Bioprocess system extending a biological process

Abstract

The present invention relates to a flexible, scalable up-stream pharmaceutical product Process System for expression of Mabs and mixing for mRNA bases consisting of multiple process containers, a liquid dividing manifold, a fluid conveying device, a solids separation unit.

Description

[0001] TITLE

[0002] Bioprocess System extending a biological process

[0003] TECHNICAL FIELD

[0004] The present invention relates to a flexible and scalable Process System for manufacturing of pharmaceuticals. Comprising multiple process containers, for upstream expression of Mabs and mixing of mRNA, comprising a manifold, sets of valves, multiple pumps, and a pre-downstream filtration I retention system. Combined highly suitable for mRNA mixing application or cultivation or fermentation of micro-organisms in liquid media for expression of biological material. And methods for operating said Process System in a true-continuous “broth train” or “liquid train” mode accommodation cost efficient capacity adjustment and flexible scaling up I down from low to high levels rapidly.

[0005] BACKGROUND OF THE INVENTION

[0006] In pharmaceutical bioreactors and fermenters, the goal is to cultivate or ferment microorganisms for production of biological materials or biomass for medical applications. Such as monoclonal antibodies or regenerative medicine for therapeutic applications or drugs or more traditional expression from living cells or micro-organisms of molecules for therapeutic purposes or as drugs. Lately upstream processing of liquids like Polymerase and a DNA template obtaining mRNA for vaccines manufacturing have become popular.

[0007] The re-usable, non-disposable Stirred-Tank-Reactor (STR) introduced in the 1980ties being either a bioreactor or fermenter is typical of the size from few liters and from the 90ties several cubic meter fabricated from Stainless Steel and high-quality glass parts. Typical volumes for lab scale STR systems matured later to 1 -20 liter comprising a glass housing I container hanging under a removable Stainless Steel head plate and the entire STR suspending in a three-leg metal structure. The head-plate contain ports for sensors, sampling, gas exchange, media exchange, and a centrally oriented shaft for external mounted servo motor connection in order to ensure liquid media agitation inside the STR. Temperature are adjusted most often by an electrical heating blanket or water jacket surrounding the STR. The disposable Stirred-Tank-Reactor were considered a novelty, and perhaps even a passing trend, as recent as mid OOties. As the Single-Use-Bioreactors (SUB) and Single-Use-Fermenter (SUF) have matured, their market acceptance for single-use equipment in general has expanded rapidly. The move to disposable production equipment is, in many instances, driven by cross contamination problems, cost reduction in steam sterilization and cleaning requirements, improved plant flexibility, reduced costs of product and faster time to market for the end product.

[0008] PRIOR ART

[0009] • US 2009 / 0311776 from Millipore Corp describes a pre-sterilized, disposable bioreactor made of two parts being the rigid plastic top cover and a rigid plastic body with one or more fluid ports in the housing body, said top cover integrates the sensor ports and all the ports having a cap;

[0010] • US 2015 / 0299644A1 from BioSana in Australia describes a method operating multiple bioreactors and the separated cells are fed back into the bioreactor. Discontinuous fed batch cell culturing of mammalian cells with the use of alternating bioreactors via a switching unit (40) based on bi-direction pumps. No bioreactor outlet valves or manifolds are mentioned for fluid control. Manufacturing or mixing of mRNA is not mentioned.

[0011] • EP 2674480 from DASGIP Information and Process Technology GmbH describes small disposable bioreactors (SUBs) designed to fit in a parallel block. The product BioBlock perform the temperature control and alternatively also drive of the stirring or agitation device. The BioBlock is designed to operate 4 STRs or SUBs in parallel encapsulating the bottom part of the SUB container.

[0012] • US 20180155667 from Stobbe describes the CellRetention perfusion system combining one pumping element, one membrane element and alternation of the broth from and returned back to the SUB.

[0013] Three different technologies, processes are broadly in use world-wide:

[0014] 1 . The STR known as a bioreactor, or a Single-Use-Bioreactor (SUB) operated in batch mode with micro-organism like mammalian cells is a one-week process known as up-stream batch cultivation. 2. The STR know as a fermenter, or a Single-Use-Fermenter (SUF) operated in batch mode with micro-organism like yeast or bacteria is a few days process known as up-stream batch fermentation.

[0015] 3. The STR designed for cultivation or fermentation though recently used as a contemporary mixer of liquids for mRNA batch manufacturing.

[0016] Currently global societies are looking after methods, technologies, processes able to decrease final product prices. It’s here continuous and semi-continuous processes becomes important and able to increase the process operations time from short time batches to weeks or months of processing. This without terminating the processes and start from scratch again and again with all the problems around contamination, cleaning, certification, etc.

[0017] The most common upstream process is batch - based and equipment typically a process container ranging from litres to several m3. Operating in batch mode limited from hours to a week followed by the entire liquid broth volume to be processed downstream instantly. This lacking flexibility and total harvest requires an expensive instant available down-stream volume capacity. Such as retention systems, separation systems, filter systems removing various particulate matter obtaining a liquid product ready for polishing into a concentrated final product. Far from a flexible and scalable continuous processing when the upstream liquid product is delivered to down-stream in batch mode.

[0018] A - Scalable and true-continuous up-stream fermentation technologies and methods in SUFs do not exist as of today. And so-to-speak continuous down-stream processes as well. The present invention combines multiple SUFs I process containers in a “broth train” system with fermentation taking place one SUF after the other (sequential operation in a rotating order) supplying down-stream with a constant mass-flow of broth. Each SUFs after harvest, supplied the broth product may be re-used, repeatable used. Requiring significant lower investment in equipment and facilities = lowering product cost. Offering scalability and flexibility able to supply demands in case of a pandemic. B - Scalable, flexible and true-continuous up-stream cultivation and methods in SLIBs I process containers do not exist as of today (except semi-continues perfusion systems). And so-to-speak continuous down-stream processes as well. The present invention combines multiple SLIBs in a “broth train” system with cultivation taking place in individua SLIBs after the other (sequential operation in a rotating order) supplying down-stream with a constant supernatant supply. Each SLIBs after harvest, supplied the broth product, may be re-used, repeatable used in a sterile manner = flexibility and lower cost of product.

[0019] C - Scalable, flexible true-continuous supply of mRNA by mixing of liquids for vaccine manufacturing and therapeutics do not exist as of today. Technologies and methods in mixers are performed in isolated batches. The present invention combines multiple Single-Use-Mixers in a “liquid train” establishing a constant flow of mixed liquids cost efficient and for further downstream processing. Offering an efficient scalable and flexible platform accommodating rapid up I down capacity changes as to needs while maintaining quality and regulatory compliance. The scalability and flexibility needed to supply demands in case of a pandemic.

[0020] SHORT PRESENTATION OF THE INVENTION

[0021] A Process System for processing a biological material contained in multiple process containers comprising a liquid processed into a liquid pharmaceutical product comprising:

[0022] • said two or more process containers comprising each an inlet port and a liquid outlet port,

[0023] • said two or more process containers comprising an outlet port valve device,

[0024] • a liquid receiving and liquid conveying manifold comprising two or more liquid inlet ports and one or more liquid outlet ports,

[0025] • two or more electronical controlled pumping devices comprising a first inlet port and a second outlet port,

[0026] • a separation device comprising a liquid inlet port said device separating the liquid pharmaceutical product into retentate and into permeate I supernatant conveyed via an outlet port. The present invention relates in a first aspect to a Process System for processing a biological material comprising: a) at least two process containers adapted for processing the biological material each container comprising an inlet port adapted for providing a liquid comprising a biological material to the process container and an outlet port adapted for providing a liquid harvest, wherein each process container comprises a valve device controlling liquid flow out of the outlet port, b) a manifold comprising at least two liquid inlet ports wherein each inlet port is in liquid communication with the outlet port of each container, and at least one liquid outlet port, c) at least one first electronical controlled pumping device adapted for conveying liquid from the first process container and at least one second electronical controlled pumping device adapted for conveying liquid from the second process container, d) at least one third electronical controlled pumping device adapted for conveying liquid from the at least one liquid outlet port of the manifold, and e) a separation device comprising a liquid inlet port in liquid communication with the at least one liquid outlet port of the manifold, and at least one liquid outlet port, wherein said separation device is adapted for separating the liquid harvest into retentate and into permeate conveyed via the at least one liquid outlet port.

[0027] In an embodiment the valve device controlling liquid flow out of the outlet port of the process container is a mass flow controlling device.

[0028] In a further embodiment the at least one first electronical controlled pumping device is a fluid pumping device comprising a first inlet port in communication with the outlet port adapted for providing a harvest liquid of the first process container, and a second outlet port in communication with a first liquid inlet port of the manifold.

[0029] In a still further embodiment the at least one second electronical controlled pumping device is a fluid pumping device comprising a first inlet port in communication with the outlet port adapted for providing a harvest liquid of the second process container, and a second outlet port in communication with a second liquid inlet port of the manifold.

[0030] In a further embodiment the at least one third electronical controlled pumping device is a fluid pumping device comprising a first inlet port in communication with the at least one liquid outlet port of the manifold and a second outlet port in communication with the liquid inlet port of the separation device.

[0031] In a still further embodiment the at least one third electronical controlled pumping device is a fluid pumping device comprising a first inlet port in communication with the liquid outlet port of the separation device and a second outlet port adapted for conveying the permeate from the liquid outlet port of the separation device.

[0032] In a further embodiment the separation device comprises two liquid outlet ports, one first liquid outlet port for conveying retentate and one second outlet port for conveying permeate.

[0033] In a still further embodiment the process system further comprising a Process-Control- System (PCS) comprising multiple analogues and / or digital input channels collecting data from multiple sensors and acting via multiple analogues and / or digital output channels controlling the Process System activities and selecting the process containers outlet valves.

[0034] In a further embodiment the separation device is an electronically controlled centrifuge.

[0035] In a still further embodiment each of the process containers is equipped with an agitation device.

[0036] In a further embodiment the process system further comprising a second manifold comprising a liquid inlet port adapted to receive permeate liquid from the at least one liquid outlet port of the separation device, and at least two liquid outlet ports. In a still further embodiment an electronical controlled pumping device operates in a closed loop guided by a laser sensor which output signal is forwarded to the PCS and said activity controlled by said PCS. Preferably, said electronical controlled pumping device is selected from at least one of the first, second or third electronical controlled pumping device. Typically, the first, the second and the third electronical controlled pumping device all operates in a closed loop guided by a laser sensor which output signal is forwarded to the PCS and said activity controlled by said PCS.

[0037] In a further embodiment the at least one electronical controlled pumping device activity is controlled by a PCS and a program stored in a memory of said PCS.

[0038] In a still further embodiment the process system further comprising a downstream operation unit facilitating the permeate conveyed via the at least two liquid outlet port of said second manifold is subjected to further down-stream process steps to provide the processed biological material.

[0039] In a further embodiment the process system is for continuous processing of biological material contained in a liquid media comprising at least two fluid tight process containers operating sequentially in parallel each with a liquid media volume processing said biological material into a broth independently comprising microorganisms suspended in said liquid media volumes.

[0040] In a still further embodiment the process system is for continuous processing of biological material contained and processed in a liquid in one or more fluid tight process containers comprising one or more of: a. an agitation devise, b. an inlet port connected to a liquid supply, c. a port connected a sensor, d. an inlet port connected to a gas supply, e. an outlet port for product harvest, f. an outlet port general purposes gas exchange, g. an external assembly of hoses and connectors, wherein said fluid tight process containers are customized to support biological activity of a biological material in a liquid.

