambers without capability for internal stage-wise compartmentalization or concurrent multistage operation.
[6] Perfusion systems have been developed that enhance fed-batch production by improving volumetric productivity and cell quality. Perfusion culture, enabled by cell retention devices, allows for continuous feeding and removal of media while retaining high-density cells in the bioreactor. This mode of operation can achieve higher cell densities than traditional fed-batch culture, potentially accelerating seed expansion while reducing the number of seed stages required.
[7] However, perfusion systems can introduce new operational challenges. These may include shear stress caused by continuous recirculation, membrane fouling or clogging in retention devices, and the need for precise nutrient balance to prevent metabolite accumulation or nutrient depletion. Moreover, scaling up high-density perfusion cultures from seed to production scale can present difficulties due to changes in volume, mixing patterns, and mass transfer characteristics. The complex setup may require skilled operators, increase process validation burden, and demand higher initial capital investment and additional facility space.
[8] Various prior-art attempts have been made to improve upstream efficiency. For example, US 12 227 723 B2 and its earlier publication US 2017/0369828 A1 describe a vertically extended variable-diameter bioreactor which allows for gradual scale-up of culture volume within a single vessel by utilizing conical or stepped cylindrical geometries. While the design intends to optimize aspect ratios through geometric tapering, it introduces substantial mechanical and operational challenges. To sustain uniform mixing across these disparate diameters, the design requires multiple impellers of different diameters mounted at different heights on a single shaft. In practice, this necessitates delivering different RPMs to each impeller to balance shear and mixing efficiency, which is technically impractical with a single-shaft drive system. Operating all impellers at the same RPM either compromises mixing at the smaller lower zone or imparts excess shear in the wider upper zone, potentially damaging shear-sensitive cells. Additionally, the vertical vessel design also results in shadow zones with poor fluid dynamics.
[9] Further, US 8 556 111 B2 discloses a flexible cuboidal bio-container with an off-centre bottom port, and US 8 556 107 B2 describes a support tote with slot-and-plate features for simplified installation and drainage. Though effective for single-use deployment, these designs are limited to a single internal chamber and offer no capability for internal stage-wise compartmentalization, inoculation control, or concurrent multistage operation. As such, users must still operate multiple seed vessels independently and perform external aseptic transfers, preserving many of the legacy process inefficiencies.
[10] Similarly, US 2003/0113915 A1 discloses a bioreactor containing a small internal inoculation well within a stainless-steel vessel, intended to eliminate early-stage shake flask expansion. While this improves workflow for initial thaw and early culture expansion, it is restricted to a single early-stage compartment and requires traditional vessel-to-vessel transfers beyond the initial step. Moreover, the system does not support independent control of agitation, aeration, or sensing per stage, nor does it offer concurrent multistage operation within a shared vessel.
[11] Several patents, including US20200377850A1 and WO2015095809A1, describe perfusion systems that enhance fed-batch production by improving volumetric productivity and cell quality. The biopharmaceutical industry has increasingly adopted perfusion-based seed bioreactors, particularly at the N-1 stage. Perfusion culture, enabled by cell retention devices such as hollow fiber TFF (HF-TFF), alternating tangential flow (ATF) systems, or membrane-based filters allows for continuous feeding and removal of media while retaining high-density cells in the bioreactor. This mode of operation can achieve cell densities up to 50 times higher than traditional fed-batch culture, accelerating seed expansion while reducing the number of seed stages required.
[12] Hence, there remains a critical need for a bioreactor system that can integrate two or more upstream seed stages and the final production stage into a single container, where each compartment can operate independently under precise process control, yet allow sequential inoculation via selectively openable partitions without requiring external transfers. Such a system would significantly reduce facility footprint, turnaround time, consumable usage, and contamination risk, delivering a practical and superior alternative to the prior art.
[13] It has been appreciated that a bioreactor system is needed that overcomes one or more of these problems.
OBJECT OF INVENTION
[14] The main object of the present invention is to provide a multistage stirred-tank bioreactor system capable of integrating multiple upstream seed stages (e.g., N-3, N-2, N-1) together with the final production stage (N) within a single vessel, thereby eliminating the need for separate seed bioreactors and inter-vessel transfers.
[15] Another object of present invention is to reduce overall manufacturing cost, process footprint, and contamination risk while simplifying workflow and compressing the seed-train expansion process into a compact and closed disposable system.
[16] Further object of present invention is to overcome the mechanical and operational limitations of prior systems that rely on complex geometries, single-compartment designs, or non-scalable inoculation solutions, by offering a robust, modular and scalable architecture adaptable to various upstream bioprocessing modes including batch, fed-batch, and perfusion.
[17] Further object of present invention is to offer alternative geometrical arrangements including bottom-to-top, top-to-bottom and radial (cluster) compartment layouts to accommodate diverse process volumes and facility constraints.
[18] Further object of present invention is to enable operation in batch, fed-batch, perfusion or continuous modes for the cultivation of mammalian, insect, plant or microbial cells and the production of recombinant proteins, viral vectors, vaccines and other biologics.
[19] Further object of present invention is to simplify qualification, automation and scale-up by using a single controller to supervise multiple compartments running concurrently or sequentially under independent set-points.
[20] Further object of present invention is to facilitate rapid installation and disposal or cleaning by integrating the multistage functionality into a single vessel that is mounted and removed as a unit.
[21] Further object of present invention is to eliminate the necessity for external tubing and manual fluidic connections required for transfer of fluid from one vessel to another.
[22] These and other objects of the invention will become apparent from the detailed description, drawings and claims that follow.

SUMMARY
[23] The present invention provides a multistage stirred-tank bioreactor system for the cultivation of cells and production of cell-derived biological materials such as monoclonal antibodies, recombinant proteins, viral vectors, and vaccines. The invention integrates multiple seed stages, such as N-3, N-2, and N-1, along with the production stage, within a single bio-container that is internally partitioned into discrete compartments separated by selectively openable partitions. Each compartment is equipped with its own agitation and aeration means, allowing independent operation and control of process parameters such as mixing time, power input per unit volume (P/V), and mass transfer coefficient (kLa) to maintain appropriate cell culture condition in each compartment. The system supports both top-to-bottom and bottom-to-top inoculation strategies, depending on the relative arrangement of compartments, to enable sequential inoculum transfer without external tubing or aseptic handling. The vessel may be fabricated from rigid or flexible materials and mounted within a reusable skid or tote providing structural support and utility access. By consolidating the entire seed-train into one closed system, the invention significantly reduces footprint, consumable usage, and risk of contamination. It also simplifies bioreactor setup, reduces turnaround time, and enables high-density cultivation in perfusion or fed-batch modes. The design allows for scalable operation while preserving bioprocess performance across all stages. The invention thus offers a compact, modular, and cost-effective solution for upstream bio-manufacturing.
BRIEF DESCRIPTION OF DRAWINGS
[24] Embodiments of the invention will be described, by way of example, with reference to the following drawings, in which:
[25] The accompanying drawings illustrate various embodiments and operational configurations of the multistage bioreactor system described in the present invention. These figures are intended to aid in the understanding of the invention and are not necessarily drawn to scale.
[26] FIG. 1 illustrates a conventional serial seed-train bioreactor setup, highlighting the multiple standalone bioreactors used in traditional upstream processing.
[27] FIG. 2 illustrates a schematic diagram of a multistage bioreactor, highlighting top-to-bottom compartment configuration integrating upstream seed compartments (N-2, N-1) with production compartment (N) according to present invention.
[28] Fig. 3a illustrates multi stage bioreactor vessel in empty state with selectively openable partitions of the seed and production compartments closed according to present invention.
[29] Fig. 3b illustrates multi stage bioreactor vessel in full-scale operation where seed compartments (N-2, N-1) and production compartment (N) are filled with culture medium and the actuated partitions create one fluidically connected, well-mixed culture chamber according to present invention.
[30] Fig. 4 illustrates a schematic diagram showing the top-to-bottom configuration for a multistage bioreactor where the container is loaded onto its skid in an empty, gamma-sterilized state according to present invention.
[31] FIG. 5 illustrates sequential operating steps of the multistage bioreactor according to present invention.
[32] FIG. 6 illustrates a top-to-bottom embodiment of a 5000 L multistage bioreactor integrating upstream seed compartments (N-2 = 50L and N-1 = 500L) with production compartment (N = 5000L) according to present invention.
[33] FIG. 7 illustrates a top-to-bottom embodiment of a 1000 L multistage bioreactor integrating upstream seed compartments (N-2 = 10 L and N-1 = 100 L) with production compartment (N = 1000 L) according to present invention.
[34] FIG. 8 illustrates a top-to-bottom embodiment of a 5000 L multistage bioreactor integrating upstream seed compartments (N-4 = 0.5 L, N-3 = 5 L, N-2 = 50 L, N-1 = 500 L) with production compartment (N = 5000 L) according to present invention.
[35] FIG. 9 illustrates a multistage bioreactor with hollow-fiber tangential-flow filtration (HFTFF) or disposable ATF modules into seed compartments according to present invention.
[36] FIG. 10 illustrates a benchtop multistage bioreactor configuration for laboratory process-development use according to present invention.
[37] Fig 11 illustrates a schematic diagram of a seed-train process using two multistage bioreactor containers in sequence, enabling expanded integration according to present invention.
[38] FIG. 12 illustrates a schematic diagram of the multistage bioreactor system implementing a bottom-to-top compartmental arrangement, wherein seed compartments are located below the production compartment according to present invention.
[39] FIG. 13 illustrates a top view of the multistage bioreactor container; wherein seed compartments are positioned radially around a central production compartment (N) and arranged in a clustered configuration within a single vessel according to present invention. Common reference numerals are used throughout the figures to indicate similar features.
DETAILED DESCRIPTION OF INVENTION
[40] The following description sets forth exemplary aspects of the present disclosure. It should be recognized, however, that such description is not intended as a limitation on the scope of the present disclosure. Rather, the description also encompasses combinations and modifications to those exemplary aspects described herein.
[41] The present disclosure relates to a multistage bioreactor system and to methods for the cultivation, expansion, and production of biologic material including, but not limited to, mammalian, insect, plant, and microbial cells, as well as cell-derived products such as antibodies, recombinant proteins, viral vectors, vaccines, and other therapeutic or diagnostic compounds. While the invention will be described below with reference to specific structural embodiments and process flow sequences, it is to be understood that the invention is not confined to the precise arrangements illustrated in the accompanying drawings. Rather, the invention is capable of implementation in numerous alternative forms, geometries, and operating modes within the scope of the appended claims.
[42] For ease of reference, certain terms and phrases used throughout this specification are defined herein. The term "biologic material" includes any cellular or viral matter, living or inactivated, as well as any substances, extracts, mixtures, products, or assemblies derived from, or corresponding to, such matter. Representative, non-limiting examples include bacterial, yeast, fungal, insect, plant, or mammalian cells; transformed or genetically engineered cell lines; viral particles; expressed proteins; monoclonal antibodies; plasmid DNA; mRNA; or other biopolymers of interest. Where appropriate the biologic material may be suspended in a "culture medium" that is, an aqueous nutritive solution containing salts, buffers, vitamins, amino acids, sugars, and other reagents suitable for sustaining growth, differentiation, or continued viability of the biologic material.
[43] The phrases "cell culture," "culture," and "cultivation" are used interchangeably to denote the maintenance of cells, tissues, or viral systems in an artificial in-vitro environment under conditions favorable to growth or product expression. The term "bioreactor" encompasses any vessel, container, or assembly in which such cell culture or cultivation occurs. The term "disposable" (or "single-use") refers to a material or component intended to be employed in a single processing cycle and thereafter discarded, in contrast to systems designed for multiple cleaning-in-place (CIP) and sterilization-in-place (SIP) cycles.
[44] As used herein, "polymer" or "polymeric material" is intended in its broadest sense to include natural, synthetic, homopolymeric, copolymeric, block, graft, random, alternating, atactic, isotactic, syndiotactic, crystalline or amorphous materials, including blends and modifications thereof. The invention is not limited to any particular polymer type or molecular architecture and may utilize rigid or flexible thermoplastic resins, elastomers, or thermosets, as well as metallic or composite materials where advantageous.
[45] Unless expressly limited otherwise, the singular forms "a," "an," and "the" should be interpreted to encompass both singular and plural referents. The term "about" is used to indicate a margin of ± 10 % of the stated value, unless the context dictates otherwise.
[46] The term "Compartment" identifies an internal sub-volume of the container, bounded in part by fixed walls and in part by an openable partition separating it from an adjacent compartment.
[47] In this context, a "compartment" is any distinct reaction chamber within the overall multistage bioreactor container (2), defined by both its fixed structural boundaries and by dynamic partitions that can be opened or closed to control fluid flow between neighboring chambers. Each compartment's fixed walls may consist of sections of rigid shell or flexible film with welded seams, and they establish the primary shape, working volume, and mechanical integrity of that stage. For example, the bottom of each compartment will be formed by the container's floor or a film layer heat-sealed at its periphery, and the sidewalls will be formed by adjacent film panels or rigid panels joined by sanitary welds or gaskets.
[48] The phrase "Selectively openable partition" (5) (or simply "partition") is a hermetic barrier maintained in a closed (sealing) configuration during independent operation of adjacent compartments and actuated, via mechanical, magnetic, pneumatic, hydraulic, or manual means, to enable controlled fluid communication between compartments. The openable partition (5) constitutes the other bounding element of a compartment. It is normally closed, ensuring that each compartment can be run under its own process conditions, but can be selectively actuated (via pneumatic bladder, magnetic latch, frangible seal, etc.) to create a conduit through which mature culture from an "upstream" compartment flows into a "downstream" compartment. When closed, these partitions (5) let one compartment be filled with media and cells and be operated as bioreactor vessel to grow cells in seed stage, yet the other compartment can be completely or partially empty.
[49] By combining fixed walls and selectively openable barriers (5) in each stage, the multistage bioreactor design transforms what would otherwise be a single bioreactor volume into multiple, sequentially activated culture zones, each a "compartment", that together compress the entire seed-train and production workflow into one integrated vessel.
[50] Unless the context clearly indicates otherwise, all numerical ranges specified herein (e.g., aspect ratio, mixing time, kLa) should be construed as inclusive of the stated end-points and internally disclosive of every sub-range contained therein.
[51] The present disclosure provides an integrated and highly versatile multistage stirred-tank bioreactor system, referred to throughout as the "multistage bioreactor", intended for the cultivation of biological material ranging from mammalian, insect, plant, fungal, and microbial cells to assorted viral cultures and derivatives thereof.
[52] Now, as shown in Fig. 2, the multistage bioreactor system according to present invention comprises a containerized multistage bioreactor vessel (container) (2) that is internally divided into multiple discrete culture zones. These zones include one or more seed compartments (3) and a production compartment (4) located adjacent to the seed compartment, each compartment functioning as an independent bioreactor stage while sharing a common outer shell. Said seed compartment may include compartments such N-3 (3c), N-2 (3b) and N-1 (3a). The compartments may be arranged in a top-to-bottom configuration, with seed compartments (3) positioned above the bottom level of the production compartment (4). For instance, the N-2 compartment (3b) (initial compartments) is placed adjacent and above the bottom level of adjacent N-1 compartment (3a), similarly N-1 compartment (3a) (intermediate compartments) is placed above the bottom level of adjacent N production (final) compartment (4).
[53] The seed compartments (3) may be configured for initial cell expansion and growth, providing controlled environments for the early stages of cell cultivation. These compartments may vary in size and number depending on the specific requirements of the bioprocess. In some aspects, multiple seed compartments of increasing volumes may be incorporated to facilitate a gradual scale-up progression. The production compartment (4), typically larger in volume than the seed compartments, may be designed for the final stage of cell cultivation where the desired biological product is expressed and accumulated. This compartment may be optimized for high-density cell culture and efficient product formation. Each compartment may be equipped with its own set of process control elements, such as agitation mechanisms, aeration systems, and sensors, allowing for independent regulation of critical parameters like temperature, pH, dissolved oxygen, and nutrient levels. This compartmentalization enables tailored cultivation conditions for each stage of the bioprocess within a single container (2).
[54] These fluidically isolated culture zones, or compartments, are separated by selectively openable partitions (5) that maintain sterile isolation under normal operating conditions and can be actuated to enable in-situ transfer of inoculum from one stage to the next, thereby eliminating the necessity for external tubing, manual fluidic connections, or intermediate seed vessels. The approach simultaneously compresses the conventional seed-train workflow, reduces the cumulative footprint of upstream equipment, and mitigates the contamination risks historically associated with inter-vessel transfers.
[55] The selectively openable partitions (5) between adjacent compartments may allow for controlled transfer of cell culture from one stage to the next. These partitions (5) may be designed with various mechanisms, such as valves, sliding gates, or dissolvable membranes, that can be actuated when predetermined cultivation milestones are reached.
[56] In some implementations, the system may incorporate additional features like sampling ports, perfusion capabilities, or in-situ product harvesting mechanisms within the production compartment (4). The overall design may aim to maintain sterility throughout the cultivation process while providing flexibility for different cell lines and bioprocessing strategies
[57] The container fixed wall and the selectively openable partitions (5) of the multistage bioreactor (also called together "the container wall and partitions") may be constructed from a variety of materials selected to meet specific process, sterilization, and operational requirements. In single-use or disposable embodiments, the container wall and partitions (5) may be formed from a multilayer polymeric film having an inner contact layer of biocompatible polyethylene or ethylene–vinyl acetate, one or more intermediate gas-barrier layers of ethylene vinyl alcohol, and an outer structural layer of oriented polyester or nylon for mechanical reinforcement and structural integrity. Typical film thicknesses range from about 250 µm to about 800 µm, providing sufficient tensile and burst strength to sustain hydrostatic head pressures at working volumes up to 10000 liters. When supplied as a pre-sterilized disposable assembly, the film tolerates gamma-irradiation doses of up to 50 kGy without significant loss of mechanical integrity or barrier performance. In reusable embodiments, the container wall may comprise polished, electropolished or passivated stainless-steel alloys (for example, 316L), titanium alloys, or rigid thermoplastics such as USP Class VI polycarbonate or high-performance polysulfone, each capable of withstanding repeated steam-in-place sterilization cycles (e.g., 121 °C for 30 minutes) without degradation. Alternatively, the plastic rigid wall container can also be supplied as single-use disposable unit for bioprocessing use.
[58] In some embodiments, the bioreactor vessel according to the present invention may comprise a single seed compartment (3) adjacent to the production compartment (4). This configuration may be suitable for processes that require fewer expansion stages or for applications where a more streamlined seed train is desired.
[59] As used herein, the term "initial compartment (3')" refers to the compartment at which the process for cell inoculation initiates. This compartment typically receives the initial cell culture and serves as the starting point for the expansion process.
[60] As used herein, the term "intermediate compartment (3")" refers to the compartment(s) located between the initial seed compartment and the production compartment (4) as shown in drawings. These intermediate compartments may facilitate gradual scale-up of the cell culture, allowing for controlled expansion of the cell population before transfer to the production compartment (4).
[61] In embodiments with multiple seed compartments (3), the system may include an initial seed compartment (3a) where the cell inoculation process begins, one or more intermediate compartments for progressive expansion, and a production compartment (4) where the final product is generated. This arrangement allows for a flexible and scalable approach to cell culture, accommodating various bioprocess requirements and cell line characteristics.
[62] The multistage bioreactor system may be configured to support different combinations of compartments, ranging from a simple two-compartment setup (one seed compartment (3) and one production compartment (4)) to more complex arrangements with multiple intermediate stages. This flexibility enables users to optimize the system for specific bioprocessing needs, cell types, and desired production scales.
[63] The container (2) includes selectively openable partitions (5) separating adjacent compartments. These partitions (5) allow for fluidic isolation between compartments during independent operation and enable controlled transfer of culture between compartments when opened. Each compartment is equipped with independent agitation mechanisms. The agitation mechanisms are visible as impeller (6) assemblies mounted within each chamber. These independent agitation systems allow for compartment-specific mixing and suspension of cells. The multistage bioreactor system incorporates independent aeration systems for each compartment. Arrays of sparger (7) elements are positioned at different levels within the compartments to provide oxygen and other gases necessary for cell growth and metabolism. Multiple sensor ports are integrated into the compartment walls. These sensor ports enable the installation of independent sensors for each compartment, allowing for monitoring and control of critical process parameters such as pH, dissolved oxygen, and temperature.
[64] The arrangement of compartments and integration of independent control systems within a single container (2) structure enables in-situ inoculation capability without external transfers. As cultures in upper seed compartments (3) reach desired densities, the selectively openable partitions (5) can be actuated to allow controlled transfer of inoculum to adjacent seed compartment (3) or production compartment (4) pre-filled with fresh culture medium, eliminating the need for external fluid handling steps.
[65] The multistage bioreactor system according to present invention maintains a unified vessel structure while preserving independent control capabilities for each compartment. This configuration provides a compact footprint for the bioreactor system while maintaining proper fluid dynamics and mixing capabilities across all compartments.
[66] Figure 3 illustrates a multistage bioreactor vessel in two different states: empty and in full-scale operation.
[67] Figure 3(a) shows the multistage bioreactor vessel in an empty state. The selectively openable partitions (5) of each compartment are shown in a closed condition, maintaining fluidic isolation between the compartments (3a).
[68] Figure 3(b) depicts the multistage bioreactor vessel in use at full scale. In this state, all compartments, including two seed compartments (3) and one production compartment (4), are filled with appropriate volumes of media. The selectively openable partitions (5) are shown in an actuated open condition. This configuration merges all three compartments, creating a fluidically connected, larger culture chamber. The open partitions (5) allow for the seamless transfer of culture between compartments without external interventions.
[69] The transition from the empty state to the full-scale operation state demonstrates the versatility of the multistage bioreactor system. The ability to maintain separate compartments during initial stages and then progressively merge them into a single, well-mixed culture chamber allows for efficient seed-train compression and scalable bioprocessing within a unified vessel.
[70] Each compartment is equipped with a dedicated agitation assembly and an independent aeration circuit, enabling physical process parameters to be matched to the metabolic needs and shear sensitivity of the culture at that specific stage. Impellers (6) may take the form of pitched-blade turbines, marine-style hydrofoils, or axial-flow profiled paddles; blade diameters, blade counts, and pitch angles can vary among compartments because the invention is not bound by any requirement for geometric similarity across stages. Shafts may be top-mounted and sealed through sterile mechanical seals, bottom-mounted through magnetically coupled drives, or laterally mounted through welded bosses if the vessel design calls for side entry. Gas transfer is furnished through spargers (7) tailored to stage volume: sintered micro-porous rings, drilled-hole manifolds of varying drill density, or drop-in microsparger rods positioned near or below the impeller to maximize the entrainment of gas bubbles into the mixing patterns of the reactor. The system is further provisioned with instrument ports that accept pH sensors, dissolved-oxygen sensors, thermowells for resistance temperature detectors, capacitance-based biomass probes, and pressure transducers, each terminated in sanitary or weldable film grommets that are located so as to minimize shadow zones and afford accurate feedback across the full range of working volumes.