[0041] In a further embodiment the process system is for continuous processing of biological material contained and processed in a liquid comprising one or more over-moulded silicone manifold systems conveying said liquid comprising one or more ports conveying said liquid or for connection of a sensor.

[0042] In a still further embodiment the process system is for continuous processing of biological material contained and cultivated in a liquid media, comprising an electronically controlled centrifuge for separation of solids from said broth liquids into retentate (solids) and permeate (liquids).

[0043] In a further embodiment the process system is for processing of biological material contained in a liquid characterised in that means for process control is performed by a customizable Process-Control-System (PCS), said PCS is characterized by multiple individual PCS devices selected from one or more of: a. a device comprising multiple configurable electric inlets and outlets for process control and data collection and a Programmable-Logic-Control (PLC) with process software stored in memory, b. a device comprising multiple configurable inlets and outlets for process control and data collection and a Programmable-Logic-Control (PLC) with process software stored in memory, c. an electrical inlet device connected to a LASER sensor for process control and a Programmable-Logic-Control (PLC) with process software stored in memory, d. a computer and display being a Human-Machine-Interface (HMI) for data collection and presentation, e. a Local-Area-Network (LAN) device connecting the above devices, wherein said Process-Control-System comprises multiple analogue and digital input channels for measuring, collecting data from a range of sensors, and various pneumatic, gas, electronic analogue as well as digital output to a variety of actuators, motors, etc. In a second aspect the present invention concerns a method of operating a Process System according to anyone of the above first aspect or the embodiments hereof converting a liquid biological material into a continuous liquid harvest flow facilitating: a. at least two fluid tight process containers each comprising a liquid inlet and a liquid outlet, said fluid tight process containers filled with a liquid processing said liquids independently into a liquid product, b. each fluid tight process container operates in batch mode sequentially, one fluid tight process container after each other arranged in such a manner that one second fluid tight container process biological material into a liquid while one first fluid tight container with processed and ready to harvest liquid is emptied and said harvested liquid conveyed to a manifold via a valve device and or a pump, c. each process container liquid outlet comprises a mass flow regulating valve device and or a pump device controlling mass flow from said process container to said manifold, d. said manifold comprise multiple connectors for sequentially liquid product inlet flow from multiple process containers, said manifold connects multiple process containers and pumps and components together into a Process System circuit, said manifold convey said liquid from multiple batch process containers via a continuous liquid outlet flow port to a centrifuge, e. said manifold convey liquid to said centrifuge in a continuous mode, f. said centrifuge receives and process the liquid product dynamically and continuously by centrifugal forces into retentate (solids) and permeate (liquids) ready for further down-stream processing.

[0044] In an embodiment the method is for converting a liquid biological material into a continuous liquid harvest flow facilitating: a. at least two fluid tight process containers each comprising a liquid inlet and a liquid outlet, said fluid tight process containers filled with a liquid processing said liquids independently into a liquid product, b. each fluid tight process container operates in batch mode sequentially, one fluid tight process container after each other arranged in such a manner that one second fluid tight container process biological material into a liquid while one first fluid tight container with processed and ready to harvest liquid is emptied and said harvested liquid conveyed to a manifold via a valve device and or a pump, c. each process container liquid outlet comprises a mass flow regulating valve device and or a pump device controlling mass flow from said process container to said manifold, d. said manifold comprise multiple connectors for sequentially liquid product inlet flow from multiple process containers, said manifold connects multiple process containers and pumps and components together into a Process System circuit, said manifold convey said liquid from multiple batch process containers via a continuous liquid outlet flow port to a first stage filter system, e. said manifold convey liquid to said filter system in a continuous mode, f. said filter system receives and process the liquid product continuously by separation solids (retentate) and permeate (liquids) ready for further downstream processing.

[0045] BRIEF DESCRIPTION OF THE FIGURES

[0046] Figure 1 illustrate a multi batch continuous Process System with 3 out of 8 optional process containers set as example limited by the manifold ports. Two diaphragm SUPs convey broth, liquid to the inlet of the separation unit, and permeate outlet directed to a triple liquid manifold with 3 membrane filters for down-stream clarification. Retentate regarded as waste in the example.

[0047] Figure 2 illustrate a multi batch continuous Process System with 1 shown out of 8 optional process containers set as example of the broth manifold. Two diaphragm SUPs as broth inlet, for the separation unit, and permeate outlet directed to a triple liquid manifold with 3 membrane filters for clarification. Retentate regarded as waste.

[0048] Figure 3 illustrate a single perfusion setup in which the single process container delivers sequentially broth to the pump before the separation device and receives a part of the concentrated broth from the separation device, a centrifuge, retentate outlet. The concentrated broth divided into two streams - one returned for the SUB and one as waste. Figure 4 illustrates a fully functional continuous up-stream Process System including half of the down-stream separation process and the customizable Process-Control- System. Multiple process containers shown out of the option of 6 process containers as the chosen manifold design. After the manifold a series of pumps convey the broth into the centrifuge and convey further the purified liquid to the manifold followed by membrane filters.

[0049] DETAILED PRESENTATION OF THE INVENTION

[0050] The presented invention provides a scalable Process System supporting biological activity in fluid tight process containers, a manifold system, a pumping system, a broth component separation system and a process control system.

[0051] Such as bioreactors or fermenters processing a biological process by micro-organisms contained in liquid media expressing a product converting the media into a broth volume. Or lately mixing various liquids into mRNA base for vaccine manufacturing. All up-stream processes followed by a preparing economic feasible down-stream process - until final purification of the Supernatant.

[0052] Within the field of cell I micro-organisms culture as applied to bio pharmaceutical processes there exists a need to separate the produced broth into a product free from micro-organisms, debris, waste substances, etc. The desired product from the cell culture may be a molecular species that the micro-organisms excrete into the media, a molecular species that remains within the micro-organisms which needs to be released by an intermediate process bursting the micro-organisms, or it may be the micro-organisms itself. At production scale, the initial stages of cell culture process typically take place in bioreactors or fermenter, which may be operated in either batch or perfusion mode. Intermediate variations such as repeated batch are practiced as well. In all cases the product must eventually be separated from other process components prior to final purification and product formulation. Product harvest is a general up-stream term applied to the micro-organisms I cell separation process from the broth I Supernatant. Clarification is a down-stream term denoting separation wherein a cell-free liquid is the objective. Cell recovery is a term often applied to separations wherein a cell concentrate is the objective. The increasing need from the pharma industry and requirement from our society for lower cost pharma product must be based on continuous processing and as of writing continuous processing are still in its infancy.

[0053] Definitions relevant for the present invention are:

[0054] • The term “a” or “an” as used herein means one or more or at least one.

[0055] • The term “alternating” as used herein describes a liquid flow of bi-directional direction. One specific and identical volume moving forth and backwards, no valves for flow direction control is included.

[0056] • The term “assembly” as used herein means a collection of pre-assembled hoses, clamps, tube fittings, small bottles. Which makes it easier for the end-user to speed up the prep time of the STR or SUB or SUF or SUM. In general, useful for noncustomized Single-Use-Bioreactors not delivered ready to use.

[0057] • The term “ATF” as used by the industry means Alternating-Tangential-Flow which is a concept, a method where the diaphragm pumps specific volume is constant and this volume is alternating, changed back and forth (forward and reverse).

[0058] • The term “back-flow” is used to describe a harvest portion return flow, decreasing the TMP, Trans Membrane-Pressure somewhat helping high velocity flow along or transverse to the membrane inlet side in order to remove deposits. Back-flow not to be confused with the term “back-flush” being widely used in the (rigid) membrane industry though not feasible on elastic membranes made from polymeric materials.

[0059] • The term “batch operation” as used herein refers to an operation method with constant Working Volume (WV) to which no fresh media is added and no used media and / or liquid removed, typically lasting less than one week when cultivating micro-organisms such as CHO cells and a few days fermenting yeast or bacteria. Characterized by a well-known and fixed process termination.

[0060] • The term “biological material” as used herein describes organic compounds, tissue, cellular components, body compatible fluids, biomass, bio-composites, biocompatible materials, antibodies in general, DNA, mRNAs, proteins, molecules proteins, for therapeutic purposes and the like which may be suspended in a liquid.

[0061] • The term “bioreactor” or “process container” as used herein means a physical device, in which biologically active environment is suitable for cultivation of micro- organism performing a desired process suspended in liquid media agitated by an impeller. The specifically designed process container performs equally well as a fermenter or a mixer.

[0062] • The term “broth” as used herein means the liquid as the result of a biological process, the cultivation, the fermentation. Broth is the non-filtered liquid, a slurry, the cultivated “soup” in a bioreactor or fermented “soup” consisting of a mix of micro-organisms, debris, media with remaining nutrients, waste, the planned product like proteins and / or micro-organisms, water, etc. The broth originates from the up-stream process followed by the multi-step down-stream processes.

[0063] • The term “centrifuge” as used herein means a continuous operating device that uses centrifugal force to subject a specimen to a specified high G-force increasing settling speed of a particle or isolate suspended particles from their surrounding medium. For example, to separate various components, solids from a liquid. This is achieved by spinning the broth across a disc at high speed within a container, thereby separating specimens of different densities into two or more different final results like retentate and permeate. A centrifuge is a +90% effective separation devices able to separate contaminants larger then few pm size from liquids of the broth. In the pharma industry the continuous operating centrifuge for clarification can replace most depth filters (with limited lifetime) separating micro-organisms, debris, various particles, micro carriers, etc from the broth continuously. Centrifuges are also available as electronically controlled Single-Use-Centrifuges (SUC), pre-sterilized modules to minimize contamination risks and mitigate the comparatively complex and expensive cleaning, validation, and commissioning of stainless-steel components.

[0064] • The term “broth train” as used herein means, is based on an affordable batch cultivation or fermentation liquid tight process containers strategy. Generating multiple broth volumes from multiple bioreactors (SUBs) or fermenters (SUFs) establishing a continues harvest by generating the “broth train”. Reduced batch volumes harvested one after each other reduces investments and contamination risks in the invented semi-continuous operating mode and reduce down-stream process equipment capacity and cost. • The term “CHO” refers to Chinese Hamster Ovary cells being a “micro-organism” and very popular mammalian cell line, expression platform of proteins for the pharma industry.

[0065] • The term “chromatography” as used herein is the final purification for biosimilar monoclonal antibody therapeutics and mRNA. Protein A chromatography is typically a batch process. Continuous multicolumn chromatography has several benefits, including reduced buffer use, increased productivity, and small footprint but still in its infancy.

[0066] • The term “container” or ’’process container” as used herein means a hollow housing, a body with an internal reservoir, which may be open or closed, such as without limitation a STR, a beaker, a flask, a bottle, a tube, a vessel, a tank, a rigid plastics SUB or SUF or SUM, a polymeric material film bag with a wall forming the reservoir. The container when operating as for instance a Stirred-Tank-Reactor is typically arranged with a vertical wall and a horizontal bottom wall pointing downwards, so that liquid or fluid in the container is predominantly maintained inside the container during operation mode. The container may be of cylindrical design, or non-cylindrical design such as conical design or of square box shaped design, a flexible film bag or combinations hereof.

[0067] • The term “Container Volume” (CV) as used herein refer to total volume of the process container or housing body when used as a bioreactor or a fermenter or a mixer.

[0068] • The term “continuous” as used here in refer to an uninterrupted process. Like if the harvest step from a SUB is performed without major interruptions, without stopping. We refer further to “batch” or “fed-batch” or “semi-continuous” or “true-continuous” processing being the terms of difference operation modes.