[71] The selectively openable partitions (5) that separate adjacent compartments are central to the operational utility of the multistage bioreactor. In single-use constructions, the partitions (5) are typically fabricated from the same multilayer film used in the container wall, heat-seamed along their peripheral edge to create leak-tight boundaries capable of withstanding routine agitation-induced turbulence as well as transient pressure imposed by the fluid filled in the upstream seed compartment (3) when the downstream receiving compartment is empty fluidically. Actuation can be executed by embedding neodymium magnetic pucks along the free edge of the partition (5); an external magnetic crank disengages these pucks from a ferromagnetic seat, allowing the film curtain to roll upward or fold laterally, thus establishing hydraulic communication between compartments. In rigid metal vessels, the partition (5) may consist of a thin stainless-steel plate clamped against a gasketed ledge by a bayonet mechanism; rotation of an external actuator unlocks the plate and allows it to swing on a hinge or drop into an integrated recess. Alternative actuation schemes may rely on pneumatic bladders that, when inflated, press a flexible diaphragm against a seat, achieving closure; deflation creates a void that permits the diaphragm to peel away under the influence of liquid pressure, thereby effecting opening. A further variant employs frangible welds that rupture when a calibrated internal pressure differential is achieved, ensuring a fail-safe opening event without external intervention. In all implementations, the partitions (5) are designed so their open or closed status is discernible by the control system, either through reed switches, inductive proximity sensors, or pressure differential readings, thereby enabling recipe logic to interlock downstream operations on partition state.
[72] In a preferred embodiment, the multistage bioreactor container (2) features a substantially square or cuboidal geometry, which facilitates efficient spatial utilization and modular integration of upstream seed compartments (3) along the planar sidewalls of the main production compartment (4). The system is configured such that the production compartment (4) integrates at least one seed compartment (3) along one of its vertical sidewalls. This first seed compartment (3), in turn, incorporates at least one smaller secondary seed compartment along one of its own sidewalls, forming a nested, hierarchical arrangement of cultivation chambers. Each seed compartment (3) is geometrically configured so that, upon selective opening of the partition (5) separating it from the adjacent chamber, the upper fluid levels align across compartments, ensuring hydraulic continuity and uniform surface height to facilitate seamless inoculum transfer. This volumetric equilibrium is achieved by elevating the bottom surface of each upstream compartment relative to the one it feeds into, allowing each to contain an appropriate working volume while maintaining a common fluid level when merged. The square or cuboidal configuration of each compartment, including the production, seed, and sub-seed chambers, supports predictable mixing dynamics and manufacturing consistency, while the planar sidewalls of each compartment further enable the modular installation of additional upstream compartments as needed. This tiered architecture allows for compact, vertically and laterally integrated seed-train compression, all housed within a single structurally supported vessel that minimizes footprint, handling complexity, and contamination risks.
[73] For example, FIG. 4 illustrates a schematic diagram showing a 2000 L multistage bioreactor in an empty, gamma-sterilized state. The container (2) is loaded onto a skid-like supporting structure (8) that provides a stable platform for mounting the bioreactor and integrating it with utility systems. The multistage bioreactor is configured in a top-to-bottom arrangement, with seed compartments (3) positioned above the production compartment (4), enabling sequential scale-up within a unified vessel structure.
[74] The multistage bioreactor vessel mounting on the skid (8) facilitates rapid installation and removal of the multistage bioreactor system as a single unit. This integrated design incorporates connections for utilities such as power, gas, and liquid handling systems. In this empty state, all selectively openable partitions (5) between compartments are shown in a closed condition, maintaining fluidic isolation between the compartments.
[75] The operational sequence of this top-to-bottom embodiment begins with the N-2 compartment (3b) (the smallest seed bioreactor compartment) being filled with 20 litres of culture medium and inoculated to an initial viable-cell density of approximately 0.3 million viable cells per ml, derived from a previous N-3 expansion or directly from a working cell bank. During a 3 to 4 day cell growth phase, the compartment is agitated at a rotational speed optimized to achieve appropriate mixing conditions, while air and oxygen flows are modulated to maintain desired dissolved oxygen saturation. Nutrient feeds, comprising concentrated amino acid and glucose mixtures, are dosed using peristaltic pumps to maintain glucose above 1 g/L and glutamine above 2 mmol.
[76] Upon reaching a target cell density of around 3.0 million cells per mL, sterile medium is introduced into the adjacent N-1 compartment (3a), filling it to a predetermined working volume of 200L while the partition (5) between seed compartments (3) remains sealed. The partition (5) between N-1 and N-2 compartment is then actuated using the magnetic latch, allowing the culture from the N-2 seed compartment (3b) to mix with the fresh medium of the N-1 compartment (3a), achieving a blended seeding density of around 0.3 million cells per mL in the N-1 stage with a cumulative working volume of 220L.
[77] Agitation rpm and aeration set-points in both compartments are adjusted to their respective operating windows, with impeller (6) tip speed preferably remaining below 1 m/s in the smaller N-2 zone to mitigate shear, while the larger N-1 zone is driven at a higher power-per-volume ratio to compensate for greater tank diameter. When the N-1 stage has matured to around 3.0 million cells per mL, a similar operation is performed to fill 2000L fresh media in the production compartment (4) and to inoculate it, culminating in 2220 liters’ of production culture seeded at approximately 0.3 million cells per mL, all without any external fluid transfer.
[78] Once an inoculum transfer is completed, the partitions (5) remain locked in the open position, effectively merging the two volumes into one larger, well-mixed culture chamber. Because gravitational potential and fluid velocity is sufficient to initiate flow and mixing, the invention alleviates the need for positive displacement pumps, thereby further safeguarding cells from shear and reducing ancillary equipment costs. The agitation (rpm) set points and sparger (7) flow rates for each of the merged compartments are set and controlled by a process controller system to account for increased volumes and modified hydrodynamic properties.
[79] The process for cultivation of cells in multistage bioreactor vessel according to present invention is shown in Fig. 5. Fig 5 illustrates sequential operating steps of the multistage bioreactor system, enabling efficient seed-train compression and automated cultivation of cells within a single vessel.
[80] In the first step, shown in Figure 5(a), the sterile, single-use multistage bioreactor container (2) according to present invention is mounted onto a dedicated support platform or skid (8). The container (2) is secured in place with all internal compartments maintained in an isolated state via selectively openable partitions (5).
[81] The second step, depicted in Figure 5(b), involves the operation of the N-2 seed compartment (3b). This compartment, representing the initial stage of the seed-train cascade, is charged with an appropriate volume of culture medium (Shown by arrow A) and inoculated with a defined concentration of viable cells. The N-2 compartment (3b) operates as an independent bioreactor stage, equipped with dedicated agitation and aeration controls to maintain optimal physicochemical parameters. The culture expands over a pre-defined interval, typically 3-5 days, to reach a target cell density.
[82] Figure 5(c) illustrates the third step, which is the inoculation of the N-1 seed compartment (3a). Upon achieving the desired culture maturity in the N-2 compartment (3b), the adjacent N-1 compartment (3a) is filled with fresh, sterile culture medium (Shown by arrow B). A control system actuates the selectively openable partition (5) separating the N-2 (3b) and N-1 (3a) compartments based on predetermined cell density thresholds. This actuation enable the partition (5) to open for establishing fluidic continuity between the compartments, enabling the mature culture from the N-2 stage to flow into and mix with the medium in the N-1 compartment (3a), effectuating in-situ inoculation. Following this transfer, both merged and unified compartments continue to operate independently with respect to agitation and gas-transfer regulation, preserving compartment-specific process control during the growth phase of the N-1 seed stage.
[83] The final step, shown in Figure 5(d), involves the inoculation of the production compartment (4) (N-stage). Following the maturation of the unified seed culture of N-2 and N-1 to a target cell density, the production compartment (4) is prefilled with sterile medium (Shown by arrow C). The control system then actuates the selectively openable partition (5) between the N-1 (3a) and N (4) compartments through a designated mechanism. At this stage, all compartments are unified. This actuation permits the transfer of combined N-1 and N-2 culture to mix into the production chamber, resulting in inoculation of the production-stage bioreactor. Similar to previous transitions, each compartment retains independent operational controls, enabling modulation of aeration and agitation parameters during the subsequent production phase.
[84] This controlled, sequential operation enables in-situ seed train compression without manual intervention. When the recipe-based process control and automation is deputed vial an advanced control system, it manages the entire process, from initial setup through final production, based on predefined cell density thresholds and process parameters. By eliminating the need for external transfers and manual operations between seed stages, the multistage bioreactor system reduces overall upstream processing time compared to conventional seed train processes.
[85] It is within the scope of the present invention to use the multiple compartments within a container according to present invention to function as a separate bioreactor for the cultivation of distinct cell types simultaneously. The compartments are designed to operate independently of each other, each providing the necessary controlled environment for the specific cell type being cultured. Notably, in this scenario, the partitions between the compartments remain unactuated, thereby preventing the unification or merging of the compartments with adjacent ones.
[86] FIG. 6 illustrates a top-to-bottom embodiment of a 5000 L multistage bioreactor integrating upstream seed compartments (3) (N-2 (3b) = 50L and N-1 (3a) = 500L) with production compartment (4) (N = 4450L). The system comprises a container (2) with multiple compartments arranged in a vertical configuration, all integrated within a single vessel structure.
[87] The production compartment (4), positioned at the bottom of the arrangement, has a volume of approximately 4500 L. This compartment is designed for the final stage of cell cultivation and product formation.
[88] Above the production compartment (4), the system incorporates two upstream seed compartments (3). The N-1 seed compartment (3a) has a volume of 450 L and is used for expanding the cell culture to a scale suitable for inoculating the production compartment (4).
[89] The topmost compartment in the arrangement is the N-2 seed compartment (3b) with a volume of 50 L, serving as the starting point for the seed train process. These compartments are fabricated along contiguous side-walls of the main vessel.
[90] Selectively openable partitions (5) separate adjacent compartments. These partitions (5) are designed such that, upon actuation, the hydraulic heads of the merged compartments align to a common fluid level, enabling controlled transfer of culture between compartments.
[91] The volumetric relationships between the compartments follow a geometric progression. The 50 L N-2 compartment (3b) expands to the 500 L merged volume in N-1 compartment (3a), representing a 10-fold increase in volume. Similarly, the transition from the 500 L N-1 compartment (3a) to the 4500 L production compartment (4) represents approximately a 10-fold increase in volume.
[92] In operation, the N-2 compartment (3b) is first charged with culture medium and inoculated to a cell density of approx. 0.3 million cells per mL. When the culture reaches a target density of about 3 million cells per mL, the partition (5) separating N-2 (3b) and N-1 (3a) is opened via its magnetic or mechanical actuator. This action blends the matured 50 L inoculum with 450 L of freshly dispensed medium in the 500 L N-1 compartment (3a), yielding a composite working volume seeded at around 0.3 million cells per mL.
[93] Upon reaching comparable maturity in the N-1 stage, the second partition (5) is actuated to merge the 500 L inoculum into 4500 L of pre-filled production medium in the N compartment (4). This establishes a production-scale culture of approximately 0.3 million cells per mL without any external fluid transfer.
[94] The multistage bioreactor system enables in-situ cascade inoculation through the selective opening of partitions (5). This design eliminates the need for external transfers between seed stages and the production stage, maintaining a closed system throughout the entire process.
[95] The top-to-bottom configuration allows for gravity-assisted transfer of culture between compartments when partitions (5) are opened. This arrangement simplifies the inoculation process and reduces the risk of contamination associated with external fluid handling.
[96] Each compartment is equipped with independent agitation, aeration, and sensing systems. These systems allow for compartment-specific control of critical process parameters throughout the seed train and production stages.
[97] This embodiment demonstrates the scalability of the multistage bioreactor system to pilot-plant volumes exceeding 5000 L while preserving seed-train compression, closed-system sterility, and proportional seed ratios, thereby extending the operational and economic advantages previously detailed for smaller-scale installations.
[98] FIG. 7 illustrates a schematic representation of a multistage bioreactor system dimensioned for a nominal production-stage working volume of approximately 1000 L. The system comprises a container (2) with multiple compartments arranged in a top-to-bottom configuration, integrating two upstream seed compartments (3) with a production compartment (4).
[99] The production compartment (4), positioned at the bottom of the arrangement, has a volume of approximately 1000 L. This compartment serves as the final stage for cell cultivation and product formation.
[100] Above the production compartment (4), the system incorporates two upstream seed compartments (3). The intermediate seed compartment, designated as N-1 (3a), has a volume of 90 L. This compartment is used for expanding the cell culture to a scale suitable for inoculating the production compartment (4).
[101] The topmost compartment in the arrangement is the initial seed compartment, designated as N-2 (3b), with a volume of 10 L. This compartment serves as the starting point for the seed train process.
[102] The volumetric relationships between the compartments follow a geometric progression. The 10 L N-2 compartment (3b) expands and merges to form the 100 L N-1 compartment (3a), representing a 10-fold increase in volume. Similarly, the transition from the 100 L N-1 compartment (3a) to the 900 L production compartment (4) represents a 10-fold increase in volume.
[103] Selectively openable partitions (5) separate adjacent compartments. These partitions (5) enable controlled transfer of culture between compartments when actuated.
[104] The operating sequence of this multistage bioreactor system begins with the 10 L N-2 compartment (3b) being charged with culture medium and inoculated to a cell density of 0.3 million cells per mL. When the culture expands to about 3 million cells per mL, the N-1 compartment (3a), pre-filled with 90 L of fresh medium, is fluidically merged with the matured N-2 inoculum by actuating the selectively openable partition (5). This action yields 100 L of N-1 culture uniformly seeded at around 0.3 million cells per mL.
[105] Upon attainment of the target density in the N-1 compartment (3a), a second partition (5) is actuated to blend the 100 L inoculum with 900 L of pre-dispensed production medium in the primary compartment. This establishes a 1000 L production culture at the desired seeding density without any external transfers.
[106] Each compartment in the multistage bioreactor system is equipped with independent agitation and aeration loops. These systems are automatically re-tuned after each partition-opening event, preserving stage-appropriate hydrodynamic and gas-transfer conditions throughout the entire cascade.
[107] The multistage bioreactor system enables in-situ cascade inoculation through the selective opening of partitions (5). This design eliminates the need for external transfers between seed stages and the production stage, maintaining a closed system throughout the entire process.
[108] The top-to-bottom configuration allows for gravity-assisted transfer of culture between compartments when partitions (5) are opened. This arrangement simplifies the inoculation process and reduces the risk of contamination associated with external fluid handling.
[109] FIG. 8 illustrates a multistage bioreactor system configured for a total production volume of 5000 L. The system comprises three geometrically proportionate seed compartments (3) arranged in a vertically stacked, top-to-bottom configuration within a single vessel structure.
[110] The topmost compartment in FIG. 8, designated as N-3 (3c), has a volume of 5 L and serves as the initial seed compartment (3’). Adjacent to the N-3 compartment (3c), the N-2 compartment (3b) has a volume of 50 L and adjacent to the N2-compartment (3b), N-1 compartment (3a) is positioned having a volume of 500L. Said N-2 and N-1 compartment function as intermediate seed compartments (3”).
[111] The volumetric relationships between the compartments follow a geometric progression. Each subsequent compartment increases in volume by a factor of 10 compared to the compartment above. This scaling allows for efficient expansion of the cell culture as the process progresses from the initial seed stage to the final production stage.
[112] The N production compartment (4), representing the production stage, is shown at the bottom of FIG. 8 with a volume of 5000 L. This compartment provides the full production capacity of the system.
[113] Each compartment is delineated from the next by a selectively openable partition (5) actuated, for example, by magnetic latch, pneumatic diaphragm, or mechanical linkage, that permits sterile fluid communication only upon command. Operation proceeds as a closed, in-situ cascade: the N-3 compartment (3c) is inoculated at 0.3 million cells per mL and cultured to a predetermined maturity, whereupon its partition (5) is opened to blend the 5 L inoculum with 45 L of fresh medium pre-charged in N-2(3b), yielding a uniformly seeded 50 L culture at the same cell density. The sequence is iteratively repeated through N-2?N-1, each time preserving the 1:10 volumetric ratio and the target seeding density, until the matured 500 L N-1 culture is merged with 4500 L of production medium in the 5000 L compartment.
[114] Each compartment is delineated from the next by a selectively openable partition (5) actuated, for example, by magnetic latch, pneumatic diaphragm, or mechanical linkage, that permits sterile fluid communication only upon command. Operation proceeds as a closed, in-situ cascade: the N-3 compartment (3c) is inoculated at 0.3 million cells per mL and cultured to a predetermined maturity, whereupon its partition (5) is opened to blend the 5 L inoculum with 45 L of fresh medium pre-charged in N-2 (3b), yielding a uniformly seeded 50 L culture at the same cell density. The sequence is iteratively repeated through N-3?N-2 and N-2?N-1, each time preserving the 1:10 volumetric ratio and the target seeding density, until the matured 500 L N-1 culture is merged with 4500 L of production medium in the 5000 L compartment (4). Independent agitation, aeration, and environmental-control loops for each stage are automatically reconfigured after every partition-opening event, thereby maintaining stage-appropriate hydrodynamics while compressing the conventional seed train from five discrete vessels to a single, compact, vertically integrated module, and eliminating all external transfers and associated contamination risks.
[115] Conversely, large-scale multistage bioreactor can be produced with cumulative volumes exceeding five thousand litres by increasing dimensions and optionally employing reinforced film envelopes that integrate fibre or woven-mesh substrates for tear resistance.
[116] FIG. 9 illustrates a multistage bioreactor system incorporating cell retention devices in the seed compartments (3). The system integrates cell separation device like hollow-fiber tangential-flow filtration (HF-TFF) or disposable alternating tangential flow (ATF) modules into the seed compartments (3), enhancing cell concentration capabilities and reducing seed stage floor requirements.
[117] The cell retention devices, such as HF-TFF systems or ATF systems, are connected to at least one seed compartment (3) of the multistage bioreactor. These devices enable the implementation of perfusion culture techniques within the seed compartments (3), allowing for increased cell densities and extended culture durations.
[118] In the HF-TFF configuration, a hollow fiber cartridge is fluidically connected to a seed compartment (3). The HF-TFF system recirculates the culture through the hollow fiber membrane, retaining cells within the compartment while allowing for the continuous removal of spent media and metabolic byproducts. This process facilitates the maintenance of optimal nutrient levels and the removal of waste products, supporting high-density cell growth.
[119] Because the system can operate in perfusion mode, each compartment may optionally incorporate a closed cell-retention loop; a hollow-fibre cartridge (HTTFF) mounted on the skid (8) side panel recirculates culture through HFTFF or an ATF system with its appropriate filter cartridge and vacuum diaphragm pump is fluidically connected to the bioreactor compartments, allowing for net removal of spent media filtrate while retaining cells in the respective compartment under closed, sterile conditions. The compartments need not be uniformly scaled; a high-density perfusion N-1 stage occupying only ten per cent of total working volume may be adequate to seed a large production compartment (4) if perfusion cell-retention technology is employed to reach cell densities exceeding seventy million cells per milliliter.
[120] For Example, as described in figure-9, the Seed compartment (3) includes HFTFF for cell concentration before seeding production stage reactor hence drastically reduce the seed stage floor requirement. In perfusion embodiments, disposable ATF cassettes may be attached and integrated into the seed compartments (3) via sterile weldless connectors. The cell-free permeate called "Filtrate" is directed to waste or nutrient recycling, while the cell-rich retentate returns to the compartment. This perfusion configuration enables achieving 50–80 million cells per mL in N-1 (3a) and/or N-2 (3b), thereby further reducing inoculation volume required for N production compartment (4) and shortening overall seed-train duration and footprint of the multistage bioreactor system.
[121] In a further embodiment, each compartment of the multistage bioreactor system, is equipped with an integrated, non-invasive or invasive cell density sensor, enabling real-time monitoring of viable cell concentration. These sensors may utilize capacitance-based technology, optical density measurements, or fluorescence detection methods to provide continuous, accurate assessment of cell growth without requiring physical sampling of the culture. The bioreactor system is operable in a fully automated, recipe-driven mode, wherein a programmable control unit continuously monitors the sensor output and triggers automated actuation of the inter-compartmental partition (5) when the seed culture in a given compartment reaches a pre-defined threshold, typically around 3 million cells per mL. The control system may incorporate predictive algorithms that analyze growth rate trends to anticipate when the threshold will be reached, enabling precise timing of partition (5) actuation and medium preparation in the receiving compartment.
[122] Upon reaching this set point, the corresponding partition (5) is selectively opened via magnetic, pneumatic or mechanical actuation, resulting in controlled and sterile transfer of inoculum into the next downstream compartment (either a larger seed stage or the final production compartment (4)), which has been pre-filled with fresh medium. The actuation mechanism may include redundant safety features to prevent premature or unintended opening, such as dual-confirmation signals from independent sensors and mechanical interlocks that verify proper alignment and readiness of both compartments before permitting transfer. The system may also incorporate flow sensors or visual confirmation systems to verify successful transfer between compartments.
[123] This operation achieves a targeted seeding density of approximately 0.3 million cells per mL in the receiving compartment, preserving the volumetric scaling ratio and process consistency. The control system automatically adjusts agitation speeds, gas flow rates, and temperature profiles in both the donor and recipient compartments to optimize mixing during the transfer process and to establish appropriate environmental conditions for the newly combined culture volume. The entire cascade comprising inoculation, growth, and transfer across all stages is thus executed in a fully automated, closed-loop manner, with no manual intervention, greatly enhancing sterility assurance, operational efficiency, and reproducibility in large-scale cell culture bioprocessing.
[124] The automated monitoring and transfer system may further incorporate adaptive feedback control, wherein the timing and conditions of each transfer are optimized based on the specific growth characteristics observed in each batch. This capability enables the system to compensate for batch-to-batch variability in cell line performance, media quality, or environmental conditions, ensuring consistent outcomes across production campaigns. Additionally, the system maintains comprehensive electronic records of all sensor readings, transfer events, and adjusting process parameters like agitation speed, gas flow rate, and temperature, facilitating process validation, troubleshooting, and regulatory compliance in GMP manufacturing environments.
[125] Yet in another embodiment of the invention as illustrated in Figure 10, while prior examples refer to multistage bioreactor systems comprising compartment volumes ranging from 20 L to 2000 L, the same architecture may be scaled down to benchtop configurations for laboratory and process development use. In such embodiments, the bioreactor vessel comprises successive compartments of approximately 20 mL (N-2 (3b)), 200 mL (N-1 (3a)), and 2 L (N (4)), proportionally mirroring the volumetric ratios and seeding densities of the commercial-scale system. These compact multistage units serve as scale-down models, capable of emulating the hydrodynamic, mass-transfer, and shear characteristics encountered in large-scale operations, thus enabling predictive process development and optimization.
[126] Furthermore, these compact, integrated multistage bioreactor systems can be effectively utilized for controlled cell expansion during master and working cell bank preparation, wherein early-stage cultures are amplified under closed-system, aseptic conditions to ensure traceability, genetic stability, and contamination-free processing. When configured with an integrated perfusion module, these benchtop bioreactors enable the generation of ultra-high cell density cultures, thereby enhancing the efficiency and scalability of cell banking workflows. This facilitates the production of consistent, high-quality seed stocks that can be cryo-preserved and subsequently used for robust and reproducible inoculation of large-scale upstream bioprocesses, ensuring seamless transition from development to commercial manufacturing.
[127] FIG. 11 illustrates a multistage bioreactor system configured for closed-loop seed development from extremely small volumes to large-scale production volumes. The system comprises a series of interconnected multistage bioreactor vessels of increasing size, enabling seamless scale-up of cell cultures without external transfers.
[128] The system integrates small-volume multistage bioreactor containers (2) with larger-scale multistage bioreactor vessels (2) through fluidic connections (closed, sterile transfer line). For example, a small-volume multistage bioreactor (2) can be fluidically attached to one or more large-scale multistage bioreactor vessels with production volumes of 2000 L or 5000 L. This integration enables the establishment of a closed-loop seed development process from the smallest seed volumes to production-scale operations.
[129] For instance, cells initially cultured in a 20 mL compartment of a small-volume multistage bioreactor can be expanded to 2 L, then transferred to intermediate volumes before ultimately inoculating a 2000 L or 5000 L production compartment (4) in a large-scale multistage bioreactor under sterile condition. This arrangement allows for the cultivation of cells from initial cell count of 0.25 million viable cells per ml to reach peak cell density of 2.5 million viable cells per ml while maintaining consistent cell density parameter across all stages of the process.