[0069] • The term “Cross-Flow-Filter” (CFF) as used herein is a membrane filter device, a separating device which allow a liquid and in liquid suspended selectable components to pass the separating device within the liquid volume onto the other side of the device by crossing, passing the device membrane with certain specification eliminating desired suspended components not to pass the separating device. The device has a first entrance for the broth and a second exit for the retentate. In between the broth first entrance and the retentate second exit further a third permeate exit for the product, the filtrate, the harvest which has passed the membrane.

[0070] • The term “cultivation” or “culturing” refers to hosting of micro-organism, such as mammalian cells, in a bioreactor for production purposes, such as expression of a product, antibody, mAbs, receptors, enzymes, proteins, vaccines, recombinant, vectors by said micro-organisms or proliferation of cells by said micro-organisms.

[0071] • The term “depth filter” refer to a variety of filters devices that use a thick, porous filtration medium to retain, particles suspended in a liquid, throughout the filter medium. The filter media pores may be progressive and arranged in a manner such that sections with a larger pore size are closer to the inlet path or port, capturing particles of a larger size and pore sizes decreases approaches the outlet path. Opposite of collecting particles on the surface of the medium such as a thin membrane with selected identical pore sizes. These depth filters are commonly used when the fluid to be filtered contains a high load of larger particles. Relative to other types of filters, depth filters can retain a large mass of larger particles before becoming clogged.

[0072] • The term “deposit” or “membrane coating” or “filter cake” as used herein refers to a process where solute, particles, solids, debris, micro-organisms deposit onto a membrane surface, such as a membrane filter surface or even into membrane pores in a way that degrades the membrane's performance and increase trans- membrane-pressure (TMP) to undesired high levels. Such deposit may be removed by increased shear force, back washing by a high velocity of the broth in flow direction or the opposite direction, inverting the liquid direction from filtering process for washing purposes.

[0073] • The term “diaphragm” or “diaphragm pump” as used herein is a round sheet of elastic material preferably with a sealing arrangement on the circumference. The diaphragm operates inside a dome shaped housing separating the wetted, liquid side from the drive fluid side. The drive fluid pressure expands controlled the diaphragm which then convey the fluid. The drive fluid, drive gas pressure is desirable controlled in a loop with a LASER sensor, a PLC and a proportional valve. The diaphragm pump may also function as a valve regulating a fluid passage.

[0074] • The term “disposable” refers to a product manufactured often from synthetic or polymeric materials preferably at low cost and to be scrapped after use. The here presented Process System is further plastic foil bagged tightly and pre-sterilized before and ready for use.

[0075] • The term “exterior facility” (area) means laboratory, production facility, testing facility in which room(s) any Process System may be in use. In the exterior facility both up-stream and the down-stream processes may take place.

[0076] • The term “electrical” or “electronically” as used here in also refers to methods or means supplying energy to electricalmotors with excitation for rotation control purpose and / or rotor orientation purposes driving devices such as impellers, rotors or the like. Electrical motors may be RPM controlled (Revolutions-per-Minute) by electronically means stabilising or altering various parameters.

[0077] • The term “fed-batch” operation as used herein refers to a bioreactor or fermenter which start the process with a minimum media volume to which fresh medium is added and no liquid removed until after termination of the process as the process container max Working Volume is reached. A minimum of media volume in the housing body are inoculated with micro-organism and appear as seed train until the maximum WV is reached prior to process termination. Typical operation time is 2 - 3 times longer than batch operation.

[0078] • The term ’’fermenter” as used herein means a physical device, a process container, suitable for fermentation of micro-organism performing a fermentation process when suspended in liquid media and agitated by a turbine.

[0079] • The term “fermentation” as used herein refers to hosting of micro-organisms, such as living single-celled organisms, prokaryotes, bacteria, yeast for industrial purposes in a fermenter expressing a product.

[0080] • The term “filter device” as used here in refer to a Cross-Flow-Filter (CFF) or a depth filter or a membrane filter. The CFF can also be a Hollow-Fibre-Filter module (HFF).

[0081] • The term “fluid” refers to a gas or a liquid, a gas such as air or nitrogen at variable volume or a liquid such as water and / or oil at constant volume or a mixture of gases and liquids.

[0082] • The term “fluid conveying device” as used herein is a pump able to convey a liquid or gas (the fluid) from one point to another point. Such devices can be a “Single- Use-Pump” (SUP) a diaphragm pump, a centrifugal pump, a peristaltic pump, a piston pump. • The term “fluid tight container” refer to a bioreactor, a fermenter, a mixer, a STR, a process container and other vessel for various uses.

[0083] • The term “glass” as used herein refers to transparent silica based amorphous brittle and solid material often with excellent corrosion resistance.

[0084] • The term “harvest” as used herein refer to the product part (such as a protein) of the broth being the expected product generated by micro-organisms cultivated in a bioreactor or fermented in a fermenter. The harvest (the filtrate, the permeate) may be separated from the broth via various filtration methods or by centrifuges in the down-stream processes. When permeate is removed by the separation device the broth is then concentrated semi-finished product.

[0085] • The term “hollow fibre module” or “Hollow-Fibre-Filters” (HFF) as used herein refer to a device made from an outer rigid wall tube with end covers housing inside a bundle of thin wall tubes fabricated from porous elastic material such as polyethersulfone or other polymers. The bundle of tubes, lumens are sealed, cast into end covers separating the tube inside from the tube outside and hereby appearing as a Cross-Flow-Filter (CFF). Pore size range from kDalton to half a micron. Pore size of 200 nm (0.2 pm) is considered to be a sterile barrier.

[0086] • The term “impeller” refers to a fluid-agitating device equipped with blades or vanes rotating inside a liquid media filled container for agitation, mixing, pumping, liquid circulation purposes.

[0087] • The term “irradiation” is the process by which an object is exposed to radiation. If administered at appropriate levels, all of these forms of radiation can be used to sterilize objects, a technique used in the production of medical instruments and disposables. The irradiation dose is usually measured in Gray. Where 1 kGy and is the radiation required to deposit 1 joule in 1 kilo matter. Levels of exposure for single-use pharmaceutical objects are often less than 40 kGy.

[0088] • The term “liquid train” as used herein refer to a product, a harvest from multiple operating in parallel one after the other Single-Use-Mixers (SUMs) hereby passing a manifold establishing a continues product, harvest generating the “liquid train” for further down-stream processing.

[0089] • The term “manifold” as used herein means a device or component connecting multiple fluid conveying pipes, tubes, hoses, channel inlets and outlets. Manifold inlet and outlet ports may be connected to valve devices, to pumps, to aseptic or sterile connectors allowing sterile connections between components of the process system to take place inside a non-sterile laboratory environment, facility. Furthermore, sensors may be mounted on, connected to the manifold collecting process data like pressure, temperature, etc. The purpose of said manifold is fluid distribution such as collection from one or more sources conveyed to or between one or more receivers. A manifold, connected to a reservoir will benefit from such liquid volume and a gas volume depending on gravity or pressure, will generate a constant continuous liquid outlet flow.

[0090] • The term “mass flow” control device as found herein are used interchangeable with a diaphragm pump or a valve.

[0091] • The terms “media”, “growth media”, “liquid media” are herein used interchangeable and refers to a sterile complex liquid mixture. Containing mostly water, carbon sources, additives such as; vitamins, hormones, growth factors, animal serum, antibiotics, antioxidants, antifoams, cell stabilizers and other components intended for cultivation of “micro-organisms” in the up-stream process. Some media are serum based, some are serum free, animal free, and protein free often described as chemically defined media. During cultivation, fermentation the micro-organism will convert a carbon source, nutrients and oxygen into “the broth” by biological activity and create, produce product and various debris - in total named the “broth”.

[0092] • The term “membrane” refers to a boundary layer, which serves as a selective barrier and remains impermeable to specific or desired particles, molecules, or substances when exposed to the action of a driving force (like pressure supplied by a pump). Porous membranes are manufactured from a variety of flexible and rigid materials such as polymers, ceramics and metals. Appear further in the technical term as a “Cross-Flow-Filter (CFF), a sterile filter, membrane filter, depth filter device”. The membrane clarifies a broth or permeate from a centrifuge and allow a fraction to pass known as harvest or supernatant.

[0093] • The term “membrane filter” as used herein refer to nano-filtration and micro-filtration systems separating various fractions of a fluid. The expression “Sterile Filtration” in the pharma industry is 200 nm 1 0.2 pm pore size able to resist micro-organisms to pass such semi-permeable membrane. The membrane media is typically a thin sheet of organic polymeric material enclosed inside a polymeric material housing with various inlets, outlets according to the process. The final liquid after clarification is also known as cell free supernatant. Can be either of or combined TFF, CFF or HFF systems.

[0094] • The term “membrane fouling” as used herein refer to the effect when solids, cells, cell parts, cell membranes, aggregates, etc creates a layer, a biofilm, a cake of debris on the porous membrane inlet surface. This effect requires higher TMP in order to overcome the increased resistance on the combined membrane and deposits and keep desired constant flux. Membrane deposits can be removed by high shear forces, high broth velocity and / or backflush, backwash.

[0095] • The term “micro-carrier” refers to a micro-organism supporting device or growth body allowing cultivation of adherent micro-organisms. Size range typical from 100 to 1.000 pm composed by porous gelatine, collagen, cellulose or polymeric materials or glass and may further be functionalized with one or more coatings.

[0096] • The terms “micro-organism” or “cells” or “biological cells” or “biological material” as used herein are used interchangeable and is typically divided into: 1. living singlecelled organisms, microbes such as; fungus, algae, moss, plankton, yeast, protozoa, eukaryotes, archaea, micro animals, extremophiles and plant cells or the like - 2. adherent or semi adherent or suspended living cells such as animal cells, insect cells, mammalian cells, human cells, stem cells - 3. prokaryotes and a variety of bacteria such as E.coli or the like - most of the above genetically modified to solve specific tasks and product needs.

[0097] • The term “mRNA” as used herein refer to synthetic manufactured messenger-RNA (ribo-nucleic-acid) or self-amplifying-mRNA or saRNA or LNP able to pass the mammalian cell membrane and cause said cell to create a protein, which in turn could directly treat a disease or function as a vaccine. mRNA is typically produced in a 1 or 2 step in-vitro synthesis through an enzymatic reaction between mixed fluids in a process container (typical Polymerase and a DNA template) followed by a multi-step purification platform including Tangential-Flow-Filtration and chromatography.

[0098] • The term “perfusion” or “cell I micro-organism retention perfusion” mode operation as used herein refers to the semi-continuous operation method or principle for a STR or a SUB where the media is sequentially exchanged, fresh nutrients sequentially added, used media / harvest removed throughout the culture period. The micro-organisms cultivation in the STR, SUB last typically 4 - 8 times longer than batch and 2 - 4 times longer than fed-batch cultivation time.

[0099] • The term “permeate” as used herein refers to specific parts of a mixture, part of a broth, part of a feedstock having passed by size selection through a membrane or by gravity differences having passed a centrifuge as liquid product. Also known as filtrate, clarified, purified - the product being an antibody, a protein, a Monoclonal- Anti-Body (Mabs), mRNA or the like.

[0100] • The term “permeable membrane” as used herein refer to a porous wall, a “membrane” allowing a liquid and selected particles (size separation) to pass the membrane and certain particles not to pass the porous wall, barrier, separation wall.

[0101] • The term “ports” as used herein refers to holes, access ponts anywhere in a wall allowing attachment of suitable fittings or relevant sensors or general connections selected from the group of; PG 13,5 threaded ports, locking ports, press-in ports, or ports involving Luer-Loc fitting, TRI-clamp port, connecting fittings, sterile fittings, ports for hoses, tubes, hose barbs, etc.

[0102] • The term “PG 13.5” is a classical Re-Useable-Sensor (RUS) size or port size with a threaded mechanical connection. PG is the technical standard term known as Stahl-Panzer-Rohr-Gewinde.

[0103] • The term “PCS” or “Process-Control-System” as used herein refer to a digitally operating control system used for automation of pharmaceutical processes. Most or all PCS are based on a Programmable-Logic-Control (PLC), or even a Personal Computer (PC) integrating a Central-Processing-Unit (CPU) with calculating power. The PLC is an industrial computer with various Input & Output (I / O) modules or points. Input from a variety of sensors, switches and output for various actuators, motors, valve, regulators, solenoids for process control purposes. The PCS integrate software with algorithms, instructions, diagnostics, process recipe stored in memory for managing, analysis of the cultivation, fermentation or other biological process for continuous and in real-time analysis and process alignment, corrections according to a process recipe. The PCS connects to a Human-Machine-Interface (HMI) monitor for surveillance purposes and recipe programming.

[0104] • The term “PLC” or “Programmable-Logic-Control” as used herein is a various Input & Output (I / O) platform with input from various sensors and output for various actuators for process control purposes. The PLC and PCS integrates “software”, algorithms, process recipe in build-in memory for managing, analysis of the cultivation or fermentation process for on-going process alignment according to the process recipe. The PCS correspond with a variety of sensors, actuators, motors, passive thermal elements, lamps, etc in order to alter, adjust the process parameters continuously.

[0105] • The term “plastics” or “polymers” refer to numerous organic and synthetic polymers such as PolyEthylene, Polypropylene, Polystyrene, Polyvinylchloride, synthetic rubber, phenol formaldehyde resin, Neoprene, Nylon, PolyAcrylonitrile, Silicone and thermoplastic polymers such as Polycarbonate, PolyMethylMethAcrylate, PolyAmides, ABS, PolyEtherSulfone or the like.

[0106] • The term “PTF” as used herein refer to the new industrial terminology and means Pulsating-Tangential-Flow being the here presented invention. The PFT process involves a pump with a first inlet valve and a second outlet valve in order to insure one-directional liquid flow.

[0107] • The term “pump” as used herein refers to a device able to convey a fluid via tubes or hoses between a variety of containers, process containers, bioreactors, vessels, reservoirs, bags, etc. Such a pump may be controlled by electric means and / or electronic devices able to adjust Revolutions-per-M inute (RPM), volume, pressure in said fluid flow. Such pump may be a diaphragm pump with one or more elastic diaphragms, a peristaltic pump, a centrifugal pump. A pump may be manufactured from metals and / or polymers. Any of the pumps may be a Single-Use-Pump. A pumps may operate as a valve device in such a manner as in a passive stage to fluid will pass.

[0108] • The term “reservoir” as used herein refer to at fluid tight vessel or container preferably under sterile conditions to hold intermediate variable fluid volumes, said reservoir may operate under controlled pressure. A reservoir may even out different liquid volumes from a semi-continuous flow, velocities and convert to a continuous stream of liquids.

[0109] • The term “retentate” as used herein refers to the parts, micro-carriers, particles, “micro-organisms”, debris of a mixture within the broth, feedstock that is held back by a separation devise, and do not pass the separation device as to its size, shape, weight or charge. Such as separated out of a broth by centrifugation. • The term “scalable” as used herein refers to the feature when CV and W are not locked into fixed glass container dimensions. Further refer to that the ratio between container diameter and height can be altered accommodating end-user requirements.

[0110] • The term “seed train” widely used in the industry generating sufficient product by starting with a small batch process container. After the first cultivation or fermentation has reach a desired target of micro-organisms, glycose and lactate content the entire broth volume is transferred to a several times larger vessel with media for further batch continuation. And so-on until the max volume said process container can accommodate is reached and the process is terminated. The problem is that the final large broth volume needs transfer to downstream which then require similar volume capacity.

[0111] • The term “semi-continuous” refers to multiple often identical process steps with interruption in between the individual steps (discontinuous) sequential available and connected one after the other. Individual sequential batch volumes may be converted to “semi-continuous” by a “reservoir”. Semi-continuous processing is characterized by a range of multiple fixed process terminations.

[0112] • The term “sensor” as used here refers to devices able to measure on-line quality process variables associated within a given process, such as the level of pH, dissolved oxygen (DO), biomass I cell density, capacitance, opacity, conductivity, dissolved carbon dioxide, lactate, glucose, glutamine, glutamate, ammonia, pressure, liquid level, fluid mass-flow, velocity, temperature, viscosity, etc. Sensor to measure SUP activity may be proximity, distance being mechanical, optical, electrical (such as triangular laser sensor, a pressure sensitive level sensor, or capacitance or ultra-sonic based distance sensors) or Raman sensors. Sensors in general available as either Re-Useable-Sensors (RUS) or Single-Use-Sensors (SUS).

[0113] • The term “separation device” as used herein refers to a range of different systems capable of dividing elements. Such as a centrifuge, a depth filter, a membrane filter, etc based on a variety of principles.

[0114] • The term “single-use” as used herein refers to a product designed for use only once and to be disposed after use typically delivered “pre-sterilized” and ready to use, such as the “Single-Use-Bioreactor” (SUB) or “Single-Use-Mixer” or “Single-Use- Sensors” (SUS) or “Single-Use-Centrifuge” (SUC) and possible combined also with an “assembly” of hoses and connecters.

[0115] • The term “Single-Use-Centrifuge” (SUC) is a pre-sterilized polymeric module designed to minimize contamination risks and mitigate the comparatively complex and expensive cleaning, validation, and commissioning of stainless-steel components. Functionality is based on mass differences of materials introduced to the centrifuge and contained various components exposed for G-forces separating said various components into one or more concentrates at various liquid outlets.

[0116] • The term “Single-Use-Sensor” (SUS) as used herein refer to a disposable device able to on-line measure analysts, process conditions, fluid concentrations and deliver a signal, such as an electrical signal relative to the concentration measured. The in-expensive SUS body is designed primarily from polymeric materials to be pre-installed in a SUB or SUF for simultaneous sterilization all enclosed in dual film bags for convenience to the end-user who then avoid sterilization before use.

[0117] • The term “Single-Use-Bioreactor” (SUB) as used herein refer to film bag(s) or rigid plastic vessels, process containers equipped for the purpose performing as a bioreactor and preferable pre-installed with an agitating impeller device and one or more “Single-Use-Sensor’s” all manufactured from disposable materials and presterilized and hereby ready for use eliminating the traditional in-house heat, high temperature sterilization in an autoclave, etc.

[0118] • The term “Single-Use-Fermenter” (SUF) as used herein refer to film bag(s) or rigid plastics fermenter and preferable pre-installed with an agitating turbine device and one or more “Single-Use-Sensors” all manufactured from disposable materials and sterilized and hereby ready for use eliminating the traditional in-house heat sterilization.

[0119] • The term “Single-Use-Mixer” or “SUM” as used herein refer to a fluid tight process container including means for liquid connection, for agitation, for harvest used to accurately mix several fluids into a complex fluid like mRNA.

[0120] • The term “Single-Use-Pump” (SUP) as used herein refer to a fluid conveying device manufactured at least partly from disposable materials. Such as a peristaltic pump or a centrifugal pump or a tube pump or a diaphragm pump or a piston pump or a direct gas-to-liquid surface driven air column pump or the like comprising disposable wetted part and re-usable non-wetted parts and a valve for flow direction control.

[0121] • The term “software” or “process controlling software” as used herein refer to; 1 Systems Software being a set of instructions, algorithms, routines and data set that tell a computers or PLCs Central-Processing-Unit (CPU) how to execute, perform specific tasks. All software programs are stores in memory communicating with the CPU of the “PLC” or “Programmable-Logic-Control” used in all “PCS” or “Process- Control-Systems”; 2. Some Application Software is proprietary to specific PCSs and a few Application Software are open in such way they can be used on a broad range of PLC platforms. Such as Lucullus from Swiss SecureCell and DeltaV from USA based Emerson.

[0122] • The term “Stainless Steel” as used herein refers to an alloyed metal based mostly on nickel, chromium, vanadium, carbon, and steel characterized with at least excellent corrosion resistance.

[0123] • The term “sterilization” as used herein refer to any process that eliminates (removes) or kills (deactivates) all forms of life and other biological agents. Sterilization can be achieved with one or more of the following: heat, chemicals, irradiation, high pressure, and filtration.

[0124] • The term “sterilized” as used herein refers to a product enclosed in a plastic film bag and exposed to sterilization methods which ensure the bag content is sterile. The product is supplied in said film bag(s) to the end-user ready to open and use. The end-user hereby avoids the troublesome heat sterilization similar to classical re-usable equipment processed in an autoclave.

[0125] • The term “sterile filter” as used herein refers to a porous membrane device where the membrane barrier exhibit pores of max 200 nm, nano-meter size. Said membrane prohibit biological matter to pass and hereby ensure that one side is kept under sterile conditions.

[0126] • The term “Stirred-Tank-Reactor” (STR) is a widely used expression and as used herein refer to a bioreactor or fermenter with a process “Container Volume” (CV) integrating an aeration device and agitation or mixing device(s) for forced exchange of nutrient and gas with the “micro-organisms” within said media. STR are manufactured predominantly from stainless steel and glass. • The term “Supernatant” or permeate as used herein principle are the clear liquid obtained after; 1 . density separation like in a centrifuge of a broth, 2. particle size separation after a set of membrane filters - the liquid broth cleaned from debris and micro-organism components for further cleaning processes.

[0127] • The term “suspension” or “suspended” as used herein refers to particles, artificial particles, micro-carriers, micro-organism depending on being preferably homogeneous suspended or mobilized in liquid (in the broth) in the STR or SUB or “process container”.

[0128] • The term “Tangential-Flow-Filter” (TFF) as used herein is a device which allow selectable components under pressure to pass from one liquid volume into another liquid volume crossing a “membrane” (Cross-Flow-Filter) eliminating some components to pass the membrane. Such membranes or filter devices may be based on screens, porous material sheets integrated into cassettes or shaped as flat or round plate, tubes, corrugated tubes or stacked hollow fibres into a cartridge.

[0129] • The term “top cover” as used herein refers to the upper, typically head plate, such as flat metal disc for the classical STR. The top cover is without limitation selected from the dominant Stainless Steel I glass STR design supplied by Sartorius, Applikon, Finesse, Broadley-James and app 20 other suppliers since the 70ties. Number of PG 13,5 ports available effected by the top cover diameter and in general being three to four ports. The top cover may take other shapes than the flat disc and be equipped with one or more ports.

[0130] • The term “TMP” or “Trans-Mem brane-Pressure” as used herein describes an excellent indicator of membrane fouling or performance. Accumulated cells, debris, particles on the membrane surface. TMP increases to compensate for the membrane fouling at constant flux.

[0131] • The term “true-continuous processing” as used herein describes equipment supporting a process delivering constantly a product with no fixed process termination.

[0132] • The term “valve” or a “valve device” as used herein refers to a fluid flow, mass flow regulating device or actuator with one or more inlets and one or more outlets. A valve will regulate, direct or control a mass flow, a pressure, a volume of a fluid (gases, liquids, media, broth) by opening, closing, or partially opening / closing or obstructing the area of a passageway. Pinch valves employ an elastic tubing or hose and an external moving actuator, device that directly press a part of the tubing walls against a fixed wall forcing the tubing walls together reducing the tube free opening. Valves may be controlled manually, pneumatically, electrically by external controlling means such as from a PLC. Valves are found as on / off valves or as controlling valves such as proportional valves with plunger position feedback allowing a controlled desired opening of a passageway between fully closed and fully open. A valve device may be a pump able to operate dynamically and proportionally regulate a liquid mass flow and or pressure such as a LASER guided diaphragm pump. A valve device may be a clamp compressing the tube wall against each other regulation the internal passage of a fluid.