[130] The connection between multiple multistage bioreactors is achieved through sterile transfer lines that maintain the closed nature of the system. This configuration enables the inoculation and initiation of large-scale production in multistage bioreactors or other stirred tank bioreactor vessels directly from small-scale seed cultures, eliminating the need for intermediate expansion steps in separate vessels.
[131] The closed-loop configuration supports the cultivation of various cell types, including mammalian cells, insect cells, plant cells, or microbial cells, and can be operated in batch, fed-batch, or perfusion modes. This provides flexibility for different bioprocessing strategies while maintaining the integrity of the closed system from the smallest seed volumes to production scale.
[132] By connecting a series of multistage bioreactors of different scales, the system enables a continuous, aseptic transfer of cell cultures from initial micro scale seed volumes (20 mL, 50 mL) to final production volumes (2000 L, 5000 L). This configuration minimizes contamination risks associated with open transfers and supports efficient scale-up of bioprocesses while reducing the overall footprint and complexity of the seed train process.
[133] Now, in some embodiments, no partitions (5) are required due to the configuration benefits and no valve or latch to be installed to open the partitions (5). Such embodiment is shown in Fig. 12 wherein the multistage bioreactor system according to present invention implements a bottom-to-top compartmental arrangement within a single container (2) structure. In said embodiment, the container (2) includes a N-production compartment (4) positioned at the top of the arrangement. Below the N-production compartment (4), the N-1 seed compartment (3a) is formed bellow the bottom level of the production compartment (4). At the bottom of the arrangement, the N-2 seed compartment (3b) is formed below the bottom level of the N-1 seed compartment (3a). This configuration allows for a sequential scale-up process within a single vessel.
[134] The N-production compartment (4) has a larger volume than the N-1 seed compartment (3a) and the N-2 seed compartment (3b). The N-1 seed compartment (3a) has a larger volume than the N-2 seed compartment (3b). These volume differences facilitate the progressive expansion of cell culture from smaller to larger scales.
[135] Each compartment of the multistage bioreactor (2) may be equipped with their individual agitation system, aeration system and process sensors. Process control is performed through a centralized programmable logic controller that hosts individual parameter loops for each compartment. Dissolved-oxygen set-points may be regulated through a cascade control that manipulates aeration-gas flow, agitation speed, or overlay gas composition. pH is modulated via sparged carbon dioxide or base addition through peristaltic pumps connected to sterile single-use delivery lines. Temperature set-points for seed stages may deviate by one or two degrees Celsius from production stage set-points to accelerate or temper growth kinetics; the system thus supports differential thermal control through compartment-specific jacket zones or through circulated heat-transfer fluid loops regulated by electrically actuated control valves.

[136] The lower bottom cavity forms the smallest seed compartment (3b) with 20L volume contained in the compartment and having a magnetically coupled impeller (6) to agitate the culture. Now in operation, the N-2 seed compartment (3b) may be inoculated with cells at a predetermined cell density parameter. The cells are cultivated in the N-2 seed compartment (3b) until a target cell density is reached. Upon reaching a target cell density of around 3.0 million cells per mL in smallest seed compartment (3b), another 180L media is dispensed in the N-1 seed compartment (3a) where the fresh media mixes with the seed culture of N-2 compartment (3b) immediately upon entering the compartment. This brings the total N-1 seed compartment (3a) culture volume to 200L, inoculated with around 0.3 million cells per mL. When the N-1 stage has matured to approximately 3.0 million cells per mL, a similar operation is performed to fill 1800L fresh media in production compartment (4) and the production compartment (4) is inoculated with 2000 liters of production culture seeded at approximately 0.3 million cells per mL, all without any external fluid transfer.
[137] The bottom-to-top configuration of the multistage bioreactor system enables a streamlined seed-train process, allowing for the cultivation of cells from an initial cell count to a maximum production volume within a single, integrated vessel. This arrangement supports the scaling of culture volume from smaller volumes in the seed stages to larger volumes in the production stage, facilitating efficient bioprocess development and production.
[138] According to another embodiment, FIG. 13 illustrates a cluster architecture of the multistage bioreactor system. The cluster architecture arranges seed compartments (3) e.g. N-1 (3a), N-2 (3b) and N-3 (3c) radially around a central production compartment (4). An intermediate seed compartment N-1 (3a) and an initial seed compartment N-2 (3b) are positioned in a radial configuration surrounding the central production compartment (4). This radial arrangement of seed compartments (3) shortens internal conduit lengths between compartments compared to linear configurations.
[139] The cluster architecture confers an appreciably compact footprint for the multistage bioreactor system. The reduced footprint provides an advantage in retrofit scenarios where plant floor area may be constrained. The radial configuration enables efficient transfer of culture between seed compartments (3) and the production compartment (4). Selectively openable partitions (5) between adjacent compartments allow for controlled inoculation from seed stages to the production stage without external transfers. Each seed compartment (3) and the production compartment (4) include independent agitation mechanisms, aeration systems, and sensors. This allows for compartment-specific control of critical process parameters such as the cell density parameter, mixing, and gas transfer while maintaining a unified vessel structure. The cluster architecture supports scaling from the initial cell count through intermediate culture volumes in the seed compartments (3) to the maximum production volume in the central compartment. The system enables progressive expansion of culture volume from smaller volumes in the initial seed compartment n-2 (3b) to larger volumes in the intermediate seed compartment n-1 (3a) and ultimately to the production volume in the central compartment (4).
[140] In-situ seed-train compression using the multistage bioreactor system according to present invention provides a number of tangible advantages when assessed against conventional seed-train methodology. By confining multiple expansion stages in a single container (2), the number of seed bioreactor systems, CIP and SIP cycles, and validation documentation related to separate vessels is curtailed. Case study modelling at a two-thousand-litre final production volume predicts a reduction in clean-room footprint of approximately 35% versus a comparable facility employing discrete N-2 and N-1 reactors. Turnaround time between batches, defined as the interval from bioreactor harvest to re-inoculation, is projected to fall by as much as 66% because the container (2) remains stationary on its skid (8) and requires no disassembly or transport between process suites. Contamination risk, measured as historical batch failure frequency, declines because the elimination of tubing disconnections removes common ingress points for adventitious agents. Capital expenditure is likewise favorably impacted; one skid-mounted centralized controller suffices for multiple compartments, whereas traditional seed trains often employ a separate control cabinet per vessel. Consumable cost benefits accrue as well: because partitions (5) are internal to a single bag, the number of disposable tubing sets, sterile connectors, and single-use sensor kits are strongly reduced.
[141] It follows from the foregoing that the multistage bioreactor system furnishes a coherent, modular, and scalable means for executing seed-train compression while delivering robust process control and maintaining sterility throughout the entire expansion cascade. By enabling inoculation transfers to occur internally through validated partition-opening events, it obviates numerous manipulation points that historically contribute to batch failure. Reductions in clean-room real estate and controller capital, coupled with rapid campaign changeover enabled by disposability, translate directly into lower cost of goods and accelerated facility depreciation schedules. The invention therefore satisfies longstanding industrial objectives of flexibility, contamination risk mitigation, and economic efficiency, all while conforming to demanding regulatory guidance concerning single-use technology, closed processing, and real-time process monitoring.
[142] Features of any of the examples or embodiments outlined above may be combined to create additional examples or embodiments without losing the intended effect. It should be understood that the description of an embodiment or example provided above is by way of example only, and various modifications could be made by one skilled in the art. Furthermore, one skilled in the art will recognize that numerous further modifications and combinations of various aspects are possible. Accordingly, the described aspects are intended to encompass all such alterations, modifications, and variations that fall within the scope of the appended claims.
[143] The dimensions and values disclosed herein are not to be understood as being strictly limited to the exact numerical values recited. Instead, unless otherwise specified, each such dimension is intended to mean both the recited value and a functionally equivalent range surrounding that value. For example, a dimension disclosed as "40 wt.%" is intended to mean "about 40 wt.%".
[144] A number of implementations have been described. Nevertheless, it will be understood that various modifications may be made without departing from the spirit and scope of the disclosure. Accordingly, other implementations are within the scope of the following claims.
[145] Features of any of the examples or embodiments outlined above may be combined to create additional examples or embodiments without losing the intended effect. It should be understood that the description of an embodiment or example provided above is by way of example only, and various modifications could be made by one skilled in the art. Furthermore, one skilled in the art will recognize that numerous further modifications and combinations of various aspects are possible. Accordingly, the described aspects are intended to encompass all such alterations, modifications, and variations that fall within the scope of the appended claims.
[146] 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
[147] The following example is provided solely to illustrate certain embodiments of the invention and is not intended to limit the scope of the claims.
[148] Example 1: Comparative evaluation of multistage bioreactor versus conventional stirred-tank single-use bioreactors for CHO cell expansion and production
[149] Purpose and Study Design: The purpose of this study was to evaluate the cell culture performance of the multistage bioreactor vessel relative to conventional single-use stirred-tank bioreactors operated in a standard seed-train configuration. The key hypothesis was that the multistage bioreactor vessel, which integrates N-2, N-1, and production stages within a single disposable container and enables in-situ inoculation via partition opening, would demonstrate superior or at-par process performance in terms of viable cell density (VCD), culture viability, metabolic efficiency, and recombinant protein productivity. Identical CHO cell banks, chemically defined media, and process operating parameters were used across both systems to ensure comparability.
[150] Materials and Methods: The multistage bioreactor vessel was configured with three fluidically isolated compartments corresponding to N-2 (2 L), N-1 (20 L), and N (200 L) stages, arranged horizontally within a closed-loop single-use bag system with magnetic impeller agitation and integrated spargers. The comparator process employed three independent cylindrical SUBs at matching scales. Both platforms were inoculated from a common working cell bank of CHO suspension cells cultivated in a chemically defined, protein-free basal medium. All reactors were operated under identical environmental conditions: temperature 37°C, pH 7.2 ± 0.2, and dissolved oxygen (DO) at 40%.
[151] In the multistage bioreactor system, transfers from the N-2 to N-1 and from N-1 to N compartments were carried out via selective opening of internal partitions, allowing seamless inoculum cascading without external tubing or operator intervention. In the STR setup, inoculation steps were executed using sterile tubing and manual aseptic connections. Daily samples were collected from all compartments for analysis of VCD, cell viability, residual glucose, lactate, and monoclonal antibody titer.
[152] Results: In the seed stages, the multistage bioreactor system showed at par performance with STR SUBs in terms of VCD. By Day 3, the N-2 compartment (2 L) in multistage bioreactor reached 5.16 ± 0.21 million cells per mL, compared to 5.19 ± 0.17 million cells per mL in the corresponding STR reactor. In the N-1 (20 L) compartment, VCD reached 4.39 ± 0.26 million cells per mL in multistage bioreactor versus 4.20 ± 0.14 million cells per mL in the STR, indicating equivalent exponential-phase growth.
[153] At the 200 L production scale, the multistage bioreactor culture achieved a peak VCD of 14.54 million cells per mL on Day 7, compared to 13.66 million cells per mL in the STR, representing a 6% improvement. The VCD in multistage bioreactor remained higher throughout the late exponential and stationary phases, suggesting prolonged metabolic competence and growth sustainability.
[154] Viability profiles likewise favored the integrated system. By Day 14, the multistage bioreactor production culture maintained 82% viability, compared to 77% in the STR control. Viability decline post-Day 10 was more gradual in multistage bioreactor, indicating a less stressful environment and better support for late-phase cellular health.
[155] Recombinant protein expression also showed a significant advantage in multistage bioreactor. The final monoclonal antibody titer on Day 14 was 1104mg/L versus 994 mg/L in the STR. Productivity rates diverged markedly after Day 10, with the multistage bioreactor vessel sustaining expression momentum for a longer duration.
Parameter Multistage bioreactor 2L STR 2L Multistage bioreactor 20L STR 20L Multistage bioreactor 200L STR 200L
Peak Cell Density (million cells per mL) 5.16 5.19 4.39 4.20 14.54 13.66
% Viability at Transfer / Harvest 98.4% 97.6% 98.11% 99.27% 82.2% 77.4%
Peak Lactate conc. (g/L) 0.35 0.27 0.29 0.41 2.43 2.77
Titer (mg/L) NA NA NA NA 1104 994

[156] Metabolic Profile: Residual glucose concentrations in both systems was maintained consistently above 1 g/L throughout the run, with fewer glucose spikes or dips compared to STRs. Lactate accumulation in the multistage bioreactor production stage showed equivalent concentrations, indicating efficient oxygen transfer, more stable pH control, and reduced metabolic stress as observed with STR.
[157] Discussion: The observed at par performance and improvements in VCD, viability, and protein yield in the multistage bioreactor system are attributed to several critical design advantages: (1) Closed, in-situ inoculation that avoids hold times, transfer lines, and shear shocks; (2) Compartment-specific agitation and aeration, which maintain optimal mixing and oxygenation at each scale without compromising shear; and (3) Integrated seed-train design, which reduces process fragmentation and operator error.
[158] The data confirm that physical consolidation of N-2, N-1, and N stages into a single vessel does not compromise process performance, on the contrary, it enhances scalability, sterility, and process economy. The elimination of sterile transfers and vessel changeovers simplifies validation, reduces labour, and minimizes contamination risk.
[159] Conclusion: This example provides quantitative evidence that the multistage bioreactor system improves upstream bioprocess outcomes compared to conventional STR-based seed-train setups. The system supports accelerated seed expansion, higher viable cell densities, improved late-stage viability, enhanced metabolic efficiency, and increased product titer. These findings validate the multistage bioreactor platform as a robust, closed, and scalable alternative to traditional modular bioreactor chains, with the potential to compress the upstream timeline, reduce cost, and improve bio-production efficiency in GMP environments.
[160] Example 2: End-to-End CHO cell expansion using two multistage bioreactor systems from vial revival to 2000?L production
[161] Purpose and Study Design: This example describes a closed, fully automated upstream bioprocess utilizing two integrated multistage bioreactor systems for the complete seed-train expansion and production of CHO suspension cells. The objective was to demonstrate full traceability, sterility maintenance, scalability, and production consistency in a system configured for end-to-end bioprocessing from thawing a cryovial to 2000?L final culture volume.
[162] Materials and Methods: The upstream process was divided across two multistage bioreactor vessels. The first system was configured with 0.02?L, 0.2?L, and 2?L compartments for vial revival and early-stage seed expansion (designated N-5 to N-3). The second multistage bioreactor vessel included 20?L (N-2), 200?L (N-1), and 2000?L (N) compartments for intermediate seed development and final production.
[163] The cell line used was a CHO suspension line from a master cell bank (MCB). A chemically defined, protein-free commercial medium was used at all stages without modification. Environmental control parameters were standardized: temperature 37?°C, pH 7.2?±?0.2 (regulated by CO2 sparging and bicarbonate buffering), and DO at 35% (maintained via air and oxygen sparging). All compartments had independently controlled agitation and aeration.
[164] Automated partition opening and transition steps were triggered by pre-set viable cell density (VCD) and culture time parameters. There were no manual fluid transfers or aseptic connections between seed stages or between the two multistage bioreactor systems. Sampling was performed daily for VCD, viability, glucose, lactate, and monoclonal antibody titer (measured every 48 hours during production by ELISA).
[165] Results: In the first multistage bioreactor vessel, all seed stages achieved consistent and reproducible VCDs. The 0.02?L, 0.2?L, and 2?L compartments each reached approximately 5?million cells per mL by Day -3. Overlap of growth curves across three batches demonstrated tight control and fidelity in micro-to-small scale transitions.
[166] The second multistage bioreactor vessel likewise showed consistent expansion across intermediate stages. The 20?L and 200?L compartments reached VCDs of approx. 5?million cells per mL within 3–4 days post-inoculation. The automated, sealed transition between the two multistage bioreactor systems preserved inoculum quality and timing, confirming the viability of a modular yet fully integrated end-to-end upstream system.
[167] In the 2000?L production compartment, peak VCD was reached by Day 7 at approximately 13.9?million cells per mL. This was followed by a gradual decline to 7.6?million cells per mL by Day 14. Cell viability remained above 95% until Day 8 and above 80% at harvest, indicating a low-shear, well-controlled environment within the flexible-film vessel.
[168] Metabolic Profile: Residual glucose levels dropped to ~1.3?g/L by Day 5 and were maintained thereafter through glucose bolus feeding. Glucose concentrations gradually increased after Day 10 as cell metabolism declined during the stationary phase. Lactate levels peaked at approximately 2.78?g/L on Day 6–7 and declined steadily, indicating a metabolic shift from glycolysis to oxidative metabolism.
[169] Discussion: The dual multistage bioreactor approach demonstrated successful end-to-end seed-train compression and continuity from a cryovial inoculation in 20?mL compartment to 2000?L production scale compartment. Across all seed stages and batches, consistent VCDs (~5?million cells per mL by Day 3) and high viability were achieved. The transition from one vessel to another occurred without open handling or delay, eliminating hold steps, intermediate equipment, and associated contamination risks.
[170] Conclusion: This experiment confirms that the multistage bioreactor platform enables a fully closed, modular, and scalable upstream bioprocess. From cryovial thaw to 2000?L harvest, it maintains sterility, consistency, and scalability across all stages.
[171] Example 3: Evaluation and performance of seed intensification strategy to inoculate 2000?L multistage bioreactor
[172] Purpose and Study Design: This example demonstrates the use of a seed-intensification strategy leveraging the multistage bioreactor platform to support time-efficient inoculation of a 2000?L production compartment of multistage bioreactor having the N-1 seed compartment of 50L and N-2 compartment of 1L, bypassing conventional intermediate stages such as 10?L, 20?L, or 200?L vessels. The primary objective was to validate whether high-density perfusion cultures in small-scale multistage bioreactor vessels could generate robust inoculum for large-scale production, thereby compressing the seed-train timeline and reducing operational complexity.
[173] Materials and Methods: The seed expansion workflow began with CHO suspension cells thawed from a qualified working cell bank and adapted in 500?mL shake flasks. Following initial expansion, the culture was transferred to a 1?L multistage bioreactor vessel equipped with a hollow-fiber tangential flow filtration (HF-TFF) module and operated under perfusion mode. The culture was intensified to =?21 million cells per mL over 5 days.
[174] The high-density inoculum was then transferred into a 50?L multistage bioreactor vessel (also configured with HF-TFF)and grown under equivalent perfusion conditions to a density of =?24?million cells per mL. Both seed stages were operated at 37?±?0.5?°C, pH 7.2?±?0.2 (regulated by CO2 and bicarbonate), and DO at 35% (via air and O2 sparging and agitation).
[175] The 2000?L production stage was pre-filled with chemically defined, serum-free medium and inoculated with approx. 0.5?million cells per mL directly from the 50?L seed vessel. The production run was conducted in batch mode with glucose-stat feeding (20% glucose) to maintain =1?g/L residual glucose. Samples were taken daily to monitor viable cell density (VCD), viability, glucose and lactate concentrations, and monoclonal antibody titer.
[176] Results: The 1?L perfusion seed reproducibly achieved VCDs of 21?million cells per mL within 5 days across all replicates. Cell viability at the end of this stage remained above 95%. The 50?L perfusion seed reached comparable high densities (24 million cells per mL) under the same 5-day culture period, with excellent viability maintained throughout. These values confirmed the suitability of both seed compartments for high-efficiency intensification, enabling elimination of intermediate scale-up vessels.
[177] In the 2000?L production stage, peak VCD of 12.6 million cells per mL and were attained by Days 6 in fed batch mode. A gradual decline followed during the stationary phase, with final cell densities of 7.1?million cells per mL by Day 14. Cell viability remained =?95% through Day 8, tapering to 81% by the end of the run, consistent with expected performance in fed-batch operations using high-inoculum strategies.
[178] Metabolic Profile: Glucose levels were stably maintained above 1?g/L throughout the production phase via periodic bolus feeding. Lactate concentrations peaked around Day 6–7 at ~2.4?g/L and subsequently declined, suggesting a metabolic shift from glycolytic to oxidative pathways typical of CHO cultures under oxygen-rich, well-mixed conditions.
[179] Discussion: The data demonstrate that high-density perfusion cultivation in small-volume multistage bioreactor compartments enables elimination of conventional intermediate seed stages, such as the 10?L and 200?L reactors often used between 1?L and 2000?L scales. The integration of HF-TFF modules within the multistage bioreactor system provided a reliable means of retaining cells and enriching culture density while maintaining cell health. The direct inoculation of a 2000?L production bioreactor from a 50?L intensified seed was accomplished in a closed, contamination-free workflow without line sterilization, reducing handling risk and operator burden.
[180] The production-stage growth curves and titer profiles confirm that inoculum generated through multistage bioreactor seed intensification performs equivalently toor better than standard seed-train derived cultures, while offering major time and equipment savings. Viability, VCD, and metabolic data were tightly controlled and consistent across all three runs, validating reproducibility of the platform and workflow.
[181] Conclusion: This example confirms the effectiveness of a seed-intensified multistage bioreactor platform for direct inoculation of large-scale production bioreactors. The process eliminated at least two conventional seed stages, reduced campaign duration by 4–5 days, and maintained excellent performance in cell growth, viability, and product expression.
[182] Example4: Process verification at 50L scale multistage bioreactor against standard process developed with conventional 5?L stirred-tank bioreactors
[183] Purpose and Study Design: This example presents a process verification study to evaluate the performance of a 50?L multistage bioreactor single-use bioreactor (SUB) configured with integrated N-2 (0.5?L), N-1 (5?L), and N (50?L) compartments. The objective was to assess upstream growth, viability, productivity, and metabolite control for a CHO-based monoclonal antibody (mAb) process and compare outcomes against conventional benchmarks: a 5?L stirred-tank reactor (STR) and 500?mL shake flasks. All systems followed identical media compositions, culture durations, and control parameters across three replicates.
[184] Materials and Methods: The test cell line was a stably expressing CHO-IgG clone cultivated in fed-batch mode using CD-CHO™ basal medium, EfficientFeed™ C (7.5% v/v), and glucose bolus additions (20% stock) to maintain residual glucose between 0.5–4?g/L. Temperature was held at 36.5?°C for the first four days and reduced to 32.5?°C from Day 5 onward. DO was maintained at 40% air saturation; pH was controlled at 7.1 ± 0.2 via CO2 overlay and NaHCO3 buffering.
[185] The multistage bioreactor workflow included: 1) Vial revival in 20ml using 125?mL shake flasks, 2) Transfer to 500?mL flasks (150?mL working volume), 3) Inoculation of the 0.5?L compartment at 0.4?million cells per mL, 4) Sequential expansion into 5?L and 50?L compartments. Comparator runs were conducted using a 5?L jacketed glass STR and 500?mL baffled shake flasks, applying scaled feed volumes and identical control settings.
[186] Results: The multistage bioreactor 50?L system achieved a mean peak VCD of 17.8 ± 0.4?million cells per mL, comparable to the 5?L STR (19.4?million cells per mL) and significantly higher than the shake flasks (13.5 million cells per mL). Exponential growth was maintained through Day 8–9 in all multistage bioreactor replicates. Cell viability remained above 94% through Day 9 and concluded at 81 ± 4% on Day 14, matching the STR benchmark (83%) and surpassing the shake flask control (72%).Average harvest titer in the multistage bioreactor was 1823 ± 78?mg/L, corresponding to 95% of the STR titer (1910?mg/L) and exceeding shake flask yields (~1070?mg/L) by =70%. Titer increased from 730?mg/L on Day 8 to peak values by Day 14 across all replicates. Glucose levels were tightly controlled across all platforms. The multistage bioreactor maintained residual glucose at 1.21 ± 0.32?g/L, consistent with STR performance (1.06?g/L), and superior to the shake flask minimum (0.18?g/L).Lactate accumulation peaked at 1.79 ± 0.09?g/L in the multistage bioreactor, similar to STR (1.71?g/L) and lower than shake flask cultures (1.42?g/L), indicating efficient pH control and oxidative metabolism in the scaled bioreactor. No pH excursions outside the 7.1 ± 0.2 range were recorded in any multistage bioreactor runs. DO levels were stably maintained via a ramped agitation strategy ranging from 58 rpm to 119 rpm (tip speed =1.0?m/s).