[0133] • The term “Working Volume” (WV) as used herein refers to the media volume, process fluid, the broth in which a cultivation takes place inside a process container. Or relevant WV for a mixing process in a process container. Further the “head space volume” + Working Volume = Container Volume (CV) or Vessel Volume (W).

[0134] DETAILED DESCRIPTION

[0135] All process container such as SUBs or SUFs or SUMs used today operate in batch mode - the presented invention breaks the batch mode processing barrier by adding one or more of the following technologies:

[0136] I. operating two or more batch, fed-batch, perfusion SUB or SUF or SUM process containers in parallel one after the other controlled by a set of valves and a manifold creating a continuous harvest flow I supply from up-stream,

[0137] II. select a first separation process by one of: o reducing the required down-stream process capacity by multiple batch depth filters operating in parallel one after the other controlled by a set of valves and a manifold, o reducing the down-stream cost by exchanging the use of batch depth filters with a continuous processing centrifuge,

[0138] III. introducing a set of valves and a manifold combining one or more process containers with a broth I liquid conveying setup followed by multiple cleaning processes,

[0139] IV. pre-assemble, pre-sterilize (irradiated) all parts for much faster turnaround, The present invention creates the required continuous product flow from both upstream as well as half down-stream only limited by the number of manifold connections, pumps and valves.

[0140] The invented fully I true continuous up-stream cultivation I fermentation I mixing platform and methods in process containers do not exist as of today. The present invention combines multiple SLIBs or SLIFs or SLIMs arranged in rotating concept for a “broth train” I “liquid train” harvest. Cultivation or fermentation or mixing taking place one after the other (sequential operation in a rotating order) supplying down-stream with a constant product supply. SLIBs or SLIFs or SLIMs after having supplied the product may be re-used, repeatable used in a sterile manner.

[0141] What’s known as “semi-continuous processing” in SLIBs as of today is maxed out before typical 6-8 weeks. The max time the best surviving micro-organisms are allowed to support like mAbs cultivation. Where the duration is just increased from one week batch to 8 weeks perfusion. Hereby any semi-continuous process of today is far from the true Continuous Process. The present invention offers no time limits and hereby is a true Continuous Process.

[0142] The invented Process System offer several advantages:

[0143] 1 . Continuous operation, when based on a central manifold, allowing the end-user to repeat the cultivation, fermentation, mixing process even based on series batch operation. Re-use of the glass / steel or steel STR is business wise considered optimal. Re-use of a SUB or a SUF or a SUM after a first and intended only first successful batch run may not be considered optimal.

[0144] 2. Extent cell densities further than 10-15 mio cells / ml in batch to 10-30 mio cells / ml in fed-batch to 30-150 mio cells / ml in semi-fed-batch perfusion by adding fresh media into the process in the fluid tight containers,

[0145] 3. Introduce a continuous operating broth separation step right after the cultivation, fermentation, mixing,

[0146] 4. Reduce the use of and cost of the down-stream dept filters and membrane filter usage significantly, 5. Introduce a semi-continuous down-stream process before chromatography polishing,

[0147] 6. Furthermore, the production time can the extended compared to for example batch or fed batch processes. Also, as compared to a batch or fed-batch process, it is possible is use a set of smaller process containers. Use of smaller fluid tight process container is of advantage as this reduces the equipment and facility related investments.

[0148] Further benefits of the presented Process System offer end-users, freedom, lower cost of manufacturing and new features such as:

[0149] • Eliminate traditional time-consuming steam sterilization as the invention is preassembled, pre-sterilized with all necessary components ready for use right out of the box for lower cost of use and higher throughput in laboratories;

[0150] • Eliminate cross contamination as to the pre-assembled and pre-sterilized concept beneficial to both Research & Development as well as full scale production;

[0151] • Minimizes instalment time as the Process System integrate a SUP, a centrifuge or a filter system outside of multiple process container performing the rotating principle obtaining the desired Continuous Processing;

[0152] • Un-parallel control of broth velocities and broth harvest volume and cycles;

[0153] • Any process container such as a STR, a SUB, a SUF, a SUM is obtainable from CerCell A / S from Denmark who offer rigid plastics customized SUB, SUF, SUM products ranging in volumes from 0.5 to 50 liter. Foil based SUB, SUF “big bags” are available for a range of suppliers such as ABEC Inc, USA in volumes up to 6 m3,

[0154] • The presented Process System enables rapid evaluation of multiple microorganism cultures or mixing processes performance, increasing productivity with significant savings on materials and labour.

[0155] The pharmaceutical industry desires cultivation, fermentation and mixing platforms supporting increased volumetric efficiency, more early on-line analyst measures all in a single-use design with capability intended for semi-continuous operation. Micro-organisms like E. coli is an important species in the fields of biotechnology, where E. coli serve as the host organism in fermenters for the majority of work with recombinant DNA. E. Coli may require high pressure exposure to retrieve the product from inside of the E. Coli. Such intermediate step is not part of the present invention but to be inserted in the process step after the SUF.

[0156] The problem of blocking membrane filters (CFF and HFF) in semi-continuous biomaterial production systems is significant. Dominant perfusion systems suppliers have not yet solved this problem. Prohibiting successful introduction of bioprocess equipment based on the latest FDA acceptance of continuous perfusion for mAbs expression for lower cost of product.

[0157] The present invention solves the current drawbacks both by introduction a continuously operating separation device, like a centrifuge removing the predominant portion of solids without blockage and a method of using this centrifuge, both described herein. Said continuously operating separation centrifuge, can separate +95% of the solids above few pm size out of the broth completely reducing fouling, blockage of the first set of down-stream filter systems significantly.

[0158] The present invention relates to a Process System comprising a process container such as a SUB for cultivation or a SUF for fermenting of biomass (broth) or a SUM for mixing liquids creating mRNA and consist of selectable components (below briefly described) such as:

[0159] • A process container comprising a range of ports, mechanical connected with or an integrated part of a hollow plastic container (rigid plastics or made from foil) with an interior reservoir optionally with one or more side wall ports. The process container may have a range of selectable diameter or cross section (such as round, cylindrical or non-cylindrical such as square design) and heights offering a desired interior reservoir and respective volume;

[0160] • Optionally a draft tube stator arranged vertical inside said process containers operating with none or one or more baffles arranged perpendicular and radially to said draft tube stator exterior space said draft tube stator having an interior space housing fluid motion, agitation means. The interior reservoir fluid agitation device(s) create a typically radial swirl of media which the tube stator convert into axial fluid movement and more efficient mixing axial vortexes;

[0161] • Optionally a baffle arranged vertical inside said container operating as a SUB extending inwards from the container side wall;

[0162] • A fluid motion, agitating device arranged on a rotating shaft attached to component comprising magnets and a bearing in contact with the container bottom for magnetic power transfer through the bottom wall driven by external magnetic means;

[0163] • A fluid motion, agitation device arranged on a rotating shaft penetrating the container top via a bearing I sealing arrangements for kinetic power input;

[0164] • A tube with controlled holes, a porous sparger wall part arranged vertical inside said container with the purpose of supplying a mixed gas addition to insure for the micro-organisms safe aeration in selectable bubble sizes;

[0165] • A range of ports in the SUB, SUF for various inlet and outlets, sensors, etc. In particular a broth, liquid harvest outlet port;

[0166] • Fluid communication for the broth, liquid of the container and a first (possible a valve) manifold, by a pumping device, said manifold inlet port directs the broth via valves, aseptic connectors in order to facilitate the broth harvest;

[0167] • Single-Use-Pump devices, said SUP devises is a broth conveying I direction I volume control device in order to facilitate the pumping operation, the Clio diaphragm pumps from www.pumpcell.com operate also as a valve and is a massflow controlled pump able to convey a specific volume and thus leave like 5 % broth volume in the SUB of SUF for a possible process continuation;

[0168] • Broth communication from the SUP to the separation device (like a SUC) producing a concentrated retentate (for possible reuse and returned partly to a SUB or SUF following presses) and a liquid permeate for further down-stream processing;

[0169] • Alternatively the SUM communication of liquids is directed to a fist filtering step.

[0170] • The SUB and SUF process containers may further operate as reservoir for the broth to be harvested said broth volume, mass flow adapted to the SUC capacity, similar reservoirs may even out difference in fluid volumes before and after the SUC;

[0171] • Liquid permeate communication from the SUC into a second manifold, said manifold multiple outlet ports direct the liquid via valves, aseptic connectors (as needed) to; • Down-stream with selectable variable capacity for further clarifying, polishing steps, like multiple membrane filter, single-use Cross-Flow-Filter (CFF) devices in selectable use for the practical period until TMP is considered too high and the next in line and ready to filter is connected, replaced via valve devices;

[0172] Manufactured from materials considered to be disposable and supplied sterilized to the end-user as cost efficient single-use apparatuses.

[0173] The fluid tight process container (STR, SUB, SUF, SUM) provides an interior space for sterile operation with exterior walls.

[0174] Dimension of the rigid plastics process container, housing assembly range 10 to 500 mm in diameter and height from 10 to 1 .000 mm.

[0175] Dimension of the flexible plastic’s fluid tight process container, like a bag, range from few liters up to 2,000 liter or more.

[0176] The invented Process System further comprise a variety of additional arranged devices and features to expand the simple batch operation to the more advantageous fed-batch and even perfusion mode operation. Such as one or more; hollow bodies appearing as reservoirs, Cross-Flow-Filter devices, aeration devices, instrumentation, sensors, liquid conveying devices, valves, actuators, fluid accumulators and the like for regulating process means.

[0177] In addition, the invented Process System comprises, a fluid tight process container, a liquid conveying device, a pumping device and a broth separating device, and a membrane filter device for final small particle removal operating in perfusion mode.

[0178] Broth from the biomaterial producing fluid tight process container is conveyed by gravity or a first SUP to a first manifold providing multiple inlets leading the broth to the SUC or filter. Optionally various valves, sterile connectors, sterile filters, etc will allow an upgrade or variant of the manifold.

[0179] Sterile filters exist in many configurations and sizes. Though advantageous to mount as the barrier towards the environment surrounding the present invention. Fluid connections as well as transfer lines between parts of the present invention may require a sterile filter is present. Such sterile filter devices ensures the process environment is kept under sterile condition at all times.

[0180] Such broth separation device able to separate broth solids from liquid preferably operating in continuous mode. Which in many ways exclude membrane filters. In one aspect of the invention, a centrifuge for separating a broth into at least two components is provided. Such a centrifuge comprises a chamber (or processing chamber) and a rotating device for receiving the broth to be centrifuged and a means for controlling the progress of the broth separation located at the SUC chamber. The separation of the harvested broth results in at least a first component (solids) and a second component (liquids) that are separated from each other. The Revolutions-Per-Minute (RPM) of the rotating part expose the introduced broth for a desirable G-force and the increased gravity allow collection of the two fractions individually by internal design. Such centrifuges (preferable of single-use design) are available from Carr Biosystems in Florida, USA or from Alfa Laval in Sweden.