Parameter Shake-flask (max) 5 L STR (max) Multistage bioreactor 50 L (mean of 3 runs)
Peak VCD (million cells per mL) 13.5 (Day 7) 19.4 (Day 9) 17.8 ± 0.4 (Day 7–8)
Viability at Day 14 (%) 72 83 81 ± 4
Harvest titer (mg/L) 1 070 1 910 1 823 ± 78
Residual Glucose (min) (g/L) 0.18 1.06 1.21 ± 0.32
Peak Lactate (g/L) 1.42 1.71 1.79 ± 0.29

[187] Discussion& Conclusion: The multistage bioreactor 50?L system demonstrated performance parity with conventional 5?L STRs in terms of VCD, viability, and mAb productivity. This process verification confirms that the 50?L multistage bioreactor integrated bioreactor supports cGMP-compliant upstream operations and can effectively replace traditional STR-based seed trains. The system achieved comparable cell growth, viability, and antibody production in a closed, space-efficient format.
[188] Example 5: Evaluation of 200L multistage bioreactor Performance for Growth of CHO and HEK293 Suspension Cell Lines
[189] Purpose and Study Design: This example investigates the capability and versatility of a 200?L multistage bioreactor system for supporting high-density fed-batch cultivation of two different mammalian suspension cell lines: CHO and HEK293. The study aimed to verify the system’s compatibility across distinct cellular phenotypes and to assess growth kinetics and viability consistency across three independent batches per cell line.
[190] Materials and Methods: The multistage bioreactor system consisted of integrated 2?L (N-2), 20?L (N-1), and 200?L (N) compartments arranged in a top-to-bottom configuration. Both CHO and HEK293 cell lines were thawed from working cell banks and initially expanded in 250?mL shake flasks using chemically defined, protein-free media.
[191] Cells were sequentially scaled into the 2?L and 20?L compartments before transfer into the 200?L production stage via selectively openable internal partitions, enabling closed, in-situ inoculation. Three production-scale batches were conducted for each cell line under the same environmental and feeding conditions of A) Temperature: 36.5?°C (reduced to 32.5?°C on Day 5), B) pH: 7.1 ± 0.2 (controlled via CO2 overlay and NaHCO3), C) %DO: 35% of air saturation (regulated by variable-speed agitation and O2 sparging), D) Feeding: Bolus addition of glucose (to maintain =1?g/L) and commercially available Feed additive.
[192] Daily sampling was performed to measure VCC (million cells per mL) and cell viability (%) using trypan blue exclusion on an automated cell counter.
[193] Results: Across three batches, the CHO cultures demonstrated consistent growth kinetics, reaching peak VCCs of: 13.6?million cells per mL on Day 7, 14.2?million cells per mL on Day 8, 13.9?million cells per mL on Day 7 respectively in Batch 1,2 and 3. Viability remained above 95% through Day 7 and concluded at 80–84% by Day 14.
[194] HEK293 cell culture also showed robust growth, with peak VCCs of7.4?million cells per mL on Day 6, 8.3?million cells per mL on Day 7, 7.9?million cells per mL on Day 7 respectively in Batch 1,2 and 3. Viability was maintained above 94% through Day 6–7 and ended between 78–82% at Day 14.
[195] Discussion& Conclusion: The multistage bioreactor 200?L system consistently supported high-density cultivation of both CHO and HEK293 suspension cell lines. Growth kinetics and viability profiles were highly reproducible across batches, affirming the platform’s suitability for multi-product facilities. The consistent peak VCCs (~13–14?million cells per mL) and viability (=80% at harvest) demonstrate that the system can meet industry benchmarks for late-stage upstream processes. The use of integrated compartments with independent agitation and aeration maintained optimal shear conditions and oxygen transfer rates tailored to each cell line. The closed, partition-based inoculation between stages minimized handling, eliminated transfer steps, and maintained sterility throughout the process.
[196] Example 6: Transient transfection of HEK293 cells to produce monoclonal antibody in a 50?L multistage bioreactor.
[197] Purpose and Study Design: This example demonstrates the feasibility of using multistage bioreactor system for the transient expression of monoclonal antibodies (mAbs) using HEK293 suspension cells and PEI/DNA mediated transient transfection protocol. The objective was to assess the system’s suitability for high-efficiency gene delivery, cell viability maintenance, and transient recombinant protein expression under scalable fed-batch conditions.
[198] Materials and Methods: Suspension-adapted HEK293F cells were cultured in SFM supplemented with 4?mM L-glutamine. Initial expansion was performed in 125?mL and 500?mL shake flasks before inoculating the 0.5?L seed compartment of the multistage bioreactor system. Cells were expanded sequentially through 5?L (N-1) and 50?L (N) compartments by opening the selectively openable partitions between compartments at a target density of ~2.5?million cells per mL. Transient transfection was carried out in the 50?L production compartment using PEI (25 kDa) complexed with a dual plasmid DNA system encoding a heavy and light chain of a human IgG1 monoclonal antibody. The PEI/DNA complex was prepared at a 3:1 mass ratio (PEI:DNA), with a final DNA concentration of 1?µg/mL and added to the bioreactor at a VCD of 3.0?million cells per mL. Transfection enhancing additives (e.g., sodium butyrate, valproic acid) were tested in parallel runs. The culture was maintained for 7 days post-transfection under the conditions: 1) Temperature: 36.5?°C, 2) pH: 7.0 ± 0.2, 3) DO: 40%, 4) Agitation: 80–120 rpm (variable, maintaining tip speed <1.0?m/s), 5) Feeding: bolus addition of glucose and chemically defined feed on Days 3 and 5. Samples were taken daily for VCD, viability, glucose, lactate, and antibody titer (measured by Protein A HPLC).
[199] Results: Transfection was successful in the production run. Peak VCD observed on Day 4 at approx. 5.5?million cells per mL. Viability remained above 85% for the first 5 days and declined gradually to 76–78% by Day 7. The mAb titer in control (no enhancer) was 250 ± 18?mg/L, with sodium butyrate (2 mM) it was 345 ± 22?mg/L and with valproic acid (2.5 mM) it was327 ± 19?mg/L. Glucose levels declined to ~1?g/L by Day 5 and were maintained by daily bolus additions. Lactate peaked at ~1.9?g/L and decreased thereafter, consistent with a post-transfection metabolic shift.
[200] Discussion: The results confirm that the multistage bioreactor 50?L system effectively supports large-scale transient transfection workflows, combining upstream seed expansion, PEI/DNA-mediated gene delivery, and protein expression in one closed, single-use platform. The selective partitioning feature eliminated the need for separate transfection vessels, reducing equipment usage and contamination risk.
[201] Example 7: Laboratory to commercial scale transfer using geometrically similar multistage bioreactor vessels
[202] Purpose and Study Design: This example evaluates the scalability and reproducibility of upstream CHO cell culture performance across geometrically scaled multistage bioreactor vessels, from 2?L laboratory scale to 2000?L commercial production scale. The objective was to demonstrate that consistent cell growth kinetics, viability, and productivity can be achieved by maintaining geometric similarity, agitation ratios, and gas transfer parameters across all vessel volumes. The study was conducted across five scales: 2?L, 20?L, 50?L, 200?L, and 2000?L, each using a multistage bioreactor vessel of proportionally matched design.
[203] Materials and Methods: All vessels used the same single-use multistage bioreactor configuration, with a cylindrical-conical geometry, dedicated impellers and spargers per compartment, and independent environmental controls. Aspect ratios, impeller-to-tank diameter ratios, and working volume-to-surface area ratios were appropriately adjusted across scales. A CHO cell line expressing a recombinant IgG1 antibody was cultivated using identical chemically defined media, feeds, and control parameters. Each culture was seeded at 0.3 million cells per mL and operated under the following conditions: 1) Temperature: 36.5?°C, 2) pH: 7.1 ± 0.2, 3) DO: 40% air saturation, 4) Agitation: scale-adjusted RPM to maintain tip speed ~0.6–0.9?m/s, 5) Feeding: bolus additions on Days 3, 5, and 7. Each scale was operated in fed-batch mode for 14 days. Daily sampling included viable cell concentration (VCC), viability (%), glucose/lactate, and final antibody titer.
[204] Results: Growth Performance: Peak VCCs were consistent across scales, with variation within ±8%.
Scale Peak VCC
(million cells per mL) Harvest Viability (%) Titres (mg/L)
2?L 14.2 83 1050
20?L 14.5 81 994
50?L 14.0 84 1186
200?L 13.8 82 1130
2000?L 14.1 80 1079

[205] Final IgG titres ranged from 994 to 1186?mg/L across all scales. The highest titer (1186?mg/L) was observed at the 200?L scale. No statistically significant difference was observed between different volume multistage bioreactors with respect to batch productivity. Mixing time (to <95% tracer homogenization) ranged from 25–70 seconds depending on volume, with <15% deviation across scales. The kLa values ranged from 12–26 h?¹, with uniform oxygen transfer maintained through sparger scaling and impeller speed adjustments.All cultures exhibited similar glucose and lactate trends, with glucose levels maintained above 1?g/L and lactate peaking around Day 6 (1.8–2.9?g/L). No pH or DO instability was observed at larger scales, affirming process control robustness.
[206] Discussion& Conclusion: The results confirm that geometric similarity, mixing profile, maintained tip speed, and proportional aeration/agitation strategies enable predictable process performance across 2?L to 2000?L multistage bioreactor scales. This validates the platform’s design for scale-down modelling, process development, and commercial production within a single geometry family. Reproducibility of growth and titer demonstrates that process validation data from development scale (e.g., 2L or 20L) runs can be reliably extrapolated to 2000?L manufacturing without the need for re-optimization. This facilitates technology transfer, accelerates regulatory approval, and reduces validation burden.
[207] Example 8: Expansion of CAR-T cells in a 20L multistage bioreactor system
[208] Purpose and Study Design: This example evaluates the use of a 20L multistage single-use bioreactor for the closed, stepwise expansion of genetically modified CAR-T cells from initial transduction to therapeutic dose scale. The objective was to demonstrate the system’s ability to support efficient, seed-train compression and T-cell proliferation within a closed, sterile environment, while maintaining cell phenotype, viability, and functional activity.
[209] Materials and Methods: The multistage bioreactor was configured with four serially connected compartments 1) N-3: 250?mL compartment, 2) N-2: 1?L compartment, 3) N-1: 5?L compartment, 4) N: 20?L production compartment. Each compartment was equipped with its own agitation and gas delivery system, enabling independent control of shear, mixing, and oxygenation. The vessel was constructed from USP Class VI polymer film and pre-sterilized via gamma irradiation. All media exchanges, transfers, and sampling were performed within the closed system.
[210] Process Overview: Peripheral blood mononuclear cells (PBMCs) from healthy donors were isolated via Ficoll gradient separation. CD3? T cells were activated in the 250?mL compartment (N-3) using anti-CD3/CD28 magnetic beads and expanded for 48 hours in proprietary T-cell expansion medium. Cells were transduced with a lentiviral vector encoding a third-generation anti-CD19 CAR construct at an appropriate MOI. Upon reaching a viable cell density of ~2.5million cells per mL, the contents of N-3 were transferred to N-2 by opening the selectively openable partition. This process continued sequentially into N-1 and N compartments as cell numbers expanded. Environmental conditions were maintained as 1) Temperature: 37?°C, 2) pH: 7.1 ± 0.2 (via bicarbonate buffering and CO2 overlay), 3) DO: 50% air saturation, 4) Agitation: low shear, variable-speed impeller at 25–55 rpm, 5) Media exchange: partial volume replaced on Days 4, 6, and 8.The final culture was harvested on Day 10 and analyzed for cell yield, viability, transduction efficiency, memory phenotype, and cytokine profile.