[0181] Permeate from the first and continuous operation capable separating device (SUC) is conveyed over a second manifold providing multiple outlets leading the permeate to one or more or a series of filter systems. Which provides the required separation specification of the task. The manifold after SUP exit provides multiple choices via valves to convey the liquid from the SUC to next set of down-stream process steps. Dept filter and or membrane filter according to the process requirements. By arranging the manifold prior to the filter steps and use pressure sensors to determine the pressure drop over the filter in use. One obtains a continuous down-stream operation mode. The PCS, PLC is able to determine the termination of one filter and introduction of the next filter in line. Hereby obtaining a continuous operating filtration system. Optionally various valves, sterile connectors, etc will allow an upgrade or variant of the manifold.

[0182] Depth filters are the typical fist down-stream process step with a thick, porous filtration medium retaining, capturing remaining solid particles suspended in the liquid, throughout the filter medium. The filter media pores may be progressive and arranged in a manner such that sections with a larger pore size are closer to the inlet path or port, capturing particles of a larger size and pore sizes decreases approaches. Depth filters have limited lifetime as to the block up with solids and as to such not for a continuous process. Highly advantageous to replace with a centrifuge and or a manifold, pressure sensors and PLC control for regular exchange allowing continuous operation.

[0183] Membrane filter, like a CFF, HFF may be manufactured from flexible semi-permeable flat material or as Hollow-Fibers bundled into a cartridge. Depth filters are not capable of continuous operation. Eventually over time depth principle filters will be blocked with solids and need replacement. Some membrane filters are able to operate semi- continuous in a setup where the collected particles creating a so-called filter cake can be removed mechanically by flushing or back flushing.

[0184] The CFF, HFF are arranged in liquid contact with the process liquid. Which allows at least a part of the expressed and desired protein product from the processes suspended in the liquid to pass over the membrane and hereby be converted to permeate, harvested product. The remaining suspended debris inside the CFF are collected on the membrane inlet side operating as a dead-end filter. Or the filter cake to be removed sequentially by flushing at controlled high fluid velocities or back flushing.

[0185] The present invented Disposable Process System supports numerous requirements and facilitate a variety of rigid or semi-rigid or flexible products used for fabrication of basic parts. Materials such as polymers to form one or more parts of the invention are, but not limited to materials such as; polycarbonates, polyesters, nylons, polyamide, PTFE resins and other fluoropolymers, acrylic and methacrylic resins and copolymers, polysulphones, polyethersulphones, polyarylsulphones, polystyrenes, polyetherimides, polyethylene terephthalates, polyvinyl chlorides, chlorinated polyvinyl chlorides, ABS and its alloys and blends, polyolefins, preferably polyethylenes such as linear low density polyethylene, low density polyethylene, high density polyethylene, and ultrahigh molecular weight polyethylene and copolymers thereof, polypropylene and copolymers thereof and metallocene generated polyolefins. Or partly, semi rigid materials such as silicone, rubber and otherwise elastic materials. In a first embodiment the invented Process System provides a series of fluid tight process containers manufactured from disposable materials suitable for microorganism cultivation or fermentation or mixing in a liquid media. The purpose of the present invention is to extent the use of broadly used one week batch SLIBs or SLIFs to months of continuous biological material manufacturing.

[0186] The key to the simplest setup is a set of manifolds, a continuous operating broth processing centrifuge unit (SUC) and a set of selectable multiple down-stream filter systems all controlled via valves by a PCS.

[0187] The manifolds may be equipped with a variety of valves, silicone hoses, aseptic connectors allow for connection of multiple SLIBs and SLIPs and filter system on both sides of the SUC.

[0188] Hereby the conventional and well proven batch cultivation or fermentation gives rise to the invented efficient continuous manufacturing. Completely avoiding the durability problems associated with semi-continuous perfusion associated with membrane fouling based on the US patent 06544424 from year 2000 by Shevitz. We recognise that Shevitz opened up the marked through a major jump from batch to perfusion.

[0189] In a second embodiment the invented Process System provides a fluid tight first process container according to the invention manufactured from disposable materials suitable for micro-organism cultivation in a liquid loop comprising various ports arranged on said first process container exterior wall for implementing sensors measuring process parameters. Further various tubes, hoses, ports pass though the outer wall of which some extending to and or from the first process container interior volume for various fluid communication with external devices. Such as a broth harvest outlet for connecting to a first SUP, multi batch continuous Process System with one SUB or SUF shown out of 8 optional SUBs or SUFs set as example by the broth manifold. The first diaphragm SUP convey broth from the SUB or SUF and pump to the first manifold. Second diaphragm SUPs convey broth to the inlet of the particle separation unit, and a third diaphragm SUP convey semi-cleaned permeate outlet directed to a triple liquid manifold port with 3 replaceable membrane filters for further clarification and TMP checked by pressure sensors. Retentate regarded as waste and discharged. The 8-inlet port manifold allow multiple SUBs or SUFs connected operation in a sequence suitable with respect to the process parameters and optimum use of the separation unit.

[0190] The “broth train” process start up comprises a first fluid tight process container. A second fluid tight process container upstart is delayed. A third fluid tight process container upstart is further delayed. A fourth fluid tight process container upstart further delayed and so forth. The time of broth harvest follows with intervals of delay every following fluid tight process container upstart. The “broth train” sequence is adjusted to ensure the broth harvest flow and the first cleaning process (SUC or a filter) capacity, and further downstream capacities is optimised for semi-continuous operation.

[0191] In a third embodiment the invented Process System provides a fluid tight first process container suitable for micro-organism perfusion mode operation comprising various ports for implementing sensors measuring various process parameters. And a broth exit port arranged on the first container exterior wall, a perfusion I semi-continuous setup in which the single SUB (more SUBs to be added around the manifold receives a part of the concentrated broth from the separation device, a centrifuge (SUC) with retentate outlet to a triple retentate manifold. The SUB may operate partly as fed-batch over 1-2 weeks with limited retentate return and fresh media addition after harvest. Or as single or multiple perfusion SUBs in parallel with fresh media addition each day. SUBs equipped with various sensors to measure various analytes; DO, pH, biomass, Glycose, Lactate, temperature, level, pressure. Agitation of fluids in the SUB via impellers driven by a servo motor. Liquid addition for pH control. Fresh liquid media inlet. Sample option via a tube into the media, broth. Close to bottom an outlet for broth harvest connected to a first SUP. The SUB broth outlet connected via a first SUP convey the broth to the SUC broth inlet. SUC outlet permeate pass a second SUP which convey the SUC clarified now liquid to a triple manifold equipped with aseptic connectors and valves followed by selectable multiple membrane filters for further product clarification. Membrane filter protected against fouling the very low solids content in the liquid retentate from the SUC. The concentrated broth retentate outlet from the SUC are divided into two streams. One optional broth part is returned to the SUB supporting the continuous cultivation, fermentation + new fresh media added to the SUB. A set of pressure sensors and manifold valves replace the first membrane filter with the second when eventually the TMP is too high. And one part of the concentrated broth is discharged as waste.

[0192] For a fed-batch SUB cultivation start with a desired volume of liquid media in order to covers at least sensors and agitation devices in the SUB + inoculated according to the desired recipe. When desired biomass and Working Volume (WV) has reached its potential, this first of the following fed-batch SUB “broth train” target is reached and the process is harvested to the invented Process System principle.

[0193] Practical process container sizes used in the present inventions is based on rigid plastic SUBs and SUFs and SUMs from Danish company www.cercell.com with dimension based on standard diameter ranging 82, 110, 137, 150, 200, 250, 300 mm. When combined with heights of 100, 250, 350, 450, 600 mm Vessel Volume sizes range from 0.5 to 50 liter is created.

[0194] The present invention also allows extended use of perfusion-SUBs designed for semi- continuous cultivation - Multiple Perfusion-SUBs, like from Danish www.perfusecell.com, with upstart and operating one after each other in the “broth train”. Offering vastly extended operation time with fresh cell line inoculation regularly for practically unlimited period of time, independent of the here presented invention operate a mix of batch, fed-batch and I or perfusion,

[0195] Possible process duration limitations are connection of further irradiated SUBs or SUFs and a filter system. The more port the manifold offers the more SUBs or SUFs and filter systems can be added. The are several methods to overcome such limitations. Blocking on a silicone hose can be performed by a valve or equipment adding force to and collapse a short stainless steel tube piece around the platinum cured silicone hose. Methods and equipment for welding thermoplastic silicone hoses are also common practise.

[0196] Practical operational cell density range 20 - 100 x 10E6 cells I millilitre preferably measured with an on-line biomass sensor available from Swiss company Hamilton. High cell density in general means higher productivity. Longer cultivation time means more throughput, less time spend in starting the cultivation per run means lower cost. A conventional batch process last one week plus days for cleaning and process restart. It takes 7-10 days of batch cultivation before the harvest process can start. Continuous processing time of 5-8 weeks is relevant with 5-10 times the cell density to batch and harvest starts after one week operation.

[0197] The preferred SUP in the embodiment is a drive gas operated diaphragm pump LASER sensor guided (US patent - US 10,288,060) for position determination of said elastic element. The diaphragm able to expand to a desired shape and I or return to a desired shape when exposed to drive gas pressure ranging from controlled vacuum to controlled overpressure. Movement of the flexible and I or elastic element with drive gas on one side and process liquid on the other side separates the non-sterile, nonwetted side with the wetted and sterile side. Furthermore, the SUP may take different shapes and be arranged anywhere outside as well as inside the container or part the rigid fluid tight process container wall or partially outside and inside on the container wall.

[0198] Methods currently in common use for after harvest separations include one or more of batch, fed-batch, intermittent, continuous, and continuous centrifugation, semicontinues depth filtration and membrane filtration. Historically, centrifuges for cell harvest of large volumes of cell culture at production scale are complex multiple use systems that require Clean-In-Place (CIP) and Steam-In-Place (SIP) technology to provide an aseptic environment to prevent contamination by micro-organisms. Recently lab scale and small-scale centrifuges for continuous operation processes have become available. These small-scale centrifuges are even based on presterilized, single-use fluid path components (SUC).

[0199] In a fourth embodiment the invented Process System provides optimized use of Single- Use-Mixer for mRNA manufacturing. Ultimately, the cost of mRNA is greatly influenced by the quantity of RNA per dose, production titres and production scale. Hereby continues manufacturing is highly desirable. In terms of upstream manufacturing for novel mRNA drug products the here presented scalable, flexible dynamic, ramping up capacity in simplified continuous Process System based on multi process containers is desirable. mRNA is a synthetic manufactured messenger-RNA or self-amplifying-mRNA and produced in a 1 or 2 step in-vitro synthesis through a recombinant or multistep enzymatic reactions between mixed liquids (typical Polymerase and a DNA template) followed by a multi-step purification platform including Tangential-Flow-Filtration and chromatography.

[0200] Typical upstream process starts by mixing Polymerase and a DNA template followed by TFF delivered to downstream being chromatography and multi-step TFF. mRNA is produced in a cell-free system and uses no animal derived raw materials. Cell-derived impurities or adventitious contaminations are thus absent, which makes the manufacturing of these molecules safe. The reaction mixture contains not only the desired product, but also a number of impurities. The typical batch process is composed of a 2-step enzymatic reaction in continues form, followed by enzyme recycling using tangential flow filtration strategies and two multimodal chromatography steps. One in bind-elute mode for the intermediate purification and a second in flowthrough mode for polishing. The removal of small size impurities can also be achieved while concentrating or diaf iltrating solutions by tangential flow filtration (TFF) Formulation is achieved using a third tangential flow filtration module.

[0201] The “liquid train” process starts up with a first fluid tight process container. A second fluid tight process container upstart is delayed. A third fluid tight process container upstart is further delayed. A fourth fluid tight process container upstart further delayed and so forth. The time of broth harvest follows with intervals of delay similar to every fluid tight process container upstart. The “liquid train” sequence is adjusted to ensure the SUM harvest flow and the first cleaning process (SUC or a filter) capacity, and further downstream capacities is optimised for continuous operation.

[0202] OPERATION METHODS

[0203] A method of operating the Process System producing a biological product cultivated biological material in a continuous process facilitating at least two process containers comprising a broth or liquid outlet, said SUBs filled with liquid media and microorganisms processing independently said biological material into a broth, each SUB operates independently in batch mode over 2-12 days. Each of at least two SLIBs is the “broth train” or “liquid train” in such a manner that one first SUB process biological material into a broth while one second SUB with processed and ready to harvest broth is emptied and said harvested broth conveyed via a manifold by a SUP. Said manifold comprise multiple valves and connectors for broth flow controlling purposes, said manifold connects components together into a circuit, said manifold convey a broth to a SUP. The SUB vessel further operates as a reservoir supporting a steady supply of broth volume controlled by the SUP conveyed to the SUC. Said SUP is a pump or a pneumatically operated diaphragm pump and controlled by a laser senor for diaphragm position said SUP able to operate both as a valve and a pump for regulation purposes as well as conveying broth to said SUC. Said SUC receives and process the broth by centrifugal forces into retentate (solids) and permeate (liquids) ready for further downstream processing. After the first SUB is partly emptied for broth new fresh media and biological material is added and the cultivation process repeated processing said biological material in fresh liquid media into a broth. At least two SUBs or multiple SUBs in the “broth train” is processing in batch cultivation mode and one or more SUBs in harvest mode organised for continuous SUC operation.

[0204] A method of operating a “broth train” Process System producing a biological product fermenting biological material in a continuous process facilitating at least two process containers I SUFs, comprising a broth outlet, said SUF filled with liquid media processing independently said biological material, micro-organism based, into a broth, each SUF operates independently in batch mode over 2-5 days. Said harvested broth conveyed via a manifold by a SUP, said manifold comprise multiple valves and connectors for broth flow controlling purposes, said manifold connects components together into a circuit, said manifold convey a broth to a SUP, said SUP is pneumatically operated and controlled by a laser sensor for diaphragm position said SUP able to operate both as a valve and a pump for regulation purposes as well as conveying broth to said SUC. The SUF vessel also operates as the reservoir for steady supply of broth volume controlled by the SUP conveyed to the SUC. Said SUC receives and process the steady broth volume received by centrifugal forces into retentate (solids) and permeate (liquids) ready for further down-stream processing, after the second SUF is emptied for broth new fresh media and biological material is added and the fermentation process repeated processing said biological material in fresh liquid media into a broth, at least one SUF or multiple SUFs in parallel batch fermentation mode and one or more SUFs in harvest mode organised for continuous SUC operation.

[0205] The Single-Use-Mixer as process container designed for mixing fluids for mRNA drug substance manufacturing is a relatively simple vessel with the drain, product harvest outlet being the most important for minimum dead volume.

[0206] The embodiments of the invented Process System are preferably connected to, controlled, operated by the Cronus-PCS from www.cronus-pcs.com. Integrating the process information into the process recipe controlling all the process variables:

[0207] • the PCS is connected to multiple sensors integrated in said process container and SUP and SUC from which the PCS continuously collects data of process variables.

[0208] • the Process System operational parameters are constantly altered by said PCS in communication with various actuators, motors, devices integrated internally in said process container, SUP, SUC or external devices to said Disposable Process System for process parameter control.

[0209] • The PCS software for process control examples are such as Lucullus from www.securecell.com in Switzerland or DeltaV distributed control system from www.emerson.com in USA.

[0210] The Process System benefit from interaction with latest PCS technologies and in particular from more advanced fully configurable PCS products from www.cronus- pcs.com. Able to comprise selectable controls for various In & Out channels such as:

[0211] • various electronic analogue as well as digital input channels for measuring, collecting data from a range of sensors.

[0212] • various pneumatic, gas, electronic analogue as well as digital output to a variety of actuators, motors, etc.

[0213] DESCRIPTION TO THE FIGURES

[0214] Figure 1 illustrate a multi batch true-continuous Process System with three out of eight optional process containers 1 , 2, 3 connected to a first broth manifold 4 with 8 inlets. The process containers in the examples are obtained from www.cercell.com. The process containers in the “broth train” are identical and equipped with various sensors 26 to measure various analytes; DO, pH, biomass, Glycose, Lactate, temperature, level, pressure. Agitation of fluids in the process containers via an impeller 5 driven by a servo motor 6 from the bottom. Liquid addition for pH control 7 and fresh liquid media inlet 8. Sample option 9 via a tube into the media, broth. Close to bottom an outlet 10 for broth harvest with a SUP 11 a with laser sensor 45. Second process containers 2 harvest port equipped with the SUP 11 b on process containers 2. Two diaphragm SUPs 12 and 13 convey harvest inlet to the separation unit 14 and SUP 13 with laser sensor 45 at permeate outlet liquid directed to second liquid manifold 15 follow by aseptic connecters 25, regulating valves 20 and 3 membrane filters 16, 17, 18 for down-stream clarification. Concentrated retentate outlet 19 originated from the SUC separation unit 14 regarded as waste. Operation of process containers 1 , 2, 3 and SUC 14 and manifolds 4, 15 with multiple valves 20, 21 in a sequence suitable with respect to the process parameters and for optimum use of the SUC separation unit 14. Sensors 22, 23, 24 measure pressure for system optimization.

[0215] Figure 2 illustrate a simplified multi batch true-continuous Process System with one process container 1 shown out of eight optional process container inlet ports by the manifold 4 design. The process container 1 is equipped with a harvest outlet valve 21 . The first of 6 illustrated diaphragm SUP 11 a convey harvested liquid from the process container 1 harvest port 10 via valve 21 and pump 11 a to the first manifold 4. Second diaphragm SUP 12 convey harvested liquid from the first manifold 4 to the separation unit 14, and a third diaphragm SUP 13 convey from separation unit 14 outlet directed to a second liquid manifold 15 with 3 replaceable membrane filters 16, 17, 18 for clarification and TMP checked by pressure sensors 22, 23, 24. Retentate from port 19 regarded as waste and discharged. The eight-port inlet manifold 4 allow multiple process containers 1 operation in a sequence controlled by one valve 21 on port 10 on each process container 1 suitable with respect to the process parameters and optimum use of a separation unit 14. Reservoirs and PCS not shown.

[0216] Figure 3 illustrate a perfusion I true-continuous principal setup in which the single process container 1 deliver broth from port 10 directly to mass flow controlling SUP 11 (more non illustrated process containers added to an eight-port manifold 4) said manifold 4 receives selectively the harvest liquid sequentially from each of eight process containers 1. From manifold 4 outlet SUP 12 receives and deliver to the separation device 14 of which retentate outlet to a SUP 13 convey liquid to retentate manifold 15. The SUB 1 may operate partly as fed-batch over 1 -2 weeks with no retentate return and fresh media addition to reach appropriate VW from which the “broth train” process can start. Said process containers equipped with various sensors to measure various analytes; DO, pH, biomass, Glycose, Lactate, temperature, level, pressure all interacting with the PCS. Agitation of fluids in the process container via impeller 5 driven by a servo motor 6 from the bottom. Liquid addition for pH control 7 and fresh liquid media inlet 8 supplied by peristaltic pumps 29. Sample port 9 via a tube connects to the media, broth inside process container 1 . Close to bottom outlet port 10 for broth harvest connected to a first SUP 11. The process container 1 broth outlet 10 connected via a first diaphragm SUP 11 with laser sensor 45 convey the broth over the manifold 4 to the SUP 12 and separation device 14 broth inlet. The separation unit 14 outlet permeate pass a second diaphragm SUP 13 which convey the by the clarified liquid to manifold 15 equipped with three aseptic connectors 25 and valves 20 followed by selectable multiple membrane filters 16, 17, 18 for further product clarification. The concentrated broth retentate outlet from the separation unit 14 pass a SUP 26 and divided into concentrate return to the process container 1 and discharge

[0217] 19 controlled by two valves 27, 28. One broth part is return to the process container 1 via valve 27 supporting the continuous cultivation, fermentation + new fresh media added to the process container 1 via port 8. Second concentrated broth outlet 19 and valve 28 is considered discharged. A set of pressure sensors and three manifold valves

[0218] 20 replace the first membrane filter 16 with the second set 17 and third 18 according to when the TMP is too high. Reservoirs even out various volumes with various system capacities and PCS not shown. Pressure sensors 22, 23, 24, interact with the PCS.

[0219] Figure 4 illustrates a fully functional true-continuous up-stream Process System including half the down-stream separation process and the customizable Process- Control-System. Multiple process containers, here four process containers in principle shown 1 , 2, 3, 4, out of the option of six process containers as the chosen manifold 4. Around the manifold 4 a series of pumps 11 a,b,c,d, 12, convey the broth into the manifold 4 and 12 into the centrifuge SUP 13 convey the purified liquid to the manifold 15 followed by aseptic connecters and valves 20 and membrane filters 16, 17. The first process container 1 (2nd, 3rd, 4this identical) equipped with various sensors 26 for DO, pH, biomass, Glycose, Lactate, temperature, level, pressure. Agitation of fluids in the process container 1 via an impeller 5 driven by a servo motor 6 from the bottom. Liquid addition for pH control 7. Fresh liquid media inlet 8 via an aseptic connector 25. Sample option 9 via a deep tube into the media, broth. Close to bottom an outlet 10 for liquid harvest to first SUP 11 .

[0220] The manifold 4 has in this example six inlet and one outlet. Outlet connected to second SUP 11 b connected to the centrifuge 14 broth inlet. The centrifuge 14 has a retentate outlet valve 28 for concentrated broth to be dumped 19. SUC 14 permeate pass SUP 13 which convey the clarified now liquid to second manifold 15 equipped with aseptic connectors 25 and valves 20 followed by membrane filters 16, 17 for product clarification and outlet ports via aseptic connectors 25.

[0221] The figure illustrates the invented Process System controlled by the latest technology Process-Control-System (PCS) comprising multiple individual PLCs for advanced process control. The illustrated PCS is configurable and as such differs from any PCS on the market. The illustrated Cronus-PCS (www.cronus-pcs.com) is based on multiple individual devices I units (# 30 to 39) designed for one specific purpose and each integrating a mini PLC. Combined the Cronus-PCS units becomes the customizable PCS platform for the desired purpose. Maximum freedom allowing the end-user to select software for input and output according to requirements, such as:

[0222] • The Sipylus 35 sensor unit which is a 6-channel configurable sensor module. One for each process container 1 , 2, 3, 4 is required,

[0223] • The first SUP drive unt Clotho 38, 39 units are dual channel for the laser guided SUPs 11 s, 12, 13 based on drive gas supplied by pneumatic pressure below and above atmospheric pressure source 44. The SUPs 11 s, 12, 13 integrates each a laser sensor 45 for accurate diaphragm position sensing. The diaphragm SUPs 11 s, 12, 13 are driven by drive gas supply from Clotho 38, 39,

[0224] • The Cilix 36 servo motor drive unit is a dual channel intelligent BLDC (Brush-Less- Direct-Current) or STEP electronically motor drive for motor 6. Both channels for agitation by impeller 5 and multiple process containers for agitation to be driven by further Cilix 36 units, • The Harmonia unit 33 unit is a dual channel intelligent thermal control of electrical heating elements and systems,

[0225] • The Camus Mass-Flow-Controller unit 37 is a dual channel quadruple gas channel mass-flow controller regulating gas composition and mass for each of the SLIBs,

[0226] • The second Clotho 39 unit is similar to 38 and a PLC driven dual diaphragm SUP for media and other liquids control,

[0227] • The HERA unit 34 family is a portfolio of stand-alone peristaltic pumps of different capacity including each a PLC and electronically controlled driver and motors such as peristaltic pump 29 for base liquids for pH control,

[0228] • The Cassiopeia unit 32 is an intelligent multi-channel intelligent LAN 43 switch including both a Linux operating CPU and a DHCP (Dynamic Host Configuration Protocol) server able to deliver IP (Internet Protocol) numbers facilitating easy connection to any Cronus-PCS (# 30 to 39) unit and additional process control 31 unit such as for the SUC and multiple pinch valves 20.