[211] Results: Across batches, the system supported consistent expansion from total ~8million input cells to >55billion total CAR-T cells within 10 days. Viability remained above 90% at all stages; final harvest viability was 85%. Transduction Efficiency measured by CAR surface expression (FACS) was 62–68% at Day 10. Following antigen stimulation (CD19? target), cells secreted high levels of IFN-?, IL-2, and TNF-a, consistent with potent effectors function.
[212] Discussion: This example demonstrates that the multistage bioreactor 20?L multistage system can consolidate the CAR-T seed train and expansion into a single closed device, eliminating the need for multiple bags, flasks, and manual transfers. Each compartment supported robust T-cell proliferation under gentle, tuneable conditions. The ability to cascade compartments without exposing the culture reduces contamination risk and operator burden.
[213] Moreover, the stage-specific control of agitation and oxygenation enabled gentle mixing and high viability, while maintaining effective gene delivery and functional output. The phenotype distribution and cytokine responses were consistent with clinical-grade CAR-T products.
[214] Conclusion: The 20?L multistage bioreactor system with four integrated compartments enabled the complete closed-loop expansion of CAR-T cells from initial activation through harvest, delivering high yields of functional, viable, transduced T cells in a GMP-aligned format. The system provides a scalable, contamination-resistant solution for personalized immunotherapy manufacturing and can be deployed for autologous or allogeneic workflows.
[215] Example 9: Comparative evaluation of CHO cell expansion in two 50L multistage bioreactors using rigid elephant-ear impeller versus flexible-film impeller designs
[216] Purpose and Study Design: This example compares the hydrodynamic performance and biological outcomes of two differently configured 50?L multistage bioreactor systems used for CHO cell expansion. One system (Bioreactor-A) employed a rigid, bottom-mounted elephant-ear impeller, while the second system (Bioreactor-B) used a flexible-film impeller design with three radially oriented film sleeves mounted on a central hub. The study evaluated viable cell density (VCD), viability and mixing performance over a 14-day fed-batch run.
[217] Materials and Methods: Each 50?L multistage bioreactor system contained three compartments configured as: N-2 (0.5?L), N-1 (5?L), N (50?L). A CHO cell line expressing a monoclonal IgG1 was used across all runs. Cells were seeded at 0.4?million cells per mL in the N-2 compartment and expanded into N-1 and N stages by in-situ partition opening. Both vessels used identical chemically defined medium and feeding strategies. The Bioreactor-A used a rigid elephant-ear impeller (4-blade, 45° pitch) mounted 100?mm from the base. Bioreactor-B used a flexible-film impeller with three heat-welded, radially oriented film sleeves, actuated by a magnetic bottom drive. The control parameters are 1) Temperature: 36.5?°C (shift to 32.5?°C on Day 5), 2) pH: 7.1 ± 0.2, 3) DO: 40% air saturation, 4) Agitation: variable speed, tip speed =0.8?m/s, 5) Feed: Efficient Feed™ on Days 3, 5, and 7.
[218] Results: Both systems supported robust CHO cell expansion. Peak VCDs, viability and productivity in the 50?L production compartment were:
Bioreactor type VCC Viability IgG titres Mixing time
Bioreactor-A (elephant-ear): 14.1 million cells per mL 82% by Day 14 1265 mg/L 35 ± 2 seconds
Bioreactor-B (film-sleeve): 13.8?million cells per mL 85% by Day 14 1158 mg/L 44 ± 3 seconds
[219] Discussion& Conclusion: Both impeller configurations supported effective CHO cell expansion and monoclonal antibody production in a multistage format. The elephant-ear impeller offered slightly faster mixing and higher oxygen transfer, due to rigid blade geometry and more pronounced axial flow. However, the flexible-film impeller system demonstrated comparable biological outcomes, with marginally higher late-stage viability and simpler fabrication. The flexible design also reduced dead zones at lower working volumes and exhibited quieter, low-shear operation, making it advantageous for shear-sensitive cell types.
[220] Reference numerals
Conventional stand alone bioreactors (1)
Multi stage bioreactor vessel (Container) (2)
Seed Compartment (3)
Initial seed compartment (3’)
Intermediate seed compartment (3”)
N-1 seed compartment (3a)
N-2 seed compartment (3b)
N-3 seed compartment (3c)
Production compartment (4)
selectively openable partition (5)
impeller (6)
sparger (7)
skid-like supporting structure (8)
,CLAIMS:We claim,
1. A multistage bioreactor system, comprising:
a container (2) configured for cultivation of a biological material;
wherein said container (2) is internally subdivided to form two or more compartments (3, 4) including an initial compartment (3’) and a final compartment (4); each said compartment (3, 4) define an internal volume filled with a culture media to cultivate cells therein;
wherein each compartment (3, 4) is equipped with agitation means, aeration means, process-sensing means, and fluid conduits that permit independent control of mixing, mass transfer and environment.
2. The multistage bioreactor system of claim 1, wherein the each compartment (3) is unified with the adjacent compartment when a predetermined cell density is reached in the compartment.
3. The multistage bioreactor system of claim 1, wherein the container comprises at least one intermediate compartment (3”) adjacently located between the initial compartment (3') and the final compartment (4) enabling sequential in-situ expansion.
4. The multistage bioreactor system of claim 1, wherein the final compartment (4) possesses the internal volume greater than the initial and intermediate compartments so as to maintain a volumetric ratio of 1: 2–1: 20 between successive stages.
5. The multistage bioreactor system as claimed in claim 1, wherein a selectively openable partition (5) is disposed between adjacent compartments, the partition hermetically sealing the compartments in a closed state and, upon actuation, establishing sterile fluid communication for in-situ transfer of culture medium and/or cells.
6. The multistage bioreactor system as claimed in claim 5, wherein the selectively openable partition (5) is configured to be actuated by a mechanism selected from magnetic latch, pneumatic bladder, mechanical linkage, hydraulic drive or frangible seal for transfer of the culture media from one compartment to the adjacent compartment.
7. The multistage bioreactor system as claimed in claim 5, wherein partition (5) is fabricated from a heat seamed multilayer film or a thin stainless-steel plate clamped against a gasketed ledge by a bayonet mechanism to permit reuse in stainless-steel vessels..
8. The multistage bioreactor system as claimed in claim 1, wherein the agitation means comprising impellers (6) chosen from pitched-blade turbine, elephant-ear impeller, hydrofoil blade or flexible-film impeller and is magnetically coupled to an external drive to maintain a closed envelope..
9. The multistage bioreactor system as claimed in claim 1, wherein the aeration means comprises stage-specific spargers (7) selected from sintered micro-porous rings, drilled-hole manifolds or micro-sparger rods.
10. The multistage bioreactor system as claimed in claim 1, wherein the process sensing means including pH sensors, dissolved oxygen sensors, temperature sensors and cell density sensors.
11. The multistage bioreactor system as claimed in claim 1, further comprising a programmable control system operatively connected to the sensing means and the selectively openable partitions, the programmable control system configured to monitor one or more parameters (temperature, pH, dissolved oxygen, viable-cell density) and to actuate the selectively openable partitions when a desired cell density threshold is met in the compartment.
12. The multistage bioreactor system as claimed in claim 1, wherein at least one compartment (3, 4) includes a perfusion loop with a cell-retention device selected from hollow-fiber tangential-flow filtration, alternating tangential-flow filtration, membrane-based filters or internal spin filters.
13. The multistage bioreactor system as claimed in claim 1, wherein the container (2) is fabricated from a flexible polymeric material having multiple layers including an inner contact layer, one or more intermediate gas-barrier layers, and an outer structural layer.
14. The multistage bioreactor system as claimed in claim 1, wherein the container (2) is fabricated from a rigid material selected from the group consisting of stainless steel, titanium alloys, and rigid thermoplastics.
15. The multistage bioreactor system as claimed in claim 1, wherein the compartments (3, 4) are arranged in a top-to-bottom, bottom-to-top or radial cluster configuration.
16. The multistage bioreactor system as claimed in claim 1, wherein the system is configured to operate in a mode selected from the group consisting of batch, fed-batch, and perfusion modes and supports cultivation of mammalian, insect, plant or microbial cells for production of recombinant proteins, monoclonal antibodies, viral vectors or vaccines.
17. The multistage bioreactor system as claimed in claim 1, wherein two or more multistage container (2) are fluidically connected in series so that an upstream container feeds culture media to the initial compartment (3’) of a downstream container without breaching sterility.
18. A method for cell cultivation by a multistage bioreactor system, comprising:
providing a multistage container (2) having an initial compartment (3’) and a final compartment (4), wherein the compartments are separated by selectively openable partitions (5);
charging the initial compartment (3’) with culture medium and inoculating with a starter cell suspension;
inoculating cell within the initial compartment (3’);
cultivating the cells in the initial compartment (3’) until a predetermined cell density is reached;
actuating and opening the selectively openable partition (5) to flow and merge the contents of the initial compartment (3’) with the culture media filled into the adjacent compartment (4);
unifying the compartments and continuing cultivation within the container to express the desired biologic product.
19. The method as claimed in claim 18, wherein the container (2) comprises an at least one intermediate compartment (3”) adjacently located between the initial compartment (3') and the final compartment (4), and the method further comprises:
cultivating the cells in the initial compartment (3’) until a first predetermined cell density is reached;
unifying the initial compartment (3’) and the intermediate compartment (3”) by actuating and opening the selectively openable partition (5) between the initial and the intermediate compartment;
merging the culture media of the initial compartment (3’) with the culture media filled in the intermediate compartment (3”);
cultivating the cells until a second predetermined cell density is reached before opening the selectively openable partition (5) between the intermediate compartment (3”) and the final compartment (4);
unifying initial, intimidate and final compartment by actuating and opening the selectively openable partition (5) between the intermediate compartment (3”) and the final compartment (4);
merging the culture media filled in the final compartment (4) with combined culture media of the initial and intimidate compartment (3’, 3”);
cultivating the cells within the container until the final cell density is reached.
20. The method as claimed in claim 18, further comprising independently controlling agitation in each compartment using impellers (6), independently controlling aeration in each compartment using spargers (7) and independently monitoring and controlling pH, dissolved oxygen, and temperature in each compartment.

21. The method as claimed in claim 18, wherein at least one compartment (3, 4) is operated in perfusion mode using a hollow-fiber or ATF cell-retention loop to generate high cell densities during cell expansion before partition actuation.
22. The method as claimed in claim 18, wherein the compartment (4) is maintained in fed-batch mode with bolus or in perfusion mode with continuous nutrient additions to keep residual glucose above 1 g L?¹ and to maintain appropriate level of nutrient to generate high cell densities.
23. The method as claimed in claim 18, wherein at least one compartment (3) is operated 1 °C to 3 °C warmer than the final compartment (4) to shorten doubling time during early expansion.
24. The method as claimed in claim 18, further comprising:
monitoring cell density in at least one compartment (3) using a non-invasive cell density sensor;
comparing the monitored cell density to a predetermined threshold value; and
automatically actuating the selectively openable partition (5) between the the adjacent compartment when viable-cell density in the upstream compartment exceeds 2 × 106 cells mL?¹ to 5 × 106 cells mL?¹, as measured by a non-invasive capacitance or optical sensor.
25. The method as claimed in claim 18, wherein the non-invasive cell density sensor is selected from the group consisting of capacitance-based sensors, optical density sensors, and fluorescence-based sensors.
26. The method as claimed in claim 18, further comprising dynamically adjusting agitation speed, gas-flow rate in response to sensor feedback after each partition opens.

Dated 8th Day of August, 2025