[0229] The entire set of Cronus-PCS and the SUC control 31 connected via LAN 43 and switches to a PC 30 running a selectable process controlling software like Lucullus or DeltaV via OPC-UA software platform. The figure is principal, and the single page is not enough to illustrate the total setup. No reservoirs are shown, no connection from pressure sensors 22, 23, 24 is shown, connections to some sensors not shown.

[0230] The following table illustrates the “broth train” or “liquid train” process principle in which process containers specific for the purpose is prepared and started and introduced into and after the “broth train” or “liquid train” principle. Each process container harvested one after each other, potentially re-introduced back into the “broth train”. The principle works equally well for cultivation or for fermentation or for mixing based on relevant equipment and time intervals between process container start up and harvest.

[0231] The table illustrates the “broth train” principle, example of the Process System based on fermentation in fluid tight process containers. For cultivation in fluid tight process containers the number of days is increased with a factor 2-5. For mixing of liquids for mRNA manufacturing the process time is even shorter compared to fermentation.

[0232] Multiple process containers arranged for fermentation operation partly one after each other in such a manner that (2-day fermentation duration example) the “broth train” harvest volumes appear in a true-continues manner for relatively steady harvest volumes adapted to the SUC (single SUC in the example) capacity:

[0233] I. one first process containers process biological material into 25 liter batch broth starts day 1 ,

[0234] II. one second SUF in a similar process starts day 2,

[0235] III. day 3 - 95% of the first process containers broth volume is harvested and conveyed to following steps and the SUC according to the SUC capacity for processing and conveyed further into downstream, fresh sterile media is added and the 5% remaining broth containing micro-organism re-establish fermenting condition and continues,

[0236] IV. day 4 - 95% of the second process containers broth volume is harvested and conveyed to following steps and the SUC according to the SUC capacity for processing and conveyed further into downstream, fresh sterile media is added and the 5% remaining broth containing micro-organism re-establish fermenting condition and continues,

[0237] V. day 5 - 95% of the first process containers broth volume is harvested and conveyed to following steps and with a volume per time to the SUC according to the SUC capacity for processing and conveyed further into downstream, fresh sterile media is added and the 5% remaining broth containing micro-organism re-establish fermenting condition and continues,

[0238] Hereby the true-continuously “broth train” or “liquid train” offer the desired product at affordable investments. Number of process containers and their capacities adjusted to one or more SUC capacities. Reservoirs arrange appropriately reduce potential sequentially larger volume adjustments to more steady flow conditions.

[0239] In general, the presented “broth train” Process System design, choices of components, assembling is unlimited and illustrated with a configurable PCS package associated with the presented true-continuous Process System embodiment. Depending on the micro-organism culture or mixing platform chosen the here presented true-continuous Process System is a cost-efficient solution involving significant reduced investment in up-stream facilities - compared to Stainless Steel and big bag systems. And lower investment in particular beneficial for the down-stream cleaning now not forced to operate in sequential batch mode, but in semi-continuous mode.

[0240] While the present invention has been described in connection with particular embodiments thereof, it will be understood by those skilled in the art that many changes and modifications may be made without departing from the scope of the invention as defined by the appending claims.

Claims

We claim:1 . A Process System for processing a biological material comprising: a) at least two process containers adapted for processing the biological material each container comprising an inlet port adapted for providing a liquid comprising a biological material to the process container and an outlet port adapted for providing a liquid harvest, wherein each process container comprises a valve device controlling liquid flow out of the outlet port, b) a manifold comprising at least two liquid inlet ports wherein each inlet port is in liquid communication with the outlet port of each container, and at least one liquid outlet port, c) at least one first electronical controlled pumping device adapted for conveying liquid from the first process container and at least one second electronical controlled pumping device adapted for conveying liquid from the second process container, d) at least one third electronical controlled pumping device adapted for conveying liquid from the at least one liquid outlet port of the manifold, and e) a separation device comprising a liquid inlet port in liquid communication with the at least one liquid outlet port of the manifold, and at least one liquid outlet port, wherein said separation device is adapted for separating the liquid harvest into retentate and into permeate conveyed via the at least one liquid outlet port.

2. The process System according to claim 1 wherein the valve device controlling liquid flow out of the outlet port of the process container is a mass flow controlling device.

3. The process System according to claim 1 or 2 wherein the at least one first electronical controlled pumping device is a fluid pumping device comprising a first inlet port in communication with the outlet port adapted for providing a harvest liquid of the first process container, and a second outlet port in communication with a first liquid inlet port of the manifold.

4. The process System according to anyone of claims 1 -3 wherein the at least one second electronical controlled pumping device is a fluid pumping device comprising a first inlet port in communication with the outlet port adapted for providing a harvest liquid of the second process container, and a second outlet port in communication with a second liquid inlet port of the manifold.

5. The process System according to anyone of claims 1 -4 wherein the at least one third electronical controlled pumping device is a fluid pumping device comprising a first inlet port in communication with the at least one liquid outlet port of the manifold and a second outlet port in communication with the liquid inlet port of the separation device.

6. The process System according to anyone of claims 1 -4 wherein the at least one third electronical controlled pumping device is a fluid pumping device comprising a first inlet port in communication with the liquid outlet port of the separation device and a second outlet port adapted for conveying the permeate from the liquid outlet port of the separation device.

7. The process System according to anyone of claims 1 -6 wherein the separation device comprises two liquid outlet ports, one first liquid outlet port for conveying retentate and one second outlet port for conveying permeate.

8. The process System according to anyone of claims 1 -7 further comprising a Process-Control-System (PCS) comprising multiple analogues and / or digital input channels collecting data from multiple sensors and acting via multiple analogues and / or digital output channels controlling the Process System activities and selecting the process containers outlet valves.

9. The process System according to anyone of claims 1 -8 wherein the separation device is an electronically controlled centrifuge.

10. The process System according to anyone of claims 1-9 wherein each of the process containers is equipped with an agitation device.

11. The process System according to anyone of claims 1-10 further comprising a second manifold comprising a liquid inlet port adapted to receive permeate liquid from the at least one liquid outlet port of the separation device, and at least two liquid outlet ports.

12. The process System according to anyone of claims 1 -11 wherein an electronical controlled pumping device operates in a closed loop guided by a laser sensor which output signal is forwarded to the PCS and said activity controlled by said PCS.

13. The process System according to anyone of claims 1 -12 wherein the at least one electronical controlled pumping device activity is controlled by a PCS and a program stored in a memory of said PCS.

14. The process System according to anyone of claims 1-13 further comprising a downstream operation unit facilitating the permeate conveyed via the at least two liquid outlet port of said second manifold is subjected to further down-stream process steps to provide the processed biological material.

15. The Process System according to anyone of claims 1-14 for continuous processing of biological material contained in a liquid media comprising at least two fluid tight process containers operating sequentially in parallel each with a liquid media volume processing said biological material into a broth independently comprising micro-organisms suspended in said liquid media volumes.

16. The Process System according to anyone of claims 1-15 for continuous processing of biological material contained and processed in a liquid in one or more fluid tight process containers comprising one or more of: h. an agitation devise,i. an inlet port connected to a liquid supply, j. a port connected a sensor, k. an inlet port connected to a gas supply, l. an outlet port for product harvest, m. an outlet port general purposes gas exchange, n. an external assembly of hoses and connectors, wherein said fluid tight process containers are customized to support biological activity of a biological material in a liquid.

17. The Process System according to anyone of claims 1 -16 for continuous processing of biological material contained and processed in a liquid comprising one or more over-moulded silicone manifold systems conveying said liquid comprising one or more ports conveying said liquid or for connection of a sensor.

18. The Process System according to anyone of claims 1-17 for continuous processing of biological material contained and cultivated in a liquid media, comprising an electronically controlled centrifuge for separation of solids from said broth liquids into retentate (solids) and permeate (liquids).

19. The Process System according to anyone of claims 1 -18 for processing of biological material contained in a liquid characterised in that means for process control is performed by a customizable Process-Control-System (PCS), said PCS is characterized by multiple individual PCS devices selected from one or more of: f. a device comprising multiple configurable electric inlets and outlets for process control and data collection and a Programmable-Logic-Control (PLC) with process software stored in memory, g. a device comprising multiple configurable inlets and outlets for process control and data collection and a Programmable-Logic-Control (PLC) with process software stored in memory, h. an electrical inlet device connected to a LASER sensor for process control and a Programmable-Logic-Control (PLC) with process software stored in memory, i. a computer and display being a Human-Machine-Interface (HMI) for data collection and presentation,j. a Local-Area-Network (LAN) device connecting the above devices, wherein said Process-Control-System comprises multiple analogue and digital input channels for measuring, collecting data from a range of sensors, and various pneumatic, gas, electronic analogue as well as digital output to a variety of actuators, motors, etc.

20. A method of operating a Process System according to anyone of claims 1-19 converting a liquid biological material into a continuous liquid harvest flow facilitating: g. at least two fluid tight process containers each comprising a liquid inlet and a liquid outlet, said fluid tight process containers filled with a liquid processing said liquids independently into a liquid product, h. each fluid tight process container operates in batch mode sequentially, one fluid tight process container after each other arranged in such a manner that one second fluid tight container process biological material into a liquid while one first fluid tight container with processed and ready to harvest liquid is emptied and said harvested liquid conveyed to a manifold via a valve device and or a pump, i. each process container liquid outlet comprises a mass flow regulating valve device and or a pump device controlling mass flow from said process container to said manifold, j. said manifold comprise multiple connectors for sequentially liquid product inlet flow from multiple process containers, said manifold connects multiple process containers and pumps and components together into a Process System circuit, said manifold convey said liquid from multiple batch process containers via a continuous liquid outlet flow port to a centrifuge, k. said manifold convey liquid to said centrifuge in a continuous mode, l. said centrifuge receives and process the liquid product dynamically and continuously by centrifugal forces into retentate (solids) and permeate (liquids) ready for further down-stream processing.

21. A method of operating a Process System according to anyone of claims 1-19 converting a liquid biological material into a continuous liquid harvest flow facilitating: g. at least two fluid tight process containers each comprising a liquid inlet and a liquid outlet, said fluid tight process containers filled with a liquid processing said liquids independently into a liquid product, h. each fluid tight process container operates in batch mode sequentially, one fluid tight process container after each other arranged in such a manner that one second fluid tight container process biological material into a liquid while one first fluid tight container with processed and ready to harvest liquid is emptied and said harvested liquid conveyed to a manifold via a valve device and or a pump, i. each process container liquid outlet comprises a mass flow regulating valve device and or a pump device controlling mass flow from said process container to said manifold, j. said manifold comprise multiple connectors for sequentially liquid product inlet flow from multiple process containers, said manifold connects multiple process containers and pumps and components together into a Process System circuit, said manifold convey said liquid from multiple batch process containers via a continuous liquid outlet flow port to a first stage filter system, k. said manifold convey liquid to said filter system in a continuous mode, l. said filter system receives and process the liquid product continuously by separation solids (retentate) and permeate (liquids) ready for further downstream processing.

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